Anti-CD74 antibody, antibody-drug conjugate, and preparation method therefor and use thereof

Abstract

Provided are an anti-CD74 antibody or an antigen-binding fragment thereof, a preparation method therefor and the use thereof, and an antibody-drug conjugate, and a preparation method therefor and the use thereof. The anti-CD74 antibody can specifically bind to CD74. The antibody-drug conjugate has application prospects in the treatment of immune diseases.

Description

Anti-CD74 antibodies, antibody-drug conjugates, their preparation methods and uses Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an antibody that specifically binds to CD74 or its antigen-binding fragment, its preparation method and uses, and also to an antibody-drug conjugate, its preparation method and uses. Background Technology

[0002] CD74 is a non-polymorphic type II transmembrane glycoprotein involved in promoting B cell growth and survival, participating in the physiological processes of various immune system cells, and mediating the development of many diseases, including autoimmune diseases, tumors, and metabolic diseases. Under normal circumstances, the immune system is in a state of immune tolerance to self-antigens. In patients with systemic lupus erythematosus (SLE), excessive apoptotic products are not cleared in time, or there is a defect in apoptosis clearance. Apoptotic products and neutrophil traps bind to nucleic acid sensors such as Toll-like receptors (TLRs), activating plasmacytoid dendritic cells, which secrete type I interferon. Type I interferon further activates lymphocytes. With the help of T cells, B cells differentiate into plasma cells, producing autoantibodies. Autoantibodies bind to self-antigens, forming immune complexes that deposit in tissues, activating complement, promoting the expression of chemokines and cytokines, recruiting more inflammatory cells to infiltrate, and activating neutrophils, lymphocytes, and monocytes / macrophages, causing tissue inflammation and ultimately leading to organ damage. T lymphocytes and B lymphocytes play crucial roles in the pathogenesis of SLE. Apoptotic and damaged cells, acting as antigens, are presented to T cells via antigen-presenting cells, leading to the production of various cytokines such as TNF-α, IL-6, IFN-γ, and IL-10. These cytokines and CD40L activate autoreactive B cells, producing autoantibodies, a hallmark of SLE. These autoantibodies cause organ damage through immune complex deposition, complement and neutrophil activation, and altered cell function, resulting in apoptosis and cytokine production. Furthermore, autoreactive B cells stimulated by autoantigens in SLE cannot be easily eliminated, and B cells can also function as antigen-presenting cells, presenting intrinsically soluble antigens to T cells, thereby activating T cells. This mutual activation of B cells and T cells leads to further autoimmune diseases. CD74 regulates the development of T and B cells, dendritic cell (DC) motility, macrophage inflammation, and thymic selection. CD74 plays a vital role in the pathogenesis of SLE, and there is potential for developing SLE therapeutics targeting CD74.

[0003] Macrophage migration inhibitory factor (MIF) is a key cytokine closely related to autoimmune and inflammatory diseases, attracting activated immune cells from the surrounding tissue to inflamed tissue. CD74, as a high-affinity receptor for MIF, can mediate its biological effects. Lupus nephritis is one of the most common clinical manifestations of SLE, and the MIF-CD74 signaling pathway is involved in its pathogenesis. Renal tubular epithelial cells express CD74 on their surface and also secrete low levels of MIF. In the event of acute kidney injury, the expression of MIF and CD74 increases to meet the inflammatory response at the site of injury. CD74 is widely expressed on myeloid antigen-presenting cells and B cells associated with the pathogenesis of SLE and can be internalized in large quantities with high internalization efficiency. Conjugation of CD74 antibodies to radionuclides or other cytotoxic agents provides the possibility of directly delivering drugs to the target site, thereby limiting the exposure of normal tissues to toxic agents and improving treatment efficiency.

[0004] Currently, there are no marketed monoclonal antibody drugs targeting CD74, and few antibodies are in clinical development. Milatuzumab (hLL1) is a humanized IgG1κ antibody that reacts with the cell surface epitope of human CD74. Multiple preclinical studies have shown that Milatuzumab can lead to the generation of free radical oxygen and loss of mitochondrial membrane potential, as well as reduce CD20 / CD74 aggregates and cell adhesion, thereby inhibiting B cell growth and survival. Clinically, Milatuzumab is used to treat multiple myeloma, but its Phase II clinical trial has been terminated due to insufficient efficacy. Therefore, there is still a need to develop monoclonal antibody drugs with high activity and efficacy targeting CD74, or to develop antibody-drug conjugates using monoclonal antibody drugs.

[0005] Glucocorticoids are steroidal compounds, a type of adrenocortical hormone, that regulate genes involved in development, metabolism, and immune responses in the human body. Physiological doses of glucocorticoids play important regulatory roles in the body, such as raising blood glucose, promoting protein breakdown, and retaining sodium while excreting potassium. Exceeding physiological doses, glucocorticoids exhibit a wide range of pharmacological effects, particularly in anti-inflammatory, immunosuppressive, and anti-shock effects. Glucocorticoids cross the cell membrane into the cytoplasm and bind to glucocorticoid receptors (GR) to form hormone receptor complexes. These complexes are then activated and enter the cell nucleus, where they regulate gene transcription through direct or indirect pathways. In the direct pathway, the hormone receptor complex binds to the GR response element (GRE) on DNA, initiating or inhibiting the transcription of related response genes' mRNA, such as inhibiting the transcription of genes like TNF-α, IL-2, and IL-6, and initiating the transcription of IκB (which can inhibit NF-κB activation) mRNA. The indirect pathway mainly exerts anti-inflammatory and immunosuppressive effects through interactions with other transcription factors, such as binding to transcription factors like NF-κB and AP, preventing them from binding to target genes and inhibiting gene expression.

[0006] Synthetic glucocorticoids are a class of potent small-molecule drugs used to treat inflammatory and immune diseases. Currently marketed and widely used glucocorticoids include prednisone, prednisolone, betamethasone, dexamethasone, fluticasone propionate, and budesonide. Glucocorticoids can be used to control inflammatory and allergic diseases such as systemic lupus erythematosus, asthma, rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, long-term supraphysiological doses can lead to various side effects, such as damage to the cardiovascular system, central nervous system, endocrine system, eyes, gastrointestinal tract, skeletal muscle, and skin. These side effects limit the dosage and duration of use for patients, and their therapeutic potential needs to be further explored. Using monoclonal antibodies to prepare monoclonal antibody-glucocorticoid conjugates can, on the one hand, reduce the frequency of administration and improve the comfort of patients with autoimmune diseases; on the other hand, the high targeting specificity of antibodies can be used to achieve precise drug delivery, reducing the dosage of glucocorticoids and mitigating the side effects associated with their use.

[0007] AbbVie disclosed an immunoconjugate of a glucocorticoid receptor agonist (US20220354959A1). Glucocorticoids have a hydrophobic steroid nucleus structure, and this conjugate has undergone hydrophilic modification on the steroid nucleus. However, its linker-payload molecule still requires organic solvents for solubilization when conjugating with antibodies, and the resulting conjugate has a low DAR value and unsatisfactory product homogeneity. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, one object of the present invention is to provide an anti-CD74 antibody that has a strong binding force to CD74 and excellent endocytic ability.

[0009] Another objective of this invention is to provide an antibody-drug conjugate that has the advantages of being able to be coupled in a pure water system without organic solvents, having a high DAR value, good uniformity, excellent biological activity, good targeting, and being easy to prepare.

[0010] The first aspect of the present invention provides an anti-CD74 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein,

[0011] The heavy chain variable region includes VH CDR1, VH CDR2 and VH CDR3 as shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 respectively; and the light chain variable region includes VL CDR1, VL CDR2 and VL CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 respectively.

[0012] In some embodiments of the present invention, the anti-CD74 antibody or its antigen-binding fragment is of animal origin, such as mouse origin, or is a chimeric antibody; more preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:1, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:5, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5.

[0013] In some embodiments of the invention, the anti-CD74 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, and its heavy chain variable region further comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising a human IgG1 or IgG4 heavy chain FR region. The light chain variable region further comprises a human light chain FR region.

[0014] In some embodiments of the present invention, the anti-CD74 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 9 to 13, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 9 to 13, and / or the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 14 to 16, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 14 to 16.

[0015] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:9, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:9, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:14.

[0016] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:9, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:9, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:15, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:15.

[0017] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:9, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:9, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:16, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:16.

[0018] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:10, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:10, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:14.

[0019] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:10, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:10, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:15, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:15.

[0020] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:10, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:10, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:16, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:16.

[0021] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:11, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:11, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:14.

[0022] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:11, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:11, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:15, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:15.

[0023] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:11, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:11, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:16, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:16.

[0024] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:12, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:12, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:14.

[0025] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:12, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:12, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:15, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:15.

[0026] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:12, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:12, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:16, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:16.

[0027] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:13, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:13, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:14.

[0028] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:13, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:13, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:15, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:15.

[0029] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD74 antibody or its antigen-binding fragment is as shown in SEQ ID NO:13, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:13, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:16, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:16.

[0030] In some embodiments of the invention, the anti-CD74 antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, and / or further comprises a light chain constant region of human κ, λ chains or variants thereof.

[0031] In some embodiments of the present invention, the antigen-binding fragment is selected from Fab, Fv, scFv, and F(ab'). 2 , dsFv and dAb.

[0032] A second aspect of the present invention provides a biomaterial, which may be:

[0033] (1) A nucleic acid molecule that encodes the anti-CD74 antibody or its antigen-binding fragment as described above; preferably, the nucleic acid molecule is a DNA molecule or an RNA molecule; those skilled in the art can deduce the DNA or RNA sequence encoding the amino acid sequence based on the anti-CD74 antibody or its antigen-binding fragment disclosed in this application, and set appropriate expression elements for it so that the DNA or RNA molecule can express the antibody or its antigen-binding fragment of the present invention.

[0034] (2) A vector containing nucleic acid molecules as described above; preferably, the vector is an expression vector;

[0035] (3) Host cells containing nucleic acid molecules or vectors as described above.

[0036] A third aspect of the present invention provides a method for preparing the anti-CD74 antibody or its antigen-binding fragment thereof, comprising:

[0037] (1) Chemical synthesis method: prepared by synthesizing the anti-CD74 antibody or its antigen-binding fragment according to the present invention;

[0038] (2) Bio-preparation method: culturing host cells as described above; further comprising isolating antibodies from the obtained culture; and purifying the antibodies.

[0039] A fourth aspect of the invention provides a composition comprising the antibody or antigen-binding fragment thereof of the invention, or biological material. Preferably, the composition is a pharmaceutical product, further comprising a pharmaceutically acceptable carrier or excipient.

[0040] The fifth aspect of the invention provides the use of the anti-CD74 antibody of the invention or its antigen-binding fragment, or biological material, or composition thereof, in the preparation of a medicament for treating immune diseases.

[0041] In another aspect, the present invention also provides a method for preventing or treating immune diseases, the method comprising administering to a subject a preventive or therapeutically effective amount of an anti-CD74 antibody or its antigen-binding fragment as described in any of the preceding claims, or a biological material, or a composition.

[0042] In another aspect, the present invention also provides an anti-CD74 antibody as described in any of the preceding claims, or an antigen-binding fragment thereof, or a biological material, or a composition thereof, for use as a medicament. In one embodiment, it is used as a medicament for treating immune diseases.

[0043] In some embodiments of the present invention, the immune disease is selected from liver fibrosis, type 1 diabetes, systemic lupus erythematosus, and Alzheimer's disease.

[0044] A sixth aspect of the present invention provides an antibody-drug conjugate with a structure as shown in formula (I) or formula (I'), or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate thereof.

[0045] Wherein, Ab represents anti-CD74 antibody or its antigen-binding fragment;

[0046] L1 represents the connector unit;

[0047] L1' represents the extension unit;

[0048] L3 represents the connection unit;

[0049] D represents a drug unit containing glucocorticoid drugs;

[0050] n represents an integer or decimal number from 1 to 8;

[0051] L2 represents the structure shown in equation (Ⅱ):

[0052] In formula (II), A1 and A2 independently represent -C(O)-, -NR1-, -NR1C(O)-, -C(O)NR1-, -O- or -S-;

[0053] La represents a C1-C6 straight-chain or branched alkylene group substituted by Lb (the Lb mentioned here is the Lb shown in formula (II), that is, La is substituted by only one Lb and the substitution relationship is shown in formula (II)).

[0054] Lb represents an unsubstituted or substituted C2-C12 straight-chain or branched alkylene group, wherein one or more -CH2- can optionally be replaced by one or more of -C(O)-, -NR1-, -NR1C(O)-, -C(O)NR1-, -C(O)O-, -OC(O)-, -O-, -S-. When Lb represents a substituted group, the substituent is selected from one or more of halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy.

[0055] B indicates

[0056] R1 represents hydrogen or C1-C6 alkyl;

[0057] 1 indicates connection to L1 or L1', and 2 indicates connection to L3.

[0058] In some embodiments of the present invention, Ab may represent an anti-CD74 antibody or its antigen-binding fragment as described above.

[0059] In some embodiments of the present invention, in formula (Ⅱ), A1 and A2 each independently represent -C(O)- or -NH-;

[0060] La represents a C1-C4 straight-chain or branched alkylene group substituted by Lb;

[0061] Lb represents -Lc-NHC(O)-Ld- or -Lc-C(O)NH-Ld-, where Lc is connected to La and Ld is connected to B. Lc represents a straight-chain or branched alkylene group from C2 to C6, and Ld represents an unsubstituted or amino-substituted straight-chain or branched alkylene group from C2 to C4.

[0062] B indicates

[0063] In some preferred embodiments, L2 represents one of the following structures:

[0064] 1 indicates connection to L1 or L1', and 2 indicates connection to L3.

[0065] In some embodiments of the present invention, L1 represents -A3-Le-A4-, A3 is connected to Ab, and A4 is connected to L2 or L1';

[0066] A3 indicates 1 indicates connection to Ab, 2 indicates connection to Le;

[0067] Le represents a C1-C6 straight-chain or branched alkylene group;

[0068] A4 represents -C(O)-, -NR1-, -NR1C(O)-, or -C(O)NR1-;

[0069] R1 represents hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C4 alkyl.

[0070] In some preferred embodiments, L1 represents one of the following structures:

[0071] 1 indicates connection to Ab, and 2 indicates connection to L2 or L1'.

[0072] In some embodiments of the present invention, L1' represents the structure shown in equation (Ⅲ):

[0073] In formula (Ⅲ), A5, A6, and A7 each independently represent -C(O)-, -NR1-, -NR1C(O)-, or -C(O)NR1-;

[0074] Lf represents a C1-C6 straight-chain or branched alkylene group substituted by Lg;

[0075] Lg represents an unsubstituted or substituted C1-C12 straight-chain or branched alkylene group. When Lg represents a substituted group, the substituent is selected from one or more of halogen, hydroxyl, cyano, amino, C1-C6 alkyl, halo-C1-C6 alkyl, and C1-C6 alkoxy.

[0076] R1 represents hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C4 alkyl;

[0077] 1 indicates connection to L1, and 2 indicates connection to L2.

[0078] In some preferred embodiments, in formula (Ⅲ), A5, A6, and A7 each independently represent -C(O)- or -NH-;

[0079] Lf represents a C1-C4 straight-chain or branched alkylene group substituted by Lg;

[0080] Lg represents an unsubstituted C1-C6 straight-chain or branched alkylene group.

[0081] In some preferred embodiments, L1' represents the following structure:

[0082] 1 indicates connection to L1, and 2 indicates connection to L2.

[0083] In some embodiments of the invention, L3 represents a peptide residue consisting of 1 to 6 amino acids selected from one or more of valine, citrulline, glycine, phenylalanine, alanine, isoleucine, lysine, serine, glutamic acid, proline, and aspartic acid, wherein the amino acids are unsubstituted or are each independently substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, C1-6 alkyl, halogenated C1-6 alkyl, and C1-6 alkoxy.

[0084] In some preferred embodiments, L3 represents a peptide residue consisting of one to four amino acids selected from valine, citrulline, glycine, phenylalanine, alanine, isoleucine, lysine, serine, glutamic acid, and aspartic acid.

[0085] In some preferred embodiments, L3 represents a peptide residue composed of the following amino acids: -glycine-, -glycine-glycine-, -glycine-glycine-glycine-, -glycine-glutamic acid-, -glycine-valine-, -glycine-alanine-, -glycine-citrulline-, -valine-citrulline-, -valine-alanine-, or -glycine-glycine-phenylalanine-glycine-.

[0086] In some further preferred embodiments, L3 represents one of the following structures:

[0087] In some embodiments of the present invention, the glucocorticoid drug is selected from one or more of prednisone, prednisolone, betamethasone, dexamethasone, fluticasone propionate, budesonide, and beclomethasone. A pharmaceutical unit containing a glucocorticoid drug refers to a pharmaceutical unit D whose structure contains the basic structure or active structure of a glucocorticoid drug, and may also include a connecting portion to allow the drug to be linked to the connecting unit without affecting its release and biological function.

[0088] In some preferred embodiments, D represents the structure shown in equation (IV-1), equation (IV-2), or equation (IV-3):

[0089] In formula (IV-1), R2 and R3 each independently represent hydrogen, halogen, hydroxyl, cyano, amino, C1-C6 alkyl, halo-C1-C6 alkyl or C1-C6 alkoxy.

[0090] p and q each independently represent 0, 1, 2, 3 or 4;

[0091] R4 and R5 each independently represent hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C4 alkyl;

[0092] R6 represents hydrogen or halogen, preferably hydrogen, F or Cl;

[0093] X represents -C(R1)2-, -C(O)-, -NR1-, -NR1C(O)-, -C(O)NR1-, -S(O)-, -S(O)2-, -O-, or -S-;

[0094] Y1 represents -C(O)-, -NR1-, -NR1C(O)-, or -C(O)NR1-;

[0095] R1 represents hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C4 alkyl;

[0096] In formula (IV-2), R7 represents a C1 to C6 alkyl group;

[0097] R8 represents hydrogen or halogen, preferably hydrogen, F or Cl;

[0098] Z represents an unsubstituted or substituted C1-C6 straight-chain or branched alkylene group. When Z represents a substituted group, the substituent is selected from one or more of halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy. Preferably, Z represents an unsubstituted C1-C4 straight-chain or branched alkylene group.

[0099] Y2 represents -C(O)-, -NR1-, -NR1C(O)-, or -C(O)NR1-;

[0100] R1 represents hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C4 alkyl;

[0101] In formula (IV-3), R9 represents hydrogen, halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0102] m represents 0, 1, 2, 3 or 4;

[0103] R 10 Indicates C1 to C6 alkyl groups;

[0104] W represents a C1-C6 straight-chain or branched alkylene group, wherein one or more of -CH2- can be replaced by one or more of -C(O)-, -NR1-, -NR1C(O)-, -C(O)NR1-, -C(O)O-, -OC(O)-, -O-, -S-; preferably, W represents -(CH2)r-OC(O)-, wherein r represents 1, 2, 3 or 4;

[0105] Y3 represents -C(O)-, -NR1-, -NR1C(O)-, or -C(O)NR1-;

[0106] R1 represents hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C4 alkyl.

[0107] In some preferred embodiments, D represents one of the following structures:

[0108] In some preferred embodiments of the present invention, the antibody-drug conjugate is selected from:

[0109] Wherein, Ab represents an anti-CD74 antibody or its antigen-binding fragment, the heavy chain variable region sequence of which is shown in SEQ ID NO:13 and the light chain variable region sequence of which is shown in SEQ ID NO:15; further, the anti-CD74 antibody or its antigen-binding fragment comprises a human IgG1 heavy chain constant region and a human κ chain light chain constant region.

[0110] n represents an integer or decimal number from 4 to 8, preferably an integer or decimal number from 6 to 8.

[0111] In some of the most preferred embodiments of the present invention, the antibody-drug conjugate is selected from:

[0112] Wherein, Ab represents an anti-CD74 monoclonal antibody, which contains a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 15, as well as a human IgG1 heavy chain constant region and a human κ chain light chain constant region; the drug-to-antibody ratio is 8.

[0113] In some other preferred embodiments according to the invention, the antibody-drug conjugate is selected from:

[0114] Wherein, Ab represents an anti-CD74 monoclonal antibody, which contains a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 15, as well as a human IgG1 heavy chain constant region and a human κ chain light chain constant region; the drug-to-antibody ratio is 6.

[0115] A seventh aspect of the invention provides a drug-linker derivative, or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate thereof, with a structure as shown in formula (V) or formula (V').

[0116] Where L0 represents the leaving group when reacting with antibody Ab; when L1 contains At that time, it forms together with L0.

[0117] In some preferred embodiments, the leaving group represented by L0 is selected from halogens or MeSO2-, for example from Br or MeSO2-;

[0118] L1, L1', L2, L3 and D are each independently defined as described in the sixth aspect of the present invention.

[0119] In some preferred embodiments of the present invention, the drug-linker derivative is selected from:

[0120] The eighth aspect of the present invention provides a method for preparing the antibody-drug conjugate described in the sixth aspect of the present invention, wherein the preparation method includes the following steps:

[0121] S1: The disulfide bonds of antibody Ab are reduced using a reducing agent;

[0122] S2: The reduced antibody Ab is coupled with the drug-linker derivative described in the seventh aspect of the present invention to obtain the antibody.

[0123] In some preferred embodiments, the reducing agent is selected from diphenylphosphinoacetic acid.

[0124] According to the above preparation process, those skilled in the art can understand that L1 connects with the thiol group contained in the antibody Ab itself after the disulfide bond is opened to form -S-. In other words, the "-S-" between L1 and Ab is not an additional external sulfur atom, but comes from Ab itself.

[0125] A ninth aspect of the present invention provides a pharmaceutical composition comprising:

[0126] (1) The antibody-drug conjugate, or its stereoisomer, pharmaceutically acceptable salt or solvate, as described in the sixth aspect of the present invention, or the drug-linker derivative, or its stereoisomer, pharmaceutically acceptable salt or solvate, as described in the seventh aspect of the present invention.

[0127] (2) Optionally, one or more other pharmaceutically active ingredients; and

[0128] (3) Pharmaceutically acceptable carriers and / or excipients.

[0129] The tenth aspect of the present invention provides a use, namely, the use of the antibody-drug conjugate, or its stereoisomer, pharmaceutically acceptable salt or solvate, as described in the sixth aspect of the present invention, the drug-linker derivative, or its stereoisomer, pharmaceutically acceptable salt or solvate, as described in the seventh aspect of the present invention, or the pharmaceutical composition described in the ninth aspect of the present invention, in the preparation of a medicament for treating autoimmune diseases.

[0130] In another aspect, the present invention also provides a method for preventing or treating autoimmune diseases, the method comprising administering to a subject a preventive or therapeutically effective amount of the antibody-drug conjugate, or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate thereof, as described in the sixth aspect of the present invention; the drug-linker derivative, or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate thereof, as described in the seventh aspect of the present invention; or the pharmaceutical composition described in the ninth aspect of the present invention.

[0131] In another aspect, the present invention also provides, as a medicament, the antibody-drug conjugate described in the sixth aspect of the present invention, or its stereoisomers, pharmaceutically acceptable salts or solvates, the drug-linker derivative described in the seventh aspect of the present invention, or its stereoisomers, pharmaceutically acceptable salts or solvates, or the pharmaceutical composition described in the ninth aspect of the present invention. In some preferred embodiments, it is used as a medicament for treating autoimmune diseases.

[0132] In some embodiments of the invention, the autoimmune disease is selected from systemic lupus erythematosus, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, rheumatoid arthritis, asthma, or psoriasis.

[0133] In some preferred embodiments, the autoimmune disease is selected from systemic lupus erythematosus.

[0134] The technical solution provided by this invention has the following advantages:

[0135] (1) The present invention designs a novel conjugate linker structure, which introduces phosphate ester groups on its side chain, which can effectively improve the hydrophilicity of the linker. This enables the drug-linker to be efficiently coupled with the monoclonal antibody to obtain a uniform antibody-drug conjugate with a high DAR value. In some cases, the coupling reaction can even be carried out under conditions without organic solvents, thus simplifying the process, reducing costs, and greatly improving production efficiency.

[0136] (2) The antibody-drug conjugate provided by the present invention has a low proportion of dimers in the formulation solution and excellent drug thermal stability, thus having the potential to prepare high-concentration subcutaneous formulations.

[0137] (3) The anti-CD74 antibody provided by this invention has significant specific binding to CD74, high binding affinity, and excellent endocytosis ability, showing good prospects for drug development. Using the anti-CD74 antibody provided by this invention to prepare antibody-drug conjugates allows the resulting conjugates to achieve rapid endocytosis in target cells, thus resulting in better targeting.

[0138] (4) The antibodies and antibody-drug conjugates provided by the present invention have excellent anti-inflammatory and immune cell proliferation inhibition effects, and therefore have good application prospects in the treatment of immune diseases (especially systemic lupus erythematosus). Attached Figure Description

[0139] Figure 1 shows the evaluation results of the binding activity of the chimeric antibody CD74 isolated from mouse No. 1 by single B cells; (A) antibody binding activity with human CD74, (B) summary of the evaluation results of antibody binding activity with human CD74.

[0140] Figure 2 shows the results of evaluating the binding activity of the CD74 chimeric antibody isolated from mouse No. 2 by single B cells.

[0141] Figure 3 shows the detection results of the candidate mouse chimeric antibody CD74 antibody binding activity (left) and endocytic activity (right).

[0142] Figure 4 shows the SDS-PAGE analysis results of the expression of humanized anti-CD74 monoclonal antibody.

[0143] Figure 5 shows the results of ELISA detection of the binding activity of human-mouse chimeric antibody CD74 and humanized anti-CD74 monoclonal antibody to CD74 protein.

[0144] Figure 6 shows the percentage of binding activity of humanized anti-CD74 monoclonal antibody to CD74 protein on the surface of Raji cells (left) and mean fluorescence intensity (MFI) (right) as detected by flow cytometry.

[0145] Figure 7 shows the percentage of endocytic activity of humanized anti-CD74 monoclonal antibody detected by flow cytometry (top) and mean fluorescence intensity (MFI) (bottom).

[0146] Figures 8A-8C show the LC-MS detection results of different ADCs.

[0147] Figures 9A-9C show the SEC-HPLC detection results for different ADCs.

[0148] Figure 10 shows the results of inflammatory factor inhibition in Test Example 1.

[0149] Figure 11 shows the B cell proliferation inhibition results in Test Example 2.

[0150] Figure 12 shows the ear thickness measurement results of the model mouse in test example 3.

[0151] Figure 13 shows the measurement results of anti-dsDNA antibodies in the model mice in test example 3. Detailed Implementation

[0152] the term

[0153] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields.

[0154] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of.”

[0155] The three-letter and single-letter codes for amino acids used in this article are as described in J. biol. chem, 243, p3558 (1968).

[0156] The term “and / or”, used alone or in combination in this document, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y” and should be used to provide clear support for both meanings or either meaning.

[0157] The terms "antibody" or "immunoglobulin," used alone or in combination herein, refer to immunoglobulin molecules that are typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, and ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain consists of a variable region (VH) and a constant region (CH). The constant region consists of three domains (CH1, CH2, and CH3). The light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of the heavy / light chain pairs form the antigen-binding sites.

[0158] The terms "complementarity-determining region" or "CDR" used alone or in combination in this article refer to the amino acid residues in the antibody variable region that are primarily responsible for binding to the antigen. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as those defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991), the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering rule (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc. For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system.

[0159] Unless otherwise stated, the variable region and CDR sequence in the embodiments of the present invention are governed by the "Kabat" numbering rule. Although a numbering system (such as Kabat) is used to define amino acid residues in a specific implementation, the corresponding technical solutions of other numbering systems are considered equivalent.

[0160] The terms "framework region" or "FR" residues, used alone or in combination herein, refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above. The term "antibody" is not limited to any particular method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0161] The term “antigen-binding fragment” as used alone or in combination herein refers to a polypeptide fragment containing a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as an “antigen-binding moiety.” See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, which contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Exemplary examples include (i) a Fab fragment having VL, CL, VH, and CH1 junctions. (ii) A domain having a disulfide bond between the heavy and light chains; (iii) A Fab' fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iv) An Fd fragment having VH and CH1 domains; (v) An Fd' fragment having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) An Fv fragment having VL and VH domains of one arm of the antibody; (vi) A dAb fragment consisting of a VH domain; (vii) (viii) Hingeless antibodies, which contain at least VL, VH, CL, and CH1 domains but lack a hinge region; (viii) F(ab)2 fragments, which are bivalent fragments containing two Fab' fragments linked by disulfide bridges in the hinge region; (ix) Single-chain antibody molecules (scFv) refer to molecules containing a variable region (VH) of the antibody heavy chain and a variable region (VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH; x) "Diabodies" have two antigen-binding sites, including a heavy chain variable domain (VH) and a light chain variable domain (VL) linked together in the same polypeptide chain; (xi) "Linear antibodies" contain a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptide form a pair of antigen-binding regions; (xii) dsFv refers to a fragment formed by replacing one amino acid residue in each of VH and VL with a cysteine ​​residue via an SS bond between the cysteine ​​residues.

[0162] The term "full-length antibody," used alone or in combination herein, refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain that, in the N-terminal to C-terminal direction, comprises a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. Preferably, the "full-length heavy chain" is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0163] The terms “Fc region” or “fragment crystallizable region” as used alone or in combination in this article are used to define the C-terminal region of the antibody heavy chain, including the native Fc region and the modified Fc region.

[0164] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0165] The terms "monoclonal antibody," "monoclonal antibody," and "mAb" used individually or in combination herein have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules, except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any specific method.

[0166] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al., Nature 352:624-628, 1991, or Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0167] Antibodies can be purified using known techniques, such as affinity chromatography with protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column and purified by immunoaffinity chromatography to achieve immunospecific antibody purification. For purification of immunoglobulins, see, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0168] As used alone or in combination herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase sequence homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Typically, at least one or two, but usually all three (heavy and / or light immunoglobulin chains) of the receptor CDR of the humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDR is referred to as the "donor," and the immunoglobulin providing the framework is referred to as the "receptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., mouse) antibody, and the receptor framework can be a naturally occurring human framework, or a sequence having approximately 85%, 90%, 95%, 99%, or higher sequence identity compared to it. Humanized antibodies typically retain the intended properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc. Donor antibodies can be mouse, rat, rabbit, or non-human primate antibodies with the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).

[0169] The term "vector," used alone or in combination herein, refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids, phage particles, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site. Based on the given nucleic acid sequence, those skilled in the art can easily construct a suitable vector that enables the nucleic acid to be replicated or expressed.

[0170] The term "host cell" as used alone or in combination herein refers to cells that can be used to introduce the vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0171] The term "antigen," used alone or in combination herein, refers to a molecule or molecular moiety that can be selectively recognized by antigen-binding protein molecules (e.g., antibodies) or bound by a binding agent. An antigen may have one or more epitopes that can interact with different antigen-binding protein molecules (e.g., antibodies).

[0172] The terms "capable of specific binding," "specific binding," or "binding," used alone or in combination in this article, refer to the ability of an antibody to bind to a particular antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8The equilibrium dissociation constant (KD) of an antibody to an antigen or epitope (M or less) is used to determine its binding antigen or epitope. In some embodiments, the KD of an antibody to an antigen is 10% or less (e.g., 1%) of the KD of the antibody to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or its epitope may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous) (such as humans or monkeys, e.g., cynomolgus macaques (Macaca fascicularis) (cynomolgus, cyno) or chimpanzees (Pan troglodytes) (chimpanzee, chimp) or marmosets (Callithrix jacchus) (commonmarmoset, marmoset).

[0173] The term "non-binding" as used alone or in combination herein means that the antibody cannot bind to an antigen or its epitope within that antigen in the manner described above for specific binding. For example, when the antibody binds at approximately 1 × 10⁻⁶... -6 M or a larger equilibrium dissociation constant (KD) binds to the antigen or its epitope within the antigen.

[0174] The terms “peptide” and “protein” as used individually or in combination herein are used interchangeably to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical analogs of naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0175] The term "sequence identity," used alone or in combination herein, refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions when optimally aligned. During alignment, gaps may be introduced where necessary to achieve the maximum percentage of sequence identity, but any conserved substitutions are not considered part of sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0176] As used herein, "C1-Cn" includes C1-C2, C1-C3, ..., C1-Cn. For example, the term "C1-C6" refers to a group having 1 to 6 carbon atoms, meaning the group contains 1, 2, 3, 4, 5, or 6 carbon atoms. Therefore, for example, "C1-C4 alkyl" refers to an alkyl group containing 1 to 4 carbon atoms, where the alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Numerical ranges in this document, such as "1-6" or "1-12," refer to integers within a given range.

[0177] The term "alkyl" as used alone or in combination herein refers to a saturated aliphatic hydrocarbon that is optionally substituted with a straight chain or optionally substituted with a branched chain. "alkyl" as used herein preferably has 1 to 6 carbon atoms, for example, 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, and hexyl. When the term "alkyl" is used in this document and a numerical range is specified, for example, "C1 to C6 alkyl" refers to an alkyl group that can be composed of 1, 2, 3, 4, 5, or 6 carbon atoms. The term alkyl in this document also includes cases where no numerical range is specified. Alkyl groups can be substituted or unsubstituted.

[0178] The term "alkyl" as used in this article refers to an alkyl group linked to other groups, such as alkoxy or haloalkyl groups, and is defined in the same way as when used alone.

[0179] The term "alkylene" as used alone or in combination herein refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. "alkylene" as used herein is preferably C1-C12 alkylene, more preferably C1-C6 alkylene. Non-limiting examples of alkylene groups include methylene, 1,1-ethylene, 1,2-ethylene, 1,1-propylene, 1,2-propylene, 1,3-propylene, 1,4-butylene, etc. Alkylenes may be substituted or unsubstituted.

[0180] The term "alkoxy" as used alone or in combination herein refers to an alkyl group as defined above, which is attached to a parent molecule via an oxygen atom. C1-C6 alkoxy groups include, for example, C1-C3 or C1-C4 alkoxy groups. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkoxy groups may be substituted or unsubstituted.

[0181] The term "haloalkyl" as used alone or in combination herein refers to an alkyl group in which one or more, or even all, of the hydrogen atoms are replaced by a halogen. C1-C6 haloalkyl includes, for example, C1-C3 or C1-C4 haloalkyl. Non-limiting examples of haloalkyl include trifluoromethyl, trichloromethyl, etc. Haloalkyl can be substituted or unsubstituted.

[0182] The term “halogen” as used alone or in combination in this article refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0183] The term "hydroxyl group" as used alone or in combination in this article refers to -OH.

[0184] The term "cyano" as used alone or in combination in this article refers to -CN.

[0185] The term "amino" as used alone or in combination in this article refers to -NH2.

[0186] When no linking site is specified, the groups used in this article can be linked to other groups in any order. For example, when the group "--NR1C(O)-" is linked to Q, it can be represented as "QC(O)NR1-" or "Q-NR1C(O)-".

[0187] In the structural fragments used in this article, if they contain numbers such as "1", "2", or "3", it means that the structural fragment is connected to the groups at both ends in a specific order, and the number indicates the direction of connection; if there is no number, it means that the structural fragment can be connected to the groups at both ends in different orders.

[0188] The term "substituted" as used alone or in combination herein refers to one or more hydrogen atoms in a group, preferably 1 to 6, more preferably 1 to 3 hydrogen atoms, which are independently substituted by the corresponding number of substituents. Those skilled in the art can determine possible or impossible substitutions without much effort (through experiment or theory). For example, an amino or hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0189] As used alone or in combination herein, the terms "antibody-drug conjugate" or "antibody-drug conjugate" (ADC) refer to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a linker unit. The antibodies or antibody fragments described herein can be conjugated to effector molecules in any manner. For example, antibodies or antibody fragments can be attached to toxic drugs chemically or recombinantly. Chemical methods for preparing conjugates or conjugates are known in the art. Methods for conjugating antibodies or antibody fragments to drugs must be able to link the antibody to the toxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

[0190] The term "Drug-to-Antibody Ratio" (DAR), used alone or in combination herein, refers to the average number of drugs conjugated to each antibody in an ADC. It can range from, for example, from about 1 to about 8 drugs conjugated to each antibody, such as selected from the ranges of 2–8, 2–7, 2–6, 2–5, 2–4, 3–4, 3–5, 5–6, 5–7, 5–8, 6–8, etc. For example, the DAR value can be the average of 1, 2, 3, 4, 5, 6, 7, and 8.

[0191] The term “pharmaceutically acceptable” as used alone or in combination herein means a substance that does not affect the biological activity or properties of the compounds or conjugates of the present invention and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological reaction or interacting adversely with any component contained in the composition.

[0192] As used alone or in combination herein, the term "pharmaceutically acceptable salt" means a salt obtained by reacting a compound or conjugate of the present invention with a free acid or free base, wherein the free acid is obtained by reacting with a non-toxic inorganic or organic base, and the free base is obtained by reacting with a non-toxic inorganic or organic acid. It can be obtained using standard procedures well known in the art. Suitable salts are listed in Remingtong's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977).

[0193] As used alone or in combination herein, the term "solvent" refers to a physical aggregate of the compound or conjugate of the present invention formed by solvation with one or more solvent molecules, the physical aggregate including varying degrees of ions and covalent bonds, such as hydrogen bonds. "Hydrate" is a solvate with water (H₂O) molecules as a solvent.

[0194] The compounds or conjugates of the present invention may contain asymmetric or chiral centers, and thus exist in different stereoisomer forms. It is contemplated that all stereoisomer forms of the compounds or conjugates of the present invention, including but not limited to diastereomers, enantiomers, sterically hindered isomers, and geometric (conformal) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention. Unless otherwise stated, the structures described in this invention also include all isomers of this structure (e.g., diastereomers, enantiomers, sterically hindered isomers, and geometric (conformal) isomers; for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, sterically hindered isomers of biphenyl structures (see *Basic Organic Chemistry* (Second Edition), Vol. 1, Xing Qiyi et al., pp. 104-105); PAC, 1996, 68, 2193. (*Basic terminology of stereochemistry* (IUPAC Recommendations 1996, on page 2201)), and (Z) and (E) conformational isomers. Therefore, individual stereoisomers of the compounds of this invention, as well as mixtures of enantiomers, diastereomers, sterically hindered isomers, and geometric (conformal) isomers, are all within the scope of this invention.

[0195] The carbon, hydrogen, oxygen, sulfur, nitrogen, F, Cl, Br, I, etc. involved in the compounds or conjugates of the present invention include their isotopic forms. The carbon, hydrogen, oxygen, sulfur, or nitrogen involved in the compounds or conjugates of the present invention may optionally be further replaced by one or more of their corresponding isotopes, wherein the isotopes of carbon include... 12 C 13 C and 14 C, the isotopes of hydrogen include protium (H), deuterium (D, also called heavy hydrogen), and tritium (T, also called superheavy hydrogen), and the isotopes of oxygen include 16 O、 17 O and 18 O, isotopes of sulfur include 32 S, 33 S, 34 S and 36 S, nitrogen isotopes include 14 N and 15 N, isotopes of fluorine include 17 F and 19 F, isotopes of chlorine include 35 Cl and 37 Cl, isotopes of bromine include 79 Br and 81 Br.

[0196] As used alone or in combination herein, the term "pharmaceutical composition" refers to a bioactive compound or conjugate optionally mixed with at least one pharmaceutically acceptable chemical component, including but not limited to carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, excipients, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, humectants, osmotic pressure regulators, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, encapsulating agents, humectants, absorbents, flocculants and anti-flocculators, filter aids, release inhibitors, etc.

[0197] The terms “subject” or “patient” as used alone or in combination herein refer to animals, including human patients in need of treatment. In some respects, this document may also be applied in veterinary practice to any mammal or other animal in need of such immune-targeted therapy, including, but not limited to, non-human primates, dogs, felines, pigs, horses, and any other animals.

[0198] The term "treatment" and other similar synonyms, used alone or in combination herein, include relieving, reducing, or improving symptoms of a disease or condition; preventing other symptoms; improving or preventing the underlying metabolic causes of symptoms; inhibiting a disease or condition, such as preventing its progression; alleviating a disease or condition; improving a disease or condition; relieving symptoms caused by a disease or condition; or stopping the symptoms of a disease or condition. Furthermore, the term includes the purpose of prevention. The term also includes achieving therapeutic and / or preventative effects.

[0199] The term "therapeutic effective amount" as used alone or in combination herein is intended to include an amount of antibody or a conjugate thereof sufficient to achieve treatment of a disease (e.g., by attenuating, improving, or maintaining the existing disease or symptoms of one or more diseases) when administered to a patient for the treatment of a disease related to the CD74 target or pathway. "Prophylactic effective amount" refers to an amount of antibody or a conjugate thereof sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject who has not yet experienced or exhibited symptoms of a disease related to the CD74 target or pathway but may be susceptible to the disease. Improving the disease includes slowing its progression or reducing the severity of subsequent disease development.

[0200] The "therapeutic effective dose" or "preventive effective dose" can vary depending on the type of reagent used for the antibody or its conjugate, how the reagent is administered, the disease and its severity, and medical history, age, weight, family history, genetic composition, stage of the CD74-mediated pathological process, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0201] The term “room temperature” as used alone or in combination in this article refers to 25±5℃.

[0202] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0203] Unless otherwise specified, all raw materials or reagents used in the embodiments of the present invention are commercially available products.

[0204] In the embodiments of the present invention, the reversed-phase chromatographic column used in the chemical synthesis is an UltimatePrep C18 (10 μm, 21.2 × 250 mm).

[0205] In the embodiments of the present invention, the chromatographic column used for ADC molecular purity analysis (SEC-HPLC) is: XBridge Protein BEH SEC (7.8×300mm, 3.5μm, 200A).

[0206] In the embodiments of the present invention, all parts of the eluent are volume ratios or volume percentages, and other percentages are mass percentages unless otherwise specified.

[0207] The abbreviations used in the embodiments of this invention have the following meanings:

[0208] (I) Synthesis of glucocorticoid derivatives

[0209] Example 1: Synthesis of Compound TM-1

[0210] The specific synthesis steps are as follows:

[0211] Step 1. Synthesis of Compound 1

[0212] A solution of tert-butyl(3-(4,4,5,5-tetramethylH,3,2-dioxaborborono-2-yl)phenyl)carbamate (4.3 g, 13.4 mmol), 4-bromomethylbenzaldehyde (4.0 g, 20.1 mmol), potassium carbonate (9.3 g, 67.0 mmol), and Pd(dpff)Cl2 (0.4 g, 0.5 mmol) in THF (84 mL) was stirred at 80 °C for 16 h. The mixture was then cooled, diluted with water (200 mL), and extracted with EtOAc (3 × 100 mL). The combined organic extracts were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography (PE / EtOAc: 4 / 1) to give compound 1 as a white solid (3.5 g, yield: 85%).

[0213] LCMS:(M-56+42) +=297.2.

[0214] 1 H NMR (400MHz, CDCl) δ9.95 (s, 1H), 7.79 (d, J = 10.1Hz, 2H), 7.32 (d, J = 11.2Hz, 2H), 7.26-7.2 7(m,1H),7.21-7.22(m,2H),6.83(d,J=9.1Hz,1H),6.53(s,1H),4.01(s,2H),1.51(s,9H).

[0215] Step 2. Synthesis of Compound 2

[0216] In a round-bottom flask, 16-α-hydroxyprednisolone (3.3 g, 8.8 mmol), (3-(4-formylbenzyl)phenyl)carbamate tert-butyl ester (compound 1) (3.0 g, 9.6 mmol), and MgSO4 (3.2 g, 26.3 mmol) were added. The solid was suspended in acetonitrile (88 mL), and the mixture was cooled to 0 °C. Then, methanesulfonic acid (3.9 mL, 43.8 mmol) was added dropwise. After returning to room temperature, the reaction turned pink after 10–20 min, and the starting material was completely consumed after 1 h. The solvent was filtered, and the filtrate was concentrated. The residue was purified by reversed-phase chromatography using gradient elution (acetonitrile / H2O (containing 0.1% FA) = 5%–95%) to give compound 2 as a white solid (1.2 g, yield: 25%).

[0217] LCMS:(M+H) + =570.1.

[0218] 1H NMR (400MHz, DMSO-d6) δ7.36(d,J=8.0Hz,2H),7.31(d,J=10.1Hz,1H),7.22–7.17(m,2H),6.92–6.84(m,1H),6.39–6. 30(m,3H),6.16(dd,J=10.1,1.6Hz,1H),5.93(s,1H),5.75(s,1H),5.40(s,1H),5.07(t,J=5.9Hz,1H),4.97–4.87(m, 3H),4.77(d,J=3.1Hz,1H),4.50(dd,J=19.5,6.3Hz,1H),4.29(s,1H),4.17(dd,J=19.4,5.6Hz,1H),3.74(s,2H),2.6 2–2.52(m,1H),2.36–2.26(m,1H),2.15–2.00(m,2H),1.78–1.64(m,4H),1.39(s,3H),1.10–0.98(m,2H),0.86(s,3H).

[0219] Step 3. Synthesis of Compound 3

[0220] (S)-2-amino-5-(tert-butoxy)-5-oxopentanoic acid (10.0 g, 49.2 mmol) and 2,5-dioxopyridin-1-yl((9H-fluorene-9-yl)methoxy)carbonyl)glycine (16.17 g, 41 mmol) were dissolved in 1,4-dioxane / H₂O (130 mL / 130 mL). NaHCO₃ (6.89 g, 82 mmol) was added to the solution, and the mixture was stirred at room temperature for 16 h. The pH of the reaction mixture was adjusted to 2-3 with citric acid solution. Extraction was performed with ethyl acetate (150 mL × 3). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to give compound 3 as a white solid (22.80 g, yield: 96%).

[0221] LCMS:(M+Na) + =505.5.

[0222] Step 4. Synthesis of Compound 4

[0223] Compound 3 (2.2 g, 4.57 mmol) and compound 2 (2.6 g, 4.57 mmol) were dissolved in THF (20 mL). EEDQ (2.3 g, 9.14 mmol) was added to the solution, and the reaction mixture was stirred at room temperature for 16 h. After the reaction was stopped, the mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (EtOAc elution) to give compound 4 as a white solid (4.1 g, yield: 44%).

[0224] LCMS:(M+Na) + =1056.7.

[0225] Step 5. Synthesis of Compound 5

[0226] Compound 4 (5.5 g, 5.32 mmol) was dissolved in DCM (30 mL), and diethylamine (1.166 g, 15.97 mmol) was added to the reaction solution. The mixture was stirred at room temperature for 3 h. The solution was purified by silica gel column chromatography (DCM / MeOH = 10 / 1 elution) to give compound 5 as a white solid (4 g, yield: 93%).

[0227] LCMS:(M+H) + =812.6.

[0228] Step 6. Synthesis of Compound 6

[0229] 2 g (6.8 mmol) of tert-butyloxycarbonyl-L-serine benzyl ester was dissolved in dichloromethane / tetrahydrofuran (1 / 1) (15 mL), and N,N-diisopropylphosphamide ditert-butyl ester (3.8 g (13.6 mmol) and tetrazolium (949 mg (13.6 mmol)) were added. The mixture was stirred at room temperature for 2 h. 30% hydrogen peroxide (922 mg (27.1 mmol) was slowly added dropwise under ice bath. After the addition was complete, the reaction mixture was stirred at room temperature for 3 h. The reaction was monitored by LCMS. After the reaction was completed, 10% sodium sulfite solution (30 mL) was added to the reaction solution under ice bath to quench the reaction. The mixture was extracted with ethyl acetate (50 mL × 3), and the organic phase was washed with 10% sodium sulfite solution (40 mL × 2). The organic phases were combined and concentrated under reduced pressure. The mixture was purified using a reversed-phase chromatography column (acetonitrile / H2O (containing 0.1% FA) = 5%-95%, gradient elution) to give compound 6 as a yellow oil (2.5 g, yield: 75.8%).

[0230] LCMS:(M+H) + =488.3.

[0231] 1H NMR (400MHz, DMSO-d6) δ7.38–7.33(m,5H),7.30(d,J=8.3Hz,1H),5.15(s,2H),4.41–4.34(m,1H),4.17–4.07(m,2H),1.38(d,J=4.1Hz,27H).

[0232] Step 7. Synthesis of Compound 7

[0233] Compound 6 (2.5 g, 5.2 mmol) was dissolved in anhydrous ethanol (20 mL), and palladium on carbon catalyst (250 mg, 10%) was added. The mixture was purged with hydrogen three times, and the reaction was carried out at room temperature under a hydrogen atmosphere with stirring for 2 h. The reaction was monitored by LCMS. After the reaction was completed, the reaction solution was filtered through diatomaceous earth and concentrated under reduced pressure to obtain compound 7 as a pale yellow oil (2.0 g, yield: 98%), which was directly used in the next step of the reaction.

[0234] LCMS:(M+H) + =398.3.

[0235] Step 8. Synthesis of Compound 8

[0236] The compound N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine (10 g, 21.4 mmol) was dissolved in N,N-dimethylformamide (60 mL), and benzyl bromide (4.4 g, 25.6 mmol) and anhydrous potassium carbonate (3.5 g, 25.6 mmol) were added. The mixture was stirred overnight at room temperature. The reaction was monitored by LCMS. After the reaction was completed, water (80 mL) was added to the reaction solution to quench the reaction, and the mixture was extracted with ethyl acetate (100 mL × 3), washed with saturated sodium chloride solution (100 mL × 3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:anhydrous methanol = 50:1) to give compound 8 as a white solid (10 g, yield: 83.3%).

[0237] LCMS:(M+Na) + =581.4.

[0238] 1H NMR (400MHz, DMSO-d6) δ7.90(d,J=7.5Hz,2H),7.82(d,J=7.7Hz,1H),7.72(d,J=7.5Hz,2H),7.42(t,J=7.5Hz,2H),7.36–7.31(m,7H),6.77(t,J=5. 3Hz,1H),5.13(s,2H),4.35–4.27(m,2H),4.22(t,J=6.9Hz,1H),4.13–4.0 6(m,1H),2.89(s,2H),1.76–1.61(m,2H),1.37(s,9H),1.36–1.20(m,4H).

[0239] Step 9. Synthesis of Compound 9

[0240] Compound 8 (10 g, 17.9 mmol) was dissolved in dichloromethane (100 mL), and trifluoroacetic acid (5 mL) was added. The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the crude product was dissolved in dichloromethane. This concentration process was repeated three times to obtain compound 9 (10 g, crude product), which was directly used in the next reaction.

[0241] LCMS:(M+H) + =459.4.

[0242] 1 H NMR (400MHz, DMSO-d6) δ7.90(d,J=7.5Hz,2H),7.81(d,J=7.8Hz,1H),7.71(d,J=7.3Hz,2H),7.43(t,J=7.4Hz,2H),7.36–7.30(m,7H),5.14(s,2 H),4.40–4.27(m,2H),4.23(t,J=6.8Hz,1H),4.11–4.06(m,1H),2.77(d ,J=6.6Hz,2H),1.77–1.61(m,2H),1.56–1.48(m,2H),1.40–1.32(m,2H).

[0243] Step 10. Synthesis of Compound 10

[0244] Compound 7 (2 g, 5.04 mmol) was dissolved in N,N-dimethylformamide (20 mL), and compound 9 (2.3 g, 5.04 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.3 g, 6.05 mmol), and N,N-diisopropylethylamine (1.95 g, 15.1 mmol) were added. The mixture was stirred at room temperature for 2 h, and the reaction was confirmed to be complete by LCMS. The reaction solution was directly purified by reversed-phase chromatography (acetonitrile / H2O (containing 0.1% FA) = 5%-95%, gradient elution) to obtain compound 10 as a yellow oil (1.1 g, yield: 26.2%).

[0245] LCMS:(M+Na) + =860.3.

[0246] 1 H NMR (400MHz, DMSO-d6) δ7.99(t,J=5.4Hz,1H),7.90(d,J=7.5Hz,2H),7.80(d,J=7.7Hz,1H) ,7.71(d,J=7.5Hz,2H),7.42(t,J=7.4Hz,2H),7.35–7.33(m,4H),7.32(s,2H),7.30(s,1H), 6.77(d,J=8.1Hz,1H),5.12(s,2H),4.33–4.27(m,2H),4.26–4.10(m,3H),4.00–3.88(m,2H) ,3.06–3.00(m,2H),1.72–1.61(m,2H),1.38(s,27H),1.33–1.28(m,2H),1.27–1.22(m,2H).

[0247] Step 11. Synthesis of Compound 11

[0248] Compound 10 (1.1 g, 1.3 mmol) was dissolved in ethyl acetate / tetrahydrofuran (3 / 1) (12 mL), and palladium on carbon catalyst (100 mg) was added. The mixture was purged with hydrogen three times, and the reaction was carried out at room temperature under a hydrogen atmosphere with stirring for 4 h. The reaction was confirmed to be complete by LCMS. The reaction solution was filtered through diatomaceous earth and concentrated under reduced pressure to obtain compound 11 as a pale yellow oil (620 mg, yield: 63%), which was used directly in the next step of the reaction.

[0249] LCMS:(M+H) + =748.4.

[0250] Step 12. Synthesis of Compound 12

[0251] Compound 11 (620 mg, 0.830 mmol) and compound 5 (673 mg, 0.830 mmol) were dissolved in N,N-dimethylformamide (10 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (315 mg, 0.830 mmol) and N,N-diisopropylethylamine (268 mg, 2.07 mmol) were added. The mixture was stirred at room temperature for 2 h, and the reaction was monitored by LCMS. The reaction solution was directly purified by reversed-phase chromatography (acetonitrile / H2O (containing 0.1% FA) = 5%-95%, gradient elution) to obtain compound 12 as a pale yellow solid (450 mg, yield: 35.2%).

[0252] LCMS:(M+Na) + =1563.7.

[0253] 11H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.19 (s, 1H), 7.99 (d, J = 7.0 Hz, 2H), 7.88 (d, J = 7.5 Hz, 2H), 7.74–7.69 (m, 2H), 7.55 (d, J = 7.3 Hz, 1H), 7.44 (t, J = 5.5 Hz, 3H), 7.41 (s, 1H), 7.38 (d, J = 2.7 Hz, 1H), 7.36 (s, 1H), 7.33 (s, 1H), 7.31 (s, 1H), 7.29 (s, 1H), 7.23 (s, 1H), 7.21 (s, 1H), 7.18 (d, J = 7.4 Hz, 1H), 6.91 (d, J = 7.7 Hz, 1H), 6.76 (d, J = 7.7 Hz, 1H), 6.18–6.13 (m, 1H), 5.92 (s, 1H), 5.39 (s, 1H), 5.08 (t, J = 5.9 Hz, 1H), 4.91 (d, J = 5.1 Hz, 1H), 4.78 (d, J = 2.7 Hz, 1H), 4.52–4.46 (m, 1H), 4.42–4.35 (m, 2H), 4.32–4.28 (m, 2H), 4.25 (d, J = 6.8 Hz, 2H), 4.22 (s, 1H), 4.20 (d, J = 5.7 Hz, 2H), 4.15 (d, J = 5.6 Hz, 1H), 3.97–3.95 (m, 1H), 3.95–3.91 (m, 2H), 3.88 (s, 2H), 3.76–3.73 (m, 2H), 3.06–3.01 (m, 2H), 2.34–2.27 (m, 2H), 2.26–2.21 (m, 2H), 2.19 (d, J = 6.2 Hz, 1H), 2.16–2.13 (m, 1H), 1.85–1.79 (m, 2H), 1.78–1.75 (m, 2H), 1.74–1.70 (m, 2H), 1.68–1.64 (m, 2H), 1.63–1.59 (m, 2H), 1.38 (d, J = 1.2 Hz, 27H), 1.33 (s, 9H), 1.24 (s, 3H), 1.06–0.98 (m, 2H), 0.86 (s, 3H).

[0254] Step 13. Synthesis of Compound 13

[0255] Compound 12 (450 mg, 0.29 mmol) was dissolved in N,N-dimethylformamide (10 mL), and diethylamine (43 mg, 0.58 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:anhydrous methanol = 15:1) to obtain compound 13 as a yellow solid (280 mg, yield: 72.7%).

[0256] LCMS:(M+H) + =1319.9.

[0257] 1H NMR (400MHz, DMSO-d6) δ9.88(s,1H),8.13(d,J=7.7Hz,1H),7.97(t,J=5.6Hz,1H),7.46(d,J=9.2Hz,2H),7.38(d ,J=8.1Hz,2H),7.31(d,J=10.0Hz,1H),7.24–7.15(m,4H),6.92(d,J=7.8Hz,1H),6.77(d,J=7.9Hz,1H),6.18–6.1 2(m,1H),5.93(s,1H),5.39(s,1H),5.08(t,J=6.1Hz,1H),4.92(d,J=5.1Hz,1H),4.78(d,J=3.0Hz,1H),4.53–4. 45(m,1H),4.42–4.35(m,1H),4.31–4.27(m,1H),4.21–4.13(m,2H),4.00–3.96(m,1H),3.95–3.91(m,1H),3.89(d ,J=3.9Hz,2H),3.78–3.72(m,2H),3.18–3.14(m,1H),3.05–3.00(m,2H),2.69–2.66(m,1H),2.34–2.31(m,1H),2 .30–2.27(m,1H),2.26–2.19(m,2H),2.19–2.09(m,2H),2.07(s,1H),2.02(d,J=7.3Hz,1H),1.96–1.91(m,1H),1. 84–1.80(m,1H),1.79–1.75(m,2H),1.75–1.69(m,2H),1.69–1.65(m,1H),1.64–1.60(m,1H),1.59–1.52(m,2H),1 .38(d,J=1.3Hz,18H),1.38(s,9H),1.37(s,9H),1.24(s,3H),1.09–1.03(m,1H),1.02–0.98(m,1H),0.86(s,3H).

[0258] Step 14. Synthesis of Compound 14

[0259] Compound 13 (385 mg, 0.29 mmol) was dissolved in N,N-dimethylformamide (10 mL), and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-alkynic acid (78 mg, 0.29 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (111 mg, 0.29 mmol), and N,N-diisopropylethylamine (94 mg, 0.73 mmol) were added. The mixture was stirred at room temperature for 2 h, and the reaction was monitored by LCMS. After the reaction was completed, the reaction solution was directly purified by reversed-phase chromatography (acetonitrile / H2O (containing 0.1% FA) = 5%-95%, gradient elution) to obtain compound 14 as a white solid (140 mg, yield: 30.6%).

[0260] LCMS:(M+H) + =1570.7.

[0261] 1 H NMR (400MHz, DMSO-d6) δ9.10 (s, 1H), 8.29–8.08 (m, 1H), 7.98 (t, J = 6.8Hz, 1H), 7.54–7. 41(m,2H),7.39–7.28(m,3H),7.24–7.16(m,4H),6.95–6.74(m,1H),6.73–6.59(m,1H), 6.20–6.09(m,1H),5.96–5.85(m,1H),5.42–5.36(m,1H),5.34–5.31(m,1H),4.92–4.76 (m,1H),4.61–4.42(m,1H),4.40–4.21(m,3H),4.20–4.11(m,2H),4.05–3.97(m,2H),3.9 1–3.86(m,3H),3.75–3.70(m,2H),3.66–3.60(m,2H),3.58–3.50(m,3H),3.40(d,J=2.9 Hz,4H),2.68–2.66(m,2H),2.34–2.32(m,3H),2.26–2.18(m,1H),2.04–2.02(m,1H),2. 01(s,1H),1.99(s,2H),1.98–1.94(m,2H),1.86–1.75(m,4H),1.73–1.59(m,3H),1.49– 1.42(m,4H),1.40–1.35(m,36H),1.33(d,J=1.2Hz,4H),1.06–0.98(m,2H),0.85(s,3H).

[0262] Step 15. Synthesis of compound TM-1

[0263] Compound 14 (140 mg, 0.09 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. After the reaction was completed, the reaction solution was directly purified by reversed-phase chromatography (acetonitrile / H2O (containing 0.1% FA) = 5%-95%, gradient elution) to obtain compound TM-1 as a white solid (29 mg, yield: 25.0%).

[0264] LCMS:(M+H) + =1301.5.

[0265] 1 H NMR (400MHz, DMSO-d6) δ10.09(s,1H),9.11(s,2H),8.79(s,1H),8.31(d,J=7.0Hz,2H),8.14(d,J=8.2Hz, 1H),7.53(s,1H),7.47(d,J=7.9Hz,1H),7.37(d,J=8.2Hz,2H),7.31(d,J=10.1Hz,1H),7.23(d,J=8.1Hz, 2H),7.18(t,J=8.0Hz,2H),6.89(d,J=8.0Hz,1H),6.18–6.14(m,1H),5.93(s,1H),5.39(s,1H),4.91(d,J =5.2Hz,1H),4.49(d,J=19.4Hz,1H),4.41–4.34(m,1H),4.29(d,J=3.2Hz,1H),4.20–4.14(m,2H),4.02–3. 94(m,2H),3.88(s,2H),3.84–3.76(m,2H),3.73–3.67(m,2H),3.40(s,3H),3.02–2.96(m,2H),2.56(d,J= 7.0Hz,1H),2.52(s,1H),2.38(d,J=7.4Hz,2H),2.35(s,1H),2.34–2.31(m,2H),2.30–2.23(m,4H),2.15–2 .06(m,2H),2.04–1.97(m,3H),1.82(d,J=7.5Hz,2H),1.78(d,J=7.8Hz,2H),1.75(s,1H),1.73–1.68(m,2 H),1.67–1.62(m,2H),1.62–1.57(m,2H),1.39(s,3H),1.13–1.06(m,1H),1.05–0.99(m,2H),0.85(s,3H).

[0266] Example 2 Synthesis of compound TM-2

[0267] The specific synthesis steps are as follows:

[0268] Step 1. Synthesis of Compound 15

[0269] Compound 13 (500 mg, 0.38 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-glutamic acid (70 mg, 0.19 mmol) were dissolved in DMF (1 mL). HOBT (77 mg, 0.57 mmol) and DCC (117 mg, 0.57 mmol) were added, and the mixture was stirred at 50 °C for 3 h. After the reaction was complete, H2O (80 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (50 mL × 3), and the organic phase was washed with brine (50 mL × 3). After drying with anhydrous Na2SO4, the phase was concentrated and purified by silica gel column chromatography (DCM / MeOH = 15 / 1) to give compound 15 as a white solid (320 mg, yield: 29%).

[0270] LCMS:(M / 2+Na) + =1509.1.

[0271] Step 2. Synthesis of Compound 16

[0272] Compound 15 (320 mg, 0.11 mmol) was dissolved in DMF (20 mL), and diethylamine (16 mg, 0.22 mmol) was added. The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure to obtain compound 16, which was used directly in the next step of the reaction.

[0273] LCMS:(M / 2+H) + =1376.0.

[0274] Step 3. Synthesis of Compound 17

[0275] Compound 16 (296 mg, 0.11 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexynic acid (29 mg, 0.11 mmol) were dissolved in DMF (10 mL), and HATU (41 mg, 0.11 mmol) and DIEA (35 mg, 0.27 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was purified by gradient elution using a reversed-phase column (ACN / H2O (0.1% FA) = 10%–60%) to give compound 17 as a white solid (50 mg, yield: 15.5%).

[0276] LCMS:(M / 2+H) + =1523.1.

[0277] Step 4. Synthesis of compound TM-2

[0278] Compound 17 (30 mg, 0.01 mmol) was dissolved in triisopropylsilane (2 mL), and TFA (2 mL) was added. The mixture was stirred at room temperature for 5 min. The reaction solution was freeze-dried, and the resulting mixture was dissolved in DMF (1 mL). The mixture was purified by high performance liquid chromatography (ACN / H2O (0.1% FA)) to give compound TM-2 as a white solid (4.2 mg, yield: 17.1%).

[0279] LCMS:(M / 2+H) + =1232.5.

[0280] 1H NMR(400MHz,DMSO-d6)δ10.03–9.94(m,2H),9.08(d,J=0.5Hz,2H),8.60–8.55(m,2H),8.52–8.48(m,1H),8.39–8.34(m,2H),8.18(d,J=9.4Hz,1H),8.15–8.09(m,2H),7.50–7.47(m,2H),7.46–7.44(m,2H),7.43–7.39(m,2H),7.38(s,1H),7.36(s,2H),7.32(s,1H),7.30(s,1H),7.23(s,1H),7.21(s,2H),7.20(s,2H),7.19(s,2H),7.17(s,2H),7.08(s,1H),6.90–6.87(m,2H),6.17(d,J=1.8Hz,1H),6.15(d,J=2.0Hz,1H),6.06(s,1H),5.92(s,2H),5.91(s,1H),5.39(s,2H),5.28(d,J=6.9Hz,1H),4.91(d,J=4.9Hz,2H),4.78–4.73(m,2H),4.69–4.61(m,2H),4.52(s,1H),4.47(s,1H),4.39–4.33(m,4H),4.29(d,J=2.0Hz,4H),4.25–4.24(m,1H),4.20(s,2H),4.15(s,2H),4.11(d,J=5.7Hz,2H),4.08–4.06(m,2H),4.04(s,2H),3.99(s,2H),3.97–3.95(m,2H),3.88(s,4H),3.83(s,2H),3.75–3.72(m,4H),3.65(s,3H),2.68–2.66(m,1H),2.34–2.32(m,2H),2.28(d,J=2.4Hz,2H),2.27–2.26(m,2H),2.24(s,2H),2.13–2.09(m,2H),2.03–1.99(m,4H),1.87–1.85(m,2H),1.83–1.80(m,4H),1.77(d,J=0.8Hz,4H),1.74–1.72(m,2H),1.72–1.70(m,2H),1.68–1.64(m,4H),1.63–1.58(m,4H),1.38(s,6H),1.24(s,4H),1.07–1.01(m,4H),1.01–0.98(m,2H),0.98–0.93(m,2H),0.85(s,6H).

[0281] Example 3 Synthesis of compound TM-3

[0282] The specific synthesis steps are as follows:

[0283] Step 1. Synthesis of Compound 18

[0284] Under nitrogen protection, Boc-Gly-Gly-Phe-Gly-OH (29 g, 66.44 mmol, 1.0 eq) was dissolved in 300 mL of N,N-dimethylformamide. Then, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (30.3 g, 79.73 mmol, 1.2 eq) and N,N-diisopropylethylamine (17.2 g, 132.89 mmol, 2.0 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 5 min, and then 4-aminobenzyl alcohol (9.0 g, 73.09 mmol, 1.1 eq) was added at room temperature. The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: water (containing 10 mM NH4HCO3) and acetonitrile (30% acetonitrile, reaching 80% concentration within 15 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 18 (21 g, yield: 55%).

[0285] MS m / z[M+H]+(ESI):542.25.

[0286] 1 H NMR (400MHz, DMSO-d6) δ9.80(s,1H),8.42(t,J=5.9Hz,1H),8.20(d,J=7.9Hz,1H),7.94(t,J=5.6Hz,1H ),7.60-7.52(m,2H),7.31-7.23(m,6H),7.22-7.15(m,1H),7.00(t,J=6.0Hz,1H),5.10(t,J=5.7Hz,1H) ,4.57-4.48(m,1H),4.43(d,J=5.7Hz,2H),3.95-3.84(m,2H),3.84-3.74(m,1H),3.62(dd,J=16.7,5.4 Hz,1H),3.54(d,J=6.0Hz,2H),3.08(dd,J=13.9,4.5Hz,1H),2.81(dd,J=13.8,9.7Hz,1H),1.38(s,9H).

[0287] Step 2. Synthesis of Compound 19

[0288] Compound 18 (21.0 g, 38.77 mmol, 1.0 eq) was dissolved in 210 mL of trifluoroacetic acid / dichloromethane (v / v, 1:3) and stirred at 0 °C for 1 h under nitrogen protection. The reaction mixture was concentrated under reduced pressure in an ice bath. The crude product was purified by high performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: water (containing 10 mM NH4HCO3) and acetonitrile (10% acetonitrile reached 100% within 20 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 19 as a white solid (12 g, yield: 66.59%).

[0289] MS m / z[M+H]+(ESI):442.05.

[0290] Step 3. Synthesis of Compound 20

[0291] Under nitrogen protection, compound 19 (12.0 g, 27.18 mmol, 1.0 eq) was dissolved in 60 mL of N,N-dimethylformamide. Then, N,N-diisopropylethylamine (10.5 g, 81.55 mmol, 3.0 eq) and O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (15.5 g, 40.77 mmol, 1.5 eq) were added to the reaction mixture. The mixture was stirred at 0 °C for 5 min, followed by the addition of Fmoc-Lys(Boc)-OH (15.3 g, 32.62 mmol, 1.2 eq in 60 mL of N,N-dimethylformamide). The resulting mixture was stirred at room temperature for 1 h and then diluted with 100 mL of water. The aqueous layer was extracted with 200 mL of dichloromethane and washed with 100 mL of sodium chloride aqueous solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: water (containing 10 mM NH4HCO3) and acetonitrile (30% acetonitrile, reaching 80% concentration within 15 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 20 as a white solid (13.0 g, yield: 50.97%).

[0292] MS m / z[M+H]+(ESI):892.25.

[0293] 1H NMR (400MHz, DMSO-d6) δ9.79 (s, 1H), 8.42 (t, J = 5.9Hz, 1H), 8.23-8.11 (m, 2H), 8.05 (t, J = 5.8Hz, 1H), 7.89 (d, J = 7. 5Hz,2H),7.72(t,J=7.1Hz,2H),7.54(dd,J=19.4,8.2Hz,3H),7.44-7.38(m,2H),7.32(dd,J=7.5,1.2Hz,2H),7.28 -7.20(m,6H),7.20-7.15(m,1H),6.77(t,J=5.4Hz,1H),5.11(t,J=5.7Hz,1 H),4.56-4.48(m,1H),4.43(d,J=5.6Hz,2H),4.33-4.19(m,3H),4.02-3.84 (m,3H),3.84-3.69(m,3H),3.65(dd,J=16.7,5.5Hz,1H),3.07(dd,J=13.8, 4.5Hz, 1H), 2.84 (dd, J=22.6, 12.4Hz, 3H), 1.68-1.45 (m, 2H), 1.36 (s, 13H).

[0294] Step 4. Synthesis of Compound 21

[0295] Benzyl 3-hydroxypropionate (20 g, 110.986 mmol, 1.0 eq) was dissolved in 200 mL of dichloromethane. Under nitrogen protection, [bis(tert-butoxy)phosphonyl]diisopropylamine (46.2 g, 166.47 mmol, 1.5 eq) and 1H-imidazolium-4,5-dicarboxynitrile (14.4 g, 122.08 mmol, 1.1 eq) were added separately at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was poured into 200 mL of saturated sodium bicarbonate aqueous solution, and the resulting mixture was extracted with 2 × 200 mL of dichloromethane. The combined organic layers were washed with 200 mL of sodium chloride aqueous solution. The organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. Hydrogen peroxide (12.58 g, 110.99 mmol, 1.0 eq) was added to 200 mL of acetonitrile solution. The resulting mixture was stirred at 25 °C for 2 h. The combined organic layers were extracted with 2 × 200 mL of dichloromethane and washed with 200 mL of saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography under the following conditions: column: C18 silica gel; mobile phase: water (containing 10 mM NH4HCO3) and acetonitrile (30% acetonitrile, reaching 100% concentration within 15 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 21 as a pale yellow oil (23.0 g, yield: 52.8%).

[0296] MS m / z[M+H]+(ESI):373.25.

[0297] Step 5. Synthesis of Compound 22

[0298] Compound 21 (23.0 g, 61.755 mmol, 1.0 eq) was dissolved in 230 mL of tetrahydrofuran, and 10% Pd / C (wt / wt = 10%, 2.3 g) was added. The flask was evacuated and rinsed five times with hydrogen. The resulting solution was stirred at room temperature for 2 h. The solution was filtered and washed with tetrahydrofuran. The filtrate was concentrated under reduced pressure to give compound 22 as a white solid (16.0 g, yield: 82%).

[0299] MS m / z[MH]-(ESI):281.10.

[0300] 1 H NMR(400MHz, DMSO-d6)δ12.38(s,1H),4.13-3.93(m,2H),2.56(t,J=6.1,1.1Hz,2H),1.43-1.36(m,18H).31P NMR(121MHz,DMSO-d6)δ-10.24.

[0301] Step 6. Synthesis of Compound 23

[0302] Compound 20 (11.0 g, 12.332 mmol, 1.0 eq) was dissolved in 130 mL of trifluoroacetic acid / dichloromethane (v / v, 1:5), and nitrogen gas was bubbled through the solution at 0 °C. The resulting solution was stirred at 0 °C for 1 h. The reaction solution was concentrated under reduced pressure in an ice bath to obtain compound 23 as a yellow oil (crude product) (11.0 g), which was used for the next reaction without further purification.

[0303] MS m / z[M+H]+(ESI):792.25.

[0304] Step 7. Synthesis of Compound 24

[0305] Under nitrogen protection, compound 23 (4.7 g, 16.669 mmol, 1.2 eq) was dissolved in 60 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (26.7 g, 83.34 mmol, 6.0 eq), 1-hydroxybenzotriazole (2.3 g, 16.67 mmol, 1.2 eq), and O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (7.9 g, 20.84 mmol, 1.5 eq) were added to the solution. The mixture was stirred at 0 °C for 5 min, and then compound 22 (11.0 g, 13.891 mmol, 1.0 eq) was added. The resulting mixture was stirred at room temperature for 1 h and then diluted with 100 mL of water. The aqueous layer was extracted with 200 mL of dichloromethane and washed with 100 mL of sodium chloride aqueous solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: water (containing 10 mM NH4HCO3) and acetonitrile (30% acetonitrile, reaching 80% concentration within 15 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 24 as a white solid (5.0 g, yield: 32.4%).

[0306] MS m / z[M+H]+(ESI):1056.40.

[0307] 1 H NMR(300MHz, DMSO-d6)δ9.78(s,1H),8.41(t,J=5.6Hz,1H),8.22-8.11(m,2H),8.05(t,J=5.7Hz,1H),7.91(dd,J=14.3,6.6Hz ,3H),7.72(t,J=6.6Hz,2H),7.54(dd,J=13.9,8.2Hz,3H),7.46-7.38(m,2H),7.36-7.29(m,2H),7.28-7.14(m,7H),5.10(t,J= 5.7Hz,1H),4.57-4.48(m,1H),4.43(d,J=5.7Hz,2H),4.32-4.19(m,3H),4.10-3.93(m,3H),3.88(t,J=6.1Hz,2H),3.83-3.60 (m,4H),3.11-2.97(m,3H),2.82(dd,J=13.4,10.0Hz,1H),2.39(t,J=6.2Hz,2H),1.58(dd,J=27.2,14.2Hz,2H),1.38(s,22H).

[0308] 31P NMR(121MHz,DMSO-d6)δ-10.21.

[0309] Step 8. Synthesis of Compound 25

[0310] Compound 24 (5.0 g, 4.734 mmol, 1.0 eq) was dissolved in 65 mL of diethylaniline / N,N-dimethylformamide (v / v, 1:40) under nitrogen protection at 0 °C. The resulting solution was stirred at room temperature for 1 h. The reaction mixture was slowly poured into 500 mL of diethyl ether at 0 °C, the solid was collected by filtration, and dried under vacuum at 25 °C to give compound 25 as a white solid (3.1 g, yield: 74.6%).

[0311] MS m / z[M+H]+(ESI):834.30.

[0312] Step 9. Synthesis of Compound 26

[0313] Under nitrogen protection, compound 25 (3.1 g, 3.717 mmol, 1.0 eq) was dissolved in 40 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (960.9 mg, 7.434 mmol, 2.0 eq) and 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxopyrrolidone-1-yl)hexanoate (1.3 g, 4.089 mmol, 1.1 eq) were added to the solution. The reaction mixture was stirred at room temperature for 1 h, then diluted with water. The aqueous layer was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: water (containing 0.05% FA) and acetonitrile (30% acetonitrile, reaching 100% concentration within 15 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 26 as a white solid (2.0 g, yield: 49.8%).

[0314] MS m / z[M+H]+(ESI):1027.35.

[0315] 1H NMR (300MHz, DMSO-d6) δ9.78(s,1H),8.41(t,J=5.8Hz,1H),8.15(q,J=6.8,5.6Hz,2H),8.03(t,J=5.6Hz,1H),7.97-7.88 (m,2H),7.56(d,J=8.3Hz,2H),7.28-7.15(m,7H),7.00(s,2H),5.10(t,J=5.7Hz,1H),4.47(dd,J=24.2,5.5Hz,3H),4.24- 4.13(m,1H),4.07-3.97(m,2H),3.96-3.83(m,2H),3.82-3.58(m,4H),3.10-2.95(m,3H),2.82(dd,J=13.9,9.5Hz,1H),2. 38(t,J=6.2Hz,2H),2.10(t,J=7.3Hz,2H),1.61(d,J=8.0Hz,1H),1.46(t,J=7.5Hz,5H),1.38(s,20H),1.27-1.11(m,4H).

[0316] 31 P NMR(121MHz,DMSO-d6)δ-10.23.

[0317] Step 10. Synthesis of Compound 27

[0318] Under nitrogen protection, a solution of R-budesonide (1.8 g, 4.181 mmol, 1.0 eq) in 20 mL of dichloromethane was mixed with di(4-nitrophenyl) carbonate (1.4 g, 4.599 mmol, 1.1 eq), triethylamine (846.1 mg, 8.362 mmol, 2.0 eq), and 4-dimethylaminopyridine (51.1 mg, 0.418 mmol, 0.1 eq). The reaction mixture was stirred at 0 °C for 15 min. The resulting mixture was diluted with 10 mL of water, and the aqueous layer was extracted with 2 × 30 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography under the following conditions: column: C18 silica gel; mobile phase: water (containing 0.05% FA) and acetonitrile (30% acetonitrile, reaching 100% within 15 min); UV wavelength: 254 nm; the fraction was concentrated under reduced pressure to obtain compound 27 as a white solid (1.5 g, yield: 57.8%).

[0319] MS m / z[M+H]+(ESI):596.15.

[0320] Step 11. Synthesis of Compound 28

[0321] Compound 26 (2.0 g, 1.947 mmol, 1.0 eq) and 4-dimethylaminopyridine (475.8 mg, 3.89 mmol, 2.0 eq) were dissolved in 20 mL of pyridine under nitrogen protection at 0 °C. The resulting solution was stirred at room temperature for 1 h, and the mixture was diluted with 10 mL of water. The aqueous layer was extracted with 2 × 30 mL of dichloromethane, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography under the following conditions: column: C18 silica gel; mobile phase: water (containing 0.05% FA) and acetonitrile (30% acetonitrile, reaching 100% concentration within 15 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 28 as a white solid (850 mg, yield: 23.6%).

[0322] MS m / z[M+H]+(ESI):1483.70.

[0323] 1 H NMR(300MHz,DMSO-d6)δ9.92(s,1H),8.42(t,J=5.9Hz,1H),8.21–8.10(m,2H),8.03(t ,J=5.7Hz,1H),7.97-7.88(m,2H),7.64(d,J=8.4Hz,2H),7.40-7.13(m,8H),6.99(s,2H ),6.17(dd,J=10.1,1.9Hz,1H),5.92(d,J=1.8Hz,1H),5.08(d,J=24.8Hz,3H),4.89-4. 77(m,2H),4.71(d,J=3.5Hz,1H),4.64(t,J=4.4Hz,1H),4.57-4.47(m,1H),4.31(s,1H) ,4.24-4.14(m,1H),4.08-3.94(m,2H),3.94-3.81(m,2H),3.81-3.59(m,4H),3.37(d, J=7.0Hz,2H),3.13-2.95(m,3H),2.83(dd,J=13.8,9.6Hz,1H),2.39(t,J=6.1Hz,2H),2 .29(d,J=12.4Hz,1H),2.12(d,J=7.6Hz,2H),2.00(d,J=16.4Hz,1H),1.80(s,2H),1.63 -1.43(m,10H),1.38(s,25H),1.30-1.10(m,6H),1.07-0.90(m,3H),0.91-0.80(m,6H).

[0324] 31P NMR(121MHz,DMSO-d6)δ-10.24.

[0325] Step 12. Synthesis of compound TM-3

[0326] Compound 28 (850 mg, 0.573 mmol, 1.0 eq) was dissolved in 8.5 mL of trifluoroacetic acid / dichloromethane (v / v, 1:10) under nitrogen protection at 0 °C, and the resulting solution was stirred at room temperature for 20 min. After the reaction was complete, the reaction mixture was concentrated under reduced pressure in an ice bath. The crude product was purified by high performance liquid chromatography under the following conditions: column: C18 silica gel; mobile phase: water (containing 10 mM NH4HCO3) and acetonitrile (10% acetonitrile to 100% within 15 min); UV wavelength: 254 nm. The fraction (300 mL) was directly lyophilized to give compound TM-3 as a white solid (350.3 mg, yield: 44.1%).

[0327] MS m / z[MH]-(ESI):1369.50.

[0328] 1H NMR(400MHz,DMSO-d6)δ10.13(s,1H),8.74(s,1H),8.56(d,J=26.8Hz,2H),8.35-8.20(m,2H),7.91(t,J=5.5Hz,1H),7.68(d,J=8.4Hz,2H),7.36-7.21(m,7H),7.17(td,J=7.1,6.6,3.0Hz,2H),6.99(s,2H),6.17(dd,J=10.1,1.9Hz,1H),5.92(s,1H),5.15-5.02(m,3H),4.81(d,J=18.0Hz,1H),4.74-4.62(m,2H),4.44-4.36(m,1H),4.31(d,J=3.5Hz,1H),4.24-4.16(m,1H),3.96-3.85(m,4H),3.78(dd,J=16.6,6.0Hz,2H),3.70(t,J=5.0Hz,3H),3.64(d,J=5.2Hz,1H),3.37(d,J=7.0Hz,3H),3.10-3.00(m,3H),2.95-2.87(m,1H),2.37-2.26(m,3H),2.16-2.06(m,3H),1.99(d,J=12.5Hz,1H),1.80(s,2H),1.63-1.42(m,11H),1.41-1.25(m,9H),1.21-1.12(m,2H),1.03-0.91(m,2H),0.91-0.82(m,6H).

[0329] 31 P NMR(121MHz,DMSO-d6)δ-0.472.

[0330] 13C NMR(101MHz,DMSO-d6)δ203.901,185.619,172.939,172.910,172.034,171.524,170.636,170.387,169.826,169.568,168.250,156 .947,154.657,139.610,138.422,134.878,130.288,129.647,129.520,128.551,127.578,126.721,122.165,119.506,104.234,97 .623,81.720,69.696,68.498,55.478,55.200,53.247,49.901,46.203,44.128,43.412,42.668,42.511,38.611,37.713,37.459,35.319,34.901,34.335,33.478,31.831,31.637,30.364,29.055,28.247,26.280,25.174,23.007,21.263,16.935,16.861,14.336.

[0331] Example 4 Synthesis of compound TM-4

[0332] The specific synthesis steps are as follows:

[0333] Step 1. Synthesis of Compound 29

[0334] Under nitrogen protection, L-phenylalanine benzyl ester hydrochloride (30.0 g, 102.82 mmol, 1.0 eq) was dissolved in 300 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (26.6 g, 205.632 mmol, 2.0 eq.), Boc-Gly-Gly-Phe-Gly-OH (28.7 g, 123.39 mmol, 1.2 eq.), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (29.6 g, 154.22 mmol, 1.5 eq.), and 1-hydroxybenzotriazole (20.8 g, 154.22 mmol, 1.5 eq.) were added to the solution. The reaction mixture was stirred at room temperature for 2 h. The resulting mixture was diluted with 500 mL of water, the aqueous layer was extracted with 300 mL of ethyl acetate and washed with 200 mL of brine, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: 10 mM ammonium bicarbonate aqueous solution and acetonitrile (acetonitrile concentration increased from 30% to 80% within 15 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 29 as a white semi-solid (37.0 g, yield: 71%).

[0335] MS m / z[M+H]+(ESI):470.30.

[0336] 1 H NMR (300MHz, DMSO-d6) δ8.38(d,J=7.6Hz,1H),7.97(t,J=5.7Hz,1H),7.40-7.17(m,10H),7.01(t,J=6.0Hz,1H),5.12 -5.01(m,2H),4.53(dd,J=8.2,6.3Hz,1H),3.80-3.64(m,2H),3.56(d,J=6.0Hz,2H),3.09-2.89(m,2H),1.38(s,9H).

[0337] Step 2. Synthesis of Compound 30

[0338] Compound 29 (27.0 g, 57.50 mmol, 1.0 eq) was dissolved in 270 mL of trifluoroacetic acid / dichloromethane (v / v = 1:3) under nitrogen protection at 0 °C. The resulting solution was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure in an ice bath to give 21.0 g of compound 30 as a yellow oil (crude), which could be used for the next reaction without further purification.

[0339] MS m / z[MH]-(ESI): 368.10.

[0340] Step 3. Synthesis of Compound 31

[0341] Fmoc-Lys(Boc)-OH (29.3 g, 62.53 mmol, 1.1 eq) was dissolved in 150 mL of N,N-dimethylformamide at 0 °C. O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (32.3 g, 85.27 mmol, 1.5 eq) was added to the solution. Compound 30 (21.0 g, 56.85 mmol, 1.0 eq) was dissolved in 100 mL of N,N-dimethylformamide and then added to the above reaction solution. Then, N,N-diisopropylethylamine (14.7 g, 113.69 mmol, 2.0 eq) and 1-hydroxybenzotriazole (9.2 g, 68.22 mmol, 1.2 eq) were added at 0 °C. The resulting mixture was stirred at room temperature for 1 h, diluted with 500 mL of water, and the aqueous layer was extracted with 300 mL of ethyl acetate and washed with 200 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: 10 mM ammonium bicarbonate aqueous solution and acetonitrile (acetonitrile concentration increased from 30% to 80% within 15 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 31 as a white solid (25.0 g, yield: 50%).

[0342] MS m / z[M+H]+(ESI):820.20.

[0343] 1 H NMR (300MHz, DMSO-d6) δ8.37(d,J=7.6Hz,1H),8.15(t,J=5.6Hz,1H),8.03(t,J=5.8Hz,1H),7. 89(d,J=7.5Hz,2H),7.76-7.66(m,2H),7.51(d,J=7.9Hz,1H),7.45-7.39(m,2H),7.36-7.28(m ,5H),7.28-7.15(m,7H),6.76(s,1H),5.13-4.98(m,2H),4.58-4.45(m,1H),4.33-4.16(m,3H) ,3.97(d,J=6.2Hz,1H),3.83-3.62(m,4H),3.10-2.81(m,4H),1.70-1.47(m,2H),1.36(s,13H).

[0344] Step 4. Synthesis of Compound 33

[0345] Under nitrogen protection at 0 °C, Boc-Ser-OBzl (25.0 g, 84.65 mmol, 1.0 eq) was dissolved in 250 mL of dichloromethane. [bis(tert-butoxy)phosphonyl]diisopropylamine (35.2 g, 126.97 mmol, 1.5 eq) and 1H-imidazolium-4,5-dicarboxynitrile (11.0 g, 93.11 mmol, 1.1 eq) were added to the solution. The resulting mixture was stirred at room temperature for 1 h, and then poured into 300 mL of saturated sodium bicarbonate aqueous solution. The resulting mixture was extracted with 2 × 300 mL of dichloromethane, and the combined organic layers were washed with 300 mL of brine. The organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. Under nitrogen protection, hydrogen peroxide (19.2 g, 169.30 mmol, 2.0 eq, 30%) was added to the crude product dissolved in 250 mL of acetonitrile at 0 °C. The resulting mixture was stirred at room temperature for another 2 h. The combined organic layers were extracted with 2 × 300 mL of dichloromethane and washed with 300 mL of saturated sodium chloride aqueous solution. The organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography under the following conditions: column: C18 silica gel; mobile phase: 10 mM ammonium bicarbonate aqueous solution and acetonitrile (30% acetonitrile, reaching 100% concentration within 15 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 33 as a pale yellow oil (26.0 g, yield: 59%).

[0346] MS m / z[M+H]+(ESI):488.30.

[0347] 1 H NMR (300MHz, DMSO-d6) δ7.42-7.24(m,6H),5.15(s,2H),4.44-4.33(m,1H),4.20-4.01(m,2H),1.38(d,J=2.8Hz,27H).

[0348] 31 P NMR(121MHz,DMSO-d6)δ-10.02.

[0349] Step 5. Synthesis of Compound 34

[0350] Compound 33 (26.0 g, 53.33 mmol, 1.0 eq) was dissolved in 260 mL of tetrahydrofuran. Pd / C (w / w = 10%, 2.6 g) was added to the solution, and inert hydrogen gas was injected. The resulting solution was stirred at room temperature for 2 h. The solution was filtered and washed with tetrahydrofuran. The filtrate was concentrated under reduced pressure to give compound 34 as a white solid (18.0 g, yield: 79%).

[0351] MS m / z[MH]-(ESI):396.15.

[0352] 1 H NMR (300MHz, DMSO-d6) δ6.94 (d, J = 8.2Hz, 1H), 4.27-4.17 (m, 1H), 4.15-4.00 (m, 2H), 1.39 (d, J = 5.3Hz, 27H).

[0353] 31 P NMR(121MHz,DMSO-d6)δ-10.05.

[0354] Step 6. Synthesis of Compound 32

[0355] Compound 31 (25.0 g, 30.50 mmol, 1.0 eq) was dissolved in 250 mL of trifluoroacetic acid / dichloromethane (v / v = 1:3), and nitrogen gas was introduced at 0 °C. The resulting reaction solution was stirred at 0 °C for 1 h, and the reaction mixture was concentrated under reduced pressure in an ice bath to obtain 21.0 g of compound 32 as a yellow oil (crude).

[0356] MS m / z[M+H]+(ESI):720.15.

[0357] Step 7. Synthesis of Compound 36

[0358] Compound 34 (13.9 g, 35.00 mmol, 1.2 eq) was dissolved in 100 mL of N,N-dimethylformamide at 0 °C. O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (16.6 g, 43.76 mmol, 1.5 eq) was added to the solution. Compound 32 (21.0 g, 29.17 mmol, 1.0 eq) was dissolved in 150 mL of N,N-dimethylformamide and the pH was adjusted to be greater than 7 with N,N-diisopropylethylamine. The solutions were then mixed. N,N-diisopropylethylamine (7.5 g, 58.35 mmol, 2.0 eq) and 1-hydroxybenzotriazole (4.7 g, 35.00 mmol, 1.2 eq) were added to the reaction mixture. The resulting mixture was stirred at room temperature for 1 h. The resulting mixture was diluted with 0.0 mL of water, and the aqueous layer was extracted with 300 mL of ethyl acetate and washed with 200 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: 10 mM ammonium bicarbonate aqueous solution and acetonitrile (acetonitrile concentration increased from 30% to 80% within 15 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 36 as a white solid (17.0 g, yield: 49%).

[0359] MS m / z[M+H]+(ESI):1099.60.

[0360] 1 H NMR (300MHz, DMSO-d6) δ8.37(d,J=7.6Hz,1H),8.14(t,J=5.7Hz,1H),8.07-7.97(m,2H),7.89(d,J=7.5Hz,2H ),7.72(t,J=6.7Hz,2H),7.52(d,J=7.8Hz,1H),7.41(dd,J=8.0,6.8Hz,2H),7.36-7.27(m,5H),7.27-7.14(m, 7H),6.77(d,J=8.2Hz,1H),5.14-4.96(m,2H),4.59-4.44(m,1H),4.31-4.12(m,4H),4.08-3.87(m,3H),3.72( t,J=4.7Hz,4H),3.09-2.89(m,4H),1.58(dd,J=26.9,13.3Hz,2H),1.40-1.35(m,26H),1.25(d,J=9.2Hz,5H).

[0361] 31 P NMR(121MHz,DMSO-d6)δ-10.08.

[0362] Step 8. Synthesis of Compound 37

[0363] Compound 36 (17.0 g, 15.47 mmol, 1.0 eq) was dissolved in 170 mL of tetrahydrofuran. Pd / C (w / w = 10%, 1.7 g) was added to the solution, and inert hydrogen gas was injected. The resulting solution was stirred at room temperature for 2 h. The solution was filtered and washed with tetrahydrofuran. The filtrate was concentrated under reduced pressure to give compound 37 as a white solid (13.0 g, yield: 75%).

[0364] MS m / z[MH]-(ESI):1007.45.

[0365] Step 9. Synthesis of Compound 38

[0366] Beclomethasone (25.0 g, 61.14 mmol, 1.0 eq) and p-toluenesulfonic acid (1.3 g, 7.34 mmol, 0.12 eq) were placed in an oven-dried round-bottom flask and dried after azeotropic treatment with toluene. The mixture was dissolved in 250 mL of anhydrous tetrahydrofuran, and triethyl orthopropionate (12.9 g, 73.36 mmol, 1.2 eq) was added dropwise. The reaction mixture was stirred for 1.5 h at room temperature under argon protection. After confirming the absence of starting substrate by TLC, 400 mL of 3 mol / L hydrochloric acid and 50 mL of methanol were added, and the mixture was stirred for 2 h at room temperature. The solvent was removed under reduced pressure, the reaction mixture was alkalized with saturated sodium bicarbonate, and extracted with ethyl acetate. The combined organic layers were dried over brine and the solvent was removed under reduced pressure to give the crude product. The crude product was washed with cold dichloromethane to give compound 38 as a white solid (24.5 g, yield: 78%).

[0367] MS m / z[M+H]+(ESI):465.25.

[0368] 1H NMR(300MHz,DMSO-d6)δ7.30(d,J=10.1Hz,1H),6.23(dd,J=10.1,1.9Hz,1H),5.99(s,1 H),5.57(d,J=4.5Hz,1H),4.94(t,J=6.2Hz,1H),4.40(s,1H),3.90(d,J=5.8Hz,2H),2. 76-2.53(m,3H),2.42-2.27(m,3H),2.19-2.00(m,2H),1.92-1.75(m,2H),1.64-1.45(m ,5H),1.29(d,J=7.2Hz,3H),1.15(d,J=11.1Hz,1H),1.03(t,J=7.4Hz,3H),0.87(s,3H).

[0369] Step 10. Synthesis of Compound 39

[0370] Compound 38 (24.5 g, 52.69 mmol, 2.0 eq) was dissolved in 250 mL of dichloromethane at 0 °C. Then, methyl 2-{[(9H-fluorene-9-ylmethoxy)carbonyl]amino}acetamido)acetate (9.7 g, 26.35 mmol, 1.0 eq) and p-toluenesulfonic acid (2.7 g, 15.81 mmol, 0.6 eq) were added at 0 °C. The mixture was stirred at room temperature for 30 min, and the solvent was removed under reduced pressure. The residue was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: 10 mM ammonium bicarbonate aqueous solution and acetonitrile (30% acetonitrile to 100% gradient, 20 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 39 as a white solid (7.5 g, yield: 15%).

[0371] MS m / z[M+H]+(ESI):773.40.

[0372] 1H NMR (300MHz, DMSO-d6) δ8.72(t,J=6.6Hz,1H),7.90(d,J=7.4Hz,2H),7.72(d,J=7.4Hz,2H),7.61(t,J=6.0Hz,1H),7.42(t,J=7. 4Hz,2H),7.37-7.24(m,3H),6.21(dd,J=10.1,1.9Hz,1H),5.99(d,J=1.8Hz,1H),5.50(d,J=4.4Hz,1H),4.62-4.50(m,2H),4.40( s,1H),4.33-4.18(m,3H),4.07-3.90(m,2H),3.63(d,J=6.1Hz,2H),2.74-2.53(m,3H),2.40-2.28(m,3H),2.11(d,J=8.1Hz,1H) ,2.04(s,1H),1.89-1.75(m,2H),1.59(s,5H),1.26(d,J=7.2Hz,3H),1.15(t,J=11.2Hz,1H),1.02(t,J=7.5Hz,3H),0.88(s,3H).

[0373] Step 11. Synthesis of Compound 40

[0374] Compound 39 (7.5 g, 9.70 mmol, 1.0 eq) was dissolved in 75 mL of N,N-diethylaniline / N,N-dimethylformamide (v / v = 1:40) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h. The solvent was removed under reduced pressure, and the residue was purified by high performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: 10 mM ammonium bicarbonate aqueous solution and acetonitrile (10% acetonitrile to 100% gradient, 20 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 40 as a white solid (4.2 g, yield: 72%).

[0375] MS m / z[M+H]+(ESI):551.40.

[0376] Step 12. Synthesis of Compound 41

[0377] Compound 37 (9.3 g, 9.15 mmol, 1.2 eq) was dissolved in 20 mL of N,N-dimethylformamide. O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.3 g, 11.43 mmol, 1.5 eq) was added to the solution under nitrogen protection at 0 °C. Compound 40 (4.2 g, 7.62 mmol, 1.0 eq) was dissolved in 20 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (2.0 g, 15.24 mmol, 2.0 eq) was added at 0 °C. The two solutions were then mixed at 0 °C, followed by the addition of 1-hydroxybenzotriazole (1.3 g, 9.15 mmol, 1.2 eq) at 0 °C. The resulting mixture was stirred at room temperature for another 1 hour. The mixture was then diluted with 100 mL of water, and the aqueous layer was extracted with 50 mL of ethyl acetate and washed with 50 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: 10 mM ammonium bicarbonate aqueous solution and acetonitrile (from 20% acetonitrile to 100% acetonitrile over 20 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 41 as a white solid (5.8 g, yield: 45%).

[0378] MS m / z[M+H]+(ESI):1542.15.

[0379] 1H NMR (300MHz, DMSO-d6) δ8.59(t,J=6.7Hz,1H),8.31(d,J=6.4Hz,1H),8.14(d,J=8.4 Hz,2H),8.05-7.96(m,2H),7.89(d,J=7.7Hz,2H),7.72(t,J=6.6Hz,2H),7.55(dd,J =22.3,7.5Hz,1H),7.45-7.36(m,2H),7.36-7.28(m,3H),7.27-7.13(m,6H),6.77(d ,J=8.4Hz,1H),6.21(dd,J=10.1,1.9Hz,1H),5.99(s,1H),5.53(d,J=4.4Hz,1H),4.6 1-4.47(m,3H),4.40(s,1H),4.31-4.12(m,4H),4.06-3.88(m,5H),3.69(dd,J=19.4 ,5.6Hz,6H),3.06(d,J=14.1Hz,3H),2.85-2.53(m,4H),2.41-2.26(m,3H),2.12(s, 1H),2.04(d,J=9.4Hz,1H),1.91-1.74(m,2H),1.57(d,J=13.1Hz,6H),1.41-1.35(m ,29H),1.27(d,J=7.3Hz,4H),1.20-1.07(m,2H),1.03(t,J=7.5Hz,3H),0.88(s,3H).

[0380] 31 P NMR(121MHz,DMSO-d6)δ-10.08.

[0381] Step 13. Synthesis of Compound 42

[0382] Compound 41 (5.8 g, 3.76 mmol, 1.0 eq) was dissolved in 60 mL of N,N-diethylaniline / N,N-dimethylformamide (v / v = 1:40). The reaction mixture was stirred at 0 °C for 1 h. The solvent was removed under reduced pressure, and the residue was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: 10 mM ammonium bicarbonate aqueous solution and acetonitrile (10% acetonitrile to 100% gradient, 20 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure. Compound 42 was given as a white solid (3.35 g, yield: 61%).

[0383] MS m / z[M+H]+(ESI):1320.15.

[0384] Step 14. Synthesis of Compound 43

[0385] Fmoc-Gly-OH (830.1 mg, 2.79 mmol, 1.1 eq) was dissolved in 20 mL of N,N-dimethylformamide. O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.92 g, 5.08 mmol, 2.0 eq) was added to the solution. Compound 42 (3.35 g, 2.54 mmol, 1.0 eq) was dissolved in 20 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (656.1 mg, 5.08 mmol, 2.0 eq) was added at 0 °C. The two solutions were then mixed, and 1-hydroxybenzotriazole (411.5 mg, 3.05 mmol, 1.2 eq) was added at 0 °C. The resulting mixture was stirred at room temperature for 1 h and diluted with 100 mL of water. The aqueous layer was extracted with 50 mL of ethyl acetate and washed with 40 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: 10 mM ammonium bicarbonate aqueous solution and acetonitrile (from 20% acetonitrile to 100% acetonitrile over 20 min); UV wavelength: 254 nm. The fraction was concentrated under reduced pressure to give compound 43 as a white solid (2.5 g, yield: 57%).

[0386] MS m / z[M+H] + (ESI): 1599.75.

[0387] 1H NMR (400MHz, DMSO-d6) δ8.61(t,J=6.7Hz,1H),8.35(t,J=5.8Hz,1H),8.23(t,J=5.8Hz,1H) ,8.14(d,J=8.1Hz,1H),8.05-7.96(m,3H),7.89(d,J=7.5Hz,2H),7.71(d,J=7.4Hz,2H),7. 53(t,J=6.1Hz,1H),7.42(dd,J=7.4,1.1Hz,2H),7.35-7.22(m,7H),7.21-7.15(m,1H),6.7 9(d,J=8.4Hz,1H),6.21(dd,J=10.1,1.9Hz,1H),6.02-5.96(m,1H),5.54(d,J=4.4Hz,1H), 4.63-4.48(m,3H),4.40(d,J=4.0Hz,1H),4.34-4.12(m,5H),4.05-3.87(m,4H),3.82-3.56 (m,8H),3.11-2.95(m,3H),2.80(dd,J=13.8,9.7Hz,1H),2.75-2.53(m,3H),2.40-2.29(m, 3H),2.16-2.01(m,2H),1.90-1.77(m,2H),1.67(d,J=6.7Hz,1H),1.62-1.47(m,6H),1.42- 1.35(m,29H),1.27(d,J=7.3Hz,5H),1.18-1.09(m,1H),1.03(t,J=7.5Hz,3H),0.88(s,3H).

[0388] 31 P NMR(162MHz,DMSO-d6)δ-10.12.

[0389] Step 15. Synthesis of Compound 44

[0390] Compound 43 (2.4 g, 1.50 mmol, 1.0 eq) was dissolved in 25 mL of N,N-dimethylformamide under nitrogen protection at 0 °C. Piperidine (1.3 g, 15.00 mmol, 10.0 eq) was added to the solution, and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and the residue was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: 10 mM ammonium bicarbonate aqueous solution and acetonitrile (10% acetonitrile to 100% within 20 min); UV wavelength: 254 nm. The fraction was extracted with ethyl acetate and concentrated under reduced pressure to give compound 44 as a white solid (1.6 g, yield: 72.02%).

[0391] MS m / z[M+H]+(ESI):1376.65.

[0392] Step 16. Synthesis of Compound 45

[0393] At 0 °C, 2-bromoacetic acid (193.7 mg, 1.39 mmol, 1.2 eq) and ethyl 2-ethoxy-1,2-dihydroquinoline-1-carboxylate (574.7 mg, 2.32 mmol, 2.0 eq) were dissolved in 20 mL of N,N-dimethylformamide, and then compound 44 (1.6 g, 1.16 mmol, 1.0 eq) was added at 0 °C. The reaction mixture was stirred at room temperature for 2 h, and the solvent was removed under reduced pressure. The residue was purified by high-performance liquid chromatography (HPLC) under the following conditions: column: C18 silica gel; mobile phase: 3 mM ammonium bicarbonate aqueous solution and acetonitrile (10% acetonitrile to 100% acetonitrile over 20 min); UV wavelength: 254 nm. The fraction was extracted with ethyl acetate and concentrated under reduced pressure to give compound 45 as a white solid (1.12 g, yield: 60%).

[0394] MS m / z[MH] - (ESI): 1496.25.

[0395] 1 H NMR (300MHz, DMSO-d6) δ8.60(s,1H),8.41(d,J=33.8Hz,2H),8.25-8.06(m,3H),7.98(s,2H),7.25(d,J=5.6Hz,6H),6.78(d,J=8 .2Hz,1H),6.21(d,J=10.0Hz,1H),5.99(s,1H),5.53(d,J=4.3Hz,1H),4.55(d,J=7.5Hz,3H),4.40(s,1H),4.17(d,J=28.5Hz,3H) ,4.08-3.88(m,6H),3.85-3.53(m,8H),3.06(d,J=17.8Hz,3H),2.86-2.57(m,4H),2.43-2.26(m,3H),2.07(d,J=10.1Hz,2H),1. 83(s,2H),1.60(s,6H),1.38(d,J=3.0Hz,27H),1.27(d,J=7.3Hz,5H),1.18(t,J=7.1Hz,3H),1.04(t,J=7.5Hz,3H),0.89(s,3H).

[0396] 31 P NMR(162MHz,DMSO-d6)δ-10.12.

[0397] Step 17. Synthesis of compound TM-4

[0398] Compound 45 (500 mg, 0.33 mmol, 1.0 eq) was dissolved in hexafluoroisopropanol (6 mL) at room temperature. The reaction mixture was stirred at 40 °C for 12 h. The solvent was removed under reduced pressure. The residue was purified by high performance liquid chromatography under the following conditions: column: C18 silica gel; mobile phase: water and acetonitrile (10% acetonitrile reached 80% within 20 min); UV wavelength: 254 nm. The product was freeze-dried to obtain compound TM-4 as a white solid (152 mg, yield: 35%).

[0399] MS m / z[MH] - (ESI): 1284.05.

[0400] 1 H NMR (300MHz, DMSO-d6) δ8.95-8.57(m,2H),8.57-7.99(m,6H),7.36-7.12(m,6H),6.21(d,J=10.0Hz,1H),5. 99(s,1H),4.61-4.37(m,4H),4.31-4.18(m,1H),4.16-3.88(m,7H),3.85-3.51(m,11H),3.30(d,J=7.0Hz,3H ),3.09(dd,J=13.7,4.0Hz,2H),3.01-2.82(m,2H),2.75-2.56(m,3H),2.42-2.30(m,3H),2.19-2.00(m,2H), 1.84(dd,J=13.5,7.5Hz,2H),1.58(d,J=11.9Hz,6H),1.46-1.10(m,8H),1.04(t,J=7.5Hz,3H),0.89(s,3H).

[0401] 31 P NMR(121MHz,DMSO-d6)δ-0.002.

[0402] Example 5: Synthesis of Compound TM-5

[0403] The specific synthesis steps are as follows:

[0404] Step 1. Synthesis of Compound 46

[0405] Compound 7 (22 g, 55.4 mmol) and benzyl ((benzyloxy)carbonyl)-L-lysine benzyl ester (27 g, 60.9 mmol) were dissolved in DMF (100 mL). HATU (27 g, 72 mmol) and DIEA (27 mL, 166.2 mmol) were added to the solution. The resulting mixture was stirred at room temperature for 2 h, and the solvent was removed under reduced pressure. The residue was purified by C18 column chromatography using a gradient elution of [ACN / H2O (containing 0.05% FA)] (5%-60%) to give compound 46 as a colorless oil (25 g, yield: 62%).

[0406] LCMS:(M+H) + =750.5.

[0407] Step 2. Synthesis of Compound 47

[0408] Pd / C (500 mg, 10% by weight) was added to a solution of compound 46 (5 g, 6.6 mmol) in MeOH (50 mL). H2 was injected into the mixture three times, and the mixture was stirred at room temperature for 2 h under hydrogen (balloon pressure). The mixture was filtered, and the filtrate was concentrated to obtain compound 47 (3.6 g, yield: 70%) as a white solid, which could be used for the next step without further purification.

[0409] LCMS:(M+H) + =526.4.

[0410] 1 H NMR(400MHz, CDCl3)δ8.18(s,2H),8.00(s,1H),6.01(s,1H),4.45(s,1H),4.31(s,1H),4.15–3.99(m,1H),3.62(s,1H),3 .29(s,1H),3.17(s,1H),2.65–2.18(m,3H),1.95–1.78(m,2H),1.46(s,9H),1.45(s,9H),1.42(s,9H),1.38–1.26(m,2H).

[0411] Step 3. Synthesis of Compound 48

[0412] To a mixture of compound 47 (1.8 g, 3.4 mmol) in DCM (10 mL), DIEA (1.1 mL, 6.8 mmol) and succinimide 6-(maleimide)hexanoate (1.0 g, 3.4 mmol) were added, and the mixture was stirred at room temperature for 15 min. The resulting mixture was concentrated under vacuum at room temperature, and the residue was purified by C18 column chromatography with a gradient elution of [ACN / H2O (containing 0.05% FA)] (5%–60%) to give compound 48 as a white solid (1.5 g, yield: 60%).

[0413] LCMS:(M+H) + =719.5.

[0414] 1 H NMR (400MHz, CDCl3) 1 H NMR (400MHz, CDCl3) δ6.69(s,2H),6.69–6.64(m,1H),5.79(d,J=7.7Hz,1H),4.57–4.52(m,1H),4.37–4.32(m,2H),4.05–3.98(m,1H),3.51(t,J=7 .2Hz,2H),3.40–3.16(m,2H),2.23(t,J=7.4Hz,2H),1.89–1.74(m,2H),1 .68–1.53(m,6H),1.49(d,J=3.1Hz,18H),1.45(s,9H),1.37–1.26(m,4H).

[0415] Step 4. Synthesis of Compound 49

[0416] Compound 48 (1.6 g, 2.2 mmol) was dissolved in DCM (20 mL). EDCI (845 mg, 4.4 mmol) and HOSu (506 mg, 4.4 mmol) were added to the solution. The reaction was stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (EtOAc elution) to obtain compound 49 as a white solid (1.3 g, yield: 66%).

[0417] LCMS:(M+H) + =816.6.

[0418] Step 5. Synthesis of Compound 50

[0419] Compound 5 (750 mg, 0.924 mmol) was dissolved in DCM (10 mL), and compound 49 (753 mg, 0.924 mmol) was added to the solution. The resulting mixture was stirred at room temperature for 70 min, concentrated, and purified by reversed-phase chromatography (ACN / H2O (containing 1% FA) = 70%) to give compound 50 as a white solid (560 mg, yield: 40%).

[0420] LCMS:(M+Na) + =1534.9.

[0421] Step 6. Synthesis of compound TM-5

[0422] Compound 50 (360 mg, 0.238 mmol) was dissolved in DCM (9 mL), and TFA / DCM (1 / 4, v / v, 1.8 mL) was added to the solution. The resulting mixture was stirred for 2 h. The mixture was concentrated and purified by high performance liquid chromatography (ACN / H2O (containing 1% TFA) = 40%) to obtain compound TM-5 as a white solid (122 mg, yield: 41%).

[0423] LCMS:(M+H) + =1244.3.

[0424] 1H NMR (400MHz, DMSO-d6) δ9.96 (s, 1H), 8.56–8.34 (m, 2H), 8.05 (dd, J = 11.1, 7.6Hz, 2H), 7 .49(s,1H),7.45(d,J=8.0Hz,1H),7.38(d,J=8.1Hz,2H),7.31(d,J=10.1Hz,1H),7.23(d ,J=8.1Hz,2H),7.18(d,J=7.9Hz,1H),6.99(s,2H),6.90(d,J=7.5Hz,1H),6.16(dd,J=1 0.1,1.8Hz,1H),5.93(s,1H),5.40(s,1H),4.92(d,J=5.2Hz,1H),4.49(d,J=19.4Hz,1H) ,4.38(d,J=4.7Hz,1H),4.29(s,1H),4.20(s,1H),4.14(d,J=6.7Hz,1H),4.08–3.99(m, 2H),3.99–3.82(m,5H),3.71(s,3H),3.39–3.32(m,14H),3.23–3.14(m,6H),3.04(d,J=6 .6Hz,3H),2.28(dd,J=22.6,13.3Hz,4H),2.20–2.05(m,4H),1.99(s,2H),1.91–1.56(m, 8H),1.55–1.36(m,11H),1.31(s,3H),1.10(ddd,J=26.8,13.2,7.9Hz,5H),0.86(s,3H).

[0425] Example 6: Synthesis of Compound TM-6

[0426] The specific synthesis steps are as follows:

[0427] Step 1. Synthesis of Compound 51

[0428] Methyl 3-hydroxypropionate (1 g, 9.6 mmol) and imidazole (980 mg, 14.4 mmol) were dissolved in THF (30 mL). TBDPSCl (2.9 g, 10.5 mmol) was added to the above reaction solution, and the mixture was stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (PE / EtOAc = 20 / 1 elution) to obtain compound 51 as a colorless oily product (3 g, yield: 93%).

[0429] 1H NMR (400MHz, CDCl3) δ7.72–7.64(m,4H),7.46–7.37(m,6H),3.95(t,J=6.4Hz,2H),3.68(s,3H),2.56(t,J=6.3Hz,2H),1.03(s,9H).

[0430] Step 2. Synthesis of Compound 52

[0431] Compound 51 (3 g, 8.7 mmol) was dissolved in MeOH / H2O (30 mL / 10 mL), and LiOH (2.6 g, 43.8 mmol) was added to the above solution. The mixture was stirred at room temperature for 2 h. The reaction mixture was acidified with hydrochloric acid aqueous solution (1 N) to pH = 5 and extracted with ethyl acetate (50 mL × 2). The organic layer was washed with brine (30 mL × 2), dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (PE / EtOAc = 5 / 1 to 1 / 1 elution) to give compound 52 as a white solid (3.4 g, yield: 80%).

[0432] 1 H NMR (400MHz, CDCl3) δ7.71–7.66(m,4H),7.42–7.35(m,6H),3.94(t,J=6.3Hz,2H),2.59(t,J=6.3Hz,2H),1.06–1.03(m,9H).

[0433] Step 3. Synthesis of Compound 53

[0434] Compound 52 (3 g, 9.1 mmol) and benzyl ((benzyloxy)carbonyl)-L-lysine benzyl ester (4.5 g, 10.2 mmol) were dissolved in DMF (40 mL). HATU (4.5 g, 11.8 mmol) and DIEA (9 mL, 54.6 mmol) were added to the above solution. The resulting mixture was stirred at room temperature for 12 h, diluted with H2O (200 mL), and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over anhydrous Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (PE / EtOAc = 1 / 1 elution) to give compound 53 as a colorless oil (4.8 g, yield: 70%).

[0435] 1H NMR (400MHz, CDCl3) δ7.64(m,4H),7.48–7.26(m,16H),6.47(s,1H),5.56(d,J=8.1Hz,1H),5.22–5.03(m,4H),4.45–4.31(m,1H),3.91(t,J=5 .7Hz,2H),3.26–3.15(m,2H),2.38(t,J=5.7Hz,2H),1.90–1.78(m,1H) ,1.75–1.63(m,1H),1.55–1.41(m,2H),1.40–1.29(m,2H),1.04(s,9H).

[0436] Step 4. Synthesis of Compound 54

[0437] Compound 53 (7.5 g, 11.0 mmol) was dissolved in THF (100 mL), and TBAF (1 M, 22 mL) was added to the solution. The reaction was stirred at room temperature for 2 h. The resulting mixture was concentrated under vacuum, and the residue was purified by C18 column chromatography with a gradient elution of [ACN / H2O (containing 0.05% FA)] (5%-60%) to give 3.4 g of compound 54 as a colorless oil (yield: 88%).

[0438] LCMS:(M+H) + =443.3.

[0439] Step 5. Synthesis of Compound 55

[0440] To a mixture of compound 54 (900 mg, 2.0 mmol) in anhydrous DCM (20 mL), N,N-diethylphosphamide di-tert-butyl ester (1.1 g, 4.0 mmol) and tetrazolium (472 mg, 6.0 mmol) were added. The mixture was stirred at room temperature for 2 h, and then 35% aqueous hydrogen peroxide solution (3 mL) was added at 0 °C. The mixture was stirred at room temperature for 16 h. The mixture was concentrated under vacuum, and the residue was purified by C18 column chromatography using a gradient elution of [ACN / H2O (containing 0.05% FA)] (5%-60%) to give compound 55 as a colorless oil (400 mg, yield: 31%).

[0441] LCMS:(M+H+Na) + =635.5.

[0442] 1H NMR (400MHz, CDCl3) δ7.40–7.28(m,10H),6.20(s,1H),5.59(d,J=8.0Hz,1H),5.17(q,J=12.2Hz,2H),5.10(s,2H),4.44–4.33(m,1H) ,4.28–4.13(m,2H),3.24–3.12(m,2H),2.52(t,J=5.9Hz,2H),1.92–1.80(m,2H),1.73–1.66(m,2H),1.46(s,18H),1.38–1.30(m,2H).

[0443] Step 6. Synthesis of Compound 56

[0444] Pd / C (100 mg, 10% by weight) was added to a solution of compound 55 (700 mg, 1.1 mmol) in MeOH (10 mL). H2 was injected into the mixture three times, and the mixture was stirred at room temperature for 4 h under H2 (balloon pressure). The mixture was filtered, and the filtrate was concentrated to obtain compound 56 as a white solid (400 mg, yield: 88%), which could be used for the next step without further purification.

[0445] LCMS:(M+H) + =411.3.

[0446] Step 7. Synthesis of Compound 57

[0447] To a mixture of compound 56 (350 mg, 0.85 mmol) in DCM (15 mL), DIEA (220 mg, 1.7 mmol) and 6-(maleimide)hexanoic acid succinimide ester (263 mg, 0.85 mmol) were added. The mixture was stirred at room temperature for 30 min. The resulting mixture was concentrated under vacuum. The residue was purified by C18 column chromatography and eluted with a gradient of [ACN / H2O (containing 0.05% FA)] (5%-60%) to give compound 57 as a colorless oily substance (400 mg, yield: 70%).

[0448] LCMS:(M+H) + =604.5.

[0449] 1H NMR (400MHz, CDCl3) δ6.69(s,2H),6.32(t,J=5.5Hz,1H),4.62–4.49(m,1H),4.24–4.16(m,2H),3.54–3.49(m,2H),3.38–3.23(m,2 H),2.52(t,J=5.4Hz,2H),2.23(t,J=7.5Hz,2H),1.89–1.74(m,2H),1.70–1.53(m,6H),1.49(d,J=4.1Hz,18H),1.40–1.28(m,4H).

[0450] Step 8. Synthesis of Compound 58

[0451] (tert-Butoxycarbonyl)-L-valine-L-alanine (25.00 g, 86.70 mmol) was dissolved in THF (250 mL), and p-aminobenzyl alcohol (10.68 g, 86.70 mmol) and EEDQ (42.88 g, 173.40 mmol) were added. The reaction mixture was stirred at room temperature for 3 h. The resulting mixture was concentrated under vacuum, and the residue was purified by silica gel column chromatography (eluting with PE / EtOAc = 5 / 1 to 1 / 1) to give compound 58 as a white solid (24.2 g, yield: 71%).

[0452] LCMS:(M+H) + =394.3.

[0453] 1 H NMR (400MHz, DMSO-d6) δ9.93(s,1H),8.05(d,J=7.0Hz,1H),7.53(d,J=8.4Hz,2H),7.24(d,J=8.4Hz,2H),6.73(d,J=8.8Hz,1H),5.10(t,J =5.7Hz,1H),4.43(d,J=5.6Hz,3H),3.90–3.76(m,1H),1.99–1.89(m,1H),1.39(s,9H),1.30(d,J=7.0Hz,3H),0.85(d,J=20.2,6.7Hz,6H).

[0454] Step 9. Synthesis of Compound 59

[0455] Compound 58 (21 g, 53.37 mmol) was dissolved in THF (210 mL), and DIEA (13.80 g, 106.74 mmol) and di(p-nitrobenzene) carbonate (32.47 g, 106.74 mmol) were added. The resulting mixture was stirred at room temperature for 16 h, the reaction mixture was concentrated, and purified by silica gel column chromatography (eluting with PE:EA = 1:1) to give compound 59 as a yellow solid (25 g, yield: 84%).

[0456] LCMS:(M+Na) + =581.5.

[0457] Step 10. Synthesis of Compound 60

[0458] Compound 59 (2.00 g, 3.58 mmol) and budesonide (1.62 g, 3.58 mmol) were dissolved in DCM (20 mL), and DIEA (972 mg, 7.16 mmol) and DMAP (46 mg, 0.36 mmol) were added. The mixture was stirred at room temperature under N2 protection for 16 h. The resulting mixture was concentrated under vacuum, and the residue was purified by silica gel column chromatography (eluting with PE / EtOAc = 5 / 1 to 1 / 1) to give compound 60 as a white solid (2.17 g, yield: 71%).

[0459] LCMS:(M+H) + =850.5.

[0460] 1H NMR (400MHz, DMSO-d6) δ10.07(s,1H),8.11–8.06(m,1H),7.61(d,J=8.5Hz,2H),7.38–7.29(m,3H),6.72(d,J=8.8Hz,1H),6.17( d,J=10.1,1.7Hz,1H),5.92(s,1H),5.22–5.09(m,3H),5.05(t,J=6.0Hz,1H),4.84(d,J=7.7,3.5Hz,1H),4.80–4.72(m,1H),4.7 2–4.62(m,1H),4.44(d,J=6.9Hz,1H),4.33–4.26(m,1H),3.92–3.78(m,1H),2.29(d,J=11.8Hz,1H),1.95(d,J=6.5Hz,1H),1.85 –1.79(m,2H),1.63–1.51(m,4H),1.49–1.44(m,1H),1.39(s,12H),1.31(d,J=7.0Hz,4H),1.03–0.93(m,2H),0.90–0.81(m,12H).

[0461] Step 11. Synthesis of Compound 61

[0462] Compound 60 (3 g, 3.5 mmol) was added to a solution of 1,4-epoxyhexacyclohexane hydrochloride (40 mL), stirred at room temperature for 2 h, the mixture was filtered, and the filtrate was concentrated to obtain 2.5 g of compound 61 as a yellow solid (hydrochloride), which could be used in the next step without further purification.

[0463] LCMS:(M+H) + =750.6.

[0464] Step 12. Synthesis of Compound 62

[0465] DIEA (105 mg, 0.81 mmol) and HATU (133 mg, 0.35 mmol) were added to a mixture of compound 57 (161 mg, 0.27 mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature for 10 min. Then, compound 61 (200 mg, 0.27 mmol) was added. The reaction mixture was directly purified by C18 column chromatography with a gradient elution of [ACN / H2O (containing 0.05% FA)] (5%-60%) to give compound 62 as a brown solid (160 mg, yield: 45%).

[0466] LCMS:(M+Na) + =1357.7.

[0467] Step 13. Synthesis of compound TM-6

[0468] Compound 62 (160 mg, 0.120 mmol) was dissolved in TFA / DCM (1 / 5, v / v, 5 mL), stirred at room temperature for 1 h, and the reaction mixture was concentrated under reduced pressure. The residue was purified by high performance liquid chromatography by elution with water and acetonitrile (10%-100%) containing 0.1% TFA to give compound TM-6 as a white solid (33 mg, yield: 25%).

[0469] LCMS:(M+H) + =1223.8.

[0470] 1 H NMR (400MHz, DMSO-d6) δ10.03(s,1H),8.18(d,J=7.0Hz,1H),7.95(d,J=8.1Hz,1H),7.88(s,1H),7.66(s,1H),7.64–7.57(m,2H),7.33(t,J=8.9Hz, 3H),7.00(s,2H),6.17(d,J=9.9Hz,1H),5.92(s,1H),5.27–5.07(m,3H), 5.05(s,1H),4.88–4.71(m,2H),4.64(s,1H),4.43–4.28(m,2H),4.30–4.1 3(m,3H),4.04–3.96(m,2H),3.36(t,J=6.7Hz,3H),3.17(s,1H),3.01(s, 2H),2.40(t,J=6.2Hz,2H),2.34–2.27(m,1H),2.10(s,3H),2.02–1.89(m, 2H),1.86–1.74(m,2H),1.56(s,4H),1.47(s,6H),1.42–1.33(m,6H),1.3 3–1.24(m,6H),1.24–1.15(m,3H),1.02–0.92(m,2H),0.92–0.78(m,12H).

[0471] Example 7 Synthesis of Compound TM-7

[0472] The specific synthesis steps are as follows:

[0473] Step 1. Synthesis of Compound 63

[0474] Compound 57 (126 mg, 0.18 mmol) was added to a mixture of compound 5 (100 mg, 0.12 mmol) in DCM (5 mL). The mixture was stirred at room temperature for 2 h, concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography by elution with a gradient of water and acetonitrile (10%-100%) containing 0.1% FA to give compound 63 as a yellow solid (85 mg, yield: 49%).

[0475] LCMS:(M+H) + =1397.9.

[0476] Step 2. Synthesis of compound TM-7

[0477] The solution of compound 63 (85 mg, 0.04 mmol) in TFA / DCM (1 / 5, v / v, 5 mL) was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography by elution with water and acetonitrile (10%-100%) containing 0.1% TFA as a gradient to give compound TM-7 as a yellow solid (15 mg, yield: 28%).

[0478] LCMS:(M+H) + =1230.2.

[0479] 1 H NMR (400MHz, CD3OD_SPE) δ7.47–7.39(m,3H),7.35(d,J=8.1Hz,2H),7.24–7.15(m,3H),6.92(d,J=7.6Hz,1H),6.74(s,2H),6.23(d,J=10.2Hz,1 H),6.00(s,1H),5.43(s,1H),5.03(d,J=5.3Hz,1H),4.61(d,J=19.4Hz, 1H),4.53–4.43(m,1H),4.41(s,1H),4.31(d,J=19.3Hz,1H),4.17(s,2H ),4.01–3.76(m,4H),3.42(t,J=7.0Hz,2H),3.18(s,2H),2.71–2.58(m, 1H),2.52(s,2H),2.47–2.32(m,3H),2.29–2.16(m,4H),2.15–1.99(m,2 H),1.99–1.93(m,1H),1.88–1.65(m,6H),1.62–1.33(m,12H),1.29–1.2 1(m,2H),1.16–1.08(m,1H),1.04(dd,J=11.2,3.4Hz,1H),0.97(s,3H).

[0480] (II) Antibody Preparation and Screening

[0481] Example 8: Preparation of anti-CD74 human-mouse chimeric antibody

[0482] The antibody gene specifically recognizing human CD74 protein was obtained using mouse single-cell RT-PCR technology, and an in vitro expression vector was constructed. Finally, the antibody was obtained using a CHO transient expression system. The specific methods are as follows: First, animal immunization was performed. Five 8-10 week old female Balb / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected and immunized with human CD74 antigen (Baiying Biotechnology; all antigens used in the following experiments were from Baiying Biotechnology). For the initial immunization, Freund's complete adjuvant (Sigma) was used; subsequently, Freund's incomplete adjuvant (Sigma) was used as a recall stimulus. Each mouse received 100 μg of antigen per immunization, with a 3-week interval between immunizations, administered via multiple subcutaneous injections. A small amount of serum sample from the immunized animals was collected and coated with human CD74-his protein onto an ELISA plate at 1 μg / mL, 100 μL per well, incubated overnight at 4°C. The plate was washed three times the following day with PBST (Thermo Fisher Scientific). Block with 2% BSA (Thermo Fisher Scientific), 200 μL per well, incubate at 37°C for 2 h. Remove the blocking solution, blot dry the plate, and add diluted animal serum sample, 100 μL per well, incubate at 37°C for 1 h. Wash the plate three times with PBST, add mouse secondary antibody (Thermo Fisher Scientific) to detect mouse serum, 100 μL per well, incubate at 37°C for 1 h. Wash the plate three times with PBST. Add substrate TMB 100 μL / well for color development, and stop the reaction with 1M HCl, 50 μL per well. Measure OD450 and detect antibody CD74 titer. Based on the immunization results, two mice with a good immune response were selected to obtain spleen and lymph node cells. Memory B cells that specifically bind to human CD74 protein were isolated by flow cytometry. cDNA was obtained by single-cell reverse transcription PCR, followed by two rounds of nested-PCR to obtain the variable genes of the antibody heavy and light chains in single cells. The paired antibody heavy chain variable region genes were sequenced to obtain the antibody gene's base sequence. An antibody expression vector was constructed, and the obtained antibody variable region genes were incorporated into the antibody expression vector containing the human constant region. This vector was then transiently transfected into CHO cells to express the antibody. The CHO cell supernatant was collected, and the antibody was purified by affinity chromatography using protein A to obtain a high-purity antibody. The obtained antibodies were functionally evaluated to screen for effective antibodies.

[0483] Flow cytometry results showed that hCD74-specific memory B cells accounted for approximately 1% of IgG-positive B cells, consistent with expectations. The positive rate of matched antibody genes obtained from single-cell reverse transfection was approximately 60-70%. A total of 280 CD74-specific memory B cells were sorted from two mice, and the matched antibody genes were cloned into human IgG1 heavy chain expression vectors and human IgK light chain expression vectors, respectively. The sequenced plasmids were transiently transfected into ExpiCHO cells (Thermo Fisher Scientific) in 6-well plates (2 mL). After 5 days of transfection, the cell supernatant was collected and coated with human CD74-his protein in ELISA plates (1 μg / mL, 100 μL per well), incubated overnight at 4°C. The plates were washed three times the following day with PBST. Blocking was performed with 2% BSA (200 μL per well), incubated at 37°C for 2 hours. The blocking solution was removed, the plates were dried, and diluted serum samples were added (100 μL per well), incubated at 37°C for 1 hour. Wash the plate three times with PBST, then add 100 μL of secondary antibody (Thermo Fisher Scientific) to each well and incubate at 37°C for 1 h. Wash the plate three times with PBST again. Add 100 μL of substrate TMB per well for color development, and stop the reaction with 1M HCl (50 μL per well). Measure OD450 to detect antibody binding activity. Two mice, numbered 1 and 2, had 17 and 15 cell lines respectively exhibiting human antigen binding activity (as shown in Figures 1 and 2).

[0484] Example 9: Screening of human-mouse chimeric CD74 antibodies

[0485] Based on the ELISA binding activity results of Example 8, 16 antibodies with high binding activity were selected for flow cytometry affinity detection and endocytosis activity detection to screen chimeric antibodies.

[0486] The flow cytometry affinity assay was performed as follows: Raji cells (ATCC) were aspirated from the culture vessel and collected into centrifuge tubes. The cells were centrifuged at 300g for 3 min, the culture medium was discarded, and the cells were resuspended in 0.2% BSA PBS. The cells were centrifuged again at 300g for 3 min to remove the supernatant. Pre-cooled 0.2% BSA PBS was added to the centrifuge tubes for resuspending and cell counting, ensuring a cell concentration of 1×10⁻⁶ cells / mL. 6Cells / mL: Add 50 μL of cells to each well of a 96-well plate and pre-chill on ice. Prepare a 45 μg / mL antibody dilution in 0.2% BSA PBS in another 96-well plate. Add the diluted antibody to each well containing 50 μL of cells. Vortex to mix and incubate on ice for 30 min. Cell washing: Centrifuge the cell suspension at 1000g for 3 min, discard the supernatant, resuspend in 200 μL of pre-chilled 0.2% BSA PBS, centrifuge at 1000g for 3 min, discard the supernatant. Cell incubation with secondary antibody: Dilute the secondary antibody (goat anti-mouse AF647, Thermo Fisher Scientific) 1:300 with pre-chilled 0.2% BSA PBS; add 50 μL of the secondary antibody dilution to each well, vortex to mix, and incubate on ice for 30 min. Cell suspensions in 96-well plates were centrifuged at 1000g for 3 min, the supernatant was discarded, and 200 μL of pre-chilled 0.2% BSA PBS was added for resuspending. Cells were then centrifuged at 1000g for 3 min, and the supernatant was discarded. Cells in each well of the 96-well plate were resuspended in 160 μL of pre-chilled 0.2% BSA PBS and analyzed by flow cytometry. Flow cytometry affinity assays showed that five antibodies (antibodies 2, 3, 6, 9, and 10) exhibited high binding activity to the CD74 protein on the surface of Raji cells (Figure 3, left).

[0487] The method for detecting endocytic activity is as follows:

[0488] Raji cells (ATCC) in culture were aspirated from the culture vessel and collected into centrifuge tubes. The tubes were centrifuged at 300g for 3 min, the culture medium was discarded, and the cells were resuspended in 0.2% BSA PBS. The cells were then centrifuged at 300g for 3 min to remove the supernatant. Pre-cooled 0.2% BSA PBS was added to the centrifuge tubes for resuspending and cell counting, ensuring a cell concentration of 1×10⁻⁶ cells / mL. 6 Add 0.6 × 10⁻⁶ cells / mL to a 1.5 mL EP tube. 5Cells were centrifuged to remove the supernatant. The primary antibody was diluted to 0.6 mL with pre-chilled complete medium, resulting in a final concentration of 10 μg / mL. The secondary antibody (goat anti-human AF647, Thermo Fisher Scientific) was diluted 1:300 with pre-chilled complete medium. Cell incubation with primary antibody: The diluted antibody was added to the pellet of a 1.5 mL EP tube, resuspended, and incubated on ice for 30 mL. The cell suspension was centrifuged at 300 g for 3 min to remove the supernatant, resuspended in 600 μL of pre-chilled complete medium, and centrifuged at 300 g for 3 min to remove the supernatant. The cell pellet was resuspended in 0.6 mL of pre-chilled complete medium, and the cell suspension was evenly distributed into six 1.5 mL EP tubes, labeled 0 h and 3 h, two tubes each, and incubated at 4 °C and 37 °C, respectively. Cell incubation at 37 °C: The EP tubes labeled 0 h and 3 h were incubated at 37 °C for 0 h and 3 h, respectively. Secondary antibody incubation: Centrifuge EP tubes at 300g for 3 min to remove supernatant. Add 100 μL of secondary antibody (Thermo Fisher Scientific) dilution buffer to each tube and mix well. Incubate EP tubes on ice for 30 min. Centrifuge cell suspension at 300g for 3 min to remove supernatant. Resuspend in 600 μL of pre-chilled complete culture medium and centrifuge to remove supernatant. Resuspend cells in EP tubes labeled 0h in 200 μL of pre-chilled complete culture medium and analyze using flow cytometry. After incubation at 3h 4℃ and 3h 37℃, perform secondary antibody incubation, wash, and analyze. Endocytosis activity assay results showed that all five antibodies could mediate the endocytosis of CD74 protein (Figure 3, right).

[0489] The positive control antibody selected for the experiment was Milatuzumab (hLL1), a humanized monoclonal antibody drug targeting human CD74 developed by Immunomedics, with its amino acid sequence referenced in Patent No.: US 8,846,002 B2. Based on the experimental results, antibody number 9, with higher endocytic activity than hLL1, was selected for subsequent humanization. This antibody is the anti-CD74 human-mouse chimeric antibody 2m46.

[0490] The sequence of antibody 2m46, after sequencing, is shown below:

[0491] Heavy chain variable region (VH):

[0492] VH CDR1: SYNMH (SEQ ID NO:2)

[0493] VH CDR2:VIYPGNGDTSYNQKFKG(SEQ ID NO:3)

[0494] VH CDR3:YYGNYEVDY(SEQ ID NO:4)

[0495] Light chain variable region (VL):

[0496] VL CDR1:KSSQSLLYSNGKTYLN(SEQ ID NO:6)

[0497] VL CDR2:LVSKLDS(SEQ ID NO:7)

[0498] VL CDR3: VQGTHFPRT (SEQ ID NO: 8).

[0499] Example 10 Preparation of Humanized Antibody

[0500] Based on the heavy chain variable region sequence of the human-mouse chimeric antibody CD74 antibody 2m46, a comparison was made with the human antibody gene sequence library (NCBI Ig BLAST) to identify corresponding sequences of human germline antibodies with similar heavy chain variable region sequences to those of the human-mouse chimeric antibody 2m46. Specifically, heavy chains FR1, FR2, and FR3 were found to contain 19 non-human sites (V gene), and FR4 contained 1 non-human site (V gene).

[0501] Based on the framework sequence optimization, five humanized antibody heavy chain variable region sequences were designed:

[0502] huVH1:

[0503] huVH2:

[0504] huVH3:

[0505] huVH4:

[0506] huVH5:

[0507] Based on the comparison of the light chain variable region sequence of the human-mouse chimeric antibody CD74 antibody 2m46 with the human antibody gene sequence library (NCBI Ig BLAST), corresponding sequences of human germline antibody variable regions similar to the light chain variable region sequence of human-mouse chimeric antibody 2m46 were identified. Among them, light chains FR1, FR2, and FR3 were found to contain 10 non-human sites (V gene), while FR4 contained 0 non-human sites (V gene).

[0508] Based on the framework sequence optimization, three humanized antibody light chain variable region sequences were designed:

[0509] huVL1:

[0510] huVL2:

[0511] huVL3:

[0512] The humanized antibodies obtained by this invention have the following combinations:

[0513] Table 1 Humanized anti-CD74 antibody combinations

[0514] Example 11 Expression of anti-CD74 humanized monoclonal antibody

[0515] Expression vectors containing the heavy chain constant region (CH) and light chain constant region (CL) of human IgG1 were provided by the Patrick Wilson laboratory. The vector sequences are available in NCBI GenBank: FJ475055 and FJ475056. Based on the humanized heavy chain variable region sequences in Table 1, five humanized heavy chain variable region genes and three light chain variable region genes were synthesized and constructed into the pcDNA3.4 expression vector (Thermo Fisher Scientific) containing the constant regions of hIgG1 and hIgκ. Fifteen humanized antibodies were expressed via transient transfection into CHO suspension cells and purified using protein A. All 15 humanized antibodies were effectively expressed, with expression levels in transiently transfected CHO cells ranging from 1 to 2 mg / 10 mL. SDS-PAGE analysis showed that the purity and monomer content of the 15 humanized antibodies were both above 95% (see Figure 4).

[0516] Example 12 ELISA Validation of Humanized Antibody Binding Activity

[0517] To investigate the binding ability of different humanized antibodies to proteins, the binding activity of recombinant antibodies to soluble recombinant human CD74 protein (Sino Biological, Cat: 11091-H07H) was verified using ELISA. The assay method was as follows: Human CD74 protein was coated onto an ELISA plate at a concentration of 0.5 μg / mL, 100 μL per well, and incubated overnight at 4°C. The next day, the plate was washed three times with PBST and blocked with 2% BSA, 200 μL per well, and incubated at 37°C for 2 h. The blocking solution was removed, the plate was dried, and antibody samples were loaded. Antibody samples were serially diluted 2-fold starting at 4 μg / mL, 100 μL per well, and incubated at 37°C for 1 h. The plate was washed three times with PBST, and Anti-Human IgG (Fc specific) antibody (Thermo Fisher Scientific) was diluted 1:8000, 100 μL per well, and incubated at 37°C for 1 h. Wash the plate three times with PBST. Add 100 μL of TMB substrate per well for color development, and finally add 1M HCl to stop the reaction, 50 μL per well. Measure the absorbance at 450 nm using a microplate reader and process the data. The results are shown in Figure 5, indicating that all 15 humanized antibodies have high binding activity against CD74, and there is no significant difference compared with the chimeric antibody 2m46.

[0518] Example 13 Humanized Antibody Affinity Detection

[0519] The affinity of the antibodies for human CD74 (Sino Biological, Cat: 11091-H07H) was tested using the Bio-Layer Interferometry (BLI) platform (Table 2). Positive control antibodies hLL1 and 2m46 were selected as controls. The results showed that all antibodies could bind efficiently to hCD74, with Hu-14 antibody exhibiting the highest affinity, 4.8 times that of hLL1, and slightly better than 2m46.

[0520] Table 2. Determination of affinity of humanized antibodies

[0521] Example 14: Binding activity of humanized antibody against cell surface CD74

[0522] Four humanized antibodies (Hu-11, Hu-12, Hu-14, and Hu-15) and the chimeric antibody 2m46 were selected. The binding activity of these four humanized antibodies to CD74 protein on the surface of Raji cells (ATCC) was detected using the FACS method. Cells were washed and resuspended with flow cytometry buffer. Specifically, the antibodies were serially diluted 5-fold starting at 100 μg / mL. Equal volumes of antibody and cells were mixed and incubated on ice for 1 h. Cells were washed three times with flow cytometry buffer, collected, and incubated on ice for 30 min in the dark with FITC anti-human IgG (Thermo Fisher Scientific). Cells were washed once with flow cytometry buffer, then once with PBS, and incubated on ice for 30 min in the dark with Fixed Viability Stain 780 (Thermo Fisher Scientific). Cells were washed twice with flow cytometry buffer and resuspended. Analytical analysis was performed using a BD Fortessa flow cytometer. The results showed that all antibodies were able to bind to proteins naturally formed on the surface of Raji cells. Among them, Hu-14 and 2m46 had higher binding activity to proteins naturally formed on the surface of Raji cells and were superior to hLL1 (Figure 6).

[0523] Example 15 Humanized Antibody Internalization Activity Experiment

[0524] The FACS method was used to verify whether humanized antibodies could still mediate rapid endocytosis of CD74 protein. Raji cells (ATCC) were incubated with antibodies and antigens to allow all antigens on the cell surface to bind to the antibodies. They were then incubated at 4°C and 37°C for 0 h and 3 h respectively. After each incubation period, the cells were immediately fixed with 4% PFA. Finally, the cells were uniformly stained with FITC anti-human IgG (Thermo Fisher Scientific) and detected by flow cytometry. If the antibody could mediate receptor endocytosis, at 37°C, with increasing incubation time, the endocytosis of the antigen-antibody complex on the cell surface increased, while the surface antigen-antibody complex gradually decreased, and the amount of fluorescently bound secondary antibody also decreased. At 4°C, the cells were considered to be in a resting state and not mediating endocytosis. Finally, by calculating the MFI and the proportion of positive cells, if the rate of decrease in MFI and the proportion of positive cells was faster at 37°C than at 4°C, endocytosis was considered to be present. The results in Figure 7 show that all four humanized antibodies could mediate CD74 endocytosis, with endocytosis efficiency similar to the positive control hLL1.

[0525] (III) Preparation and property analysis of ADC

[0526] Example 16 Preparation of anti-CD74 antibody-glucocorticoid conjugate

[0527] Diphenylphosphoacetic acid (2.9 to 3.0 equivalents) was mixed with 20 mg of CD74 monoclonal antibody (concentration 10 mg / mL) in PBS buffer solution at pH 7 (the anti-CD74 monoclonal antibody is Hu-14 antibody, whose heavy chain variable region sequence is SEQ ID No: 13, light chain variable region sequence is SEQ ID No: 15, heavy chain constant region is the human IgG1 heavy chain constant region, and light chain constant region is the light chain constant region of the human κ chain) and incubated overnight at 0°C for reduction. The glucocorticoid derivative TM-1 (10 equivalents) prepared in Example 1 was dissolved in 1×PBS and added to the partially reduced anti-CD74 monoclonal antibody solution to continue conjugation at room temperature for 2 h. After the reaction, a 100 kDa ultrafiltration tube (Thermo Scientific) was used. TM Centrifuge for 10 min, then elute and purify using an Akta Avant 150, 50 mL molecular sieve gel column. The elution system is citrate buffer (formulation: citrate dihydrate: 5.59 mg, citrate monohydrate: 0.21 g, sucrose 60 g, plus purified water to a total mass of 1 kg).

[0528] Following the same method described above, anti-CD74 antibody-glucocorticoid conjugates were prepared using glucocorticoid derivatives TM-2 to TM-7 prepared in Examples 2 to 7, respectively. Among them, the conditions for preparing ADC for TM-2 were completely consistent with those for TM-1, and no organic solvent was required. The other compounds and the comparative compound (as shown in Figure 8C) required the use of the organic solvent N,N-dimethylacetamide (DMA) as a solubilizer during coupling, with a DMA ratio of 5% (volume ratio). The other coupling conditions were the same as those for TM-1.

[0529] The aforementioned different anti-CD74 antibody-glucocorticoid conjugates were numbered Hu-14-TM1-ADC to Hu-14-TM7-ADC, as well as a control ADC (prepared by conjugating AbbVie's linker payload with the anti-CD74 monoclonal antibody Hu-14, the linker payload being prepared according to US20220354959A1), and were subjected to the following tests:

[0530] (1) DAR value detection

[0531] ADC sample preparation: Take the ADC sample into a 1.5 mL EP tube and dilute it to 100 μL with PBS buffer (the final concentration of ADC is 1 mg / mL). Add 1 μL of PNGase F enzyme to the diluted sample and incubate it in a metal incubator at 37 °C for 18 h. Then add 100 μL of 8 M guanidine hydrochloride solution and 50 μL of 0.5 M DTT solution, and incubate it in a water bath at 37 °C for 30 min. Centrifuge at 12000 rpm for 5 min, and take 200 μL of the supernatant into a vial for later use.

[0532] LC / MS detection conditions:

[0533] Chromatographic conditions: Acquity UPLC system (Waters Corporation, USA); chromatographic column: ACQUITY BEH Phenyl 1.7μm 2.1×50mm column (Waters Corporation, USA). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The elution program is shown in the table below. The column temperature was 75℃, the TUV was 214nm, and the injection volume was 5μL.

[0534] Mass spectrometry conditions: Synapt G2-Si QTOF (Waters Corporation, USA); ESI source, positive ion mode, MS scanning mode.

[0535] (2) SEC-HPLC detection

[0536] Sample preparation: Take 1 mL of mobile phase and place it in a vial as blank.

[0537] Take 100 μL of sample, transfer it into a sample vial, and place it in the HPLC sample tray.

[0538] Testing conditions:

[0539] Mobile phase: (0.2M potassium dihydrogen phosphate + 0.25M potassium chloride, pH = 6.95 ± 0.05): isopropanol = 85:15 (v / v);

[0540] Chromatographic column: XBridge Protein BEH SEC (7.8×300mm, 3.5μm, 200A);

[0541] Flow rate: 0.5 mL / min;

[0542] Column temperature: 25℃;

[0543] Sample tray temperature setting: 12℃;

[0544] Detection wavelength: 280nm;

[0545] Reference wavelength: 360nm;

[0546] Washing time: 35 min;

[0547] Sample loading amount: 100 μg.

[0548] (3) Thermodynamic (DSF) detection

[0549] The testing instrument was a NANOTEMPER-Prometheus NT.48.

[0550] Dilute the sample to be tested to 1 mg / mL with NB005 diluent. Equilibrate the test solution to room temperature, fill the capillary tube with the sample, and place it in the capillary groove of the injector. Cover with the capillary retainer, then seal both ends of the capillary tube with capillary adhesive. Close the capillary injector and start the test according to the conditions set in the table below.

[0551] Figures 8A-8C show the LC-MS DAR values ​​of different anti-CD74 antibody-glucocorticoid conjugates. Figure 8A shows the LC-MS results of the ADC prepared using TM-1 (Hu-14-TM1-ADC), with DAR = 8; Figure 8B shows the LC-MS results of the ADC prepared using TM-5 (Hu-14-TM5-ADC), with DAR = 6.4; and Figure 8C shows the LC-MS results of the control ADC, with DAR = 4.1.

[0552] Therefore, the glucocorticoid drug-linker derivative prepared by this invention has good hydrophilicity. When conjugated with anti-CD74 monoclonal antibody, the ADC obtained under the same conjugation conditions (equivalent reducing agent, same reaction temperature, same linker-payload small molecule equivalent) has a high DAR value. Furthermore, the LC-MS detection results in Figure 8 show that the ADC product prepared by TM-1 of this invention is a homogeneous DAR8 component with excellent homogeneity. Moreover, TM-1 can also achieve conjugation with antibody under organic solvent-free conditions, which is helpful for research and development in the CMC stage.

[0553] Figures 9A-9C show the SEC-HPLC results of different anti-CD74 antibody-glucocorticoid conjugates. Among them, the ADC prepared using TM-1 has the best purity, reaching 99.4%.

[0554] Tables 3 and 4 show the thermodynamic (DSF) data for different anti-CD74 antibody-glucocorticoid conjugates.

[0555] Table 3

[0556] Table 4

[0557] T m (melting point temperature) and T agg (Aggregation initiation temperature) is a characteristic temperature reflecting protein thermal denaturation and aggregation. m The higher the value, the better the conformational stability of the protein. agg With T m The difference reflects the protein's tendency to aggregate, T monset (denaturation initiation temperature) indicates the temperature at which a protein begins to undergo conformational changes.

[0558] The test results in Tables 3 and 4 show that both ADCs prepared using TM-1 and TM-5 exhibit excellent thermal stability, especially the ADC prepared using TM-1, whose T... m T agg The values ​​were all significantly higher than the control ADC (AbbVie).

[0559] Drug efficacy test

[0560] Animal source:

[0561] The mice used were BALB / c-hCD74 mice, derived from Jicui Pharmaceutical Technology Co., Ltd. The BALB / c-hCD74 mouse model was developed using gene editing technology in BALB / c background mice. This strain can express human-mouse chimeric CD74 protein on the surface of mouse immune cells. This protein possesses the extracellular region of human CD74 while retaining the transmembrane and intracellular regions of mouse CD74. This model is expected to express humanized CD74 at a physiological level in mice and correctly mediate the CD74 signaling pathway. Therefore, the BALB / c-hCD74 mouse model is suitable for evaluating the efficacy and safety of CD74-targeting drugs such as human CD74 inhibitors and CD74-ADCs. B cells were extracted from 8-10 week old mice and used for proliferative experiments.

[0562] Test Example 1: Inflammation Suppression Experiment

[0563] The expression and release of inflammatory cytokines in human PBMCs stimulated with the anti-CD74 antibody-glucocorticoid conjugate prepared in the above examples were detected.

[0564] Human primary peripheral blood mononuclear cells (PBMCs) (purchased from Heyousheng) were washed in 50 mL PBS, resuspended in FBS containing 5% DMSO, aliquoted, and frozen in liquid nitrogen until use. The PBMCs were thawed and resuspended in RPMI medium supplemented with 2% FBS and 1% penicillin-streptomycin, at a density of 1.5 × 10⁶ cells per well. 5 The cells were seeded into a cell assay plate. The groups were set up as follows: Group 1: no LPS stimulation, no other reagents; Group 2: LPS stimulation, PBS; Group 3: LPS stimulation, anti-CD74 monoclonal antibody (final concentration 0.3 nM, abbreviated as "mab" group); Group 4: LPS stimulation, ADC (final concentration 0.3 nM). Following the above group settings, PBS, anti-CD74 monoclonal antibody (Hu-14), and ADC were added to the corresponding groups, and the cells were incubated for 4 hours. Finally, LPS (20 ng / mL) was added for overnight stimulation. The next day, the assay plate was rotated at 1000 rpm for 5 min, and 100 μL of supernatant culture medium was directly transferred to another 96-well plate. The following kits were used to analyze the concentrations of IL-6, TNF-α, and IFN-γ: IL-6 Human Uncoated ELISA Kit (Invitrogen, 88-7066-88), TNF-α Human Uncoated ELISA Kit (Invitrogen, 88-7346-88), and IFN-γ Human Uncoated ELISA Kit (Invitrogen, 88-7316-88). The results are shown in Figure 10.

[0565] Figure 10 shows that the ADC prepared in this invention has a significant anti-inflammatory effect at the cellular level.

[0566] Test Example 2B Cell Inhibition Experiment

[0567] 1. In vitro isolation and culture of B cells

[0568] After preparing single-cell suspensions from spleen and lymph node cells of 8-10 week old mice, red blood cells were removed using erythrocyte lysis buffer. Cells were then resuspended in enrichment buffer (0.5% FBS, 2mM EDTA in 1×PBS) and counted. The suspensions were then diluted to 1×10⁻⁶ cells / mL. 8 Cells / mL, transferred to a 5mL flow cytometry tube for later use. Add STEMCELL EasySep to the flow cytometry tube. TMThe Mouse B Cell Isolation Kit (Catalog #19854) contains 20 μL of FcR blocker and 50 μL of Isolation Cocktail, which are incubated at room temperature for 10 min. After 10 min, RapidSpheres are vortexed. TM Mix the magnetic beads for 30 seconds, then add 75 μL of the magnetic beads to a flow cytometry tube. Gently pipette to mix, and incubate at room temperature for 2.5 min. Bring the volume of the flow cytometry tube to 2.5 mL, and gently pipette up and down 2-3 times. Place the flow cytometry tube on a magnetic rack and incubate at room temperature for 2.5 min. Remove the magnetic rack and slowly tilt to pour the liquid into a new 15 mL centrifuge tube. Centrifuge and count the cells to obtain mouse B cells. Dilute the isolated mouse B cells to 1 x 10⁻⁶ using B cell culture medium (1640 medium (source culture), containing 10% FBS, 1% Glutamine, β-ME). 5 Cells / 200μL. Seed cells in 96-well plates, 200μL per well, for a total of four groups: Group 1: no LPS stimulation; Groups 2 to 4: LPS lipopolysaccharide (20ng / mL) stimulation. PBS, anti-CD74 monoclonal antibody (final concentration 1nM, referred to as "mab" group), and ADC (final concentration 1nM) were added to Groups 2, 3, and 4 respectively. Incubate at 37℃, 5% CO2 for 3 days.

[0569] 2. B cell proliferation markers

[0570] The isolated B cells were washed twice with PBS, diluted to 1×10^6 / mL, and 1 μL of CellTrace was added. TM CTV reagent, incubate at 37°C in the dark for 20 min. After 20 min, add 5 times the volume of complete culture medium, incubate at 37°C in the dark for 5 min, then centrifuge at 400g.

[0571] 3. Flow cytometry detection of cell proliferation

[0572] Cells in 96-well plates were washed with FACS buffer (PBS containing 2% FBS and 2mM EDTA), centrifuged, resuspended in 350μL FACS buffer, and analyzed using a flow cytometer with a 405nm excitation and emission filter. The results are shown in Figure 11.

[0573] Figure 11 shows that the ADC prepared in this invention can significantly inhibit the proliferation of B cells at the cellular level.

[0574] Test Example 3: TLR agonist-induced SLE model mouse experiment

[0575] The TLR agonist-induced mouse model of systemic lupus erythematosus (SLE) is a commonly used model for human SLE. The anti-CD74 monoclonal antibody used in this invention is a humanized antibody that does not recognize the mouse CD74 antigen target. Therefore, BALB / c background CD74 humanized transgenic mice (purchased from Jicui Pharmaceutical, female, catalog number T054480) were selected. The experimental steps are as follows:

[0576] a. Model induction

[0577] Starting on Day 0, mice were divided into two groups according to their body weight: a normal control group (n=4) and a model group (n=16). Mice in the model group received 5% imiquimod cream on the inner side of their right ear three times a week for eight weeks.

[0578] b. Animal grouping and administration

[0579] In week 4, the animals were divided into two groups, G2 and G3, based on dsDNA levels and body weight, and administered the drugs accordingly. The specific design is shown in Table 5.

[0580] Table 5

[0581] Note: BIW, twice a week; ip, intraperitoneal injection; solvent: citrate buffer (formulation: citrate dihydrate: 5.59mg, citrate monohydrate: 0.21g, sucrose 60g, plus purified water to a total mass of 1kg); mouse weight calculated as 25g.

[0582] c. Administration time and method

[0583] G1 (Normal group) did not receive imiquimod induction, but received intraperitoneal injection of the solvent twice a week starting from week 4; G2 (Model group) was induced from day 0, and received intraperitoneal injection of the solvent twice a week starting from week 4; G3 (ADC administration group) was induced from day 0, and received Hu-14-TM1-ADC intraperitoneally starting from week 4.

[0584] d. Ear thickness measurement

[0585] The thickness of the right ear was measured once a week, and the results are shown in Figure 12.

[0586] e. Serum autoantibody marker detection (abnormal antibody SLE biomarker)

[0587] Blood samples were collected every 28 days for testing. The assay was performed using a mouse anti-double-stranded DNA antibody IgG (dsDNA) enzyme-linked immunosorbent assay kit (ELK Biotechnology, ELK8414). The testing procedure is as follows:

[0588] 1. After the kit and sample have equilibrated at room temperature, add 100 μL of standard working buffer (diluted serially according to the instructions) or 100 μL of sample to each well and incubate at 37°C for 80 minutes.

[0589] 2. Discard the liquid in the microplate, add 200 μL of wash buffer to each well, and wash 3 times. After patting dry, add 100 μL of biotinylated antibody working solution to each well and incubate at 37°C for 50 minutes.

[0590] 3. Discard the liquid in the microplate, add 200 μL of wash buffer to each well, and wash 3 times. After patting dry, add 100 μL of HRP enzyme working solution to each well and incubate at 37°C for 50 minutes.

[0591] 4. Discard the liquid in the microplate, add 200 μL of washing buffer to each well, and wash 5 times. After patting dry, add 90 μL of TMB to each well and incubate at 37°C for 20 min.

[0592] 5. Add 50 μL of stop solution to each well, take an immediate reading at 450 nm, calculate the results, and the detection results are shown in Figure 13.

[0593] The results in Figures 12 and 13 show that the anti-CD74 antibody-glucocorticoid conjugate prepared in this invention has excellent anti-inflammatory effects. In a TLR agonist-induced mouse model, the 10 mg / kg dose of ADC showed significant efficacy, rapidly reducing ear inflammation and significantly decreasing ear thickness after administration. In addition, the anti-ds-DNA antibody index was also significantly lower than that in the Model group.

[0594] Furthermore, the ADC of this invention has been observed to effectively downregulate the expression of inflammatory factors such as IL-6, TNF-α, and IFN-γ in SLE model mice, alleviate the symptoms of excessive proteinuria in SLE model mice, and significantly inhibit excessive splenic immunity. Therefore, the anti-CD74 antibody-glucocorticoid conjugate prepared in this invention has great potential to be developed into a drug for the treatment of autoimmune diseases (especially systemic lupus erythematosus).

[0595] Unless otherwise specified, the terms used in this invention have the meanings commonly understood by those skilled in the art.

[0596] The embodiments described in this invention are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Those skilled in the art can make various other substitutions, changes and improvements within the scope of this invention. Therefore, this invention is not limited to the above embodiments, but is only defined by the claims.

Claims

An anti-CD74 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein, the heavy chain variable region comprises a VH CDR1, a VH CDR2 and a VH CDR3 as set forth in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively; and the light chain variable region comprises a VL CDR1, a VL CDR2 and a VL CDR3 as set forth in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The anti-CD74 antibody or antigen-binding fragment thereof of claim 1, wherein, the anti-CD74 antibody or the antigen-binding fragment thereof is of animal origin or is a chimeric antibody; preferably, the amino acid sequence of the heavy chain variable region of the antibody or the antigen-binding fragment thereof is set forth in SEQ ID NO: 1, or has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 5, or has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 5; or, the anti-CD74 antibody or the antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is set forth in any one of SEQ ID NO: 9-13, or has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NO: 9-13; and the amino acid sequence of the light chain variable region is set forth in any one of SEQ ID NO: 14-16, or has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NO: 14-16; preferably, the anti-CD74 antibody or the antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, and / or further comprises a light chain constant region of human kappa, lambda chain or a variant thereof; Further preferably, said antigen binding fragment is selected from the group consisting of Fab, Fv, scFv, F(ab') 2 , dsFv and dAb. a biological material, which is: (1) a nucleic acid molecule encoding the anti-CD74 antibody or the antigen-binding fragment thereof according to claim 1 or 2; (2) a vector containing the nucleic acid molecule of (1); preferably, the vector is an expression vector; (3) a host cell containing the nucleic acid molecule of (1) or the vector of (2). a method for preparing the anti-CD74 antibody or the antigen-binding fragment thereof according to claim 1 or 2, which is: (1) a chemical synthesis method: synthesizing according to the amino acid sequence of the anti-CD74 antibody or the antigen-binding fragment thereof according to claim 1 or 2; (2) a biological preparation method: culturing the host cell of claim 3; further, it also includes isolating the antibody from the obtained culture; and purifying the antibody. a method for preparing the anti-CD74 antibody or the antigen-binding fragment thereof according to claim 1 or 2, which is: (1) a chemical synthesis method: synthesizing according to the amino acid sequence of the anti-CD74 antibody or the antigen-binding fragment thereof according to claim 1 or 2; (2) a biological preparation method: culturing the host cell of claim 3; further, it also includes isolating the antibody from the obtained culture; and purifying the antibody. A composition comprising the anti-CD74 antibody or antigen-binding fragment thereof according to claim 1 or 2, or the biomaterial according to claim 3; preferably, the composition is a medicament, further comprising a pharmaceutically acceptable carrier or excipient. Use of the anti-CD74 antibody or antigen-binding fragment thereof according to claim 1 or 2, or the biomaterial according to claim 3, or the composition according to claim 5, for the manufacture of a medicament for the treatment of an immune disease; preferably, the immune disease is selected from the group consisting of liver fibrosis, type I diabetes, systemic lupus erythematosus and Alzheimer's disease. An antibody-drug conjugate of the structure shown as Formula (I) or Formula (I’), or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein Ab represents an anti-CD74 antibody or antigen-binding fragment thereof; preferably, Ab represents an anti-CD74 antibody or antigen-binding fragment thereof according to claim 1 or 2; L1 represents a linker unit; L1' represents an extension unit; L3 represents a linking unit; D represents a drug unit comprising a glucocorticoid drug; n represents an integer or a decimal number from 1 to 8; L2represents a structure represented by formula (II): in formula (II), A1, A2 each independently represents -C(O)-, -NR1-, -NR1C(O)-, -C(O)NR1-, -O- or -S-; La represents a C1-C6 straight chain or branched chain alkylene substituted with Lb; Lb represents an unsubstituted or substituted C2-C12 straight chain or branched chain alkylene, in which one or more -CH2- is optionally replaced with one or more of -C(O)-, -NR1-, -NR1C(O)-, -C(O)NR1-, -C(O)O-, -OC(O)-, -O-, -S-, and when Lb represents a substituted group, the substituents are selected from one or more of halogen, hydroxy, cyano, amino, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy; B represents R1 represents hydrogen or C1-C6 alkyl; 1 represents attachment to L1 or L1', and 2 represents attachment to L3. The anti-body-drug conjugate according to claim 7, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, wherein, in formula (II), A1, A2 each independently represents -C(O)- or -NH-; La represents a C1-C4 straight chain or branched chain alkylene substituted with Lb; Lb represents -Lc-NHC(O)-Ld- or -Lc-C(O)NH-Ld-, Lc is attached to La, and Ld is attached to B, Lc represents a C2-C6 straight chain or branched chain alkylene, and Ld represents an unsubstituted or amino-substituted C2-C4 straight chain or branched chain alkylene; B represents Preferably, L2represents one of the following structures: 1 represents attachment to L1 or L1', and 2 represents attachment to L3. The antibody-drug conjugate according to claim 7 or 8, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein, L1 represents -A3-Le-A4-, A3 is attached to Ab, and A4 is attached to L2 or L1'; A3represents 1 represents attachment to Ab, and 2 represents attachment to Le; Le represents a C1-C6 straight chain or branched chain alkylene; A4 represents -C(O)-, -NR1-, -NR1C(O)- or -C(O)NR1-; R1 represents hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C4 alkyl; Preferably, L1represents one of the following structures: 1 represents attachment to Ab, and 2 represents attachment to L2 or L1'; and / or L1' represents a structure represented by formula (III): in formula (III), A5, A6, A7 each independently represents -C(O)-, -NR1-, -NR1C(O)- or -C(O)NR1-; Lf represents a C1-C6 straight chain or branched chain alkylene group substituted with Lg; Lg represents an unsubstituted or substituted C1-C12 straight chain or branched chain alkylene group, when Lg represents a substituted group, the substituent is selected from one or more of halogen, hydroxy, cyano, amino, C1-C6 alkyl, halo C1-C6 alkyl, C1-C6 alkoxy; R1 represents hydrogen or C1-C6 alkyl, preferably represents hydrogen or C1-C4 alkyl; 1 represents connection with L1, 2 represents connection with L2; Preferably, in formula (III), A5, A6, A7 each independently represents -C(O)- or -NH-; Lf represents a C1-C4 straight chain or branched chain alkylene group substituted with Lg; Lg represents an unsubstituted C1-C6 straight chain or branched chain alkylene group; More preferably, L1' represents the following structure: 1 represents connection with L1, 2 represents connection with L2; and / or L3 represents a peptide residue consisting of 1 to 6 amino acids selected from one or more of valine, citrulline, glycine, phenylalanine, alanine, isoleucine, lysine, serine, glutamic acid, aspartic acid, which are unsubstituted or each independently substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, C1-6 alkyl, halo C1-6 alkyl, C1-6 alkoxy; Preferably, L3 represents a peptide residue consisting of 1 to 4 amino acids selected from one or more of valine, citrulline, glycine, phenylalanine, alanine, isoleucine, lysine, serine, glutamic acid, aspartic acid; More preferably, L3 represents a peptide residue consisting of -glycine-, -glycine-glycine-, -glycine-glycine-glycine-, -glycine-glutamic acid-, -glycine-valine-, -glycine-alanine-, -glycine-citrulline-, -valine-citrulline-, -valine-alanine-, or -glycine-glycine-phenylalanine-glycine-; Further preferably, L3represents one of the following structures: and / or the glucocorticoid drug is selected from one or more of prednisone, prednisolone, betamethasone, dexamethasone, fluticasone propionate, budesonide, beclometasone; Preferably, D represents a structure according to formula (IV-1), (IV-2) or (IV-3): In formula (IV-1), R2, R3 each independently represents hydrogen, halogen, hydroxy, cyano, amino, C1-C6 alkyl, halo C1-C6 alkyl, or C1-C6 alkoxy; p, q each independently represents 0, 1, 2, 3, or 4; R4, R5 each independently represents hydrogen or C1-C6 alkyl, preferably represents hydrogen or C1-C4 alkyl; R6 represents hydrogen or halogen, preferably represents hydrogen, F, or Cl; X represents -C(R1)2-, -C(O)-, -NR1-, -NR1C(O)-, -C(O)NR1-, -S(O)-, -S(O)2-, -O-, or -S-; Y1 represents -C(O)-, -NR1-, -NR1C(O)-, or -C(O)NR1-; R1represents hydrogen or C1-C6alkyl, preferably hydrogen or C1-C4alkyl; In formula (IV-2), R7 represents C1-C6 alkyl; R8 represents hydrogen or halogen, preferably represents hydrogen, F, or Cl; Z represents unsubstituted or substituted C1-C6 straight chain or branched chain alkylene, when Z represents a substituted group, the substituent is selected from one or more of halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy; preferably, Z represents unsubstituted C1-C4 straight chain or branched chain alkylene; Y2 represents -C(O)-, -NR1-, -NR1C(O)- or -C(O)NR1-; R1represents hydrogen or C1-C6alkyl, preferably hydrogen or C1-C4alkyl; In formula (IV-3), R9 represents hydrogen, halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; m represents 0, 1, 2, 3 or 4; R 10 represents C1-C6alkyl; W represents C1-C6 straight chain or branched chain alkylene, wherein one or more -CH2- is optionally replaced with one or more of -C(O)-, -NR1-, -NR1C(O)-, -C(O)NR1-, -C(O)O-, -OC(O)-, -O-, -S-; preferably, W represents -(CH2)r-OC(O)-, wherein r represents 1, 2, 3 or 4; Y3 represents -C(O)-, -NR1-, -NR1C(O)- or -C(O)NR1-; R1 represents hydrogen or C1-C6 alkyl, preferably represents hydrogen or C1-C4 alkyl; More preferably, D represents one of the following structures: The antibody-drug conjugate according to any one of claims 7-9, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein, The antibody drug conjugate is selected from the group consisting of: wherein, Ab represents an anti-CD74 antibody or an antigen binding fragment thereof, the heavy chain variable region sequence of which is shown as SEQ ID NO: 13, and the light chain variable region sequence of which is shown as SEQ ID NO: 15; further, the anti-CD74 antibody or the antigen binding fragment thereof comprises a human IgG1 heavy chain constant region and a light chain constant region of human κ chain; n represents an integer or a decimal number of 4-8, preferably an integer or a decimal number of 6-8. A drug-linker derivative of the structure shown as Formula (V) or Formula (V’), or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein L0represents a leaving group upon reaction with the antibody Ab, preferably selected from halogen or MeSO2-, more preferably selected from Br or MeSO2-; when L1comprises At that time, it forms together with L0. L1, L1’, L2, L3 and D are each independently as defined in any one of claims 7-10. The method of any one of claims 7-10, wherein the antibody-drug conjugate is prepared by the method of any one of claims 1-6. The preparation method comprises the following steps: S1: reducing the disulfide bond of the antibody Ab using a reducing agent; S2: coupling the reduced antibody Ab with the drug-linker derivative of claim 11 to obtain the antibody-drug conjugate; Preferably, the reducing agent is selected from diphenylphosphinoacetic acid. A pharmaceutical composition, wherein, The pharmaceutical composition comprises: (1) the antibody-drug conjugate of any one of claims 7-10, or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, or the drug-linker derivative of claim 11, or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof; (2) optionally one or more other pharmaceutically active ingredients; and (3) a pharmaceutically acceptable carrier and / or excipient. The use of the antibody-drug conjugate of any one of claims 7-10, or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, the drug-linker derivative of claim 11, or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, or the pharmaceutical composition of claim 13 in the manufacture of a medicament for treating an autoimmune disease. Preferably, said autoimmune disease is selected from systemic lupus erythematosus, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, asthma, rheumatoid arthritis or psoriasis.

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