CN114642739A - Antibody drug conjugate targeting B7-H3, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a B7-H3 targeted antibody drug conjugate, and a preparation method and application thereof. The invention provides an antibody drug conjugate shown in formula I, which has good targeting property, good inhibition effect on tumor cells positively expressing B7-H3, good drug forming property and good safety. The antibody drug conjugate has the inhibiting effect of B7-H3, and also has a good inhibiting effect on at least one of NCI-N87, A375, LN-229, PA-1, MDA-MB-468, Calu-6 and Hs-700T cells.

Description

Antibody drug conjugate targeting B7-H3, and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a B7-H3 targeted antibody drug conjugate, and a preparation method and application thereof.

Background

B7-H3, also known as CD276, was first reported in 2001 (Chapoval AI et al, Nat Immunol 2001, 2 (3): 269-274), was not considered to belong to the Pachylomorphin and myelin oligodendrocyte glycoprotein due to the absence of a header structure and B30.2 domain, and was identified as belonging to the B7 family, a member of the immunoglobulin superfamily (Chapoval AI et al, Nat Immunol 2001, 2 (3): 269-274), in contrast to other family members such as PD-L1, B7-H4, CD80, CD86, etc.: B7-H3 exists in humans as two distinct variants, namely 2IgB7-H3 and 4IgB7-H3, where 4IgB7-H3 is an exon duplication of 2IgB7-H3, and in humans as 4IgB7-H3 (Sun M et al, The Journal of Immunology 2002, 168 (12): 6294-6297; Ling V et al, Genomics 2003, 82 (3): 365-377; Steinberger P et al, J IMMUNOL 2004, 172 (4): 2352-2359), while in mice only The 2IgB7-H3 structure (nM et al, The Journal of Immunology 2002, 168 (12): 6294-6297) is present. The research result shows that 2IgB7-H3 of natural mouse and 4IgB7-H3 of human show similar functions and no functional difference (Ling V et al, Genomics 2003, 82 (3): 365-.

Although mRNA levels of B7-H3 are widely expressed, for example, high levels of mRNA of B7-H3 can be detected in various tissues and organs of human body, including heart, liver, placenta, prostate, testis, uterus, pancreas, small intestine and colon, the protein expression level is relatively limited to non-immune cells such as resting fibroblasts, endothelial cells, osteoblasts and amniotic fluid stem cells, and induced antigen-presenting cells and NK cell surfaces (Hofmeyer KA et al, Proc Natl Acad Sci US A2008, 105 (30): 10277 and 10278; YIKH et al, Immunol Rev 2009, 229 (1): 145 and 151; Picardae et al, CLIN CANCER RES 2016, 22 (14): 3425 and 3431). B7-H3 protein level is low in expression in normal healthy tissues, for example, B7-H3 can be detected in liver, lung, bladder, testis, prostate, breast, placenta, lymph organ tissues of normal human body, while B7-H3 protein is over-expressed in a large number of malignant tumors and is a marker antigen of tumor cells, and researches show that B7-H3 can be highly expressed in various cancers such as prostate cancer, ovarian cancer, colorectal cancer, renal cell carcinoma, non-small cell lung cancer, pancreatic cancer, melanoma, gastric cancer, bladder cancer, malignant glioma and osteosarcoma, especially in various cancers such as head and neck cancer, renal cancer, brain glioma and thyroid cancer (Roth TJ et al, CANCER RES2007, 67 (16): 7893-7900; Zang x et al, MODERN PATHOL 2010, 23 (8): 1104-1112; IngetsVA et al, INTJCANCER2012, 131 (11): 2528 2536; sun J et al, Cancer Immunology, immunotherpy 2010, 59 (8): 1163-1171; crispen PL et al, CLIN CANCER RES 2008, 14 (16): 5150-; zhang G et al, LUNG CANCER 2009, 66 (2): 245-249; yamato I et al, BrJ Cancer 2009, 101 (10): 1709-1716; tekle C et al, INT J CANCER2012, 130 (10): 2282-2290; KatayamaA et al, INTJONCOL 2011, 38 (5): 1219-1226; WuCP et al, World J Gastroenterol2006, 12 (3): 457-; WuD et al, ONCOLLETT2015, 9 (3): 1420-1424), B7-H3 is expressed not only on tumor cells but also on tumor neovascular endothelial cells, and is a very broad-spectrum tumor marker antigen. High expression of the B7-H3 protein can promote cancer progression, and is associated with poor prognosis and poor survival benefit for patients.

Although early research results show that B7-H3 can stimulate the function of activated T cells, promote the proliferation of CD4 and CD8 cells and the secretion of IFN-gamma, intensive research has shown that B7-H3 is a negative regulatory molecule of T cells as an immune check point, and mainly plays the roles of inhibiting the function of the T cells and down regulating the activity of the T cells. Both Woong-Kyung Suh and Durbaka V.R.Prasad studies showed that murine B7-H3 protein significantly inhibited CD4, CD8 cell proliferation dose-dependently (SuhW et al, NATIMMINOL 2003, 4 (9): 899-. Judith Leitner et al also showed that human 4Ig-B7-H3Ig and 2Ig-B7-H3Ig both inhibited T cell proliferation in vitro and inhibited secretion of the relevant cytokines (IFN-. gamma., IL-2, IL-10, IL-13) by CD4 and CD8 cells (Leitner J et al, EURJIMMUNOL 2009, 39 (7): 1754-1764), and further analysis indicated that B7-H3 mediated inhibition of T cell proliferation primarily by inhibition of IL-2 production. While antibodies that target neutralizing B7-H3 in mice significantly promoted The progression of Experimental Autoimmune Encephalomyelitis (EAE) and promoted The proliferation of CD4 cells, objectively demonstrated that B7-H3 inhibits The function of T cells in vivo (Prasad DVR et al, The Journal of Immunology2004, 173 (4): 2500-. In the Woong-Kyung Sub study B7-H3 deficient mice also showed earlier development of experimental autoimmune encephalomyelitis (caused by Th1 cells) compared to wild type mice under immune EAE conditions, suggesting that B7-H3 mainly inhibits Th1 cells (SuhW et al, NAT IMMUNOL 2003, 4 (9): 899-906). As mentioned above, B7-H3 is controversial about T cell function, but promotion of T cell function by B7-H3 is only found in mouse studies at present, while promotion of T cell function by human B7-H3 is not reported for a while, although the receptor of B7-H3 is not determined, B7-H3 is considered as a negative regulatory molecule of T cells by the main theory of the academia at present.

Based on the fact that B7-H3 can inhibit the activity of T cells so as to mediate escape immune surveillance of tumor cells, it is effective to block the binding of B7-H3 and unknown receptors so as to mediate T cell activation and inhibit the activity of the tumor cells, for example, the existing clinical results of Enoblituzumab show that the T cells have different degrees of remission on different tumors and have better curative effects, but a plurality of patients still have disease progression, so that the single development of monoclonal antibodies aiming at B7-H3 still has larger clinical unsatisfied requirements, and the existing clinical results show that the anti-tumor effect of the monoclonal antibodies needs to be further improved.

Antibody-drug conjugates (ADCs) are a new generation of Antibody-targeted therapeutic drugs, mainly applied to the treatment of cancer tumors. The ADC Drug is composed of three parts of a small molecule cytotoxic Drug (Drug), an Antibody (Antibody) and a Linker (Linker) for connecting the Antibody and the cytotoxic Drug, wherein the small molecule cytotoxic Drug is combined on an Antibody protein by a chemical coupling method. The ADC drug specifically recognizes and guides the small molecule drug to reach the cancer cell target point expressing the cancer specific antigen by using the antibody, and enters the cancer cell through the endocytosis effect. The linker moiety is cleaved under the action of intracellular low pH environment or lysosomal protease to release small molecule cytotoxic drugs, thereby achieving the effect of specifically killing cancer cells without damaging normal tissue cells. Therefore, the ADC drug combines the characteristics of targeting specificity of the antibody and high toxicity of the small molecular toxin to cancer cells, and the effective Therapeutic dose window (Therapeutic window) of the drug is greatly expanded. Clinical researches prove that ADC drugs have high drug effect and are relatively stable in blood, can effectively reduce the toxicity of small-molecule cytotoxic drugs (chemical drugs) to the circulation system and healthy tissues, and are the research and development hot spots of the current international anticancer drugs.

Disclosure of Invention

The invention aims to overcome the defect of single species of the existing antibody drug conjugate, and provides a B7-H3 targeted antibody drug conjugate, and a preparation method and application thereof.

The antibody drug conjugate provided by the invention has good targeting property, good inhibition effect on tumor cells positively expressing B7-H3, good drug forming property and good safety. The antibody drug conjugate has the inhibiting effect of B7-H3, and also has a good inhibiting effect on at least one of NCI-N87, A375, LN-229, PA-1, MDA-MB-468, Calu-6 and Hs-700T cells.

The invention solves the technical problems through the following technical scheme.

The invention provides an antibody drug conjugate shown as a formula I or a pharmaceutically acceptable salt;

wherein Ab is B7-H3 antibody; m is 2-8;

the amino acid sequence of the light chain in the B7-H3 antibody is shown in a sequence table SEQ ID NO:1, the amino acid sequence of the heavy chain is shown as the sequence table SEQ ID NO:2 is shown in the specification;

d is

R²And R⁵Are each independently H, C₁-C₆Alkyl or halogen;

R³and R⁶Are each independently H, C₁-C₆Alkyl or halogen;

R⁴and R⁷Are each independently C₁-C₆An alkyl group;

R¹is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl radical, C₁～C₆Alkyl or C₃～C₁₀A cycloalkyl group; r is as described^1-1、R^1-2And R^1-3Are each independently C₁～C₆An alkyl group;

L₁independently one or more of a phenylalanine residue, an alanine residue, a glycine residue, an isoleucine residue, a leucine residue, a proline residue, and a valine residue; p is 2-4;

wherein n is independently 1-12, and c is a carbonyl group and L₁Is connected with the f end and the L₃The d ends of the two are connected;

L₃is composed of

Wherein the b terminal is connected to the Ab, and the d terminal is connected to the L₂Are connected.

In a preferred embodiment of the present invention, in the antibody conjugate drug, some groups have the following definitions, and the definition of the non-mentioned group is as described in any one of the above (the content of this paragraph is hereinafter referred to as "in a preferred embodiment of the present invention"):

said L₃The b-terminus of (a) is preferably linked to a thiol group on the antibody in thioether form. To be provided with

For the purpose of example only,

the connection form with cysteine residue in the antibody is

In a preferred embodiment of the invention, when said R is²And R⁵Are each independently C₁-C₆When alkyl, said C₁～C₆The alkyl group is preferably C₁～C₄The alkyl group is more preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and most preferably a methyl group.

In a preferred embodiment of the invention, when said R is²And R⁵When each is independently a halogen, the halogen is preferably fluorine, chlorine, bromine or iodine, and more preferably fluorine.

In a preferred embodiment of the invention, when said R is³And R⁶Are each independently C₁-C₆When alkyl, said C₁～C₆The alkyl group is preferably C₁～C₄The alkyl group is more preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and most preferably a methyl group.

In a preferred embodiment of the invention, the term "A" or "B" refers to a compound of formula (I)R is as described³And R⁶When each is independently a halogen, the halogen is preferably fluorine, chlorine, bromine or iodine, and more preferably fluorine.

In a preferred embodiment of the present invention, when said R is⁴And R⁷Are each independently C₁-C₆When alkyl, said C₁～C₆The alkyl group is preferably C₁～C₄The alkyl group is more preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and most preferably an ethyl group.

In a preferred embodiment of the invention, R²And R⁵Are each independently C₁-C₆An alkyl group.

In a preferred embodiment of the invention, R³And R⁶Each independently is a halogen.

In a preferred embodiment of the invention, R⁴And R⁷Is ethyl.

In a preferred embodiment of the invention, D is

In a preferred embodiment of the present invention, when D is

When the antibody drug conjugate is a conjugate of

In a preferred embodiment of the present invention, when said R is¹Is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆When alkyl, said C₁～C₆The alkyl group is preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and more preferably an ethyl group.

In a preferred embodiment of the invention, when said R is¹To be multiple-NR^1-1R^1-2Substituted C₁～C₆When an alkyl group is used, the plural is two or three.

In a preferred embodiment of the present invention, when said R is^1-1And R^1-2Each independently is C₁～C₆When alkyl, said C₁～C₆The alkyl group is preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and more preferably a methyl group.

In a preferred embodiment of the invention, when said R is¹Is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆When alkyl, said-NR^1-1R^1-2preferably-N (CH)₃)₂。

In a preferred embodiment of the invention, when said R is¹Is one-NR^1-1R^1-2Substituted C₁～C₆When alkyl, said is substituted by one-NR^1-1R^1-2Substituted C₁～C₆The alkyl group is preferably

In a preferred embodiment of the invention, when said R is¹Is represented by one or more R^1-3S(O)₂-substituted C₁～C₆When alkyl, said C₁～C₆The alkyl group is preferably C₁～C₄The alkyl group is more preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and most preferably an ethyl group.

In a preferred embodiment of the present invention, when said R is¹To be multiple of R^1-3s(O)₂-substituted C₁～C₆When an alkyl group is used, the plural is two or three.

In a preferred embodiment of the present invention, when said R is^1-3Is C₁～C₆When there is alkyl, theC₁～C₆The alkyl group is preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and more preferably a methyl group.

In a preferred embodiment of the invention, when said R is¹Is to be an R^1-3S(O)₂-substituted C₁～C₆When alkyl, said is substituted by one R^1-3S(O)₂-substituted C₁～C₆Alkyl is

In a preferred embodiment of the invention, when said R is¹Is C₁～C₆When alkyl, said C₁～C₆The alkyl group is preferably a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl group, most preferably a methyl group.

In a preferred embodiment of the present invention, m is an integer (e.g., 2, 3, 4, 5, 6, 7 or 8) or a non-integer, preferably 4 to 8, more preferably 6 to 8, further preferably 7 to 8, still further preferably 7.4 to 7.85, e.g., 7.47, 7.48, 7.52, 7.62, 7.64, 7.65, 7.67, 7.72, 7.78, 7.83 or 7.85.

In a preferred embodiment of the present invention, L is₁Preferably one or more of phenylalanine residue, alanine residue, glycine residue and valine residue, more preferably valine residue and/or alanine residue, said plurality is preferably two or three, and said p is preferably 2.

In a preferred embodiment of the present invention, said (L)₁)_pPreferably, it is

Wherein the g terminal is through the carbonyl group and said L₂Are connected with the c terminal of the terminal.

In a preferred embodiment of the present invention, n is preferably 8 to 12, such as 8, 9, 10, 11 and 12, and further such as 8 or 12.

In a preferred embodiment of the invention, the term "A" or "B" refers to a compound of formula (I)R is as described^1-1、R^1-2And R^1-3Independently is preferably C₁～C₆When alkyl, said C₁～C₆Alkyl is preferably C₁～C₄The alkyl group is more preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and most preferably a methyl group.

In a preferred embodiment of the present invention, R is¹Is represented by one or more-NR^i-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-38(O)₂-substituted C₁～C₆Alkyl, or, C₁～C₆An alkyl group; further preferably by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, or substituted by one or more R^1-3S(O)₂-substituted C₁～C₆An alkyl group; most preferably by one or more R^1-3S(O)₂-substituted C₁～C₆An alkyl group.

In the present invention, when Ab is B7-H3 antibody, the B7-H3 antibody is the residue of B7-H3 antibody (a group formed by substituting hydrogen on a sulfhydryl group in the B7-H3 antibody).

In a preferred embodiment of the present invention, the compound represented by formula I is any one of the following schemes:

the first scheme is as follows:

R²and R⁵Are each independently C₁-C₆An alkyl group;

R³and R⁶Each independently is halogen, D is preferably

R¹Is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～₆Alkyl, or, C₁～C₆An alkyl group;

L₁is one or more of phenylalanine residue, alanine residue, glycine residue and valine residue;

scheme II:

R²and R⁵Are each independently C₁-C_aAn alkyl group;

R³and R⁶Each independently is halogen, D is preferably

m is 7 to 8, m is,

R¹is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～2₆Alkyl, or, C₁～C₆An alkyl group;

L₁independently a valine residue and/or an alanine residue;

the third scheme is as follows:

d is

R¹Is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl, or, C₁～C₆An alkyl group;

and the scheme is as follows:

d is

m is 7 to 8, m is,

R¹is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl, or, C₁～C₆An alkyl group;

L₁independently a valine residue and/or an alanine residue.

In a preferred embodiment of the present invention, the antibody drug conjugate is preferably

In a preferred embodiment of the present invention, L is₂Preferably, it is

In a preferred embodiment of the invention, Ab is B7-H3 antibody; d is

L₁Is valine residue and/or alanine residue, p is 2, (L)₁) p is preferably

R¹Is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl or C₁～C₆Alkyl, preferably by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, or substituted by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl, further preferably substituted by one or more R^1-3s(O)₂-substituted C₁～C₆An alkyl group; said R^1-1、R^1-2And R^1-3Independently is C₁～C₄Alkyl, preferably methyl; said group consisting of one or more-NR^1-1R^1-2Substituted C₁～C₆The alkyl group is preferably

Said by one or more R^1-3S(O)₂-substituted C₁～2₆The alkyl group is preferably

L₂Is composed of

L₃Is composed of

In a preferred embodiment of the present invention, the antibody drug conjugate is preferably any one of the following compounds:

wherein Ab is B7-H3 antibody, and m is 7.47, 7.48, 7.52, 7.62, 7.64, 7.65, 7.67, 7.72, 7.78, 7.83 or 7.85.

In a preferred embodiment of the present invention, the antibody drug conjugate is preferably any one of the compounds shown below:

ab is B7-H3 antibody, m is preferably 7.64;

ab is B7-H3 antibody, m is preferably 7.67;

ab is B7-H3 antibody, m is preferably 7.83;

ab is B7-H3 antibody, m is preferably 7.65;

ab is B7-H3 antibody, m is preferably 7.78;

ab is B7-H3 antibody, m is preferably 7.62;

ab is B7-H3 antibody, m is preferably 7.48;

ab is B7-H3 antibody, m is preferably 7.52;

ab is B7-H3 antibody, m is preferably 7.47;

ab is B7-H3 antibody m is preferably 7.85; or

Ab is B7-H3 antibody, m is preferably 7.72.

The invention also provides a preparation method of the antibody drug conjugate, which comprises the following steps: carrying out coupling reaction on a compound shown as a formula II and Ab-hydrogen as shown in the specification;

wherein, L is₁、L₂、L₃、R¹P and Ab are as defined above.

In the present invention, the conditions and operations of the coupling reaction may be those conventional in the art.

The invention also provides a pharmaceutical composition, which comprises a substance X and a pharmaceutic adjuvant, wherein the substance X is the antibody drug conjugate or the pharmaceutically acceptable salt thereof.

In the pharmaceutical composition, the above substance x may be used in a therapeutically effective amount.

The invention also provides an application of the substance X or the pharmaceutical composition in preparing B7-H3 protein inhibitors.

The invention also provides application of the substance x or the pharmaceutical composition in preparing a medicament for treating and/or preventing tumors, wherein the tumors are preferably B7-H3 positive tumors. The B7-H3 positive tumor is preferably one or more of B7-H3 positive lung cancer, ovarian cancer, melanoma, pancreatic cancer, breast cancer, brain glioma, prostate cancer and gastric cancer.

In certain embodiments of the invention, in the lung cancer, the lung cancer cells are NCI-1703 cells or Calu-6 cells;

in certain embodiments of the invention, in the ovarian cancer, the ovarian cancer cells are PA-1 cells;

in certain embodiments of the invention, in the melanoma, the melanoma cells are a375 cells;

in certain embodiments of the invention, in the pancreatic cancer, the pancreatic cancer cells are Hs-700T cells;

in certain embodiments of the invention, in the breast cancer, the breast cancer cells are MDA-MB-468 cells;

in certain embodiments of the invention, in said brain glioma, the brain glioma cells are LN-229 cells;

in certain embodiments of the invention, in the gastric cancer, the gastric cancer cells are NCI-N87 cells.

Unless otherwise indicated, the following terms appearing in the specification and claims of the invention have the following meanings:

the medicinal auxiliary materials can be auxiliary materials widely adopted in the field of medicine production. The excipients are used primarily to provide a safe, stable and functional pharmaceutical composition and may also provide methods for dissolving the active ingredient at a desired rate or for promoting the effective absorption of the active ingredient after administration of the composition by a subject. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients may include one or more of the following excipients: buffers, chelating agents, preservatives, co-solvents, stabilizers, excipients and surfactant colorants, flavors and sweeteners.

The term "pharmaceutically acceptable" means that the salts, solvents, excipients, etc., are generally non-toxic, safe, and suitable for use by the patient. The "patient" is preferably a mammal, more preferably a human.

The term "pharmaceutically acceptable salt" refers to salts prepared from the compounds of the present invention with relatively nontoxic, pharmaceutically acceptable acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral forms of such compounds with a sufficient amount of a pharmaceutically acceptable base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to: lithium salt, sodium salt, potassium salt, calcium salt, aluminum salt, magnesium salt, zinc salt, bismuth salt, ammonium salt, and diethanolamine salt. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent. The pharmaceutically acceptable acid includes inorganic acids including, but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like. The pharmaceutically acceptable acids include organic acids including, but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, saccharic acid, formic acid, ethanesulfonic acid, pamoic acid (i.e. 4, 4' -methylene-bis (3-hydroxy-2-naphthoic acid)), amino acids (e.g. glutamic acid, arginine), and the like. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they may be converted to base addition salts or acid addition salts. See, in particular, Berge et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19(1977), or, Handbook of Pharmaceutical Salts: properties, Selection, and Use (P.Heinrich Stahl and Camille G.Wermuth, ed., Wiley-VCH, 2002).

The natural or native sequence of B7-H3 may be isolated from nature or may be prepared by recombinant DNA techniques, chemical synthesis, or a combination of these and similar techniques.

Antibodies are herein to be interpreted in their broadest sense and specifically bind to a target, such as a carbohydrate, polynucleotide, fat, polypeptide, etc., through at least one antigen recognition region located in the variable region of the immunoglobulin molecule. Specifically included are intact monoclonal antibodies, polyclonal antibodies, bispecific antibodies, and antibody fragments, so long as they possess the desired biological activity.

Antibodies of the invention can be prepared using techniques well known in the art, such as hybridoma methods, recombinant DNA techniques, phage display techniques, synthetic techniques, or combinations thereof, or other techniques known in the art.

Description of the term "drug-antibody conjugation ratio" (i.e. DAR). L-D is a group reactive with the conjugation site on the antibody, L is a linker, D is a cytotoxic agent further conjugated on the antibody linked to L, D is Dxd in the present invention, the number of DAR finally conjugated D per antibody is represented by m or m can also represent the number of individual antibodies conjugated D. In some embodiments, m is actually an average value between 2 and 8, 4 and 8, or 6 and 8, or m is some integer of 2, 3, 4, 5, 6, 7, or 8; in some embodiments, m is an average of 2, 4, 6, or 8; in other embodiments, m is an average of 2, 3, 4, 5, 6, 7, or 8.

Linker refers to a direct or indirect linkage between an antibody and a drug. Attachment of the linker to the mAb can be accomplished via a number of means, such as via surface lysines, reductive coupling to oxidized carbohydrates, and via reduction of cysteine residues released by interchain disulfide bonds. A variety of ADC ligation systems are known in the art, including hydrazone, disulfide and peptide-based ligation.

The term "treatment" or its equivalent when used with reference to, for example, cancer, refers to a procedure or process for reducing or eliminating the number of cancer cells in a patient or alleviating the symptoms of cancer. "treating" cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will actually be eliminated, that the number of cells or disorders will actually be reduced or that the symptoms of the cancer or other disorder will actually be alleviated. Generally, methods for treating cancer are performed even with a low likelihood of success, but are still considered to induce an overall beneficial course of action, given the patient's history and estimated survival expectations.

The term "prevention" refers to a reduced risk of acquiring or developing a disease or disorder.

The term "cycloalkyl" refers to a saturated cyclic hydrocarbon radical having three to twenty carbon atoms (e.g., C)₃-C₆Cycloalkyl), including monocyclic cycloalkyl. Cycloalkyl groups contain 3 to 20 carbon atoms, preferably 3 to 10 carbon atoms, more preferably 3 to 6 carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloOctyl, cyclononyl, and cyclodecyl.

The term "alkyl" refers to a straight or branched chain alkyl group having the indicated number of carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, and the like.

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

The term "heteroaryl" refers to an aryl (or aromatic ring) containing 1, 2, 3, or 4 heteroatoms independently selected from N, O and S, which may be a monocyclic aromatic system, such as furyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, and the like.

The term "aryl" refers to any stable monocyclic or bicyclic carbocyclic ring wherein all rings are aromatic. Examples of the above aryl unit include phenyl or naphthyl.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

Unless otherwise specified, the room temperature in the present invention means 20 to 30 ℃.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

1. the antibody drug conjugate containing the B7-H3 antibody provided by the invention has good targeting property and good inhibition effect on various tumor cells expressing B7-H3 and the like.

2. In vivo studies show that the antibody drug conjugate has better in vitro cytotoxicity and in vivo anti-tumor activity.

3. The antibody drug conjugate has good solubility and good drug forming property, has no abnormal phenomena such as precipitation and the like in the coupling preparation process, and is very beneficial to the preparation of the antibody drug conjugate.

Drawings

FIG. 1 shows the construction of light and heavy chain expression vectors of FDA016 antibody, wherein Ab-L is the antibody light chain and Ab-H is the antibody heavy chain.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Description of abbreviations:

PCR polymerase chain reaction

CHO Chinese hamster ovary cells

HTRF homogeneous time-resolved fluorescence

PB phosphate buffer

EDTA ethylene diamine tetraacetic acid

TECP tris (2-carboxyethyl) phosphine

DMSO dimethyl sulfoxide

DMF N, N-dimethylformamide

HATU 2- (7-benzotriazol oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate

V/V V/V, volume ratio

UV ultraviolet visible light

ELISA enzyme-linked immunosorbent assay

BSA bovine serum albumin

rpm revolutions per minute

FBS fetal bovine serum

Example 1: preparation of B7-H3 antibody

In the invention, a monoclonal antibody FDA016 with high affinity and specific targeting B7-H3 is selected, and the amino acid sequence of the light chain is shown as SEQ ID NO:1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO:2, respectively. FDA016 has its light and heavy chain nucleotide sequence obtained by means of whole gene synthesis (suzhou jinzhi). Separately constructed into pV81 vector (as shown in FIG. 1) by two enzyme cleavages of EcoR I and Hind III (available from TAKARA), and transformed into Trans1-T1 competent cells (available from King Kokino gold, Beijing) by ligationBiology, cargo number: CD501-03), from which clones were picked for PCR identification and checked, and sequencing confirmed, and culture-amplified positive clones were extracted from the plasmids to obtain an antibody light chain eukaryotic expression plasmid FDA016-L/pV81 and an antibody heavy chain eukaryotic expression plasmid FDA016-H/pV81, which were expressed using XbaI (purchased from Takara, cat No.: 1093S), transforming to CHO cells (purchased from ATCC) adapted to suspension growth by electric shock, connecting the cells after electric shock to 96-well plates by 2000-5000 cells/well, measuring expression quantity by HTRF method (homogeneous phase time-resolved fluorescence) after culturing for 3 weeks, selecting the first ten cells from the cells, amplifying, and freezing. Resuscitating a cell into a 125ml shake flask (culture volume 30m1), 37 deg.C, 5.0% CO₂Shaking culture at 130rpm, 3 days later expanding to 1000ml shake flask (culture volume 300ml), 37 deg.C, 5.0% CO₂Shaking culture at 130rpm, supplementing a supplemented medium with an initial culture volume of 5-8% every other day from the fourth day, culturing until 10-12 days, finishing the culture, centrifuging the harvest solution at 9500rpm for 15min, removing cell precipitates, collecting supernatant, filtering with a 0.22 μm filter membrane, and purifying the treated sample by using a MabSelect affinity chromatography column (purchased from GE company) to obtain the antibody FDA 016.

The light chain amino acid sequence of FDA016 is shown below:

SEQ ID NO：1：

the amino acid sequence of FDA016 is as follows:

SEQ ID NO：2：

example 2: synthesis of linker-drug conjugates

Example 2-1: synthesis of LE12

Synthesis of intermediate 2:

(S) -2-azidopropionic acid (10g, 86.9mmol) and 4-aminobenzyl alcohol (21.40g, 173.8mmol) were dissolved in 300mL of a mixed solvent of dichloromethane and methanol (volume ratio 2: 1), 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (21.49g, 86.9mmol) was added, and after 5 hours at room temperature, the solvent was evaporated under reduced pressure, and then the resulting crude product was purified by silica gel column chromatography [ dichloromethane: ethyl acetate ═ 1: 1(v/v) ] to give intermediate 2(16.3g, yield 85%), ESI-MSm/z: 221(M + H).

Synthesis of intermediate 3:

intermediate 2(15g, 68.2mmol) and bis (p-nitrophenyl) carbonate (22.82g, 75.02mmol) were mixed and dissolved in 200mL of anhydrous N, N-dimethylformamide, and 25mL of triethylamine was added to react at room temperature for 2 hours. After the reaction of the starting materials was monitored by LC-MS, methylamine hydrochloride (6.91g, 102.3mmol) was added and the reaction was continued at room temperature for 1 hour. After the reaction was completed, most of the solvent was distilled off under reduced pressure, and then 200mL of water and 200mL of ethyl acetate were added, the organic phase was collected after liquid separation, the organic phase was dried and concentrated, and the obtained crude product was purified by silica gel column chromatography [ dichloromethane: ethyl acetate ═ 10: 1(v/v) ] to obtain intermediate 3(18.9g, yield 100%), ESI-MS m/z: 278(M + H).

Synthesis of intermediate 5:

mixing the intermediate 3(10g, 36.1mmol) and paraformaldehyde (1.63g, 54.2mmol), dissolving in 150mL of anhydrous dichloromethane, slowly adding trimethylchlorosilane (6.28g, 57.76mmol), reacting at room temperature for 2 hours to obtain a crude solution of the intermediate 4, monitoring the reaction by sampling and adding methanol to quench the solution, filtering the reaction solution after the reaction is finished, adding tert-butyl glycolate (9.54g, 72.2mmol) and triethylamine (10mL, 72.2mmol) into the filtrate, and continuing to react at room temperature for 2 hours. After the completion of the reaction, most of the solvent was removed by distillation under reduced pressure, and the obtained crude product was purified by silica gel column chromatography [ petroleum ether: ethyl acetate ═ 3: 1(v/v) ] to obtain intermediate 5(11.2g, yield 74%), ESI-MS m/z: 422(M + H).

Synthesis of intermediate 6:

intermediate 5(10g, 23.8mmol) was dissolved in 80mL of anhydrous tetrahydrofuran, 80mL of water was added, followed by tris (2-carboxyethylphosphine) hydrochloride (13.6g, 47.6mmol), and the reaction was carried out at room temperature for 4 hours. After the reaction was completed, tetrahydrofuran was distilled off under reduced pressure, followed by extraction with ethyl acetate, the resulting organic phase was dried, the solvent was distilled off under reduced pressure, and purification was performed by silica gel column chromatography [ dichloromethane: methanol ═ 10: 1(v/v) ] to obtain intermediate 6(8.1g, yield 86%), ESI-MS m/z: 396(M + H).

Synthesis of intermediate 8:

intermediate 6(5g, 12.7mmol) was dissolved in 60mL of a mixed solvent of dichloromethane and methanol (v/v: 2: 1), and 3mL of trifluoroacetic acid was slowly added thereto, followed by reaction at room temperature for 30 minutes. After the reaction, water and ethyl acetate of the same volume are added, the organic phase is dried and concentrated, and the obtained crude product is directly used in the next step.

The crude product obtained in the above step was dissolved in 50mL of anhydrous N, N-dimethylformamide, and Fmoc-valine hydroxysuccinimide ester (8.3g, 19.1mmol) and triethylamine (5mL) were added to the solution to react at room temperature for 2 hours. After the reaction was completed, most of the solvent was removed by distillation under the reduced pressure, and the obtained crude product was purified by silica gel column chromatography [ dichloromethane: methanol ═ 10: 1(v/v) ] to obtain intermediate 8(5.4g, yield 64%), ESI-MSm/z: 661(M + H).

Synthesis of intermediate 9:

the intermediate 8(1g, 1.5mmol) and Exatecan mesylate (0.568g, 1mmol) were mixed in 30mL of anhydrous N, N-dimethylformamide, and 2mL of 2- (7-benzotriazole oxide) -N, N' -tetramethyluronium hexafluorophosphate (1.14g, 3.0mmol) and triethylamine were added and reacted at room temperature for 2 hours. After the reaction was completed, the solvent was removed by distillation under the reduced pressure, and the crude product was purified by silica gel column chromatography [ chloroform: methanol ═ 10: 1(v/v) ] to obtain intermediate 9(0.94g, yield 87%), ESI-MS m/z: 1078(M + H).

Synthesis of compound LE 12:

intermediate 9(1g, 0.929mmol) was dissolved in 20mL of anhydrous DMF and 0.5mL of 1, 8-diazabicycloundec-7-ene was added and reacted at room temperature for 1 hour. After the starting material had reacted, succinimidyl 6- (maleimido) hexanoate (428.5mg, 1.39mmol) was added directly and stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography [ chloroform: methanol ═ 8: 1(v/v) ] to give the title compound (0.7g, yield 73%), ESI-MS m/z: 1035(M + H).

Example 2-2: synthesis of Compound LE13

Synthesis of intermediate 14

After commercially available intermediate 12(267mg, 0.8mmol) was mixed with paraformaldehyde (50mg, 1.6mmol), dissolved in 20mL of anhydrous dichloromethane, chlorotrimethylsilane (0.3mL, 3.4mmol) was slowly added, and the reaction was carried out at room temperature for 2 hours after the completion of the addition. Then, the reaction was monitored by liquid chromatography-mass spectrometry after sampling and quenching with methanol, after the reaction was completed, the reaction solution was filtered, then tert-butyl glycolate (211mg, 1.6mmol) and dipyridamole 0.5mL were added to the filtrate, the reaction was continued at room temperature for about 2 hours, most of the solvent was removed by distillation under reduced pressure after the reaction was completed, and the obtained crude product was purified by silica gel column chromatography [ dichloromethane: methanol ═ 20: 1(v/v) ] to obtain intermediate 14(260mg, yield 68%), ESI-MS m/z: 479(M + H).

Synthesis of intermediate 15

Intermediate 14(238mg, 0.50mmol) was dissolved in 6mL of a mixed solvent of dichloromethane and methanol (v/v ═ 2: 1), and 0.3mL of trifluoroacetic acid was slowly added to the solution to react at room temperature for 30 minutes. After the reaction, water and ethyl acetate of the same volume are added, the organic phase is dried and concentrated, and the obtained crude product is directly used in the next step.

Synthesis of intermediate 16

The crude product obtained in the above step was mixed with Exatecan mesylate (170mg, 0.30mmol) in 5mL of anhydrous N, N-dimethylformamide, and 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (341mg, 0.90mmol) and triethylamine (0.60 mL) were added and reacted at room temperature for 2 h. After the reaction was completed, the solvent was removed by distillation under the reduced pressure, and the obtained crude product was purified by silica gel column chromatography [ chloroform: methanol ═ 10: 1(v/v) ] to obtain intermediate 16(210mg, 83%), ESI-MS m/z: 840(M + H).

Synthesis of intermediate 17

Intermediate 16(100mg, 0.12mmol) was dissolved in 15mL of anhydrous tetrahydrofuran, 3mL of water was added, and then 0.3mL of a1 mol/L aqueous solution of triethylphosphine was added, and the reaction was carried out at room temperature for 4 hours. After completion of the reaction, the reaction solution was distilled under reduced pressure to remove tetrahydrofuran, sodium bicarbonate was added to the remaining aqueous solution to adjust pH to neutral, dichloromethane was then added for extraction, the obtained organic phase was dried and the solvent was distilled off under reduced pressure, and the obtained crude product was purified by silica gel column chromatography [ dichloromethane: methanol ═ 10: 1(v/v) ] to obtain intermediate 17(69mg, yield 71%), ESI-MS m/z: 814(M + H).

Synthesis of Compound LE13

Intermediate 17(120mg, 0.15mmol) obtained according to the synthesis method of the above step was mixed with a commercially available raw material MC-V (102mg, 0.33mmol) in 40mL of dichloromethane, a condensing agent 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (82mg, 0.33mmol) was added, the mixture was reacted overnight at room temperature, the solvent was evaporated under reduced pressure after the reaction was completed, and the obtained crude product was purified by silica gel column chromatography [ dichloromethane: methanol ═ 10: 1(V/V) ] to obtain compound LE13(116mg, yield 70%), ESI-MS m/z: 1106.5(M + H).

Examples 2 to 3: synthesis of Compound LE14

Synthesis of intermediate 19

Commercially available intermediate 18(300mg, 0.8mmol) was dissolved in 20mL of anhydrous dichloromethane in admixture with paraformaldehyde (50mg, 1.6mmol), and trimethylchlorosilane (0.3mL, 3.4mmol) was slowly added and reacted at room temperature for 2 hours, with the reaction monitored by liquid chromatography after sample quenching with methanol. After the reaction was completed, the reaction mixture was filtered, tert-butyl glycolate (211mg, 1.6mmol) and triethylamine (0.22m, 1.6mmol) were added to the filtrate, the reaction was continued at room temperature for about 2 hours, most of the solvent was removed by distillation under reduced pressure after the completion of the reaction, and the resulting crude product was subjected to silica gel column chromatography [ dichloromethane: methanol ═ 20: 1(v/v) ]]Purification afforded intermediate 19(349mg, 85% yield), ESI-MSm/z: 514(M + H) of the reaction mixture,¹H NMR(400MHz，CDCl₃)δ8.13(s，1H)，7.56(d，J＝7.5Hz，2H)，7.35(s，2H)，5.14(s，2H)，4.91(s，2H)，4.25(q，J＝7.1Hz，1H)，3.99(d，J＝42.5Hz，2H)，3.85(t，J＝6.2Hz，2H)，3.40(dd，J＝18.5，7.6Hz，2H)，2.89(d，J＝48.6Hz，3H)，1.65(d，J＝6.8Hz，3H)，1.46(s，9H)。

synthesis of intermediate 20

Intermediate 19(257mg, 0.50mmol) was dissolved in 6mL of a mixed solvent of dichloromethane and methanol (v/v. about.2: 1), and 0.3mL of trifluoroacetic acid was slowly added thereto to react at room temperature for 30 minutes. After the reaction, water and ethyl acetate of the same volume are added, the organic phase is dried and concentrated, and the obtained crude product is directly used in the next step.

The resulting crude product and Exatecan mesylate (170mg, 0.30mmol) were mixed in 5mL of anhydrous N, N-dimethylformamide, and 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (341mg, 0.90mmol) and triethylamine (0.60 mL) were added and reacted at room temperature for 2 h. After the reaction, the solvent was removed by distillation under the reduced pressure, and the crude product was subjected to silica gel column chromatography [ dichloromethane: methanol ═ 20: 1(v/v)]Purification afforded intermediate 20(212mg, 81% yield), ESI-MS m/z: 875(M + H).¹H NMR(400MHz，CDCl₃)δ8.27(d，J＝34.7Hz，1H)，7.63-7.35(m，5H)，7.21-7.10(m，1H)，5.71-5.48(m，2H)，5.24-4.95(m，3H)，4.95-4.72(m，4H)，4.45(s，1H)，4.33-3.97(m，3H)，3.75(s，2H)，3.39-2.99(m，4H)，2.76(d，J＝15.3Hz，3H)，2.43-2.15(m，5H)，2.04(s，1H)，1.94-1.75(m，2H)，1.62(d，J＝6.6Hz，3H)，1.11-0.89(m，3H)。

Synthesis of intermediate 21

Intermediate 20(77mg, 0.09mmol) was dissolved in 12mL of anhydrous tetrahydrofuran, 3mL of water was added, and then 0.3mL of a1 mol/L aqueous solution of triethylphosphine was added, and the reaction was carried out at room temperature for 4 hours. After the reaction, the tetrahydrofuran is removed by distillation under reduced pressure, sodium bicarbonate is added into the residual water solution to adjust the pH value to be neutral, then dichloromethane is added for extraction, the obtained organic phase is dried and the solvent is removed by distillation under reduced pressure, and the obtained crude product is chromatographed by a silica gel column (dichloromethane: methanol ═ 10: 1(v/v)]Purification afforded intermediate 21(53mg, 69% yield), ESI-MS m/z: 849(M + H).¹H NMR(400MHz，DMSO)δ8.52(s，1H)，7.79(d，J＝10.8Hz，1H)，7.67-7.55(m，2H)，7.47-7.21(m，3H)，6.51(s，1H)，5.60(s，1H)，5.52-5.32(m，2H)，5.30-5.11(m，2H)，5.11-4.94(m，2H)，4.94-4.74(m，2H)，4.02(s，2H)，3.81-3.66(m，2H)，3.60-3.35(m，4H)，3.24-3.08(m，2H)，2.94(d，J＝30.8Hz，3H)，2.39(s，3H)，2.28-2.04(m，2H)，2.00-1.73(m，2H)，1.22(d，J＝6.6Hz，3H)，0.96-0.70(m，3H)。

Synthesis of Compound LE14

Intermediate 21(134mg, 0.16mmol) and the commercially available starting material MC-V (102mg, 0.33mmol) were mixed in 40mL of dichloromethane, the condensing agent 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (82mg, 0.33mmol) was added, the reaction was carried out overnight at room temperature, the solvent was evaporated under reduced pressure after the completion of the reaction, and the crude product was subjected to silica gel column chromatography [ dichloromethane: methanol ═ 10: 1(V/V)]Purification afforded compound LE14(137mg, 75% yield), ESI-MSm/z: 1141.4(M + H).¹H NMR(400MHz，DMSO)δ9.97(s，1H)，8.52(s，1H)，8.27-8.09(m，1H)，7.88-7.70(m，2H)，7.63-7.51(m，2H)，7.28(s，3H)，6.99(s，2H)，6.51(s，1H)，5.59(s，1H)，5.50-5.32(m，2H)，5.17(s，2H)，4.98(s，2H)，4.85(d，J＝17.3Hz，2H)，4.43-4.33(m，1H)，4.21-4.12(m，1H)，4.03(s，2H)，3.74-3.64(m，2H)，3.20-3.03(m，3H)，3.02-2.84(m，4H)，2.36(s，3H)，2.23-2.09(m，4H)，2.01-1.90(m，1H)，1.90-1.78(m，2H)，1.55-1.39(m，4H)，1.30(d，J＝6.7Hz，3H)，1.23-1.11(m，2H)，0.93-0.77(m，9H)。

Examples 2 to 4: synthesis of Compound LE15-LE20

Intermediate VI can be prepared starting from Fmoc-L-valine-L-alanine by substituting methylamine hydrochloride in step b with the corresponding commercially available amino compound, according to steps a and b of the synthesis of intermediate 3 in example 2-1. The subsequent steps, starting from intermediate VI, follow the same procedures as steps c, d, f and h of example 2-1 to give intermediate Ix, which is similar to intermediate 9, which is then treated according to the same procedures as steps i and j of example 6 to remove the amino protecting group and then condensed with different commercially available maleimides to give the final product. The structures of the amino compound and maleimide used are shown in Table 1. Compound LE 15: off-white solid, ESI-MS m/z: 1121.2(M + H); compound LE 16: light yellow solid, ESI-MS m/z: 1167.1(M + H); compound LE 17: yellow solid, ESI-MS m/z: 1132.3(M + H); compound LE 18: light yellow solid, ESI-MS m/z: 1305.4(M + H); compound LE 19: light yellow solid, ESI-MS m/z: 1307.4(M + H); compound LE 20: light yellow solid, ESI-MS m/z: 1337.6(M + H).

TABLE 1 intermediates used in the synthesis of LE15-LE20

Examples 2 to 5: synthesis of Compounds LE21 and LE22

Synthesis of Compound DXD-1

A mixture of commercially available Exatecan mesylate (0.568g, 1mmol) and commercially available 2- (tert-butyldimethylsiloxy) acetic acid (CAS: 105459-05-0, 0.38g, 2mmol) was dissolved in 20mL of anhydrous dichloromethane, and the condensing agent HATU (0.76g, 2mmol) and 1mL of pyridine were added and stirred at room temperature for 2 hours. After the reaction is finished, the solvent is evaporated to dryness under reduced pressure, and the obtained crude product is subjected to column chromatography [ dichloromethane: methanol ═ 50: 1(v/v)]Purification afforded the title compound DXD-1(0.55g, 90% yield), ESI-MS m/z: 608.1(M + H).¹H NMR(400MHz，CDCl₃)δ7.73(d，J＝10.5Hz，1H)，7.64(s，1H)，7.05(d，J＝9.2Hz，1H)，5.80-5.62(m，2H)，5.41-5.14(m，4H)，4.29-4.15(m，2H)，4.08-4.03(m，1H)，3.27-3.07(m，2H)，2.45(s，3H)，2.38-2.28(m，2H)，1.96-1.81(m，2H)，1.04(t，J＝7.4Hz，3H)，0.80(s，9H)，0.11(s，3H)，0.03(s，3H).

Preparation of intermediate V

Intermediate V was prepared by following the procedure for the preparation of compound 4 in example 2-1, replacing methylamine hydrochloride in step b with the corresponding commercially available amino compound.

Synthesis of LE21-LE22

Intermediate V was reacted with DXD-1, followed by treatment with 10% trifluoroacetic acid/dichloromethane solution to give intermediate X, which was then reacted according to subsequent steps e, g, i and j of compound 5 in example 2-1: and reducing the intermediate x to obtain an amino compound, condensing the obtained amino compound and Fmoc-valine hydroxysuccinimide ester, removing the Fmoc protecting group of amino in the obtained product, and reacting the obtained amino product with 6- (maleimide) hexanoic acid succinimide ester to obtain a final product. Compound LE 21: yellow solid, ESI-MS m/z: 1141.2(M + H); compound LE 22: yellow solid, ESI-MS m/z: 1106.6(M + H).

Examples 2 to 6: synthesis of Compound LS13

With reference to the synthesis of LE15 in examples 2-4, SN-38 (7-ethyl-10-hydroxycamptothecin) and intermediate VII (R)¹Methylsulfonyl ethyl), deprotection, condensation and the like to obtain a compound LS 13:¹H NMR(400MHz，DMSO)δ9.92(d，J＝22.4Hz，1H)，8.14(s，1H)，8.08(d，J＝9.1Hz，1H)，7.81(d，J＝8.0Hz，1H)，7.70-7.50(m，3H)，7.47(d，J＝7.2Hz，1H)，7.34(d，J＝7.2Hz，1H)，7.27(s，1H)，7.20(s，1H)，6.98(s，2H)，6.51(s，1H)，5.61(s，2H)，5.48-5.35(m，2H)，5.27(s，2H)，5.10(d，J＝20.6Hz，2H)，4.36(s，1H)，4.21-4.07(m，1H)，3.84(s，2H)，3.48(s，2H)，3.21-2.92(m，6H)，2.25-2.04(m，2H)，2.04-1.78(m，3H)，1.55-1.36(m，4H)，1.36-1.10(m，9H)，0.95-0.71(m，10H)。

examples 2 to 7: synthesis of Compound GGFG-Dxd

The compound GGFG-Dxd was prepared by reference to the known synthetic methods reported in WO2015146132A 1. ESI-MS m/z: 1034.5(M + H) in the form of a,¹H-NMR(400MHz，DMSO-d₆)δ8.61(t，J＝6.4Hz，1H)，8.50(d，J＝8.5Hz，1H)，8.28(t，J＝5.1Hz，1H)，8.11(d，J＝7.5Hz，1H)，8.05(t，J＝5.7Hz，1H)，7.99(t，J＝5.9Hz，1H)，7.77(d，J＝11.0Hz，1H)，7.31(s，1H)，7.25-7.16(m，5H)，6.98(s，2H)，6.51(s，1H)，5.59(dt，J＝7.4，4.1Hz，1H)，5.41(s，2H)，5.20(s，2H)，4.64(d，J＝6.1Hz，2H)，4.53-4.40(m，1H)，4.02(s，2H)，3.74-3.37(m，8H)，3.18-3.00(m，2H)，3.04-2.97(m，1H)，2.77(dd，J＝13.5，9.4Hz，1H)，2.38(s，3H)，2.19(dd，J＝14.9，8.5Hz，2H)，2.11-2.05(m，2H)，1.86(dd，J＝14.0，6.7Hz，2H)，1.45(s，4H)，1.20-1.14(m，2H)，0.87(t，J＝7.1Hz，3H).

example 3: preparation of antibody drug conjugates

The antibody FDA016 of B7-H3 prepared according to the method of example 1 was replaced into 50mM PB/1.0mM EDTA buffer (pH7.0) using a G25 desalting column, 12 equivalents of TECP was added, and the mixture was stirred at 37 ℃ for 2 hours to completely open the inter-antibody disulfide bonds, followed by adjusting the pH of the reduced antibody solution to 6.0 using phosphoric acid and lowering the temperature of the water bath to 25 ℃ to prepare for the coupling reaction. The linker-drug conjugates LE12-LE22, LS13 and GGFG-Dxd prepared as described in example 2 above were dissolved in DMSO, 12 equivalents of the linker-drug conjugate were pipetted therefrom and added dropwise to the reduced antibody solution, and DMSO was further added thereto to a final concentration of 10% (v/v), and the reaction was stirred at 25 ℃ for 0.5 hour, and after completion of the reaction, the sample was filtered using a 0.22um membrane. Purifying by using tangential flow ultrafiltration system to remove unconjugated small molecules, wherein the buffer solution is 50mMPB/1.0mMEDTA solution (pH6.0), adding sucrose with final concentration of 6%, and storing in-20 deg.C refrigerator. The absorbance values were measured at 280nm and 370nm, respectively, using the UV method to calculate the DAR value, and the results are shown in Table 2 below.

The coupling reactions were carried out according to the same procedures as those described in the example above, samples were prepared according to the highest DAR (i.e. excess coupling), the occurrence of precipitates was observed when each coupling reaction occurred, and the proportion and recovery of the polymer after completion of each coupling reaction were calculated, and the results are also shown in table 2.

TABLE 2 conjugation protocol for the preparation of different Antibody Drug Conjugates (ADC)

"/" indicates no calculated recovery

In practical research, the linker-drug conjugate GGFG-Dxd is also precipitated when being coupled with other antibodies, and the proportion of the polymer is high, so that the linker-drug conjugate has no universality. Most of the linker-drug conjugates in the technical scheme are tried to be coupled with different antibodies including F016 without generating precipitates, and the proportion of the polymer is in a normal range, so that the antibody-drug conjugate has good solubility and druggability, and the preparation of the antibody-drug conjugate is facilitated without generating precipitates in the coupling process.

Effect example 1: evaluation of in vitro killing Activity of antibody drug conjugates

Calu-6(ATCC) cells were selected as cell lines for the in vitro activity assay of experiments2000 cells per well were seeded in 96-well cell culture plates and cultured for 20-24 hours. The antibody drug conjugate prepared according to the method of example 3 was prepared into sample solutions of 11 concentration gradients of 1000, 166.7, 55.6, 18.6, 6.17, 2.06, 0.69, 0.23, 0.08, 0.008 and 0nM using L15 cell culture medium containing 10% FBS, 100. mu.l/well of the diluted sample solution was added to the cell-seeded culture plate, and the plate was incubated at 37 ℃ with 5% CO₂Adding the mixture after the culture in an incubator for 144 hours

The results of the calculation of IC50 after the data was read by a SpectraMaxL microplate reader (OD570nm, 2s interval reading) are shown in Table 3.

Using the same method as described above, each antibody drug conjugate was tested for cytotoxic killing activity against a number of tumor cells NCI-N87, A375, Hs-700T, LN-229, MDA-MB-468, PA-1 and Raji, respectively, purchased from ATCC, and the results are shown in Table 3. As can be seen from the results in Table 3, the antibody drug conjugate provided by the invention has excellent in vitro killing activity on cells such as Calu-6, NCI-N87, A375, Hs-700T, LN-229, MDA-MB-468 and PA-1. And the prepared ADC has no killing activity on Raji negative cells, which shows that the prepared ADC has specific targeted killing activity.

TABLE 3 in vitro killing Activity of antibody drug conjugates

Effect example 2: in vitro plasma stability test

This example evaluates the stability of antibody conjugate drugs prepared according to the method of example 3 in human plasma. Specifically, in this example, the antibody-conjugated drug of example 3 was added to human plasma, and the free drug was extracted by placing the drug in a water bath at 37 ℃ for 1, 3, 7, 14, 21, and 28 days with an internal standard (irinotecan is used as an internal standard substance) and then measuring the amount released by high performance liquid chromatography, and the results are shown in table 4.

TABLE 4 stability evaluation of different ADCs in human plasma

The result of the plasma stability shows that the antibody drug conjugate provided by the invention has good plasma stability.

Effect example 3: in vitro enzyme digestion assay for linker-drug conjugates

The linker-drug conjugates (LE14 and GGFG-Dxd) were incubated with cathepsin B in three different pH (5.0, 6.0, 7.0) buffers, samples were taken at different time points into hplc-ms and percent drug release was determined by external standard method (DXD as external standard). The results of the experiment (as shown in Table 5) show that GGFG-Dxd cleaves at a slower rate over the pH range used, whereas the linker-drug conjugate LE14 employed in the present invention cleaves rapidly over the pH range of 5.0 to 7.0.

TABLE 5 cleavage of LE14 and GGFG-Dxd in vitro at different pH

Effect example 4: in vitro enzyme digestion experiment of FDA016-LS13

NCI-N87 cell line was selected as an experimental cell line, and after incubating the samples in cathepsin B system (100mM sodium acetate-acetic acid buffer, 4mM dithiothreitol, pH 5.0) at 37 ℃ for 4 hours, the resulting samples were diluted with a medium to various concentrations, and 8 concentrations (1.5-10 fold) were set according to SN-38 concentration of 70nM to 0.003nMDilution), change in killing (inhibiting) ability of the cell line was observed for 144 hours, and the cell line was examined by

Luminescence Cell Viability Assay chemiluminescent staining, reading fluorescence data and calculating IC50 values.

The enzyme digestion sample obtained by incubating the cathepsin B system for 4 hours at 37 ℃ is precipitated by proper amount of ethanol to remove protein, the generated micromolecular compound is detected and released by high performance liquid chromatography, the release rate in 4 hours is determined by taking equivalent SN-38 as reference, and the result shows that the release rate reaches 99%.

Experimental results (shown in Table 6) show that the cytotoxic activity of FDA016-LS13 after enzyme digestion is almost the same as that of equivalent SN-38, and also show that FDA016-LS13 almost completely releases SN-38 and plays a role under the action of cathepsin B, while unpredictable changes can occur when FDA016-LS13 is endocytosed into lysosomes, so that SN-38 cannot effectively play a role.

TABLE 6 change in killing activity of NCI-N87 cell line before and after digestion of FDA016-LS13 with cathepsin B system

Effect example 5: test for antitumor Activity of FDA016-LE14 in Calu-6 human Lung cancer model

6-8 week old female Balb/c nude mice were selected for right dorsal cervical subcutaneous injection of 5X 10 in 100ul PBS⁶Human lung cancer cell (Calu-6), when the tumor grows to the average volume of 150-³At this time, the mice were randomly divided into 5 groups according to the tumor size and the body weight of the mice, and 6 animals were administered to the mice per group, i.e., a placebo group, 5mg/kg FDA016-GGFG-Dxd group, 10mg/kg FDA016-GGFG-Dxd group, 5mg/kg FDA016-LE14 group, and 10mg/kg FDA016-LE14 group, intraperitoneally and once per week. The body weight and tumor volume of the experimental animals were measured twice a week and the survival status of the animals during the experiment was observed. The results are shown in Table 7, when the mice in the placebo group were at the end of the administrationMean tumor volume 2107.51mm³. Test drug 5.0mg/kg FDA016-GGFG-Dxd treatment group with 14 th balance of mean tumor volume 72.35mm after dosing is finished³10mg/kg of the FDA016-GGFG-Dxd treatment group had a 14 th balance mean tumor volume of 3.28mm after the end of dosing³. Test drug 5.0mg/kg FDA016-LE14 treatment group with 14 th balance mean tumor volume 50.48mm after dosing is finished³10mg/kg of FDA016-LE14 treatment group with a 14 th day mean tumor volume of 0.00mm after the end of the administration³. The experimental result shows that FDA016-LE14 has better in-vivo anti-tumor activity, and all experimental mice have no death condition and no weight loss condition, which indicates that FDA016-LE14 has good safety.

TABLE 7 antitumor Activity of FDA016-LE14 in Calu-6 human Lung cancer model

Note: group 01 is blank control group; group 02 was 5mg/kg FDA 016-GGFG-Dxd; 03 is 10mg/kg FDA 016-GGFG-Dxd; group 04 is 5mg/kg FDA016-LE14 group; group 05 was FDA016-LE14 at 10 mg/kg.

SEQUENCE LISTING

<110> Shanghai Compound Dangjiang biomedical corporation

<120> B7-H3 targeted antibody drug conjugate, and preparation method and application thereof

<130> P20017416C

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 215

<212> PRT

<213> Homo sapiens

<400> 1

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Ile Tyr Ser Tyr

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Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val

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Tyr Asn Thr Lys Thr Leu Pro Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala

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Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

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Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

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Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

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Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

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Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

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Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

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Ser Phe Asn Arg Gly Glu Cys

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<210> 2

<211> 447

<212> PRT

<213> Homo sapiens

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

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Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Thr Ile Asn Ser Gly Gly Ser Asn Thr Tyr Tyr Pro Asp Ser Leu

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg His Asp Gly Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

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Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

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Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

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Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

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Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

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Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

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Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

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Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

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Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

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Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

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Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

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Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

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Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

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Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

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Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

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Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

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Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

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Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

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Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

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Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

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Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

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His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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Claims (11)

1. An antibody drug conjugate shown in formula I or a pharmaceutically acceptable salt thereof;

wherein Ab is B7-H3 antibody; m is 2-8;

the amino acid sequence of the light chain in the B7-H3 antibody is shown as a sequence table SEQ ID NO. 1, and the amino acid sequence of the heavy chain is shown as a sequence table SEQ ID NO. 2;

d is

R²And R⁵Are each independently H, C₁-C₆Alkyl or halogen;

R³and R⁶Are each independently H, C₁-C₆Alkyl or halogen;

R⁴and R⁷Are each independently C₁-C₆An alkyl group;

R¹is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl radical, C₁～C₆Alkyl or C₃～C₁₀A cycloalkyl group; said R^1-1、R^1-2And R^1-3Are each independently C₁～C₆An alkyl group;

L₁independently one or more of a phenylalanine residue, an alanine residue, a glycine residue, an isoleucine residue, a leucine residue, a proline residue, and a valine residue; p is 2-4;

L₂is composed of

Wherein n is independently 1-12, and c is a carbonyl group and L₁Is connected with the f end and the L₃The d ends of the two are connected;

L₃is composed of

Wherein the b terminal is connected to the Ab, and the d terminal is connected to the L₂Are connected.

2. The antibody drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

when said R is²And R⁵Are each independently C₁-C₆When alkyl, said C₁～C₆Alkyl is C₁～C₄An alkyl group;

and/or, when said R is²And R⁵When each is independently halogen, the halogen is fluorine, chlorine, bromine or iodine;

and/or, when said R is³And R⁶Are each independently C₁-C₆When alkyl, said C₁～C₆Alkyl is C₁～C₄An alkyl group;

and/or, when said R is³And R⁶When each is independently halogen, the halogen is fluorine, chlorine, bromine or iodine;

and/or, when said R is⁴And R⁷Are each independently C₁-C₆When alkyl, said C₁～C₆Alkyl is C₁～C₄An alkyl group;

and/or, said L₃The b terminal of (a) is linked to a thiol group on the antibody in a thioether form;

and/or, when said R is¹Is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆When alkyl, said C₁～C₆Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably ethyl;

and/or, when said R is¹To be multiple-NR^1-1R^1-2Substituted C₁～C₆When alkyl, said plurality is two or three;

and/or, when said R is^1-1And R^1-2Each independently is C₁～C₆When alkyl, said C₁～C₆Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl;

and/or, when said R is¹Is represented by one or more R^1-3S(O)₂-substituted C₁～C₆When alkyl, said C₁～C₆Alkyl is C₁～C₄An alkyl group, preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, more preferably an ethyl group;

and/or, when said R is¹To be multiple of R^1-3S(O)₂-substituted C₁～C₆When alkyl, said plurality is two or three;

and/or, when said R is^1-3Is C₁～C₆When alkyl, said C₁～C₆Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl;

and/or, when said R is¹Is C₁～C₆When alkyl, said C₁～C₆Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl;

and/or m is an integer or a non-integer, preferably 4 to 8, more preferably 6 to 8, and further preferably 7 to 8;

and/or, said p is 2;

and/or n is 8-12;

and/or, when said R is^1-1、R^1-2And R^1-3Independently is C₁～C₆When alkyl, said C₁～C₆Alkyl is C₁～C₄An alkyl group.

3. The antibody drug conjugate of claim 2, or a pharmaceutically acceptable salt thereof, wherein:

when said R is²And R⁵Are each independently C₁-C₆When alkyl, said C₁～C₆Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl;

and/or, when said R is²And R⁵When each is independently halogen, said halogen is fluorine;

and/or, when said R is³And R⁶Are each independently C₁-C₆When alkyl, said C₁～C₆Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl;

and/or, when said R is³And R⁶When each is independently halogen, said halogen is fluorine;

and/or, when said R is⁴And R⁷Are each independently C₁-C₆When alkyl, said C₁～C₆Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably ethyl;

and/or, when said R is¹Is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆When alkyl, said-NR^1-1R^1-2is-N (CH)₃)₂；

And/or, when said R is¹Is one-NR^1-1R^1-2Substituted C₁～C₆When alkyl, said radical is substituted by one-NR^1-1R^1-2Substituted C₁～C₆Alkyl is

And/or, when said R is¹Is represented by an R^1-3S(O)₂-substituted C₁～C₆When alkyl, said is substituted by one R^1-3S(O)₂-substituted C₁～C₆Alkyl is

And/or said m is from 7.4 to 7.85, such as 7.47, 7.48, 7.52, 7.62, 7.64, 7.65, 7.67, 7.72, 7.78, 7.83 or 7.85;

and/or, said (L)₁)_pIs composed of

Wherein the g terminal is through the carbonyl group and said L₂The end c is connected;

and/or, said n is 8, 9, 10, 11 and 12;

and/or, when said R is^1-1、R^1-2And R^1-3Independently is C₁～C₆When alkyl, said C₁～C₆Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

4. The antibody drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

R²and R⁵Are each independently C₁-C₆An alkyl group;

and/or, R³And R⁶Each independently is halogen;

and/or, R⁴And R⁷Is ethyl;

and/or, said L₁Is one or more of phenylalanine residue, alanine residue, glycine residue and valine residue, preferably valine residue and/or alanine residue, and the plurality is preferably two or three;

and/or, said R¹Is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl, or, C₁～C₆An alkyl group; preferably by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, or substituted by one or more R^1-3S(O)₂-substituted C₁～C₆An alkyl group; more preferably by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl radical；

Preferably, D is

5. The antibody drug conjugate of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein the antibody drug conjugate is according to any one of the following schemes:

the first scheme is as follows:

R²and R⁵Are each independently C₁-C₆An alkyl group;

R³and R⁶Each independently is halogen, D is preferably

R¹Is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl, or, C₁～C₆An alkyl group;

L₁is one or more of phenylalanine residue, alanine residue, glycine residue and valine residue;

scheme II:

R²and R⁵Are each independently C₁-C₆An alkyl group;

R³and R⁶Each independently is halogen, D is preferably

m is 7 to 8, m is,

R¹is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl, or, C₁～C₆An alkyl group;

L₁independently a valine residue and/or an alanine residue;

and a third scheme is as follows:

d is

R¹Is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl, or, C₁～C₆An alkyl group;

and the scheme is as follows:

d is

m is 7-8;

R¹is represented by one or more-NR^1-1R^1-2Substituted C₁～C₆Alkyl, by one or more R^1-3S(O)₂-substituted C₁～C₆Alkyl, or, C₁～C₆An alkyl group;

L₁independently a valine residue and/or an alanine residue.

6. The antibody drug conjugate of claim 1, wherein the antibody drug conjugate is any one of the following compounds:

wherein Ab is B7-H3 antibody, and m is 7.47, 7.48, 7.52, 7.62, 7.64, 7.65, 7.67, 7.72, 7.78, 7.83 or 7.85.

7. The antibody drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the antibody drug conjugate is any one of the compounds shown below:

ab is B7-H3 antibody, m is preferably 7.64;

ab is B7-H3 antibody, m is preferably 7.67;

ab is B7-H3 antibody, m is preferably 7.83;

ab is B7-H3 antibody, m is preferably 7.65;

ab is B7-H3 antibody, m is preferably 7.78;

ab is B7-H3 antibody, m is preferably 7.62;

ab is B7-H3 antibodyBody, m is preferably 7.48;

ab is B7-H3 antibody, m is preferably 7.52;

ab is B7-H3 antibody, m is preferably 7.47;

ab is B7-H3 antibody m is preferably 7.85; or

Ab is B7-H3 antibody, m is preferably 7.72.

8. A method of preparing an antibody drug conjugate of any one of claims 1 to 7 comprising the steps of: carrying out coupling reaction on a compound shown as a formula II and Ab-hydrogen as shown in the specification;

9. a pharmaceutical composition comprising substance X and a pharmaceutical excipient, wherein substance X is the antibody drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, and the amount of substance X is preferably a therapeutically effective amount.

10. Use of substance X, or a pharmaceutical composition according to claim 9, wherein substance X is an antibody drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, in the preparation of a B7-H3 protein inhibitor.

11. Use of substance X, or a pharmaceutical composition according to claim 9, in the manufacture of a medicament for the treatment and/or prevention of a tumour, said substance X being an antibody drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof; the tumor is preferably a B7-H3 positive tumor; the B7-H3 positive tumor is preferably one or more of B7-H3 positive lung cancer, ovarian cancer, melanoma, pancreatic cancer, breast cancer, brain glioma, prostate cancer and gastric cancer; the lung cancer cell is preferably NCI-1703 cell or Calu-6 cell; the ovarian cancer cells are preferably PA-1 cells; the melanoma cells are preferably A375 cells; the pancreatic cancer cells are preferably Hs-700T cells; the breast cancer cells are preferably MDA-MB-468 cells; the brain glioma cells are preferably LN-229 cells; the gastric cancer cells are preferably NCI-N87 cells.

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