CN116036303A - Antibody-drug conjugate and preparation method and application thereof - Google Patents

Antibody-drug conjugate and preparation method and application thereof Download PDF

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CN116036303A
CN116036303A CN202310035642.4A CN202310035642A CN116036303A CN 116036303 A CN116036303 A CN 116036303A CN 202310035642 A CN202310035642 A CN 202310035642A CN 116036303 A CN116036303 A CN 116036303A
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antibody
compound
trastuzumab
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王雷
刘海东
尚晓
韩泰熙
陈祝
赵柏腾
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Profoundbio Suzhou Co Ltd
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Abstract

The invention provides an antibody-drug conjugate, a preparation method and application thereof. The structural formula of the antibody-drug conjugate is

Description

Antibody-drug conjugate and preparation method and application thereof
Technical Field
The invention belongs to the field of medicines, and particularly relates to an antibody-medicine conjugate and a preparation method and application thereof.
Background
An antibody-drug conjugate (antibody drug conjugate, ADC) is a monoclonal antibody or antibody fragment linked to a small molecule cytotoxic drug with biological activity via a chemical linker. Compared with the traditional chemotherapy, the novel medicine based on the antibody can effectively deliver the cytotoxic medicine to the tumor target cells, has high specificity of the antibody and high toxicity of the cytotoxic medicine to the tumor, so that the antibody-medicine conjugate can more accurately bind to the tumor cells and reduce the damage to normal cells.
The ADC comprises three components, an antibody, a linker and a cytotoxic drug. By targeting specific antigens, ADCs are able to penetrate into tumor tissue and be engulfed by tumor cells into the enzyme solution, releasing the cytotoxic drug. Although there are currently nearly 15 ADC drugs on the market, the ADC still needs to be further optimized in terms of structural design. For example, an ideal linker should remain stable in the circulatory system and effectively release the payload (e.g., cytotoxic drug) in the tumor. However, existing linkers typically release the payload unspecifically and inevitably lead to off-target toxicity. It can be seen that linker design plays a key role in regulating the stability of ADC in systemic circulation and the efficiency of payload release in tumors, greatly affecting Pharmacokinetic (PK), therapeutic and toxicity profile of ADC.
Human epidermal growth factor receptor 2 (HER 2, also known as ERBB 2) is a member of the epidermal growth factor receptor family (EGFR). The HER2 gene is located on chromosome 17q21 and the encoded product is a transmembrane glycoprotein with tyrosine kinase activity and has a molecular weight of about 185kD, also known as p185.HER2 promotes invasion and metastasis of cancer cells by autophosphorylating intracellular tyrosine residues to activate them, thereby activating downstream signaling, by forming homodimers or heterodimers with other EGFR receptors HER1 (EGFR, erbB-1), HER3 (ErbB-3), HER4 (ErbB-4).
Currently, marketed anti-HER 2 targeted drugs mainly include: 1. monoclonal antibodies, e.g., trastuzumab (trastuzumab), pertuzumab (Pertuzumab); 2. ADC, for example, trastuzumab emtansine (T-DM 1), trastuzumab deruxtecan (T-DXd). There remains a need for an anti-HER 2 targeted drug, especially an ADC drug, that is more therapeutic and more capable of inhibiting tumor growth.
Disclosure of Invention
According to one aspect of the present specification there is provided an antibody-drug conjugate, the conjugate having the structure shown in formula Ia:
Figure BDA0004049198190000011
wherein, the mAb is trastuzumab (anti-HER 2 antibody); thiol-linker-irinotecan conjugate LD038 obtained by reduction of the conjugate via inter-chain disulfide bond of the mAb
Figure BDA0004049198190000021
Generating Michael addition to obtain the product; n is between 1 and 10.
In some embodiments, n is between 5 and 10.
In some embodiments, n is 8.
In some embodiments, the linker is a cleavable hydrophilic linker.
According to another aspect of the present specification, there is provided a method of preparing an antibody-drug conjugate as described above, the method comprising:
a) Preparation of the linker-irinotecan conjugate LD038
Figure BDA0004049198190000022
b) And (3) after the trastuzumab is reduced, carrying out a coupling reaction with the LD038 to obtain the antibody-drug conjugate.
In some embodiments, the coupling reaction with LD038 after the reduction of trastuzumab to obtain the antibody-drug conjugate may comprise: and the sulfhydryl group obtained after the inter-chain disulfide bond of the trastuzumab is reduced is subjected to Michael addition with the LD038.
In some embodiments, the preparing the linker-irinotecan conjugate LD038 comprises:
i) Synthesis of Compound 38-6
Figure BDA0004049198190000031
ii) combining the compound 38-6 with the compound 38-7
Figure BDA0004049198190000032
Reacting to obtain the connector 38-9 of the irinotecan
Figure BDA0004049198190000033
And +.>
iii) Combining said compound 38-9 with MC-OSu
Figure BDA0004049198190000034
And (3) reacting to obtain the LD038.
In some embodiments, the compound 38-6 is prepared by the compound 38-1
Figure BDA0004049198190000035
N-hydroxysuccinimide (HOSu), compound 38-3
Figure BDA0004049198190000036
And D-glucose.
In some embodiments, the compound 38-1 is mixed with the N-hydroxysuccinimide (HOSu), dichloromethane and EDCl are added and reacted at room temperature to give the compound 38-2
Figure BDA0004049198190000041
Mixing the compound 38-2, DIPEA and DMF, adding the compound 38-3, and reacting at room temperature to obtain a compound 38-4
Figure BDA0004049198190000042
Fmoc removal of the compound 38-4 to give compound 38-5
Figure BDA0004049198190000043
Mixing the compound 38-5, the D-glucose, acetic acid and methanol, heating to 50 ℃, reacting for 30 minutes, and adding NaCNBH 3 Reacting at 50 ℃ for 16 hours to obtain the compound 38-6.
According to another aspect of the present specification there is provided a pharmaceutical composition comprising an antibody-drug conjugate as described above, together with one or more pharmaceutically acceptable excipients, diluents or carriers.
According to a further aspect of the present specification there is provided the use of said antibody-drug conjugate or said pharmaceutical composition in the manufacture of a medicament for the treatment of HER2 overexpressing cancer.
In some embodiments, the cancer is selected from breast cancer, ovarian cancer, and pancreatic cancer.
Drawings
The present specification will be further elucidated by way of example embodiments, which will be described in detail by means of the accompanying drawings. The embodiments are not limiting, in which like numerals represent like structures, wherein:
FIG. 1 is a chromatogram of a measurement of PRO1102 content using SEC-HPLC as shown in some embodiments of the present disclosure;
FIG. 2 is a chromatogram of a determination of trastuzumab-deruxtech (8) conjugate content using the SEC-HPLC method according to some embodiments of the present disclosure;
FIG. 3 is a chromatogram of a determination of trastuzumab-vedotin (4) conjugate content using the SEC-HPLC method according to some embodiments of the present disclosure;
FIG. 4 is a graph of the detection of binding activity of PRO1102, trastuzumab and other trastuzumab conjugates to breast ductal carcinoma cell HCC1954 according to some embodiments of the present disclosure;
FIG. 5 is a graph of the detection of binding activity of PRO1102, trastuzumab and other trastuzumab conjugates to human ovarian cancer cells SKOV-3, as illustrated in some embodiments of the present disclosure;
FIG. 6 is a graph of the detection of binding activity of PRO1102, trastuzumab and other trastuzumab conjugates to breast cancer cells JIMT-1 according to some embodiments of the present disclosure;
FIG. 7 is a graph of the detection of binding activity of PRO1102, trastuzumab and other trastuzumab conjugates to human pancreatic cancer cells Capan-1 according to some embodiments of the present disclosure;
FIG. 8 is a graph of the results of detection of the internalization rate of trastuzumab in a plurality of tumor target cells as illustrated in some embodiments of the present disclosure;
FIG. 9 is a graph of the results of the detection of the internalization rate of PRO1102 in a plurality of tumor target cells according to some embodiments of the present disclosure;
FIG. 10 is a graph of cytotoxicity detection results of PRO1102, trastuzumab and other trastuzumab conjugates on breast ductal carcinoma cells HCC1954 according to some embodiments of the present disclosure;
FIG. 11 is a graph of cytotoxicity detection results of PRO1102, trastuzumab and other trastuzumab conjugates on human ovarian cancer cells SK-OV-3 as illustrated in some embodiments of the present specification;
FIG. 12 is a graph of cytotoxicity detection results of PRO1102, trastuzumab and other trastuzumab conjugates on breast cancer cells JIMT-1 according to some embodiments of the present disclosure;
FIG. 13 is a graph of cytotoxicity detection results of PRO1102, trastuzumab and other trastuzumab conjugates on human pancreatic cancer cells Capan-1 according to some embodiments of the present disclosure;
fig. 14 is a tumor growth curve of PRO1102, trastuzumab and other trastuzumab conjugates shown in some embodiments of the present disclosure following subcutaneous implantation of nude mice against breast ductal carcinoma cells HCC 1954;
FIG. 15 is a tumor growth curve of PRO1102, trastuzumab and other trastuzumab conjugates shown in some embodiments of the present specification following subcutaneous transplantation of nude mice against human ovarian cancer cells SK-OV-3;
fig. 16 is a tumor growth curve of PRO1102, trastuzumab and other trastuzumab conjugates shown in some embodiments of the present specification following subcutaneous transplantation of nude mice against breast cancer cells JIMT-1;
FIG. 17 is a graph showing tumor growth curves of PRO1102, trastuzumab and other trastuzumab conjugates following subcutaneous transplantation of nude mice against human pancreatic cancer cells Capan-1 according to some embodiments of the present disclosure;
Fig. 18 is a graph of the Pharmacokinetics (PK) of PRO1102, trastuzumab and other trastuzumab conjugates in rats, according to some embodiments of the present disclosure.
Detailed Description
In order to more clearly illustrate the technical solutions of the embodiments of the present specification, the drawings that are required to be used in the description of the embodiments will be briefly described below. It is apparent that the drawings in the following description are only some examples or embodiments of the present specification, and it is possible for those of ordinary skill in the art to apply the present specification to other similar situations according to the drawings without inventive effort.
As used in this specification and the claims, the terms "a," "an," "the," and/or "the" are not specific to a singular, but may include a plurality, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that the steps, elements and/or materials specifically identified are included, but they do not constitute an exclusive list, and may include other steps, elements and/or materials.
Some common abbreviations and english words in this application have the following meanings:
EDCl: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride
HOSu: n-hydroxysuccinimide
DCM: dichloromethane (dichloromethane)
Fmoc: 9-fluorenylmethoxycarbonyl
Boc: boc-group
DIPEA: n, N-diisopropylethylamine
DMF: n, N-dimethylformamide
DEA: diethanolamine (DEA)
D-glucose: d-glucose
NaCNBH 3 : sodium cyanoborohydride
MeOH: methanol
THF: tetrahydrofuran (THF)
HATU:2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate
TFA: trifluoroacetic acid
DMSO: dimethyl sulfoxide
EDTA: ethylenediamine tetraacetic acid
TCEP: tris (2-carboxyethyl) phosphine
LCMS: liquid chromatography-mass spectrometry instrument
SMCC-DM1: small molecules comprising a linker 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) linked to the cytotoxic drug maytansine (DM 1)
Mc-vcMAE: small molecules linked by the linker maleimidocaproyl (Mc), the cleavable dipeptide valine-citrulline (vc), and the cytotoxic drug monomethyl auristatin E (MMAE)
Definition of some terms in this application:
the terms "compound/conjugate of the present specification", "antibody-drug conjugate of the present invention" refer to compounds represented by formula Ia. This compound was named PRO1102 or trastuzumab-LD038. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compound of formula Ia.
The term "pharmaceutically acceptable salt" refers to salts of a compound of formula Ia with an acid or base that are suitable for use as a medicament. Pharmaceutically acceptable salts include inorganic and organic salts.
The term "drug" refers to a cytotoxic drug, specifically a chemical molecule that is capable of having a strong disruption of its normal growth within a tumor cell. Cytotoxic drugs can in principle kill tumor cells at sufficiently high concentrations, but due to lack of specificity, they can also cause apoptosis in normal cells, leading to serious side effects. The term includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes, cytotoxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably cytotoxic drugs. The cytotoxic drug may be maytansine, auristatin, anthracycline, camptothecin analogues, etc. Camptothecin analogs act as topoisomerase I (TOP 1) inhibitors, inhibiting DNA synthesis in vivo, and exerting antitumor effects. Exemplary camptothecin analogs include topotecan (topotecan), irinotecan (irinotecan; CPT-11), belotecan (belotecan), irinotecan (exatecan), deluximab (deruxtecan), and the like. In some embodiments, the drug is irinotecan, which has the structural formula
Figure BDA0004049198190000061
The term "linker" refers to a chemical structural fragment or bond that is linked at one end to an antibody and at the other end to a drug, or can be linked to other linkers before being linked to a drug.
The term "antibody" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. For example, the light chains of antibodies can be divided into kappa and lambda light chains. Heavy chains can be divided into μ, δ, γ, α and ε, which define the isotype of antibodies as IgM, igD, igG, igA and IgE, respectively. In the light and heavy chains, the variable region is linked to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). VH and VL regions can be further divided into hypervariable regions (known as Complementarity Determining Regions (CDRs)) separated by relatively conserved regions (known as Framework Regions (FR)). Each VH and VL is typically composed of 3 CDRs and 4 FR in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the N-terminal to the C-terminal. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. Antibodies may have different antibody isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subtypes), igA1, igA2, igD, igE, or IgM antibodies.
The antibody in the antibody-drug conjugate in this specification refers to an anti-HER 2 antibody. In some embodiments, the anti-HER 2 antibody is trastuzumab (trastuzumab).
The term "DAR value" refers to the average number of drugs attached to each antibody in the molecule of formula (Ia), and may also be expressed as the ratio of the amount of drug to the amount of antibody. In some embodiments of the present application, the DAR value is denoted as n. Illustratively, n may be any integer or fractional value between 1-10, e.g., 1, 3, 5, 7, 8, 9, 10, etc. The DAR value per ADC molecule after the coupling reaction can be characterized using conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays, and HPLC.
In the present invention, the term "pharmaceutically acceptable" component refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.
The terms "about" and "about" may describe a range of values within a certain value, such as adding or subtracting 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, etc. of the value. For example, the term "about 150mm 3 "may include 135mm 3 To 165mm 3 。
"between a-b" means any number between a and b, including decimal or integer numbers. Between a-b, there are included the boundary values a and b. For example, "between 1-10" means any number between 1 and 10, e.g., 1, 5, 6.5, 8, 10, etc.
One aspect of the present description provides an antibody-drug conjugate. The structure is shown in formula Ia
Figure BDA0004049198190000071
Wherein, the mAb is trastuzumab which is an anti-HER 2 antibody; thiol-linker-irinotecan conjugate LD038 produced by reduction of the conjugate via interchain disulfide bond of the mAb
Figure BDA0004049198190000072
Generating Michael addition to obtain the product; n is between 1 and 10.
In some embodiments, n has a value between 5 and 10. In some embodiments, n has a value between 6 and 9. In some embodiments, n has a value between 7 and 9. In some embodiments, n has a value of 8.
In some embodiments, the antibody-drug conjugate (ADC) of formula Ia consists of a linker, the DNA topoisomerase i inhibitor irinotecan, and the HER2 antibody trastuzumab.
In the ADC drug design, the proper linker can fully balance the interaction force between the antibody and the small molecule, and the produced ADC has better stability and can reach the expected effect of the ADC drug. In some embodiments of the present disclosure, the linker linking the irinotecan and trastuzumab is a cleavable hydrophilic linker, which can increase the hydrophilicity of the antibody-drug conjugate due to the more hydroxyl groups contained in the linker, thereby controlling the aggregation of ADC, and allowing the ADC drug with high load (dar=8) to have good physicochemical properties. The linker comprises a valine-citrulline (Val-Cit) dipeptide structure
Figure BDA0004049198190000081
The dipeptide structure can prevent small molecule drugs from being released prematurely, and the stability of the antibody-drug conjugate in the in vivo circulatory system is improved.The dipeptide structure exhibits a broad sensitivity to a variety of cathepsins, including cathepsin B, cathepsin K, cathepsin L, and the like. Cathepsin B is highly expressed in tumor cell specificity, and thus, the dipeptide structure in the cleavable hydrophilic linker can release irinotecan inside the tumor.
In some embodiments, the cleavable hydrophilic linker further comprises a long-chain polyethanol structure connected by an amide linking group, the linking group forms an amide bond after reaction, and a conjugate formed by the amide bond is relatively stable and is not easy to break in the systemic circulation, so that the stability of the ADC can be improved, the treatment window is enlarged, and the tolerance of the organism to drugs is improved.
Irinotecan is a DNA topoisomerase i inhibitor. Topoisomerase inhibitors act on topoisomerase enzymes and exert inhibitory activity on many tumors. DNA topoisomerase is a key enzyme for DNA replication and plays an important role in cell transcription, recombination and repair. Compared with normal cells, the content and activity of topoisomerase in tumor cells are obviously improved, so that the topoisomerase is an excellent action target point of the antitumor drug, for example, camptothecins, podophyllotoxins, doxorubicin and the like all take the topoisomerase as the target point, and the DNA replication is interfered to play an antitumor role. As a water-soluble camptothecin derivative, irinotecan has excellent antitumor function.
Trastuzumab is a recombinant DNA-derived humanized IgG 1-type monoclonal antibody developed against the P185 glycoprotein expressed by the cell-regulated nuclear HER2 gene, the light chain variable region of which consists of a murine portion, which recognizes the P185 glycoprotein, while the heavy chain fixed region and the majority of the light chain region are both human portions. Trastuzumab selectively binds to P185 glycoprotein after entering the human body, inhibiting HER2 positive tumor cell growth. Trastuzumab is a potential medium for antibody-dependent cell-mediated cytotoxicity (ADCC), has anti-tumor effects, and can also increase the sensitivity of tumor cells to chemotherapy, thereby increasing the therapeutic effect of chemotherapy.
The antibody-drug conjugate of the specification takes the monoclonal antibody as a carrier to efficiently transport the small molecule cytotoxicity drugs into target tumor cells in a targeting manner, thereby playing an anti-tumor role. The antibody-drug conjugates of the present disclosure may be used to treat HER2 over-expressed cancers, which may include breast cancer, ovarian cancer, lung cancer, pancreatic cancer, gastric cancer, colon cancer, endometrial cancer, colorectal cancer, and the like. The cancer is preferably breast, ovarian and pancreatic cancer.
In another aspect of the present disclosure, a method of preparing an antibody-drug conjugate as described above is provided. The method may comprise the steps of:
a) Preparation of the linker-irinotecan conjugate LD038
Figure BDA0004049198190000082
b) And (3) after the trastuzumab is reduced, carrying out a coupling reaction with the LD038 to obtain the antibody-drug conjugate.
In some embodiments, the coupling reaction with LD038 after the reduction of trastuzumab to obtain the antibody-drug conjugate may comprise: the thiol obtained by reducing the inter-chain disulfide bond of trastuzumab is subjected to Michael addition with LD038. In some embodiments, one of the reduced sulfhydryl groups of the disulfide bond undergoes a Michael addition reaction with LD038.
In some embodiments, the preparation of the linker-irinotecan conjugate LD038 comprises the steps of:
i) Synthesis of Compound 38-6
Figure BDA0004049198190000091
ii) combining the compound 38-6 with the compound 38-7
Figure BDA0004049198190000092
Reacting to obtain the connector 38-9 of the irinotecan
Figure BDA0004049198190000093
And
iii) Combining said compound 38-9 with MC-OSu
Figure BDA0004049198190000094
And (3) reacting to obtain the LD038.
In some embodiments, the compound 38-6 is prepared by the compound 38-1
Figure BDA0004049198190000095
N-hydroxysuccinimide (HOSu), compound 38-3 +.>
Figure BDA0004049198190000096
And D-glucose.
In some embodiments, the compound 38-1 is mixed with the N-hydroxysuccinimide (HOSu), dichloromethane and EDCl are added and reacted at room temperature to give the compound 38-2
Figure BDA0004049198190000101
Mixing the compound 38-2, DIPEA and DMF, adding the compound 38-3, and reacting at room temperature to obtain a compound 38-4
Figure BDA0004049198190000102
Fmoc removal of the compound 38-4 to give compound 38-5
Figure BDA0004049198190000103
Mixing the compound 38-5, the D-glucose, acetic acid and methanol, heating to 50 ℃, reacting for 30 minutes, and adding NaCNBH 3 Reacting at 50 ℃ for 16 hours to obtain the compound 38-6. In some embodiments, naCNBH is added 3 After 4 hours of reaction at 50 ℃, naCNBH can be added 3 And D-glucose was reacted for a further 12 hours.
Coupling trastuzumab and irinotecan through a linker according to the preparation method, wherein the binding activity and internalization activity of the obtained ADC product on tumor target cells are not affected; compared with the drug conjugate of the same type of trastuzumab-deluximab, for example, trastuzumab-deruxtecan conjugate (trastuzumab-deruxtecan (8), the specific preparation method is shown in example 2, trastuzumab-monomethyl auristatin E conjugate (trastuzumab-vedotin (4), the specific preparation method is shown in example 4 and trastuzumab-maytansine conjugate (trastuzumab-emtansine (4), the specific preparation method is shown in example 3), and trastuzumab-LD038 (PRO 1102) synthesized by the preparation method of the antibody-drug conjugate of the specification has certain advantages in the aspects of cytotoxicity to tumor cells in vitro, antitumor activity in rats and pharmacokinetics in vivo.
For more details on the preparation method, reference may be made to the contents of example 1. It should be noted that the person skilled in the art can perform the expanded or contracted production according to the preparation method of the present invention, for example, if it is desired to mass-produce antibody-drug conjugates, the amount of each reagent can be increased and each reaction condition can be adaptively modified, and the replacement of each reagent in the method or the modification or replacement of the concentration and ratio thereof are all within the scope of the present invention.
According to another aspect of the present specification there is provided a pharmaceutical composition comprising an antibody-drug conjugate of formula Ia as described above, together with one or more pharmaceutically acceptable excipients, diluents or carriers.
The pharmaceutical composition comprises a safe and effective amount of the antibody-drug conjugate. The effective amount may vary depending on the mode of administration and the severity of the condition to be treated, etc. The selection of a preferred effective amount can be determined (e.g., by clinical trials) based on various factors (e.g., the patient's weight, the patient's immune status, the route of administration, pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.).
In some embodiments, the excipient is an adjunct to the pharmaceutical composition other than the primary drug, which may also be referred to as an adjuvant. Excipients may include, but are not limited to, binders, fillers, disintegrants, lubricants, for example; a base portion in a semisolid formulation ointment, cream; preservative, antioxidant, correctant, aromatic, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, colorant, etc. in liquid preparation.
Diluents are used to increase the weight and volume of a pharmaceutical composition (e.g., in tablet form). Diluents may include, but are not limited to, inorganic salts such as starch, lactose, calcium, microcrystalline cellulose, and the like.
The pharmaceutically acceptable carrier may include a coating, a capsule, a microcapsule, a nanocapsule, or the like, or any combination thereof. In some embodiments, the carrier may protect the critical ingredients in the composition while reducing or avoiding deactivation or decomposition of the critical ingredients under some negative conditions (e.g., oxidation, denaturation by strong acids or bases, etc.). For example, enzymes or relatively low pH values in gastric juice may cause the breakdown or inactivation of key components. The carrier may help maintain or enhance the efficacy of the pharmaceutical composition by protecting key ingredients in the composition.
In some embodiments, the carrier may be used for controlled release of a key ingredient (e.g., irinotecan). Controlled release may include, but is not limited to, slow release, sustained release, targeted release, and the like. For example, the carrier may comprise a hydrogel capsule, microcapsule or nanocapsule made of collagen, gelatin, chitosan, alginate, polyvinyl alcohol, polyethylene oxide, starch, cross-linked starch, and the like, or any combination thereof.
In some embodiments, the pharmaceutically acceptable carrier may include a dispersion medium (e.g., a solvent), a coating, a buffer, a stabilizing formulation, an isotonic and absorption delaying agent, and the like. Exemplary pharmaceutically acceptable carriers can include phosphate buffered saline solutions, water, emulsions (e.g., oil/water emulsions), various types of wetting agents, sterile solutions, gels, bioabsorbable matrix materials, and the like, or other suitable materials, or any combination thereof.
The pharmaceutical composition may be administered to a subject, e.g., a human or animal, suffering from HER2 overexpressing cancer. The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles and solvents that may be used are water, ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated to form a microemulsion by adding it to a mixture of water and glycerol. The injection or microemulsion may be injected into the patient's blood by local bolus injection. In some embodiments, the composition may be stored at a suitable temperature, which may include room temperature (about 20 ℃), 4 ℃, -20 ℃, -80 ℃, and the like. The composition may also be formulated in a variety of forms for storage and transport, such as powders. The powder may be a sterile powder to which a solvent may be added and mixed well before use to prepare a solution for injection or topical application.
According to a further aspect of the present specification there is provided the use of said antibody-drug conjugate or said pharmaceutical composition in the manufacture of a medicament for the treatment of HER2 overexpressing cancer. In some embodiments, HER2 overexpressing cancers may include breast cancer, ovarian cancer, lung cancer, pancreatic cancer, gastric cancer, colon cancer, endometrial cancer, colorectal cancer, and the like. In some embodiments, the cancer is preferably breast, ovarian, and pancreatic cancer.
Examples
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Example 1 preparation of PRO1102 (trastuzumab-LD 038) antibody drug conjugate
(1) Synthesis of LD038
Figure BDA0004049198190000111
Step 1, compound 38-1 (650 mg,0.774 mmol) and N-hydroxysuccinimide (HOSu) (177.98 mg,1.548 mmol) were added to a reaction flask, and dried dichloro was addedMethane (8 mL), stirring at room temperature, EDCl (296.69 mg, 1.268 mmol) was added, stirring at room temperature until the reaction was complete, water was added, repeated extraction with DCM twice (10 mL. Times.2). The extracts were combined, dried over anhydrous sodium sulfate, filtered, concentrated to dryness to give 38-2 (552 mg,0.589mmol, 76.12%) which was used directly in the next reaction, LCMS: M/z= 959.4 (M+Na) + 。
Figure BDA0004049198190000112
Step 2, compound 38-2 (300 mg, 0.356 mmol) and DIPEA (138.22 mg,1.071 mmol) were added to a reaction flask, dried DMF (2 mL) was added, stirring at RT, compound 38-3 (87.97 mg, 0.356 mmol) was added to the reaction flask, stirring at RT was carried out until the reaction was complete, the reaction solution was directly purified by reverse phase preparation (40 g C18 column, mobile phase was acetonitrile and 0.01% TFA in water), the purified preparations were combined, concentrated and lyophilized to give Compound 38-4 (260 mg,0.243mmol, yield 68.14%), LCMS ((M-100)/2+H) + ＝484.9。
Figure BDA0004049198190000121
/>
Step 3, compound 38-4 (260 mg,0.243 mmol), diethylamine and acetonitrile were added to a reaction flask, and the reaction was stirred at room temperature for 2 hours, the reaction mixture was directly purified by reverse phase preparation (12 g C18 column, mobile phase was acetonitrile and 0.01% TFA aqueous solution), and the purified preparations were combined, concentrated and lyophilized to give Compound 38-5 (170 mg,0.201mmol, yield 82.54%). LCMS, ESI M/z= 846.6 (m+h) + 。
Figure BDA0004049198190000122
Step 4, compound 38-5 (170 mg,0.201 mmol), D-glucose (217.08 mg,1.206 mmol), acetic acid (1.21 mg, 0.020mmol) and methanol (5 mL) were added to a reaction flask, heated to 50℃and reacted at this temperature for 30 minutes, addedInto NaCNBH 3 (75.98 mg,1.206 mmol) and the reaction was continued at this temperature for 4 hours. Adding NaCNBH 3 (75.98 mg,1.206 mmol) and D-glucose (217.08 mg,1.206 mmol), at 50℃overnight. Methanol was removed to give an aqueous solution, which was purified by reverse phase preparation to give 38-6 (106 mg,0.090mmol, yield 44.92%). LCMS, ESI M/z= 537.9 ((M-100)/2+H) + 。
Figure BDA0004049198190000123
Step 5, compound 38-6 (250 mg,0.213 mmol), HATU (121.45 mg,0.319 mmol), DIPEA (82.41 mg,0.639 mmol) and DMF (2 mL) were added to a reaction flask, the reaction was stirred at room temperature for 5 minutes, compound 38-7 (178.88 mg,0.213 mmol) was added, the reaction was stirred at room temperature for 2 hours, the reaction was complete, and the reaction solution was directly purified by reverse phase preparation (40 g C18 column, acetonitrile and 0.01% TFA aqueous solution as mobile phase) to give Compound 38-8 (270 mg,0.135mmol, yield 63.48%). LCMS, ESI M/z= 666.6 (M/3+H) + ，999.2(M/2+H) + 。
Figure BDA0004049198190000131
Step 6, compound 38-8 (120 mg,0.060 mmol), dichloromethane and TFA (2 mL) were added to a reaction flask, reacted at room temperature for 1 hour, the starting material disappeared, aqueous sodium bicarbonate solution was added, the reaction was continued at room temperature with stirring for 1 hour, all trifluoroacetate was destroyed, the aqueous phase after methylene chloride was removed by concentration, and purification by reverse phase preparation (C18 column, mobile phase acetonitrile and 0.01% TFA aqueous solution) gave Compound 38-9 (80 mg,0.042mmol, yield 70.19%). LCMS, ESI M/z= 633.2 (M/3+H) + ，949.2(M/2+H)。
Figure BDA0004049198190000132
Step 7, compound 38-9 (20 mg,0.01 mmol), DIPEA (4.08 mg,0.032 mmol)And dry DMF (1 mL) in a reaction flask, stir at room temperature, slowly drop a solution of compound 38-10 (4.88 mg,0.016 mmol) in DMF (1 mL) into the reaction solution, react overnight at room temperature, and complete the reaction, the reaction solution is directly purified by reverse phase preparation (C18 column, mobile phase acetonitrile and 0.01% TFA in water) to give compound LD038 (PB 038) (11 mg,0.005mmol, yield 49.91%), white solid LCMS, M/z= 697.7 (M/3+H) + . The nuclear magnetic data of LD038 are as follows:
1 HNMR(400MHz，DMSO-d6):δ10.03(s，1H)，8.19-8.11(m，2H)，8.07(d，J＝8.8Hz，1H)，7.96(d，J＝7.6Hz，1H)，7.82-7.77(m，2H)，7.66(d，J＝8.4Hz，1H)，7.60(d，J＝8.4Hz，2H)，7.36(d，J＝8.0Hz，2H)，7.32(s，1H)，7.00(s，2H)，6.53(s，1H)，5.99(t，J＝5.6Hz，1H)，5.45-5.43(m，6H)，5.30-5.24(m，3H)，5.08(s，2H)，4.84-4.74(m，2H)，4.65-4.49(m，4H)，4.45-4.35(m，3H)，4.27-4.17(m，2H)，4.04-3.95(m，2H)，3.80-3.77(m，2H)，3.71-3.67(m，2H)，3.62-3.55(m，9H)，3.53-3.43(m，44H)，3.27-3.21(m，2H)，3.16-3.07(m，2H)，3.07-2.93(m，6H)，2.38(s，3H)，2.29(t，J＝6.4Hz，2H)，2.23-2.13(m，2H)，2.13-2.08(m，2H)，2.00-1.82(m，4H)，1.73-1.54(m，4H)，1.54-1.40(m，7H)，1.40-1.30(m，4H)，1.30-1.14(m，5H)，0.90-0.81(m，9H)ppm。
(2) Preparation of PRO1102 (trastuzumab-LD 038) conjugate
To 20mg of trastuzumab antibody buffer, 50mM phosphate and 5mM EDTA, pH=6.9, was added 10 equivalents of aqueous TCEP-HCl. The reaction was carried out at 25℃for 2 hours. Excess TCEP was removed by ultrafiltration with 50mM phosphate 5mM EDTA ph=6.9 buffer. 9.5 equivalents of 20mg/mL of LD038 in water was added to the reduced antibody and the coupling reaction was reacted at 25℃for 2 hours. Excess LD038 and its byproducts were removed by ultrafiltration with 50mM phosphate and 5mM EDTA, ph=6.9 buffer, and the trastuzumab-LD038 conjugate after ultrafiltration was stored in 20mM histidine solution containing 6% sucrose, 0.02% tween 20, ph=5.6. The purity of the trastuzumab-LD038 conjugate was finally 98% by SEC-HPLC and the DAR value by LCMS was 8.0. The resulting trastuzumab-LD038 conjugate was designated PRO1102.
Example 2 preparation of trastuzumab-deruxtech (8) conjugate
To 20mg of trastuzumab antibody buffer, 50mM phosphate and 5mM EDTA, pH=6.9, was added 10 equivalents of aqueous TCEP-HCl. The reaction was carried out at 25℃for 2 hours. Excess TCEP was removed by ultrafiltration with 50mM phosphate 5mM EDTA, ph=6.9 buffer. 9.5 equivalents of 20mg/mL of a solution of deruxecan in DMSO was added to the reduced antibody and the coupling reaction was reacted at 25℃for 8 hours. Excess deruxtecan and its byproducts were removed by ultrafiltration with 50mM phosphate and 5mM EDTA, ph=6.9 buffer, and the trastuzumab-deruxtecan (8) conjugate after ultrafiltration was stored in 20mM histidine solution with 6% sucrose, 0.02% tween 20, ph=5.6, and the purity of the trastuzumab-deruxtecan (8) conjugate was 96% as measured by SEC-HPLC and the DAR value was 6.5 as measured by LCMS.
Example 3 preparation of trastuzumab-emtansine (4)
To 20mg of trastuzumab antibody buffer, which is 50mM phosphate and 5mM EDTA, was added 12 equivalents of SMCC-DM1 in DMSO, pH=7.5. The reaction was carried out at 25℃for 5 hours. Excess SMCC-DM1 and other byproducts were removed by ultrafiltration with 50mM phosphate, 5mM EDTA, and buffer at ph=6.9. Trastuzumab-emtansine (4) obtained by ultrafiltration was stored in 20mM histidine solution with ph=5.6 containing 6% sucrose, 0.02% tween 20. The purity of trastuzumab-emtansine (4) was finally 98% by SEC-HPLC and the DAR value by LCMS was 4.1.
Example 4 preparation of trastuzumab-vedotin (4) conjugate
To 20mg of trastuzumab antibody buffer was added 2.2 equivalents of aqueous TCEP-HCl, 50mM phosphate and 5mM EDTA, ph=6.9. The reaction was carried out at 25℃for 2 hours. 5.0 equivalents of 20mg/mL Mc-vcmMAE in DMSO was added to the reduced antibody and the coupling reaction was allowed to react at 25℃for 2 hours. The excess Mc-vcMMAE and its byproducts were removed by ultrafiltration with 50mM phosphate and 5mM EDTA, ph=6.9 buffer, and the trastuzumab-vedotin (4) conjugate obtained by ultrafiltration was stored in 20mM histidine solution containing 6% sucrose, 0.02% tween 20, ph=5.6. The purity of the trastuzumab-vedotin (4) conjugate was finally 97% by SEC-HPLC and the DAR value was 3.7 by LCMS.
Example 5 in vitro binding Activity of antibody-drug conjugate to HER 2-positive tumor cell lines
(1) Cell culture
HCC1954 (kobai, cat# CBP 60374) is a human breast cancer cell, SK-OV-3 (kobai, cat# CBP 60291) is a human ovarian cancer cell, JIMT-1 (kobai, cat# CBP 60378) is a human breast cancer cell, all purchased from kobai organisms. Raji (ATCC, commodity catalog number CCL-86) is a lymphoma cell, purchased from ATCC, and cap-1 is a human pancreatic cancer cell, given away to friends. Using CO 2 Constant temperature incubator at 37℃with 5% CO 2 These cell lines were cultured under conditions. HCC1954 and Raji were cultured in RPMI1640 (Gibco, cat# 11875093) medium containing 10% FBS (Cell max, cat# SA 211.02), SK-OV-3 was cultured in McCoy's5a Modified (Gibco, cat# 16600082) medium containing 10% FBS (Cell max, cat# SA 211.02), JIMT-1 was cultured in DMEM (Gibco, cat# C11995500 BT) medium containing 10% FBS (Cell max, cat# SA 211.02), and Capan-1 was cultured in IMDM (Gibco, cat# 12440053) medium containing 20% FBS (Cell max, cat# SA 211.02). Cells were subcultured every 1-2 days, with 0.25% trypsin being used to digest the cells at passage and ensure reasonable passage density. When the cells are in the logarithmic growth phase and the number meets the assay requirements, the cells are collected and counted for subsequent cell assays.
(2) Tumor cell line copy number determination
The target copy number was detected by QIFKIT (DAKO, K0078), cells were labeled with a mouse monoclonal antibody targeting the target antigen, and the set-up and calibration microspheres in the cells and kit were labeled in parallel with a fluorescein-conjugated anti-mouse antibody. Fluorescein was related to the number of mouse monoclonal antibody molecules bound to cells and microspheres, samples were analyzed on a flow cytometer, and copy numbers were calculated according to standard curve formulas. The copy number of HER2 molecules on the surface of the tumor cell lines examined is shown in table 1.
TABLE 1 target copy number in tumor cell lines
Cell lines Tumor type Copy number of HER2 (×10) 3 per cell)
HCC1954 Breast cancer 365
SK-OV-3 Ovarian cancer 467
JIMT-1 Breast cancer 70
Capan-1 Pancreatic cancer 33
Raji Lymphoma carcinoma of lymph 0.18
As can be seen from Table 1, the SK-OV-3 cells had the greatest copy number and HCC1954 cells had lower Raji copy numbers. Thus, HCC1954, SKOV-3HER2, JIMT-1, and Capan-1 were used as HER2 positive tumor cell lines (tumor target cells) for subsequent cell assays.
(3) Assessment of binding Activity by flow cytometry
The binding activity of antibody trastuzumab or antibody-drug conjugate (ADC) prepared in examples 1-4 to HER2 positive tumor cell lines HCC1954, SK-OV-3, JIMT-1, cap-1 was assessed by flow cytometry (Beckman, cytoflex). At 3X 10 on V-shaped bottom 96-well plate 5 Cells/well were seeded and then incubated with 100 μl of trastuzumab or trastuzumab conjugate in a gradient dilution. After incubation at 4℃for 30 min, the cells were washed twice with PBS, stained with 100. Mu.L of 1:200 diluted PE-anti-human Fc in FACS buffer (1 XPBS with 1% BSA), and then incubated at 4℃for 30 min. After the incubation, the cells were washed twice with PBS and flow cytometric analysis was performed.
The results are shown in fig. 4-7 and table 2, and the binding activity of pro1102 and other trastuzumab conjugates on multiple tumor target cells is similar to trastuzumab, indicating that none of these trastuzumab conjugates after conjugation altered the binding activity of trastuzumab.
TABLE 2trastuzumab and antibody-drug conjugates thereof for EC against multiple tumor cell lines 50
Figure BDA0004049198190000151
Example 6 internalization Rate study of antibody-drug conjugate PRO1102 in HER 2-positive tumor cell lines
Internalization ability of trastuzumab and its conjugate PRO1102 in HER2 positive tumor cell lines was detected using a flow cytometer. Cell culture as described in example 5 (1) above, 3X 10 was performed using FACS buffer (1 XPBS, 0.1% BSA) containing 10. Mu.g/mL trastuzumab or trastuzumab conjugate PRO1102 5 The individual cells were incubated at 4℃for 30 minutes. The cells were then washed at 4 ℃ to remove unbound material and placed on ice or transferred to 37 ℃ as required. PE-anti-human Fc at 4 DEG CCells were stained at progressive time points (0 h, 0.5h, 1h, 2h, 3h, 4 h) for 30 minutes and analyzed using a flow cytometer.
The calculation method of the internalization rate is as follows: the MFI of the cell surface bound antibody at each time point at 37 ℃ was subtracted from the Mean Fluorescence Intensity (MFI) of the cell surface bound antibody at time point 0 at 4 ℃ and then divided by the MFI of the cell surface bound antibody at time point 0 at 4 ℃.
The results, shown in figures 8-9, of pro1102, were similar to trastuzumab in internalization capacity in multiple tumor target cells, indicating that the internalization activity of trastuzumab was not altered following conjugation.
EXAMPLE 7 cytotoxicity Studies of antibody-drug conjugates of the invention on HER 2-positive tumor cell lines
Cell culture was as described in example 5 (1) above. The day prior to the addition of trastuzumab or trastuzumab conjugate of examples 1-4, cells were harvested and plated onto 96-well pure white flat bottom culture plates. The following day, cells were exposed to a test sample ranging from 100 μg/mL to 0.05 μg/mL (3-fold gradient dilution) of trastuzumab or trastuzumab conjugate. The culture plate is cultured for 96-120 hours at 37 ℃. Subsequently, 40. Mu.L of Cell-titer Glo (CTG) was added to each well of the plate, and after 5 minutes of incubation, luciferase was read for analysis using a microplate reader. All readings were normalized to the percentage of viable cells in untreated control wells and IC was calculated by Prism software 50 Values.
The results are shown in FIGS. 10-13 and Table 3. By IC in Table 3 50 The data shows that PRO1102 using TOP1 inhibitor as the pharmacophore is equivalent to trastuzumab-deruxecan (8) in terms of cell activity, and trastuzumab-emtansine (4) and trastuzumab-vedotin (4) using tubulin inhibitor as the pharmacophore have strong activity, which is related to the activity of the pharmacophore used in ADC drugs.
TABLE 3 IC of trastuzumab and antibody-drug conjugates thereof for various tumor cell lines 50
Figure BDA0004049198190000152
Figure BDA0004049198190000161
EXAMPLE 8 in vivo anti-tumor Activity study of antibody-drug conjugate on HER 2-positive tumor cell lines in mouse xenograft model
In vivo anti-tumor activity of trastuzumab conjugates (trastuzumab-deruxecan (8), trastuzumab-emtansine (4), trastuzumab-vedotin (4) and PRO 1102) was evaluated against tumor cell lines HCC1954 (breast cancer), SK-OV-3 (ovarian cancer), JIMT-1 (breast cancer) and cap-1 (pancreatic cancer) transplanted nude mice subcutaneously.
Tumor cells suspended in 0.1mL of medium were injected to establish tumor models (see table 4). Tumor growth was observed daily after tumor inoculation, and the average tumor size was selected to be approximately 150mm 3 Is divided into groups using hierarchical randomization according to tumor volume. The day after randomization (randomization day defined as D0) was initiated and mice received a single (day 0) intravenous injection at the doses listed in table 5.
Table 4 experimental parameters table of tumor model
Figure BDA0004049198190000162
The study was conducted according to the study protocol approved by the Institutional Animal Care and Use Committee (IACUC). At routine monitoring, all effects of tumor growth and dosing on animal behavior were examined, for example, activity, food intake and water intake (by visual inspection), weight gain/loss (2 or 3 weight measurements per week), eye/hair dullness, and any other abnormal effects. Death and observed clinical signs were recorded. It will be observed that the disease is continuously worsening or that the tumor size exceeds 3000mm 3 Is euthanized.
The primary endpoint was to observe whether the tumor growth could be delayed or mice cured. Tumor size was measured in both directions using calipers and volume (mm) was calculated using the following formula 3 )：V＝0.5×a×b 2 Wherein a and b are the length of the tumor respectivelyDiameter and minor diameter.
Tumor volumes between groups were analyzed using one-way ANOVA. Comparison was made with vehicle group using Tukey post hoc test.
All data were plotted and analyzed using software graphpadprism 8.4.2. P < 0.05 is considered statistically significant.
Table 5trastuzumab conjugate dosing regimen in 4 mouse human tumor xenograft models
Figure BDA0004049198190000163
Figure BDA0004049198190000171
FIGS. 14-17 are tumor growth curves after subcutaneous implantation of HCC1954, SK-OV-3, JIMT-1, and Capan-1, respectively. PRO1102 showed stronger tumor suppression in multiple tumor models than trastuzumab-deruxecan (8), trastuzumab-emtansine (4), trastuzumab-vedotin (4). No significant tumor growth inhibition was shown by trastuzumab-emtansine (4) for JIMT-1 and Capan-1, whereas PRO1102 inhibited tumor growth significantly more than trastuzumab-deruxecan (8). The inhibition of tumor growth by pro1102 was significantly stronger for Capan-1 than for other conjugates and trastuzumab.
According to the preparation method of the antibody-drug conjugate provided by the embodiment of the specification, the linker-irinotecan conjugate LD038 is obtained, and LD038 is coupled to the target HER2 antibody to obtain the antibody-drug conjugate PRO1102, so that the killing power of the antibody-drug conjugate to tumor target cells is improved on the basis of not changing the affinity of the antibody.
Example 9 in vivo Pharmacokinetic (PK) study of antibody-drug conjugates in rats
Male Sprague Dawley rats (n=3 rats/group) received 3mg/kg trastuzumab and conjugates thereof (trastuzumab-deruxecan (8), trastuzumab-vedotin (4), PRO 1102) for intravenous administration. Orbital blood was collected from each rat at 10 minutes, 4 hours, 1 day, 4 days, 7 days, 10 days, 14 days, and 21 days post-dose. Total anti-concentration in plasma was measured by an in-house developed assay and calculated using winnonlin8.2 software.
The results are shown in FIG. 18.PRO1102 exhibited excellent PK characteristics comparable to the unconjugated parent trastuzumab.
The antibody-drug conjugate PRO1102 in the embodiment of the specification has good targeting function and good plasma stability, cannot be quickly and clearly seen by plasma, can effectively deliver small-molecule drugs into tumor target cells, and the adopted cleavable hydrophilic linker can enable the prepared antibody-drug conjugate PRO1102 to be similar to the PK behavior of the naked anti-trastuzumab, has similar elimination half-life and clearance rate, has good curative effect and can be further used for later clinical research.
It will be appreciated by those skilled in the art that the above examples are illustrative of the invention and are not to be construed as limiting the invention. Any modifications, equivalent substitutions and variations, etc., which are within the spirit and principles of the present invention, are intended to be included within the scope of the present invention.

Claims (12)

1. An antibody-drug conjugate, characterized in that the conjugate has a structure represented by formula Ia,
Figure FDA0004049198180000011
wherein,,
mAb is the anti-HER 2 antibody trastuzumab;
thiol-linker-irinotecan conjugate LD038 obtained by reduction of the conjugate via inter-chain disulfide bond of the mAb
Figure FDA0004049198180000012
Generating Michael addition to obtain the product;
n is between 1 and 10.
2. The antibody-drug conjugate of claim 1, wherein n is between 5-10.
3. The antibody-drug conjugate of claim 5, wherein n is 8.
4. The antibody-drug conjugate of any one of claims 1-3, wherein the linker is a cleavable hydrophilic linker.
5. A method of preparing an antibody-drug conjugate of claim 1, comprising the steps of:
a) Preparation of linker-irinotecan conjugate LD038
Figure FDA0004049198180000021
b) And (3) after the trastuzumab is reduced, carrying out a coupling reaction with the LD038 to obtain the antibody-drug conjugate.
6. The method of claim 5, wherein the step of performing a coupling reaction with LD038 after reducing trastuzumab to obtain the antibody-drug conjugate comprises:
and the sulfhydryl group obtained after the inter-chain disulfide bond of the trastuzumab is reduced is subjected to Michael addition with the LD038.
7. The method of preparing an antibody-drug conjugate according to claim 5, wherein the preparing the linker-irinotecan conjugate LD038 comprises:
i) Synthesis of Compound 38-6
Figure FDA0004049198180000022
ii) combining the compound 38-6 with the compound 38-7
Figure FDA0004049198180000023
Reacting to obtain the connector 38-9 of the irinotecan
Figure FDA0004049198180000031
And +.>
iii) Combining said compound 38-9 with MC-OSu
Figure FDA0004049198180000032
And (3) reacting to obtain the LD038.
8. The method of preparing an antibody-drug conjugate according to claim 7, wherein the compound 38-6 is prepared by the compound 38-1
Figure FDA0004049198180000033
N-hydroxysuccinimide (HOSu), compound 38-3 +.>
Figure FDA0004049198180000034
And D-glucose.
9. The method for producing an antibody-drug conjugate according to claim 8, wherein,
the compound 38-1 is mixed with the N-hydroxysuccinimide (HOSu), methylene dichloride and EDCl are added, and the mixture is reacted at room temperature to obtain the compound 38-2
Figure FDA0004049198180000035
Mixing the compound 38-2, DIPEA and DMF, adding the compound 38-3, and reacting at room temperature to obtain a compound 38-4
Figure FDA0004049198180000036
Fmoc removal of the compound 38-4 to give compound 38-5
Figure FDA0004049198180000041
And
Mixing the compound 38-5, the D-glucose, acetic acid and methanol, heating to 50 ℃, reacting for 30 minutes, and adding NaCNBH 3 Reacting at 50 ℃ for 16 hours to obtain the compound 38-6.
10. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1-4, and one or more pharmaceutically acceptable excipients, diluents or carriers.
11. Use of an antibody-drug conjugate according to any one of claims 1-4 or a pharmaceutical composition according to claim 10 in the manufacture of a medicament for the treatment of HER2 overexpressing cancer.
12. The use according to claim 11, wherein the cancer is selected from breast cancer, ovarian cancer and pancreatic cancer.
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CN115364238A (en) * 2022-08-16 2022-11-22 山东大学 Atlizumab-MMAD conjugate and preparation method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115364238A (en) * 2022-08-16 2022-11-22 山东大学 Atlizumab-MMAD conjugate and preparation method and application thereof
CN115364238B (en) * 2022-08-16 2024-02-02 山东大学 Atuzumab-MMAD conjugate and preparation method and application thereof

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