Fc-III-4C polypeptide-polymer-drug conjugate and preparation method and application thereof

Abstract

The present invention relates to a Fc-III-4C polypeptide-polymer-drug conjugate and a preparation method and application thereof. The main chain of the Fc-III-4C polypeptide-polymer-drug conjugate is polyglutamic acid / aspartic acid, and the side carboxyl group is bonded to the Fc-III-4C polypeptide and a cytotoxic drug or an immunomodulatory drug, which is obtained by bonding the Fc-III-4C polypeptide and the cytotoxic drug or the immunomodulatory drug to the side chain carboxyl group of polyglutamic acid / aspartic acid. The present invention also provides an antibody-polymer-drug conjugate, which is obtained by blending a monoclonal antibody with an Fc-III-4C polypeptide-polymer-drug conjugate in an aqueous solution. The present invention also provides the application of the antibody-polymer-drug conjugate in the preparation of anticancer drugs. The antibody-polymer-drug conjugate of the present invention is convenient to prepare and does not affect the structure of the antibody Fab segment, and has the advantages of controllable DAR, strong universality, and easy storage and transportation.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an Fc-III-4C polypeptide-polymer-drug conjugate and a preparation method and application thereof. Background Art

[0002] Improving the delivery efficiency of drugs at the tumor site has always been an urgent problem to be solved in the field of cancer treatment. In recent years, nanomedicines that passively target tumors through the permeation and retention effect (EPR effect) have improved certain drug tumor enrichment, but due to the diversity and heterogeneity of tumors, the improvement of the tumor delivery efficiency of passively targeted nanomedicines is still small.

[0003] Compared with passively targeted nanomedicines, actively targeted nanomedicines greatly improve the tumor delivery efficiency of drugs through active recognition of tumor sites. They mainly include small molecule targeted preparations, monoclonal antibodies, antibody-drug conjugates, and targeted nano-drug delivery systems. Among them, based on the precise recognition of certain tumors by monoclonal antibodies, antibody-drug conjugates (ADCs), known as "magic bullets", have been widely studied and have significant therapeutic effects. ADC drugs are composed of three parts: antibodies, linkers, and cytotoxic drugs. After highly active small molecule drug payloads are chemically connected to monoclonal antibodies through cleavable or non-cleavable linkers, the cytotoxic drugs are delivered to the tumor by using the specific recognition of certain antigens on the tumor by monoclonal antibodies. Currently, 14 anti-tumor ADC drugs based on targets such as tumor proteins HER2, Trop2, and CD22 are on the market. For example, the best-selling ADC drug T-DM1 is composed of anti-HER2 monoclonal antibodies and the tubulin inhibitor DM1, with a drug-antibody ratio (DAR) of 3.5. Through the specific recognition of anti-HER2 monoclonal antibodies for tumors with high HER2 protein expression, cytotoxic drugs are selectively delivered to the tumor site, achieving good anti-tumor effects.

[0004] However, the current preparation and purification process of ADC drugs still needs to be improved. Existing ADC drugs are obtained by directly combining the amino group or exposed thiol group on the antibody with the drug payload. The preparation time is often more than 2 days. During the preparation process, the drug payload will randomly combine with the antigen-binding fragment (Fab segment) or crystallizable segment (Fc segment) of the antibody. The combination of the drug and the Fab segment will have a potential impact on the tumor antigen recognition function of the monoclonal antibody. In addition, the presence of hydrophobic drug payloads often causes ADC drugs to precipitate and become ineffective after long-term storage. Summary of the invention

[0005] In view of this, the present invention aims to solve the technical problems in the prior art and provide an Fc-III-4C polypeptide-polymer-drug conjugate and a preparation method and application thereof. The Fc-III-4C polypeptide-polymer-drug conjugate of the present invention and an antibody are used to prepare an antibody-polymer-drug conjugate. This method is convenient to prepare and does not affect the structure of the antibody Fab segment. It has the advantages of controllable DAR, strong universality, and easy storage and transportation.

[0006] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0007] The present invention provides an Fc-III-4C polypeptide-polymer-drug conjugate having a structure shown in formula (I):

[0008]

[0009] In formula (I), R1 is a C2 to C10 straight chain alkyl group, a C3 to C10 branched chain alkyl group, or a C6-C20 aryl group;

[0010] R2 is hydroxy, alkoxy which may be substituted by a functional group, or Fc-III-4C;

[0011] R3 is a hydrogen ion or a cation;

[0012] R4 is a hydroxyl group, an alkoxy group which may or may not be substituted by a functional group, or a cytotoxic drug, or an immunomodulatory drug;

[0013] R5 is a hydrogen atom, a C2 to C10 straight chain acyl group, or a C3 to C10 branched chain acyl group;

[0014] L1, L2, L3 are independently selected from -CH2- or -CH2CH2-;

[0015] n is the degree of polymerization, 10≤n≤300; x, y, z are percentages, x+y+z=1, x>0, y>0, z>0.

[0016] Preferably, R1 is a C3-C8 straight-chain alkyl group, a C5-C8 branched-chain alkyl group or a C8-C15 aryl group; R2 is selected from a hydroxyl group or Fc-III-4C; R3 is selected from a hydrogen atom, a metal cation or an organic cation; R4 is selected from a hydroxyl group, or any one of monomethyl auristatin E (Val-Cit-PAB-MMAE), maytansine, paclitaxel, vinorelbine, docetaxel, hydroxycamptothecin, gemcitabine, cytarabine, tegafur, methotrexate, epirubicin, pirarubicin, idarubicin, mitomycin, mitoxantrone, ifosfamide, dacarbazine, cisplatin and oxaliplatin; R5 is selected from a hydrogen atom, a formyl group, an acetyl group, a propionyl group or a butyryl group.

[0017] Further preferably, the R1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, phenyl, naphthyl, biphenyl or anthracenyl, and the R3 is selected from a hydrogen atom, a sodium ion, a potassium ion, an ammonium ion or a positively charged amino acid ion.

[0018] Further preferably, the R2 is Fc-III-4C, the R4 is Val-Cit-PAB-MMAE, and the Fc-III-4C polypeptide-polymer-drug conjugate has a structure shown in formula (II):

[0019]

[0020] Still more preferably, in formula (II), n=160, x=0.025, x is the ratio of the number of grafts with Fc-III-4C, y=0.875, z is the ratio of the number of grafts with Val-Cit-PAB-MMAE, z=0.1.

[0021] The present invention also provides a method for preparing an Fc-III-4C polypeptide-polymer-drug conjugate, comprising the following steps:

[0022] The Fc-III-4C polypeptide-polymer-drug conjugate is obtained by bonding the side chain carboxyl group of the polymer compound to an Fc-III-4C polypeptide having a high affinity for the immunoglobulin Fc segment, and then bonding a cytotoxic drug or an immunomodulatory drug to the remaining side chain carboxyl group of the polymer compound;

[0023] The polymer compound is polyglutamic acid, polyaspartic acid or a glutamic acid-aspartic acid copolymer.

[0024] Preferably, the molar ratio of the side chain carboxyl groups in the polymer compound to the Fc-III-4C polypeptide is 3 to 300:1, and the molar ratio of the side chain carboxyl groups in the polymer compound to the cytotoxic drug or immunomodulator drug is 3 to 300:1.

[0025] The present invention also provides an antibody-polymer-drug conjugate, which is obtained by mixing a monoclonal antibody containing an Fc segment and an Fc-III-4C polypeptide-polymer-drug conjugate in an aqueous solution.

[0026] Preferably, the molar ratio of the Fc-III-4C polypeptide to the Fc segment-containing monoclonal antibody in the Fc-III-4C polypeptide-polymer-drug conjugate is 1 to 2:1;

[0027] The Fc segment-containing monoclonal antibody is an antibody selected from the group consisting of anti-PDL1, Trop2, HER2, PD1, CTLA-4, OX40, OX40L, CD16, 4-1BB, EGFR, CD3, CD3ε, CD19, CD28, CD33, CD22, CD79b, BCMA, MET, EpCAM, CD13, CD73, CD20 and Nectin-4.

[0028] The present invention also provides an application of an antibody-polymer-drug conjugate in the preparation of anticancer drugs.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The Fc-III-4C polypeptide-polymer-drug conjugate of the present invention is used with an antibody to prepare an antibody-polymer-drug conjugate. This method is convenient to prepare and does not affect the antibody Fab segment structure. It has the advantages of controllable DAR, strong universality, and easy storage and transportation.

[0031] The Fc-III-4C polypeptide-polymer-drug conjugate prepared by the present invention can be combined with a variety of different types of antibodies and cytotoxic drugs, thereby preparing the antibody-polymer-drug conjugate simply and efficiently.

[0032] The present invention provides a universal, simple and convenient method for preparing antibody-polymer-drug conjugates. The antibody-polymer-drug conjugates prepared by the present invention can target tumor sites and increase the high enrichment of toxic drugs at tumor sites. They can be used in the fields of preparing antibody drugs for cancer treatment and anti-infective drugs, and have a wide range of application fields and extremely high clinical application prospects.

[0033] The Fc-III-4C polypeptide-polymer-drug conjugate of the present invention can be stored separately from the antibody and directly prepared when used, thereby avoiding the risk of aggregation of traditional ADC drugs during storage and transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0035] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the polyglutamic acid PLG obtained in Example 1 of the present invention.

[0036] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of Fc-PLG obtained in Example 2 of the present invention.

[0037] Figure 3 This is the drug loading amount of Fc-PLG-MMAE obtained in Example 3 of the present invention.

[0038] Figure 4 This is the particle size distribution of Fc-PLG-MMAE obtained in Example 3 of the present invention and IgG-PLG-MMAE and aPDL1-PLG-MMAE obtained in Example 4.

[0039] Figure 5 The IgG-PLG-MMAE and aPDL1-PLG-MMAE obtained in Example 4 of the present invention were evaluated for in vitro MC38 cytotoxicity using a CCK-8 cell proliferation assay and were grouped into: Fc-PLG-MMAE, IgG-PLG-MMAE and aPDL1-PLG-MMAE.

[0040] Figure 6 The aPDL1+IgG-PLG-MMAE and aPDL1-PLG-MMAE obtained in Example 4 of the present invention were used to treat MC38 tumor-bearing mice, including treatment regimen (A), tumor volume changes of subcutaneous tumor-bearing mice (B), mouse weight changes (C), and tumor volume changes of mice in each group (D). The groups in the figure are: PBS group, 2.5 mg·kg -1 aPDL1+IgG-PLG-MMAE (2.17 mg kg - 1 aPDL1,2.31mg·kg -1 IgG and 0.08mg·kg -1 MMAE) group, 1.25mg·kg -1 aPDL1-PLG-MMAE (1.09 mg kg -1 PDL1 and 0.04 mg kg -1 MMAE) group and 2.5 mg·kg -1 aPDL1-PLG-MMAE (2.17 mg kg -1 PDL1 and 0.08 mg kg -1 MMAE) group.

[0041] Figure 7 H&E analysis and TUNEL staining analysis of tumors in MC38 tumor-bearing mice treated with aPDL1+IgG-PLG-MMAE and aPDL1-PLG-MMAE obtained in Example 4 of the present invention (A) and H&E analysis of normal tissues (heart, liver, spleen, lung, and kidney) (B).

[0042] Figure 8 This is a graph showing the systemic toxicity evaluation of MC38 tumor-bearing mice after treatment with aPDL1+IgG-PLG-MMAE and aPDL1-PLG-MMAE obtained in Example 4 of the present invention. DETAILED DESCRIPTION

[0043] The inventive concept of the present invention is: the present invention provides an Fc-III-4C polypeptide-polymer-drug conjugate and preparation, antibody-polymer-drug conjugate and application. The Fc-III-4C polypeptide-polymer-drug conjugate of the present invention has a main chain of polyglutamic acid / aspartic acid (PLG / A), and a side carboxyl group is bonded to an Fc-III-4C polypeptide and a cytotoxic drug such as MMAE, wherein the grafted MMAE can also be replaced by grafting other different types of cytotoxic drugs. The present invention bonds an Fc-III-4C polypeptide having a high affinity for the Fc segment of an immunoglobulin (antibody) through a side chain carboxyl group of polyglutamic acid / aspartic acid (PLG / A), and then bonds a cytotoxic drug or an immunomodulatory drug to the remaining side chain carboxyl group of polyglutamic acid / aspartic acid (PLG / A) to obtain an Fc-III-4C polypeptide-polymer-drug conjugate. Since the dissociation constant (Kd) of the Fc-III-4C polypeptide to human immunoglobulin IgG (including antibodies) is as high as 2.45 nM, and it does not affect the activity of the antibody. Therefore, the monoclonal antibody and the Fc-III-4C polypeptide-polymer-drug conjugate are incubated in a phosphate buffer to prepare an antibody-polymer-drug conjugate. The preparation of the Fc-III-4C polypeptide-polymer-drug conjugate of the present invention can be combined with different types of antibodies containing Fc segments and grafted with different types of cytotoxic drugs.

[0044] A large number of studies have shown that tumor cells can upregulate the expression of PD1 protein (PDL1 and PDL2), and PDL1 protein is the main PD1 protein used by tumor cells, so PDL1 protein shows a high expression level in the tumor site. Therefore, the specific binding of anti-PDL1 monoclonal antibodies and the like with the PDL1 protein in the tumor site can promote the large-scale enrichment of anti-PDL1 monoclonal antibodies on the surface of tumor cells. Monomethyl auristatin E (MMAE) is a synthetic derivative of sea dolitoxin 10, which has a strong anti-tumor effect. Like vinblastine, it belongs to the anti-microtubule class of drugs, and plays an effective mitotic inhibitory effect by inhibiting tubulin polymerization, but its toxicity is about 200 times that of vinblastine. Therefore, the present invention provides an antibody-polymer-drug conjugate (aPDL1-PLG-MMAE) in which a drug load of MMAE or the like is combined with an Fc-III-4C polypeptide-polymer-drug conjugate (Fc-PLG-MMAE) and an anti-PDL1 monoclonal antibody. Through the specific binding of anti-PDL1 antibody to PDL1 protein in the tumor site, MMAE is released after being degraded by intracellular lysosomes after being endocytosed, inhibiting tubulin polymerization and playing an effective mitotic inhibitory role, promoting tumor cell death. The feasibility of the preparation method of antibody-polymer-drug conjugate and the effectiveness of the drug are verified through relevant examples.

[0045] The present invention provides an Fc-III-4C polypeptide-polymer-drug conjugate, the main chain of which is polyglutamic acid / aspartic acid (PLG / A), and the side carboxyl groups are bonded to the Fc-III-4C polypeptide and the cytotoxic drug or immunomodulator drug, and has a structure shown in formula (I):

[0046]

[0047] In formula (I), R1 is a C2 to C10 straight chain alkyl group, a C3 to C10 branched chain alkyl group, or a C6-C20 aryl group;

[0048] R2 is hydroxy, alkoxy which may be substituted by a functional group, or Fc-III-4C;

[0049] R3 is a hydrogen ion or a cation;

[0050] R4 is a hydroxyl group, an alkoxy group which may or may not be substituted by a functional group, or a cytotoxic drug, or an immunomodulatory drug;

[0051] R5 is a hydrogen atom, a C2 to C10 straight chain acyl group, or a C3 to C10 branched chain acyl group;

[0052] L1, L2, L3 are independently selected from -CH2- or -CH2CH2-;

[0053] n is the degree of polymerization, 10≤n≤300; x, y, z are percentages, x+y+z=1, x>0, y>0, z>0.

[0054] Preferably, R1 is a C3-C8 straight-chain alkyl group, a C5-C8 branched alkyl group or a C8-C15 aryl group. Further preferably, R1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, phenyl, naphthyl, biphenyl or anthracenyl.

[0055] Preferably, the R2 is selected from hydroxyl or Fc-III-4C, and further preferably, the R2 is Fc-III-4C.

[0056] Preferably, R3 is selected from a hydrogen atom, a metal cation or an organic cation, and further preferably, R3 is selected from a hydrogen atom, a sodium ion, a potassium ion, an ammonium ion or a positively charged amino acid ion.

[0057] Preferably, R4 is selected from any one of hydroxyl, or monomethyl auristatin E (Val-Cit-PAB-MMAE), maytansine, paclitaxel, vinorelbine, docetaxel, hydroxycamptothecin, gemcitabine, cytarabine, tegafur, methotrexate, epirubicin, pirarubicin, idarubicin, mitomycin, mitoxantrone, ifosfamide, dacarbazine, cisplatin and oxaliplatin, and further preferably, R4 is monomethyl auristatin E (Val-Cit-PAB-MMAE).

[0058] Preferably, R5 is selected from a hydrogen atom, a formyl group, an acetyl group, a propionyl group or a butyryl group.

[0059] Still further preferably, the R2 is Fc-III-4C, and the R4 is Val-Cit-PAB-MMAE;

[0060] The Fc-III-4C polypeptide-polymer-drug conjugate has a main chain of polyglutamic acid / aspartic acid (PLG / A), and a side carboxyl group is bonded to the Fc-III-4C polypeptide and MMAE, and has a structure shown in formula (II):

[0061]

[0062] R1, R3, R5, x, y, z, n, and L1, L2, L3 in formula (II) are consistent with those defined in formula (I) and are not further described here.

[0063] Further preferably, the material of the Fc-III-4C polypeptide-polymer-drug conjugate is polyglutamic acid homopolymer (PLG), Fc-III-4C polypeptide with generally high affinity to the crystallizable segment (Fc segment) of immunoglobulin (antibody), and monomethyl auristatin E (MMAE), a highly cytotoxic drug. In formula (II), n=160, x=0.025, x is the proportion of the number of grafts with Fc-III-4C, y=0.875, z is the proportion of the number of grafts with Val-Cit-PAB-MMAE, and z=0.1.

[0064] The present invention also provides a method for preparing an Fc-III-4C polypeptide-polymer-drug conjugate, comprising the following steps:

[0065] The Fc-III-4C polypeptide with high affinity to the Fc segment of immunoglobulin (antibody) is bonded to the side chain carboxyl group of polyglutamic acid / aspartic acid (PLG / A), and then a cytotoxic drug or an immunomodulatory drug is bonded to the remaining side chain carboxyl group of polyglutamic acid / aspartic acid (PLG / A) to obtain an Fc-III-4C polypeptide-polymer-drug conjugate.

[0066] The polyglutamic acid / aspartic acid represents polyglutamic acid, polyaspartic acid or a glutamic acid-aspartic acid copolymer.

[0067] Preferably, the molar ratio of the side chain carboxyl groups in the PLG / A to the Fc-III-4C polypeptide is 3 to 300:1, and the molar ratio of the side chain carboxyl groups in the PLG / A to the cytotoxic drug or immunomodulator drug is 3 to 300:1; further preferably, the molar ratio of the side chain carboxyl groups in the PLG / A to the Fc-III-4C polypeptide is 40:1, and the molar ratio of the side chain carboxyl groups in the PLG / A to the cytotoxic drug or immunomodulator drug is 10:1.

[0068] The present invention also provides an antibody-polymer-drug conjugate, which is obtained by mixing a monoclonal antibody containing an Fc segment and an Fc-III-4C polypeptide-polymer-drug conjugate in an aqueous solution.

[0069] Preferably, the monoclonal antibody containing the Fc segment is incubated with the Fc-III-4C polypeptide-polymer-drug conjugate in a phosphate buffer for 3 to 5 hours to obtain the antibody-polymer-drug conjugate. The advantages are: first, the preparation method of the antibody-polymer-drug conjugate is simple and fast; second, the binding of the Fc-III-4C polypeptide to the antibody will not affect the Fab segment structure of the antibody, that is, it will not affect the antigen recognition effect of the antibody; finally, the Fc-III-4C polypeptide-polymer-drug conjugate and the antibody can be stored separately and directly prepared when used, avoiding the risk of aggregation of traditional ADC drugs during storage and transportation.

[0070] Preferably, the molar ratio of the Fc-III-4C polypeptide to the Fc segment-containing monoclonal antibody in the Fc-III-4C polypeptide-polymer-drug conjugate is 1 to 2:1; further preferably, the molar ratio of the two is 2:1.

[0071] The Fc segment-containing monoclonal antibody is an antibody selected from the group consisting of anti-PDL1, Trop2, HER2, PD1, CTLA-4, OX40, OX40L, CD16, 4-1BB, EGFR, CD3, CD3ε, CD19, CD28, CD33, CD22, CD79b, BCMA, MET, EpCAM, CD13, CD73, CD20 and Nectin-4.

[0072] Further preferably, in the antibody-polymer-drug conjugate of the present invention, the Fc segment-containing monoclonal antibody is an anti-PDL1 antibody (aPDL1), and the cytotoxic drug is monomethyl auristatin E (Val-Cit-PAB-MMAE).

[0073] Preferably, in the antibody-polymer-drug conjugate of the present invention, the molar ratio of the monoclonal antibody containing the Fc segment to the cytotoxic drug is 1:8.

[0074] The present invention also provides an application of an antibody-polymer-drug conjugate in the preparation of anticancer drugs.

[0075] The present invention also provides a method for preparing an antibody-polymer-drug conjugate for inhibiting tumor growth, wherein the subject is a mammal or a tumor cell of the mammal, and the mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. The primate is preferably a monkey, an ape or a Homo sapiens. The subject may be a patient suffering from a tumor, or an ex vivo tumor cell of a patient suffering from a tumor. Based on this, the present invention has found that the antibody-polymer-drug conjugate has a more obvious tumor treatment effect.

[0076] According to the present invention, after replacing the type of monoclonal antibody and the cytotoxic drug, the antibody-polymer-drug conjugate can also be used for the treatment of one or more of the tumors including nasal and paranasal sinus malignant tumors, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, testicular malignant tumors, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytic carcinoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma and multiple myeloma.

[0077] The present invention uses a series of in vitro experiments to prepare antibody-polymer-drug conjugates with uniform particle size distribution. In vivo experiments have found that aPDL1-PLG-MMAE can significantly inhibit tumor growth in a mouse MC38 colon cancer tumor model and has little effect on the weight of the mice. This provides a new treatment idea and strategy for the clinical treatment of cancer and has a good application prospect.

[0078] The present invention provides an Fc-III-4C polypeptide-polymer-drug conjugate and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0079] The present invention does not limit the source of the above Fc-III-4C, which can be commercially available or prepared by methods well known to those skilled in the art; preferably, it can be prepared according to the method disclosed in CN 107556385A, and its amino acid sequence is CDCAWHLGELVWCTC.

[0080] To further illustrate the present invention, the following describes in detail an Fc-III-4C polypeptide-polymer-drug conjugate and its preparation, an antibody-polymer-drug conjugate and its application provided by the present invention in combination with the examples.

[0081] The reagents used in the following examples are all commercially available.

[0082] Example 1: Preparation of poly(L-glutamic acid) homopolymer (PLG)

[0083] 10.0 g of γ-benzyl-L-glutamate-N-internal carboxylic anhydride monomer (BLG-NCA) was dissolved in 100.0 mL of anhydrous dichloromethane (DCM) under anhydrous and oxygen-free conditions, and 21.0 mg of n-hexylamine was added after stirring and dissolved, and stirred in an oil bath environment at 30°C for 72 hours. Subsequently, 890.0 μL of acetic anhydride was added to react for 12 hours. After the reaction was completed, the reaction solution was precipitated with 1.0 L of ether, and the lower layer polymer was obtained by centrifugation and then re-washed with ether, and vacuum dried at room temperature for 12 hours to obtain the intermediate product poly(γ-benzyl-L-glutamate) (PBLG).

[0084] 5.0 g of PBLG was dissolved in 50.0 mL of dichloroacetic acid at 30°C, and 15.0 mL of a 33% hydrogen bromide-glacial acetic acid solution was added while stirring. After stirring at 30°C for 1.5 hours, the reaction solution was precipitated into 700.0 mL of ether and centrifuged. The precipitate was re-dissolved in N,N-dimethylformamide (DMF), dialyzed with deionized water, and freeze-dried to obtain poly(L-glutamic acid) homopolymer (PLG). Nuclear magnetic resonance analysis (such as Figure 1 ). The results show that the chemical shift of 0.79ppm is the methyl (CH3-) signal peak on the main chain (a), the chemical shift of 1.22ppm is the 4 methylene (-CH2-CH2-CH2-CH2-) signal peak on the main chain (b), the chemical shift of 4.25ppm is the second methyl (-CH-) signal peak on the main chain (c), the chemical shift of 2.21ppm is the methylene (-CH2-) signal peak connected to the carbonyl group on the side group (e), and the chemical shift of 1.77~2.01ppm is the methylene (-CH2-) signal peak connected to the main chain on the side group (d). The degree of polymerization of PLG calculated by NMR is 160.7.

[0085] Example 2: Preparation of Fc-PLG

[0086] 100.0 mg PLG and 12.3 mg N, N'-disuccinimidyl carbonate (DSC) were dissolved in 20.0 mL anhydrous DMF, 7.3 mg triethylamine was added and reacted in an anhydrous and oxygen-free environment at 30°C for 12 hours, and then 33.4 mg Fc-III-4C polypeptide was added and continued for 6 hours. The reactant was placed in a dialysis bag (Mw = 3.5 kDa) and dialyzed with DMF for 12 hours, and then dialyzed in ultrapure water with a pH value of 6.8 to 7.4 for 72 hours. After freeze-drying, PLG grafted Fc-III-4C polypeptide material (Fc-PLG) was obtained, and nuclear magnetic resonance analysis was performed using deuterated water as solvent. Figure 2 The characteristic signal peaks of Fc-III-4C polypeptide (peaks d, e, f, g, h, i, j) are at chemical shifts of 6.71 to 7.60 ppm. According to NMR calculations, four Fc-III-4C polypeptides were grafted. The results showed that the Fc-III-4C polypeptide was successfully grafted to the PLG side chain, and the number of Fc-III-4C polypeptide grafts could be controlled during the synthesis process.

[0087] Example 3: Preparation of Fc-PLG-MMAE

[0088] 20.0 mg Fc-PLG and 7.4 mg DSC were dissolved in 20.0 mL anhydrous DMF, 4.3 mg triethylamine was added and reacted in an anhydrous and oxygen-free environment at 30 ° C for 12 hours, and then 16.0 mg of cathepsin B responsive MMAE prodrug (Val-Cit-PAB-MMAE) was added and continued to react for 24 hours. The reaction solution was placed in a dialysis bag (Mw = 7.0 kDa) and dialyzed with DMF for 12 hours, and then dialyzed in ultrapure water with a pH value of 6.8 to 7.4 for 72 hours. The Fc-PLG-MMAE product was obtained after freeze-drying. The MMAE grafting amount was then tested by HPLC. The specific method is as follows: Fc-PLG-MMAE (1.0 mg mL) was dissolved in 30.0 μL morpholineethanesulfonic acid (MES) buffer (10.0 mM morpholineethanesulfonic acid sodium salt, 40.0 μM dithiothreitol). -1 ) and incubated at 37°C for 10 minutes. Then, human cathepsin B (20.0 ng·μL) was added to the above 30.0 μL MES buffer. -1), continued to incubate at 37°C for 24 hours, and treated with EDTA-free protease inhibitor cocktail. All samples were analyzed using an Elite Classical HPLC system equipped with a Supersil ODS2 column (4.6mm*250mm, 5μm). The elution conditions were as follows: mobile phase A: water (0.1% formic acid); mobile phase B: acetonitrile (0.1% formic acid); mobile gradient A: B from 75:25 to 1:99, elution time 6.8 minutes; flow rate: 0.5mL·min -1 The results are as follows Figure 3 As shown, the drug loading of MMAE in Fc-PLG-MMAE is 26.34±0.97%, indicating that MMAE is successfully grafted onto the side chain of Fc-PLG, and the number of MMAE grafts can be controlled during the synthesis process.

[0089] Example 4: Preparation of Antibody-Polymer-Drug Conjugates

[0090] Fc-PLG-MMAE and different antibodies (IgG antibody or anti-PDL1 antibody) were incubated in a phosphate buffer with a pH value of 7.4 for 4 hours at a molar ratio of 2:1. The specific method was as follows: 0.14 mg of Fc-PLG-MMAE was dissolved in 1.0 mL of phosphate buffer with a pH value of 7.4, and 1.0 mg of IgG antibody or 108.5 μL of aPDL1 antibody (8.67 mg mL -1 ), incubated at 4°C for 4 hours. The antibody-polymer-drug conjugates IgG-PLG-MMAE and aPDL1-PLG-MMAE were obtained by utilizing the high affinity of the Fc-III-4C polypeptide on Fc-PLG-MMAE to the Fc segment of monoclonal antibodies (IgG, aPDL1).

[0091] The obtained IgG-PLG-MMAE and aPDL1-PLG-MMAE were subjected to dynamic light scattering analysis to determine the hydrodynamic radius of the particles formed in water. Figure 4 , 0.5 mg·mL prepared in Example 4 -1 IgG-PLG-MMAE and 0.5 mg mL - 1 The dynamic light scattering results of aPDL1-PLG-MMAE showed that the hydrodynamic radii of IgG-PLG-MMAE and aPDL1-PLG-MMAE were 122.7±2.0nm and 110.6±6.5nm, respectively.

[0092] Example 5: In vitro cytotoxicity test of antibody-polymer-drug conjugates

[0093] The cytotoxicity of Fc-PLG-MMAE, IgG-PLG-MMAE and aPDL1-PLG-MMAE on MC38 cells was investigated by CCK-8 cell proliferation assay. 5.0×10 3 cells, incubated overnight, and then added the required concentrations of Fc-PLG-MMAE, IgG-PLG-MMAE, and aPDL1-PLG-MMAE. After incubation for 24 hours, 20.0 μL of CCK-8 solution was added to each well and incubated for another hour. The absorbance of each well at 450 nm was measured using an ELISA reader. Figure 5 As shown in Figure 2, aPDL1-PLG-MMAE has a strong cytotoxic effect on MC38 cells, IC 50 The results show that aPDL1-PLG-MMAE has a significant tumor cell recognition effect on MC38 cells with high expression of PDL1 protein.

[0094] Example 6: Anti-tumor effect of antibody-polymer-drug conjugate on MC38 mouse colon cancer model

[0095] C57BL / 6 mice (6 weeks old, weighing about 20 g, 20 mice) were selected as experimental animals, and mouse MC38 colon cancer cells were subcutaneously injected to construct a mouse colon cancer tumor model and to evaluate the in vivo tumor inhibition effect. Figure 6 As shown in (A), when the tumor grows to about 110 mm 3 The time was recorded as day 0, and the tumor-bearing mice were randomly divided into 4 groups: PBS group, 2.5 mg·kg -1 aPDL1+IgG-PLG-MMAE (2.17 mg kg -1 aPDL1,2.31mg·kg -1 IgG and 0.08mg·kg -1 MMAE) group, 1.25mg·kg -1 aPDL1-PLG-MMAE (1.09 mg kg -1 aPDL1 and 0.04 mg kg -1 MMAE) group and 2.5 mg·kg -1 aPDL1-PLG-MMAE (2.17 mg kg -1 aPDL1 and 0.08 mg kg -1 The drug was injected through the tail vein on days 0, 3, 6, and 9, and the tumor volume and body weight were monitored every 3 days until the tumor grew to about 1800 mm. 3, and conduct pharmacodynamic and safety evaluation. The tumor volume was calculated using the following formula:

[0096] The formula for calculating the mouse tumor volume is: V = (a × b 2 ) / 2, tumor inhibition rate TSR = (Ac-Ax) / Ac×100%. Where a is the long diameter of the tumor, b is the short diameter of the tumor; Ac is the average tumor volume of the control group, and Ax is the average tumor volume of the treatment group.

[0097] like Figure 6 (B) with Figure 6 (D) Compared with the PBS group and aPDL1+IgG-PLG-MMAE (2.5 mg kg -1 ) group, aPDL1-PLG-MMAE (1.25 mg kg -1 ) group and aPDL1-PLG-MMAE (2.5 mg kg -1 ) group could effectively inhibit tumor growth. At the end of treatment, aPDL1-PLG-MMAE (1.25 mg kg -1 ) group and aPDL1-PLG-MMAE (2.5 mg kg -1 ) groups had tumor inhibition rates of 80.6% and 92.3%, respectively, which were significantly higher than those of the IgG-PLG-MMAE group without targeting effect (TSR: 29.2%). At the end of tumor inhibition on day 21, aPDL1-PLG-MMAE (2.5 mg kg -1 The tumor volume of mice in the treated group was 157.2±40.3mm 3 , was intravenously injected with aPDL1-PLG-MMAE (2.5 mg kg -1 ) group had better therapeutic effect, which indicates that aPDL1 can target tumor cells, increase the enrichment of nanoparticles in tumors, and improve the ability of nanoparticles to kill tumor cells.

[0098] At the same time, if Figure 6 (C), aPDL1-PLG-MMAE (1.25 mg·kg -1 ) group and aPDL1-PLG-MMAE (2.5 mg kg -1 ) group had no significant weight loss, like the PBS group and the aPDL1+IgG-PLG-MMAE group, indicating that the antibody-polymer-drug conjugate used was safe and well tolerated.

[0099] Example 7: Immunohistochemical analysis

[0100] After the tumor inhibition experiment, H&E staining and TUNEL staining were performed on the mouse tumor and normal tissues, and the anti-tumor effect and toxic side effects of the antibody-polymer-drug conjugate were analyzed by immunohistochemistry. Figure 7 As shown in (A), in the H&E stained sections, the tumors in the PBS group had large nucleoli, rich nucleoplasm, and high cell density, indicating strong vitality and proliferation activity. The IgG-PLG-MMAE group showed slight killing of tumor cells, which was due to the passive targeting effect of the drug, which enriched a small amount in the tumor and released MMAE to kill the tumor. The aPDL1-PLG-MMAE group, which has an active tumor targeting effect, showed large-area tumor cell necrosis, and the higher the dose, the stronger the killing effect. In the TUNEL stained sections, the tumor cell density in the PBS group was large and there was little cell apoptosis; the IgG-PLG-MMAE group showed a small amount of tumor cell necrosis; and the aPDL1-PLG-MMAE group showed large-area tumor cell apoptosis, and the number of apoptotic cells increased with the increase in drug concentration. This is due to the specific recognition of aPDL1-PLG-MMAE on MC38 cells with high levels of PDL1 expression, which is consistent with the H&E results. At the same time, Figure 7 As shown in (B), normal tissues (heart, liver, spleen, lung, and kidney) of all groups showed no obvious cell morphological changes, indicating that aPDL1-PLG-MMAE has good safety.

[0101] Example 8: Biochemical index detection

[0102] After the tumor inhibition experiment, mouse plasma was collected for liver and kidney function tests to evaluate whether each treatment group had potential toxic side effects. Figure 8 As shown, compared with the PBS healthy control group, the aPDL1+IgG-PLG-MMAE (2.5 mg kg -1 ) group, aPDL1-PLG-MMAE (1.25 mg kg -1 ) group and aPDL1-PLG-MMAE (2.5 mg kg -1 ) groups after treatment, there were no significant differences in the levels of alanine aminotransferase (ALT), alkaline phosphatase (AKP), uric acid (UA), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CRE), and all were within the normal range, indicating that no liver or kidney damage occurred in each treatment group and that the drug has the potential for further clinical application.

[0103] In summary, the present invention provides a universal Fc-III-4C polypeptide-polymer-drug conjugate, which can be easily and conveniently prepared by connecting the Fc-III-4C polypeptide to the antibody, and an antibody-polymer-drug conjugate targeting the PDL1 protein on the surface of mouse tumor cells is prepared, and its value in anti-tumor is verified. The present invention bonds the Fc-III-4C polypeptide with high affinity to the Fc segment of immunoglobulin (antibody) through the side chain carboxyl group of polyglutamic acid / aspartic acid (PLG / A), and then bonds the cytotoxic drug or immunomodulatory drug to the remaining side chain carboxyl group of polyglutamic acid / aspartic acid (PLG / A) to obtain the Fc-III-4C polypeptide-polymer-drug conjugate. Finally, the monoclonal antibody and the Fc-III-4C polypeptide-polymer-drug conjugate are incubated in a phosphate buffer for 3 to 5 hours to obtain the antibody-polymer-drug conjugate. This method has the following advantages: first, the preparation method of antibody-polymer-drug conjugate is simple and rapid; second, the binding of Fc-III-4C polypeptide to antibody will not affect the Fab segment structure of the antibody, that is, it will not affect the antigen recognition effect of the antibody; finally, Fc-III-4C polypeptide-polymer-drug conjugate and antibody can be stored separately and prepared directly when used, avoiding the risk of aggregation of traditional ADC drugs during storage and transportation.

[0104] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An Fc-III-4C polypeptide-polymer-drug conjugate, characterized in that: It has the structure shown in formula (II): (II), R1 is a C2 to C10 straight chain alkyl group, a C3 to C10 branched chain alkyl group, or a C6-C20 aryl group; R3 is a hydrogen ion or a cation; R5 is a hydrogen atom, a C2 to C10 straight chain acyl group, or a C3 to C10 branched chain acyl group; L1, L2, L3 are independently selected from -CH2- or -CH2CH2-; n is the degree of polymerization, 10≤n≤300; x, y, z are percentages, x+y+z=1, x>0, y>0, z>0.

2. The Fc-III-4C polypeptide-polymer-drug conjugate according to claim 1, characterized in that: The R1 is a C3-C8 straight-chain alkyl group, a C5-C8 branched-chain alkyl group or a C8-C15 aryl group; the R3 is selected from a hydrogen atom, a metal cation or an organic cation; the R5 is selected from a hydrogen atom, a formyl group, an acetyl group, a propionyl group or a butyryl group.

3. The Fc-III-4C polypeptide-polymer-drug conjugate according to claim 1, characterized in that: The R1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, phenyl, naphthyl, biphenyl or anthracenyl, and the R3 is selected from a hydrogen atom, a sodium ion, a potassium ion, an ammonium ion or a positively charged amino acid ion.

4. The Fc-III-4C polypeptide-polymer-drug conjugate according to claim 1, characterized in that: In formula (II), n=160, x=0.025, x is the ratio of the number of grafts to Fc-III-4C, y=0.875, z is the ratio of the number of grafts to Val-Cit-PAB-MMAE, z=0.

1.

5. A method for preparing the Fc-III-4C polypeptide-polymer-drug conjugate according to any one of claims 1 to 4, characterized in that: The following steps are involved: The Fc-III-4C polypeptide-polymer-drug conjugate is obtained by bonding the side chain carboxyl group of the polymer compound to an Fc-III-4C polypeptide having a high affinity to the Fc segment of immunoglobulin, and then bonding the Val-Cit-PAB-MMAE drug to the remaining side chain carboxyl group of the polymer compound; The polymer compound is polyglutamic acid, polyaspartic acid or a polyglutamic acid-aspartic acid copolymer.

6. The method for preparing the Fc-III-4C polypeptide-polymer-drug conjugate according to claim 5, characterized in that: The molar ratio of the side chain carboxyl group in the polymer compound to the Fc-III-4C polypeptide is 3 to 300:1, and the molar ratio of the side chain carboxyl group in the polymer compound to the Val-Cit-PAB-MMAE drug is 3 to 300:

1.

7. An antibody-polymer-drug conjugate, characterized in that: The method is obtained by mixing a monoclonal antibody containing an Fc segment with the Fc-III-4C polypeptide-polymer-drug conjugate described in any one of claims 1 to 4 in an aqueous solution.

8. The antibody-polymer-drug conjugate according to claim 7, characterized in that: The molar ratio of the Fc-III-4C polypeptide to the Fc segment-containing monoclonal antibody in the Fc-III-4C polypeptide-polymer-drug conjugate is 1 to 2:1; The Fc segment-containing monoclonal antibody is an anti-PDL1 or anti-PD1 antibody.

9. Use of the antibody-polymer-drug conjugate according to claim 8 in the preparation of anti-colon cancer drugs.

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