Antibody-immunomodulatory drug conjugates with high dar values and methods of making and using the same

Abstract

The present application relates to the field of biopharmacy, in particular to antibody-immunomodulatory drug conjugate with high DAR value and its preparation method and application. The present application provides antibody-immunomodulatory drug conjugate, including immunomodulatory drug conjugate and monoclonal antibody. The present application provides a kind of simple and convenient method for preparing antibody-immunomodulatory drug conjugate with universality. Fc-III-4C-PEG-PLG prepared by the present application can be combined with a variety of different antibodies and a variety of different drugs, so as to simply and efficiently prepare antibody-high molecular drug conjugate. The antibody-high molecular immunomodulatory drug conjugate prepared by the present application can be up to 30, which can be used for cancer treatment, self-immune disease treatment and other fields, has wide application field and extremely high clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and more particularly to antibody-immunomodulatory drug conjugates with high DAR values, their preparation methods, and applications. Background Technology

[0002] Based on the precise recognition of certain tumor cells by antibodies, antibody-drug conjugates (ADC drugs) have been extensively studied. In recent years, a number of ADC drugs targeting targets such as HER2, Trop2, EGFR and BCMA have been launched, making the market very hot.

[0003] Taking the HER2 target as an example, Roche first launched Hercelium, loaded with the highly toxic microtubule inhibitor DM1, with a drug-to-antibody ratio (DAR, the number of small molecule drugs attached to each antibody) of approximately 3.5, and global sales reached US$1.946 billion in 2020. Subsequently, Daiichi Sankyo launched DS8201, loaded with the highly toxic topoisomerase I inhibitor Dxd, with a DAR of approximately 8.0. Compared to Hercelium, DS8201 has a higher DAR and higher drug-to-cell membrane penetration, resulting in an objective response rate more than twice that of Hercelium. Rongchang Bio's product, Aidixi, loaded with the highly toxic microtubule inhibitor MMAE, is the first original ADC drug developed in my country. The launch of this drug greatly stimulated the enthusiasm of Chinese pharmaceutical companies and research institutions for the research and development of ADC drugs.

[0004] However, the available targets for ADC drugs are currently limited, leading to a crowded market. Therefore, to expand the cancer indications for ADC drugs and broaden the patient population and market size, it is urgent to explore novel targets for ADC drugs. PDL1 protein is highly expressed in some tumors but expressed at extremely low levels in stable human tissues. Bolt Biotherapeutics, based on its proprietary ISACs conjugation platform, has recently pioneered the development of ADC drugs based on PDL1 antibodies, which are currently in the preclinical development stage. Furthermore, PDL1 antibodies, as therapeutic antibody drugs, are already included in the national medical insurance catalog. Their good efficacy and relatively low price make them suitable as targets for further development of ADC drugs, indicating significant research and translational potential and a broad market prospect.

[0005] Meanwhile, considering the impact of drug loading on antibody structure and water solubility, the DAR of currently marketed ADC drugs is below 10, and the efficiency of drug delivery to tumors is limited by the limited number of drugs that can be loaded onto a single antibody. Therefore, many companies are actively developing methods to enhance DAR. Mersana Therapeutics, based on its proprietary Dolaflexin conjugate platform, has launched multiple pipelines, and its next-generation ADC drugs have achieved a DAR of up to 24, approximately three times that of DS8201. In February 2022, it reached a collaboration agreement with Johnson & Johnson. However, it is evident that the degree of DAR enhancement is still insufficient to meet the effective drug concentrations at the tumor site required for patients with low target expression. Limited by the low DAR value, the low drug targeting efficiency at the tumor site makes it difficult for current ADC drugs to carry other types of drugs besides highly toxic drugs, especially immunomodulatory drugs that can activate the patient's own anti-tumor immune response by modulating the tumor immune microenvironment.

[0006] Therefore, developing a novel antibody-drug conjugate platform with universal antibody applicability, which can significantly increase the DAR value of ADC drugs through a simple and rapid preparation method, can precisely deliver immunomodulatory drugs to the tumor site. By activating the patient's own anti-tumor immune response, it can alleviate the tumor immunosuppressive microenvironment and improve the therapeutic effect, which will expand the patient population and market size, provide patients with new drug options, and has a huge market potential. Summary of the Invention

[0007] In view of this, the present invention provides antibody-immunomodulatory drug conjugates with high DAR values, their preparation methods, and applications. The present invention provides a universally applicable, simple, and convenient method for preparing antibody-immunomodulatory drug conjugates. The Fc-III-4C-PEG-PLG prepared by the present invention can bind to various different antibodies and drugs, thus enabling simple and efficient preparation of antibody-polymer-drug conjugates. The antibody-polymer-immunomodulatory drug conjugates prepared by the present invention can achieve a DAR as high as 30, and can be used in cancer treatment, autoimmune disease treatment, and other fields, possessing broad application areas and extremely high clinical application prospects.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides antibody-immunomodulatory drug conjugates, including immunomodulatory drug conjugates and monoclonal antibodies; the immunomodulatory drug conjugates include macromolecular drugs and linkers;

[0010] The polymeric drug includes any of the following items and drugs;

[0011] (I) Polyglutamic acid; or

[0012] (II), polyaspartic acid.

[0013] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, the drug is grafted to the side carboxyl group of the polyglutamic acid or the polyaspartic acid.

[0014] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, the grafting rate of the drug grafted on the polyglutamic acid is greater than 20%.

[0015] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, the structural formula of the polymer drug is represented by formula I;

[0016]

[0017] wherein:

[0018] R1 is the repeating unit -[OCH2CH2] m -, 1≤m≤20;

[0019] R2 is a fragment that binds the drug;

[0020] R3 is selected from H or a cation;

[0021] R4 is selected from H, a linear acyl group having 2 to 10 carbon atoms or a branched acyl group having 3 to 10 carbon atoms;

[0022] L1 and L2 are independently selected from -CH2- or -CH2CH2-;

[0023] x is the content of the repeating unit, 0<x<1; y is the degree of polymerization, 10≤y≤500;

[0024] preferably, the structural formula of the polymer drug is represented by formula II;

[0025]

[0026] wherein:

[0027] R1 is the repeating unit -[OCH2CH2] m -, 1≤m≤20;

[0028] R3 is selected from H or a cation;

[0029] R4 is selected from H, a linear acyl group having 2 to 10 carbon atoms or a branched acyl group having 3 to 10 carbon atoms;

[0030] L1 and L2 are independently selected from -CH2- or -CH2CH2-;

[0031] x is the content of the repeating unit, 0<x<1; y is the degree of polymerization, 10≤y≤500.

[0032] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, the drug comprises: an original drug with an amino group or a hydroxyl group, or a modified prodrug.

[0033] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, the drug comprises: an original drug with an amino group or a hydroxyl group.

[0034] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, the drug comprises: an antitumor drug with an amino group or a hydroxyl group.

[0035] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, the drug comprises: the antitumor immunological drug R848 with an amino group.

[0036] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, the drug comprises one or more of doxorubicin, camptothecin or paclitaxel.

[0037] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, R1 is the repeating unit -[OCH2CH2] m -, m=3.

[0038] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, R3 is selected from: H, a metal cation or an organic cation.

[0039] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, R3 is selected from: H, sodium ion, potassium ion, ammonium ion or positively charged amino acid ion.

[0040] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, R4 is selected from: H, acetyl or propionyl.

[0041] In some embodiments of the present invention, in the above antibody-immunomodulatory drug conjugate, x is the content of the repeating unit, x=0.2; y is the degree of polymerization, y=150.

[0042] In some embodiments of the present invention, the preparation method of the polymer drug in the above antibody-immunomodulatory drug conjugate comprises the following steps: mixing the polyglutamic acid or polyaspartic acid, PyBop, DIPEA and the drug, dialyzing and freeze-drying to obtain the polymer drug.

[0043] In some embodiments of the present invention, in the above preparation method, the mixing time is 3 days and the temperature is 35°C.

[0044] In some embodiments of the present invention, the dialysis in the above preparation method includes a first dialysis and a second dialysis; the first dialysis uses DMF and lasts for 12 hours; the second dialysis uses ultrapure water and lasts for 72 hours.

[0045] In some embodiments of the present invention, the preparation method of the above-mentioned preparation method includes the following steps:

[0046] S1: Mix benzyl glutamate NCA monomer, anhydrous N,N-dimethylformamide and 1-amino-11-azido-3,6,9-trioxaundecan, and then mix with acetic anhydride to obtain reaction solution 1;

[0047] S2: Take the reaction solution 1 and precipitate it in diethyl ether, filter, wash, and dry to obtain the intermediate product;

[0048] S3: Dissolve the intermediate product and mix it with a hydrogen bromide / glacial acetic acid solution to obtain reaction solution 2;

[0049] S4: Take the reaction solution 2 and precipitate it in diethyl ether, centrifuge, collect the precipitate, redissolve it, dialyze it, freeze dry it to obtain the polyglutamic acid or the polyaspartic acid.

[0050] In some embodiments of the present invention, the mixing time of the benzyl glutamate NCA monomer, anhydrous N,N-dimethylformamide and 1-amino-11-azido-3,6,9-trioxaundecan in the preparation method S1 described above is 72 h, and the temperature is 30 °C.

[0051] In some embodiments of the present invention, the mixing time with acetic anhydride in the preparation method S1 described above is 6 hours.

[0052] In some embodiments of the present invention, the drying temperature in the preparation method S2 described above is 20-30°C and the drying time is 24 hours.

[0053] In some embodiments of the present invention, in the above preparation method S2, the intermediate product is azide-poly(γ-phenylmethyl-L-glutamate).

[0054] In some embodiments of the present invention, the concentration of the hydrogen bromide or glacial acetic acid solution in the preparation method S3 described above is 33% (mass percentage).

[0055] In some embodiments of the present invention, the mixing temperature in the preparation method S3 described above is 30°C and the mixing time is 1 hour.

[0056] In some embodiments of the present invention, the resolution in preparation method S4 above uses DMF.

[0057] In some embodiments of the present invention, the dialysis described in preparation method S4 above uses deionized water.

[0058] In some embodiments of the present invention, in the above-mentioned antibody-immunomodulatory drug conjugate, the molar ratio of the immunomodulatory drug conjugate to the monoclonal antibody is 1:1; or

[0059] The molar ratio of the polymeric drug to the Linker is 1:1; or

[0060] The molar ratio of the polyglutamic acid or polyaspartic acid to the drug is 1:30.

[0061] In some embodiments of the present invention, in the above-mentioned antibody-immunomodulatory drug conjugate, the monoclonal antibody comprises: a monoclonal antibody containing an Fc fragment; or

[0062] The Linker includes an Fc-III-4C polypeptide modified with a DBCO group.

[0063] In some embodiments of the present invention, the monoclonal antibody in the above-mentioned antibody-immunomodulatory drug conjugate includes an aPDL1 antibody.

[0064] In some embodiments of the present invention, the Fc-III-4C polypeptide in the above-mentioned antibody-immunomodulatory drug conjugate exhibits a generally high affinity for the immunoglobulin (antibody) constant region (Fc). Any peptide exhibiting a generally high affinity for the immunoglobulin (antibody) constant region (Fc) can be used as a linker; this invention does not limit such applications and all are within the scope of protection of the present invention.

[0065] In some embodiments of the present invention, the preparation method of the Linker in the above-mentioned antibody-immunomodulatory drug conjugate includes the following steps: taking the Fc-III-4C polypeptide and DBCO-PEG 45 -NHS was mixed in the absence of anhydrous DMF, dialyzed, and lyophilized to obtain the Linker.

[0066] In some embodiments of the present invention, the mixing time for the above-mentioned antibody-immunomodulatory drug conjugate is 1 day, and the temperature is 35°C.

[0067] In some embodiments of the present invention, in the above-mentioned antibody-immunomodulatory drug conjugate, the dialysis includes a first dialysis and a second dialysis; the first dialysis uses DMF and lasts for 12 hours; the second dialysis uses ultrapure water and lasts for 72 hours.

[0068] In some embodiments of the present invention, the preparation method of the above-mentioned antibody-immunomodulatory drug conjugate includes the following steps: taking the polymeric drug or the polymeric drug obtained by the preparation method and the Linker or the Linker obtained by the preparation method, mixing them in the presence of anhydrous DMSO, dialyzing, and lyophilizing to obtain the immunomodulatory drug conjugate.

[0069] In some embodiments of the present invention, the mixing temperature of the above-mentioned antibody-immunomodulatory drug conjugate is 37°C and the time is 1 day.

[0070] In some embodiments of the present invention, in the above-mentioned antibody-immunomodulatory drug conjugate, the dialysis includes a first dialysis and a second dialysis; the first dialysis uses DMF and lasts for 12 hours; the second dialysis uses ultrapure water and lasts for 72 hours.

[0071] In some embodiments of the present invention, in the above-mentioned antibody-immunomodulatory drug conjugate, the amino acid sequence of the linker is as shown in SEQ ID NO:1: CDCAWHLGELVWCTC.

[0072] In some embodiments of the present invention, in the above-mentioned antibody-immunomodulatory drug conjugate, the linker has the chemical formula shown in Formula III;

[0073]

[0074] Where: R5 is the repeating unit - [OCH2CH2] n- , 2≤n≤60.

[0075] In some embodiments of the present invention, in the above-mentioned antibody-immunomodulatory drug conjugate, R5 in the linker is a repeating unit - [OCH2CH2]. n- n = 45.

[0076] In some embodiments of the present invention, the antibody-immunomodulatory drug conjugate described above has the structural formula shown in Formula IV.

[0077]

[0078] in:

[0079] R1 is the repeating unit - [OCH2CH2] m -,1≤m≤20;

[0080] R3 is selected from H or cations;

[0081] R4 is selected from H, straight-chain acyl groups of C2-C10 or branched-chain acyl groups of C3-C10;

[0082] R5 is a repeating unit -[OCH2CH2] n- , 2≤n≤60;

[0083] L1 and L2 are each independently selected from -CH2- or -CH2CH2-;

[0084] x is the content of the repeating unit, 0<x<1; y is the degree of polymerization, 10≤y≤500.

[0085] The present invention also provides a preparation method of the above-mentioned antibody-immunomodulatory drug conjugate, which comprises mixing the immunomodulatory drug conjugate and the monoclonal antibody to obtain the antibody-immunomodulatory drug conjugate.

[0086] In some embodiments of the present invention, in the above preparation method, the molar ratio of the immunomodulatory drug conjugate to the monoclonal antibody is 1:1.

[0087] In some embodiments of the present invention, in the above preparation method, the mixing time is 12 h and the temperature is 4°C.

[0088] The present invention also provides use of the above-mentioned antibody-immunomodulatory drug conjugate or the antibody-immunomodulatory drug conjugate obtained by the above preparation method in preparation of an antitumor drug.

[0089] In some embodiments of the present invention, in the above use, the tumor includes one or more of malignant tumor of nasal cavity and paranasal sinus, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, intracranial tumor, 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, renal cancer, prostate cancer, bladder cancer, testicular malignant tumor, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytic carcinoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma or multiple myeloma.

[0090] The present invention provides an antibody-immunomodulatory drug conjugate, comprising an immunomodulatory drug conjugate and a monoclonal antibody; the immunomodulatory drug conjugate comprises a polymeric drug and a Linker; the polymeric drug comprises any of the following and a drug: (I) polyglutamic acid; or (II) polyaspartic acid, and a preparation method and use of the antibody-immunomodulatory drug conjugate.

[0091] The beneficial effects of the present invention include:

[0092] (1) Convenient reaction conditions: The antibody-polymer drug conjugate product can be obtained by stirring the Linker-PLG-drug conjugate and the monoclonal antibody in an aqueous environment for 4 hours.

[0093] (2) Does not affect antigen-binding fragment (Fab region): The drug load in existing ADC drugs will randomly bind to the amino or thiol groups in the Fab region or Fc region of the monoclonal antibody, which may have a potential impact on the antigen recognition ability of the monoclonal antibody. Using the affinity of the peptide to the Fc region to bind the monoclonal antibody does not affect its Fab structure.

[0094] (3) Wide range of monoclonal antibody and drug selection: Any monoclonal antibody containing the Fc region can be combined with any drug containing a modifiable functional group;

[0095] (4) High drug-antibody ratio (DAR): The DAR value of the product obtained based on this technology can reach twice that of the existing highest DAR ADC product;

[0096] (5) Easy to store and transport: The monoclonal antibody and the Linker-PLG-drug conjugate are stored separately, avoiding the risk of aggregation during the storage and transportation of traditional ADC drugs. Attached Figure Description

[0097] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0098] Figure 1 The 1H NMR spectrum of polyglutamic acid (PLG) obtained in Example 1 of this invention is shown.

[0099] Figure 2 The 1H NMR spectrum and UV absorption spectrum of PLG / R848 obtained in Example 2 of this invention are shown; wherein: A shows the 1H NMR spectrum; B shows the UV absorption spectrum;

[0100] Figure 3 The following are the 1H NMR spectrum and MALDI-TOF-MS of the Linker obtained in Example 3 of this invention; wherein: A shows the 1H NMR spectrum; B shows the 1H NMR spectrum; C shows the MALDI-TOF-MS.

[0101] Figure 4 The 1H NMR spectrum and UV absorption spectrum of Linker-PLG / R848 obtained in Example 4 of this invention are shown; wherein: A shows the 1H NMR spectrum; B shows the UV absorption spectrum;

[0102] Figure 5This invention illustrates the particle size distribution and potential of PLG, Linker-PLG / R848, and aPDL1-PLG / R848 obtained in Examples 1, 4, and 5; wherein: A represents the particle size distribution; B represents the corresponding particle size distribution. Figure 5 -A represents the specific numerical value; C indicates the electric potential; D indicates the corresponding value. Figure 5 The specific value of -B;

[0103] Figure 6 The aPDL1-PLG / R848 obtained in Example 5 of this invention is used to activate dendritic cells in vitro, and its changes are analyzed by flow cytometry; wherein: A represents cd11c + MHCII + The proportion of positive cells; B indicates the corresponding percentage. Figure 6 -A represents the specific value; C indicates CD80. + CD86 + The proportion of positive cells; D indicates the corresponding Figure 6 The specific value of -B;

[0104] Figure 7 This diagram illustrates the treatment regimen, changes in subcutaneous tumor volume, changes in mouse weight, and mouse survival time after treating MC38 colon cancer C57BL / 6 mice with aPDL1-PLG / R848 obtained in Example 5 of this invention. Wherein: A represents the treatment regimen; B represents changes in tumor volume; C represents changes in mouse weight; and D represents mouse survival time. All Groups I-VI in the diagram refer to the PBS, aPDL1, PLG / R848, Mix (aPDL1&PLG / R848), and aPDL1-PLG / R848 groups, respectively. Detailed Implementation

[0105] This invention discloses antibody-immunomodulatory drug conjugates with high DAR values, their preparation methods, and applications.

[0106] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0107] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0108] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0109] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0110] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0111] This invention provides antibody-immunomodulatory drug conjugates with high DAR values, their preparation methods, and applications.

[0112] Preferably, the polymer material of the antibody-drug conjugate is polyglutamic acid homopolymer (PLG), the linker is an Fc-III-4C polypeptide (modified with a DBCO group) that has a universally high affinity for the constant region (Fc) of immunoglobulins (antibodies), and the drug is rethimod (R848).

[0113] Preferably, the molar ratio of PLG to rethimod (R848) in the antibody-polymer-drug conjugate is 1:30.

[0114] Preferably, the prepared antibody-polymer drug conjugate is aPDL1 containing the Fc fragment and Linker-PLG / R848.

[0115] Preferably, the molar ratio of Linker-PLG / R848 to aPDL1 in the antibody-pharmaceutical conjugate aPDL1-PLG / R848 is 1:1.

[0116] The method for preparing antibody-drug conjugates provided by this invention is not limited to the two methods mentioned above, and can also be used to prepare other antibody-polymer drug conjugates.

[0117] The antibody-pharmaceutical conjugate prepared in this invention can be combined with any drug containing a hydroxyl or amino group, such as doxorubicin, camptothecin, or paclitaxel.

[0118] The antibody polymer drug conjugate prepared by the present invention can bind any monoclonal antibody containing an Fc fragment, such as an anti-PD-L1 monoclonal antibody.

[0119] Preferably, the tumor comprises one or more of malignant tumor of nasal cavity and paranasal sinus, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, intracranial tumor, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and peripancreatic ampullary carcinoma, biliary tract cancer, renal cancer, prostate cancer, bladder cancer, testicular malignant tumor, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrohistiocytic carcinoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma and multiple myeloma.

[0120] The antibody-polymer drug has a structure shown in Formula I.

[0121]

[0122] In Formula I, R1 is the repeating unit -[OCH2CH2] m -, specifically 1≤m≤20, preferably m=3; R2 is a drug-binding fragment, specifically an original drug with an amino group or a hydroxyl group (or a modified prodrug); preferably an original drug with an amino group or a hydroxyl group; more preferably an anti-tumor drug with an amino group or a hydroxyl group; in specific embodiments, the anti-tumor immunodrug R848 with an amino group is preferred;

[0123] R3 is H or a cation; preferably H, a metal cation or an organic cation; more preferably H, a sodium ion, a potassium ion, an ammonium ion or a positively charged amino acid ion;

[0124] R4 is H, a linear acyl group of C2-C10 or a branched acyl group of C3-C10; preferably H, an acetyl group or a propionyl group.

[0125] L1 and L2 are each independently selected from -CH2- or -CH2CH2-;

[0126] x is the content of the repeating unit, 0<x<1; y is the degree of polymerization, 10≤y≤500; in some specific embodiments, x=0.2, y=150, and the structural formula is shown in Formula II;

[0127]

[0128] In Formula II, the selection of R1, R2, R4, L1 and L2 is the same as that in the structure of Formula I, which will not be repeated herein.

[0129]

[0130] In Formula III, R5 is the repeating unit -[OCH2CH2] n-, 2≤n≤60; in some specific embodiments, n=45.

[0131] The present invention does not limit the source of the above-mentioned Fc-III-4C, and it can be prepared by commercially available products or by methods 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.

[0132]

[0133] In Equation IV, the selection of R1, R2, R4, L1, and L2 is the same as that in Equations I, II, and III, and will not be elaborated here.

[0134] This invention provides a method for preparing an antibody-polymer drug conjugate and its application, comprising polymer drugs with structures of formulas I and II, referred to herein as polymer drugs. In some specific embodiments, the polymer drug is PLG / R848 or PLG / MMAE. It also comprises a structure of formula III, referred herein as Linker. In some specific embodiments, it is a Linker. Finally, it comprises a structure of formula IV, referred herein as Linker-polymer drug. In some specific embodiments, it is Linker-PLG / R848 or Linker-PLG / MMAE.

[0135] The antibody-drug conjugates consist of an antibody, a linker, and a drug polymer. Since the Fc-III-4C peptide in the linker has a dissociation constant (Kd) of only 2.45 nM for human immunoglobulin IgG (including antibodies), it does not affect antibody activity. Furthermore, the antibody Fc fragment is highly conserved. Therefore, Linear r-PLG / R848 or Linker-PLG / MMAE can bind to antibodies containing the Fc fragment to prepare antibody-drug conjugates.

[0136] This invention also provides a method for preparing an antibody drug to inhibit tumor growth, wherein the object is a mammal or tumor cells of said mammal, preferably a rodent, even-toed ungulate, perissodactyl, lagomorph, or primate. The primate is preferably a monkey, ape, or Homo sapiens. The object can be a patient suffering from a tumor, or isolated tumor cells from a patient suffering from a tumor. Based on this, this invention has found that bispecific antibody drugs have a more significant tumor therapeutic effect.

[0137] In Examples 1 to 7 of this invention, all raw materials and reagents used can be purchased from the market.

[0138] The present invention will be further illustrated below with reference to the embodiments:

[0139] Example 1: Preparation of azide-poly(L-glutamic acid) homopolymer (PLG)

[0140] 1.0 g of benzyl glutamate NCA monomer (BLG-NCA) was dissolved in 10.0 mL of anhydrous N,N-dimethylformamide (DMF). After stirring and dissolving, 4.0 mg of 1-amino-11-azido-3,6,9-trioxaundecan was added. The mixture was sealed and stirred at 30 °C for 72 hours. Acetic anhydride was then added and the reaction was continued for 6 hours. After the reaction was completed, the resulting reaction solution was precipitated into 1.0 L of diethyl ether. The solution was then filtered and washed with diethyl ether, and dried under vacuum at room temperature (25 °C ± 5 °C) for 24 hours to obtain the intermediate product azido-poly(γ-phenylmethyl-L-glutamate) (PBLG).

[0141] 1.0 g of the N3-PBLG prepared above was dissolved in 10 mL of dichloroacetic acid. Under stirring, 3 mL of a 33% (w / w) hydrogen bromide / glacial acetic acid solution was added, and the mixture was stirred at 30 °C for 1 hour. The resulting reaction solution was then settled into 1.0 L of diethyl ether. After centrifugation, the precipitate was reconstituted with DMF, dialyzed against deionized water, and lyophilized to obtain a zirconia-terminated poly(L-glutamic acid) homopolymer (PLG).

[0142] Nuclear magnetic resonance analysis (NMR) was performed on the prepared poly(L-glutamic acid) homopolymer (e.g. Figure 1 As shown in the figure, using deuterated water as the deuteration reagent (with the addition of sodium deuterium oxide), the results showed that the chemical shift of 4.35 ppm was the signal peak of the methine group on the main chain, the chemical shift of 2.30 ppm was the signal peak of the methylene group attached to the carbonyl group on the side group, and the chemical shifts of 2.07 ppm and 1.96 ppm were the signal peaks of the methylene group attached to the main chain on the side group. According to NMR calculations, the degree of polymerization of the obtained azido-poly(L-glutamic acid) was 150.

[0143] Example 2: Preparation of PLG / R848

[0144] 50.0 mg of the azide-terminated poly(L-glutamic acid) homopolymer (PLG) obtained in Example 1 was dissolved in 10.0 mL of anhydrous DMF. 54.7 mg of PyBop, 13.6 mg of DIPEA, and 37.0 mg of retinoic acid (hereinafter referred to as R848) were added. After reacting at 35°C for 3 days, the reactants were dialyzed against DMF for 12 hours, and then against ultrapure water for 72 hours. The product was lyophilized, and the resulting PLG / R848 was analyzed by nuclear magnetic resonance using deuterated water (with added sodium deuterium oxide) as the solvent. The results are as follows: Figure 2 As shown in Figure A, the results indicate that R848 was successfully grafted onto the PLG side chain, and this is shown in the UV absorption spectrum (as in Figure A). Figure 2As shown in B), after being grafted onto the PLG chain, its maximum ultraviolet absorption wavelength shifts from 320nm to 327nm.

[0145] Example 3: Preparation of Linker

[0146] 5.3 mg Fc-III-4C peptide was mixed with 5.0 mg DBCO-PEG 45 -NHS was dissolved in 3.0 mL of anhydrous DMF and reacted at 35°C for 1 day. The reactants were then dialyzed against DMF for 12 hours, followed by dialyzed against ultrapure water for 72 hours. The product was lyophilized, and the resulting linker was analyzed by NMR using deuterated water as the solvent. (e.g., ...) Figure 3 A and Figure 3 (as shown in B), DBCO-PEG 45 The chemical shifts of 2.8 ppm and 7.25–7.55 ppm in NHS represent the four hydrogens on the methylene group of N-hydroxysuccinimide (NHS) and the diphenylcyclooctylene (DBCO), respectively. Figure 3 (as shown in B), Figure 3 The disappearance of the peak at 2.88 ppm in the 1H NMR spectrum of Linker A indicates successful synthesis of Linker. MALDI TOF-MS analysis showed the following results: Figure 3 As shown in Figure C, the molecular weight of the Linker reaches 3700–4000, which is consistent with the above NMR results.

[0147] Example 4: Preparation of Linker-PLG / R848

[0148] 10.0 mg of Linker (terminally containing a DBCO group) obtained in Example 3 and 50.0 mg of PLG / R848 (main chain containing an azide group) obtained in Example 2 were dissolved in DMSO under anhydrous conditions and reacted at 37°C for 1 day. The reactants were first dialyzed against DMF for 12 hours, and then against ultrapure water for 72 hours. The product was lyophilized, and the resulting Linker-PLG / R848 was analyzed by NMR using deuterated water as the solvent. The results are as follows: Figure 4 As shown in Figure A, the results indicate that the Linker was successfully bonded to the PLG / R848, and UV analysis shows (as shown in Figure A). Figure 4 As shown in Figure B, ultraviolet characteristic absorption peaks appear at both 290 nm and 327 nm.

[0149] Example 5: Preparation of aPDL1-PLG / R848

[0150] Utilizing the high affinity of the Fc-III-4C peptide on Linker-PLG / R848 obtained in Example 4 for the Fc region of monoclonal antibodies, the antibody-pharmaceutical conjugate aPDL1-PLG / R848 was prepared under aqueous solution incubation conditions. Reaction conditions: The molar ratio of Linker-PLG / R848 to aPDL1 (brand: BioXcell, trade code: BE0101) was 1:1, and incubation was carried out at 4°C for 12 hours.

[0151] Dynamic light scattering analysis and Zeta potential analysis were performed on the obtained aPDL1-PLG / R848 to determine the hydrodynamic radius of the particles formed in water. Figure 5 A and Figure 5 B represents the dynamic light scattering results of aPDL1-PLG / R848 in water at a concentration of 0.5 mg / mL. The figure shows that the mechanical radius is between 100 and 160 nm. Figure 5 C and Figure 5 D represents the Zeta potential analysis result; the potential of aPDL1-PLG / R848 is -9.8 eV.

[0152] Example 6a: Effect of PDL1-PLG / R848 on the activation of dendritic cells in vitro

[0153] Evaluation of the effect of in vitro activation of dendritic cells: Bone marrow cells were extracted from the femur and tibia of C57BL / 6 mice (6 weeks old, female) and differentiated into unactivated (quiescent) dendritic cells under the induction of GM-CSF and IL-4. After 7 days of cell culture, the cells were divided into 5 groups: Group IV consisted of PBS, aPDL1 (47.0 μg / mL), R848 (3.0 μg / mL), Mix (aPDL1 (47.0 μg / mL) & PLG / R848 (3.0 μg / mL)), and the aPDL1-PLG / R848 group obtained in Example 5, with drug concentrations of 3.0 μg / mL R848 and 47.0 μg / mL aPDL1 antibody (volume 25.0 μL). After 1 day of incubation, flow cytometry analysis was performed using CD11c, MHCII, CD80, and CD86 fluorescent dyes.

[0154] The effect of aPDL1-PLG / R848 on activating dendritic cells was analyzed by flow cytometry. Dendritic cells play a role in presenting antigens to naive T cells during anti-tumor immunity. Activated dendritic cells stimulate T cells to kill tumor cells by upregulating their surface proteins MHCII, CD80, and CD86. Figure 6The results shown are flow cytometry results of the above proteins on the surface of dendritic cells. It can be seen that the expression level of MHCII protein in the aPDL1-PLG / R848 group (Group V) is 3.4 times higher than that in the PBS group (Group I), and the co-expression of CD80 and CD86 proteins is 3.7 times higher than that in the PBS group (Group I).

[0155] Example 7a: Therapeutic effect of PDL1-PLG / R848 on MC38 colon cancer tumor model

[0156] Evaluation of in vivo tumor inhibition effect: 30 C57BL / 6 mice (7-8 weeks old, weighing approximately 18g) were selected as experimental animals. A colon cancer tumor model was established by subcutaneous injection of mouse MC38 colon cancer cells. The tumors grew to an average size of 40-50mm. 3 Animals were divided into five groups: Group IV consisted of the PBS group, the aPDL1 group (100 μg / animal), the PLG / R848 group (R848: 6.28 μg / animal), the Mix(aPDL1+PLG / R848) group (aPDL1 antibody 100 μg / animal and R848 6.28 μg / animal), and the aPDL1-PLG / R848 group obtained in Example 5. Day 0 was designated as the starting day. Drugs were administered via tail vein injection on days 0, 3, 6, and 9, with dosages of antibody 100 μg / animal and R848 6.28 μg / animal. Tumor volume and body weight were monitored every 3 days until day 40 for pharmacodynamic evaluation. Tumor volume and tumor inhibition rate were calculated using the following formula:

[0157] Tumor volume: V=(a×b) 2 ) / 2

[0158] Tumor inhibition rate: TSR = [(Ac-Ax) / Ac] × 100%

[0159] 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.

[0160] like Figure 7 B and Figure 7 As shown in Figure E, compared with the control group (PBS, Group I), the tumor inhibition rates of the aPDL1 treatment group (Group II) and the PLG / R848 treatment group (Group III) were 36.5% and 1.1%, respectively. The tumor inhibition rate of the aPDL1 and PLG / R848 mixed group (MIX, Group IV) was 50.8%, while the aPDL1-PLG / R848 treatment group (Group V) achieved the best tumor growth inhibition effect, with a tumor inhibition rate (TSR%) of 86.8% at the end of treatment. This indicates that antibody-pharmaceutical conjugates can improve the effect of simply mixing the two drugs.

[0161] like Figure 7 C and Figure 7 As shown in F, no significant weight changes were observed in any of the treatment groups during the treatment phase, indicating that the drug is safe.

[0162] like Figure 7 As shown in Figure D, compared with the control group, the survival time of the experimental group was significantly prolonged. After 40 days, the survival rate of the aPDL1-PLG / R848 group was 83.33%, while the survival rate of other control groups was less than 20%, demonstrating that the aPDL1-PLG / R848 drug can effectively improve the survival of MC38 colon cancer C57BL / 6 mice.

[0163] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody-immunomodulatory drug conjugate, characterized in that, Comprising an immunomodulatory drug conjugate and a monoclonal antibody; wherein the immunomodulatory drug conjugate comprises a polymer drug and a Linker, and the structural formula of the immunomodulatory drug conjugate is shown as Formula IV; Formula IV; Wherein: R1 is the repeating unit - [OCH2CH2] m -,1≤m≤20; R3 is selected from H or a cation; R4 is selected from H, a linear acyl group having 2 to 10 carbon atoms or a branched acyl group having 3 to 10 carbon atoms; R5 is the repeating unit - [OCH2CH2] n- , 2≤n≤60; L1 and L2 are each independently selected from -CH2- or -CH2CH2-; x is the content of the repeating unit, 0<x<1; y is the degree of polymerization, 10≤y≤500; The monoclonal antibody is aPDL1.

2. The method for preparing the antibody-immunomodulatory drug conjugate as described in claim 1, characterized in that, Mixing the immunomodulatory drug conjugate and the monoclonal antibody to obtain the antibody-immunomodulatory drug conjugate.

3. Use of the antibody-immunomodulatory drug conjugate according to claim 1 or the antibody-immunomodulatory drug conjugate obtained by the preparation method according to claim 2 in the preparation of an anti-tumor drug; The tumor is colon cancer.

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