A protein-polysaccharide-drug conjugate and its application

Abstract

The present invention provides a protein-polysaccharide-drug conjugate and its application, wherein the structure of the protein-polysaccharide-drug conjugate is shown in Formula I. The protein-polysaccharide-drug conjugate provided by the present invention has a high drug-antibody ratio, has no unstable ester bond connection part, enhances its stability in the body circulation, avoids the toxic and side effects caused by non-targeted drug release, and at the same time, by connecting the antibody to the end group of the polysaccharide and the small molecule drug to the side chain of the polysaccharide, it is ensured that one polymer molecule can only be connected to one antibody molecule, so that the product structure is easy to determine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a protein-polysaccharide-drug conjugate and its application, and in particular to a protein-polysaccharide-drug conjugate with good killing effect and its application. Background Art

[0002] Antibody-drug conjugates are a class of therapeutic agents that connect antibodies to drugs via linkers. They have the characteristics of high specificity of antibodies and high toxicity of cytotoxins to tumors. In the clinical treatment of tumor targeted therapy, some antibody-drug conjugates have been actively studied (Ricart AD, and Tolcher AW, 2007, Nature Clinical Practice, 4, 245-255; Krop et al., 2010, Journal of clinical oncology, 28, 2698-2704). However, existing antibody-drug conjugates have shown some limitations, such as the inability to deliver sufficient concentrations of drugs to the target site. To overcome this problem, many scientific researchers and developers have tried to increase the drug-antibody ratio of antibody-drug conjugates. Due to the hydrophobic characteristics of most highly active cytotoxins, simply increasing the drug-antibody ratio often causes the antibody-drug conjugate to be too hydrophobic and produce aggregation, which in turn causes the antibody-drug conjugate to be quickly cleared in vivo with many toxic side effects.

[0003] Mersana has developed and invented a protein-polymer-drug conjugate, which connects multiple hydrophobic drugs to antibodies by introducing a linker of a class of polymers, poly-1-hydroxymethylvinyl hydroxyformal (PHF), to achieve an antibody-drug conjugate with a high drug-antibody ratio without affecting the physicochemical properties of the antibody or drug (patent number: CN10374804B, US8685383B2). In this patent, the drug or small molecule is connected to the PHF backbone by esterification of the hydroxyl groups on the polymer PHF, that is, the drug or small molecule is connected to the polymer through an ester bond. However, the ester bond of this connection is unstable in vivo and can be cleaved at multiple enzymatic sites, which will reduce the anti-tumor activity and increase the risk of toxicity caused by premature non-targeted release of the drug. Beijing Norin Biotechnology has also developed an antibody-drug conjugate based on PHF polymer linkers (patent number: CN109563215B, US20190083634), which makes the drug molecule more stable when connected to the PHF skeleton. However, in the structure of the antibody-drug conjugate, because both the drug molecule and the protein are connected to the side chain of the polymer molecule, there is a possibility that one protein is connected to multiple polymers and one polymer may be connected to multiple antibody molecules, which is difficult to determine in terms of structure. In addition, the polymer PHF needs to be prepared artificially, which will also increase the cost of preparing the protein-polymer-drug conjugate.

[0004] Therefore, how to provide an antibody-drug conjugate with low toxicity, good stability, easy-to-determine structure and good therapeutic effect has become an urgent problem to be solved. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a protein-polysaccharide-drug conjugate and its application, and in particular to provide a protein-polysaccharide-drug conjugate with good killing effect and its application. The protein-polysaccharide-drug conjugate provided by the present invention has a high drug-antibody ratio, has no unstable ester bond connection part, enhances its stability in the body circulation, avoids the toxic and side effects caused by non-targeted drug release, and at the same time, by connecting the antibody to the end group of the polysaccharide and the small molecule drug to the side chain of the polysaccharide, it is ensured that one polymer molecule can only be connected to one antibody molecule, making the product structure easy to determine.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides a protein-polysaccharide-drug conjugate, wherein the structure of the protein-polysaccharide-drug conjugate is as shown in Formula I:

[0008]

[0009] Wherein n1 is an integer from 1 to 300, n2 is an integer from 1 to 60, and x is an integer or decimal from 1 to 10.

[0010] Linker1 is selected from substituted or unsubstituted C1-C6 alkylenethio groups; up to 4 carbon atoms in the C1-C12 alkylenethio groups are replaced by any one or at least two of nitrogen atoms, oxygen atoms, sulfur atoms, amide groups, ethanol groups or polyethylene glycol groups.

[0011] Linker2 is selected from Wherein m1 and m2 are selected from integers of 2-10;

[0012] A drug selected from the class of microtubule inhibitors;

[0013] Selected from monoclonal antibodies.

[0014] In the above structure, the groups where n1 and n2 are located are not strictly distributed in the order shown in the structural formula. The structural formula only shows the specific structure of the groups where n1 and n2 are located. In the compounds defined in the present invention, the groups where n1 and n2 are located are randomly distributed with respect to each other, and the present invention does not impose too many restrictions on this.

[0015] The protein-polysaccharide-drug conjugate with a specific structure introduces hyaluronic acid as a linker to connect multiple hydrophobic drug molecules and antibodies, which can effectively improve biodegradability and biocompatibility, has the advantages of being non-toxic and non-immunogenic, and the raw materials can be easily obtained commercially; and there is no unstable ester bond connection part in the structure, which enhances its stability in the body circulation and avoids the toxic side effects caused by non-targeted drug release; at the same time, by connecting the antibody to the end group of the polysaccharide and the small molecule drug to the side chain of the polysaccharide, it is ensured that one polymer molecule can only be connected to one antibody molecule, and one Antibody molecules can be connected to multiple polymer molecules, which not only ensures that the product has a high drug-antibody ratio and thus has excellent therapeutic effects, but also makes the product structure relatively clearer and easier to characterize. In contrast, the existing patented technologies (patent numbers: CN10374804B, US8685383B2, CN109563215B, US20190083634) make it difficult to determine and characterize the product structure because one antibody can be connected to multiple polymer molecules, and one polymer molecule can also be connected to multiple antibody molecules; at the same time, the synthesis process is simple and convenient for production and preparation.

[0016] The above protein-polysaccharide-drug conjugate can be prepared by conventional methods in the art, and the present invention does not impose too many limitations on this. The following preparation method is provided as an example:

[0017] Sodium hyaluronate is mixed with propargylamine to obtain sodium hyaluronate whose terminal groups are completely functionalized with alkynyl groups, and then reacted with H2N-Linker1 to obtain sodium hyaluronate with side chain thiol functionalization and terminal alkynyl functionalization, and then mixed with azide-functionalized monoclonal antibody to obtain the protein-polysaccharide-drug conjugate.

[0018] Preferably, the substituted group includes any one of C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, hydroxyl, nitro, amino, amide or halogen.

[0019] Among them, the C1-C6 alkyl group can be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc., the C1-C6 alkoxy group can be, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, etc., the C2-C6 alkenyl group can be, for example, vinyl, propenyl, isopropenyl, etc., and the C2-C6 alkynyl group can be, for example, ethynyl, propynyl, butynyl, etc.

[0020] Preferably, the Linker1 is selected from substituted or unsubstituted C1-C6 alkylenethio groups; up to 4 carbon atoms (eg, 1, 2, 3 or 4) in the C1-C6 alkylenethio group are replaced by any one or at least two of an amide group, an ethanol group or a polyethylene glycol group.

[0021] Preferably, the Linker1 is selected from any one of the following structures:

[0022]

[0023]

[0024] The Linker1 is selected from any one of the following structures:

[0025]

[0026] Preferably, the microtubule inhibitor drugs include auristatin E, maytansine alkaloids, paclitaxel, doxorubicin, camptothecin, irinotecan, methotrexate, pyrrolobenzodiazepine, amatine, calicheamicin, topoisomerase I inhibitors, DNA binding drugs, DNA-alkylating drugs or RNA polymerase inhibitors and their derivatives.

[0027] Preferably, the monoclonal antibody includes trastuzumab, or a specific antibody or synthetic functionalized antibody for an antigen in the group consisting of EGFR, TROP2, CEACAM5, folate receptor-a, ROR1, ROR2, mesothelin, NaPi2b, 5T4, HER3, BCMA, B7H3, B7H4, Nectin-4, PTK7, PSMA, MUC1, CLDN18.2, CD56, CD30, CD33, CD79b, CD19, CD22, CD138, CD70, CD98, SLC44A4, LIV-1, fibrinogen extracellular domain B, endothelin receptor ETB or tenascin C.

[0028] Preferably, in the protein-polysaccharide-drug conjugate, the average molar ratio of drug to monoclonal antibody is 6-25.

[0029] On the other hand, the present invention also provides the use of the protein-polysaccharide-drug conjugate as described above in the preparation of anti-tumor drugs.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention provides a protein-polysaccharide-drug conjugate, which introduces hyaluronic acid as a linker to connect multiple hydrophobic drug molecules and antibodies, can effectively improve biodegradability and biocompatibility, has the advantages of being non-toxic and non-immunogenic, and the raw materials can be easily obtained commercially; and there is no unstable ester bond connection part in the structure, which enhances its stability in the body circulation and avoids the toxic side effects caused by non-targeted drug release; at the same time, by connecting the antibody to the end group of the polysaccharide and the small molecule drug to the side chain of the polysaccharide, it is ensured that one polymer molecule can only be connected to one antibody molecule, and One antibody molecule can be connected to multiple polymer molecules, which not only ensures that the product has a high drug-antibody ratio and thus has excellent therapeutic effects, but also makes the product structure relatively clearer and easier to characterize. In contrast, the existing patented technologies (patent numbers: CN10374804B, US8685383B2, CN109563215B, US20190083634) make it difficult to determine and characterize the product structure because one antibody can be connected to multiple polymer molecules, and one polymer molecule can also be connected to multiple antibody molecules; at the same time, the synthesis process is simple and convenient for production and preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the SEC spectrum of compound 25 and trastuzumab at a wavelength of 280 nm;

[0033] Figure 2 is the SEC spectrum of compound 30 and trastuzumab at a wavelength of 280 nm;

[0034] Figure 3 is the SEC spectrum of compound 34 and trastuzumab at a wavelength of 280 nm;

[0035] Figure 4 is the dose response curve of the survival % NCI-N87 cells treated with compounds 25 to 34, compound 12, compound 18, auristatin E, trastuzumab, and phosphate buffer;

[0036] Figure 5 is the dose response curve of the survival % JIMT-1 cells treated with compounds 25 to 34, compound 12, compound 18, auristatin E, trastuzumab, and phosphate buffer;

[0037] Figure 6 It is the dose response curve of the viable % MDA-MB-231 cells treated with Compound 25 to Compound 34, Compound 12, Compound 18, Auristatin E, Trastuzumab, and phosphate buffer. DETAILED DESCRIPTION

[0038] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0039] Abbreviations

[0040] The following abbreviations are used in the following synthetic schemes and examples. This list is not intended to be an exhaustive list of all abbreviations used in the application, as additional standard abbreviations that are readily apparent to those skilled in the art can also be used in the synthetic schemes and examples.

[0041] Alkyn=alkynyl

[0042] BTTAA = 2-(4-((bis((1-(2-methyl-2-propyl)-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetic acid

[0043] DAR = Drug-Antibody Ratio

[0044] DMAc=N,N-dimethylacetamide

[0045] DMTMM = 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride

[0046] g = grams

[0047] h = hours

[0048] HA = Sodium Hyaluronate

[0049] Herceptin = trastuzumab

[0050] H NMR = Hydrogen Nuclear Magnetic Resonance

[0051] kDa = kilodaltons

[0052] LC-MS = Liquid Chromatography Mass Spectrometry

[0053] MES = Morpholineethanesulfonic acid

[0054] MMAE = Auristatin E

[0055] mmol = millimole

[0056] mM = millimole per liter

[0057] mL = milliliters

[0058] mg / mL = milligrams per milliliter

[0059] MWCO = Molecular Weight Cut-off

[0060] PDA HCl=2-(2-pyridyldisulfide)ethylamine hydrochloride

[0061] TCEP = tris(2-carboxyethyl)phosphine

[0062] Example 1, Synthesis of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 12 kDa, compound 1)

[0063] First, weigh 3g of sodium hyaluronate (HA, 12kDa, 0.25mmol) and dissolve it in 150mL of sodium acetate buffer (100mM, pH 5.6), stir it at 50℃ in a three-necked round-bottom flask, and after stirring for 2h, the hyaluronic acid is completely dissolved, the solution becomes clear, and the concentration of HA is 20mg / mL. Continue to keep the HA solution at 50℃ and stir it, slowly add a certain amount of propargylamine (1.6mL, 25mmol) to the solution through a pipette, and the color of the solution becomes light yellow. Then, add a certain amount of sodium cyanoborohydride (1.58g, 25mmol) to the reaction solution. After the addition is completed, continue the reaction for 120h and control the temperature at 50℃. After the reaction is completed, the color of the reaction solution becomes lighter, and 1050mL of sodium acetate buffer (100mM, pH 5.6) is added to the reaction solution to dilute it so that the concentration of HA in the reaction mixture is 2.5mg / mL. Continue to add 22mL of sodium acetate buffer (3M, pH 5.5) to the reaction mixture for dilution so that the concentration of sodium acetate in the reaction mixture is 150mM. Then prepare ethanol (5L) 4 times the volume of the reaction solution. Slowly add the reaction mixture into ethanol for precipitation while stirring. The precipitate is purified by centrifugation. The supernatant is removed by centrifugation, and the collected precipitate is washed twice with 80v / v% ethanol and twice with ethanol. Finally, the collected precipitate is placed in a 50mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. The white Alkyn-HA solid powder (2.8g, yield: 93.3%) is collected and stored in nitrogen at -20°C. Characterization by nuclear magnetic 1H NMR shows that the end groups of hyaluronic acid are completely functionalized with alkynyl groups. Its nuclear magnetic results 1 H NMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.024.21-4.75; Alkynyl functional group: δ (ppm) 2.50-2.91.

[0064]

[0065] Compound 1

[0066] Example 2, Synthesis of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 36 kDa, compound 2)

[0067] First, weigh 3g of sodium hyaluronate (HA, 36kDa, 0.083mmol) and dissolve it in 150mL of sodium acetate buffer (100mM, pH 5.6), stir at 50℃ in a three-necked round-bottom flask, and after stirring for 2h, the hyaluronic acid is completely dissolved, the solution becomes clear, and the concentration of HA is 20mg / mL. Continue to keep the HA solution at 50℃ and stir, and slowly add a certain amount of propargylamine (0.53mL, 8.3mmol) to the solution through a pipette, and the color of the solution turns light yellow. Then, add a certain amount of sodium cyanoborohydride (0.53g, 8.3mmol) to the reaction solution. After the addition is completed, continue the reaction for 120h and the temperature is controlled at 50℃. After the reaction is completed, the color of the reaction solution becomes lighter. 1050 mL of sodium acetate buffer (100 mM, pH 5.6) is added to the reaction solution to dilute the reaction mixture so that the concentration of HA in the reaction mixture is 2.5 mg / mL. 22 mL of sodium acetate buffer (3 M, pH 5.5) is added to the reaction mixture to dilute the reaction mixture so that the concentration of sodium acetate in the reaction mixture is 150 mM. Then 4 times the volume of the reaction solution is prepared. Ethanol (5 L) is slowly added to the reaction mixture in the presence of stirring for precipitation. The precipitate is purified by centrifugation, the supernatant is removed by centrifugation, and the collected precipitate is washed twice with 80 v / v% ethanol and twice with ethanol, respectively. Finally, the collected precipitate is placed in a 50 mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. White Alkyn-HA solid powder (2.52 g, yield: 84.0%) is collected and stored at -20 ° C under nitrogen. 1H NMR characterization showed that the hyaluronic acid end groups were completely functionalized with alkynyl groups. 1 H NMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.024.21-4.75; Alkynyl functional group: δ (ppm) 2.50-2.91.

[0068]

[0069] Compound 2

[0070] Example 3, Synthesis of Sodium Hyaluronate with Side Chain Thiol Functionalization and Terminal Alkyne Functionalization (Alkyn / HA-SH, 12 kDa, Compound 3)

[0071] First, 400 mg of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 12 kDa, 1 mmol repeating unit) was weighed and dissolved in 20 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5), and stirred at 35 ° C in a three-necked round-bottom flask. After stirring for 2 hours, Alkyn-HA was completely dissolved, the solution became clear, and the concentration of HA was 20 mg / mL. The HA solution was kept at 35 ° C and stirred, and a certain amount of 2-(2-pyridyldisulfide)ethylamine hydrochloride (PDA.HCl, 111.4 mg, 0.5 mmol) was slowly added to the solution. Then, a certain amount of 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM, 83.1 mg, 0.3 mmol) was added to the reaction solution. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 35 ° C. The concentration of HA during the reaction was 20 mg / mL. After 24 hours of reaction, a certain amount of tris(2-carboxyethyl)phosphine (TCEP, 286.6 mg, 1 mmol) was added to the reaction solution, and the reaction was continued at 35° C. for 4 hours. After the reaction was completed, 140 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5) was added to the reaction solution for dilution so that the concentration of HA in the reaction mixture was 2.5 mg / mL. 8 mL of sodium acetate buffer (3 M, pH 5.5) was further added to the reaction mixture for dilution so that the concentration of sodium acetate in the reaction mixture was 150 mM. Then prepare ethanol (670mL) with a volume of 4 times that of the reaction solution. Slowly add the reaction mixture into the ethanol for precipitation while stirring. The precipitate is purified by centrifugation, and the supernatant is removed by centrifugation. The collected precipitate is washed twice with 80v / v% ethanol and twice with ethanol. Finally, the collected precipitate is placed in a 50mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. The white Alkyn / HA-SH solid powder (280mg, yield: 70.0%) is collected and stored at -20°C in a nitrogen atmosphere. Nuclear magnetic 1H NMR characterization shows that the degree of substitution of thiol groups in hyaluronic acid is 10.4%, that is, there are approximately 3.1 thiol molecules in one HA molecule. Note: The degree of substitution is defined as the number of repeating units substituted by thiol groups for every 100 repeating units. Its nuclear magnetic results 1 H NMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.02 4.21-4.75; Thiol functional group: δ (ppm) 2.59 (-CH2-CH2-SH).

[0072]

[0073] Compound 3

[0074] Example 4, Synthesis of Sodium Hyaluronate with Side Chain Thiol Functionalization and Terminal Alkyne Functionalization (Alkyn / HA-PEG4-SH, 12 kDa, Compound 4)

[0075] First, 210 mg of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 12 kDa, 0.53 mmol repeating unit) was weighed and dissolved in 10.5 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5), and stirred at 35 ° C in a three-necked round-bottom flask. After stirring for 2 hours, Alkyn-HA was completely dissolved, the solution became clear, and the concentration of HA was 20 mg / mL. The HA solution was kept at 35 ° C and stirred, and a certain amount of SPDP-PEG4-NH2 (123.4 mg, 0.265 mmol) was slowly added to the solution. Then, a certain amount of 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM, 43.6 mg, 0.16 mmol) was added to the reaction solution. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 35 ° C. The concentration of HA during the reaction was 20 mg / mL. After 24 hours of reaction, a certain amount of tris(2-carboxyethyl)phosphine (TCEP, 150.5 mg, 0.53 mmol) was added to the reaction solution, and the reaction was continued at 35° C. for 4 hours. After the reaction was completed, 73.5 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5) was added to the reaction solution for dilution so that the concentration of HA in the reaction mixture was 2.5 mg / mL. 4.5 mL of sodium acetate buffer (3 M, pH 5.5) was further added to the reaction mixture for dilution so that the concentration of sodium acetate in the reaction mixture was 150 mM. Then prepare ethanol (360 mL) with a volume 4 times that of the reaction solution. Slowly add the reaction mixture into the ethanol for precipitation while stirring. The precipitate is purified by centrifugation, and the supernatant is removed by centrifugation. The collected precipitate is washed twice with 80 v / v% ethanol and twice with ethanol. Finally, the collected precipitate is placed in a 50 mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. The white Alkyn / HA-PEG4-SH solid powder (120 mg, yield: 57.1%) is collected and stored at -20°C in a nitrogen atmosphere. 1H NMR characterization shows that the degree of substitution of thiol groups in hyaluronic acid is 10.1%, that is, there are approximately 3.1 thiol molecules in one HA molecule. Note: The degree of substitution is defined as the number of repeating units replaced by thiol groups for every 100 repeating units. Its nuclear magnetic results 1H NMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.02 4.21-4.75; Thiol functional group: δ (ppm) 2.75 (-NHCO-CH2-CH2-SH), δ (ppm) 2.54 (-NHCO-CH2-CH2-SH).

[0076]

[0077] Compound 4

[0078] Example 5, Synthesis of Sodium Hyaluronate with Side Chain Thiol Functionalization and Terminal Alkyne Functionalization (Alkyn / HA-PEG10-SH, 12 kDa, Compound 5)

[0079] First, 120 mg of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 12 kDa, 0.30 mmol repeating unit) was weighed and dissolved in 6 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5), and stirred at 35 ° C in a three-necked round-bottom flask. After stirring for 2 hours, Alkyn-HA was completely dissolved, the solution became clear, and the concentration of HA was 20 mg / mL. The HA solution was kept at 35 ° C and stirred, and a certain amount of SPDP-PEG10-NH2 (110.1 mg, 0.15 mmol) was slowly added to the solution. Then, a certain amount of 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM, 24.9 mg, 0.09 mmol) was added to the reaction solution. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 35 ° C. The concentration of HA during the reaction was 20 mg / mL. After 24 hours of reaction, a certain amount of tris(2-carboxyethyl)phosphine (TCEP, 86.0 mg, 0.3 mmol) was added to the reaction solution, and the reaction was continued at 35° C. for 4 hours. After the reaction was completed, 42 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5) was added to the reaction solution for dilution so that the concentration of HA in the reaction mixture was 2.5 mg / mL. 2.5 mL of sodium acetate buffer (3 M, pH 5.5) was further added to the reaction mixture for dilution so that the concentration of sodium acetate in the reaction mixture was 150 mM. Then prepare ethanol (210 mL) with a volume 4 times that of the reaction solution. Slowly add the reaction mixture into the ethanol for precipitation while stirring. The precipitate is purified by centrifugation, and the supernatant is removed by centrifugation. The collected precipitate is washed twice with 80 v / v% ethanol and twice with ethanol. Finally, the collected precipitate is placed in a 50 mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. The white Alkyn / HA-PEG10-SH solid powder (78.5 mg, yield: 65.4%) is collected and stored at -20 ° C in nitrogen. Nuclear magnetic 1H NMR characterization shows that the degree of substitution of thiol groups in hyaluronic acid is 7.7%, that is, there are approximately 2.3 thiol molecules in one HA molecule. Note: The degree of substitution is defined as the number of repeating units replaced by thiol groups for every 100 repeating units. Its nuclear magnetic results 1 H NMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.02 4.21-4.75; Thiol functional group: δ (ppm) 2.75 (-NHCO--CH2-CH2-SH), δ (ppm) 2.54 (-NHCO-CH2-CH2-SH).

[0080]

[0081] Compound 5

[0082] Example 6, Synthesis of Sodium Hyaluronate with Side Chain Thiol Functionalization and Terminal Alkyne Functionalization (Alkyn / HA-SH, 12 kDa, Compound 6)

[0083] First, 400 mg of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 12 kDa, 1 mmol repeating unit) was weighed and dissolved in 20 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5), and stirred at 35 ° C in a three-necked round-bottom flask. After stirring for 2 hours, Alkyn-HA was completely dissolved, the solution became clear, and the concentration of HA was 20 mg / mL. The HA solution was kept at 35 ° C and stirred, and a certain amount of 2-(2-pyridyldisulfide)ethylamine hydrochloride (PDA.HCl, 111.4 mg, 0.5 mmol) was slowly added to the solution. Then, a certain amount of 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM, 152.2 mg, 0.55 mmol) was added to the reaction solution. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 35 ° C. The concentration of HA during the reaction was 20 mg / mL. After 24 hours of reaction, a certain amount of tris(2-carboxyethyl)phosphine (TCEP, 286.6 mg, 1 mmol) was added to the reaction solution, and the reaction was continued at 35° C. for 4 hours. After the reaction was completed, 140 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5) was added to the reaction solution for dilution so that the concentration of HA in the reaction mixture was 2.5 mg / mL. 8 mL of sodium acetate buffer (3 M, pH 5.5) was further added to the reaction mixture for dilution so that the concentration of sodium acetate in the reaction mixture was 150 mM. Then prepare ethanol (670mL) with a volume 4 times that of the reaction solution. Slowly add the reaction mixture into the ethanol for precipitation while stirring. The precipitate is purified by centrifugation, and the supernatant is removed by centrifugation. The collected precipitate is washed twice with 80v / v% ethanol and twice with ethanol. Finally, the collected precipitate is placed in a 50mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. The white Alkyn / HA-SH solid powder (270mg, yield: 67.5%) is collected and stored at -20°C in a nitrogen atmosphere. Nuclear magnetic 1H NMR characterization shows that the degree of substitution of thiol groups in hyaluronic acid is 18.8%, that is, there are approximately 5.6 thiol molecules in one HA molecule. Note: The degree of substitution is defined as the number of repeating units replaced by thiol groups for every 100 repeating units. Its nuclear magnetic results 1 H NMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.02 4.21-4.75; Thiol functional group: δ (ppm) 2.59 (-CH2-CH2-SH).

[0084]

[0085] Compound 6

[0086] Example 7, Synthesis of Sodium Hyaluronate with Side Chain Thiol Functionalization and Terminal Alkyne Functionalization (Alkyn / HA-SH, 12 kDa, Compound 7)

[0087] First, weigh 200 mg of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 12 kDa, 1 mmol repeating unit) and dissolve it in 10 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5), and stir at 35 ° C in a three-necked round-bottom flask. After stirring for 2 hours, Alkyn-HA is completely dissolved, the solution becomes clear, and the concentration of HA is 20 mg / mL. Continue to keep the HA solution at 35 ° C and stir, and slowly add a certain amount of 2-(2-pyridyldisulfide)ethylamine hydrochloride (PDA.HCl, 55.7 mg, 0.5 mmol) to the solution. Then, add a certain amount of 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM, 110.7 mg, 0.8 mmol) to the reaction solution. After the addition is completed, continue the reaction for 24 hours and the temperature is controlled at 35 ° C. The concentration of HA during the reaction process is 20 mg / mL. After 24 hours of reaction, a certain amount of tris(2-carboxyethyl)phosphine (TCEP, 143.3 mg, 1 mmol) was added to the reaction solution, and the reaction was continued at 35° C. for 4 hours. After the reaction was completed, 70 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5) was added to the reaction solution for dilution so that the concentration of HA in the reaction mixture was 2.5 mg / mL. 4 mL of sodium acetate buffer (3 M, pH 5.5) was further added to the reaction mixture for dilution so that the concentration of sodium acetate in the reaction mixture was 150 mM. Then prepare ethanol (340 mL) with a volume of 4 times that of the reaction solution. Slowly add the reaction mixture into the ethanol for precipitation while stirring. The precipitate is purified by centrifugation, and the supernatant is removed by centrifugation. The collected precipitate is washed twice with 80 v / v% ethanol and twice with ethanol. Finally, the collected precipitate is placed in a 50 mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. The white Alkyn / HA-SH solid powder (180 mg, yield: 90.0%) is collected and stored at -20°C in a nitrogen atmosphere. Nuclear magnetic 1H NMR characterization shows that the degree of substitution of thiol groups in hyaluronic acid is 27.6%, that is, there are approximately 8.1 thiol molecules in one HA molecule. Note: The degree of substitution is defined as the number of repeating units substituted by thiol groups for every 100 repeating units. Its nuclear magnetic results 1H NMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.02 4.21-4.75; Thiol functional group: δ (ppm) 2.59 (-CH2-CH2-SH).

[0088]

[0089] Compound 7

[0090] Example 8, Synthesis of Sodium Hyaluronate with Side Chain Thiol Functionalization and Terminal Alkyne Functionalization (Alkyn / HA-SH, 36 kDa, Compound 8)

[0091] First, 400 mg of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 36 kDa, 1 mmol repeating unit) was weighed and dissolved in 20 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5), and stirred at 35 ° C in a three-necked round-bottom flask. After stirring for 2 hours, Alkyn-HA was completely dissolved, the solution became clear, and the concentration of HA was 20 mg / mL. The HA solution was kept at 35 ° C and stirred, and a certain amount of 2-(2-pyridyldisulfide)ethylamine hydrochloride (PDA.HCl, 111.4 mg, 0.5 mmol) was slowly added to the solution. Then, a certain amount of 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM, 22.2 mg, 0.08 mmol) was added to the reaction solution. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 35 ° C. The concentration of HA during the reaction was 20 mg / mL. After 24 hours of reaction, a certain amount of tris(2-carboxyethyl)phosphine (TCEP, 286.6 mg, 1 mmol) was added to the reaction solution, and the reaction was continued at 35° C. for 4 hours. After the reaction was completed, 140 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5) was added to the reaction solution for dilution so that the concentration of HA in the reaction mixture was 2.5 mg / mL. 8 mL of sodium acetate buffer (3 M, pH 5.5) was further added to the reaction mixture for dilution so that the concentration of sodium acetate in the reaction mixture was 150 mM. Then prepare ethanol (670mL) with a volume of 4 times that of the reaction solution. Slowly add the reaction mixture into the ethanol for precipitation while stirring. The precipitate is purified by centrifugation, and the supernatant is removed by centrifugation. The collected precipitate is washed twice with 80v / v% ethanol and twice with ethanol. Finally, the collected precipitate is placed in a 50mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. The white Alkyn / HA-SH solid powder (356mg, yield: 89.0%) is collected and stored at -20°C in nitrogen. Nuclear magnetic 1H NMR characterization shows that the degree of substitution of thiol groups in hyaluronic acid is 3.9%, that is, there are approximately 3.5 thiol molecules in a 36KDa HA molecule. Note: The degree of substitution is defined as the number of repeating units replaced by thiol groups for every 100 repeating units. Its nuclear magnetic results 1 H NMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.02 4.21-4.75; Thiol functional group: δ (ppm) 2.65 (-CH2-CH2-SH).

[0092]

[0093] Compound 8

[0094] Example 9, Synthesis of Sodium Hyaluronate with Side Chain Thiol Functionalization and Terminal Alkyne Functionalization (Alkyn / HA-SH, 36 kDa, Compound 9)

[0095] First, 400 mg of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 36 kDa, 1 mmol repeating unit) was weighed and dissolved in 20 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5), and stirred at 35 ° C in a three-necked round-bottom flask. After stirring for 2 hours, Alkyn-HA was completely dissolved, the solution became clear, and the concentration of HA was 20 mg / mL. The HA solution was kept at 35 ° C and stirred, and a certain amount of 2-(2-pyridyldisulfide)ethylamine hydrochloride (PDA.HCl, 111.4 mg, 0.5 mmol) was slowly added to the solution. Then, a certain amount of 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM, 33.3 mg, 0.12 mmol) was added to the reaction solution. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 35 ° C. The concentration of HA during the reaction was 20 mg / mL. After 24 hours of reaction, a certain amount of tris(2-carboxyethyl)phosphine (TCEP, 286.6 mg, 1 mmol) was added to the reaction solution, and the reaction was continued at 35° C. for 4 hours. After the reaction was completed, 140 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5) was added to the reaction solution for dilution so that the concentration of HA in the reaction mixture was 2.5 mg / mL. 8 mL of sodium acetate buffer (3 M, pH 5.5) was further added to the reaction mixture for dilution so that the concentration of sodium acetate in the reaction mixture was 150 mM. Then prepare ethanol (670mL) with a volume of 4 times that of the reaction solution. Slowly add the reaction mixture into the ethanol for precipitation while stirring. The precipitate is purified by centrifugation, and the supernatant is removed by centrifugation. The collected precipitate is washed twice with 80v / v% ethanol and twice with ethanol. Finally, the collected precipitate is placed in a 50mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. The white Alkyn / HA-SH solid powder (354mg, yield: 88.5%) is collected and stored at -20°C in nitrogen. Nuclear magnetic 1H NMR characterization shows that the degree of substitution of thiol groups in hyaluronic acid is 6.1%, that is, there are approximately 5.4 thiol molecules in a 36KDa HA molecule. Note: The degree of substitution is defined as the number of repeating units replaced by thiol groups for every 100 repeating units. Its nuclear magnetic results 1 H NMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.02 4.21-4.75; Thiol functional group: δ (ppm) 2.65 (-CH2-CH2-SH).

[0096]

[0097] Compound 9

[0098] Example 10, Synthesis of Sodium Hyaluronate with Side Chain Thiol Functionalization and Terminal Alkyne Functionalization (Alkyn / HA-SH, 36 kDa, Compound 10)

[0099] First, weigh 200 mg of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 36 kDa, 1 mmol repeating unit) and dissolve it in 10 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5), and stir at 35 ° C in a three-necked round-bottom flask. After stirring for 2 hours, Alkyn-HA is completely dissolved, the solution becomes clear, and the concentration of HA is 20 mg / mL. Continue to keep the HA solution at 35 ° C and stir, and slowly add a certain amount of 2-(2-pyridyldisulfide)ethylamine hydrochloride (PDA.HCl, 55.7 mg, 0.5 mmol) to the solution. Then, add a certain amount of 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM, 27.8 mg, 0.2 mmol) to the reaction solution. After the addition is completed, continue the reaction for 24 hours and the temperature is controlled at 35 ° C. The concentration of HA during the reaction process is 20 mg / mL. After 24 hours of reaction, a certain amount of tris(2-carboxyethyl)phosphine (TCEP, 143.3 mg, 1 mmol) was added to the reaction solution, and the reaction was continued at 35° C. for 4 hours. After the reaction was completed, 70 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5) was added to the reaction solution for dilution so that the concentration of HA in the reaction mixture was 2.5 mg / mL. 4 mL of sodium acetate buffer (3 M, pH 5.5) was further added to the reaction mixture for dilution so that the concentration of sodium acetate in the reaction mixture was 150 mM. Then prepare ethanol (340mL) with a volume of 4 times that of the reaction solution. Slowly add the reaction mixture into the ethanol for precipitation while stirring. The precipitate is purified by centrifugation, and the supernatant is removed by centrifugation. The collected precipitate is washed twice with 80v / v% ethanol and twice with ethanol. Finally, the collected precipitate is placed in a 50mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. The white Alkyn / HA-SH solid powder (187mg, yield: 93.5%) is collected and stored at -20°C in nitrogen. 1H NMR characterization shows that the degree of substitution of thiol groups in hyaluronic acid is 8.5%, that is, there are approximately 7.7 thiol molecules in a 36KDa HA molecule. Note: The degree of substitution is defined as the number of repeating units replaced by thiol groups for every 100 repeating units. Its nuclear magnetic results 1H NMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.02 4.21-4.75; Thiol functional group: δ (ppm) 2.65 (-CH2-CH2-SH).

[0100]

[0101] Compound 10

[0102] Example 11, Synthesis of Sodium Hyaluronate with Side Chain Thiol Functionalization and Terminal Alkyne Functionalization (Alkyn / HA-SH, 36 kDa, Compound 11)

[0103] First, weigh 200 mg of terminal alkynyl functionalized sodium hyaluronate (Alkyn-HA, 36 kDa, 1 mmol repeating unit) and dissolve it in 10 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5), and stir at 35 ° C in a three-necked round-bottom flask. After stirring for 2 hours, Alkyn-HA is completely dissolved, the solution becomes clear, and the concentration of HA is 20 mg / mL. Continue to keep the HA solution at 35 ° C and stir, and slowly add a certain amount of 2-(2-pyridyldisulfide)ethylamine hydrochloride (PDA.HCl, 55.7 mg, 0.5 mmol) to the solution. Then, add a certain amount of 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM, 41.7 mg, 0.3 mmol) to the reaction solution. After the addition is completed, continue the reaction for 24 hours and the temperature is controlled at 35 ° C. The concentration of HA during the reaction process is 20 mg / mL. After 24 hours of reaction, a certain amount of tris(2-carboxyethyl)phosphine (TCEP, 143.3 mg, 1 mmol) was added to the reaction solution, and the reaction was continued at 35° C. for 4 hours. After the reaction was completed, 70 mL of morpholineethanesulfonic acid buffer (100 mM, pH 6.5) was added to the reaction solution for dilution so that the concentration of HA in the reaction mixture was 2.5 mg / mL. 4 mL of sodium acetate buffer (3 M, pH 5.5) was further added to the reaction mixture for dilution so that the concentration of sodium acetate in the reaction mixture was 150 mM. Then prepare ethanol (340 mL) with a volume 4 times that of the reaction solution. Slowly add the reaction mixture into the ethanol for precipitation while stirring. The precipitate is purified by centrifugation, and the supernatant is removed by centrifugation. The collected precipitate is washed twice with 80 v / v% ethanol and twice with ethanol. Finally, the collected precipitate is placed in a 50 mL centrifuge tube and placed in a freeze dryer for high vacuum freeze drying. The white Alkyn / HA-SH solid powder (172 mg, yield: 86.0%) is collected and stored at -20°C in a nitrogen atmosphere. 1H NMR characterization shows that the degree of substitution of thiol groups in hyaluronic acid is 15.5%, that is, there are approximately 14.0 thiol molecules in a 36KDa HA molecule. Note: The degree of substitution is defined as the number of repeating units substituted by thiol groups for every 100 repeating units. Its nuclear magnetic results 1 HNMR (D2O): Hyaluronic acid (HA): δ (ppm) 1.86-2.01, 3.28-4.02 4.21-4.75; Thiol functional group: δ (ppm) 2.65 (-CH2-CH2-SH).

[0104]

[0105] Compound 11

[0106] Example 12, Synthesis of terminal alkynyl functionalized sodium hyaluronate-auristatin E polymer prodrug (Alkyn / HA-MMAE, 12 kDa, compound 12)

[0107] First, 20 mg of sodium hyaluronate functionalized with side chain thiol and terminal alkynyl (compound 3, Alkyn / HA-SH, 12 kDa, 0.005 mmol-SH group) was weighed and dissolved in 2 mL of phosphate buffer (5 mM, pH 7.4), and stirred at 37 ° C in a round-bottom flask. After stirring for 2 hours, Alkyn / HA-SH was completely dissolved, the solution became clear, and the concentration of Alkyn / HA-SH was 10 mg / mL. The Alkyn / HA-SH solution was kept at 37 ° C and stirred, and 2 mL of phosphate buffer was slowly added to the solution for dilution. Then, a certain amount of N, N-dimethylacetamide (DMAc, 638 μL) was added to the reaction solution, and 362 μL of the pre-prepared auristatin E load linker mother solution (vc-PAB-MMAE, mother solution concentration: 20 mM) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 37°C. The concentration of HA during the reaction was 4 mg / mL, and the volume ratio of the organic solvent DMAc was 20%. After 24 hours of reaction, the reaction mixture was filtered, and the reaction solution was purified using an ultrafiltration tube (MWCO: 3KDa). The collected sample was stored in a phosphate buffer (5mM, pH 7.4) (9.46 mg, yield: 47.3%), and the sample was stored in nitrogen at 2-8°C. UV characterization showed that the drug polymer ratio was 4.4, that is, there were approximately 4.4 on one hyaluronic acid molecule. In addition, the amount of residual free load was detected to be 0.08 mol / mol%.

[0108]

[0109] Compound 12

[0110] Example 13, Synthesis of terminal alkynyl functionalized sodium hyaluronate-auristatin E polymer prodrug (Alkyn / HA-PEG4-MMAE, 12 kDa, compound 13)

[0111] First, 20 mg of sodium hyaluronate functionalized with side chain thiol and terminal alkynyl (compound 4, Alkyn / HA-PEG4-SH, 12 kDa, 0.005 mmol-SH group) was weighed and dissolved in 2 mL of phosphate buffer (5 mM, pH 7.4), and stirred at 37 ° C in a round-bottom flask. After stirring for 2 hours, Alkyn / HA-PEG4-SH was completely dissolved, the solution became clear, and the concentration of Alkyn / HA-SH was 10 mg / mL. The Alkyn / HA-PEG4-SH solution was kept at 37 ° C and stirred, and 2 mL of phosphate buffer was slowly added to the solution for dilution. Then, a certain amount of N, N-dimethylacetamide (DMAc, 648 μL) was added to the reaction solution, and a volume of 352 μL of the pre-prepared auristatin E load linker mother solution (vc-PAB-MMAE, mother solution concentration: 20 mM) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 37°C. The concentration of HA during the reaction was 4 mg / mL, and the volume ratio of the organic solvent DMAc was 20%. After 24 hours of reaction, the reaction mixture was filtered, and the reaction solution was purified using an ultrafiltration tube (MWCO: 3KDa). The collected sample was stored in a phosphate buffer (5mM, pH 7.4) (11.6 mg, yield: 58.0%), and the sample was stored in nitrogen at 2-8°C. UV characterization showed that the drug-polymer ratio was 4.1, that is, there were approximately 4.1 on one hyaluronic acid molecule. In addition, the amount of residual free load was detected to be 0.19 mol / mol%.

[0112]

[0113] Compound 13

[0114] Example 14, Synthesis of terminal alkynyl functionalized sodium hyaluronate-auristatin E polymer prodrug (Alkyn / HA-PEG10-MMAE, 12 kDa, compound 14)

[0115] First, 20 mg of sodium hyaluronate functionalized with side chain thiol and terminal alkynyl (compound 5, Alkyn / HA-PEG10-SH, 12 kDa, 0.0038 mmol-SH group) was weighed and dissolved in 2 mL of phosphate buffer (5 mM, pH 7.4), and stirred at 37 ° C in a round-bottom flask. After stirring for 2 hours, Alkyn / HA-PEG10-SH was completely dissolved, the solution became clear, and the concentration of Alkyn / HA-SH was 10 mg / mL. The Alkyn / HA-PEG10-SH solution was kept at 37 ° C and stirred, and 2 mL of phosphate buffer was slowly added to the solution for dilution. Then, a certain amount of N, N-dimethylacetamide (DMAc, 727 μL) was added to the reaction solution, and a volume of 273 μL of the pre-prepared auristatin E load linker mother solution (vc-PAB-MMAE, mother solution concentration: 20 mM) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 37°C. The concentration of HA during the reaction was 4 mg / mL, and the volume ratio of the organic solvent DMAc was 20%. After 24 hours of reaction, the reaction mixture was filtered, and the reaction solution was purified using an ultrafiltration tube (MWCO: 3KDa). The collected sample was stored in a phosphate buffer (5mM, pH 7.4) (12.9 mg, yield: 64.5%), and the sample was stored in nitrogen at 2-8°C. UV characterization showed that the drug-polymer ratio was 3.6, that is, there were approximately 3.6 on one hyaluronic acid molecule. In addition, the amount of residual free load was not detected.

[0116]

[0117] Compound 14

[0118] Example 15, Synthesis of terminal alkynyl functionalized sodium hyaluronate-auristatin E polymer prodrug (Alkyn / HA-MMAE, 12 kDa, compound 15)

[0119] First, 20 mg of sodium hyaluronate functionalized with side chain thiol and terminal alkynyl (compound 6, Alkyn / HA-SH, 12 kDa, 0.0093 mmol-SH group) was weighed and dissolved in 2 mL of phosphate buffer (5 mM, pH 7.4), and stirred at 37 ° C in a round-bottom flask. After stirring for 2 hours, Alkyn / HA-SH was completely dissolved, the solution became clear, and the concentration of Alkyn / HA-SH was 10 mg / mL. The Alkyn / HA-SH solution was kept at 37 ° C and stirred, and 2 mL of phosphate buffer was slowly added to the solution for dilution. Then, a certain amount of N, N-dimethylacetamide (DMAc, 380 μL) was added to the reaction solution, and a volume of 620 μL of the pre-prepared auristatin E load linker mother solution (vc-PAB-MMAE, mother solution concentration: 20 mM) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 37°C. The concentration of HA during the reaction was 4 mg / mL, and the volume ratio of the organic solvent DMAc was 20%. After 24 hours of reaction, the reaction mixture was filtered, and the reaction solution was purified using an ultrafiltration tube (MWCO: 3KDa). The collected sample was stored in a phosphate buffer (5mM, pH 7.4) (10.6 mg, yield: 53.0%), and the sample was stored in nitrogen at 2-8°C. UV characterization showed that the drug polymer ratio was 6.2, that is, there were approximately 6.2 on one hyaluronic acid molecule. In addition, the amount of residual free load was detected to be 0.42 mol / mol%.

[0120]

[0121] Compound 15

[0122] Example 16, Synthesis of terminal alkynyl functionalized sodium hyaluronate-auristatin E polymer prodrug (Alkyn / HA-MMAE, 12 kDa, compound 16)

[0123] First, 20 mg of sodium hyaluronate functionalized with side chain thiol and terminal alkynyl (compound 7, Alkyn / HA-SH, 12 kDa, 0.0137 mmol-SH group) was weighed and dissolved in 2 mL of phosphate buffer (5 mM, pH 7.4), and stirred at 37 ° C in a round-bottom flask. After stirring for 2 hours, Alkyn / HA-SH was completely dissolved, the solution became clear, and the concentration of Alkyn / HA-SH was 10 mg / mL. The Alkyn / HA-SH solution was kept at 37 ° C and stirred, and 2 mL of phosphate buffer was slowly added to the solution for dilution. Then, a certain amount of N, N-dimethylacetamide (DMAc, 137 μL) was added to the reaction solution, and 863 μL of the pre-prepared auristatin E load linker mother solution (vc-PAB-MMAE, mother solution concentration: 20 mM) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 37°C. The concentration of HA during the reaction was 4 mg / mL, and the volume ratio of the organic solvent DMAc was 20%. After 24 hours of reaction, the reaction mixture was filtered, and the reaction solution was purified using an ultrafiltration tube (MWCO: 3KDa). The collected sample was stored in a phosphate buffer (5mM, pH 7.4) (7.15 mg, yield: 35.8%), and the sample was stored in nitrogen at 2-8°C. UV characterization showed that the drug polymer ratio was 8.0, that is, there were approximately 8.0 on one hyaluronic acid molecule. In addition, the amount of residual free load was detected to be 1.54 mol / mol%.

[0124]

[0125] Compound 16

[0126] Example 17, Synthesis of terminal alkynyl functionalized sodium hyaluronate-auristatin E polymer prodrug (Alkyn / HA-MMAE, 36 kDa, compound 17)

[0127] First, 42.7 mg of sodium hyaluronate functionalized with side chain thiol and terminal alkynyl (compound 8, Alkyn / HA-SH, 36 kDa, 0.0041 mmol-SH group) was weighed and dissolved in 4.27 mL of phosphate buffer (5 mM, pH 7.4), and stirred at 37 ° C in a round-bottom flask. After stirring for 2 hours, Alkyn / HA-SH was completely dissolved, the solution became clear, and the concentration of Alkyn / HA-SH was 10 mg / mL. The Alkyn / HA-SH solution was kept at 37 ° C and stirred, and 4.27 mL of phosphate buffer was slowly added to the solution for dilution. Then, a certain amount of N, N-dimethylacetamide (DMAc, 1831.8 μL) was added to the reaction solution, and a volume of 303.2 μL of the pre-prepared auristatin E load linker mother solution (vc-PAB-MMAE, mother solution concentration: 20 mM) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 37°C. The concentration of HA during the reaction was 4 mg / mL, and the volume ratio of the organic solvent DMAc was 20%. After 24 hours of reaction, the reaction mixture was filtered, and the reaction solution was purified using an ultrafiltration tube (MWCO: 3KDa). The collected sample was stored in a phosphate buffer (5mM, pH7.4) (26.1 mg, yield: 61.0%), and the sample was stored in nitrogen at 2-8°C. UV characterization showed that the drug polymer ratio was 4.9, that is, there were approximately 4.9 on one hyaluronic acid molecule. In addition, the amount of residual free load was detected to be 0.44 mol / mol%.

[0128]

[0129] Compound 17

[0130] Example 18, Synthesis of terminal alkynyl functionalized sodium hyaluronate-auristatin E polymer prodrug (Alkyn / HA-MMAE, 36 kDa, compound 18)

[0131] First, 23.1 mg of sodium hyaluronate functionalized with side chain thiol and terminal alkynyl (compound 9, Alkyn / HA-SH, 36 kDa, 0.0035 mmol-SH group) was weighed and dissolved in 2.31 mL of phosphate buffer (5 mM, pH 7.4), and stirred at 37 ° C in a round-bottom flask. After stirring for 2 hours, Alkyn / HA-SH was completely dissolved, the solution became clear, and the concentration of Alkyn / HA-SH was 10 mg / mL. The Alkyn / HA-SH solution was kept at 37 ° C and stirred, and 2.31 mL of phosphate buffer was slowly added to the solution for dilution. Then, a certain amount of N, N-dimethylacetamide (DMAc, 902 μL) was added to the reaction solution, and a volume of 253 μL of the pre-prepared auristatin E load linker mother solution (vc-PAB-MMAE, mother solution concentration: 20 mM) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 37°C. The concentration of HA during the reaction was 4 mg / mL, and the volume ratio of the organic solvent DMAc was 20%. After 24 hours of reaction, the reaction mixture was filtered, and the reaction solution was purified using an ultrafiltration tube (MWCO: 3KDa). The collected sample was stored in a phosphate buffer (5mM, pH 7.4) (17.5 mg, yield: 75.8%), and the sample was stored in nitrogen at 2-8°C. UV characterization showed that the drug polymer ratio was 5.4, that is, there were approximately 5.4 on one hyaluronic acid molecule. In addition, the amount of residual free load was detected to be 0.38 mol / mol%.

[0132]

[0133] Compound 18

[0134] Example 19, Synthesis of terminal alkynyl functionalized sodium hyaluronate-auristatin E polymer prodrug (Alkyn / HA-MMAE, 36 kDa, compound 19)

[0135] First, 22.6 mg of sodium hyaluronate functionalized with side chain thiol and terminal alkynyl (compound 10, Alkyn / HA-SH, 36 kDa, 0.0048 mmol-SH group) was weighed and dissolved in 2.26 mL of phosphate buffer (5 mM, pH 7.4), and stirred at 37 ° C in a round-bottom flask. After stirring for 2 hours, Alkyn / HA-SH was completely dissolved, the solution became clear, and the concentration of Alkyn / HA-SH was 10 mg / mL. The Alkyn / HA-SH solution was kept at 37 ° C and stirred, and 2.26 mL of phosphate buffer was slowly added to the solution for dilution. Then, a certain amount of N, N-dimethylacetamide (DMAc, 791 μL) was added to the reaction solution, and a volume of 339 μL of the pre-prepared auristatin E load linker mother solution (vc-PAB-MMAE, mother solution concentration: 20 mM) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 37°C. The concentration of HA during the reaction was 4 mg / mL, and the volume ratio of the organic solvent DMAc was 20%. After 24 hours of reaction, the reaction mixture was filtered, and the reaction solution was purified using an ultrafiltration tube (MWCO: 3KDa). The collected sample was stored in a phosphate buffer (5mM, pH 7.4) (12.8 mg, yield: 56.7%), and the sample was stored in nitrogen at 2-8°C. UV characterization showed that the drug polymer ratio was 10.7, that is, there were approximately 10.7 on one hyaluronic acid molecule. In addition, the amount of residual free load was detected to be <0.14 mol / mol%.

[0136]

[0137] Compound 19

[0138] Example 20, Synthesis of terminal alkynyl functionalized sodium hyaluronate-auristatin E polymer prodrug (Alkyn / HA-MMAE, 36 kDa, compound 20)

[0139] First, 20.2 mg of sodium hyaluronate functionalized with side chain thiol and terminal alkynyl (compound 11, Alkyn / HA-SH, 36 kDa, 0.0077 mmol-SH group) was weighed and dissolved in 2.02 mL of phosphate buffer (5 mM, pH 7.4), and stirred at 37 ° C in a round-bottom flask. After stirring for 2 hours, Alkyn / HA-SH was completely dissolved, the solution became clear, and the concentration of Alkyn / HA-SH was 10 mg / mL. The Alkyn / HA-SH solution was kept at 37 ° C and stirred, and 2.02 mL of phosphate buffer was slowly added to the solution for dilution. Then, a certain amount of N, N-dimethylacetamide (DMAc, 483 μL) was added to the reaction solution, and a volume of 527 μL of the pre-prepared auristatin E load linker mother solution (vc-PAB-MMAE, mother solution concentration: 20 mM) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 37°C. The concentration of HA during the reaction was 4 mg / mL, and the volume ratio of the organic solvent DMAc was 20%. After 24 hours of reaction, the reaction mixture was filtered, and the reaction solution was purified using an ultrafiltration tube (MWCO: 3KDa). The collected sample was stored in a phosphate buffer (5mM, pH 7.4) (13.9 mg, yield: 68.8%), and the sample was stored in nitrogen at 2-8°C. UV characterization showed that the drug polymer ratio was 13.6, that is, there were approximately 13.6 on one hyaluronic acid molecule. In addition, the amount of residual free load was detected to be <0.14 mol / mol%.

[0140]

[0141] Compound 20

[0142] Example 21, Synthesis of Azido-Functionalized Trastuzumab (Herceptin-PEG4-N3, Compound 21)

[0143] First, the trastuzumab solution was replaced into phosphate buffer (50mM, pH 6.5) through an ultrafiltration tube, and the antibody solution concentration was 20.39mg / mL by UV detection. 981μL of trastuzumab solution (20mg) was added to the reaction vessel, followed by 819μL of phosphate buffer (50mM, pH 6.5) for dilution, 157.2μL of N,N-dimethylacetamide solvent, and 42.8μL of pre-prepared NHS-PEG4-N3 linker solution (mother liquor concentration: 5mg / mL). After the addition was completed, the reaction was continued for 4h and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 10mg / mL, and the volume ratio of the organic solvent DMAc was 10%. After 4 hours of reaction, succinic acid buffer (200 mM, pH 5.0) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified and replaced with an ultrafiltration tube (MWCO: 30 KDa), and the sample was collected and stored in succinic acid buffer (20 mM, pH 5.0) (18.06 mg, yield: 90.3%), and the sample was stored at 2-8 ° C under nitrogen. LC-MS characterization showed that the degree of substitution of the azide functional group in trastuzumab was 2.56. The detection data are as follows: component 1 (antibody, molecular weight 145171.00, signal percentage 6.67%), component 2 (antibody + 1 linker, molecular weight 145443.82, signal percentage 19.20%), component 3 (antibody + 2 linkers, molecular weight 145717.09, signal percentage 26.09%), component 4 (antibody + 3 linkers, molecular weight 145989.79, signal percentage 22.67%), component 5 (antibody + 4 linkers, molecular weight 146262.53, signal percentage 14.73%), component 6 (antibody + 5 linkers, molecular weight 146535.19, signal percentage 7.25%), component 7 (antibody + 6 linkers, molecular weight 146808.90, signal percentage 2.67%), component 8 (antibody + 7 linkers, molecular weight 147079.98, signal percentage 0.72%).

[0144]

[0145] Compound 21

[0146] Example 22, Synthesis of Azido-Functionalized Trastuzumab (Herceptin-PEG4-N3, Compound 22)

[0147] First, the trastuzumab solution was replaced into phosphate buffer (50mM, pH 6.5) through an ultrafiltration tube, and the antibody solution concentration was 20.39mg / mL by UV detection. 981μL of trastuzumab solution (20mg) was added to the reaction vessel, followed by 819μL of phosphate buffer (50mM, pH 6.5) for dilution, 135.8μL of N,N-dimethylacetamide solvent, and 64.2μL of pre-prepared NHS-PEG4-N3 linker solution (mother liquor concentration: 5mg / mL). After the addition was completed, the reaction was continued for 4h and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 10mg / mL, and the volume ratio of the organic solvent DMAc was 10%. After 4 hours of reaction, succinic acid buffer (200 mM, pH 5.0) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified and replaced with an ultrafiltration tube (MWCO: 30 KDa), and the collected sample was stored in succinic acid buffer (20 mM, pH 5.0) (18.56 mg, yield: 92.8%), and the sample was stored at 2-8 ° C under nitrogen. LC-MS characterization showed that the degree of substitution of the azide functional group in trastuzumab was 3.96. The detection data are as follows: component 1 (antibody, molecular weight 145171.00, signal percentage 1.49%), component 2 (antibody + 1 linker, molecular weight 145443.82, signal percentage 6.79%), component 3 (antibody + 2 linkers, molecular weight 145717.09, signal percentage 13.92%), component 4 (antibody + 3 linkers, molecular weight 145989.79, signal percentage 20.38%), component 5 (antibody + 4 linkers, molecular weight 146262.53, signal percentage 20.66%), Component 6 (antibody + 5 linkers, molecular weight 146535.19, signal percentage 16.95%), component 7 (antibody + 6 linkers, molecular weight 146808.90, signal percentage 10.47%), component 8 (antibody + 7 linkers, molecular weight 147079.98, signal percentage 5.75%), component 9 (antibody + 8 linkers, molecular weight 147353.71, signal percentage 2.68%), component 10 (antibody + 9 linkers, molecular weight 147626.47, signal percentage 0.08%).

[0148]

[0149] Compound 22

[0150] Example 23, Synthesis of Azido-Functionalized Trastuzumab (Herceptin-PEG4-N3, Compound 23)

[0151] First, the trastuzumab solution was replaced into phosphate buffer (50mM, pH 6.5) through an ultrafiltration tube, and the antibody solution concentration was 20.39mg / mL by UV detection. 981μL of trastuzumab solution (20mg) was added to the reaction vessel, followed by 819μL of phosphate buffer (50mM, pH 6.5) for dilution, 114.4μL of N,N-dimethylacetamide solvent, and 85.6μL of pre-prepared NHS-PEG4-N3 linker solution (mother liquor concentration: 5mg / mL). After the addition was completed, the reaction was continued for 4h and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 10mg / mL, and the volume ratio of the organic solvent DMAc was 10%. After 4 hours of reaction, succinic acid buffer (200 mM, pH 5.0) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified and replaced with an ultrafiltration tube (MWCO: 30 KDa), and the sample was collected and stored in succinic acid buffer (20 mM, pH 5.0) (17.88 mg, yield: 89.4%), and the sample was stored at 2-8 ° C under nitrogen. LC-MS characterization showed that the degree of substitution of the azide functional group in trastuzumab was 5.20. The detection data are as follows: component 1 (antibody, molecular weight 145171.00, signal percentage 0%), component 2 (antibody + 1 linker, molecular weight 145443.82, signal percentage 1.74%), component 3 (antibody + 2 linkers, molecular weight 145717.09, signal percentage 6.14%), component 4 (antibody + 3 linkers, molecular weight 145989.79, signal percentage 12.25%), component 5 (antibody + 4 linkers, molecular weight 146262.53, signal percentage 17.78%), component 6 (antibody + 5 linkers, molecular weight 146535.19, signal percentage 19.47%), component 7 (antibody + 6 linkers, molecular weight 146808.90, signal percentage 17.32%), component 8 (antibody + 7 linkers, molecular weight 147079.98, signal percentage 12.42%), component 9 (antibody + 8 linkers, molecular weight 147353.71, signal percentage 7.79%), component 10 (antibody + 9 linkers, molecular weight 147626.47, signal percentage 3.73%), component 11 (antibody + 10 linkers, molecular weight 147898.96, signal percentage 1.24%), component 10 (antibody + 9 linkers, molecular weight 148171.41, signal percentage 0.13%).

[0152]

[0153] Compound 23

[0154] Example 24, Synthesis of Azido-Functionalized Trastuzumab (Herceptin-PEG4-N3, Compound 24)

[0155] First, the trastuzumab solution was replaced into sodium borate buffer (15mM, pH 8.0) through an ultrafiltration tube, and the antibody solution concentration was 23.75mg / mL by UV detection. 210.5μL of trastuzumab solution (5mg) was added to the reaction vessel, followed by 205μL of sodium borate buffer (15mM, pH 8.0) for dilution, 34.4μL of pre-prepared tris(2-carboxyethyl)phosphine solution (TCEP, mother liquor concentration: 10mM), 22.1μL of N,N-dimethylacetamide solvent, 27.9μL of pre-prepared azide sulfur bridge linker solution (mother liquor concentration: 10mg / mL), and after the addition was completed, the reaction was continued for 2h and the temperature was controlled at 37°C. The concentration of trastuzumab during the reaction was 10mg / mL, and the volume ratio of the organic solvent DMAc was 10%. After 2 hours of reaction, the terminated reaction solution was purified and replaced with an ultrafiltration tube (MWCO: 30KDa), and the collected sample was stored in succinic acid buffer (20mM, pH 5.0) (3.94mg, yield: 78.8%), and the sample was stored at 2-8°C under nitrogen. LC-MS characterization showed that the degree of substitution of the azide functional group in trastuzumab was 3.73. The detection data are component 1 (antibody, molecular weight 145171.00, signal percentage 0%), component 2 (antibody + 1 linker, molecular weight 145553.13, signal percentage 0%), component 3 (antibody + 2 linkers, molecular weight 145938.83, signal percentage 5.36%), component 4 (antibody + 3 linkers, molecular weight 146325.92, signal percentage 30.59%), component 5 (antibody + 4 linkers, molecular weight 146707.07, signal percentage 64.04%).

[0156]

[0157] Compound 24

[0158] Example 25, Synthesis of Trastuzumab-Sodium Hyaluronate-Auristatin E Antibody Drug Conjugate (Herceptin-HA-MMAE, Compound 25)

[0159] First, 107.8 μL of azide-functionalized trastuzumab solution (compound 22, Herceptin-PEG4-N3, concentration: 18.55 mg / mL) was taken out by pipette and added to the reaction vessel, and 809.6 μL of terminal alkynyl-functionalized sodium hyaluronate-auritin E solution (compound 12, Alkyn / HA-MMAE, concentration: 2.43 mg / mL) was added to the reaction vessel, followed by the addition of 333.4 μL of the pre-prepared click chemistry catalytic solution (6 mM copper sulfate, 30 mM BTTAA, 90 mM sodium ascorbate) for dilution, and 749.3 μL of phosphate buffer (50 mM, pH 7.4) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 1 mg / mL. After reacting for 24 hours, 50 μL of sodium ethylenediaminetetraacetate solution (500 mM) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified using an ultrafiltration tube (MWCO: 50 KDa) and exchanged into a phosphate buffer (50 mM, pH 7.4). The product was further purified by size exclusion chromatography using a superdex200 chromatography column (eluent: 50 mM phosphate buffer, 15% isopropanol, pH 7.4), and then purified and exchanged into a phosphate buffer (50 mM, pH 7.4) using an ultrafiltration tube (MWCO: 50 KDa) to obtain a sample stored in a phosphate buffer (50 mM, pH 7.4) (0.68 mg, yield: 34%), and the sample was stored at 2-8°C under nitrogen. Through SEC characterization, the average molar ratio of auristatin E drug to trastuzumab in the product was calculated to be 8.2 based on the absorption peak intensity at 248nm and 280nm and the extinction coefficients of the drug molecule and the antibody at the two wavelengths.

[0160] The calculation formula of its DAR value is as follows:

[0161]

[0162] Where DAR is the abbreviation of Drug-Antibody Ratio, which is the drug-antibody ratio;

[0163] A248 represents the absorption intensity of the product at 248nm;

[0164] A280 indicates the absorption intensity of the product at 280nm;

[0165] ε Ab 280nm represents the extinction coefficient of the antibody at 280nm;

[0166] ε Ab 248nm represents the extinction coefficient of the antibody at 248nm;

[0167] ε MMAE 280nm represents the extinction coefficient of auristatin E at 280nm;

[0168] ε MMAE 248 nm represents the extinction coefficient of auristatin E at 248 nm.

[0169] The SEC spectra of compound 25 and trastuzumab at a wavelength of 280 nm are shown in Figure 1 As shown in the figure, it can be seen that there is basically no naked antibody in the product, and the antibody basically participates in the reaction. In addition, the aggregate ratio of the product is very low, and the SEC purity is very high. In the existing patented technologies (patent numbers: CN10374804B, US8685383B2, CN109563215B, US20190083634), because there is an antibody that can connect to multiple polymer molecules, and at the same time a polymer molecule can connect to multiple antibody molecules, there may be aggregation and cross-linking of molecules, resulting in a high level of aggregation of product molecules, and it is difficult to determine the specific structure of the product.

[0170]

[0171] Compound 25

[0172] Example 26, Synthesis of Trastuzumab-Sodium Hyaluronate-Auristatin E Antibody Drug Conjugate (Herceptin-HA-MMAE, Compound 26)

[0173] First, 108.1 μL of azide-functionalized trastuzumab solution (compound 22, Herceptin-PEG4-N3, concentration: 18.5 mg / mL) was taken out by pipette and added to the reaction vessel, and 688.2 μL of terminal alkynyl-functionalized sodium hyaluronate-auritin E solution (compound 13, Alkyn / HA-PEG4-MMAE, concentration: 2.85 mg / mL) was added to the reaction vessel, followed by the addition of 333.4 μL of the pre-prepared click chemistry catalytic solution (6 mM copper sulfate, 30 mM BTTAA, 90 mM sodium ascorbate) for dilution, and 870.4 μL of phosphate buffer (50 mM, pH 7.4) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 1 mg / mL. After reacting for 24 hours, 50 μL of sodium ethylenediaminetetraacetate solution (500 mM) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified using an ultrafiltration tube (MWCO: 50 KDa) and exchanged into a phosphate buffer (50 mM, pH 7.4). The product was further purified by size exclusion chromatography using a superdex200 chromatography column (eluent: 50 mM phosphate buffer, 15% isopropanol, pH 7.4), and then purified and exchanged into a phosphate buffer (50 mM, pH 7.4) using an ultrafiltration tube (MWCO: 50 KDa) to obtain a sample stored in a phosphate buffer (50 mM, pH 7.4) (0.75 mg, yield: 37.5%), and the sample was stored at 2-8° C. and filled with nitrogen. Through SEC characterization, the average molar ratio of auristatin E drug to trastuzumab in the product was calculated to be 8.5 based on the absorption peak intensity at 248nm and 280nm and the extinction coefficients of the drug molecule and the antibody at the two wavelengths.

[0174]

[0175] Compound 26

[0176] Example 27, Synthesis of Trastuzumab-Sodium Hyaluronate-Auristatin E Antibody Drug Conjugate (Herceptin-HA-MMAE, Compound 27)

[0177] First, 108.1 μL of azide-functionalized trastuzumab solution (compound 22, Herceptin-PEG4-N3, concentration: 18.5 mg / mL) was taken out by pipette and added to the reaction vessel, and 691.8 μL of terminal alkynyl-functionalized sodium hyaluronate-auritin E solution (compound 14, Alkyn / HA-PEG10-MMAE, concentration: 2.84 mg / mL) was added to the reaction vessel, followed by the addition of 333.4 μL of the pre-prepared click chemistry catalytic solution (6 mM copper sulfate, 30 mM BTTAA, 90 mM sodium ascorbate) for dilution, and 866.7 μL of phosphate buffer (50 mM, pH 7.4) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 1 mg / mL. After reacting for 24 hours, 50 μL of sodium ethylenediaminetetraacetate solution (500 mM) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified using an ultrafiltration tube (MWCO: 50 KDa) and exchanged into a phosphate buffer (50 mM, pH 7.4). The product was further purified by size exclusion chromatography using a superdex200 chromatography column (eluent: 50 mM phosphate buffer, 15% isopropanol, pH 7.4), and then purified and exchanged into a phosphate buffer (50 mM, pH 7.4) using an ultrafiltration tube (MWCO: 50 KDa) to obtain a sample stored in a phosphate buffer (50 mM, pH 7.4) (0.80 mg, yield: 40.0%), and the sample was stored at 2-8° C. and filled with nitrogen. Through SEC characterization, the average molar ratio of auristatin E drug to trastuzumab in the product was calculated to be 6.3 based on the absorption peak intensity at 248nm and 280nm and the extinction coefficients of the drug molecule and the antibody at the two wavelengths.

[0178]

[0179] Compound 27

[0180] Example 28, Synthesis of Trastuzumab-Sodium Hyaluronate-Auristatin E Antibody Drug Conjugate (Herceptin-HA-MMAE, Compound 28)

[0181] First, 107.8 μL of azide-functionalized trastuzumab solution (compound 22, Herceptin-PEG4-N3, concentration: 18.55 mg / mL) was taken out by pipette and added to the reaction vessel, and 685.3 μL of terminal alkynyl-functionalized sodium hyaluronate-auritin E solution (compound 15, Alkyn / HA-MMAE, concentration: 2.87 mg / mL) was added to the reaction vessel, followed by the addition of 333.4 μL of the pre-prepared click chemistry catalytic solution (6 mM copper sulfate, 30 mM BTTAA, 90 mM sodium ascorbate) for dilution, and 873.6 μL of phosphate buffer (50 mM, pH 7.4) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 1 mg / mL. After reacting for 24 hours, 50 μL of sodium ethylenediaminetetraacetate solution (500 mM) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified using an ultrafiltration tube (MWCO: 50 KDa) and exchanged into a phosphate buffer (50 mM, pH 7.4). The product was further purified by size exclusion chromatography using a superdex200 chromatography column (eluent: 50 mM phosphate buffer, 15% isopropanol, pH 7.4), and then purified and exchanged into a phosphate buffer (50 mM, pH 7.4) using an ultrafiltration tube (MWCO: 50 KDa) to obtain a sample stored in a phosphate buffer (50 mM, pH 7.4) (0.56 mg, yield: 28.0%), and the sample was stored at 2-8°C under nitrogen. Through SEC characterization, the average molar ratio of auristatin E drug to trastuzumab in the product was calculated to be 13.3 based on the absorption peak intensity at 248nm and 280nm and the extinction coefficients of the drug molecule and the antibody at the two wavelengths.

[0182]

[0183] Compound 28

[0184] Example 29, Synthesis of Trastuzumab-Sodium Hyaluronate-Auristatin E Antibody Drug Conjugate (Herceptin-HA-MMAE, Compound 29)

[0185] First, 109.6 μL of azide-functionalized trastuzumab solution (compound 22, Herceptin-PEG4-N3, concentration: 18.25 mg / mL) was taken out by pipette and added to the reaction vessel, and 1186.7 μL of terminal alkynyl-functionalized sodium hyaluronate-auritin E solution (compound 16, Alkyn / HA-MMAE, concentration: 1.323 mg / mL) was added to the reaction vessel, followed by the addition of 333.4 μL of the pre-prepared click chemistry catalytic solution (6 mM copper sulfate, 30 mM BTTAA, 90 mM sodium ascorbate) for dilution, and 370.4 μL of phosphate buffer (50 mM, pH 7.4) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 1 mg / mL. After 24 hours of reaction, 50 μL of sodium ethylenediaminetetraacetate solution (500 mM) was added to the reaction solution to terminate the reaction, and the terminated reaction solution was purified using an ultrafiltration tube (MWCO: 50 KDa) and exchanged into a phosphate buffer (50 mM, pH 7.4). The product was further purified by size exclusion chromatography using a superdex200 chromatography column (eluent: 50 mM phosphate buffer, 15% isopropanol, pH 7.4), and then purified and exchanged into a phosphate buffer (50 mM, pH 7.4) using an ultrafiltration tube (MWCO: 50 KDa) to obtain a sample stored in a phosphate buffer (50 mM, pH 7.4) (0.39 mg, yield: 19.5%), and the sample was stored at 2-8°C under nitrogen. Through SEC characterization, the average molar ratio of auristatin E drug to trastuzumab in the product was calculated to be 24.6 based on the absorption peak intensity at 248nm and 280nm and the extinction coefficients of the drug molecule and the antibody at the two wavelengths.

[0186]

[0187] Compound 29

[0188] Example 30, Synthesis of Trastuzumab-Sodium Hyaluronate-Auristatin E Antibody Drug Conjugate (Herceptin-HA-MMAE, Compound 30)

[0189] First, 107.8 μL of azide-functionalized trastuzumab solution (compound 22, Herceptin-PEG4-N3, concentration: 18.55 mg / mL) was taken out by pipette and added to the reaction vessel, and 1595.7 μL of terminal alkynyl-functionalized sodium hyaluronate-auritin E solution (compound 17, Alkyn / HA-MMAE, concentration: 3.67 mg / mL) was added to the reaction vessel, followed by the addition of 333.4 μL of the pre-prepared click chemistry catalytic solution (6 mM copper sulfate, 30 mM BTTAA, 90 mM sodium ascorbate) for dilution, and 36.9 μL of phosphate buffer (50 mM, pH 7.4) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 1 mg / mL. After reacting for 24 hours, 50 μL of sodium ethylenediaminetetraacetic acid solution (500 mM) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified using an ultrafiltration tube (MWCO: 50 KDa) and exchanged into a phosphate buffer (50 mM, pH 7.4). The product was further purified by size exclusion chromatography using a superdex200 chromatography column (eluent: 50 mM phosphate buffer, 15% isopropanol, pH 7.4), and then purified and exchanged into a phosphate buffer (50 mM, pH 7.4) using an ultrafiltration tube (MWCO: 50 KDa) to obtain a sample stored in a phosphate buffer (50 mM, pH 7.4) (0.68 mg, yield: 34.0%), and the sample was stored at 2-8° C. and filled with nitrogen. Through SEC characterization, the average molar ratio of auristatin E drug to trastuzumab in the product was calculated to be 9.6 based on the absorption peak intensity at 248nm and 280nm and the extinction coefficient of the drug molecule and the antibody at the two wavelengths. The SEC spectrum of compound 30 and trastuzumab at a wavelength of 280nm is shown in Figure 2. Figure 2 As shown in the figure, it can be seen that there is basically no naked antibody in the product, and the antibody basically participates in the reaction. In addition, the aggregate ratio of the product is very low, and the SEC purity is very high. In the existing patented technologies (patent numbers: CN10374804B, US8685383B2, CN109563215B, US20190083634), because there is an antibody that can connect to multiple polymer molecules, and at the same time a polymer molecule can connect to multiple antibody molecules, there may be aggregation and cross-linking of molecules, resulting in a higher level of aggregation of product molecules.

[0190]

[0191] Compound 30

[0192] Example 31, Synthesis of Trastuzumab-Sodium Hyaluronate-Auristatin E Antibody Drug Conjugate (Herceptin-HA-MMAE, Compound 31)

[0193] First, 107.8 μL of azide-functionalized trastuzumab solution (compound 22, Herceptin-PEG4-N3, concentration: 18.55 mg / mL) was taken out by pipette and added to the reaction vessel, and 1240.1 μL of terminal alkynyl-functionalized sodium hyaluronate-auritin E solution (compound 18, Alkyn / HA-MMAE, concentration: 4.73 mg / mL) was added to the reaction vessel, followed by the addition of 333.4 μL of the pre-prepared click chemistry catalytic solution (6 mM copper sulfate, 30 mM BTTAA, 90 mM sodium ascorbate) for dilution, and 318.8 μL of phosphate buffer (50 mM, pH 7.4) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 1 mg / mL. After reacting for 24 hours, 50 μL of sodium ethylenediaminetetraacetic acid solution (500 mM) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified using an ultrafiltration tube (MWCO: 50 KDa) and exchanged into a phosphate buffer (50 mM, pH 7.4). The product was further purified by size exclusion chromatography using a superdex200 chromatography column (eluent: 50 mM phosphate buffer, 15% isopropanol, pH 7.4), and then purified and exchanged into a phosphate buffer (50 mM, pH 7.4) using an ultrafiltration tube (MWCO: 50 KDa) to obtain a sample stored in a phosphate buffer (50 mM, pH 7.4) (0.78 mg, yield: 39.0%), and the sample was stored at 2-8°C under nitrogen. Through SEC characterization, the average molar ratio of auristatin E drug to trastuzumab in the product was calculated to be 14.3 based on the absorption peak intensity at 248nm and 280nm and the extinction coefficients of the drug molecule and the antibody at the two wavelengths.

[0194]

[0195] Compound 31

[0196] Example 32, Synthesis of Trastuzumab-Sodium Hyaluronate-Auristatin E Antibody Drug Conjugate (Herceptin-HA-MMAE, Compound 32)

[0197] First, 108.1 μL of azide-functionalized trastuzumab solution (compound 22, Herceptin-PEG4-N3, concentration: 18.5 mg / mL) was taken out by pipette and added to the reaction vessel, and 635.6 μL of terminal alkynyl-functionalized sodium hyaluronate-auritin E solution (compound 19, Alkyn / HA-MMAE, concentration: 8.54 mg / mL) was added to the reaction vessel, followed by the addition of 333.4 μL of the pre-prepared click chemistry catalytic solution (6 mM copper sulfate, 30 mM BTTAA, 90 mM sodium ascorbate) for dilution, and 923.0 μL of phosphate buffer (50 mM, pH 7.4) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 1 mg / mL. After reacting for 24 hours, 50 μL of sodium ethylenediaminetetraacetic acid solution (500 mM) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified using an ultrafiltration tube (MWCO: 50 KDa) and exchanged into a phosphate buffer (50 mM, pH 7.4). The product was further purified by size exclusion chromatography using a superdex200 chromatography column (eluent: 50 mM phosphate buffer, 15% isopropanol, pH 7.4), and then purified and exchanged into a phosphate buffer (50 mM, pH 7.4) using an ultrafiltration tube (MWCO: 50 KDa) to obtain a sample stored in a phosphate buffer (50 mM, pH 7.4) (0.42 mg, yield: 21.0%), and the sample was stored at 2-8°C under nitrogen. Through SEC characterization, the average molar ratio of auristatin E drug to trastuzumab in the product was calculated to be 12.4 based on the absorption peak intensity at 248nm and 280nm and the extinction coefficients of the drug molecule and the antibody at the two wavelengths.

[0198]

[0199] Compound 32

[0200] Example 33, Synthesis of Trastuzumab-Sodium Hyaluronate-Auristatin E Antibody Drug Conjugate (Herceptin-HA-MMAE, Compound 33)

[0201] First, 108.1 μL of azide-functionalized trastuzumab solution (compound 22, Herceptin-PEG4-N3, concentration: 18.5 mg / mL) was taken out by pipette and added to the reaction vessel, and 781.3 μL of terminal alkynyl-functionalized sodium hyaluronate-auritin E solution (compound 20, Alkyn / HA-MMAE, concentration: 6.95 mg / mL) was added to the reaction vessel, followed by the addition of 333.4 μL of the pre-prepared click chemistry catalytic solution (6 mM copper sulfate, 30 mM BTTAA, 90 mM sodium ascorbate) for dilution, and 777.2 μL of phosphate buffer (50 mM, pH 7.4) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 1 mg / mL. After reacting for 24 hours, 50 μL of sodium ethylenediaminetetraacetate solution (500 mM) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified using an ultrafiltration tube (MWCO: 50 KDa) and exchanged into a phosphate buffer (50 mM, pH 7.4). The product was further purified by size exclusion chromatography using a superdex200 chromatography column (eluent: 50 mM phosphate buffer, 15% isopropanol, pH 7.4), and then purified and exchanged into a phosphate buffer (50 mM, pH 7.4) using an ultrafiltration tube (MWCO: 50 KDa) to obtain a sample stored in a phosphate buffer (50 mM, pH 7.4) (0.41 mg, yield: 20.5%), and the sample was stored at 2-8°C under nitrogen. Through SEC characterization, the average molar ratio of auristatin E drug to trastuzumab in the product was calculated to be 18.2 based on the absorption peak intensity at 248nm and 280nm and the extinction coefficients of the drug molecule and the antibody at the two wavelengths.

[0202]

[0203] Compound 33

[0204] Example 34, Synthesis of Trastuzumab-Sodium Hyaluronate-Auristatin E Antibody Drug Conjugate (Herceptin-HA-MMAE, Compound 34)

[0205] First, 183.5 μL of azide-functionalized trastuzumab solution (compound 24, Herceptin-PEG4-N3, concentration: 10.9 mg / mL) was taken out by pipette and added to the reaction vessel, and 648.2 μL of terminal alkynyl-functionalized sodium hyaluronate-auritin E solution (compound 12, Alkyn / HA-MMAE, concentration: 2.85 mg / mL) was added to the reaction vessel, followed by the addition of 333.4 μL of the pre-prepared click chemistry catalytic solution (6 mM copper sulfate, 30 mM BTTAA, 90 mM sodium ascorbate) for dilution, and 835 μL of phosphate buffer (50 mM, pH 7.4) was added. After the addition was completed, the reaction was continued for 24 hours and the temperature was controlled at 22°C. The concentration of trastuzumab during the reaction was 1 mg / mL. After reacting for 24 hours, 50 μL of sodium ethylenediaminetetraacetic acid solution (500 mM) was added to the reaction solution to terminate the reaction, and then the terminated reaction solution was purified using an ultrafiltration tube (MWCO: 50 KDa) and exchanged into a phosphate buffer (50 mM, pH 7.4). The product was further purified by size exclusion chromatography using a superdex200 chromatography column (eluent: 50 mM phosphate buffer, 15% isopropanol, pH 7.4), and then purified and exchanged into a phosphate buffer (50 mM, pH 7.4) using an ultrafiltration tube (MWCO: 50 KDa) to obtain a sample stored in a phosphate buffer (50 mM, pH 7.4) (0.60 mg, yield: 30.0%), and the sample was stored at 2-8°C under nitrogen. Through SEC characterization, the average molar ratio of auristatin E drug to trastuzumab in the product was calculated to be 8.2 based on the absorption peak intensity at 248nm and 280nm and the extinction coefficient of the drug molecule and the antibody at the two wavelengths. The SEC spectrum of compound 34 and trastuzumab at a wavelength of 280nm is shown in Figure 3 As shown in the figure, it can be seen that there is basically no naked antibody in the product, and the antibody basically participates in the reaction. In addition, the aggregate ratio of the product is very low, and the SEC purity is very high. In the existing patented technologies (patent numbers: CN10374804B, US8685383B2, CN109563215B, US20190083634), because there is an antibody that can connect to multiple polymer molecules, and at the same time a polymer molecule can connect to multiple antibody molecules, there may be aggregation and cross-linking of molecules, resulting in a higher level of aggregation of product molecules.

[0206]

[0207] Compound 34

[0208] Cell viability assay:

[0209] The activity of trastuzumab-sodium hyaluronate-auritin E antibody drug conjugate was evaluated using the Cell Titer-Glo luminescent cell viability assay from Promega. In this embodiment, cell viability was determined using the gastric cancer cell line NCI-N87 expressing HER2, the breast cancer cell line JIMT-1 expressing HER2, and the breast cancer cell line MDA-MB-231 expressing low levels of HER2. The cells were placed in an opaque-walled 96-well plate culture medium and allowed to adhere for 12 hours at 37°C, 5% CO2, and 95% humidity, with a cell density of 5000 cells / well per well for the three cell lines. After adhesion overnight, a series of different concentrations of test compounds or conjugates were added to the experimental wells and cultured for 72 hours at 37°C, 5% CO2, and 95% humidity. The plate was equilibrated at room temperature for 30 minutes, and a volume of Cell Titer-Glo reagent equal to the volume of cell culture medium present in each well was added. After vortexing and mixing, the plate was cultured at 20°C for 10 minutes. Luminescence data was recorded using Envision mμLtilabelreader, and GraphPad Prism 7.0 software was used to analyze the data and calculate the IC50 value and maximum inhibition rate.

[0210] The protein-polysaccharide-drug conjugates obtained in Examples 25-34 above, as well as auristatin E, trastuzumab, sodium hyaluronate and phosphate buffer were tested. The results are as follows: Figure 4-6 And the following table shows:

[0211]

[0212]

[0213]

[0214]

[0215] The results of three groups of cell experiments showed that 10 groups of trastuzumab-sodium hyaluronate-auritin E antibody drug conjugates (compounds 25 to 34) showed very good cell killing results for cell lines with high HER2 expression, medium HER2 expression or low HER2 expression, especially for cell lines with low HER2 expression, the killing effect was even better than that of cell lines with medium HER2 expression. The experimental results showed that the cell killing effect increased with the increase of the proportion of drug antibodies in the compound, and was much better than the cytotoxicity of the free load of auristatin E. Sodium hyaluronate had no killing effect in the three cell lines, showing that the product of the present invention has high safety.

[0216] The applicant declares that the present invention illustrates the protein-polysaccharide-drug conjugate and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0217] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0218] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A protein-polysaccharide-drug conjugate, It is characterized in that The structure of the protein-polysaccharide-drug conjugate is shown in Formula I: Formula I; Where n1 is an integer from 1 to 300, n2 is an integer from 1 to 60, and x is an integer or decimal from 1 to 10; Linker1 is selected from any of the following structures: , or ; Linker2 is selected from or , wherein m1 and m2 are selected from integers of 2-10; A drug selected from the class of microtubule inhibitors; Selected from monoclonal antibodies.

2. The protein-polysaccharide-drug conjugate according to claim 1, It is characterized in that The microtubule protein inhibitor drugs include auristatin E, maytansine alkaloids or paclitaxel.

3. The protein-polysaccharide-drug conjugate according to claim 1, It is characterized in that The monoclonal antibody includes trastuzumab, or a specific antibody or synthetic functionalized antibody for an antigen in the group consisting of EGFR, TROP2, CEACAM5, folate receptor-a, ROR1, ROR2, mesothelin, NaPi2b, 5T4, HER3, BCMA, B7H3, B7H4, Nectin-4, PTK7, PSMA, MUC1, CLDN18.2, CD56, CD30, CD33, CD79b, CD19, CD22, CD138, CD70, CD98, SLC44A4, LIV-1, fibrin extracellular domain B, endothelin receptor ETB or tenascin C.

4. The protein-polysaccharide-drug conjugate according to any one of claims 1 to 3, It is characterized in that In the protein-polysaccharide-drug conjugate, the average molar ratio of drug to monoclonal antibody is 6-25.

5. Use of the protein-polysaccharide-drug conjugate according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.

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