An antibody conjugate targeting pancreatic cancer and its preparation method and application

Abstract

The application discloses an antibody conjugate for targeted treatment of pancreatic cancer and a preparation method and application thereof, and relates to the technical field of biological medicines.The antibody conjugate comprises an anti-CD279 antibody, a cytotoxic drug and a linker; the cytotoxic drug is connected with the anti-CD279 antibody through the linker; and the cytotoxic drug is Mocetinostat.In the application, Mocetinostat is connected with a specific monoclonal antibody anti-PD-1 antibody through a linker, the antibody only recognizes and combines with a specific antigen which is highly expressed on the surface of tumor cells and is lowly expressed or not expressed on normal tissues.The antibody conjugate can achieve the purpose of targeting pancreatic cancer tumor cells, solve the problems of tumor chemotherapy resistance and immune escape, enhance the treatment effect of tumors, effectively reduce the toxic and side effects of small molecule drugs, and thus provide a new strategy of high efficiency and safety for the treatment of pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antibody-drug conjugate for targeted therapy of pancreatic cancer, its preparation method, and its application. Background Technology

[0002] Pancreatic cancer is a highly malignant gastrointestinal tumor with a very poor prognosis, and current treatments have limited effectiveness. The immunosuppressive nature of the tumor microenvironment and the resistance of tumor cells to chemotherapy are the main reasons for treatment failure. In recent years, immune checkpoint inhibitors (such as anti-PD-1 / PD-L1 monoclonal antibodies) have shown good efficacy in various tumors, but the response rate in pancreatic cancer is generally below 10%, partly due to the immunosuppressive microenvironment and immune escape caused by antigen presentation defects. To improve the efficacy of immunotherapy, researchers have begun to explore combination therapy strategies. Among them, the combination of histone deacetylase (HDAC) inhibitors and immune checkpoint inhibitors shows promising potential. HDAC inhibitors can alter chromatin structure, regulate gene expression, reverse T cell exhaustion, and enhance tumor immunogenicity. Studies have shown that in preclinical models, the combination of Mocetinostat and anti-PD-1 antibodies can significantly improve T cell infiltration and anti-tumor activity. Furthermore, this combination strategy has also been attempted to reverse resistance to chemotherapy drugs such as gemcitabine. However, this combination therapy has the following significant limitations, leading to the failure of clinical trials:

[0003] Mocetinostat (Moce) is a histone deacetylase inhibitor, primarily targeting class I HDACs (HDAC1, 2, 3) and class IV HDAC11. In a phase II clinical trial, Mocetinostat failed due to severe toxic side effects. Adverse reactions are common with HDAC inhibitors; excessive side effects can impact patients' quality of life, leading to medication discontinuation or dose reduction, directly affecting efficacy assessment and contributing significantly to clinical trial failures. Therefore, reducing the dosage of Mocetinostat is an urgent need in combination therapy strategies in this field.

[0004] These toxicities primarily stem from its non-selectivity of action. Although it exhibits relative selectivity for HDAC subtypes, it still cannot distinguish between cancer cells and normal healthy cells. As a pan-HDAC inhibitor, its systemic administration faces serious limitations. The toxin indiscriminately attacks rapidly dividing normal cells throughout the body, resulting in a very narrow therapeutic window—the dose required for effective anti-cancer treatment is very close to the dose that produces unacceptable toxicity. This makes it difficult to improve efficacy in monotherapy by simply increasing the dose, as the toxicity will reach an unmanageable level first, leading to severe dose-limiting toxicities (such as myelosuppression and neurotoxicity), which greatly limits its clinical administration window and therapeutic effect. Under systemic exposure conditions, it can cause serious adverse reactions such as thrombocytopenia, fatigue, and gastrointestinal toxicity, limiting its clinical application dosage and treatment duration. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody-drug conjugate for targeted therapy of pancreatic cancer, its preparation method, and its application, thereby addressing the problems existing in the prior art. This antibody-drug conjugate can target pancreatic cancer tumor cells, overcome the challenges of chemotherapy resistance and immune escape, enhance the therapeutic effect, and effectively reduce the toxic side effects of small molecule drugs, thus providing a highly efficient and safe new strategy for the treatment of pancreatic cancer.

[0006] This invention addresses the need to reduce the dosage of Mocetinostat in combination drug strategies, and considers the significant differences between antibody drugs and small molecule drugs in terms of in vivo distribution, half-life, and clearance mechanisms, making it difficult to achieve synergistic concentrations and synchronous effects at tumor sites. Furthermore, free Mocetinostat lacks tumor selectivity, and while inhibiting tumor HDAC activity, it can also affect the epigenetic state of normal cells, leading to off-target toxicity. This invention overcomes these limitations by introducing antibody-drug conjugate (ADC) technology.

[0007] ADC drugs consist of three parts: a specific targeting antibody, a highly effective effector molecule (cytotoxic drug), and a linker that connects the two and can cleave under specific conditions. This invention links Mocetinostat (as the payload / warhead) to a specific monoclonal antibody, an anti-PD-1 antibody, via a linker. This antibody recognizes and binds only to a specific antigen that is highly expressed on the surface of tumor cells but poorly expressed or not expressed in normal tissues, achieving precise drug delivery. This design offers several advantages:

[0008] (1) Targeted Delivery – Solving the Core Problem of “Non-Selectivity”: The toxicity of Mocetinostat stems from its systemic exposure. After oral administration, it spreads throughout the body via the bloodstream, indiscriminately acting on all normal tissues and cells expressing class I HDAC (such as hematopoietic stem cells and gastrointestinal mucosal cells), leading to widespread toxicity. In contrast, ADC drugs remain stable in the bloodstream with extremely low toxicity (because the "warhead" is "sealed"). Only when it reaches the tumor area, the antibody binds to the tumor cell antigen, and the entire ADC complex is internalized into the cancer cells, will Mocetinostat be efficiently released within the cells. This precisely "concentrates" the HDAC inhibitory activity within the tumor, greatly reducing systemic exposure to normal tissues.

[0009] (2) Increase local drug concentration, thereby reducing systemic dosage: To achieve effective drug concentrations within the tumor, high doses of oral Mocetinostat must be administered, which may lead to systemic toxicity. The "target-internalization" mechanism of ADCs allows a large amount of warhead drug to accumulate inside each cancer cell. Only one ADC molecule is needed to deliver multiple warhead molecules into a cancer cell. Therefore, to achieve the same intracellular killing effect, the total warhead (Mocetinostat) dose injected into the body is much lower than the dose required for single oral administration.

[0010] (3) Overcoming drug resistance and generating synergistic effects: Simultaneously achieving the dual mechanism of immune microenvironment remodeling and direct tumor killing, HDAC inhibitors such as Mocetinostat can alter chromatin structure, loosening the condensed DNA, which helps expose more drug targets and reverse certain drug resistance mechanisms. When used as the warhead of ADCs, its effect in the local tumor microenvironment can upregulate the expression of antigens targeted by the ADC antibody in cancer cells, or make cancer cells more sensitive to subsequent treatments, producing an "epigenetic priming" effect and forming a synergistic anti-cancer effect.

[0011] Based on this, the present invention provides the following solution:

[0012] This invention provides an antibody-drug conjugate for targeted treatment of pancreatic cancer, the antibody-drug conjugate comprising an anti-CD279 antibody, a cytotoxic drug, and a linker; the cytotoxic drug is linked to the anti-CD279 antibody via the linker;

[0013] The cytotoxic drug is Mocetinostat.

[0014] Furthermore, the linker contains a maleimide group; the cytotoxic drug is covalently linked to a thiol group formed after the reduction of the disulfide bond between the antibody chains via a linker containing a maleimide group.

[0015] Furthermore, the linker also contains a valine-citrulline dipeptide.

[0016] The present invention also provides a method for preparing the above-mentioned antibody conjugate, comprising the following steps:

[0017] The anti-CD279 antibody was mixed with a thiol reducing agent and then a linker-loaded molecule was added for a coupling reaction to obtain the antibody conjugate.

[0018] The linker-load molecule is obtained by linking the linker to the cytotoxic drug.

[0019] Furthermore, the thiol reducing agent is tris(2-carbonylethyl)phosphohydrochloride.

[0020] The present invention also provides a pharmaceutical composition for treating pancreatic cancer, comprising gemcitabine and the above-described antibody-drug conjugate.

[0021] The present invention also provides the use of the above-described antibody conjugates or pharmaceutical compositions in the preparation of medicaments for treating pancreatic cancer.

[0022] The present invention also provides a medicament for treating pancreatic cancer, wherein the active ingredient includes the antibody-drug conjugate described above.

[0023] Furthermore, the active ingredient of the drug also includes gemcitabine.

[0024] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0025] The present invention discloses the following technical effects:

[0026] The monoclonal antibody anti-PD1 / Mocetinostat antibody conjugate of the present invention has the following significant advantages and technical effects:

[0027] (1) Highly effective targeting of tumors and reversal of the immunosuppressive microenvironment: With the high affinity and specificity of anti-PD1 antibodies, ADC drugs can be precisely enriched in pancreatic cancer tissues and tumor-infiltrating lymphocytes with high PD-1 expression, effectively blocking the PD-1 / PD-L1 signaling pathway and relieving immunosuppression; at the same time, the HDAC inhibitor Mocetinostat is delivered to the lesion to synergistically promote the recovery of T cell function and induce tumor cell apoptosis.

[0028] (2) Significantly overcomes chemotherapy resistance and immune escape: Mocetinostat, as a histone deacetylase inhibitor, can reverse epigenetic-mediated silencing of chemotherapy resistance genes and restore tumor sensitivity to traditional chemotherapy drugs.

[0029] (3) The toxic side effects are significantly lower than those of traditional chemotherapy and single-drug combination regimens: Through the targeted delivery mechanism of ADC, Mocetinostat is mainly released in tumor cells, greatly reducing systemic exposure and thus significantly reducing drug toxic side effects.

[0030] (4) It has good drug-like properties and clinical translation prospects: The anti-PD1 antibody selected in this invention is humanized IgG1, which has low immunogenicity and long half-life; the linker LP1 has high stability in circulation and high cleavage efficiency under the action of glucuronidase expressed in tumor cells, thus realizing efficient and safe intracellular drug release.

[0031] (5) Significant economic benefits and social value: This invention solves the three major problems of targeting, drug resistance and immune escape through a one-drug-multi-target mechanism. It can potentially replace the existing multi-drug free combination regimen, reduce the treatment burden on patients, and significantly improve compliance and quality of life. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The chemical structural formula of the HDAC inhibitor Mocetinostat;

[0034] Figure 2 A schematic diagram of the synthetic route for compound WBPX1635-LP001;

[0035] Figure 3 This is a mass spectrometry analysis chromatogram of the antibody conjugate prepared in Example 1;

[0036] Figure 4 This is a schematic diagram showing the detection results of the free drug level of antibody-drug conjugates.

[0037] Figure 5 A schematic diagram showing the tumor size of mice in each group;

[0038] Figure 6 A statistical graph showing the tumor weight of mice in each group;

[0039] Figure 7 The results of serum liver and kidney function indicators (ALT, TBIL, UREA, and CREA) in mice of each group are shown in the figure.

[0040] Figure 8H&E staining images of liver and kidney tissue sections from mice in each group. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] This invention develops a novel antibody-drug conjugate (ADC) for targeted therapy of pancreatic cancer, consisting of three parts: a monoclonal antibody anti-PD1, a cytotoxic payload, and a linker. The antibody is conjugated to the cytotoxic payload via the linker, together conjugating the antibody-drug conjugate with cell targeting and cell-killing efficacy. The specific structure of this antibody-drug conjugate is as follows:

[0047] Antibody: Monoclonal antibody anti-PD1 (anti-CD279 antibody).

[0048] Cytotoxic load: Mocetinostat (chemical structure shown) Figure 1 (), which, after chemical modification, contains groups that can react with bridging agents.

[0049] Linker: A bifunctional bridging agent with highly reactive groups at both ends (such as sulfonates and haloacetamides) and a cleavable (such as enzyme-sensitive dipeptides) or non-cleavable spacer arm at the center.

[0050] Connection method: The linker simultaneously reconnects two thiol groups generated after the reduction of a disulfide bond via covalent bonds (stable CS bonds), and covalently loads two Mocetinostat molecules onto its bridging structure. All four interchain disulfide bonds of the entire antibody molecule are rebridged by this "drug-linker-drug" structure.

[0051] The monoclonal antibody anti-PD1 in this antibody-drug conjugate targets tumor cells. It specifically recognizes and binds to PD-1 molecules highly expressed on the surface of tumor cells or on immune cells in the tumor microenvironment, blocking the PD-1 / PD-L1 immunosuppressive pathway and mediating the endocytosis of the ADC for precise delivery. The small molecule drug Mocetinostat is a histone deacetylase (HDAC) inhibitor. It mainly promotes tumor cell apoptosis, differentiation, and cell cycle arrest by inhibiting HDAC activity, causing chromatin relaxation and transcriptional activation. It can also reverse the tumor immunosuppressive microenvironment and enhance the anti-tumor activity of T cells. In a specific embodiment of the invention, LP1 is used as a linker. It is a cleavable linker designed to utilize the highly expressed glucuronidase in tumor cells for specific hydrolysis. After entering the cell, LP1 is cleaved under the action of this enzyme, thereby precisely releasing the active loading molecule Mocetinostat, achieving a highly efficient intracellular killing effect. Because this linker remains relatively stable in normal blood circulation, it can effectively avoid premature drug release, thus significantly improving the safety and therapeutic index of the ADC drug. After entering the body, this ADC drug first relies on its antibody moiety (anti-PD1) to specifically recognize PD-1 molecules, targeting and accumulating on the surface of tumor cells or tumor-infiltrating lymphocytes. Through antigen-mediated endocytosis, the ADC is efficiently taken up into the cells. Subsequently, under the action of glucuronidase, which is highly expressed in tumor cells, Linker-LP1 is specifically cleaved, releasing the loaded small molecule inhibitor Mocetinostat. Mocetinostat inhibits HDAC activity, induces chromatin remodeling and gene expression regulation, ultimately leading to tumor cell apoptosis and reversing the immune tolerance microenvironment.

[0052] Example 1

[0053] A method for preparing an antibody-drug conjugate for targeted therapy of pancreatic cancer:

[0054] 1. Add 27.3 mL of binding buffer (PBS, pH 7.4) and 1.28 mL of 5 mM tris(2-carbonylethyl)phosphohydrochloride (TCEP) solution to a 125 mL bottle containing 200 mg of monoclonal antibody anti-PD1 (anti-CD279 antibody). After thoroughly mixing the reaction mixture (antibody concentration in the reaction system is 7.00 mg / mL), incubate at 22°C for 18 hours to obtain the first reaction solution.

[0055] The anti-CD279 antibody was purchased from BioXCell, product code BE0146.

[0056] 2. The activated linker MC-Val-Cit-PABC-PNP was coupled with the functionalized Mocetinostat derivative under organic base catalysis to obtain WBPX1635-LP001.

[0057] Compound WBPX1635-LP001 was synthesized via a five-step reaction, as follows: Figure 2 As shown. A white solid product was finally obtained, and its structure and purity were confirmed by LC-MS, ¹H NMR, HPLC, and SFC. The specific synthesis and characterization of compound WBPX1635-LP001 are as follows:

[0058] (1) Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (compound 3)

[0059] EEDQ (2.17 g, 8.78 mmol) was added to a 40 mL solution of dichloromethane containing (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (compound 1, 2.00 g, 4.39 mmol) and (((9H-fluorene-9-yl)methoxycarbonyl)glycine) (compound 2, 1.57 g, 5.27 mmol). The reaction mixture was stirred at 20 °C for 12 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography (hexane / ethyl acetate gradient elution) to give target compound 3 as 2.10 g of white solid, yield: 62.5%; LCMS: EB17835-87-P1A, m / z = 735.4 (M+H). +, Rt = 1.884 min, LCMS: EB17835-87-P1L, m / z =735.2 (M+H) + Rt = 0.718 min.

[0060] (2) Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (compound 4)

[0061] Compound 3 (2.1 g, 2.74 mmol) and bis(4-nitrophenyl) carbonate (3.34 g, 10.9 mmol) were dissolved in tetrahydrofuran (THF, 40 mL), and DIEA (3.55 g, 27.4 mmol, 4.78 mL) was added. The mixture was stirred at 20 °C for 12 h. The reaction was complete as detected by LC-MS (EB17835-89-P1A). After removing the solvent under reduced pressure, the residue was purified by silica gel column chromatography (SiO2, eluent: hexane / ethyl acetate = 100 / 0 - 1 / 1) to give target compound 4 as a white solid, 2.40 g (yield 89.4%). LC-MS characterization: EB17835-89-P1A: m / z = 900.5 ([M+H) + ), Rt = 2.149 min, EB17835-89-P1L: m / z = 900.3 ([M+H] + Rt = 0.820 min.

[0062] (3) Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-4-((((2(4-(((4-(pyridin-3-yl)pyrimidin-2-yl)amino)methyl)benzamido)phenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (compound 6)

[0063] Compound 4 (617 mg, 631 μmol) and N-(2-aminophenyl)-4-(((4-(pyridin-3-yl)pyrimidin-2-yl)amino)methyl)benzamide (compound 5, 250 mg, 631 μmol) were dissolved in DMF (10 mL), and HOAt (85.8 mg, 631 μmol) and DIEA (245 mg, 1.89 mmol, 330 μL) were added. The mixture was stirred at 20 °C for 12 hours. LC-MS (EB17835-99-P1B) analysis showed that approximately 16.8% of reactant 1 (i.e., compound 4) remained unreacted, and the main peak of the target product was detected. After filtration of the reaction solution, the solvent was removed under reduced pressure to obtain the residue, which was then purified by prep-HPLC (F-Welch Xtimate C18 40×200 mm, 7 µm; mobile phase: H2O (0.1% TFA)-ACN; gradient: 28%-68% B, 19 min) to give target compound 6 as 400 mg of white solid (yield 53.9%). LC-MS characterization: EB17835-99-P1B: m / z = 1157.8 ([M+H) + ), Rt = 1.871 min, EB17835-105-P1L: m / z = 1158.7 ([M+H] + ), Rt = 2.580 min.

[0064] (4) A general method for preparing (2S,3S,4S,5R,6S)-6-(2-(2-aminoacetamido)-4-((((2-(4-(((4-(pyridin-3-yl)pyrimidin-2-yl)amino)methyl)benzoylamino)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (compound 7).

[0065] Compound 6 (400 mg, 340 μmol) was dissolved in a mixed solvent of methanol (8 mL) / tetrahydrofuran (8 mL) / water (5 mL), and LiOH·H₂O (85.6 mg, 2.04 mmol) was added. The mixture was stirred at 0 °C for 3 hours. After the reaction was complete, 10% acetic acid dissolved in THF was added at 0 °C to adjust the pH to approximately 5. The solvent was removed under reduced pressure to obtain the residue, which was purified by prep-HPLC (F-Welch Xtimate C18 40×200 mm, 7 µm; mobile phase: H₂O (0.1% TFA) – ACN; gradient: 0%-38% B, 19 min) to give target compound 7 as a white solid product of 160 mg (yield 59.2%). LC-MS characterization: EB17835-109-P1A: m / z = 795.0 ([M+H) + Rt = 2.945 min.

[0066] (5) A general method for preparing (2S,3S,4S,5R,6S)-6-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propylamino)acetamido)-4-((((2-(4-(((4-(pyridin-3-yl)pyrimidin-2-yl)amino)methyl)benzamido)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (compound WBPX1635-LP001).

[0067] Compound 7 (150 mg, 189 μmol) was dissolved in DMF (7 mL) with 2,5-dioxopyrrolidone-1-yl 3-(2,5-dioxo-1H-pyrrolo-1-yl)propionate (60.3 mg, 226 μmol). DIEA (73.2 mg, 566 μmol, 98.6 μL) was added as a base, and the mixture was stirred at 20 °C for 2 hours. After the reaction was complete, the mixture was separated into water (10 mL) and ethyl acetate (10 mL), the organic phase was washed with brine (20 mL), dried (Na₂SO₄), filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (F-Welch Xtimate C18 40×200 mm, 7 µm; mobile phase: H2O (0.1% TFA) – ACN; gradient 2%–42% B, 19 min) to give the target compound WBPX1635-LP001 as 119 mg of white solid (yield 65.8%, purity 98.7%). ¹H NMR (DMSO-d6, 400 MHz): δ 9.74–2.40 ppm (multiple peaks, consistent with structural characteristics). LC-MS: EB17835-116-P1A: m / z = 946.0 ([M+H]). + ), Rt = 3.322 min, EB17835-115-P1L: m / z = 946.5 ([M+H] + Rt = 1.502 min, HPLC / SFC: meets purification requirements.

[0068] 3. Add 3.18 mL of dimethylacetamide (DMA) solution containing 10 mM WBPX1635-LP001 to the first reaction solution, mix thoroughly, and incubate at 22°C for 1 hour to carry out the coupling reaction (antibody concentration is 6.30 mg / mL during the coupling reaction) to obtain the second reaction solution. Centrifuge the second reaction solution to obtain the crude product.

[0069] 3. The crude product was subjected to ultrafiltration dialysis (using a Pellicon 3 cartridge ultrafiltration membrane, 30 kDa, 0.11 m). 2 The sample was purified by purification, and the preparation buffer was 20 mM histidine buffer (containing 8.0% w / v sucrose, pH 6.0).

[0070] 4. The purified sample was filtered through a 0.22 µm filter membrane to obtain the antibody conjugate.

[0071] The mass spectrometry analysis chromatogram of the antibody conjugate prepared in this embodiment is shown below. Figure 3The results show that the main peak molecular weight corresponds to a DAR of 7.96, and the distribution is concentrated. The free drug level detection results for this antibody-drug conjugate are shown below. Figure 4 The result shows Freedrug level < 0.09.

[0072] Example 1 of effect verification

[0073] 1. Experimental Design

[0074] In a mouse orthotopic pancreatic xenograft model, different treatments were administered using the chemotherapeutic drug gemcitabine (CAS No.: 95058-81-4), the small molecule drug Mocetinostat, the monoclonal antibody anti-PD1 (anti-CD279 antibody), and the antibody-drug conjugate (ADC) prepared in Example 1. The study evaluated whether the combination of the ADC and chemotherapy resulted in better tumor suppression compared to gemcitabine alone or in combination with the small molecule drug Mocetinostat, the monoclonal antibody, and gemcitabine. Side effects were also assessed. Grouping was as follows:

[0075] The control group (Ctrl) was given an equal volume of solvent;

[0076] Gemcitabine (Gem) 50 mg / kg;

[0077] Gemcitabine 50 mg / kg + anti-PD1 15 mg / kg + Mocetinostat 30 mg / kg;

[0078] Gemcitabine 50 mg / kg + ADC 15 mg / kg (anti-PD1=15 mg / kg, Mocetinostat≈0.75 mg / kg);

[0079] Gemcitabine 50 mg / kg + ADC 30 mg / kg (anti-PD1 = 30 mg / kg, Mocetinostat ≈ 1.5 mg / kg);

[0080] Each group had n=6, and the administration method was intraperitoneal injection.

[0081] The dosage calculation method for the antibodies and small molecule drugs included in the ADC is as follows:

[0082] ADC drug concentration is determined using the BCA method, where the standard for BCA detection is an antibody; therefore, the ADC dose is equivalent to the anti-PD1 dose. Mocetinostat concentration can be calculated using the DAR value, as shown in the following formula:

[0083] MW mAb =147154 Da, MW LP =945.90;

[0084] C LP (mg / mL) = MW LP ×[C mAb (mg / mL) / MW mAb ]×DAR.

[0085] 2. Experimental Methods

[0086] To establish an orthotopic syngeneic tumor model, mouse pancreatic tumor cells (KPCs) required for model construction were first collected. Sufficient pre-cultured cells were digested, resuspended, and counted. KPC cells were injected at a rate of 5 × 10⁶ cells per mouse. 5 Prepare a cell suspension of 30 μL in volume. Use twice the number of mice to be injected, adding 30%-50% matrix gel to the suspension and placing it on ice. Then, perform animal surgery. Anesthetize the mice with isoflurane. After complete anesthesia, use autoclaved surgical instruments to incise the abdominal skin and peritoneum. Use circular forceps to remove the spleen, which is connected to the pancreas. Stretch the pancreas and inject 30 μL of cell solution using a Hamilton's needle. After injection, return the pancreas to the mouse's abdominal cavity. Suture the abdominal incision with absorbable sutures and disinfect the area. The entire procedure is performed in a laminar flow hood, and all mice are housed in an SPF-grade animal facility.

[0087] After KPC cell injection, the drugs were administered according to the experimental groups.

[0088] Mice were euthanized after blood was collected from their eyeballs 30 days later. Tumors were collected, photographed, and their weight was recorded. Hearts, livers, spleens, lungs, and kidneys were collected, sectioned, and stained with hematoxylin and eosin (HE) to assess drug safety. Whole blood samples were incubated at room temperature for 2 hours, then centrifuged at 2-8°C and 3000 rpm for 15 min. The supernatant was immediately used to detect alanine aminotransferase (ALT), total bilirubin (TBIL), urea (UREA), and serum creatinine (CREA) to reflect liver and kidney function.

[0089] 3. Experimental Results

[0090] By comparing the size and weight of mouse tumors in each group ( Figure 5 and Figure 6It can be seen that, compared to the single-drug group ( Group ) and the three-drug free combination group ( (Group), ADC combined with gemcitabine Groups and The tumor tissue in the group showed smaller volume and weight, indicating that the ADC drug exhibited a significant tumor-suppressive effect, with the high-dose group showing a more pronounced effect. Compared to In the group where the small molecule toxic component Mocetinostat was used at a dose of 30 mg / kg, the dose of Mocetinostat carried by the ADC drug was significantly reduced (approximately 20-fold and 40-fold) in the two groups where the ADC was combined with gemcitabine. This indicates that the conjugation of anti-PD1 and Mocetinostat can significantly reduce the amount of Mocetinostat used while achieving better tumor suppression effects.

[0091] like Figure 7 As shown, there were no differences among the groups in serum liver and kidney function indicators (ALT, TBIL, UREA, CREA), and all levels were within the normal reference range. H&E staining results of liver and kidney tissue sections from each group ( Figure 8 The results showed that no significant inflammatory response occurred in the liver and kidneys in any of the groups. This indicates that all the medication regimens are safe in terms of liver and kidney function.

[0092] Based on the above experimental results, it can be concluded that the combination of anti-PD1 and Mocetinostat in this invention can significantly inhibit tumor progression in mouse models with extremely low doses of small molecule inhibitors, and has good biosafety.

[0093] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An antibody-drug conjugate for targeted therapy of pancreatic cancer, characterized in that, The antibody conjugate was obtained by conjugating an anti-CD279 antibody with a linker-loador molecule. The chemical structural formula of the linker-loaded molecule is as follows: 。 2. A method for preparing the antibody conjugate as described in claim 1, characterized in that, Includes the following steps: The anti-CD279 antibody is mixed with a thiol reducing agent and then the linker-loaded molecule is added for a coupling reaction to obtain the antibody conjugate.

3. The preparation method according to claim 2, characterized in that, The thiol reducing agent is tris(2-carbonylethyl)phosphohydrochloride.

4. A pharmaceutical composition for treating pancreatic cancer, characterized in that, Including gemcitabine and the antibody-drug conjugate of claim 1.

5. The use of an antibody conjugate as described in claim 1 or a pharmaceutical composition as described in claim 4 in the preparation of a medicament for treating pancreatic cancer.

6. A drug for treating pancreatic cancer, characterized in that, The active ingredient includes the antibody conjugate as described in claim 1; The drug also includes pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that, The active ingredient in the drug also includes gemcitabine.

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