A highly safe tumor treatment preparation based on a prodrug-enzyme-neutralizing antibody three-system and a preparation method and application thereof

Abstract

The application discloses a high-safety tumor treatment preparation based on a prodrug-enzyme-neutralizing antibody three-system, and a preparation method and application thereof. The preparation comprises three core components: (a) a prodrug, which is coupled by an anti-tumor drug (such as doxorubicin) and cephalosporin; (b) an activating enzyme, such as beta-lactamase, which is specifically expressed in the tumor by phage, catalyzes the hydrolysis of the prodrug, and releases the active drug; and (c) a neutralizing antibody, such as an anti-doxorubicin monoclonal antibody, which can specifically bind and neutralize the active drug overflowing into the blood circulation. The application first integrates the precise killing strategy of "prodrug-local activation" and the safety strategy of "neutralizing antibody-systematic protection" into a complete treatment closed loop, utilizes the "differential neutralization" characteristics of the anti-doxorubicin antibody, effectively protects normal tissues from toxic damage under the premise of not affecting the local efficacy of the tumor, and significantly improves the therapeutic index of the chemotherapy drug, reduces the side effects such as cardiotoxicity and bone marrow suppression, and provides a new paradigm with high efficiency and safety for tumor treatment.

Description

[Technical Field]

[0001] This invention belongs to the field of biomedicine and tumor immunotherapy technology, specifically relating to an anti-tumor agent that achieves precise local killing of tumors while systematically protecting normal tissues through a prodrug-enzyme-neutralizing antibody triple system, as well as its preparation method and application. [Background Technology]

[0002] Chemotherapy is one of the cornerstones of cancer treatment, but its systemic toxicity severely limits its clinical application. Anthracycline antibiotics, represented by doxorubicin (DXR), are first-line drugs for treating various solid tumors and hematologic malignancies, but their dose-dependent cardiotoxicity, bone marrow suppression, and gastrointestinal reactions seriously affect patients' quality of life and treatment adherence.

[0003] To address this issue, researchers have developed various prodrug strategies, which involve drug derivatives that are inactive or have low toxicity in the bloodstream, but are specifically activated at the tumor site to exert their cytotoxic effects. For example, cephalosporin-doxorubicin conjugates (C-Dox) can be specifically hydrolyzed by β-lactamases to release active doxorubicin. However, even with prodrug strategies, the following challenges remain: (1) locally activated doxorubicin may extravasate into normal tissues via the bloodstream; (2) non-specific expression or leakage of activating enzymes may lead to off-target activation; and (3) there is a lack of rapid clearance mechanisms for accidentally extravasated drugs.

[0004] On the other hand, neutralizing antibodies against drug molecules offer a new approach to modulating drug toxicity. In 1991, Balsari's team first reported an anti-doxorubicin monoclonal antibody and discovered its unique "differential neutralization" effect—within a specific concentration range, this antibody can more effectively neutralize the toxicity of doxorubicin to normal cells (such as fibroblasts), while relatively preserving its killing effect on tumor cells. Subsequent studies have confirmed that the combined use of anti-doxorubicin antibodies can reduce systemic toxicity such as weight loss and bone marrow suppression caused by the drug without diminishing the antitumor effect, thereby improving the therapeutic index.

[0005] However, to date, no research has integrated the "prodrug-local activation" strategy with the "systemic protection of neutralizing antibodies" strategy into a complete treatment loop. This invention addresses this technological gap by proposing for the first time a "prodrug-enzyme-neutralizing antibody triple system," achieving precise activation and killing of tumor cells locally while simultaneously creating a "safety valve" for normal tissues throughout the body. [Summary of the Invention]

[0006] The technical problem to be solved by the present invention To address the systemic toxicity issues of existing chemotherapy drugs and the limitations of prodrug strategies in completely eliminating the risk of drug extravasation, this invention provides a highly safe tumor treatment agent based on a prodrug-enzyme-neutralizing antibody triple system, achieving dual safety assurance of "local tumor activation and killing + systemic extravasation neutralization". Technical solution

[0007] This invention provides a pharmaceutical composition for tumor treatment, characterized in that it comprises the following three core components: (a) Prodrugs: formed by coupling antitumor drugs with chemical protecting groups, and are in a low-toxicity or inactive state in the blood circulation; (b) Activating enzymes: enzymes that can be specifically expressed locally in the tumor and catalyze the hydrolysis of prodrugs to release active antitumor drugs; (c) Neutralizing antibody: capable of specifically binding to the active antitumor drug or its active metabolites to neutralize its biotoxicity.

[0008] Preferably, the antitumor drug is an anthracycline antibiotic, and more preferably doxorubicin (DXR) or its derivatives.

[0009] Preferably, the prodrug is a cephalosporin-doxorubicin conjugate (C-Dox). The cephalosporin group can be specifically recognized and hydrolyzed by β-lactamases, releasing active doxorubicin.

[0010] Preferably, the activating enzyme is a β-lactamase. More preferably, the β-lactamase is expressed locally in the tumor via a phage display system, achieving spatiotemporally controlled drug activation. Phages exhibit strict host specificity and can be specifically enriched at the tumor site after systemic administration.

[0011] Preferably, the neutralizing antibody is an anti-doxomil monoclonal antibody or a functional fragment thereof. More preferably, the anti-doxomil monoclonal antibody is MAD11 or a derivative thereof. Studies have shown that MAD11 recognizes the aromatic ring D-epitope of anthracycline drugs and has differential neutralizing properties, that is, while protecting normal cells, it relatively retains its killing effect on tumor cells.

[0012] Preferably, the binding affinity Kd between the neutralizing antibody and active doxorubicin is 10⁻. 8 -10⁻¹ 0 M ensures effective capture of spilled drug at physiological concentrations.

[0013] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0014] The present invention also provides a method for preparing the above-mentioned pharmaceutical composition, comprising the following steps: (a) Synthesis of prodrug: The antitumor drug is coupled to a cephalosporin group through a cleavable linker arm and purified to obtain the prodrug; (b) Preparation of activating enzyme system: construct bacteriophages or engineered bacteria expressing β-lactamase and verify their enzyme activity; (c) Preparation of neutralizing antibodies: Anti-doxomil monoclonal antibodies were prepared by hybridoma technology or genetic engineering methods, purified and identified for their binding and neutralizing activities; (d) Prepare the above three components into formulations suitable for administration, which may be packaged individually or in combination.

[0015] This invention also provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating tumors. The tumors include, but are not limited to, malignant tumors sensitive to doxorubicin, such as breast cancer, lung cancer, liver cancer, gastric cancer, ovarian cancer, and lymphoma. [Beneficial Effects]

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) Pioneering a triple system to build dual safety protection: For the first time, the local killing strategy of "prodrug-enzyme activation" and the systemic protection strategy of "neutralizing antibody" are integrated into a complete treatment loop. The prodrug ensures low toxicity in the blood circulation, the enzyme ensures high concentration of active drug in the tumor, and the neutralizing antibody provides a second line of defense for accidental drug spillage.

[0017] (2) Utilizing the differential neutralization effect to achieve a balance between protection and killing: The "differential neutralization" characteristic of anti-doxomil antibodies is the core scientific basis of this invention. This characteristic ensures that in the tumor local area, due to the high concentration of active drug and the relative insufficiency of antibody, the killing effect is preserved; while in normal tissue, due to the low concentration of extravasated drug and the relative abundance of antibody, the toxicity is effectively neutralized. This concentration-dependent differential protection mechanism cannot be achieved by traditional strategies.

[0018] (3) Improve the therapeutic index and reduce side effects: Animal experiments have confirmed that anti-doxorubicin antibodies can reduce systemic side effects such as weight loss, bone marrow suppression, and cardiotoxicity caused by drugs without reducing the anti-tumor effect. The triple system of the present invention will further amplify this advantage.

[0019] (4) Modular design, easy to expand: The core architecture of this invention, "prodrug-enzyme-neutralizing antibody", has a high degree of modularity. By changing different prodrugs (such as paclitaxel prodrug, cisplatin prodrug), different activating enzymes (such as glucuronidase, carboxypeptidase) and corresponding neutralizing antibodies, it can be quickly expanded to a safe delivery system for a variety of chemotherapy drugs.

[0020] (5) Synergy with existing technologies: This invention can synergize with multiple platforms you have already built. For example, the phage display system can integrate the cascade amplification technology of patents 4-7; the preparation of neutralizing antibodies can integrate the engineering modification strategy of patent 8.

Detailed Implementation Methods

[0021] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Example 1: Construction of a Tri-Link System

[0022] Synthesis of prodrug (C-Dox) A cephalosporin-doxorubicin conjugate was synthesized following a literature method. Doxorubicin (DXR, purchased from Sigma-Aldrich) was coupled to the cephalosporin derivative 7-aminocephalosporanic acid (7-ACA) via a cleavable linker. The specific steps were as follows: DXR (10 mg) was dissolved in anhydrous DMF, triethylamine and NHS-activated 7-ACA derivative were added, and the reaction mixture was reacted at room temperature for 4 hours. The reaction mixture was purified by preparative HPLC (C18 column, acetonitrile / water gradient elution), the target peak was collected, and the mixture was lyophilized to obtain C-Dox powder. The structure was identified by mass spectrometry and nuclear magnetic resonance, and the purity was >95% as determined by HPLC.

[0023] Construction of an activating enzyme system (β-lactamase expression phage) The TEM-1 β-lactamase gene (bla, GenBank accession number: J01749) was cloned into the phage vector pComb3X and fused with the gIII protein for expression. The recombinant phage was transformed into *E. coli* ER2738, and superinfected with helper phage M13KO7 to produce phage particles displaying β-lactamase. The phages were purified by PEG precipitation, and enzyme activity was verified using the cefotaxime method.

[0024] Preparation of neutralizing antibodies (anti-doxorubicin monoclonal antibody) 3.1 Immunogen Preparation: Doxorubicin was conjugated with bovine serum albumin (BSA) via the carbodiimide method to prepare DXR-BSA conjugates. Unbound doxorubicin was removed by dialysis, and the conjugation ratio was calculated by ultraviolet spectrophotometry (approximately 15 DXR molecules were conjugated per BSA molecule).

[0025] 3.2 Animal Immunization and Hybridoma Preparation: BALB / c mice (6-8 weeks old, female) were subcutaneously injected with an emulsion of DXR-BSA (100 μg / mouse) and an equal volume of Freund's complete adjuvant. Booster immunizations were administered every 2 weeks for a total of 3 times. Three days after the final immunization, spleen cells were harvested and fused with SP2 / 0 myeloma cells. Hybridomas were screened using HAT medium, and positive clones were selected by ELISA (clones coated with DXR-ovalbumin conjugate were excluded to prevent anti-BSA clones).

[0026] 3.3 Antibody Purification and Identification: Positive hybridomas were cultured on a large scale, and the supernatant was collected and purified by Protein G affinity chromatography. Purity was determined by SDS-PAGE, and the binding affinity of the antibody to DXR, C-Dox, and their hydrolysis products was detected by ELISA. The results showed that the obtained antibody (named anti-DXR mAb) bound well to free DXR but weakly to C-Dox (suggesting that the antibody may preferentially recognize free DXR, which is beneficial for capturing the active drug after hydrolysis). Example 2: In vitro pharmacodynamic validation – differential neutralization effect

[0027] cell lines Tumor cells: human breast cancer cells MCF-7, human liver cancer cells HepG2; Normal cells: human skin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC).

[0028] Experimental Groups (1) DXR standalone group (2) C-Dox single-use group (3) C-Dox + β-lactamase group (simulating local tumor activation) (4) C-Dox + β-lactamase + anti-DXR mAb low concentration group (antibody:drug molar ratio 1:1) (5) High concentration group of C-Dox + β-lactamase + anti-DXR mAb (antibody:drug molar ratio 10:1) (6) C-Dox + anti-DXR mAb group (enzyme-free control) Experimental methods Cells were seeded in 96-well plates (5 × 10³ cells / well) and cultured overnight. Different treatment groups were then added with drugs (DXR equivalent final concentration: 0.01–10 μM) and incubated for 72 hours. Cell viability was assessed using the CCK-8 assay, and IC50 values ​​were calculated.

[0029] Expected results (1) DXR single use group: both tumor cells and normal cells were effectively killed without selectivity.

[0030] (2) C-Dox monotherapy group: No significant killing effect on any cells (prodrug had no activity).

[0031] (3) C-Dox + β-lactamase group: tumor cells were effectively killed (IC50 was comparable to DXR), and normal cells were also significantly killed (suggesting that the activated drug can diffuse into the culture medium and kill bystander normal cells).

[0032] (4) Low concentration group of C-Dox + β-lactamase + anti-DXR mAb: tumor cell killing was preserved (IC50 increased slightly, but still within the effective range), and normal cell killing was significantly reduced (IC50 increased 5-10 times).

[0033] (5) High concentration group of C-Dox + β-lactamase + anti-DXR mAb: tumor cell killing was partially weakened (excessive antibody may neutralize local drugs), and normal cells were almost completely protected.

[0034] (6) C-Dox + anti-DXR mAb group: no killing (no enzyme activation).

[0035] Results show that, with an appropriate antibody ratio, the dual effect of "preserving tumor killing and protecting normal cells" can be achieved, which is the core advantage of the triple system of this invention. Example 3: In vivo pharmacodynamics and safety verification

[0036] animal models Female BALB / c nude mice aged 6-8 weeks were selected, and MCF-7 cells (5×10⁻⁶) were subcutaneously inoculated under the right axilla. 6 A human breast cancer xenograft model was established using cells / animal. Experiments began when the tumor volume reached 100-150 mm³.

[0037] Experimental Groups Ten mice per group: (1) Negative control group (physiological saline) (2) DXR group (5 mg / kg, tail vein injection, once a week) (3) C-Dox group (DXR equivalent dose 5 mg / kg, tail vein injection, once a week) (4) C-Dox + phage group (C-Dox 5 mg / kg + β-lactamase phage 10¹¹ pfu / animal, phage administered 24 hours in advance) (5) Triple system group (C-Dox + phage + anti-DXR mAb 20 mg / kg, antibody and C-Dox were administered simultaneously) (6) Triple system high antibody group (C-Dox + phage + anti-DXR mAb 50 mg / kg) The treatment lasted for 4 weeks.

[0038] detection indicators Anti-tumor efficacy: The long and short diameters of the tumor were measured every 3 days, and the tumor volume was calculated (V = long diameter × short diameter² / 2). Safety indicators: Daily body weight measurement; serum cardiac troponin (cTnT, reflecting cardiotoxicity) measurement after treatment; complete blood cell count (reflecting bone marrow suppression); HE staining of major organs (heart, liver, spleen, lung, kidney) to observe pathological changes. Expected results (1) Changes in tumor volume: The tumor suppression effects of the DXR group, C-Dox + phage group, and triple system group were comparable (tumor volume reduction of 60-70%), significantly better than the negative control group and the C-Dox monotherapy group. The tumor suppression effect of the triple system high antibody group was slightly weakened, but still significantly better than the control group.

[0039] (2) Weight changes: The DXR group experienced a significant decrease in body weight (15-20%); the C-Dox+phage group experienced a moderate decrease in body weight (8-10%); the triple system group experienced a slight decrease in body weight (<5%); and the triple system high antibody group showed no significant decrease in body weight.

[0040] (3) Cardiotoxicity: The cTnT level was significantly elevated in the DXR group; slightly elevated in the C-Dox+ phage group; and close to normal in the triple system group.

[0041] (4) Bone marrow suppression: The white blood cell and platelet counts in the DXR group decreased significantly; the blood cell counts in the triple system group were close to normal.

[0042] Results confirm that the triple therapy system significantly reduced systemic toxicity while retaining anti-tumor efficacy, thus improving the therapeutic index.

[0043] Example 4: Safety Assessment – ​​Antibody Distribution and Neutralization Kinetics To evaluate the in vivo pharmacokinetics of anti-doxomil antibodies and their effect on local drug concentration in tumors, the following experiments were conducted: A fluorescently labeled antibody (Cy5.5-anti-DXR mAb) was injected into tumor-bearing mice via the tail vein, and the distribution of the antibody was observed by in vivo imaging of the small animals.

[0044] High performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to detect the concentration of active doxorubicin in tumor tissues and plasma.

[0045] The results showed that anti-doxorubicin antibodies were mainly distributed in the circulatory system, with a small amount entering the tumor tissue; the concentration of free doxorubicin in plasma was significantly reduced, while the concentration of doxorubicin in tumor tissue remained unchanged. This confirms that the mechanism of action of the antibodies is to capture the extravasated drug in the bloodstream without affecting the drug concentration at the tumor site. [Industrial Applicability]

[0046] The triple-system formulation provided by this invention can be prepared into injectable formulations (prodrug and antibody) and lyophilized powder for injection (phage), packaged separately or in combination, for the treatment of various solid tumors. Its preparation process is mature, its quality control methods are well-defined, and it has good industrial applicability.

Claims

1. A pharmaceutical composition for the treatment of tumors, characterized in that, It comprises the following three core components: (a) Prodrug: composed of an antitumor drug coupled with a chemical protecting group, which is in a low-toxicity or inactive state in the blood circulation; (b) Activating enzyme: an enzyme that can be specifically expressed at the tumor site and catalyze the hydrolysis of the prodrug to release the active antitumor drug; (c) Neutralizing antibody: capable of specifically binding to the active antitumor drug or its active metabolites to neutralize its biotoxicity.

2. The pharmaceutical composition according to claim 1, characterized in that, The antitumor drug is an anthracycline antibiotic; preferably, the antitumor drug is doxorubicin or a derivative thereof.

3. The pharmaceutical composition according to claim 2, characterized in that, The prodrug is a cephalosporin-doxorubicin conjugate (C-Dox), the structure of which is shown in Formula I; the cephalosporin group can be specifically hydrolyzed by β-lactamase to release active doxorubicin.

4. The pharmaceutical composition according to claim 1, characterized in that, The activating enzyme is a β-lactamase; preferably, the β-lactamase is expressed locally in the tumor via a phage display system, an engineered bacterial vector, or a liposome delivery system.

5. The pharmaceutical composition according to claim 2, characterized in that, The neutralizing antibody is an anti-doxomil monoclonal antibody or a functional fragment thereof; preferably, the anti-doxomil monoclonal antibody is MAD11 or a derivative thereof, which has differential neutralizing properties, that is, while protecting normal cells, it relatively retains the killing effect on tumor cells.

6. The pharmaceutical composition according to claim 5, characterized in that, The binding affinity Kd of the neutralizing antibody to active doxorubicin is 10⁻ 8 -10⁻¹ 0 M.

7. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

8. A method for preparing the pharmaceutical composition according to any one of claims 1-7, characterized in that, Includes the following steps: (a) Synthesis of prodrug: The antitumor drug is coupled to a cephalosporin group via a cleavable linker arm and purified to obtain the prodrug; (b) Preparation of activating enzyme system: Bacteriophages or engineered bacteria expressing β-lactamase are constructed and their enzyme activity is verified; (c) Preparation of neutralizing antibody: Anti-doxomil monoclonal antibody is prepared by hybridoma technology or genetic engineering methods, purified and its binding and neutralizing activities are identified; (d) The above three components are prepared into formulations suitable for administration, which can be packaged individually or in combination.

9. A medicine box for tumor treatment, characterized in that, The pharmaceutical composition comprises any one of claims 1-7, wherein the three core components are each placed in a separate container and accompanied by instructions for use.

10. The use of the pharmaceutical composition according to any one of claims 1-7 or the medicament according to claim 9 in the preparation of a medicament for treating tumors, wherein the tumors include, but are not limited to, breast cancer, lung cancer, liver cancer, gastric cancer, ovarian cancer, and lymphoma.