WO2023109704A1 - 一种抗体合成菌-纳米刺激剂杂合体及其抗肿瘤应用 - Google Patents

一种抗体合成菌-纳米刺激剂杂合体及其抗肿瘤应用 Download PDF

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WO2023109704A1
WO2023109704A1 PCT/CN2022/138176 CN2022138176W WO2023109704A1 WO 2023109704 A1 WO2023109704 A1 WO 2023109704A1 CN 2022138176 W CN2022138176 W CN 2022138176W WO 2023109704 A1 WO2023109704 A1 WO 2023109704A1
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antibody
nano
stimulator
peg
bacteria
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French (fr)
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蔡林涛
廖健洪
郑明彬
潘宏
张保珍
黄国俊
唐晓帆
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深圳先进技术研究院
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Definitions

  • the invention belongs to the field of targeted drug delivery systems, and in particular relates to a pH-responsive cytokine nano-stimulator and its application in anti-tumor therapy, especially based on the pH response of immunoglobulins such as interleukins and anti-tumor factors
  • the anti-tumor field application of nanoparticles, and the preparation method related to the nanoparticles are particularly important.
  • T cells play a decisive role in anti-tumor immunotherapy, and the number and activity of effector T cells (such as CD8+, CD4+, etc.) infiltrating into tumors are positively correlated with the therapeutic effect of solid tumors.
  • effector T cells such as CD8+, CD4+, etc.
  • clinical studies have found that some patients treated with immune checkpoint antibody drugs have a low immune response rate and poor efficacy through tumor biopsy analysis. The main reason is that there is insufficient effector T cells in the tumor lesion. Therefore, the lack of effector T cells in tumor tissue is the root cause of poor efficacy and low immune response rate of immune checkpoint antibody drugs.
  • cytokines and other immune active proteins to promote the proliferation and anti-tumor activity of effector T cells.
  • a nano drug delivery system for anti-tumor factors can be directly constructed to kill cancer cells through targeted delivery and immune stimulation of massive proliferation of effector T cells.
  • another researcher used the combined administration strategy of cytokines and PD-1 antibody for the treatment of metastatic solid tumors. The results showed that cytokines can promote the proliferation and activity of effector T cells, enhance the immune response, and weaken the solid tumors. Resistance to PD-1 antibody drugs.
  • the above strategies can solve the shortage of effector T cells and improve the therapeutic effect of PD-1 antibodies to a certain extent, they cannot solve the key issues such as the sustained effectiveness of immune checkpoint antibody drugs and the simultaneous and precise delivery of cytokines and antibody drugs. .
  • the existing immune checkpoint-related antibody drugs have defects such as off-target effects, high price, non-renewable, and prone to immune-related adverse reactions, which severely limit their clinical applicability. Therefore, how to efficiently deliver the antibody drug to the tumor lesion, how to continuously block the immune checkpoint signaling pathway, and then release the immune suppression of T cells by the tumor microenvironment for a long time, and enhance the anti-tumor effect of T cells It is a difficult problem to be solved urgently in this field, and it is also the technical problem to be solved by the present invention.
  • Cytokine nanostimulators can promote the proliferation of effector T cells at the tumor site, activate the anti-tumor activity of effector T cells, and then solve the bottlenecks of low immune response rate, easy recurrence and limited curative effect of immune checkpoint antibody drugs.
  • the traditional cocktail-style drug administration cannot make live cell drugs and nano-drugs delivered to tumor lesions synchronously. Therefore, how to realize the synchronous delivery of nanomedicine and living cell medicine is another technical problem to be solved by the present invention.
  • T cell stimulators are encapsulated in nanoparticles by using the sustained and controlled release function of nano-medicines, which can continuously stimulate the proliferation and activation of T cells after sustained release.
  • cytokines traditional chemical reactions will affect the function and activity of living cells, causing irreversible effects. Therefore, how to realize the co-delivery of nanomedicine and living cell drug without destroying the functional activity of living cell drug is another technical problem to be solved by the present invention.
  • the present invention is based on Escherichia coli Nissle 1917 (EcN) probiotics or attenuated Salmonella typhimurium (VPN20009), using genetic engineering, nanomaterial preparation and bio-orthogonal technology Construction of an antibody-synthetic bacteria-nanostimulator hybrid system capable of expressing immune checkpoint inhibitors and releasing cytokines in response to intelligence.
  • EsN Escherichia coli Nissle 1917
  • VPN20009 attenuated Salmonella typhimurium
  • the present invention also relates to an antibody-synthesizing bacterium-nano-stimulator hybrid system, which is to couple a nano-stimulator to the surface of an engineered bacterium through a mild Michael addition or a "click chemistry" reaction to prepare an antibody-synthesizing bacterium-nano-stimulator.
  • Stimulator hybrid system and its application in the field of anti-tumor immunotherapy. details as follows:
  • the present invention provides a copolymer capable of responding to tumor extracellular slightly acidic environment (pH ⁇ 6.5), said copolymer being Mal-poly (PEG n -co-His) or DBCO-poly (PEG n -co-His );
  • the preparation method of DBCO-poly(PEG n -co-His) is Michael addition reaction, wherein the molar ratio of HDDA, His, DBCO-PEG n -NH 2 is 1:0.05 ⁇ 0.95:0.05 ⁇ 0.95; preferably 1:0.85:0.15.
  • the present invention also provides a pH responsive copolymer, the pH responsive copolymer is Mal-poly(PEG 3400 -co-His), its structure example is as follows:
  • the present invention also provides a pH responsive copolymer, the pH responsive copolymer is DBCO-poly(PEG 3400 -co-His), and its structure example is as follows:
  • the present invention also provides a nano-stimulator (NPs) prepared according to the above-mentioned copolymer, which contains a maleimide (Mal)-labeled immunoactive protein nano-stimulator or a DBCO-labeled immunoactive protein nano-stimulator.
  • NPs nano-stimulator
  • the immune active protein is wrapped inside the copolymer, the immune active protein includes cytokines, and the cytokines include any one or more of the following: IL-2, IL-4, IL-5 , IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, TNF- ⁇ , IFN- ⁇ , IFN- ⁇ .
  • nano-stimulator is IL-12 nano-stimulator (INPs).
  • the present invention also provides an antibody-synthesizing bacterium-nano-stimulating agent hybrid system prepared by the above-mentioned nano-stimulator and antibody-synthesizing bacteria, wherein the antibody-synthesizing bacterium is an antibody-synthesizing bacterium labeled with a sulfhydryl group or an azide group;
  • the hybrid system is obtained through the addition of maleimide (Mal) to the sulfhydryl group Michael on the antibody-synthesizing bacteria, or the hybrid system is obtained through the "click" between DBCO and the azide group on the antibody-synthesizing bacteria. Chemistry" reaction coupling obtained.
  • the antibody-synthesizing bacteria are bacteria, preferably Salmonella typhimurium or Escherichia coli.
  • Salmonella typhimurium includes VPN20009.
  • the escherichia coli includes Nissle 1917.
  • mercapto group is from tris(2-carboxyethyl)phosphine (TCEP).
  • the azide group is from N 3 -PEG-NHS.
  • the antibody-synthesizing bacteria express immune checkpoint antibodies; preferably, the immune checkpoint antibodies include any one or more of the following: CTLA-4 antibody, PD-1 antibody, PD-L1 antibody; more preferably , the antibody is selected from a mammalian antibody, a humanized antibody, a nanobody, a chimeric antibody or a functionally active fragment thereof, and the mammal includes a human, a sheep, a mouse, a rabbit, a monkey, an orangutan, a baboon, a pig, a bovine , horses, horses, camels, etc.
  • the immune checkpoint antibodies include any one or more of the following: CTLA-4 antibody, PD-1 antibody, PD-L1 antibody; more preferably , the antibody is selected from a mammalian antibody, a humanized antibody, a nanobody, a chimeric antibody or a functionally active fragment thereof, and the mammal includes a human, a sheep, a mouse, a rabbit,
  • nano-stimulator is prepared by thin film dispersion method or microemulsion method.
  • the present invention also provides a kind of preparation method of above-mentioned nano-stimulant, adopts film dispersion method, comprises the steps:
  • the present invention also provides a preparation method of the above-mentioned copolymer, comprising the following steps: preparing by Michael addition reaction, weighing an appropriate amount of 1,6-hexanediol diacrylate (HDDA), histamine (His) and Mal- PEG n -NH 2 or DBCO-PEG n -NH 2 is mixed in a molar ratio of 1:0.05 ⁇ 0.95:0.05 ⁇ 0.95 and dissolved in an appropriate volume of dimethyl sulfoxide (DMSO), in an inert gas for 35- React at 85°C for 16-72 hours, purify and freeze-dry to obtain polymer powder, and obtain the copolymer Mal-poly(PEG n -co-His) or DBCO-poly(PEG n -co-His) after analysis and identification .
  • HDDA 1,6-hexanediol diacrylate
  • His histamine
  • DBCO-PEG n -NH 2 Mal- PEG
  • the molar ratio is preferably 1:0.85:0.15; the reaction is carried out at 55° C. for 48 hours.
  • the present invention also provides a method for preparing an antibody-synthesizing bacterium-nano-stimulator hybrid system, comprising the following steps:
  • step (c) adding the nano-stimulator according to claim 4 to the antibody-synthesizing bacteria labeled with active groups prepared in step (b), incubating at 25-45°C for 0.5-4h, and centrifuging to remove uncoupled nano-stimulators agent, after testing, the suspension of the antibody-synthesizing bacteria-nano-stimulator hybrid system is obtained.
  • the labeling active group is a sulfhydryl group or an azide group; when the labeling active group is a sulfhydryl group, the nano-stimulator is the Mal-labeled immunoactive protein nano Stimulator; when the labeling active group is an azide group, the nano-stimulator is the DBCO-labeled immunoactive protein nano-stimulator.
  • the mercapto group includes TCEP or the azide group includes N 3 -PEG-NHS.
  • the method for constructing the antibody-synthesizing bacteria includes the following steps:
  • step (c) Apply the recombinant bacteria obtained in step (b) on the resistant medium to screen for monoclonal clones, identify positively by PCR, and sequence to obtain a strain containing the recombinant plasmid, that is, an antibody-synthesizing bacterium expressing the immune checkpoint antibody .
  • the present invention also provides the application of the above-mentioned copolymer, nano-stimulator and/or antibody-synthesizing bacteria-nano-stimulator hybrid system, said application being selected from any one or more of the following:
  • the above-mentioned cells include normal cells or tumor cells.
  • the above-mentioned diseases include tumors.
  • the present invention also provides the application of the above-mentioned antibody-synthesizing bacteria-nano-stimulator hybrid system in the preparation of immunotherapeutic reagents or medicines.
  • the above-mentioned immunotherapy is aimed at tumors.
  • the present invention also provides a composition containing the above-mentioned copolymer, nano-stimulator and/or antibody-synthesizing bacteria-nano-stimulator hybrid system.
  • composition is a drug or a detection reagent.
  • composition also contains pharmaceutically or medically acceptable carriers or excipients.
  • the drug or the composition also includes anti-tumor drugs; preferably, the anti-tumor drugs include broad-spectrum anti-tumor drugs and/or targeted anti-tumor drugs; more preferably, the broad-spectrum anti-tumor drugs
  • the drug is selected from any one or more of camptothecin drugs, doxorubicin drugs, paclitaxel drugs or platinum drugs, and the anti-tumor targeted drug is selected from zanubrutinib, nilotinib, i Matinib, Vimodegib, Vemurafenib, Tesirolimus, Sunitinib, Seritinib, Regorafenib, Afatinib, Trametinib, Pranatinib, Boron Tezomib, pazopanib, axitinib, romidepsin, everolimus, ibrutinib, lenvatinib, dabrafenib, crizotinib
  • the drug or the composition also includes a polypeptide substance, and the polypeptide includes an antigen or an antibody, more preferably, the antibody is selected from the group consisting of adalimumab, cetuximab, and imomab , trastuzumab, nivolumab, daratumumab ramucirumab, nexituzumab, pembrolizumab, pembrolizumab, ofatumumab, blinatumumab Antibodies, bevacizumab, panitumumab, obinutuzumab, bentuximab, denutuzumab, tositumomab, elotuzumab, trastuzumab Any one or more of monoclonal antibody or rituximab.
  • the antibody is selected from the group consisting of adalimumab, cetuximab, and imomab , trastuzumab, n
  • the above-mentioned tumors include cancers, which include basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, Nasopharyngeal cancer, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, blastoma, head cancer, tongue cancer, oral cancer, etc.
  • cancers which include basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, Nasopharyngeal cancer, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, blastoma, head cancer, tongue cancer, oral cancer, etc.
  • the medicine or the composition is any one of oral preparations, injections, tablets, pills, powders, suspensions, granules, aerosols, emulsions, dispersions, capsules, ointments, suppositories or more; preferably, the injection includes any one or more of subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, intralymph node injection, intratumoral injection or subfoot injection.
  • the present invention has the following beneficial technical effects:
  • the invention discloses a novel targeted drug delivery system of antibody synthetic bacteria-nano-stimulator hybrid, as shown in Figure 1, which relates to the application of nano-engineered living cell preparations, especially in the field of anti-tumor immunotherapy, specifically Disclosed are a pH-responsive copolymer, a nano-stimulator, an antibody-synthetic bacterium, a hybrid system of an antibody-synthetic bacterium-nano-stimulator, and a preparation method thereof.
  • the bioactive hybrid system uses mild and efficient Michael addition or "click chemistry" reactions for chemical conjugation. Targeted colonization of tumors can be achieved through the hypoxia targeting of engineered bacteria, and immune checkpoint antibody drugs can be continuously expressed to relieve the immune suppression of T cells.
  • the nano-stimulator on the surface of the bacteria can respond to the microacid environment outside the tumor (pH ⁇ 6.5), release cytokines in a controlled manner, stimulate the proliferation and activation of T cells, and fully activate the body's anti-tumor effect through dual immune regulation of T cells. Effect, and then solve the clinical problems such as the low response rate of immune checkpoint antibody drugs and serious immune-related adverse reactions.
  • the engineered bacteria can target and colonize tumors and continuously synthesize and secrete antibodies, which can relieve the immune suppression of T cells by the tumor microenvironment, enhance T cell infiltration and improve the anti-tumor immunotherapy;
  • the surface nanoparticles of the bacteria respond to the release of immune active proteins such as cytokines, which can effectively promote the proliferation of effector T cells and anti-tumor activity.
  • the antibody synthetic bacteria hybrid system can comprehensively improve the effect of anti-tumor immunotherapy through dual immune regulation of effector T cells.
  • Nano-stimulators based on Mal-poly (PEG n -co-His) or DBCO-poly (PEG n -co-His) endow nanoparticles with the function of responding to the tumor extracellular slightly acidic environment (pH ⁇ 6.5), which can Controlled release of immune active proteins such as cytokines.
  • the engineered bacteria constructed by genetic engineering technology can not only target hypoxia and colonize tumors, but also continuously and stably express immune checkpoint antibody drugs.
  • the bioactive delivery system can be used to co-deliver immune checkpoint inhibitors and cytokines to achieve dual immune regulation of T cells, fully activate the body's anti-tumor effect, and solve the immune response of antibody drugs such as immune checkpoint inhibitors Clinical problems such as low rate and severe immune-related adverse reactions.
  • Fig. 2 is a schematic diagram of Michael addition reaction synthesis of Mal-poly(PEG 3400 -co-His) copolymer with pH response.
  • Fig. 3 is a schematic diagram of Michael addition reaction synthesis of DBCO-poly(PEG 3400 -co-His) copolymer with pH response.
  • Figure 4 is a schematic diagram of the construction of recombinant bacteria expressing immune checkpoint antibody drugs.
  • Fig. 6 is a schematic diagram of Michael addition reaction for preparing antibody-synthesizing bacteria-nano-stimulator hybrid system (Mal labeling).
  • Figure 7 is the SEM characterization (A) and Malvern particle size meter characterization (B) of the antibody synthetic bacteria-nano-stimulator hybrid system constructed based on Escherichia coli Nissle 1917 (EcN); wherein, EcN is Escherichia coli Nissle 1917, EcN/Ab. is the antibody-expressing EcN, NPs is the nano-stimulator, and NPs@Bacteria/Ab. is the constructed antibody-synthetic bacteria-nano-stimulator hybrid system.
  • EcN Escherichia coli Nissle 1917
  • EcN/Ab is the antibody-expressing EcN
  • NPs is the nano-stimulator
  • NPs@Bacteria/Ab is the constructed antibody-synthetic bacteria-nano-stimulator hybrid system.
  • Figure 8 is the pH response release curve (A) of the PD-1 antibody synthetic bacteria-interleukin 12 (IL-12) nano-stimulator hybrid system constructed based on Escherichia coli Nissle 1917 (EcN) and the engineered bacteria expressing PD- 1 Western Bloting characterization of antibody (B), wherein, EcN is Escherichia coli Nissle 1917, EcN/PD-1. is EcN expressing PD-1 antibody, INPs is IL-12 nano-stimulator, INPs@Bacteria/Ab.
  • the antibody-synthesizing bacteria-IL12 nano-stimulator hybrid system was constructed.
  • the Michael addition reaction prepared tumor extracellular pH-responsive copolymers, as shown in Figure 3.
  • the copolymer is further analyzed and identified by gel permeation chromatography/NMR.
  • IL-12 nano-stimulator by microemulsion method: 10 ⁇ g of IL-12 was dissolved in a small amount of ultrapure water, and 10 mg of Mal-poly(PEG 3400 -co-His) copolymer was dissolved in 10 times the equivalent volume of dichloromethane , mixed and stirred for 1 min, added 1% polyvinyl alcohol (PVA) and stirred for another 1 min, and finally added 10 mL of PBS and continued to stir for 30 min to obtain IL-12 nano-stimulator solutions (INPs).
  • PVA polyvinyl alcohol
  • INPs IL-12 nano-stimulator solutions
  • the surface Zeta potential, particle size and PDI were detected by DLS, the morphology was observed by TEM and SEM, and the drug loading and encapsulation efficiency were detected by IL-12 p70 ELISA kit.
  • IL-12 nano-stimulator by microemulsion method: 10 ⁇ g of IL-12 was dissolved in trace ultrapure water, and 10 mg of DBCO-poly(PEG 3400 -co-His) copolymer was dissolved in 10 times the equivalent volume of dichloromethane , mixed and stirred for 1 min, added 1% polyvinyl alcohol (PVA) and stirred for another 1 min, and finally added 10 mL of PBS and continued to stir for 30 min to obtain IL-12 nano-stimulator solutions (INPs).
  • PVA polyvinyl alcohol
  • INPs IL-12 nano-stimulator solutions
  • the surface Zeta potential, particle size and PDI were detected by DLS, the morphology was observed by TEM and SEM, and the drug loading and encapsulation efficiency were detected by IL-12 p70 ELISA kit.
  • Example 5 Preparation of recombinant Escherichia coli Nissle 1917 (EcN/PD-1) expressing PD-1 antibody
  • Example 6 Preparation of recombinant Salmonella typhimurium expressing PD-1 antibody
  • Example 8 Preparation of recombinant Salmonella typhimurium expressing CTLA-4 antibody
  • Example 9 Preparation of DBCO-labeled PD-1 antibody-synthesizing bacteria EcN-IL12 nano-stimulator hybrid system (INPs@EcN/PD-1)
  • a synthetic bacterial EcN-IL12 nano-stimulator hybrid system (INPs@EcN/PD-1) prepared by "click chemistry" reaction for PD-1 antibody, labeled with DBCO, as shown in Figure 5, includes the following steps:
  • the synthetic bacteria EcN-IL12 nano-stimulator hybrid system (INPs@EcN/PD-1) prepared by Michael addition reaction of PD-1 antibody, Mal-labeled, as shown in Figure 6, includes the following steps:

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Abstract

本发明公开了一种抗体合成菌-纳米刺激剂杂合体新型靶向给药系统，及其在抗肿瘤免疫治疗领域的应用，具体公开了pH响应共聚物、纳米刺激剂、抗体合成菌以及抗体合成菌-纳米刺激剂杂合体系统及其制备方法和应用。

Description

一种抗体合成菌-纳米刺激剂杂合体及其抗肿瘤应用 技术领域
本发明属于靶向给药系统领域，具体涉及一种具有pH响应的细胞因子纳米刺激剂和其在抗肿瘤治疗中的应用，特别是基于白细胞介素和抗肿瘤因子等免疫球蛋白的pH响应纳米粒子的抗肿瘤领域应用，以及涉及该纳米粒子的制备方法。
背景技术
近年来，以程序性死亡受体1(PD-1)、程序性死亡受体配体1(PD-L1)和细胞毒性T淋巴细胞相关蛋白4(CTLA-4)为靶点的免疫检查点抑制疗法取得了令人鼓舞的成果并成为研究热点。通过免疫检查点抑制剂阻断PD-1/PD-L1的相互作用，解除肿瘤微环境免疫抑制是抗肿瘤免疫治疗的一种有效途径。然而，由于传统免疫检查点抗体药物毒副作用大、不可再生和易产生耐药性等缺陷，在临床治疗上需多次给药，严重限制了其适用性。因此，如何提高免疫检查点抗体药物的靶向递送和持续有效性，进而克服CTLA-4或PD-1/PD-L1免疫检查点通路的抑制，增强效应T细胞的抗肿瘤活性，实现实体瘤的高效免疫治疗是现有技术需要着重解决的关键技术问题。
近期大量研究表明，T细胞在抗肿瘤免疫治疗中起决定性的作用，而浸润到肿瘤的效应T细胞(如CD8+、CD4+等)的数量和活性与实体瘤的治疗效果呈正相关。同时临床研究发现，通过肿瘤组织活检分析部分使用免疫检查点抗体药物治疗的患者免疫应答率低、疗效不佳，其主要原因是肿瘤病灶内效应T细胞不足。因此，肿瘤组织效应T细胞匮乏是导致免疫检查点抗体药物疗效差和免疫应答率低的根本原因。如何促进肿瘤部位效应T细胞的增殖、激活 效应T细胞的抗肿瘤活性，进而解决免疫检查点抗体药物免疫应答率低、易复发和疗效受限等瓶颈也是需要解决的问题。
针对肿瘤效应T细胞不足，研究者采用了细胞因子等免疫活性蛋白来促进效应T细胞的增殖和抗肿瘤活性。如直接构建一种抗肿瘤因子纳米给药系统，通过靶向递送和免疫刺激效应T细胞的大量增殖来杀灭癌细胞。而又有研究者采用细胞因子和PD-1抗体联合给药策略用于转移性实体瘤的治疗，结果表明细胞因子能促进效应T细胞的增殖和活性，增强了免疫应答，进而减弱了实体瘤对PD-1抗体药物的耐药性。诸上策略虽能一定程度上解决效应T细胞不足并提高PD-1抗体的治疗效果，但均未能解决免疫检查点抗体药物的持续有效性以及细胞因子和抗体药物的同步精准递送等关键问题。
综上，如何构建一种安全、高效的新型生物活性精准递送系统，持续克服免疫检查点抑制、促进效应T细胞的增殖和抗肿瘤活性仍然是一个巨大的挑战。
现有免疫检查点相关抗体药物由于存在脱靶效应、价格昂贵、不可再生、易产生免疫相关不良反应等缺陷，严重限制了其临床适用性。因此，如何将所述抗体药物高效地递送至肿瘤病灶部位，如何实现对免疫检查点信号通路的持续阻断，进而长效地解除肿瘤微环境对T细胞的免疫抑制，增强T细胞的抗肿瘤活性，最终实现转移性实体瘤的高效免疫治疗，是本领域亟需解决的一个难题，也是本发明拟解决的技术问题。
肿瘤组织效应T细胞匮乏是导致免疫检查点抗体药物疗效差和免疫应答率低的根本原因。细胞因子纳米刺激剂能促进肿瘤部位效应T细胞的增殖、激活效应T细胞的抗肿瘤活性，进而解决免疫检查点抗体药物免疫应答率低、易复发和疗效受限等瓶颈，然而由于代谢动力学的差异，传统的鸡尾酒式给药无法使得活细胞药物和纳米药物同步递送至肿瘤病灶。因此，如何实现纳米药物和活细胞药物的同步递送是本发明拟解决的另一个技术问题。
利用基因工程技术赋予活细胞药物的药物合成功能，是持续阻断免疫检查点通路的一种有效途径。另一方面，利用纳米药物缓控释功能将T细胞刺激剂(细胞因子)包裹在纳米粒 子中，缓释后能持续刺激T细胞的增殖和活化。然而，传统的化学反应将影响活细胞的功能和活性，造成不可逆的影响。因此，如何在不破坏活细胞药物功能活性的前提下实现纳米药物和活细胞药物的共递送是本发明拟解决的另一个技术问题。
针对肿瘤免疫检查点抑制与效应T细胞不足的科学问题，本发明基于大肠杆菌属Nissle 1917(EcN)益生菌或减毒鼠伤寒沙门氏菌(VPN20009)，采用基因工程、纳米材料制备及生物正交技术构建能表达免疫检查点抑制剂和智能响应释放细胞因子的抗体合成菌-纳米刺激剂杂合体系统。利用该生物活性递送系统的靶向穿透、免疫检查点抑制剂和细胞因子的双重免疫调控，全面激活机体抗肿瘤效应，解决免疫检查点抑制剂等抗体药物的免疫应答率低和治疗疗效差的难题，为全面提高免疫检查点抑制剂的抗肿瘤治疗提供重要理论依据和技术手段。
发明内容
针对上述归纳和总结的现有技术长期存在，却仍未能有效解决的技术问题，本发明提供了一种具有pH响应的细胞因子纳米刺激剂，及其基于白细胞介素和抗肿瘤因子等免疫球蛋白的pH响应纳米粒子的抗肿瘤领域应用。还提供了一种抗体合成菌杂合体系统，其为通过基因工程构建的免疫检查点抗体药物合成菌，及其在抗肿瘤领域的应用。最后，本发明还涉及抗体合成菌-纳米刺激剂杂合体系统，其为将纳米刺激剂通过温和的Michael加成或“点击化学”反应偶联在工程菌的表面，制备得到抗体合成菌-纳米刺激剂杂合体系统，以及在抗肿瘤免疫治疗领域的应用。具体如下：
本发明提供了一种能响应肿瘤胞外微酸环境(pH<6.5)的共聚物，所述共聚物是Mal-poly(PEG n-co-His)或DBCO-poly(PEG n-co-His)；
所述Mal-poly(PEG n-co-His)是由Mal-PEG n-NH 2，6-己二醇二丙烯酸酯(HDDA)和组胺(His)制备，其中PEG重复单元n＝1-5000；优选地，PEG重复单元n＝3400，即 Mal-poly(PEG 3400-co-His)；
所述DBCO-poly(PEG n-co-His)是由DBCO-PEG n-NH 2，6-己二醇二丙烯酸酯(HDDA)和组胺(His)制备，其中PEG重复单元n＝1-5000；优选地，PEG重复单元n＝3400，即DBCO-poly(PEG 3400-co-His)。
进一步地，所述Mal-poly(PEG n-co-His)的制备方法是采用Michael加成反应，其中HDDA、His、Mal-PEG n-NH 2的摩尔比为1:0.05～0.95:0.05～0.95；优选为1:0.85:0.15。
进一步地，所述DBCO-poly(PEG n-co-His)的制备方法是采用Michael加成反应，其中HDDA、His、DBCO-PEG n-NH 2的摩尔比为1:0.05～0.95:0.05～0.95；优选为1:0.85:0.15。
本发明还提供了一种pH响应共聚物，所述pH响应共聚物是Mal-poly(PEG 3400-co-His)，其结构示例如下：
Figure PCTCN2022138176-appb-000001
本发明还提供了一种pH响应共聚物，所述pH响应共聚物是DBCO-poly(PEG 3400-co-His)，其结构示例如下：
Figure PCTCN2022138176-appb-000002
本发明还提供了一种根据上述共聚物制备的纳米刺激剂(NPs)，所述纳米刺激剂含有马 来酰亚胺(Mal)标记的免疫活性蛋白纳米刺激剂或DBCO标记的免疫活性蛋白纳米刺激剂，所述免疫活性蛋白包裹于所述共聚物的内部，所述免疫活性蛋白包括细胞因子，所述细胞因子包括以下任一种或多种：IL-2、IL-4、IL-5、IL-10、IL-12、IL-15、IL-17、IL-18、IL-21、TNF-α、IFN-α、IFN-γ。
进一步地，所述纳米刺激剂为IL-12纳米刺激剂(INPs)。
本发明还提供了一种由上述纳米刺激剂和抗体合成菌制备的抗体合成菌-纳米刺激剂杂合体系统，所述抗体合成菌是巯基基团或叠氮基团标记的抗体合成菌；所述杂合体系统是通过马来酰亚胺(Mal)与抗体合成菌上的巯基基团Michael加成获得，或所述杂合体系统是通过DBCO与抗体合成菌上的叠氮基团的“点击化学”反应偶联获得。
进一步地，所述抗体合成菌是细菌，优选为鼠伤寒沙门氏菌或大肠杆菌。
进一步地，所述鼠伤寒沙门氏菌包括VPN20009。
进一步地，所述大肠杆菌包括Nissle 1917。
进一步地，所述巯基基团来自三(2-羧乙基)膦(TCEP)。
进一步地，所述叠氮基团来自N 3-PEG-NHS。
进一步地，所述抗体合成菌表达免疫检查点抗体；优选地，所述免疫检查点抗体包括以下任一种或多种：CTLA-4抗体、PD-1抗体、PD-L1抗体；更优选地，所述抗体选是哺乳动物源抗体、人源化抗体、纳米抗体、嵌合抗体或其功能活性片段，所述哺乳动物包括人、羊、鼠、兔、猴、猩猩、狒狒、猪、牛、马、购、骆驼等。
进一步地，所述纳米刺激剂是经过薄膜分散法或微乳液法制备。
本发明还提供了一种上述纳米刺激剂的制备方法，采用薄膜分散法，包括如下步骤：
(a)有机相中溶解所述共聚物，旋蒸除去有机相，得薄膜聚合物层；
(b)将所述所述的免疫活性蛋白和PBS加入，探头超声获得免疫活性蛋白纳米粒子；
(c)超滤或透析，得到具有Mal或DBCO修饰的核壳结构纳米粒子，即所述纳米刺激剂。
本发明还提供了一种上述纳米刺激剂的制备方法，采用微乳液法，包括如下步骤：
(a)有机相中溶解所述共聚物，加入聚乙烯醇(PVA)表面活性剂继续搅拌；
(b)缓慢滴入免疫活性蛋白溶液，继续搅拌，加入PBS并于超声探头下超声；
(c)超滤或透析，得到表面具有Mal或DBCO修饰的核壳结构纳米粒子，即所述纳米刺激剂。
本发明还提供了一种上述共聚物的制备方法，包括如下步骤：通过Michael加成反应制备，称取适量1,6-己二醇二丙烯酸酯(HDDA)、组胺(His)与Mal-PEG n-NH 2或DBCO-PEG n-NH 2按摩尔比1:0.05～0.95:0.05～0.95的比例混合并溶解在适量体积的二甲基亚砜(DMSO)中，在惰性气体中35-85℃条件下反应16-72h，提纯并冷冻干燥得到聚合物粉末，经分析、鉴定获得所述共聚物Mal-poly(PEG n-co-His)或DBCO-poly(PEG n-co-His)。
进一步地，所述摩尔比优选为1:0.85:0.15；所述反应是在55℃条件下反应48h。
本发明还提供了一种抗体合成菌-纳米刺激剂杂合体系统的制备方法，包括以下步骤：
(a)将上述的抗体合成菌菌液收集在离心管中，离心并重悬于缓冲溶液中；
(b)加入适量的标记活性基团反应，离心提取标记活性基团的抗体合成菌；
(c)将权利要求4所述的纳米刺激剂加入到步骤(b)制备的标记活性基团的抗体合成菌中，在25-45℃下孵育0.5-4h，离心除去未偶联的纳米刺激剂，经检测，获得所述抗体合成菌-纳米刺激剂杂合体系统的混悬液。
进一步地，所述标记活性基团为巯基基团或叠氮基团；当所述所述标记活性基团为巯基基团时，所述纳米刺激剂为所述的Mal标记的免疫活性蛋白纳米刺激剂；当所述所述标记活性基团为叠氮基团时，所述纳米刺激剂为所述的DBCO标记的免疫活性蛋白纳米刺激剂。
进一步地，所述巯基基团包括TCEP或所述叠氮基团包括N 3-PEG-NHS。
进一步地，所述抗体合成菌的构建方法包括如下步骤：
(a)将含有编码所述免疫检查点抗体的核苷酸序列的目的表达片段通过酶切、连接等步骤 导入表达空载体，得到重组表达载体；
(b)将所述重组表达载体转化到宿主细菌中，得到表达所述免疫检查点抗体的重组细菌
(c)将步骤(b)得到的重组菌涂布在抗性培养基上筛选单克隆，经PCR阳性鉴定，测序，获得含有重组质粒的菌株，即表达所述免疫检查点抗体的抗体合成菌。
本发明还提供了上述的共聚物、纳米刺激剂和/或抗体合成菌-纳米刺激剂杂合体系统的应用，所述应用选自如下任一种或多种：
(1)在制备药物组合物中的应用；
(2)在制备用于细胞检测的试剂中的应用；
(3)在制备用于疾病诊断或治疗的试剂中的应用；
(4)在体外筛选药物中的应用；
(5)在不以疾病诊断为目的体外检测中的应用。
进一步地，上述细胞包括正常细胞或肿瘤细胞。
进一步地，上述疾病包括肿瘤。
本发明还提供了上述的抗体合成菌-纳米刺激剂杂合体系统在制备免疫治疗试剂或药物中的应用。
进一步地，上述免疫治疗针对的是肿瘤。
本发明还提供了一种含有上述的共聚物、纳米刺激剂和/或抗体合成菌-纳米刺激剂杂合体系统的组合物。
进一步地，所述组合物是药物或检测试剂。
进一步地，所述组合物还含有药学或医学上可接受的载体或赋形剂。
进一步地，所述药物或所述组合物还包括抗肿瘤药物；优选地，所述抗肿瘤药物包括抗肿瘤广谱药物和/或抗肿瘤靶向药物；更优选地，所述抗肿瘤广谱药物选自喜树碱类药物、阿霉素类药物、紫杉醇类药物或铂类药物中任一种或多种，所述抗肿瘤靶向药物选自泽布替尼、 尼罗替尼、伊马替尼、维莫德吉、维罗非尼、替西罗莫司、舒尼替尼、赛立替尼、瑞格非尼、阿法替尼、曲美替尼、普钠替尼、硼替佐米、帕唑帕尼、阿西替尼、罗米地辛、依维莫司、依鲁替尼、乐伐替尼、达拉菲尼、克唑替尼、卡非佐米、奥斯替尼、卡博替尼、卡比替尼、吉非替尼、伏立诺他、凡德他尼、艾乐替尼、狄诺塞麦、索尼德吉、索拉非尼、博舒替尼、贝利司他、奥拉帕尼、阿柏西普、拉帕替尼、达沙替尼、帕博西尼、帕比司他或厄洛替尼中任一种或多种。
进一步地，所述药物或所述组合物还包括多肽类物质，所述多肽包括抗原或抗体，更优选地，所述抗体选自阿达木单抗、西妥昔单抗、替伊莫单抗、曲妥珠单抗、纳武单抗、达雷木单抗雷莫芦单抗、耐昔妥珠单抗、派姆单抗、派姆单抗、奥法木单抗、博纳吐单抗、贝伐珠单抗、帕尼单抗、奥宾尤妥珠单抗、本妥昔单抗、地努图希单抗、托西莫单抗、埃罗妥珠单抗、曲妥珠单抗或利妥昔单抗中任一种或多种。
进一步地，上述肿瘤包括癌，所述癌包括基底细胞癌、鳞状细胞癌、食管癌、恶性胶质瘤、膀胱癌、宫颈癌、乳腺癌、肺癌、肝癌、胃癌、结肠癌、直肠癌、鼻咽癌、胰腺癌、甲状腺癌、前列腺癌、白血病、淋巴瘤、肾脏肿瘤、肉瘤、母细胞瘤、头癌、舌癌、口腔癌等。
进一步地，所述药物或所述组合物是口服剂、注射剂、片剂、丸剂、散剂、混悬剂、颗粒剂、气雾剂、乳剂、分散剂、胶囊剂、膏剂、栓剂中任一种或多种；优选地，所述注射剂包括皮下注射、肌肉注射、腹腔注射、静脉注射、淋巴结内注射、瘤内注射或足下注射中任一种或多种。
本发明相比于现有技术，具有以下有益的技术效果：
本发明公开了一种抗体合成菌-纳米刺激剂杂合体新型靶向给药系统，如图1所示，涉及纳米工程化活细胞制剂的应用，特别是在抗肿瘤免疫治疗领域的应用，具体公开了pH响应共聚物、纳米刺激剂、抗体合成菌以及抗体合成菌-纳米刺激剂杂合体系统及其制备方法。该生物活性杂合体系统采用温和、高效的Michael加成或“点击化学”反应进行化学偶联。通 过工程菌的缺氧靶向能实现肿瘤靶向定植，并能持续表达免疫检查点抗体药物，解除T细胞的免疫抑制。与此同时，细菌表面的纳米刺激剂能响应肿瘤胞外微酸环境(pH<6.5)，可控释放细胞因子，刺激T细胞的增殖活化，通过对T细胞的双重免疫调控全面激活机体抗肿瘤效应，进而解决免疫检查点抗体药物响应率低和免疫相关不良反应严重等临床难题。
本发明技术的主要优势：(a)工程菌能靶向、定植于肿瘤并持续合成、分泌抗体，可解除肿瘤微环境对T细胞的免疫抑制，增强T细胞浸润并提高抗肿瘤的免疫疗效；(b)菌表面纳米粒子响应释放细胞因子等免疫活性蛋白，有效促进效应T细胞增殖和抗肿瘤活性。该抗体合成菌杂合体系统通过对效应T细胞的双重免疫调控，可全面提高抗肿瘤免疫治疗效果。
本发明的主要技术效果：
1)Mal或DBCO标记的纳米刺激剂能与工程菌表面标记的巯基或叠氮基团发生温和、高效的发生Michael加成或“点击化学”反应，而不影响工程菌的功能活性。
2)基于Mal-poly(PEG n-co-His)或DBCO-poly(PEG n-co-His)的纳米刺激剂赋予纳米粒子具有响应肿瘤胞外微酸环境(pH<6.5)的功能，可控释放细胞因子等免疫活性蛋白。
3)通过基因工程技术构建的工程菌不仅能缺氧靶向、定植于肿瘤，还能持续稳定的表达免疫检查点抗体药物。
4)利用该生物活性递送系统能靶向共递送免疫检查点抑制剂和细胞因子，实现对T细胞的双重免疫调控，全面激活机体抗肿瘤效应，解决免疫检查点抑制剂等抗体药物的免疫应答率低和免疫相关不良反应严重等临床难题。
附图说明
图1为抗体合成菌-纳米刺激剂杂合体系统的示意图。
图2为Michael加成反应合成具有pH响应的Mal-poly(PEG 3400-co-His)共聚物的示意图。
图3为Michael加成反应合成具有pH响应的DBCO-poly(PEG 3400-co-His)共聚物的示意图。
图4为表达免疫检查点抗体药物的重组细菌的构建示意图。
图5为“点击化学”反应制备抗体合成菌-纳米刺激剂杂合体系统(DBCO标记)的示意图。
图6为Michael加成反应制备抗体合成菌-纳米刺激剂杂合体系统(Mal标记)的示意图。
图7为基于大肠杆菌属Nissle 1917(EcN)构建的抗体合成菌-纳米刺激剂杂合体系统的SEM表征(A)和马尔文粒度仪表征(B)；其中，EcN为大肠杆菌属Nissle 1917，EcN/Ab.为表达抗体的EcN，NPs为纳米刺激剂，NPs@Bacteria/Ab.为构建的抗体合成菌-纳米刺激剂杂合体系统。
图8为基于大肠杆菌属Nissle 1917(EcN)构建的PD-1抗体合成菌-白细胞介素12(IL-12)纳米刺激剂杂合体系统的pH响应释放曲线(A)以及工程菌表达PD-1抗体的Western Bloting表征(B)，其中，EcN为大肠杆菌属Nissle 1917，EcN/PD-1.为表达PD-1抗体的EcN，INPs为IL-12纳米刺激剂，INPs@Bacteria/Ab.为构建的抗体合成菌-IL12纳米刺激剂杂合体系统。
具体实施方式
以下通过具体实施例对本发明作进一步详细说明，以使本领域技术人员能够更好地理解本发明并予以实施，但实施例并不作为本发明的限定。
以下实施例中所使用的实验方法如无特殊说明，均为常规方法。所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：Mal-poly(PEG 3400-co-His)的制备
Michael加成反应制备肿瘤胞外pH响应共聚物，如图2所示。称取一定量的1,6-己二醇二丙烯酸酯(HDDA)、组胺(His)和马来酰亚胺-聚乙二醇氨基(Mal-PEG 3400-NH 2)按摩尔比1:0.85:0.15的比例混合并溶解在一定体积的二甲基亚砜(DMSO)中，在氩气氛围55℃ 条件下反应48h，提纯并冷冻干燥得到聚合物粉末。所述共聚物进一步通过凝胶渗透色谱/核磁分析、鉴定。
实施例2：DBCO-poly(PEG 3400-co-His)的制备
Michael加成反应制备肿瘤胞外pH响应共聚物，如图3所示。称取一定量的1,6-己二醇二丙烯酸酯(HDDA)、组胺(His)和二苯基环辛炔-聚乙二醇氨基(DBCO-PEG 3400-NH 2)按摩尔比1:0.85:0.15的比例混合并溶解在一定体积的二甲基亚砜(DMSO)中，在氩气氛围55℃条件下反应48h，提纯并冷冻干燥得到聚合物粉末。所述共聚物进一步通过凝胶渗透色谱/核磁分析、鉴定。
实施例3：Mal标记的IL-12纳米刺激剂的制备
采用微乳液法制备IL-12纳米刺激剂：10μg的IL-12溶于微量超纯水中，10mg的Mal-poly(PEG 3400-co-His)共聚物溶于10倍当量体积二氯甲烷中，混合搅拌1min，加1％的聚乙烯醇(PVA)再搅拌1min，最后加入10mL PBS继续搅拌30min，即得IL-12纳米刺激剂溶液(INPs)。表面Zeta电位、粒径和PDI由DLS检测，形貌通过TEM和SEM观察，并采用IL-12 p70 ELISA试剂盒检测载药量和包封率。
实施例4：DBCO标记的IL-12纳米刺激剂的制备
采用微乳液法制备IL-12纳米刺激剂：10μg的IL-12溶于微量超纯水中，10mg的DBCO-poly(PEG 3400-co-His)共聚物溶于10倍当量体积二氯甲烷中，混合搅拌1min，加1％的聚乙烯醇(PVA)再搅拌1min，最后加入10mL PBS继续搅拌30min，即得IL-12纳米刺激剂溶液(INPs)。表面Zeta电位、粒径和PDI由DLS检测，形貌通过TEM和SEM观察，并采用IL-12 p70 ELISA试剂盒检测载药量和包封率。
实施例5：表达PD-1抗体的重组大肠杆菌Nissle 1917(EcN/PD-1)，的制备
制备表达PD-1抗体的重组大肠杆菌Nissle 1917(EcN/PD-1)，如图4所示，具体方法如 下：
(a)将含有PD-1抗体编码的核苷酸序列的目的表达片段通过酶切、连接等步骤导入表达空载体，得到重组表达载体；
(b)及将所述重组表达载体转化到大肠杆菌Nissle 1917(EcN)的宿主细菌中，得到表达所述抗体的重组细菌；
(c)涂布在抗性培养基上筛选单克隆，经PCR阳性鉴定，测序，获得含有重组质粒的菌株(EcN/PD-1)。
实施例6：表达PD-1抗体的重组鼠伤寒沙门氏菌的制备
制备表达PD-1抗体的重组鼠伤寒沙门氏菌，如图4所示，具体方法如下：
(a)将含有PD-1抗体编码的核苷酸序列的目的表达片段通过酶切、连接等步骤导入表达空载体，得到重组表达载体；
(b)及将所述重组表达载体转化到鼠伤寒沙门氏菌(VPN20009)的宿主细菌中，得到表达所述抗体的重组细菌；
(c)涂布在抗性培养基上筛选单克隆，经PCR阳性鉴定，测序，获得含有重组质粒的菌株。
实施例7：表达CTLA-4抗体的重组大肠杆菌Nissle 1917的制备
制备表达CTLA-4抗体的重组大肠杆菌Nissle 1917，如图4所示，具体方法如下：
(a)将含有CTLA-4抗体编码的核苷酸序列的目的表达片段通过酶切、连接等步骤导入表达空载体，得到重组表达载体；
(b)及将所述重组表达载体转化到大肠杆菌Nissle 1917(EcN)的宿主细菌中，得到表达所述抗体的重组细菌；
(c)涂布在抗性培养基上筛选单克隆，经PCR阳性鉴定，测序，获得含有重组质粒的菌 株。
实施例8：表达CTLA-4抗体的重组鼠伤寒沙门氏菌的制备
制备表达CTLA-4抗体的重组鼠伤寒沙门氏菌，如图4所示，具体方法如下：
(a)将含有CTLA-4抗体编码的核苷酸序列的目的表达片段通过酶切、连接等步骤导入表达空载体，得到重组表达载体；
(b)及将所述重组表达载体转化到鼠伤寒沙门氏菌(VPN20009)的宿主细菌中，得到表达所述抗体的重组细菌；
(c)涂布在抗性培养基上筛选单克隆，经PCR阳性鉴定，测序，获得含有重组质粒的菌株。
实施例9：基于DBCO标记的PD-1抗体合成菌EcN-IL12纳米刺激剂杂合体系统(INPs@EcN/PD-1)的制备
采用“点击化学”反应制备PD-1抗体的合成菌EcN-IL12纳米刺激剂杂合体系统(INPs@EcN/PD-1)，DBCO标记，如图5所示，包括如下步骤：
(a)将密度为10 8CFU/mL的大肠杆菌Nissle 1917(EcN)菌液收集在离心管中，5000rpm离心并重悬于PBS溶液中；
(b)加入一定量的N 3-PEG-NHS反应2h，5000rpm离心提取叠氮标记的菌泥；
(c)将1mL的DBCO标记的INPs加入到10 8CFU的叠氮基标记大肠杆菌Nissle 1917(EcN)中在30℃下孵育2h，差速离心除去未偶联INPs，获得PD-1抗体合成菌EcN–IL12纳米刺激剂杂合体系统(INPs@EcN/PD-1)混悬液；
(d)表面Zeta电位、粒径和PDI由DLS检测，形貌及偶联效率通过TEM和SEM观察分析；
(e)结合IL-12 p70 ELISA试剂盒检测IL-12纳米刺激剂“点击化学”反应的偶联效率。最 后，采用活/死细菌染色实验考察抗体合成菌-纳米刺激剂杂合体系统的活性影响。
实施例10：基于Mal标记的PD-1抗体合成菌EcN-IL12纳米刺激剂杂合体系统(INPs@EcN/PD-1)制备及结构、控释和生物合成表征
采用Michael加成反应制备PD-1抗体的合成菌EcN-IL12纳米刺激剂杂合体系统(INPs@EcN/PD-1)，Mal标记，如图6所示，包括如下步骤：
(a)将密度为10 8CFU/mL的大肠杆菌Nissle 1917(EcN)菌液收集在离心管中，5000rpm离心并重悬于PBS溶液中；
(b)加入一定量三(2-羧乙基)膦(TCEP)，反应15min，5000rpm离心提取巯基标记的菌泥；
(c)将1mL的Mal标记的INPs加入到10 8CFU巯基标记的大肠杆菌Nissle 1917(EcN)中在30℃下孵育2h，差速离心除去未偶联INPs，获得PD-1抗体的合成菌EcN-IL12纳米刺激剂杂合体系统(INPs@EcN/PD-1)的混悬液；
(d)表面Zeta电位、粒径和PDI由DLS检测，形貌及偶联效率通过TEM和SEM观察分析；
结果如图7所示，基于大肠杆菌属Nissle 1917(EcN)构建的抗体合成菌-纳米刺激剂杂合体系统的SEM表征(A)和马尔文粒度仪表征(B)，其中，EcN为大肠杆菌属Nissle 1917，EcN/Ab.为表达抗体的EcN，NPs为纳米刺激剂，NPs@Bacteria/Ab.为构建的抗体合成菌-纳米刺激剂杂合体系统。
(e)结合IL-12 p70 ELISA试剂盒检测IL-12纳米刺激剂Michael加成反应的偶联效率。最后，采用活/死细菌染色实验考察抗体合成菌-纳米刺激剂杂合体系统的活性影响；
结果如图8所示，基于大肠杆菌属Nissle 1917(EcN)构建的PD-1抗体合成菌-白细胞介素12(IL-12)纳米刺激剂杂合体系统的pH响应释放曲线(A)以及工程菌表达PD-1抗体 的Western Bloting表征(B)，其中，EcN为大肠杆菌属Nissle 1917，EcN/PD-1.为表达PD-1抗体的EcN，INPs为IL-12纳米刺激剂，INPs@Bacteria/Ab.为构建的抗体合成菌-IL12纳米刺激剂杂合体系统。
实施例11：基于Mal标记的CTLA-4抗体合成菌-IL-12纳米刺激剂杂合体系统的制备
(a)将密度为10 8CFU/mL的鼠伤寒沙门氏菌液(VPN20009)收集在离心管中，5000rpm离心并重悬于PBS溶液中；
(b)加入一定量的TCEP 15min，5000rpm离心提取巯基标记的菌泥；
(c)将1mL的Mal标记的NPs加入到10 8CFU的叠氮基标记的鼠伤寒沙门氏菌(VPN20009)中在30℃下孵育2h，差速离心除去未偶联NPs，获得CTLA-4抗体合成菌-纳米刺激剂杂合体系统混悬液；
(c)表面Zeta电位、粒径和PDI由DLS检测，形貌及偶联效率通过TEM和SEM观察分析；
(d)结合IL-12 p70 ELISA试剂盒检测IL-12纳米刺激剂Michael加成反应的偶联效率。最后，采用活/死细菌染色实验考察抗体合成菌-纳米刺激剂杂合体系统的活性影响。
实施例12：基于DBCO标记的CTLA-4抗体合成菌-IL-12纳米刺激剂系统的制备
(a)将密度为10 8CFU/mL的鼠伤寒沙门氏菌液(VPN20009)收集在离心管中，5000rpm离心并重悬于PBS溶液中；
(b)加入一定量的N 3-PEG-NHS反应2h，5000rpm离心提取叠氮标记的菌泥；
(c)将1mL的DBCO标记的NPs加入到10 8CFU的叠氮基标记的鼠伤寒沙门氏菌(VPN20009)中在30℃下孵育2h，差速离心除去未偶联NPs，获得CTLA-4抗体合成菌-纳米刺激剂杂合体系统混悬液；
(c)表面Zeta电位、粒径和PDI由DLS检测，形貌及偶联效率通过TEM和SEM观察分 析；
(d)结合IL-12 p70 ELISA试剂盒检测IL-12纳米刺激剂“点击化学”的偶联效率。最后，采用活/死细菌染色实验考察抗体合成菌-纳米刺激剂杂合体系统的活性影响。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (25)

  1. 一种能响应肿瘤胞外微酸环境的共聚物，所述共聚物是Mal-poly(PEG n-co-His)或DBCO-poly(PEG n-co-His)；
    所述Mal-poly(PEG n-co-His)是由Mal-PEG n-NH 2，6-己二醇二丙烯酸酯(HDDA)和组胺(His)制备，其中PEG重复单元n＝1-5000；优选地，PEG重复单元n＝3400，即Mal-poly(PEG 3400-co-His)；
    所述DBCO-poly(PEG n-co-His)是由DBCO-PEG n-NH 2，6-己二醇二丙烯酸酯(HDDA)和组胺(His)制备，其中PEG重复单元n＝1-5000；优选地，PEG重复单元n＝3400，即DBCO-poly(PEG 3400-co-His)。
  2. 根据所述权利要求1所示的共聚物，其中，所述Mal-poly(PEG n-co-His)的制备方法是采用Michael加成反应，其中HDDA、His、Mal-PEG n-NH 2的摩尔比为1:0.05～0.95:0.05～0.95；优选为1:0.85:0.15；
    所述DBCO-poly(PEG n-co-His)的制备方法是采用Michael加成反应，其中HDDA、His、DBCO-PEG n-NH 2的摩尔比为1:0.05～0.95:0.05～0.95；优选为1:0.85:0.15。
  3. 一种pH响应共聚物，所述pH响应共聚物是Mal-poly(PEG 3400-co-His)，其结构示例如下：
    Figure PCTCN2022138176-appb-100001
    或
    所述pH响应共聚物是DBCO-poly(PEG 3400-co-His)，其结构示例如下：
    Figure PCTCN2022138176-appb-100002
  4. 一种由权利要求1-3任一项所述的共聚物制备的纳米刺激剂(NPs)，所述纳米刺激剂为马来酰亚胺(Mal)标记的免疫活性蛋白纳米刺激剂或DBCO标记的免疫活性蛋白纳米刺激剂，所述免疫活性蛋白包裹于所述共聚物的内部，所述免疫活性蛋白包括细胞因子，所述细胞因子包括以下任一种或多种：IL-2、IL-4、IL-5、IL-10、IL-12、IL-15、IL-17、IL-18、IL-21、TNF-α、IFN-α、IFN-γ；
    优选地，所述纳米刺激剂为IL-12纳米刺激剂(INPs)。
  5. 一种由权利要求1-3任一项所述的共聚物或权利要求4所述的纳米刺激剂与抗体合成菌共价结合而制备的抗体合成菌-纳米刺激剂杂合体系统(NPs@Bacteria/Ab)，所述抗体合成菌是巯基基团或叠氮基团标记的抗体合成菌；所述杂合体系统是通过马来酰亚胺(Mal)与所述抗体合成菌上的巯基基团Michael加成获得，或所述杂合体系统是通过DBCO与所述抗体合成菌上的叠氮基团的“点击化学”反应偶联获得。
  6. 根据权利要求5所述的抗体合成菌-纳米刺激剂杂合体系统，所述抗体合成菌是细菌，优选为鼠伤寒沙门氏菌或大肠杆菌；更优选地，所述鼠伤寒沙门氏菌包括VPN20009；更优选地，所述大肠杆菌包括Nissle 1917(EcN)。
  7. 根据权利要求5所述的抗体合成菌-纳米刺激剂杂合体系统，所述巯基基团来自三(2-羧乙基)膦(TCEP)；所述叠氮基团来自N 3-PEG-NHS。
  8. 根据权利要求5-7任一项所述的抗体合成菌-纳米刺激剂杂合体系统，所述抗体合成菌表达免疫检查点抗体；优选地，所述免疫检查点抗体包括以下任一种或多种：CTLA-4抗 体、PD-1抗体、PD-L1抗体；更优选地，所述抗体选是哺乳动物源抗体、人源化抗体、纳米抗体、嵌合抗体或其功能活性片段，所述哺乳动物包括人、羊、鼠、兔、猴、猩猩、狒狒、猪、牛、马、购、骆驼等。
  9. 根据权利要求8所述的抗体合成菌-纳米刺激剂杂合体系统，其是PD-1/PD-L1抗体合成菌-纳米刺激剂杂合体系统(NPs@Bacteria/PD-1/PD-L1)，或PD-1/PD-L1抗体合成菌Nissle 1917-纳米刺激剂杂合体系统(NPs@EcN/PD-1/PD-L1)，或PD-1/PD-L1抗体合成菌Nissle 1917-IL12纳米刺激剂杂合体系统(INPs@EcN/PD-1/PD-L1)；
    优选地，所述的抗体合成菌-纳米刺激剂杂合体系统为PD-1抗体合成菌Nissle 1917-IL12纳米刺激剂杂合体系统(INPs@EcN/PD-1)。
  10. 一种权利要求1-3任一项所述的共聚物的制备方法，包括如下步骤：通过Michael加成反应制备，称取适量1,6-己二醇二丙烯酸酯(HDDA)、组胺(His)与Mal-PEG n-NH 2或DBCO-PEG n-NH 2按摩尔比1:0.05～0.95:0.05～0.95的比例混合并溶解在适量体积的二甲基亚砜(DMSO)中，在惰性气体中35-85℃条件下反应16-72h，提纯并冷冻干燥得到聚合物粉末，经分析、鉴定获得所述共聚物Mal-poly(PEG n-co-His)或DBCO-poly(PEG n-co-His)。
  11. 根据权利要求10所述的方法，所述摩尔比优选为1:0.85:0.15；所述反应是在55℃条件下反应48h。
  12. 一种权利要求4所述的纳米刺激剂的制备方法，采用薄膜分散法或微乳液法制备，
    所述薄膜分散法包括如下步骤：
    (a)有机相中溶解所述共聚物，旋蒸除去有机相，得薄膜聚合物层，
    (b)将所述所述的免疫活性蛋白和PBS加入，探头超声获得免疫活性蛋白纳米粒子，
    (c)超滤或透析，得到具有Mal或DBCO修饰的核壳结构纳米粒子，即所述纳米刺激剂；
    所述微乳液法包括如下步骤：
    (A)有机相中溶解所述共聚物，加入聚乙烯醇(PVA)表面活性剂继续搅拌，
    (B)缓慢滴入免疫活性蛋白溶液，继续搅拌，加入PBS并于超声探头下超声，
    (C)超滤或透析，得到表面具有Mal或DBCO修饰的核壳结构纳米粒子，即所述纳米刺激剂。
  13. 一种抗体合成菌-纳米刺激剂杂合体系统(NPs@Bacteria/Ab)的制备方法，包括以下步骤：
    (a)将权利要求5-9任一项所述的抗体合成菌菌液收集在离心管中，离心并重悬于缓冲溶液中；
    (b)加入适量的标记活性基团反应，离心提取标记活性基团的抗体合成菌；
    (c)将权利要求4所述的纳米刺激剂加入到步骤(b)制备的标记活性基团的抗体合成菌中，在25-45℃下孵育0.5-4h，离心除去未偶联的纳米刺激剂，经检测，获得所述抗体合成菌-纳米刺激剂杂合体系统的混悬液。
  14. 根据权利要求13所述的方法，所述标记活性基团为巯基基团或叠氮基团；当所述所述标记活性基团为巯基基团时，所述纳米刺激剂为所述的Mal标记的免疫活性蛋白纳米刺激剂；当所述所述标记活性基团为叠氮基团时，所述纳米刺激剂为所述的DBCO标记的免疫活性蛋白纳米刺激剂。
  15. 根据权利要求13所述的方法，所述标记活性基团为TCEP或N 3-PEG-NHS；当所述所述标记活性基团为TCEP时，所述纳米刺激剂为所述的Mal标记的免疫活性蛋白纳米刺激剂；当所述所述标记活性基团为N 3-PEG-NHS时，所述纳米刺激剂为所述的DBCO标记的免疫活性蛋白纳米刺激剂。
  16. 根据权利要求13-15任一项所述的方法，所述抗体合成菌的构建方法包括：
    (a)将含有编码所述免疫检查点抗体的核苷酸序列的目的表达片段通过酶切、连接等步骤导入表达空载体，得到重组表达载体；
    (b)将所述重组表达载体转化到宿主细菌中，得到表达所述免疫检查点抗体的重组细菌
    (c)将步骤(b)得到的重组菌涂布在抗性培养基上筛选单克隆，经PCR阳性鉴定，测序，获得含有重组质粒的菌株，即表达所述免疫检查点抗体的抗体合成菌。
  17. 根据权利要求1-3任一项所述的共聚物、权利要求4所述的纳米刺激剂和/或权利要求5-9任一项所述的抗体合成菌-纳米刺激剂杂合体系统的应用，所述应用选自如下任一种或多种：
    (1)在制备药物组合物中的应用；
    (2)在制备用于细胞检测的试剂中的应用；
    (3)在制备用于疾病诊断或治疗的试剂中的应用；
    (4)在体外筛选药物中的应用；
    (5)在不以疾病诊断为目的体外检测中的应用。
  18. 根据权利要求17所述的应用，所述细胞包括正常细胞或肿瘤细胞；所述疾病包括肿瘤。
  19. 一种权利要求5-9任一项所述的抗体合成菌-纳米刺激剂杂合体系统或权利要求13-16任一项所述的方法在制备用于免疫治疗或检测的试剂或药物中的应用，所述免疫治疗或检测的对象包括肿瘤或疑似具有肿瘤特征的细胞、组织或人。
  20. 一种含有权利要求1-3任一项所述的共聚物、权利要求4所述的纳米刺激剂和/或权利要求5-9任一项所述的抗体合成菌-纳米刺激剂杂合体系统的组合物。
  21. 根据权利要求20所述的组合物，所述组合物是药物或检测试剂。所述组合物还含有药学或医学上可接受的载体或赋形剂。
  22. 根据权利要求17-21任一项所述药物或组合物，其还包括抗肿瘤化合物；优选地，所述抗肿瘤化合物包括抗肿瘤广谱药物和/或抗肿瘤靶向药物；更优选地，所述抗肿瘤广谱药物选自喜树碱类药物、阿霉素类药物、紫杉醇类药物或铂类药物中任一种或多种，所述抗肿瘤靶向药物选自泽布替尼、尼罗替尼、伊马替尼、维莫德吉、维罗非尼、替西罗莫司、舒尼替 尼、赛立替尼、瑞格非尼、阿法替尼、曲美替尼、普钠替尼、硼替佐米、帕唑帕尼、阿西替尼、罗米地辛、依维莫司、依鲁替尼、乐伐替尼、达拉菲尼、克唑替尼、卡非佐米、奥斯替尼、卡博替尼、卡比替尼、吉非替尼、伏立诺他、凡德他尼、艾乐替尼、狄诺塞麦、索尼德吉、索拉非尼、博舒替尼、贝利司他、奥拉帕尼、阿柏西普、拉帕替尼、达沙替尼、帕博西尼、帕比司他或厄洛替尼中任一种或多种。
  23. 根据权利要求17-21任一项所述药物或组合物，其还包括多肽类物质，所述多肽包括抗原或抗体；优选地，所述抗体选自阿达木单抗、西妥昔单抗、替伊莫单抗、曲妥珠单抗、纳武单抗、达雷木单抗雷莫芦单抗、耐昔妥珠单抗、派姆单抗、派姆单抗、奥法木单抗、博纳吐单抗、贝伐珠单抗、帕尼单抗、奥宾尤妥珠单抗、本妥昔单抗、地努图希单抗、托西莫单抗、埃罗妥珠单抗、曲妥珠单抗或利妥昔单抗中任一种或多种。
  24. 根据权利要求18-19所述的应用、权利要求22-23任一项所述的药物或组合物，所述肿瘤包括癌，所述癌包括基底细胞癌、鳞状细胞癌、食管癌、恶性胶质瘤、膀胱癌、宫颈癌、乳腺癌、肺癌、肝癌、胃癌、结肠癌、直肠癌、鼻咽癌、胰腺癌、甲状腺癌、前列腺癌、白血病、淋巴瘤、肾脏肿瘤、肉瘤、母细胞瘤、头癌、舌癌或口腔癌。
  25. 根据权利要求17-24任一项所述药物或组合物，所述药物或所述组合物是口服剂、注射剂、片剂、丸剂、散剂、混悬剂、颗粒剂、气雾剂、乳剂、分散剂、胶囊剂、膏剂、栓剂中任一种或多种；优选地，所述注射剂包括皮下注射、肌肉注射、腹腔注射、静脉注射、淋巴结内注射、瘤内注射或足下注射中任一种或多种。
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