WO2016165632A1 - 一种能联合免疫细胞增强肿瘤杀伤能力的双特异性抗体及其制备方法和应用 - Google Patents

一种能联合免疫细胞增强肿瘤杀伤能力的双特异性抗体及其制备方法和应用 Download PDF

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WO2016165632A1
WO2016165632A1 PCT/CN2016/079307 CN2016079307W WO2016165632A1 WO 2016165632 A1 WO2016165632 A1 WO 2016165632A1 CN 2016079307 W CN2016079307 W CN 2016079307W WO 2016165632 A1 WO2016165632 A1 WO 2016165632A1
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cancer
virus
antibody
tumor
bispecific antibody
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PCT/CN2016/079307
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English (en)
French (fr)
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于浩洋
李正成
苏静
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深圳市中联生物科技开发有限公司
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Application filed by 深圳市中联生物科技开发有限公司 filed Critical 深圳市中联生物科技开发有限公司
Priority to EP16779607.7A priority Critical patent/EP3290440B1/en
Priority to EP24172423.6A priority patent/EP4385572A2/en
Priority to US15/548,630 priority patent/US10758625B2/en
Priority to CN201680020916.0A priority patent/CN107531790A/zh
Publication of WO2016165632A1 publication Critical patent/WO2016165632A1/zh
Priority to US16/130,031 priority patent/US11000603B2/en
Priority to US16/935,679 priority patent/US20200345854A1/en
Priority to US17/224,288 priority patent/US20210338836A1/en

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Definitions

  • the invention relates to the field of immunomedicine, in particular to a bispecific antibody capable of combining immune cells to enhance tumor targeting killing ability, a preparation method and application thereof.
  • Immunocytotherapy has undergone the development of LAK, CIK, and DC-CIK, and induces T cells (DiKat), genetically modified DC-induced T cells (AV/LV-DC-CTL), and gene modification toward antigen-loaded DCs.
  • the chimeric antigen receptor T cell (CAR-T) direction is rapidly developing.
  • the clinical trial results of B lymphoma targeting the CD19 target announced by Novartis have reached a full efficiency of 93%, highlighting the immune cell therapy resistance.
  • the prospect of cancer is also prohibitively expensive.
  • the cost of each treatment is estimated to be 500,000 US dollars, and it is currently only good for CD19 positive B lymphoma. The limitations are obvious.
  • the invention provides a bispecific antibody, a preparation method and application thereof, in particular to a bispecific antibody capable of combining tumor cells to enhance tumor targeting killing ability, a preparation method and application thereof.
  • the present invention provides a bispecific antibody capable of enhancing tumor targeting killing ability in combination with an immune cell, the bispecific antibody comprising a first antibody portion and binding of an antigen expressed on an effector T cell A second antibody portion of an antigen expressed on a target cell.
  • the second antibody portion described in the present invention may be one or more antibodies, each of which may bind to a target cell.
  • the first antibody moiety and the second antibody moiety are linked by a nanomaterial.
  • the attachment method can be achieved by using a carboxyl group on the surface of the nanoparticle and an amino group of the antibody.
  • a “multispecific antibody” is one that can simultaneously bind at least two targets having different structures (eg, two different antigens, two different epitopes on the same antigen, or a hapten and/or an antigen or epitope).
  • a “bispecific antibody” is an antibody that can simultaneously bind two targets with different structures.
  • a “multispecific antibody” or “bispecific antibody” includes multiple or two antibodies (eg, two monoclonal antibodies) that bind to different targets linked by a nanomaterial.
  • the multispecific antibody or bispecific antibody described herein may have at least one antibody that specifically binds to T cells linked by a nanomaterial and at least one specific binding produced by or associated with a diseased cell, tissue, organ or pathogen
  • An antibody to a related antigen eg, a tumor associated antigen.
  • the invention adopts the principle of immunocellular cell targeted therapy, and utilizes the specific binding ability of the antibody to connect the antibody capable of specifically recognizing the tumor cell and the tumor killer cell with the clinically usable degradable nano material. Come to form a bispecific antibody.
  • anti-cancer monoclonal antibodies most anti-cancer monoclonal antibodies are based on their inherent biological functions including: complement-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity. (ADCC) apoptosis induction, opsonophagocytosis and the like.
  • the bispecific antibody of the present invention has a side that specifically recognizes and binds to tumor cells, and the other side recognizes and binds to killer T lymphocytes, thereby helping T cells recognize tumor cells and pull them closer between tumor cells and killer T lymphocytes.
  • the physical distance facilitates the killing of tumor cells by T lymphocytes and serves to enhance the specificity and effectiveness of immune cell therapy.
  • the nanomaterials in the present invention are biodegradable nanomaterials.
  • the nanomaterials described in the present invention are generally present in the form of nanoparticles selected from conventional degradable nanomaterials, such as polylactic acid-glycolic acid, polylactic acid, polycaprolactone, and polybutylene glycol. Any one or a mixture of at least two of a diester, a polyaniline, a polycarbonate, an ethylene lactide copolymer or a glycolide caprolactone copolymer is preferred, but not limited thereto.
  • conventional degradable nanomaterials such as polylactic acid-glycolic acid, polylactic acid, polycaprolactone, and polybutylene glycol.
  • Any one or a mixture of at least two of a diester, a polyaniline, a polycarbonate, an ethylene lactide copolymer or a glycolide caprolactone copolymer is preferred, but not limited thereto.
  • the invention adopts the advantages that the nano material has sustained release and good biocompatibility, for example, PLGA is a medicinal excipient approved by the FDA, has good biocompatibility and biodegradability, is non-toxic, non-irritating, and has high strength. It is easy to process and shape, and the final decomposition product is hydrolyzed by carbon dioxide in the biological problem. It can be completely absorbed after being placed in the body for 3-6 months.
  • PLGA is a medicinal excipient approved by the FDA, has good biocompatibility and biodegradability, is non-toxic, non-irritating, and has high strength. It is easy to process and shape, and the final decomposition product is hydrolyzed by carbon dioxide in the biological problem. It can be completely absorbed after being placed in the body for 3-6 months.
  • the target cell is a B cell, a cancer cell or a pathogen cell or the like.
  • the antigen expressed on the target cell is carbonic anhydrase IX, alpha-fetoprotein, ⁇ -actinin-4, A3, A33 antibody-specific antigen, ANG2, ART-4, B7, Ba 733, BAGE , BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27
  • hyorhinis M. orale, M. arginini, Acholeplasma laidlawii, Any one of M. salivarium and M. pneumoniae, preferably CD19, CD20, CD22, CD30, CD33, CD38, CD123, Muc1, Muc16, HER2, HER3, EGFRvIII, VEGFA, Any of CEA, GPA33, GP100, ANG2, L1CAM, ROR-1, CS1, MICA or MICB.
  • the antigen expressed on the effector T cells is ADAM17, CD2, CD3, CD4, CD5, CD6, CD8, CD11a, CD11b, CD14, CD16, CD16b, CD25, CD28, CD30, CD32a, CD40, CD40L, CD44 Any one of CD45, CD56, CD57, CD64, CD69, CD74, CD89, CD90, CD137, CD177, CEACAM6, CEACAM8, HLA-DR ⁇ chain, KIR, LSECtin or SLC44A2, preferably CD2, CD3, CD4, Any of CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69 or CD90.
  • the present invention provides a method of producing a bispecific antibody according to the first aspect of the present invention, which comprises linking a nanomaterial to a first antibody moiety and a second antibody moiety.
  • the method for preparing a bispecific antibody according to the present invention comprises the following steps:
  • the nanomaterial obtained in the step (1) is linked to a mixture of the first antibody portion and the second antibody portion.
  • the preparation of the nano material comprises: completely dissolving the nano material by using a solvent, stirring, adding water to form a uniform emulsion.
  • the nano material is polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polybutylene glycol succinate, polyaniline, polycarbonate, ethylene lactide copolymer or glycolide Any one or a mixture of at least two of the ester copolymers.
  • the solvent is any one of acetone, methyl ethyl ketone, methanol, ethanol or isopropyl alcohol or a mixture of at least two.
  • the collecting of the nanomaterial comprises: collecting the prepared nanomaterial by centrifugation, then resuspending it with deionized water, and repeating the operation of washing the nanomaterial twice.
  • the activation of the nanomaterial comprises: using 1-10 mg/mL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDS) and N-hydroxysuccinyl
  • EDS 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinyl
  • the amine (NHS) mixed solvent activates the nanomaterial at room temperature for 0.5-5 h.
  • the connecting comprises: centrifuging the activated nano material, and then washing the nano material once with the ligation reaction solution, mixing the first antibody portion and the second antibody portion to be connected in equal amounts. Adding to the ligation reaction solution, and then resuspending the nano material with the ligation reaction solution containing the first antibody portion and the second antibody portion, and connecting the reaction at room temperature for 0.5-5 h. After the reaction is finished, the nano material is collected by centrifugation, and the buffer solution of Du's phosphate is used. The nanomaterial was washed twice and then resuspended in Dun's phosphate buffer solution (D-PBS) and stored at 4 ° C until use.
  • D-PBS Dun's phosphate buffer solution
  • the method for preparing the bispecific antibody of the present invention specifically comprises the following steps:
  • nanomaterials are completely dissolved to a concentration of 5 to 30 mg/mL by acetone, and the nanometers are magnetically stirred at 500 to 1500 rpm/min according to a volume ratio of acetone to deionized water of 1:4. A solution of the material and acetone is added to the deionized water to form a uniform emulsion, and then stirring is continued until the acetone is volatilized;
  • the present invention provides the use of the bispecific antibody according to the first aspect, in the manufacture of a medicament for treating, preventing or diagnosing a tumor.
  • the tumor includes, but is not limited to, liver cancer, non-small cell lung cancer, small cell lung cancer, adrenocortical carcinoma, acute (chronic B) lymphoma, myeloma, prostate cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, Cervical cancer, kidney cancer, bladder cancer and lymphoma.
  • the present invention provides a bispecific antibody according to any one of claims 1 to 3 for use in the treatment or prevention of a tumor.
  • the tumor includes, but is not limited to, liver cancer, non-small cell lung cancer, small cell lung cancer, adrenocortical carcinoma, acute (chronic B) lymphoma, myeloma, prostate cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, Cervical cancer, kidney cancer, bladder cancer and lymphoma.
  • the invention provides a method of treating a tumor, the method comprising administering to a subject a bispecific antibody of the invention.
  • the tumor includes, but is not limited to, liver cancer, non-small cell lung cancer, small cell lung cancer, adrenocortical carcinoma, acute (chronic B) lymphoma, myeloma, prostate cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, Cervical cancer, kidney cancer, bladder cancer and lymphoma.
  • the present invention has at least the following beneficial effects:
  • the bispecific antibody prepared by the invention has the characteristics of being faster, simpler and more practical, and can directly apply the monoclonal antibody that has been approved for marketing on the market. It is used for connection and practical use. Because the raw materials used are all clinically approved and listed, it can be To quickly push to clinical use;
  • the bispecific antibody of the invention has biodegradability, no genetic recombination, small side effects, high safety and low cost.
  • the specific antibody corresponding to different tumor cells can be combined, and the utility model has the advantages of convenient use and the like, and only needs to simultaneously add the bispecific antibody preparation of the invention to the CTL cell infusion induced in vitro to achieve similar targeting and high-efficiency killing.
  • the purpose of tumor cells, and the side effects produced are smaller than CAR-T therapy;
  • All materials of the present invention can be degraded into non-toxic and harmless products in the human body, and can be degraded and metabolized in a short period of time, and thus have higher safety than CAR-T.
  • Example 1 is a schematic view showing the assembly of the bispecific antibody of Example 1.
  • Example 2 is a result of evaluation of a cell killing experiment of the bispecific antibody of Example 1.
  • Figures 3-9 show the ability of other bispecific antibodies to kill tumor cells.
  • Figure 10 shows the anti-tumor efficacy of the bispecific antibody against CD3-PLGA-anti-MUCl in vivo.
  • Figure 11 is a graph showing the mortality of experimental animals in an in vivo anti-tumor experiment.
  • FIG. 1 is a schematic view showing the assembly of the bispecific antibody of this embodiment, and the specific steps of the preparation are as follows:
  • PLGA was completely dissolved in acetone to a concentration of 5 mg/mL, and a solution of PLGA and acetone was magnetically stirred at 1000 rpm/min in a volume ratio of acetone to deionized water of 1:4. Add deionized water to form a uniform emulsion, and then continue to stir until the acetone is evaporated;
  • Nanoparticles with larger particles were collected by centrifugation at 8000 rpm/min for 10 min; then smaller particles were collected by centrifugation at 15000 rpm/min for 10 min, and then resuspended in deionized water, respectively.
  • the nanoparticle is washed twice, and the larger particle nanoparticle and the smaller particle nanoparticle are respectively subjected to the following operations;
  • the evaluation results are shown in Figure 2, where * There is a significant difference in the representative T test.
  • a in Fig. 2 represents a nanoparticle-linked bispecific antibody of a larger particle (i.e., a particle collected by centrifugation at a rotational speed of 8000 rpm/min, 10 min), wherein B represents a smaller particle (i.e., a rotational speed of 8000 rpm) /min, 10 min without centrifugation, 15000 rpm / min, 10 min centrifugation of the resulting particles of the nanoparticle-linked bispecific antibody.
  • a larger particle i.e., a particle collected by centrifugation at a rotational speed of 8000 rpm/min, 10 min
  • B represents a smaller particle (i.e., a rotational speed of 8000 rpm) /min, 10 min without centrifugation, 15000 rpm / min, 10 min centrifugation of the resulting particles of the nanoparticle-linked bispecific antibody.
  • the kill rate of the control group was 7.9%; the kill rate of the larger particle nanoparticle-linked bispecific antibody group was 24.5%; the smaller particle of the nanoparticle-linked bispecific antibody The kill rate is 40.7%.
  • a bispecific antibody capable of combining immune cells to enhance tumor targeting killing ability was prepared.
  • the preparation method is as follows:
  • PLA was completely dissolved in acetone to a concentration of 15 mg/mL, and a solution of PLA and acetone was magnetically stirred at 500 rpm/min in a volume ratio of acetone to deionized water of 1:4. Add deionized water to form a uniform emulsion, and then continue to stir until the acetone is evaporated;
  • a bispecific antibody capable of combining immune cells to enhance tumor targeting killing ability was prepared.
  • the preparation method is as follows:
  • PCL was completely dissolved in acetone to a concentration of 30 mg/mL, and a solution of PCL and acetone was stirred at 1500 rpm/min in a volume ratio of acetone to deionized water of 1:4. Add deionized water to form a uniform emulsion, and then continue to stir until the acetone is evaporated;
  • anti-CD3 and anti-MUC1 bispecific antibodies (anti-CD3-PLGA-anti-MUC1) linked by PLGA to kill other tumor cells was evaluated.
  • the original medium was discarded, and the DC-CIK cell density was adjusted by using the cytokine-free X-vivo 15 medium to make a 100 ⁇ l volume DC-CIK.
  • the number of cells was 4 times that of the target cells (the effective target ratio was 4:1), and 100 ⁇ l of DC-CIK cell suspension was added to the cancer cell culture plate, and 10 ⁇ l (bispecific antibody preparation content of 0.2 mg, containing the monoclonal antibody component) was added.
  • Killing rate [1 - (experimental group - effect control group) / (target control group - blank control group)] ⁇ 100%
  • the blank control indicates the addition of the medium
  • the target control group indicates the addition of the target cells + medium
  • the effect control group indicates the addition of the effector cells + medium
  • the experimental group indicates the effector cells + target cells + medium + bispecific antibody.
  • HKC-human embryonic kidney epithelial cells A549-human non-small cell lung cancer cells; HepG2-human liver cancer cells; HT29-human colon cancer cells; AGS-human gastric adenocarcinoma cells; Hela-human cervical cancer cells; 5637-human bladder Cancer cells; OS-RC-2-human kidney cancer cells.
  • Anti-CD3 and anti-CD33 bispecific antibodies anti-CD3-PLGA-anti-CD33, anti-CD3-PLA-anti-CD33 and anti-CD-linked anti-CD33, anti-CD33 and anti-CD33 were prepared by nanoparticle-based methods using PLGA, PLA or PCL as nanomaterials. CD3-PCL-anti-CD33. The ability of the bispecific antibody of the present invention to kill tumor cells was evaluated according to the method described in Example 5.
  • the bispecific antibodies CD3-PLA-CD33 and CD3-PCL-CD33 also showed a killing rate of 83% or more for black Burkitt lymphoma cells and human B lymphocyte tumor cells.
  • Nanoparticle anti-CD8-PLGA-anti-MUCl, CD8-PLA-MUC1 and anti-CD8-PCL-linked with anti-CD8 antibody and anti-MUC1 antibody were prepared using PLGA, PLA or PCL as nanomaterials using a method similar to the above.
  • the ability of the bispecific antibody of the present invention to kill tumor cells was evaluated by the method described in Example 5.
  • HKC-human embryonic kidney epithelial cells A549-human non-small cell lung cancer cells; HepG2-human liver cancer cells; HT29-human colon cancer cells; AGS-human gastric adenocarcinoma cells; Hela-human cervical cancer cells; 5637-human bladder Cancer cells; OS-RC-2-human kidney cancer cells.
  • Bispecific antibody anti-CD8-PLA-anti-MUC1 and anti-CD8-PCL-anti-MUC1 showed for human Non-small cell lung cancer cells and human gastric adenocarcinoma cells have a kill rate of more than 95%; both human colon cancer cells and human kidney cancer cells have a kill rate of more than 92%; for human breast cancer cells, human cervical cancer cells and humans Bladder cancer cells all show a killing rate of nearly 90%, and the killing rate for human liver cancer cells is also about 80%.
  • bispecific antibodies linked by nanoparticles were prepared using methods similar to those described above: anti-CD3-PLGA-anti-CD19, anti-CD3-PLGA-anti-CD20, anti-CD3-PLGA-anti-her2, against these killing tumor cells The ability was evaluated. The results are shown in Tables 4-7 and Figures 6-9.
  • human lung adenocarcinoma A549 was inoculated under healthy armpits in healthy Balb-c nude mice (clean grade, female, four weeks old, body weight 18-22 g, purchased from Guangdong Medical Laboratory Animal Center), after inoculation for 3-4 weeks. Thirty nude mice with a tumor diameter of about 0.5 x 0.5 cm were randomly divided into 3 groups for experiments.
  • the DC-CIK group was injected with 1 ⁇ 10 6 number of DC-CIK cells each time, and the DC-CIK+ double-antibody group was injected 1 ⁇ each time. 10 6 number of DC-CIK cells and 0.4 mg (0.4 ⁇ g of monoclonal antibody total) bispecific antibody anti-CD3-PLGA-anti-MUC1, and the control group was injected with the same volume of physiological saline.
  • the volume of the recorded tumor was measured from the first day. The rate of inhibition of tumor by bispecific antibodies was calculated.
  • the bispecific antibody of the present invention kills cancer cells by T cells.
  • the ability has been improved, combined with immune cells to enhance tumor killing ability, compared with CAR-T immune cell therapy, has the characteristics of small side effects, high safety, low cost and convenient use.

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Abstract

本发明提供了一种能联合免疫细胞增强肿瘤靶向杀伤能力的双特异性抗体及其制备方法和应用。通过采用化学方法连接抗体和可降解的纳米颗粒，一个纳米颗粒上同时连接两种或两种以上多个抗体分子，其中一种抗体能够特异结合免疫细胞，另一种或几种抗体可以特异结合肿瘤细胞，从而起到增强免疫细胞靶向特异杀伤肿瘤细胞的效果。

Description

一种能联合免疫细胞增强肿瘤杀伤能力的双特异性抗体及其制备方法和应用 技术领域
本发明涉及免疫医学领域，尤其涉及一种能联合免疫细胞增强肿瘤靶向杀伤能力的双特异性抗体及其制备方法和应用。
背景技术
2008年《新英格兰医学杂志》报道1例晚期黑色素瘤的免疫治疗取得成功，该患者为全身多处转移病例，经自身CD4+T细胞治疗后所有病灶消失，经随访26个月长期生存。2010年FDA批准Dendreon Corporation(丹德里昂公司)自体免疫细胞治疗技术用于前列腺癌临床应用，2011年的诺贝尔医学奖也授予了从事肿瘤免疫治疗相关的3位科学家，预示了免疫治疗在恶性肿瘤治疗领域的广阔前景。
近几年，利用基因修饰诱导的T细胞免疫治疗肿瘤取得了良好的疗效，为肿瘤的免疫细胞治疗发展指明了新的方向。预示了免疫治疗在恶性肿瘤治疗领域的广阔前景。
免疫细胞治疗技术经历了LAK、CIK、DC-CIK的发展历程，并朝着抗原负载的DC诱导T细胞(DiKat)、基因修饰的DC诱导T细胞(AV/LV-DC-CTL)以及基因修饰的嵌合抗原受体T细胞(CAR-T)方向飞速发展，目前由诺华公布的针对CD19靶点的B淋巴瘤临床实验结果宣布完全有效率达到了93％，更是突出了免疫细胞疗法抗癌的前景。然而CAR-T因其细胞复杂的制备过程，高额的费用也同时让人望而却步，其每个疗程的成本预计是50万美元，并且目前也只是针对CD19阳性B淋巴瘤体现了良好疗效，其局限性显而易见。
如何研发一种可以加强免疫细胞疗法的特异性和有效性，并具有简单、方 便使用等优点的免疫细胞治疗技术是目前亟待解决的问题。
发明内容
本发明提供了一种双特异性抗体及其制备方法和应用，特别是一种能联合免疫细胞增强肿瘤靶向杀伤能力的双特异性抗体及其制备方法和应用。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了一种能联合免疫细胞增强肿瘤靶向杀伤能力的双特异性抗体，所述的双特异性抗体包括结合在效应T细胞上表达的抗原的第一抗体部分和结合在靶细胞上表达的抗原的第二抗体部分。
本发明中所述的第二抗体部分可以是一种或多种抗体，其均可以结合靶细胞。
本发明中，所述第一抗体部分和第二抗体部分通过纳米材料进行连接。其连接方式可采用利用纳米颗粒表面的羧基和抗体的氨基连接。
“多特异性抗体”是可以同时结合至少两个具有不同结构的靶标(例如，两个不同的抗原、在同一抗原上的两个不同表位、或半抗原和/或抗原或表位)的抗体。“双特异性抗体”是可以同时结合两个具有不同结构的靶标的抗体。本文所述的“多特异性抗体”或“双特异性抗体”包括通过纳米材料连接的结合不同靶标的多种或两种抗体(例如两种单克隆抗体)。本文所述的多特异性抗体或双特异性抗体可以具有通过纳米材料连接的至少一种特异性结合T细胞的抗体和至少一种特异性结合由患病细胞、组织、器官或病原体产生或与其相关的抗原(例如肿瘤相关抗原)的抗体。
本发明以免疫细胞靶向治疗为原则，利用抗体的特异性结合能力，将能够特异识别肿瘤细胞和肿瘤杀伤细胞的抗体用临床可用的降解的纳米材料连接起 来，从而形成双特异性抗体。与抗癌单抗相比较，大多数抗癌单抗作为治疗疾病的药物主要基于其固有的生物学功能包括：补体介导的细胞毒性作用(CDC)、抗体依赖的细胞介导的细胞毒作用(ADCC)凋亡诱导、调理吞噬等。本发明的双特异性抗体是其一边特异性识别并结合肿瘤细胞，另一边识别并结合杀伤性T淋巴细胞，从而帮助T细胞识别肿瘤细胞，并拉近肿瘤细胞和杀伤性T淋巴细胞之间的物理距离，方便T淋巴细胞杀伤肿瘤细胞，起到加强免疫细胞疗法的特异性和有效性的目的。
本发明中所述纳米材料为生物可降解纳米材料。
本发明中所述的纳米材料通常是以纳米颗粒的形式而存在，选自常规的可降解的纳米材料，例如可以是聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物，优选但不限于此。
本发明采用纳米材料具有缓释、良好的生物相容性等优点，例如PLGA是美国FDA批准的药用辅料，具有良好生物相容性和生物可降解性，无毒、无刺激性，强度高，易加工成型，在生物题内经酶解最终分解产物为水合二氧化碳，置入体内后3-6个月可完全吸收。
本发明中，所述靶细胞是B细胞、癌细胞或病原体细胞等。
优选地，所述靶细胞上表达的抗原为碳酸酐酶IX、甲胎蛋白、α-辅肌动蛋白-4、A3、A33抗体特异性抗原、ANG2、ART-4、B7、Ba 733、BAGE、BrE3-抗原、CA125、CAMEL、CAP-1、CASP-8/m、、CCCL19、CCCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、 CD38、CD40、CD40L、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD79b、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CS1、CXCR4、CXCR7、CXCL12、HIF-1α、结肠特异性抗原-p(CSAp)、CEA(CEACAM5)、CEACAM6、c-met、DAM、EGFR、EGFRvIII、EGP-1、EGP-2、ELF2-M、Ep-CAM、Flt-1、Flt-3、叶酸受体、G250抗原、GAGE、gp100、GPA33、GROB、HLA-DR、HM1.24、人绒毛膜促性腺激素(HCG)和其亚单位、HER2/neu、HER3、HMGB-1、低氧可诱导因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IL-2、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、IL-33、胰岛素样生长因子-1(IGF-1)、KC4-抗原、KS-1-抗原、KS1-4、L1CAM、Le-Y、LDR/FUT、巨噬细胞迁移抑制因子(MIF)、MAGE、MAGE-3、MART-1、MART-2、NY-ES0-1、TRAG-3、mCRP、MCP-1、MICA、MICB、MIP-1A、MIP-1B、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、胰腺癌粘蛋白、胎盘生长因子、p53、PLAGL2、前列腺酸性磷酸酶、PSA、PRAME、PSMA、P1GF、ILGF、ILGF-1R、IL-6、IL-25、ROR-1、RS5、RANTES、T101、SAGE、S100、存活蛋白、存活蛋白-2B、TAC、TAG-72、腱生蛋白、TRAIL受体、TNF-α、Tn抗原、汤姆森-弗里德里希抗原、肿瘤坏死抗原、TROP-2、VEGFA、VEGFR、ED-B纤连蛋白、WT-1、17-1A-抗原、补体因子C3、C3a、C3b、C5a、C5、血管生成标记物、bc1-2、bc1-6、Kras、cMET、致癌基因产物、HIV病毒、结核分枝杆菌(mycobacterium tuberculosis)、无乳链球菌(Streptococcus agalactiae)、抗甲氧西林性金黄色葡萄球菌(meticillin-resistant Staphylococcus aureus)、嗜肺军团菌(Legionella pneumophilia)、 酿脓链球菌(Streptococcus pyogenes)、大肠杆菌(Escherichia coli)、淋病奈瑟氏菌(Neisseria gonorrhoeae)、脑膜炎奈瑟氏菌(Neisseria meningitidis)、肺炎球菌(Pneumococcus)、新型隐球菌(Cryptococcus neoformans)、荚膜组织胞浆菌(Histoplasma capsulatum)、B型流感嗜血杆菌(Hemophilis influenzae B)、苍白密螺旋体(Treponema pallidum)、莱姆病螺旋体(Lyme disease spirochetes)、铜绿假单胞菌(Pseudomonas aeruginosa)、麻风分枝杆菌(Mycobacterium leprae)、流产布鲁氏菌(Brucella abortus)、狂犬病病毒、流感病毒、巨细胞病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、人血清细小样病毒、呼吸道合胞病毒、水痘-带状疱疹病毒、乙型肝炎病毒、丙型肝炎病毒、麻疹病毒、腺病毒、人T细胞白血病病毒、埃-巴二氏病毒、鼠白血病病毒、腮腺炎病毒、水泡性口膜炎病毒、辛德毕斯病毒、淋巴细胞性脉络丛脑膜炎病毒、疣病毒、蓝舌病病毒、仙台病毒、猫白血病毒、呼肠孤病毒、脊髓灰质炎病毒、猿猴病毒40、小鼠乳腺瘤病毒、登革热病毒、风疹病毒、西尼罗河病毒、恶性疟原虫(Plasmodium falciparum)、间日疟原虫(Plasmodium vivax)、刚地弓形虫(Toxoplasma gondii)、让氏锥虫(Trypanosoma rangeli)、克氏锥虫(Trypanosoma cruzi)、罗德西亚锥虫(Trypanosoma rhodesiensei)、布氏锥虫(Trypanosoma brucei)、曼森氏裂体吸虫(Schistosoma mansoni)、日本裂体吸虫(Schistosoma japonicum)、牛巴贝虫(Babesia bovis)、艾美耳球虫(Elmeria tenella)、旋盘尾丝虫(Onchocerca volvulus)、热带利什曼虫(Leishmania tropica)、旋毛虫(Trichinella spiralis)、小泰勒虫(Theileria parva)、泡状带绦虫(Taenia hydatigena)、羊绦虫(Taenia ovis)、牛肉绦虫(Taenia saginata)、细粒棘球绦虫(Echinococcus granulosus)、科氏中殖孔绦虫(Mesocestoides corti)、关节炎支原体(Mycoplasma arthritidis)、鼻支原体(M.hyorhinis)、口腔支原体(M.orale)、精氨酸支原体(M.arginini)、拉氏无胆甾原体(Acholeplasma laidlawii)、 唾液支原体(M.salivarium)和肺炎支原体(M.pneumoniae)中的任意一种，优选地为CD19、CD20、CD22、CD30、CD33、CD38、CD123、Muc1、Muc16、HER2、HER3、EGFRvIII、VEGFA、CEA、GPA33、GP100、ANG2、L1CAM、ROR-1、CS1、MICA或MICB中的任意一种。
优选地，所述效应T细胞上表达的抗原为ADAM17、CD2、CD3、CD4、CD5、CD6、CD8、CD11a、CD11b、CD14、CD16、CD16b、CD25、CD28、CD30、CD32a、CD40、CD40L、CD44、CD45、CD56、CD57、CD64、CD69、CD74、CD89、CD90、CD137、CD177、CEACAM6、CEACAM8、HLA-DRα链、KIR、LSECtin或SLC44A2中的任意一种，优选地为CD2、CD3、CD4、CD5、CD6、CD8、CD25、CD28、CD30、CD40、CD40L、CD44、CD45、CD69或CD90中的任意一种。
第二方面，本发明还提供了如本发明第一方面所述的双特异性抗体的制备方法，其包括将纳米材料与第一抗体部分和第二抗体部分连接。
本发明中所述的双特异性抗体的制备方法，其包括以下步骤：
(1)纳米材料的制备、收集和活化；
(2)将步骤(1)得到的纳米材料与第一抗体部分和第二抗体部分的混合物进行连接。
本发明的步骤(1)中，所述纳米材料的制备包括：利用溶剂将纳米材料完全溶解，搅拌，加水，形成均匀的乳浊液。
优选地，所述纳米材料为聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物。
优选地，所述溶剂为丙酮、丁酮、甲醇、乙醇或异丙醇中的任意一种或至少两种的混合物。
优选地，所述纳米材料的收集包括：离心收集制备的纳米材料，然后用去离子水重悬，重复操作2次洗涤纳米材料。
优选地，所述纳米材料的活化包括：利用1-10mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDS)和N-羟基琥珀酰亚胺(NHS)混合溶剂室温活化纳米材料0.5-5h。
本发明的步骤(2)中，所述连接包括：将活化后的纳米材料离心收集，然后用连接反应液洗涤纳米材料1次，将需要连接的第一抗体部分和第二抗体部分等量混合加入到连接反应液，然后用含有所述第一抗体部分和第二抗体部分的连接反应液重悬纳米材料，室温连接反应0.5-5h，反应结束后离心收集纳米材料，用杜氏磷酸盐缓冲溶液洗涤纳米材料2次，然后再重悬到杜氏磷酸盐缓冲溶液(D-PBS)中放4℃保存备用。
本发明中所述双特异性抗体的制备方法，具体包括以下步骤：
(1)纳米材料的制备：利用丙酮将纳米材料完全溶解至浓度为5～30mg/mL，按照丙酮和去离子水1∶4的体积比，在500～1500rpm/min磁力搅拌的状态下将纳米材料与丙酮的溶液加入去离子水中，形成均匀的乳浊液，然后继续搅拌至丙酮挥发；
(2)纳米材料的收集：8000～15000rpm/min离心收集制备的纳米材料，然后用去离子水重悬，重复操作2次洗涤纳米材料；
(3)纳米材料的活化：利用1～10mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合溶剂室温活化纳米材料0.5～5h；
(4)纳米材料连接抗体：将活化后的纳米材料离心收集，然后用0.1M、pH＝8.0的杜氏磷酸盐缓冲溶液洗涤纳米材料1次，将需要连接的第一抗体部分和第二抗体部分等量混合加入到连接反应液，然后用含有所述第一抗体部分和第二抗体部分的连接反应液重悬纳米材料，室温连接反应0.5～5h，反应结束后离心收集纳米材料，用杜氏磷酸盐缓冲溶液洗涤纳米材料2次，然后再重悬到杜氏磷酸盐缓冲溶液中放4℃保存备用。
第三方面，本发明还提供了如第一方面所述的双特异性抗体在制备治疗、预防或诊断肿瘤的药物中的应用。所述肿瘤包括但不限于：肝癌、非小细胞肺癌、小细胞肺癌、肾上腺皮质癌、急性(慢性B)淋巴细胞瘤、骨髓瘤、前列腺癌、乳腺癌、食管癌、胃癌、结直肠癌、宫颈癌、肾癌、膀胱癌和淋巴瘤。
第四方面，本发明还提供用于治疗或预防肿瘤的根据权利要求1-3任一项所述的双特异性抗体。所述肿瘤包括但不限于：肝癌、非小细胞肺癌、小细胞肺癌、肾上腺皮质癌、急性(慢性B)淋巴细胞瘤、骨髓瘤、前列腺癌、乳腺癌、食管癌、胃癌、结直肠癌、宫颈癌、肾癌、膀胱癌和淋巴瘤。
第五方面，本发明提供治疗肿瘤的方法，所述方法包括给受治疗者施用本发明的双特异性抗体。所述肿瘤包括但不限于：肝癌、非小细胞肺癌、小细胞肺癌、肾上腺皮质癌、急性(慢性B)淋巴细胞瘤、骨髓瘤、前列腺癌、乳腺癌、食管癌、胃癌、结直肠癌、宫颈癌、肾癌、膀胱癌和淋巴瘤。
与现有技术相比，本发明至少具有以下有益效果：
(1)与现有的采用生物法表达双特异性抗体相比，本发明制备的双特异性抗体具有更加快捷、工艺更简单实用等特点，其可以将市面上已经审批上市的单抗药物直接拿来连接实用，因所使用的原料全是临床已经审批上市的，故可 以快速推向临床使用；
(2)与基因修饰的嵌合抗原受体T细胞(CAR-T)免疫细胞疗法相比，本发明的双特异性抗体具有生物可降解，无基因重组，副作用小，安全性高，成本低，可针对不同肿瘤细胞组合对应的特异性抗体，使用方便等优点，只需在体外诱导的CTL细胞回输时同时加入本发明的双特异性抗体制剂即可起到相似的靶向和高效杀伤肿瘤细胞的目的，并且产生的副作用比CAR-T疗法小；
(3)本发明的所有材料在人体内均可降解成无毒无害产物，并且可以在短期内降解代谢，因而比CAR-T的安全性要高。
附图说明
图1是实施例1的双特异性抗体的组装示意图。
图2是实施例1的双特异性抗体的细胞杀伤实验评估结果。
图3-9显示了其他双特异性抗体杀伤肿瘤细胞的能力。
图10显示了双特异性抗体抗CD3-PLGA-抗MUC1体内抗肿瘤的效力。
图11为体内抗肿瘤实验的实验动物死亡曲线。
具体实施方式
以下将结合附图，通过具体实施方式对本发明进行详细描述。
实施例
实施例1
图1是该实施例的双特异性抗体的组装示意图，其制备的具体步骤如下：
(1)PLGA纳米颗粒的制备：利用丙酮将PLGA完全溶解至浓度为5mg/mL，按照丙酮和去离子水1∶4的体积比，在1000rpm/min磁力搅拌的状态下将PLGA与丙酮的溶液加入去离子水中，形成均匀的乳浊液，然后继续搅拌至丙酮挥发；
(2)PLGA纳米颗粒的收集：8000rpm/min离心10min收集制备得到较大颗粒的纳米颗粒；再用15000rpm/min离心10min收集得到较小颗粒的纳米颗粒，然后分别用去离子水重悬，重复操作2次洗涤纳米颗粒，较大颗粒的纳米颗粒和较小颗粒的纳米颗粒分别进行如下操作；
(3)PLGA纳米颗粒的活化：利用5mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合溶剂室温活化PLGA纳米颗粒1h；
(4)PLGA纳米颗粒连接抗体：将活化后的纳米材料离心收集，然后用0.1M、pH＝8.0的杜氏磷酸盐缓冲溶液洗涤纳米材料1次，将需要连接的CD3和Muc1单抗等量混合加入到连接反应液，然后用含有所述第一抗体部分和第二抗体部分的连接反应液重悬纳米材料，室温连接反应0.5h，反应结束后离心收集纳米材料，用杜氏磷酸盐缓冲溶液洗涤纳米材料2次，然后再重悬到杜氏磷酸盐缓冲溶液中放4℃保存备用。
实施例2
将实施例1的双特异性抗体加入T细胞和癌细胞MCF-7杀伤实验体系中，检测该双特异性抗体对T细胞杀伤癌细胞能力的提升情况，其中T细胞∶MCF-7＝4∶1，反应时间为8h，其中对照组为加等量纳米颗粒(未连接任何抗体)，结果采用3次独立重复实验结果平均值±标准差表示，其评估结果如图2所示，其中，*代表T检验比较具有显著差异。
其中，图2中的A表示较大颗粒(即采用转速为8000rpm/min，10min离心收集得到的颗粒)的纳米颗粒连接的双特异性抗体，其中的B表示较小颗粒(即采用转速为8000rpm/min，10min未离心下来，15000rpm/min，10min离心得到的颗粒)的纳米颗粒连接的双特异性抗体。
从图2可以看出，其中对照组的杀伤率为7.9％；较大颗粒的纳米颗粒连接的双特异性抗体组的杀伤率为24.5％；较小颗粒的纳米颗粒连接的双特异性抗体的杀伤率为40.7％。
实施例3
以PLA为纳米材料，制备了能联合免疫细胞增强肿瘤靶向杀伤能力的双特性抗体，其制备方法如下：
(1)PLA纳米颗粒的制备：利用丙酮将PLA完全溶解至浓度为15mg/mL，按照丙酮和去离子水1∶4的体积比，在500rpm/min磁力搅拌的状态下将PLA与丙酮的溶液加入去离子水中，形成均匀的乳浊液，然后继续搅拌至丙酮挥发；
(2)PLA纳米颗粒的收集：8000rpm/min离心收集制备的纳米颗粒，然后用去离子水重悬，重复操作2次洗涤纳米颗粒；
(3)PLA纳米颗粒的活化：利用1mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合溶剂室温活化PLA纳米颗粒0.5h；
(4)PLA纳米颗粒连接抗体：将活化后的纳米材料离心收集，然后用0.1M、pH＝8.0的杜氏磷酸盐缓冲溶液洗涤纳米材料一次，将需要连接的CD3单抗和CD19单抗等量混合加入到连接反应液，然后用含有所述第一抗体部分和第二抗体部分的连接反应液重悬纳米材料，室温连接反应5h，反应结束后离心收集纳米材料，用杜氏磷酸盐缓冲溶液洗涤纳米材料2次，然后再重悬到杜氏磷酸盐缓冲溶液中放4℃保存备用。
实施例4
以PCL为纳米材料，制备了能联合免疫细胞增强肿瘤靶向杀伤能力的双特性抗体，其制备方法如下：
(1)PCL纳米颗粒的制备：利用丙酮将PCL完全溶解至浓度为30mg/mL，按照丙酮和去离子水1∶4的体积比，在1500rpm/min磁力搅拌的状态下将PCL与丙酮的溶液加入去离子水中，形成均匀的乳浊液，然后继续搅拌至丙酮挥发；
(2)PCL纳米颗粒的收集：15000rpm/min离心收集制备的纳米颗粒，然后用去离子水重悬，重复操作2次洗涤纳米颗粒；
(3)PCL纳米颗粒的活化：利用10mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合溶剂室温活化PLGA纳米颗粒5h；
(4)PCL纳米颗粒连接抗体：将活化后的纳米材料离心收集，然后用0.1M、pH＝8.0的杜氏磷酸盐缓冲溶液洗涤纳米材料一次，将需要连接的CD3和CD20单抗等量混合加入到连接反应液，然后用含有所述第一抗体部分和第二抗体部分的连接反应液重悬纳米材料，室温连接反应2.5h，反应结束后离心收集纳米材料，用杜氏磷酸盐缓冲溶液洗涤纳米材料2次，然后再重悬到杜氏磷酸盐缓冲溶液中放4℃保存备用。
实施例5
对通过PLGA连接的抗CD3和抗MUC1双特异性抗体(抗CD3-PLGA-抗MUC1)杀伤其他肿瘤细胞的能力进行了评估。
具体而言，于96孔板中，以5000个靶细胞每孔培养12h后，弃原培养基，利用无细胞因子的X-vivo 15培养基调整DC-CIK细胞密度，使100μl体积DC-CIK细胞数量为靶细胞4倍(效靶比为4∶1)，加100μl DC-CIK细胞悬液到癌细胞培养板中，并加入10μl(双特异性抗体制剂含量为0.2mg，含单抗成分共0.2μg)制备的双特异性抗体，培养箱中孵育8h，然后加入CCK-8试剂，按照试剂说明进行孵育，用酶标仪检测450nm处吸光值。对数据进行统计分析， 按照以下公式计算DC-CIK细胞对癌细胞的杀伤率。
杀伤率＝[1-(实验组-效应对照组)/(靶对照组-空白对照组)]×100％
其中，空白对照表示加入培养基；靶对照组表示加入靶细胞+培养基；效应对照组表示加入效应细胞+培养基；实验组表示效应细胞+靶细胞+培养基+双特异性抗体。
结果显示于表1和图3。
表1.抗CD3-PLGA-抗MUC1对肿瘤细胞的杀伤能力
Figure PCTCN2016079307-appb-000001
注：HKC-人胚肾上皮细胞；A549-人非小细胞肺癌细胞；HepG2-人肝癌细胞；HT29-人结肠癌细胞；AGS-人胃腺癌细胞；Hela-人宫颈癌细胞；5637-人膀胱癌细胞；OS-RC-2-人肾癌细胞。
实施例6
使用与上述类似的方法，以PLGA、PLA或PCL为纳米材料，制备了通过纳米颗粒连接的抗CD3和抗CD33双特异性抗体抗CD3-PLGA-抗CD33、抗CD3-PLA-抗CD33和抗CD3-PCL-抗CD33。按照实施例5中所述的方法，对本发明的双特异性抗体杀伤肿瘤细胞的能力进行了评估。
结果显示于表2和图4中。
表2.抗CD3-PLGA-抗CD33对肿瘤细胞的杀伤能力
Figure PCTCN2016079307-appb-000002
注：Raji-黑人Burkitt淋巴瘤细胞；RAMOS-人B淋巴细胞瘤细胞。
双特异性抗体CD3-PLA-CD33和CD3-PCL-CD33对于黑人Burkitt淋巴瘤细胞和人B淋巴细胞瘤细胞，同样表现出83％以上的杀伤率。
实施例6
使用与上述类似的方法，以PLGA、PLA或PCL为纳米材料，制备了连接有抗CD8抗体和抗MUC1抗体的纳米颗粒抗CD8-PLGA-抗MUC1、CD8-PLA-MUC1和抗CD8-PCL-抗MUC1。用实施例5中所述的方法对本发明的双特异性抗体杀伤肿瘤细胞的能力进行了评估。
结果显示于表2和图5中。
表3.抗CD8-PLGA-抗MUC1对肿瘤细胞的杀伤能力
Figure PCTCN2016079307-appb-000003
注：HKC-人胚肾上皮细胞；A549-人非小细胞肺癌细胞；HepG2-人肝癌细胞；HT29-人结肠癌细胞；AGS-人胃腺癌细胞；Hela-人宫颈癌细胞；5637-人膀胱癌细胞；OS-RC-2-人肾癌细胞。
双特异性抗体抗CD8-PLA-抗MUC1和抗CD8-PCL-抗MUC1显示出对于人 非小细胞肺癌细胞和人胃腺癌细胞均具有95％以上的杀伤率；对于人结肠癌细胞和人肾癌细胞均具有92％以上的杀伤率；对于人乳腺癌细胞、人宫颈癌细胞和人膀胱癌细胞，其均表现出接近90％的杀伤率，对于人肝癌细胞的杀伤率也在80％左右。
实施例7
使用与上述类似的方法，制备了通过纳米颗粒连接的其他双特异性抗体：抗CD3-PLGA-抗CD19、抗CD3-PLGA-抗CD20、抗CD3-PLGA-抗her2、，对这些杀伤肿瘤细胞的能力进行了评估。结果显示于表4-7中和图6-9。
表4.抗CD3-PLGA-抗CD19对肿瘤细胞的杀伤能力
Figure PCTCN2016079307-appb-000004
注：Raji-黑人Burkitt淋巴瘤细胞；RAMOS-人B淋巴细胞瘤细胞。
表5.抗CD3-PLGA-抗CD20对肿瘤细胞的杀伤能力
Figure PCTCN2016079307-appb-000005
注：Raji-黑人Burkitt淋巴瘤细胞；RAMOS-人B淋巴细胞瘤细胞。
表6.抗CD3-PLGA-抗her2对肿瘤细胞的杀伤能力
Figure PCTCN2016079307-appb-000006
注：MCF-7-人乳腺癌细胞。
表7.抗CD3-PLGA-抗CD38对肿瘤细胞的杀伤能力
Figure PCTCN2016079307-appb-000007
注：Raji-黑人Burkitt淋巴瘤细胞。
实施例6.体内抗肿瘤实验
对本发明的双特异性抗体内抗肿瘤的效力进行了评估。具体而言，在健康Balb-c裸鼠(清洁级，雌性，四周龄，体重18-22g，购自广东省医学实验动物中心)腋下接种人肺腺癌细胞A549，接种3-4周后取瘤体直径约0.5×0.5cm的裸鼠30只随机分成3组用于实验。
于第1、3、5天分别给药一次，采用尾静脉注射方式给药，DC-CIK组每次注射1×106数量的DC-CIK细胞，DC-CIK+双抗组每次注射1×106数量的DC-CIK细胞和0.4mg(单抗含量共0.4μg)双特异性抗体抗CD3-PLGA-抗MUC1，对照组注射同等体积的生理盐水。
从第一天起开始测量记录瘤体体积。计算双特异性抗体对肿瘤的抑制率。
在观察到对照组和单独DC-CIK治疗组个体全部死亡后停止实验，总观察时间为128天。
各个组的平均肿瘤体积显示于图10中。实验结果表明：双特异性抗体抗CD3-PLGA-抗MUC1对肿瘤的抑制率达到88.73％，有60％个体(6/10)肿瘤完全消失。
就存活时间而言，当与未治疗的小鼠相比时，用单独DC-CIK治疗和用DC-CIK+双特异性抗体治疗均提供显著的存活益处(图11)。截止第105天，未治疗组小鼠全部死亡。截至第128天，单独DC-CIK治疗组小鼠全部死亡，而DC-CIK+双特异性抗体治疗组仍有6只小鼠存活。
通过上述实施例可以看出，本发明的双特异性抗体对T细胞杀伤癌细胞的 能力有所提升，能联合免疫细胞增强肿瘤杀伤能力，相比CAR-T免疫细胞疗法，具有副作用小，安全性高，成本低，使用方便等特点。
申请人声明，本发明通过上述实施例来说明本发明的工艺方法，但本发明并不局限于上述工艺步骤，即不意味着本发明必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等，在不背离本发明的理念、精神和范围的情况下，均落在本发明的保护范围和公开范围之内。

Claims (17)

  1. 一种能联合免疫细胞增强肿瘤靶向杀伤能力的双特异性抗体，其特征在于，其包括结合在效应T细胞上表达的抗原的第一抗体部分和结合在靶细胞上表达的抗原的第二抗体部分。
  2. 根据权利要求1所述的双特异性抗体，其特征在于，所述第一抗体部分和第二抗体部分通过纳米材料进行连接；
    优选地，所述纳米材料为生物可降解纳米材料；
    优选地，所述纳米材料为聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物。
  3. 根据权利要求1或2所述的双特异性抗体，其特征在于，所述靶细胞是B细胞、癌细胞或病原体细胞；
    优选地，所述靶细胞上表达的抗原为CD19、CD20、CD22、CD30、CD33、CD123、Muc1、Muc16、HER2、HER3、EGFRvIII、VEGFA、CEA、GPA33、GP100、ANG2、L1CAM、ROR-1、CS1、MICA或MICB中的任意一种；
    优选地，所述效应T细胞上表达的抗原为CD2、CD3、CD4、CD5、CD6、CD8、CD25、CD28、CD30、CD40、CD40L、CD44、CD45、CD69或CD90中的任意一种。
  4. 根据权利要求1或2所述的双特异性抗体，其特征在于，所述靶细胞是B细胞、癌细胞或病原体细胞；
    优选地，所述靶细胞上表达的抗原为碳酸酐酶IX、甲胎蛋白、α-辅肌动蛋白-4、A3、A33抗体特异性抗原、ANG2、ART-4、B7、Ba 733、BAGE、BrE3-抗原、CA125、CAMEL、CAP-1、CASP-8/m、、CCCL19、CCCL21、CD1、CD1a、 CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD79b、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CS1、CXCR4、CXCR7、CXCL12、HIF-1α、结肠特异性抗原-p(CSAp)、CEA(CEACAM5)、CEACAM6、c-met、DAM、EGFR、EGFRvIII、EGP-1、EGP-2、ELF2-M、Ep-CAM、Flt-1、Flt-3、叶酸受体、G250抗原、GAGE、gp100、GPA33、GROB、HLA-DR、HM1.24、人绒毛膜促性腺激素(HCG)和其亚单位、HER2/neu、HER3、HMGB-1、低氧可诱导因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IL-2、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、IL-33、胰岛素样生长因子-1(IGF-1)、KC4-抗原、KS-1-抗原、KS1-4、L1CAM、Le-Y、LDR/FUT、巨噬细胞迁移抑制因子(MIF)、MAGE、MAGE-3、MART-1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MICA、MICB、MIP-1A、MIP-1B、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、胰腺癌粘蛋白、胎盘生长因子、p53、PLAGL2、前列腺酸性磷酸酶、PSA、PRAME、PSMA、PlGF、ILGF、ILGF-1R、IL-6、IL-25、ROR-1、RS5、RANTES、T101、SAGE、S100、存活蛋白、存活蛋白-2B、TAC、TAG-72、腱生蛋白、TRAIL受体、TNF-α、Tn抗原、汤姆森-弗里德里希抗原、肿瘤坏死抗原、TROP-2、VEGFA、VEGFR、ED-B纤连蛋白、WT-1、17-1A-抗原、补体因子C3、C3a、C3b、C5a、C5、血管生成标记物、bc1-2、bc1-6、Kras、cMET、致癌基因产物、HIV病毒、结核分枝杆菌(mycobacterium tuberculosis)、无乳链球菌(Streptococcus agalactiae)、抗甲氧西林性金黄色葡萄球菌(meticillin-resistant Staphylococcus aureus)、嗜肺军团菌(Legionella pneumophilia)、酿脓链球菌(Streptococcus pyogenes)、大肠杆菌(Escherichia coli)、 淋病奈瑟氏菌(Neisseria gonorrhoeae)、脑膜炎奈瑟氏菌(Neisseria meningitidis)、肺炎球菌(Pneumococcus)、新型隐球菌(Cryptococcus neoformans)、荚膜组织胞浆菌(Histoplasma capsulatum)、B型流感嗜血杆菌(Hemophilis influenzae B)、苍白密螺旋体(Treponema pallidum)、莱姆病螺旋体(Lyme disease spirochetes)、铜绿假单胞菌(Pseudomonas aeruginosa)、麻风分枝杆菌(Mycobacterium leprae)、流产布鲁氏菌(Brucella abortus)、狂犬病病毒、流感病毒、巨细胞病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、人血清细小样病毒、呼吸道合胞病毒、水痘-带状疱疹病毒、乙型肝炎病毒、丙型肝炎病毒、麻疹病毒、腺病毒、人T细胞白血病病毒、埃-巴二氏病毒、鼠白血病病毒、腮腺炎病毒、水泡性口膜炎病毒、辛德毕斯病毒、淋巴细胞性脉络丛脑膜炎病毒、疣病毒、蓝舌病病毒、仙台病毒、猫白血病毒、呼肠孤病毒、脊髓灰质炎病毒、猿猴病毒40、小鼠乳腺瘤病毒、登革热病毒、风疹病毒、西尼罗河病毒、恶性疟原虫(Plasmodium falciparum)、间日疟原虫(Plasmodium vivax)、刚地弓形虫(Toxoplasma gondii)、让氏锥虫(Trypanosoma rangeli)、克氏锥虫(Trypanosoma cruzi)、罗德西亚锥虫(Trypanosoma rhodesiensei)、布氏锥虫(Trypanosoma brucei)、曼森氏裂体吸虫(Schistosoma mansoni)、日本裂体吸虫(Schistosoma japonicum)、牛巴贝虫(Babesia bovis)、艾美耳球虫(Elmeria tenella)、旋盘尾丝虫(Onchocerca volvulus)、热带利什曼虫(Leishmania tropica)、旋毛虫(Trichinella spiralis)、小泰勒虫(Theileria parva)、泡状带绦虫(Taenia hydatigena)、羊绦虫(Taenia ovis)、牛肉绦虫(Taenia saginata)、细粒棘球绦虫(Echinococcus granulosus)、科氏中殖孔绦虫(Mesocestoides corti)、关节炎支原体(Mycoplasma arthritidis)、鼻支原体(M.hyorhinis)、口腔支原体(M.orale)、精氨酸支原体(M.arginini)、拉氏无胆甾原体(Acholeplasma laidlawii)、唾液支原体(M.salivarium)和肺炎支原体(M.pneumoniae)中的任意一种；
    优选地，所述效应T细胞上表达的抗原为ADAM17、CD2、CD3、CD4、 CD5、CD6、CD8、CD11a、CD11b、CD14、CD16、CD16b、CD25、CD28、CD30、CD32a、CD40、CD40L、CD44、CD45、CD56、CD57、CD64、CD69、CD74、CD89、CD90、CD137、CD177、CEACAM6、CEACAM8、HLA-DRα链、KIR、LSECtin或SLC44A2中的任意一种。
  5. 根据权利要求1-3任一项所述的双特异性抗体的制备方法，其特征在于，其包括将纳米材料与第一抗体部分和第二抗体部分连接。
  6. 根据权利要求5所述的制备方法，其特征在于，其包括以下步骤：
    (1)纳米材料的制备、收集和活化；
    (2)将步骤(1)得到的纳米材料与第一抗体部分和第二抗体部分的混合物进行连接。
  7. 根据权利要求6所述的制备方法，
    所述纳米材料为聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物；和/或
    所述溶剂为丙酮、丁酮、甲醇、乙醇或异丙醇中的任意一种或至少两种的混合物。
  8. 根据权利要求4所述的双特异性抗体的制备方法，其特征在于，其包括将纳米材料与第一抗体部分和第二抗体部分连接。
  9. 根据权利要求8所述的制备方法，其特征在于，其包括以下步骤：
    (1)纳米材料的制备、收集和活化；
    (2)将步骤(1)得到的纳米材料与第一抗体部分和第二抗体部分的混合物进行连接。
  10. 根据权利要求9所述的制备方法，
    所述纳米材料为聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物；和/或
    所述溶剂为丙酮、丁酮、甲醇、乙醇或异丙醇中的任意一种或至少两种的混合物。
  11. 根据权利要求1-3任一项所述的双特异性抗体在制备治疗、预防或诊断肿瘤的药物中的应用。
  12. 根据权利要求4所述的双特异性抗体在制备治疗、预防或诊断肿瘤的药物中的应用。
  13. 根据权利要求11或12所述的应用，其中所述肿瘤选自肝癌、非小细胞肺癌、小细胞肺癌、肾上腺皮质癌、急性(慢性B)淋巴细胞瘤、骨髓瘤、前列腺癌、乳腺癌、食管癌、胃癌、结直肠癌、宫颈癌、肾癌、膀胱癌和淋巴瘤。
  14. 用于治疗或预防肿瘤的根据权利要求1-4任一项所述的双特异性抗体。
  15. 根据权利要求14所述的双特异性抗体，其中所述肿瘤选自肝癌、非小细胞肺癌、小细胞肺癌、肾上腺皮质癌、急性(慢性B)淋巴细胞瘤、骨髓瘤、前列腺癌、乳腺癌、食管癌、胃癌、结直肠癌、宫颈癌、肾癌、膀胱癌和淋巴瘤。
  16. 治疗肿瘤的方法，所述方法包括给受治疗者施用根据1-4任一项所述的双特异性抗体。
  17. 根据权利要求16所述的方法，其中所述肿瘤选自肝癌、非小细胞肺癌、小细胞肺癌、肾上腺皮质癌、急性(慢性B)淋巴细胞瘤、骨髓瘤、前列腺癌、 乳腺癌、食管癌、胃癌、结直肠癌、宫颈癌、肾癌、膀胱癌和淋巴瘤。
PCT/CN2016/079307 2015-04-14 2016-04-14 一种能联合免疫细胞增强肿瘤杀伤能力的双特异性抗体及其制备方法和应用 WO2016165632A1 (zh)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018137609A1 (zh) * 2017-01-24 2018-08-02 本康生物制药（深圳）有限公司 抗体偶联物、相关的药物组合物及应用
WO2018166527A1 (zh) * 2017-03-17 2018-09-20 本康生物制药（深圳）有限公司 多特异性抗体、抗体偶联物和相关的药物组合物及应用
US11000603B2 (en) 2015-04-14 2021-05-11 Benhealth Biopharmaceutic (Shenzhen) Co., Ltd. Multi-specific binding conjugate, related pharmaceutical compositions and use
CN113125732A (zh) * 2019-12-31 2021-07-16 博阳生物科技（上海）有限公司 一种白介素6的均相检测试剂盒及其应用
WO2022148736A1 (en) 2021-01-05 2022-07-14 Transgene Vectorization of muc1 t cell engager
US11525009B2 (en) 2016-06-22 2022-12-13 Benhealth Biopharmaceutic (Shenzhen) Co. Ltd. Bispecific antibody and antibody conjugate for tumor therapy and use thereof

Families Citing this family (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230132604A (ko) 2015-01-27 2023-09-15 라바 테라퓨틱스 엔.브이. Cd1d를 표적화하는 단일 도메인 항체
CN104829730A (zh) * 2015-04-14 2015-08-12 苏静 一种能联合免疫细胞增强肿瘤杀伤能力的双特异性抗体及其制备方法和应用
CN104826110B (zh) * 2015-05-19 2017-09-29 中国人民解放军第二军医大学 新一代多功能抗体纳米团簇的制备及其协同治疗应用
CN106554414B (zh) * 2015-09-18 2019-04-23 上海科济制药有限公司 抗cd19全人抗体以及靶向cd19的免疫效应细胞
WO2017123627A1 (en) * 2016-01-11 2017-07-20 Stc Biologics, Inc. Bispecific targeting agents and methods for their preparation
EP3450460B1 (en) * 2016-04-29 2023-07-12 Benhealth Biopharmaceutic (Shenzhen) Co. Ltd. Multi-specific binding conjugate, related pharmaceutical compositions and use
AU2017306426B2 (en) 2016-08-02 2024-09-26 Memorial Sloan-Kettering Cancer Center Treating metastatic cancer and model systems for metastatic disease
CN107177632B (zh) * 2017-05-27 2020-02-07 上海优卡迪生物医药科技有限公司 一种基于octs技术的髓系白血病car-t治疗载体及其构建方法和应用
MX2020005651A (es) * 2017-11-30 2020-10-28 Novartis Ag Receptor de antigeno quimerico dirigido a bcma y usos del mismo.
US20220002398A1 (en) * 2018-06-07 2022-01-06 Oncoone Research & Development Gmbh ANTI-oxMIF/ANTI-CD3 ANTIBODY FOR CANCER TREATMENT
CN110687293A (zh) * 2018-07-06 2020-01-14 中国人民解放军第四军医大学 一种用于检测胃癌抗原MG7-Ag的试剂盒及其应用
CN112469734A (zh) * 2018-08-03 2021-03-09 南京驯鹿医疗技术有限公司 表达嵌合抗原受体的t细胞、该嵌合抗原相关表达载体以及它们的应用
JP7457007B2 (ja) 2018-09-19 2024-03-27 ラヴァ・セラピューティクス・エヌ・ヴイ 二重作用ＣＤ１ｄ免疫グロブリン
WO2020083277A1 (zh) * 2018-10-22 2020-04-30 上海一宸医药科技有限公司 一种双特异性抗体
CA3117079A1 (en) * 2018-10-30 2020-05-07 Peter Maccallum Cancer Institute Bispecific polypeptides for engagement of car expressing immune cells with antigen presenting cells and uses thereof
KR102437436B1 (ko) * 2019-04-26 2022-08-30 주식회사 엠디헬스케어 스트렙토코커스 파이오제네스 세균 유래 단백질 및 이의 용도
CN110123844A (zh) * 2019-05-13 2019-08-16 天津大学 CuS矿化流感病毒的肿瘤联合治疗纳米制剂的合成方法
US20220280568A1 (en) * 2019-08-15 2022-09-08 City Of Hope Dual bispecific antibody compounds and uses thereof
CN112336873B (zh) * 2020-08-04 2022-04-19 华南理工大学 用于多特异性抗体递送的蛋白型纳米颗粒及其应用、制备方法
WO2022063302A1 (zh) * 2020-09-25 2022-03-31 克莱格医学有限公司 免疫细胞活性调节
JP2023550148A (ja) 2020-11-20 2023-11-30 シンシア・イノベーション・インコーポレイテッド がん免疫治療に用いられる武装二重ｃａｒ－ｔ組成物及び方法
CN112807447A (zh) * 2021-01-18 2021-05-18 中国药科大学 一种pd-l1靶向负载索拉菲尼plga纳米制剂及其制备方法
CN114085376B (zh) * 2021-12-02 2022-12-20 中国科学院长春应用化学研究所 一种多肽及其制备方法、双特异性抗体及其应用
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024179515A1 (en) * 2023-02-28 2024-09-06 LaNova Medicines Limited Ceacam5-dependent 4-1bb-agonistic bispecific antibodies
WO2024193253A1 (en) * 2023-03-18 2024-09-26 Xadcera Biopharmaceutical (Suzhou) Co., Ltd. Anti-her3/muc1 antibodies and uses thereof
CN116789828B (zh) * 2023-05-31 2023-11-17 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) 一种抗cd132的单克隆抗体及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004110390A2 (en) * 2003-06-17 2004-12-23 Immunomedics, Inc. Anti-cd74 immunoconjugates and methods
WO2005103081A2 (en) * 2004-04-20 2005-11-03 Genmab A/S Human monoclonal antibodies against cd20
CN104829730A (zh) * 2015-04-14 2015-08-12 苏静 一种能联合免疫细胞增强肿瘤杀伤能力的双特异性抗体及其制备方法和应用

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1470289A (zh) * 2002-07-26 2004-01-28 北京华京五方实用纳米科技开发有限公 一种高分子纳米药物载体和制剂的制备方法
CN101374546B (zh) * 2005-12-16 2016-03-23 Ibc医药公司 基于免疫球蛋白的多价生物活性装配体
CN104379169A (zh) * 2012-08-14 2015-02-25 Ibc药品公司 用于治疗疾病的t-细胞重定向双特异性抗体
US9682143B2 (en) * 2012-08-14 2017-06-20 Ibc Pharmaceuticals, Inc. Combination therapy for inducing immune response to disease
CN112079929A (zh) * 2012-11-21 2020-12-15 武汉友芝友生物制药有限公司 双特异性抗体
BR112015013444B1 (pt) * 2012-12-13 2022-11-01 Immunomedics, Inc Uso de um imunoconjugado
WO2014110591A1 (en) * 2013-01-14 2014-07-17 Fred Hutchinson Cancer Research Center Compositions and methods for delivery of immune cells to treat un-resectable or non-resected tumor cells and tumor relapse
EP2970505A4 (en) 2013-03-14 2016-10-19 California Inst Biomedical Res SPECIFIC ANTIBODIES AND USES THEREOF
WO2015035365A1 (en) * 2013-09-09 2015-03-12 The Nohns Hopkins University Targeting the m2-tumor associated macrophage for cancer therapy
US11525009B2 (en) 2016-06-22 2022-12-13 Benhealth Biopharmaceutic (Shenzhen) Co. Ltd. Bispecific antibody and antibody conjugate for tumor therapy and use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004110390A2 (en) * 2003-06-17 2004-12-23 Immunomedics, Inc. Anti-cd74 immunoconjugates and methods
WO2005103081A2 (en) * 2004-04-20 2005-11-03 Genmab A/S Human monoclonal antibodies against cd20
CN104829730A (zh) * 2015-04-14 2015-08-12 苏静 一种能联合免疫细胞增强肿瘤杀伤能力的双特异性抗体及其制备方法和应用

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11000603B2 (en) 2015-04-14 2021-05-11 Benhealth Biopharmaceutic (Shenzhen) Co., Ltd. Multi-specific binding conjugate, related pharmaceutical compositions and use
US11525009B2 (en) 2016-06-22 2022-12-13 Benhealth Biopharmaceutic (Shenzhen) Co. Ltd. Bispecific antibody and antibody conjugate for tumor therapy and use thereof
WO2018137609A1 (zh) * 2017-01-24 2018-08-02 本康生物制药（深圳）有限公司 抗体偶联物、相关的药物组合物及应用
CN110382550A (zh) * 2017-01-24 2019-10-25 本康生物制药（深圳）有限公司 抗体偶联物、相关的药物组合物及应用
EP3599250A4 (en) * 2017-01-24 2021-06-09 Benhealth Biopharmaceutic (Shenzhen) Co., Ltd. ANTIBODY CONJUGATE, ASSOCIATED PHARMACEUTICAL COMPOSITION AND APPLICATION
WO2018166527A1 (zh) * 2017-03-17 2018-09-20 本康生物制药（深圳）有限公司 多特异性抗体、抗体偶联物和相关的药物组合物及应用
CN110520442A (zh) * 2017-03-17 2019-11-29 本康生物制药（深圳）有限公司 多特异性抗体、抗体偶联物和相关的药物组合物及应用
CN113125732A (zh) * 2019-12-31 2021-07-16 博阳生物科技（上海）有限公司 一种白介素6的均相检测试剂盒及其应用
CN113125732B (zh) * 2019-12-31 2023-08-08 科美博阳诊断技术(上海)有限公司 一种白介素6的均相检测试剂盒及其应用
WO2022148736A1 (en) 2021-01-05 2022-07-14 Transgene Vectorization of muc1 t cell engager

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