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WO2024061364A9 - 一种抗4-1bb纳米抗体及其制备和应用 - Google Patents

一种抗4-1bb纳米抗体及其制备和应用 Download PDF

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WO2024061364A9
WO2024061364A9 PCT/CN2023/120848 CN2023120848W WO2024061364A9 WO 2024061364 A9 WO2024061364 A9 WO 2024061364A9 CN 2023120848 W CN2023120848 W CN 2023120848W WO 2024061364 A9 WO2024061364 A9 WO 2024061364A9
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antibody
antigen
variants
fragment
fusion protein
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PCT/CN2023/120848
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French (fr)
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WO2024061364A1 (zh
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李欣檑
曾昕
刘江海
李玲芳
杜建华
朱迪
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成都盛世君联生物技术有限公司
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Publication of WO2024061364A9 publication Critical patent/WO2024061364A9/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present application relates to the field of biomedicine technology, and specifically to an anti-4-1BB nanobody and its preparation and application.
  • 4-1BB (CD137) is one of the costimulatory members of the tumor necrosis factor receptor superfamily (TNFRSF) and is an inducible T cell surface receptor that is not only highly expressed on activated T cells, but also remains on T cells.
  • TNFRSF tumor necrosis factor receptor superfamily
  • Pollok K E, Kim S H team showed that anti-CD3 antibodies or concanavalin A (Con A) stimulated high expression of 4-1BB on splenic T cells.
  • 4-1BB's natural ligand 4-1BBL is present on activated antigen-presenting cells (APCs).
  • 4-1BB When 4-1BB binds to its natural ligand or agonistic monoclonal antibody (MoAb), it transmits a unique costimulatory signal that plays a role in T cell responses in vivo and in vitro, including stimulating T cell proliferation, upregulating survival-related genes, and producing Th1 cytokines such as IL-2, IFN- ⁇ and TNF-a.
  • IL-2 is one of the main positive growth factors of T cells.
  • High levels of IL-2 secreted by CD8+T cells play an important role in inducing cell cycle progression, producing cytokines such as IFN- ⁇ , and inducing memory CD8+T cells.
  • 4-1BB-mediated signaling plays a key role in preventing activation-induced cell death, promoting rejection of cardiac allografts and skin grafts, increasing CD8+T cell cytolytic capacity, and eradicating tumors.
  • Nanobody (Nb) is the variable region of heavy chain antibodies (IgG2 and IgG3) in camelids, and is known as the smallest antigen-binding fragment in nature. Compared with traditional full-length monoclonal antibodies (mAb, about 150KD), Nb has the advantages of small molecular weight (12-15KD), simpler structure, low immunogenicity, high tissue permeability, high stability, high solubility, low aggregation, and easy cloning. In addition, compared with similar mAb products, Nb production costs are significantly reduced and can be used by most cancer patients.
  • 4-1BB monoclonal antibodies still face many challenges, including 1) large molecular size (160-170kD), which limits their ability to penetrate solid tumors and the blood-brain barrier, and 2) the large workload involved in the in vitro eukaryotic expression and cell screening process, resulting in high production costs.
  • the present invention provides an anti-4-1BB nanobody and its preparation method and use.
  • the anti-4-1BB nanobody has the advantages of small molecular weight, simpler structure, low immunogenicity, high tissue permeability, high stability, high solubility and low aggregation, and easy cloning, and can be used to increase the cytolytic ability of CD8+T cells and treat or prevent tumors.
  • the present invention provides an anti-4-1BB antibody and a variant thereof, or an antigen-binding fragment thereof, wherein the anti-4-1BB antibody comprises three complementary determining regions CDR1, CDR2 and CDR3 of VHH named 137-1, 137-7, 137-12, 137-16, 137-18, 137-36 and 137-39 (as shown in the following table),
  • the variants are humanized variants or identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) variants.
  • the antibodies and variants thereof described herein, or antigen-binding fragments thereof are selected from camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)' 2 fragment, F(ab)' 3 fragment, Fv, scFv, bi-scFv, (scFv) 2 , miniantibodies, double-chain antibodies, three-chain antibodies, four-chain antibodies, disulfide-stabilized Fv proteins and single domain antibodies (sdAb, nanoantibodies), camel antibodies, bispecific antibodies or trispecific antibodies.
  • the present invention also provides a fusion protein comprising the antibody and variants thereof, or antigen-binding fragments thereof described herein.
  • the fusion protein described herein further comprises a tag sequence (e.g., Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence, or an additional epitope (e.g., other epitopes directed against or different from 4-1BB) or an additional antibody active fragment (e.g., antibodies or antibody active fragments directed against other epitopes or the same epitope of 4-1BB, or ligands that can bind to 4-1BB).
  • a tag sequence e.g., Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.
  • an IgG1-Fc protein sequence e.g., an additional epitope (e.g., other epitopes directed against or different from 4-1BB) or an additional antibody active fragment (e.g., antibodies or antibody active fragments directed against other epitopes or the same epitope of 4-1BB, or lig
  • the present invention also provides an antibody-drug conjugate, which comprises the antibody and variants thereof described herein, or an antigen-binding fragment thereof.
  • the drug is selected from the following: radioactive labels, 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes, biotin, streptavidin, digoxigenin, haptens, immunogenic proteins, nucleic acid molecules with sequences complementary to the target, or any combination of the foregoing; or immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents and antiparasitic agents, or any combination of the foregoing.
  • the present invention also provides an isolated polynucleotide expressing an antibody or an antigen-binding fragment thereof, characterized in that the polynucleotide is capable of expressing the antibody and its variants, or its antigen-binding fragment described herein; the polynucleotide is capable of expressing the fusion protein described herein; the polynucleotide is capable of expressing the antibody-drug conjugate described herein.
  • the present invention also provides a vector comprising the polynucleotide described herein, preferably a plasmid vector.
  • the present invention also provides a host cell, comprising the polynucleotide described herein or the vector described herein, preferably, the host cell is a eukaryotic cell.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody and variants thereof described herein, or antigen-binding fragments thereof, the fusion protein described herein, the antibody-drug conjugate described herein, and optionally, a pharmaceutically acceptable carrier.
  • the present invention also provides a use of the antibody and variants thereof described herein, or antigen-binding fragments thereof, the fusion protein described herein, and the antibody-drug conjugate described herein in the preparation of a drug for treating and/or preventing diseases associated with 4-1BB.
  • the present invention also provides a method for treating and/or preventing 4-1BB-related diseases and related symptoms, comprising administering an effective amount of the antibody and variants thereof described herein, or an antigen-binding fragment thereof, the fusion protein described herein, the antibody-drug conjugate described herein, or the pharmaceutical composition described herein to a subject.
  • the present invention also provides a method for detecting whether a sample contains T cells that highly express 4-1BB, which comprises the step of contacting the antibody and variants thereof described herein, or antigen-binding fragments thereof, and the fusion protein described herein with the sample.
  • the detection can be for diagnostic purposes or non-diagnostic purposes.
  • the present invention also provides a detection product, which comprises the antibody and variants thereof described herein, or an antigen-binding fragment thereof.
  • the product is selected from one or more of a detection reagent, a kit, a chip or a test paper.
  • the present invention further provides the following technical solutions:
  • the first aspect of the present invention provides an anti-4-1BB nanobody, wherein the heavy chain variable region of the anti-4-1BB nanobody consists of a framework region FR and a complementary determining region CDR, and the complementary determining region CDR comprises at least one of the following groups:
  • Nanobodies are the variable regions of heavy chain antibodies (IgG2 and IgG3) in camelids, and are known as the smallest antigen-binding fragments in nature. Compared with traditional full-length monoclonal antibodies (mAb, about 150KD), Nb has the advantages of small molecular weight (12-15KD), simpler structure, low immunogenicity, high tissue permeability, high stability, high solubility and low aggregation, and easy cloning. In addition, compared with mAb counterparts, the production cost of Nb is significantly reduced, and most cancer patients can use it.
  • Nbs are expected to provide a wide range of application scenarios in cancer treatment and diagnosis.
  • camels were immunized with recombinant 4-1BB antigen and a VHH phage library was constructed. After biological screening, 7 functional anti-4-1BB Nbs (such as the antibodies described in SEQ ID Nos: 1-7) were obtained.
  • the framework region FR includes at least one of the following groups:
  • framework region FR of the present invention is not limited to the above sequences, and any sequence that can achieve its function is within the protection scope of the present invention.
  • the second aspect of the present invention provides a polynucleotide, which is a polynucleotide encoding the above-mentioned anti-4-1BB nanobody.
  • the third aspect of the present invention provides a recombinant expression vector, which comprises the above-mentioned polynucleotide.
  • the expression vector includes a prokaryotic expression vector or a eukaryotic expression vector.
  • the fourth aspect of the present invention provides a recombinant host cell, which comprises the above-mentioned polynucleotide, or comprises the above-mentioned recombinant expression vector.
  • the host cell includes a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from Escherichia coli or yeast cells.
  • the host cell is selected from HEK293T cells, HEK293F cells, Expi293F cells or CHO cells.
  • the fifth aspect of the present invention provides a method for preparing the above-mentioned anti-4-1BB nanobody, comprising the following steps:
  • the culture was purified to obtain anti-4-1BB nanoantibodies.
  • purification is performed using Protein A agarose purification resin.
  • the sixth aspect of the present invention provides a bispecific antibody, which comprises the above-mentioned anti-4-1BB nanobody and a second antibody.
  • the second antibody includes but is not limited to 4-1BB nanobody, CD47 nanobody, VEGF nanobody, HER2 nanobody, EGFR nanobody, HER3 nanobody, B7H3 nanobody, TIGIT nanobody, OX-40 nanobody, CD40 nanobody or PD-L1 nanobody.
  • the seventh aspect of the present invention provides the use of the above-mentioned anti-4-1BB nanobody or bispecific antibody in the preparation of drugs for preventing and/or treating cancer and detecting 4-1BB protein.
  • cancer includes but is not limited to gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor or bladder tumor.
  • the eighth aspect of the present invention provides a pharmaceutical composition, comprising: the above-mentioned anti-4-1BB nanobody or bispecific antibody; and pharmaceutically acceptable excipients.
  • the dosage form of the pharmaceutical composition includes but is not limited to injection, powder injection, tablet or capsule.
  • a ninth aspect of the present invention provides a kit for detecting 4-1BB protein, the kit comprising: the above-mentioned anti-4-1BB nanobody or bispecific antibody; and a reagent acceptable in detection.
  • Figure 1 shows the FACS binding of VHH-hFc recombinant antibodies to human 4-1BB overexpressing 293F cells
  • FIG2 shows the IL-2 release of PBMC stimulated by VHH-hFc recombinant antibody (in the presence of OKT3);
  • FIG3 shows the IL-2 release of PBMC stimulated by VHH-hFc recombinant antibody (in the absence of OKT3).
  • the present invention discloses an anti-4-1BB nano antibody and its biomaterial and product.
  • Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.
  • Nanobody (Nb) is a naturally light chain-deficient antibody in the peripheral blood of alpacas. It contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other or even aggregate into clumps as the artificially modified single-chain antibody fragment (scFv). More importantly, the VHH structure cloned and expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody, and is the smallest unit known to bind to the target antigen.
  • VHH heavy chain variable region
  • scFv artificially modified single-chain antibody fragment
  • the framework region (FR) is the skeleton region within the variable region of an antibody. There are about 110 amino acid sequences near the N-terminus of the H and L chains of immunoglobulins that vary greatly, while the amino acid sequences of the other parts are relatively constant. Based on this, the light chain and heavy chain can be divided into the variable region (V) and the constant region (C).
  • the variable region contains the hypervariable region HVR (hypervariable region) or complementary determining region CDR (Complementarity-determining region) and the FR skeleton region. The variability of FR is lower than that of CDR.
  • Complementarity determining region The entire antibody molecule can be divided into two parts: the constant region and the variable region. In the variable region, there is a small part of the amino acid residues that change particularly strongly. The residue composition and arrangement order of these amino acids are more prone to variation, which is called the hypervariable region. There are three hypervariable regions (HVR) in the V region of the L chain and H chain. Because this part can form precise complementarity with the antigenic determinant in terms of spatial structure, the hypervariable region is also called the complementarity determining region.
  • HVR hypervariable regions
  • Bispecific antibodies artificial antibodies containing two specific antigen-binding sites, which can build a bridge between target cells and functional molecules (cells) to stimulate a directed immune response. They are a type of genetically engineered antibody and have become a hot topic in the field of antibody engineering. They have broad application prospects in tumor immunotherapy.
  • the reagents, instruments, strains or biological materials used in the present invention can all be obtained commercially.
  • the human 4-1BB extracellular domain recombinant protein (hFc tag) was injected into multiple masses at multiple points on the back of the neck of two alpacas, and the absorption of the subcutaneous injection masses was tracked to confirm the correct immunization.
  • 0.5 mg of antigen was mixed with Freund's complete adjuvant in a ratio of 1:1 and injected after emulsification, with a volume of 1 ml per alpaca;
  • Second immunization 3 weeks after the first immunization, 0.25 mg of antigen was mixed with Freund's incomplete adjuvant in a ratio of 1:1, emulsified and injected, with an injection volume of 1 mL per alpaca;
  • the third immunization 3 weeks after the second immunization, 0.25 mg of antigen was mixed with Freund's incomplete adjuvant in a ratio of 1:1, emulsified and injected, with an injection volume of 1 ml per alpaca;
  • the amplified region is the Leader-CH2 region of the alpaca heavy chain antibody gene, and the product size is 700bp and 900bp; use DNA gel electrophoresis and gel cutting to recover the 700bp PCR product; 2) Design nested inner primers (3 pairs of alpaca) and use the 700bp first round PCR product as a template for the second round of PCR amplification.
  • the amplified region is the VHH fragment of the alpaca heavy chain antibody variable region, and the product size is 400bp; Use a PCR product purification kit to purify and recover the second round PCR product.
  • the library capacity of the alpaca antibody library Lib 4-1BB Alpaca is 4.60 ⁇ 10 9 . Randomly select 20 monoclones from each plate for sequencing, and the results show that the VHH insertion efficiency of the alpaca antibody library Lib 4-1BB Alpaca is 90%.
  • the above mixture was added to a centrifuge tube containing 1 mL of NEB5 ⁇ F' bacteria, shaken at 37°C, and cultured for 1 hour; 20 ⁇ L of the culture solution in the centrifuge tube was diluted to an appropriate multiple, spread on an LB/Carb50 culture plate, placed in a 37°C biochemical incubator overnight, and used for calculation of titer and enrichment the next day; 1 ⁇ L of helper phage M13K07 (final concentration of 10 10 cells/mL) was added to the remaining culture solution, shaken at 37°C, and cultured for 1 hour; the above culture solution was transferred to 35 mL of 2YT/Carb50/Kan25 culture solution, placed in a shaker, cultured overnight at 37°C, and phages were collected to form an antibody library for each round.
  • the clones from the enrichment round were randomly selected and expanded in 96-deep-well plates. After centrifugation, the supernatant was used for Phage ELISA screening. The clones with OD values > 2 in combination with the blocking solution were selected, which were defined as positive clones, and sequenced and aligned to obtain unique sequences.
  • Primers were designed to locate the constant regions on both sides of VHH, the variable regions of the phage library were amplified, the PCR products were tested by E-gel, and the PCR library fragments were purified and recovered using a kit for NGS sequencing. The data were statistically analyzed using SPSS 2.0 and Microsoft Excel 2019, and sorted according to the number and frequency of DNA.
  • the affinity of the 7 sequences of VHH-hFc recombinant antibodies to the human and monkey 4-1BB extracellular region recombinant proteins was determined by ELISA method: the human and monkey 4-1BB extracellular region recombinant proteins (his tag) were added to the ELISA 96-well plate, 200 ng/well, and coated overnight at 4°C.
  • the VHH-hFc recombinant antibodies were diluted to different concentrations (0.014-10 ⁇ g/mL), reacted with the antigen by ELISA, and the HRP-labeled anti-VHH secondary antibody was used for color development, and the absorbance value at 450nm was measured by an enzyme reader.
  • the experimental results show that the 7 sequences of VHH-hFc recombinant antibodies can bind to the human or monkey 4-1BB extracellular region recombinant proteins, among which CD137-1, CD137-7, CD137-16, and CD137-18 ranked in the top 4 in affinity with the human or monkey 4-1BB extracellular region recombinant proteins.
  • the same experimental method was used to detect the affinity of the recombinant antibodies to the mouse 4-1BB extracellular region recombinant protein.
  • the results (Table 5) showed that the VHH-hFc recombinant antibodies of CD137-1, CD137-7, CD137-16, and CD137-18 did not bind or weakly bound to the mouse 4-1BB extracellular region recombinant protein.
  • Example 8 Affinity KD value of nanobody and antigen recombinant protein
  • Biacore was used to detect the affinity KD values of VHH-hFc recombinant antibodies of 4 sequences (CD137-1, CD137-7, CD137-16, and CD137-18) with human 4-1BB extracellular region recombinant protein: Biacore 8k set the sample chamber and flow chamber temperature to 25°C, ProteinA chip captured VHH-hFc samples as Ligand, HBS-EP+pH7.4 buffer diluted the corresponding target protein as Analytes, Analyte flow rate 30 ⁇ L/min, binding 120s, dissociation 600s. The data were analyzed by 1:1binding and Fit local Model fitting. The experimental results (Table 6) show that the VHH-hFc recombinant antibodies of the 4 sequences can bind to the human 4-1BB extracellular region recombinant protein, and the KD value is 3.3-38nM.
  • the ELISA kit was used to detect the level of IL-2 released after stimulating human PMBC with VHH-hFc recombinant antibodies of 4 sequences (CD137-1, CD137-7, CD137-16, and CD137-18): 96-well cell culture plates were coated with or without CD3 antibody OKT3, overnight at 4°C; the next day, PBMC (1X10e5/well) and different concentrations of 4-1BB recombinant antibodies were added, and the supernatant was collected after continuing to culture for 72 hours for ELISA detection of IL-2 levels.

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Abstract

涉及生物医药技术领域，具体涉及一种抗4-1BB纳米抗体及其制备和应用。该抗4-1BB纳米抗体的互补决定区包含：SEQ ID NOs：1-7中所示的CDR1、CDR2和CDR3等。该抗4-1BB纳米抗体可用于预防和/或治疗癌症。

Description

一种抗4-1BB纳米抗体及其制备和应用 技术领域
本申请涉及生物医药技术领域，具体涉及一种抗4-1BB纳米抗体及其制备和应用。
背景技术
T细胞免疫功能的有效激发需要一个共刺激过程和细胞因子环境来促进细胞周期的进展、存活和增值。4-1BB(CD137)是肿瘤坏死因子受体超家族(TNFRSF)的共刺激成员之一，是一种诱导型T细胞表面受体，它不仅在激活后的T细胞上高表达，而且还持续保留在T细胞上。Pollok K E,Kim S H团队表明，抗CD3抗体或刀豆蛋白(Concanavalin A，Con A)刺激脾T细胞上的4-1BB高表达。4-1BB的天然配体4-1BBL存在于活化的抗原递呈细胞上(APC)，当4-1BB与其天然配体或激动性单克隆抗体(MoAb)结合时，会传递一种独特的共刺激信号，在体内和体外的T细胞反应中发挥作用，包括刺激T细胞增殖，上调生存相关基因，以及产生Th1细胞因子如IL-2,IFN-γ和TNF-a等。其中，IL-2是T细胞主要的阳性生长因子之一，由CD8+T细胞分泌的高水平的IL-2在诱导细胞周期进程，产生IFN-γ等细胞因子，以及诱导记忆性CD8+T细胞方面起重要作用。此外，4-1BB介导的信号传导在预防活化诱导的细胞死亡、促进心脏同种异体移植物和皮肤移植物的排斥、增加CD8+T细胞溶细胞能力和根除肿瘤方面发挥着关键作用。
纳米抗体(Nanobody,Nb)是骆驼科动物体内重链抗体(IgG2和IgG3)的可变区域，被称为是自然界存在的最小的抗原结合片段。与传统全长单克隆抗体(mAb，约150KD)相比，Nb具有分子量小(12-15KD)，结构更简单，低免疫原性，高组织渗透性、高稳定性、高溶解性和低聚集性以及易于克隆等优点。此外，与mAb同类产品相比，Nb生产成本显著降低，大多数癌症患者都可以使用。
目前4-1BB单克隆抗体仍然面临了许多挑战，包括1)大分子尺寸(160-170kD)，限制了它们穿透实体瘤和血脑屏障的能力，以及 2)体外真核表达和细胞筛选过程涉及的工作量较大，导致生产成本高。
发明内容
有鉴于此，本发明提供了一种抗4-1BB纳米抗体及其制备方法和用途。该抗4-1BB纳米抗体具有分子量小、结构更简单、低免疫原性、高组织渗透性、高稳定性、高溶解性和低聚集性以及易于克隆等优点，且可用于增加CD8+T细胞溶细胞能力和治疗或预防肿瘤。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种抗4-1BB抗体及其变体，或其抗原结合片段，所述抗4-1BB抗体包含名称为137-1、137-7、137-12、137-16、137-18、137-36、137-39的VHH的3个互补决定区CDR1、CDR2和CDR3(如下表)，

示例性地，对于本文所述的抗体及其变体，或其抗原结合片段，所述变体为人源化变体或同一性(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％)变体。
进一步地，对于本文所述的抗体及其变体，或其抗原结合片段，其选自骆驼Ig、Ig NAR、Fab片段、Fab’片段、F(ab)’2片段、F(ab)’3片段、Fv、scFv、双-scFv、(scFv)2、微型抗体、双链抗体、三链抗体、四链抗体、二硫键稳定的Fv蛋白以及单结构域抗体(sdAb，纳米抗体)、骆驼抗体、双特异性抗体或三特异性抗体。
本发明还提供了一种融合蛋白，其包含本文所述的抗体及其变体，或其抗原结合片段。
示例性地，对于本文所述的融合蛋白，其还包含标签序列(例如Poly-His、Hemagglutinin、c-Myc、GST、Flag-tag等)或IgG1-Fc蛋白序列，或附加表位(如针对或不同于4-1BB的其他表位)或附加的抗体活性片段(如针对4-1BB的其他表位或相同表位的抗体或抗体活性片段，或能结合4-1BB的配体)。
本发明还提供了一种抗体-药物缀合物，其包含本文所述的抗体及其变体，或其抗原结合片段。
对于本文所述的抗体-药物缀合物，其中所述的药物选自如下：放射性标记物、32P、35S、荧光染料、电子致密试剂、酶、生物素、链霉亲和素、洋地黄毒苷、半抗原、免疫原性蛋白质、具有与靶标互补的序列的核酸分子，或任何前述的组合；或免疫调节化合物、抗癌剂、抗病毒剂、抗菌剂、抗真菌剂和抗寄生虫剂，或任何前述的组合。
本发明还提供了一种分离的表达抗体或其抗原结合片段的多核苷酸，其特征在于，所述多核苷酸能够表达本文所述的抗体及其变体，或其抗原结合片段；所述多核苷酸能够表达本文所述的融合蛋白；所述多核苷酸能够表达本文所述的抗体-药物缀合物。
本发明还提供了一种载体，其包含本文所述的多核苷酸，优选为质粒载体。
本发明还提供了一种宿主细胞，其包含根据本文所述的多核苷酸或本文所述的载体，优选地，所述宿主细胞是真核细胞。
本发明还提供了一种药物组合物，其包含本文所述的抗体及其变体，或其抗原结合片段，包含本文所述的融合蛋白，包含本文所述的抗体-药物缀合物，以及任选地，包含药学上可接受的载体。
本发明还提供了一种本文所述的抗体及其变体，或其抗原结合片段，本文所述的融合蛋白，本文所述的抗体-药物缀合物在制备治疗和/或预防与4-1BB相关疾病的药物中的用途。
本发明还提供了一种治疗和/或预防与4-1BB相关疾病以及相关症状的方法，其包括将有效量的本文所述的抗体及其变体，或其抗原结合片段，本文所述的融合蛋白，本文所述的抗体-药物缀合物，或本文所述的药物组合物施用至受试者。
本发明还提供了一种检测样品中是否含有高表达4-1BB的T细胞的方法，其包括将本文所述的抗体及其变体，或其抗原结合片段，本文所述的融合蛋白与样品接触的步骤，任选地，所述检测可以是诊断目的，也可以是非诊断目的。
本发明还提供了一种一种检测产品，其包含本文所述的抗体及其变体，或其抗原结合片段。
示例性地，对于本文所述的检测产品，所述产品选自检测试剂、试剂盒、芯片或试纸中一种或多种。
为了实现上述发明目的，本发明进一步提供以下技术方案：
本发明第一方面提供了一种抗4-1BB纳米抗体，该抗4-1BB纳米抗体的重链可变区由框架区FR和互补决定区CDR组成，互补决定区CDR包含如下组中的至少一组：
(1)SEQ ID NO：1中所示的CDR1、CDR2和CDR3；
(2)SEQ ID NO：2中所示的CDR1、CDR2和CDR3；
(3)SEQ ID NO：3中所示的CDR1、CDR2和CDR3；
(4)SEQ ID NO：4中所示的CDR1、CDR2和CDR3；
(5)SEQ ID NO：5中所示的CDR1、CDR2和CDR3；
(6)SEQ ID NO：6中所示的CDR1、CDR2和CDR3；
(7)SEQ ID NO：7中所示的CDR1、CDR2和CDR3。
纳米抗体(Nb)是骆驼科动物体内重链抗体(IgG2和IgG3)的可变区域，被称为是自然界存在的最小的抗原结合片段。与传统全长单克隆抗体(mAb，约150KD)相比，Nb具有分子量小(12-15KD)、结构更简单、低免疫原性、高组织渗透性、高稳定性、高溶解性和低聚集性以及易于克隆等优点。此外，与mAb同类产品相比，Nb的生产成本显著降低，大多数癌症患者都可以使用。2018年9月欧洲药品管理局已经批准了首款纳米抗体药物caplacizumab(商品名Cablivi)，主要用于治疗成人获得性血栓性血小板减少性紫癜(aTTP)。因此，Nbs有望在癌症治疗和诊断方面提供广泛的应用场景。
本研究采用重组4-1BB抗原免疫骆驼，构建VHH噬菌体文库，经生物淘筛后获得了7种功能性的抗4-1BB Nb(例如SEQ ID Nos：1-7所述的抗体)。
在本发明提供的实施例中，框架区FR包含如下组中的至少一组：
(1)SEQ ID NO：1中所示的FR1、FR2、FR3和FR4；
(2)SEQ ID NO：2中所示的FR1、FR2、FR3和FR4；
(3)SEQ ID NO：3中所示的FR1、FR2、FR3和FR4；
(4)SEQ ID NO：4中所示的FR1、FR2、FR3和FR4；
(5)SEQ ID NO：5中所示的FR1、FR2、FR3和FR4；
(6)SEQ ID NO：6中所示的FR1、FR2、FR3和FR4；
(7)SEQ ID NO：7中所示的FR1、FR2、FR3和FR4。
但本发明的框架区FR并非限定于上述序列，只要能够实现其功能的序列均在本发明保护范围之内。
本发明第二方面提供了一种多核苷酸，该多核苷酸为编码上述抗4-1BB纳米抗体的多核苷酸。
本发明第三方面提供了一种重组表达载体，重组表达载体包含上述多核苷酸。
在本发明提供的实施例中，表达载体包括原核表达载体或真核表达载体。
本发明第四方面提供了一种重组宿主细胞，重组宿主细胞包含上述多核苷酸，或者包含上述重组表达载体。
在本发明提供的实施例中，宿主细胞包括原核细胞或真核细胞。
在本发明提供的具体实施例中，宿主细胞选自大肠杆菌或酵母细胞。
在本发明提供的具体实施例中，宿主细胞选自HEK293T细胞、HEK293F细胞、Expi293F细胞或CHO细胞。
本发明第五方面提供了上述抗4-1BB纳米抗体的制备方法，包括如下步骤：
将编码上述抗4-1BB纳米抗体的多核苷酸插入表达载体中，得到重组表达载体；
将重组表达载体转入宿主细胞，得到重组宿主细胞；
培养重组宿主细胞，得到培养物；
对培养物进行纯化，获得抗4-1BB纳米抗体。
在本发明提供的实施例中，纯化为采用Protein A琼脂糖纯化树脂进行纯化。
本发明第六方面提供了一种双特异性抗体，双特异性抗体包含上述抗4-1BB纳米抗体和第二抗体。
在本发明提供的实施例中，第二抗体包括但不限于4-1BB纳米抗体、CD47纳米抗体、VEGF纳米抗体、HER2纳米抗体、EGFR纳米抗体、HER3纳米抗体、B7H3纳米抗体、TIGIT纳米抗体、OX-40纳米抗体、 CD40纳米抗体或PD-L1纳米抗体。
本发明第七方面提供了上述抗4-1BB纳米抗体或双特异性抗体在制备预防和/或治疗癌症的药物、检测4-1BB蛋白中的应用。
在本发明提供的实施例中，癌症包括但不限于胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤或膀胱肿瘤。
本发明第八方面提供了一种药物组合物，药物组合物包括：上述抗4-1BB纳米抗体或双特异性抗体；以及药学上可接受的辅料。
在本发明提供的实施例中，药物组合物的剂型包括但不限于注射剂、粉针剂、片剂或胶囊剂。
本发明第九方面提供了一种检测4-1BB蛋白的试剂盒，试剂盒包括：上述抗4-1BB纳米抗体或双特异性抗体；以及检测学上可接受的试剂。
附图说明
图1为VHH-hFc重组抗体与人4-1BB过表达293F细胞的FACS结合力；
图2为VHH-hFc重组抗体刺激PBMC的IL-2释放(OKT3存在条件下)；
图3为VHH-hFc重组抗体刺激PBMC的IL-2释放(无OKT3存在条件下)。
具体实施方式
本发明公开了一种抗4-1BB纳米抗体及其生物材料和产品，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
术语解释：
纳米抗体(Nanobody,Nb)，在羊驼外周血液中存在一种天然缺失轻链的抗体，该抗体只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，但却不像人工改造的单链抗体片段(scFv)那样容易相互沾粘，甚至聚集成块。更重要的是单独克隆并表达出来的VHH结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是已知的可结合目标抗原的最小单位。
框架区(Framework region,FR)，为抗体可变区内骨架区。在免疫球蛋白的H和L链的近N端约有110个氨基酸序列的变化很大，其他部分的氨基酸序列相对恒定，据此可将轻链和重链区分为可变区(V)和恒定区(C)。可变区内包含超变区HVR(hypervariable region)或称互补决定区CDR(Complementarity-determining region)与FR骨架区。FR的可变性要低于CDR。FR分子共四个，分别为FRl、FR2、FR3和FR4。在识别抗体时，四个FR分子卷曲使CDR分子相互靠近。
互补决定区(complementarity determining region,CDR)：整个抗体分子可分为恒定区和可变区两部分。在可变区内有一小部分氨基酸残基变化特别强烈，这些氨基酸的残基组成和排列顺序更易发生变异区域称高变区。在L链、H链的V区中有三个高变区(hypervariable regions，HVR)，该部位因在空间结构上可与抗原决定簇形成精密的互补，故高变区又称互补性决定区。
双特异性抗体：是含有2种特异性抗原结合位点的人工抗体，能在靶细胞和功能分子(细胞)之间架起桥梁应，激发具有导向性的免疫反应，是基因工程抗体的一种，现已成为抗体工程领域的热点，在肿瘤的免疫治疗中具有广阔的应用前景。
20种氨基酸的名称及缩写符号：

本发明中所用试剂、仪器、菌种或生物材料等均可通过商购获得。
下面结合实施例，进一步阐述本发明：
实施例1：抗原制备和动物免疫
1、动物免疫：
对2只羊驼的颈背部皮下、肌肉多点注射人4-1BB胞外区重组蛋白(hFc标签)成多个包块，跟踪观察皮下注射包块的吸收情况，以确认免疫正确。
首次免疫使用0.5mg抗原与弗氏完全佐剂1:1混合，乳化后注射，体积1ml/只羊驼；
第二次免疫：首次免疫3周后，使用0.25mg抗原与弗氏不完全佐剂1:1混合，乳化后注射，注射体积1mL/只羊驼；
第三次免疫：第二次免疫3周后，使用0.25mg抗原与弗氏不完全佐剂1:1混合，乳化后注射，注射体积1ml/只羊驼；
第四次免疫：三次免疫3周后，使用0.25mg抗原与弗氏不完全佐剂1:1混合，乳化后注射，注射体积1mL/只羊驼。
2、血清处理和效价检测：
在第三次和第四次免疫后一周，采集外周血2mL，分离血清。将人4-1BB胞外区重组蛋白(his标签)包被在ELISA 96孔板中，利用ELISA法测定血清中抗体效价。ELISA结果显示2只羊驼的四免血清效价>1：32000,符合建库标准。
实施例2：噬菌体展示免疫抗体库的构建
由于羊驼第四次免疫后血清效价>1：32000，表明血清中存在抗人4-1BB的高亲和力抗体。因此，我们按以下步骤构建噬菌体展示免疫抗体库：
①采集2只羊驼第四次免疫后的外周血各50mL，分离淋巴细胞PBMC，合并两只羊驼的PBMC；取2×107的PBMC，利用RNA提取试剂盒提取总RNA；取适量RNA(如3-5μg)，通过RT-PCR反转录试剂盒获得cDNA。②通过巢式PCR分步获取IgG2和IgG3重链可变区序列(纳米抗体的重链可变区域VHH)，实验步骤如下：1)设计1对特异性的巢式外侧引物，以cDNA为模板进行第一轮PCR扩增，扩增区域为羊驼重链抗体基因的Leader-CH2区域，产物大小为700bp和900bp；通过DNA凝胶电泳，切胶回收700bp PCR产物；2)设计巢式内侧引物(羊驼3对)，以700bp的第一轮PCR产物为模板进行第二轮PCR扩增，扩增区域为羊驼重链抗体可变区VHH片段，产物大小为400bp；使用PCR产物纯化试剂盒对第二轮PCR产物进行纯化回收。③通过同源重组或酶切连接的方式将重链可变区序列插入到经酶切处理的线性化噬菌粒载体VHH-libTemplate中，获得重组载体；经纯化回收后，转化超级感受态SS320细胞(含辅助噬菌体M13K07)；转化后的菌液用SOC培养基重悬、活化1小时；取少量菌液进行10倍比梯度稀释，选取适宜的稀释滴度，于LB/tet10及LB/Carb50培养板上涂板，置于37℃生化培养箱过夜，次日用于库容的计算；剩余菌液转入大体积的2YT/Carb50/Kan25液体培养基中，置于37℃摇床，过夜培养，次日收获上清，加入1/4倍体积的PEG/NaCl溶液，沉淀噬菌体后，取适量PBT溶液重悬、稀释至需要的浓度，即得到噬菌体展示免疫抗体库(-80℃保存备用)。④统计LB/Carb50平板上克隆数目，计算库容为：羊驼抗体库Lib 4-1BB Alpaca的库容为4.60×109。从平板上各随机挑取20个单克隆测序，结果表明：羊驼抗体库库Lib 4-1BB Alpaca的VHH插入效率为90％。
实施例3：抗体库的筛选
将5μg/mL的人4-1BB胞外区重组蛋白(his标签)加入96孔板(100μL/孔)，4℃包被过夜；将NEB5αF’大肠杆菌在2YT/Tet10平板划线生长，37℃孵箱过夜培养；第二天，从过夜的2YT/Tet10平板上挑取NEB5αF’单克隆，加入到3mL 2YT/Tet10液体培养基中，37℃摇菌生长至OD600＝0.8；同时，去除96孔板的抗原上清液，每孔加入200μL的1％BSA封闭，同时在空白孔加入200μL 1％BSA作为阴性对照孔，在室温下置于3D旋转振荡器2小时；之后，去除蛋白孔和对照孔的上清液，用200μLPT清洗，各加入100μL的噬菌体抗体库，在室温下置于3D旋转振荡器2hr；去除蛋白孔和对照孔的上清液，用200μL PT清洗；向孔中加入100μL 100mM HCl，室温放置5分钟；将上清液吸出，加入到1.5mL离心管中，使用1M Tris-HCl中和。将上述混合液加入到含1mL NEB5αF’菌的离心管中，摇床37℃，培养1hr；取离心管中培养液20μL进行适宜倍数的稀释，于LB/Carb50培养板上涂板，置于37℃生化培养箱过夜，次日用于滴度和富集程度的计算；剩余培养液中加入1μL辅助噬菌体M13K07(终浓度为1010个/mL)，摇床37℃，培养1hr；将上述培养液转入35mL 2YT/Carb50/Kan25培养液中，置于摇床中，37℃过夜培养，收集噬菌体形成每轮的抗体库。
重复以上操作2-3轮，直到出现噬菌体富集。若抗原结合孔在LB/Carb50培养板上的菌落数是阴性对照孔的10倍以上，视为富集成功。在本实验中，Lib 4-1BB Alpaca在第二轮筛选后，抗原结合孔的菌落数是阴性对照孔的100倍，表明富集成功。
随机挑取富集轮的克隆，在96深孔板中扩大培养，离心后使用上清进行Phage ELISA筛选。挑选与人4-1BB胞外区重组蛋白(his标签)结合OD数值：与封闭液结合OD数值>2的克隆，定义为阳性克隆，进行测序和比对，获得Unique序列。
实施例4：NGS测序
设计引物位于VHH两侧的恒定区，扩增噬菌体文库可变区，对PCR产物进行E-gel检测，再利用试剂盒纯化回收PCR文库片段用于NGS测序。所得数据利用SPSS 2.0和Microsoft Excel 2019进行统计分析，按照DNA的数目和频率进行排序。
实施例5：阳性序列和NGS高频序列的比对和统计
将phage ELISA阳性序列和NGS高频序列(Top 50)，进行比对，并按照CDR H3的差异进行分组，结果见表1。
表1.羊驼免疫获得的4-1BB纳米抗体

注：下划线区域为CDR
实施例6：纳米抗体的真核表达
对实施例5的表1内序列，共7条，进行了真核表达，实验步骤为：1)通过PCR扩增这些序列的VHH片段，使用同源重组或者酶切连接的方法将片段插入含有human IgG1 Fc标签的真核表达载体pFcIG中；电转入大肠杆菌trans5α宿主菌中，经过博莱霉素筛选，对单克隆测序获得正确的重组质粒；然后扩大培养含重组质粒的宿主菌，使用去内毒素试剂盒获得无菌无内毒素质粒；2)用无血清培养基培养HEK293F细胞；使用polyplus悬浮细胞转染试剂将重组表达质粒转入HEK293F细胞进行表达。在转染24和72小时后加入补料，在第5天收集上清，使用Protein A琼脂糖纯化树脂分离纯化抗体，置换保存在PBS的溶液中。实验结果(表2)显示：7条序列的VHH-hFc重组抗体的293F细胞瞬转表达的产量为53-231.4mg/L；SDS-PAGE电泳鉴定：137-1、137-7、137-12、137-18、137-36序列的VHH-hFc的条带大小正常，纯度>95％；137-16、137-39序列的重组表达抗体存在非特异性条带，纯度<95％。
表2.VHH-His重组抗体的真核瞬转表达结果
实施例7：纳米抗体与抗原重组蛋白的亲和力EC50
使用ELISA法，测定7条序列的VHH-hFc重组抗体与人、猴4-1BB胞外区重组蛋白的亲和力：将人、猴4-1BB胞外区重组蛋白(his标签)加入到ELISA 96孔板中，200ng/孔，4℃包被过夜。将VHH-hFc重组抗体稀释到不同的浓度(0.014-10μg/mL)，与抗原进行ELISA反应，使用HRP标记的anti-VHH二抗进行显色，用酶标仪测量450nm处的吸光度值。实验结果(表3和表4)表明：7条序列的VHH-hFc重组抗体都能结合人或者猴4-1BB胞外区重组蛋白，其中CD137-1、CD137-7、CD137-16、CD137-18与人或者猴4-1BB胞外区重组蛋白的亲和力排在前4位。使用相同的实验方法检测重组抗体与小鼠4-1BB胞外区重组蛋白的亲和力，结果(表5)显示：CD137-1、CD137-7、CD137-16、和CD137-18的VHH-hFc重组抗体不结合或者弱结合小鼠4-1BB胞外区重组蛋白。
表3.VHH-hFc重组抗体与人4-1BB(his标签)的ELISA结合力
表4.VHH-hFc重组抗体与猴4-1BB(his标签)的ELISA结合力

表5.VHH-hFc重组抗体与小鼠4-1BB(his标签)的ELISA结合力
实施例8：纳米抗体与抗原重组蛋白的亲和力KD值
使用Biacore检测4条序列(CD137-1、CD137-7、CD137-16、和CD137-18)的VHH-hFc重组抗体与人4-1BB胞外区重组蛋白的亲和力KD值：Biacore 8k设置样品仓和流通室温度25℃，ProteinA芯片捕获VHH-hFc样品作为Ligand，HBS-EP+pH7.4缓冲液稀释对应靶蛋白作为Analytes，Analyte流速30μL/min,结合120s，解离600s。数据采用1:1binding，Fit local Model拟合分析。实验结果(表6)表明：4条序列的VHH-hFc重组抗体都能结合人4-1BB胞外区重组蛋白，KD值在3.3-38nM。
表6.VHH-hFc重组抗体与人4-1BB(his标签)的SPR结合力

实施例9：纳米抗体与抗原重组蛋白过表达细胞的结合
使用流式细胞仪，检测纳米抗体与人4-1BB过表达293F细胞的亲和力：1)使用含有4-1BB全长基因的真核表达质粒转染293F细胞，继续培养24小时；2)取0.3×106个瞬转过表达人4-1BB的293F细胞，用PBS洗涤2次，然后用100μL PBS重悬；3)与10μg/mL VHH-hFc重组抗体孵育1小时；4)PBS洗涤细胞3次后，用100μL PBS重悬细胞，加入0.2μg/mL的FITC标记的Anti-human Fc抗体，孵育1小时；4)PBS洗涤细胞3次后，用300μL PBS重悬细胞，使用流式细胞仪检测荧光。实验结果(图1)表明：CD137-1、CD137-7、CD137-16、CD137-18的VHH-hFc重组抗体能够结合人4-1BB过表达293F细胞。
实施例10:PBMC激活实验
使用ELISA试剂盒检测4条序列(CD137-1、CD137-7、CD137-16、和CD137-18)的VHH-hFc重组抗体刺激人PMBC后，释放IL-2的水平：96孔细胞培养板上包被或者不包被CD3抗体OKT3，4℃过夜；第二天加入PBMC(1X10e5/well)和不同浓度的4-1BB重组抗体，继续培养72小时后收集上清，用于IL-2水平的ELISA检测。实验结果(图2、图3)表明：CD137-1、CD137-7、CD137-16、和CD137-18的VHH-hFc重组抗体，在包被OKT3的情况下，都能够刺激人PBMC释放IL-2。但是在没有包被OKT3的情况下，CD137-1、CD137-7、CD137-16、和CD137-18激活PBMC释放IL-2的能力明显下降，抗体EC50数值明显升高(表7)。另外，实验结果(图3)显示：在有无OKT3的条件下，CD137-1和CD137-18刺激PBMC后的IL-2最大释放量(Max value)数值接近，表明CD137-1和CD137-18 VHH-hFc的重组抗体在有无OKT3的条件下都可以100％激活PBMC。但是在无OKT3的条件下，CD137-7和CD137-16的VHH-hFc重组抗体不能够100％激活PBMC，这两个抗体在无OKT3的条件下的IL-2最大释放量(Max value)数值显著低于在有OKT3的条件下。这些数据表明：CD137-1和CD137-18对PBMC的100％激活是不依赖于CD3抗体，而CD137-7和CD137-16对PBMC的100％激是CD3抗体依赖的。
表7.VHH-hFc重组抗体刺激PBMC生产IL-2的EC50数值
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (11)

  1. 一种抗4-1BB抗体及其变体，或其抗原结合片段，所述抗4-1BB抗体包含名称为137-1、137-7、137-12、137-16、137-18、137-36、137-39的VHH的3个互补决定区CDR1、CDR2和CDR3，
    示例性地，对于所述的抗体及其变体，或其抗原结合片段，所述变体为人源化变体或同一性(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、 96％、97％、98％、99％、99.5％、99.9％)变体；
    或，示例性地，所述的抗体及其变体，或其抗原结合片段，其选自骆驼Ig、Ig NAR、Fab片段、Fab’片段、F(ab)’2片段、F(ab)’3片段、Fv、scFv、双-scFv、(scFv)2、微型抗体、双链抗体、三链抗体、四链抗体、二硫键稳定的Fv蛋白以及单结构域抗体(sdAb，纳米抗体)、骆驼抗体、双特异性抗体或三特异性抗体，进一步地，所述抗体是名称为137-1、137-7、137-12、137-16、137-18、137-36、137-39的抗体。
  2. 一种融合蛋白，其包含权利要求1所述的抗体及其变体，或其抗原结合片段，
    示例性地，所述的融合蛋白还包含标签序列(例如Poly-His、Hemagglutinin、c-Myc、GST、Flag-tag等)或IgG1-Fc蛋白序列，或附加表位(如针对或不同于4-1BB的其他表位)或附加的抗体活性片段(如针对4-1BB的其他表位或相同表位的抗体或抗体活性片段，或能结合4-1BB的配体)。
  3. 一种抗体-药物缀合物，其包含权利要求1所述的抗体及其变体，或其抗原结合片段。
  4. 一种分离的表达抗体或其抗原结合片段的多核苷酸，其特征在于，所述多核苷酸能够表达权利要求所述的抗体及其变体，或其抗原结合片段；所述多核苷酸能够表达本文所述的融合蛋白；所述多核苷酸能够表达本文所述的抗体-药物缀合物。
  5. 一种载体，其包含权利要求4所述的多核苷酸，优选为质粒载体。
  6. 一种宿主细胞，其包含根据权利要求4所述的多核苷酸或权利要求5所述的载体，优选地，所述宿主细胞是真核细胞。
  7. 一种药物组合物，其包含权利要求1所述的抗体及其变体，或其抗原结合片段，包含权利要求2所述的融合蛋白，包含权利要求3所述的抗体-药物缀合物，以及任选地，包含药学上可接受的载体。
  8. 权利要求1所述的抗体及其变体，或其抗原结合片段，权利要求2所述的融合蛋白，权利要求3所述的抗体-药物缀合物在制备治疗和/或预防与4-1BB相关疾病的药物中的用途。
  9. 一种治疗和/或预防与4-1BB相关疾病以及相关症状的方法，其包括将有效量的权利要求2所述的融合蛋白，权利要求3所述的抗体-药物缀合物，或权利要求7所述的药物组合物施用至受试者。
  10. 一种检测样品中是否含有高表达4-1BB的T细胞的方法，其包括将权利要求1所述的抗体及其变体，或其抗原结合片段，权利要求2所述的融合蛋白，或权利要求3所述的抗体-药物缀合物与样品接触的步骤，任选地，所述检测可以是诊断目的，也可以是非诊断目的。
  11. 一种检测4-1BB蛋白的试剂盒，其特征在于，所述试剂盒包括：权利要求1所述的抗体及其变体，或其抗原结合片段，权利要求2所述的融合蛋白，或权利要求3所述的抗体-药物缀合物；以及检测学上可接受的试剂。
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