WO2023115718A1 - 抗pd-l1与ox40双特异性抗体及其用途 - Google Patents

抗pd-l1与ox40双特异性抗体及其用途 Download PDF

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WO2023115718A1
WO2023115718A1 PCT/CN2022/080322 CN2022080322W WO2023115718A1 WO 2023115718 A1 WO2023115718 A1 WO 2023115718A1 CN 2022080322 W CN2022080322 W CN 2022080322W WO 2023115718 A1 WO2023115718 A1 WO 2023115718A1
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bispecific antibody
antibody
heavy chain
amino acid
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French (fr)
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刘浩
许文娟
徐婷
周伟
崔智强
叶洪涛
鲍文英
范清林
宋礼华
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安徽安科生物工程（集团）股份有限公司
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Definitions

  • the present invention belongs to the field of biotechnology, and specifically relates to bispecific antibodies and antibody fragments that specifically bind to OX40 and PD-L1 and compositions containing the antibodies or antibody fragments, as well as nucleic acids encoding the antibodies or antibody fragments thereof and Host cells containing it, and related uses, especially its use in treating diseases related to OX40 or PD-L1, such as cancer and the like.
  • OX40 also known as CD134, belongs to the tumor necrosis factor (TNF) receptor family and is an important co-stimulator of T cell responses.
  • TNF tumor necrosis factor
  • Trimeric OX40 binds to trimeric OX40L protein, activates downstream NF- ⁇ B, PI3K, and AKT pathways, increases the survival and expansion of effector T cells and memory T cells, and increases cytokine secretion.
  • stimulation of these co-stimulatory receptors with agonists should enhance antitumor immunity in the highly immunosuppressive tumor microenvironment.
  • Extensive mouse data also confirm the therapeutic potential of this class of drugs. For example, Genentech combined an OX40 agonist antibody with a PD-L1 antibody, and in a mouse model, 90% of colorectal cancer tumors achieved complete remission.
  • OX40 is a type I transmembrane glycoprotein mainly expressed by T cells (constitutively expressed by regulatory T cells and activated by effector T cells).
  • the ligand of OX40 is OX40L, which was originally found in HTLV-1 converted T cells, called pg34, mainly expressed in APC, and also expressed in NK cells, mast cells and activated T cells.
  • OX40 is expressed on the surface of activated T cells, mainly CD4+ T cells, with a small amount of expression on the surface of CD8+ T cells.
  • OX40 In cancer, activated T cells expressing OX40 are found in tumor infiltrating lymphocytes, and OX40 and its partner Somatic OX40-L plays a key role in the induction and maintenance of T cell responses, so OX40 has become an important target for tumor immunotherapy.
  • the body's immune response process requires the participation of various immune cells and immune molecules, and the activation of T cells is the core of the immune response.
  • T cell activation needs at least two signals, in addition to the first signal T cell recognition of MHC-peptide compounds, various signal stimuli are also necessary during the recognition process. This is the so-called dual-signal hypothesis.
  • the ligand of OX40, OX40L also known as CD252
  • OX40 causes T lymphocytes to divide the number of memory T cells during a large number of divisions, which is conducive to the smooth progress of the second immune response.
  • This biological characteristic of OX40 means that antibodies developed based on this target are expected to bring therapeutic effects to a variety of immune system diseases, including tumors, organ transplantation, asthma, rheumatoid arthritis, systemic lupus erythematosus, etc.
  • TNFR agonism requires receptor aggregation
  • improved aggregation and receptor agonism for the TNF family especially OX40 molecules
  • the present invention fulfills these and other needs.
  • PD-1/PD-L1 signaling pathway plays an important role in regulating immune tolerance, microbial infection and tumor immune escape.
  • PD1 programmed cell death 1, programmed cell death factor 1
  • T cells immune cells
  • PD-L1 a ligand of PD-1
  • Targeting immune checkpoint molecules such as PD-1 or the ligand PD-L1 can add to existing tumor treatments, and blocking the PD-1/PD-L1 signaling pathway can activate inhibited T cells to attack cancer cells.
  • PD-L1 protein has been detected in dozens of human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, and kidney cancer, and the expression of PD-L1 The level is closely related to the clinical and prognosis of patients.
  • PD-L1 is expressed in a variety of tumors, less than 50% of tumor patients respond to treatment with monoclonal antibodies against PD-1/PD-L1, so target other immune checkpoints, such as OX40 , a target that can theoretically have a more general therapeutic effect, can increase and improve the application of monoclonal antibodies in tumor therapy.
  • the purpose of the present invention is to provide an anti-OX40 and PD-L1 antibody that can enhance the immune stimulation of T effector cells and promote the secretion of cytokines, and the antibody has shown a significant tumor suppression effect in a mouse tumor model.
  • the technical solution adopted by the present invention is: a bispecific antibody, comprising IgG domains of light chain and heavy chain and a nanobody variable region part, the nanobody variable region part is connected to the The N-terminal or C-terminal of the heavy chain is connected in series with the variable regions of one or two Nanobodies; the Nanobody domain has binding specificity to the first antigen OX40, and the IgG domain has binding specificity to the second antigen PD-L1 sex.
  • the present invention uses the existing humanized anti-PD-L1 monoclonal antibody and fully human anti-OX40 monoclonal antibody to form a variety of anti-PD-L1 and fully human anti-OX40 monoclonal antibodies in the form of five structural compositions shown in Figure 1.
  • Bispecific antibodies to two antigens of OX40 and tetrameric forms of OX40 nanobodies The bispecific antibodies or nanobody tetramers (this application includes 5 kinds, specifically as follows: L52-D7H232, L52-2D7H232, L52H232-D7, L52H232-2D7 and 2D7-Fc) all have anti-PD1/PD-L1 or Blocking activity of OX40/OX40L.
  • the nanobody variable region (VHH) part can be connected to the N-terminal or C-terminal of the anti-PD-L1 antibody heavy chain; one or more anti-OX40 nanobody VHH can also be connected in series;
  • the bispecific antibody has a first binding specificity to OX40 and a second binding specificity to PD-L1, or, has a first binding specificity to PD-L1 and a second binding specificity to OX40.
  • the obtained bispecific antibodies can all bind human OX40 at the ELISA level, and can bind cynomolgus monkey OX40, but cannot bind mouse OX40; can bind PD-L1 at the ELISA level; can activate downstream signals after binding to OX40, and Has PD-L1 dependence.
  • the bispecific antibody is selected from one or more of fully human antibodies, humanized antibodies, chimeric antibodies or recombinant antibodies.
  • the nanobody is a fully human anti-human OX40 monoclonal nanobody, comprising a heavy chain variable region, and the heavy chain variable region comprises a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and the CDR3 region Respectively comprising SEQ ID NO: 34, 35, 36, any one of the amino acid sequences shown, or a homologous sequence having at least 80% sequence identity with any one thereof.
  • Said Nanobody comprises an amino acid sequence having SEQ ID No: 13, 14, 15 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% similarity to SEQ ID No: 13, 14, 15 .
  • the heavy chain is an IgG1 heavy chain of the PD-L1 antibody, and the heavy chain includes an amino acid sequence having SEQ ID No: 10, 12, 17, 20, 23, 26 or having the same sequence as SEQ ID No: 10, 12, 17 , 20, 23, 26 amino acid sequences of at least 90%, 95%, 96%, 97%, 98% similarity.
  • the light chain is the ⁇ light chain of the PD-L1 antibody, and the ⁇ light chain comprises an amino acid sequence having SEQ ID No: 9, 11, 16, 18, 19, 21, 22, 24, 25, 27 or having the same amino acid sequence as Amino acid sequences of at least 90%, 95%, 96%, 97%, 98% similarity to SEQ ID Nos: 9, 11, 16, 18, 19, 21, 22, 24, 25, 27.
  • the bispecific antibody also includes CH1, CH2, and CH3 of the framework region of human IgG1.
  • the IgG domain comprises an IgG1 constant region having the amino acid sequence of SEQ ID No: 7 or having at least 90%, 95%, 96%, 97%, 98% similarity to SEQ ID No: 7 amino acid sequence.
  • the antibody is an antibody in the form of IgG1 or an antibody in the form of IgG2 or IgG4 or an antigen-binding fragment, and its Fc domains are respectively IgG1, IgG2, IgG3, IgG4 or corresponding engineered subtypes.
  • amino acid sequences of the light chain and the heavy chain are respectively: SEQ ID No: 9 and SEQ ID No: 10 (L52H232), or SEQ ID No: 16 and SEQ ID No: 17 (L52-D7H232), or SEQ ID No : 19 and SEQ ID No: 20 (L52-2D7H232), or SEQ ID No: 22 and SEQ ID No: 23 (L52H232-D7), or SEQ ID No: 25 and SEQ ID No: 27 (L52H232-2D7).
  • the bispecific antibody of the present invention has better accelerated stability, and can maintain higher stability during repeated freezing and thawing and storage at 40°C.
  • the agonist activity of an anti-OX40 antibody is assessed by OX40 signaling (eg, detection of NFKB downstream signaling).
  • OX40 signaling eg, detection of NFKB downstream signaling.
  • the invention provides anti-OX40 antibodies or fragments thereof that increase the level of NFKB-mediated transcriptional activity as compared to IgGl control antibodies. Compared with the corresponding control IgG1, the anti-OX40 and PD-L1 bispecific antibody or fragment thereof of the present invention can increase the level of NF ⁇ B-mediated transcriptional activity several times.
  • the bispecific antibody of the present invention exhibits significant agonistic activity in the presence of human Fc ⁇ IIb cells.
  • the anti-OX40 and PD-L1 bispecific antibody of the present invention has better anti-tumor activity than known anti-OX40 antibodies and anti-PD-L1 antibodies, for example, compared to known anti-OX40 Antibody or PD-L1 antibody, the bispecific antibody of the present invention can significantly reduce tumor volume in a subject, preferably without affecting the subject's body weight.
  • the change of transaminase content is relatively low.
  • the part of the OX40 antibody in the present invention is in the VHH form of the Nanobody obtained by panning, and this form can be conveniently further converted into an Fc fusion protein by genetic engineering techniques well known to those skilled in the art.
  • the anti-PD-L1 antibody part is obtained through humanization of rabbit-derived antibody.
  • the rabbit-derived antibody has extremely high affinity activity, and the generation of immunogenicity in vivo can be reduced after humanization.
  • the bispecific antibody of the present invention is in the form of IgG1 in all evaluations, and the Fc has strong ADCC activity.
  • the preferred anti-human OX40 and PD-L1 bispecific antibody L52-2D7H232 exhibits strong tumor suppressive activity in the MC38 tumor model of mice.
  • the Fc region and human OX40 sequence involved in the present invention are directly or indirectly obtained from human body.
  • Such direct methods include, but are not limited to, genomic DNA cloning or cDNA libraries.
  • the indirect methods include, but are not limited to, synthetic or complete de novo synthesis of intact DNA based on biological information provided by Genbank or other publications or websites.
  • DNA synthesis techniques include, but are not limited to, PCR-based DNA synthesis methods.
  • a fully human anti-human huOX40 and PD-L1 bispecific antibody which can specifically bind to human huOX40 and PD-L1 antigens;
  • the double antibody consists of an IgG part and a VHH part;
  • the IgG part includes SEQ ID NO Any of the amino acid sequences shown in: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26
  • the VHH part includes any one of the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 or a homologous sequence having at least 90% sequence identity with any of them; that is, the above-mentioned 3 formats of Nanobodies: D7, D7-Fc and 2D7-Fc.
  • the method for preparing the above-mentioned bispecific antibody includes culturing the above-mentioned host cell or other expression vector, and expressing the above-mentioned bispecific antibody from the expression vector or host cell.
  • the bispecific antibody can be used to prepare a pharmaceutical composition or to make a kit or a product.
  • the prepared medicine can be used to treat cancer, and the treatment method includes administering to the subject an effective amount of the bispecific antibody or its antigen-binding fragment or the above-mentioned pharmaceutical composition; the cancer is lung cancer, colon cancer, gastric cancer, kidney cancer or liver cancer.
  • the antibody or antibody fragment of the present invention its corresponding heavy chain or light chain coding sequence can be inserted between the transcriptional and translational control sequences of the expression vector.
  • the expression vector of the present invention contains regulatory sequences such as promoters, enhancers and the like.
  • the expression vector and its control sequence should be compatible with recipient cells.
  • the expression of the bispecific antibody in the present invention can also be achieved by transient expression.
  • the expression strategy includes transfecting mammalian cells with one or more expression vectors with DNA fragments encoding the light chain and heavy chain of the antibody, so that the bispecific antibody is expressed and assembled in the recipient cells.
  • the preferred expression method is It is secreted into the culture medium, and the antibody can be recovered therefrom by methods such as chromatography well known to those skilled in the art.
  • Figure 1 is a schematic diagram of the tetramer structure of the bispecific antibody and nanobody
  • Figure 2 is the determination of the binding activity of anti-OX40 and PD-L1 bispecific antibody to OX40 antigen by ELISA method
  • Figure 3 is the determination of the binding activity of anti-OX40 and PD-L1 bispecific antibody to PD-L1 antigen by ELISA method
  • Fig. 4 is the activity detection of OX40 preferred antibody blocking OX40L and OX40 binding
  • Figure 5 is the detection of the binding activity of the preferred bispecific antibody blocking PD-L1 and PD1;
  • Figure 6-a and Figure 6-b are the detection of agonistic activity of OX40 and PD-L1 bispecific antibody
  • Figure 7 shows that the agonistic activity of preferred OX40 and PD-L1 bispecific antibodies is PD-L1 dependent
  • Figure 8-a to Figure 8-e show the SEC purity detection of OX40 and PD-L1 bispecific antibody
  • Figure 8-a is a detection map of 2D7-Fc
  • Figure 8-b is a detection map of L52-2D7H232
  • Figure 8-c is the detection map of L52H232-2D7;
  • Figure 8-d is a detection map of L52-D7H232
  • Figure 8-e is a detection map of L52H232-D7;
  • Figure 9 shows the cross-binding activities of the preferred OX40 and PD-L1 bispecific antibodies to human, murine and monkey OX40 antigens
  • Figure 10-a shows that the preferred bispecific antibody L52-2D7H232 of the present invention has a better tumor inhibitory effect in the MC38 tumor mouse model
  • Figure 10-b to Figure 10-f show the changes in tumor volume of mice in each group
  • Figure 11 records the changes in body weight of the antibody of the present invention during the treatment of tumor mice.
  • the present invention mainly relates to bispecific antibodies that specifically bind PD-L1 and OX40.
  • the bispecific antibodies include a first arm that binds to PD-L1 and a second arm that binds to OX40.
  • the bispecific antibody includes a first arm that binds to OX40 and a second arm that binds to PD-L1; the domains containing the arms include but are not limited to Fab and VHH structures, and the Fc domain can be IgG1, IgG2, IgG3, IgG4 or the corresponding Engineered subtypes.
  • the Nanobody D7 of OX40 was connected to the N-terminal or C-terminal of the PD-L1 heavy chain in a dimer or tetramer manner, respectively, and a tetrameric Fc fusion form of the Nanobody D7 was constructed; the formed bispecific All antibody forms are symmetrical structures, and the Fc segment is the wild type of IgG1.
  • the molecular forms are shown in Figure 1.
  • Example 2 PD-L1 and OX40 bispecific antibody ELISA level combined with OX40 detection
  • rhOX40-Avi-His protein Dilute the expressed and purified rhOX40-Avi-His protein to 1.0 ⁇ g/ml, pave a 96-well ELISA plate and overnight at 4°C; wash three times with TPBS (PBS+0.1% Tween 20) the next day, dissolve 3% skimmed milk powder in TPBS, Block at 37°C for 1 hour; then wash three times with TPBS, add the antibody to be tested (0.1 ⁇ M in the first well, serial 5-fold serial dilution), and incubate with shaking at room temperature for 2 hours; wash three times with TPBS, add goat-anti diluted 1:5000 Human Fc, 100 ⁇ l per well, incubated with shaking at room temperature for 30 min; washed three times with TPBS, and added 100 ⁇ l of OPD o-phenylenediamine (purchased from Sigma, Cat.
  • Example 3 PD-L1 and OX40 bispecific antibody ELISA level combined with PD-L1 detection
  • the binding of the bispecific antibody to the antigenic protein PD-L1 was detected by ELISA.
  • the bispecific antibody can block the combination of OX40L and OX40 was detected at the level of ELISA.
  • the OX40 antigen was plated on a 96-well ELISA plate at a concentration of 1 ⁇ g/ml, and left overnight at 4°C. The next day, the ELISA plate was washed 3 times with PBS+0.1% Tween20 solution, and blocked with PBS-3% skim milk powder solution at 37°C for 1 hour.
  • Example 5 Bispecific antibody blocks PD1/PD-L1 pathway signal transduction
  • CHO-PDL1-TCR and Jurkat-PD1-NFAT cells Resuscitate CHO-PDL1-TCR and Jurkat-PD1-NFAT cells, suspend the cells with medium, add 100 ⁇ l of cell suspension to the black Corning 96-well plate (to make the number of cells in the well reach 50000 cells/well), and in 80 ⁇ l of PBS buffer solution was added around the black Corning 96-well plate, and then placed in a CO2 incubator for 16-24 hours.
  • dilution buffer to dilute the antibody concentration to the highest concentration of 100 ⁇ mol, and then make a 3-fold serial dilution, a total of 9 gradients for use; absorb the CHO-PDL1-TCR cell supernatant, and prepare the prepared PDL1 monoclonal antibody dilution (40 ⁇ l per well) After incubating with the plated CHO-PDL1-TCR cells at room temperature for 20 minutes, add 40 ⁇ l Jurkat-PD1-NFAT (6.25x 105 cells/ml) suspension into the black Corning 96-well plate with a pipette gun, and then put Cultivate for 6 hours in a CO2 incubator.
  • Jurkat-PD1-NFAT 6.25x 105 cells/ml
  • the agonist activity of the bispecific antibodies of the invention can be assessed by measuring the promotion of NFKB-mediated transcriptional activation in a luciferase reporter gene assay.
  • a certain number of CHO-DG44-Fc ⁇ IIB (44F10) and Jurkat-OX40-NF ⁇ B-Luc (21C6) cells were collected and placed in a centrifuge tube, and the cells were resuspended with theoretical buffer (containing CD3 or CD28 antibody at the specified final concentration); Add the mixed cells into a 96-well plate with a white bottom, 50 ⁇ L per well; prepare antibody diluent: dilute the antibody concentration with theoretical buffer to the concentration of the first well of 0.48 ⁇ M, then make a 2-fold serial dilution, a total of 10 gradients for use, add the above-mentioned layered cells In a 96-well plate, 50 ⁇ L/well; the above cell-antibody mixed suspension was placed on a mixer and mixed for 2 minutes, then placed in a CO2 incubator
  • the preferred antibodies L52-D7H232, L52-2D7H232 and 2D7-Fc showed significant agonist activity compared with the control IgG1 antibody; but L52H232-2D7 did not show obvious agonist activity, But L52-2D7H232 showed the strongest agonist activity.
  • Example 7 Preferred bispecific antibody agonist activity is PD-L1 dependent
  • CHO-DG44-PDL1 and CHO-K1 cells After trypsinization of CHO-DG44-PDL1 and CHO-K1 cells, they were resuspended with theoretical Buffer: 1640+10% FBS to a density of 1.6x10 6 cells/ml, each 25 ⁇ l/well, that is, 40,000 cells/well, were spread on white Corning 96 orifice plate. Dilute the antibody with 1640+10% FBS medium, the initial concentration is 150nM, and 4-fold gradient dilution to 10 wells.
  • Example 8 Purity detection of the anti-OX40 and PD-L1 bispecific antibody of the present invention
  • the purity of the bispecific antibody was detected by molecular exclusion, and the results showed that the OX40 and PD-L1 bispecific antibody L52-D7H232, L52-2D7H232, L52H232-D7 and L52H232-2D7 had higher purity, and the OX40 nanobody tetramer The purity of 2D7-Fc is also higher; this is consistent with the stability of the results of symmetric bispecific antibodies.
  • Example 9 Detection of the binding of the anti-PD-L1 and OX40 bispecific antibody of the present invention to murine and cynomolgus monkey OX40
  • the anti-PD-L1 and OX40 bispecific antibody of the present invention can bind to murine and cynomolgus monkey OX40 was detected at the level of ELISA.
  • the antibody to be tested and the detection antibody were added successively, and the experimental results were analyzed according to the OD490 readings, as shown in Figure 9; the experimental results showed that the anti-PD-L1 and OX40 of the present invention
  • the bispecific antibody also has better binding activity to cynomolgus monkey OX40, but does not specifically recognize mouse OX40.
  • our laboratory also identified that the preferred PD-L1 and OX40 bispecific antibodies also did not bind to murine PD-L1.
  • Example 10 Anti-tumor activity of the antibody of the present invention
  • mice were purchased from Biocytogen Biotechnology Co., Ltd., and antibodies L52-D7H232, L52-2D7H232, L52H232 and Durvalumab and MC38-PD-L1 cells were subcutaneously injected into the mice. After the average tumor volume was 100-120 mm3 , the mice were divided into groups and IP administration was started. Bispecific antibodies and control antibodies were administered twice a week, and 2 times a week. The tumor volume was measured and the body weight of the animals was weighed for four weeks.
  • the experimental results show that the tested bispecific antibody can inhibit the growth of the tumor better than the control single-drug antibody, as shown in Figure 10 (af); the tumors of the 6 mice in the preferred antibody L52-2D7H232 group were completely complete at different times after administration. Elimination, see Figure 10-e.
  • the bispecific antibody experimental group did not significantly affect the body weight of the mice compared with the control group; that is, it is preferred that the anti-PD-L1 and OX40 bispecific antibodies have strong anti-tumor activity in mice.
  • the present invention also covers any combination of any of the embodiments described herein. Any embodiment described herein or any combination thereof applies to any PD-L1 and OX40 bispecific antibody or fragment thereof, method and use of the invention described herein.

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Abstract

提供了一种双特异性抗体，可以增强T细胞活化与增殖、促进细胞因子分泌的抗OX40和PD-L1抗体，所述抗体在小鼠肿瘤模型中表现出了显著的肿瘤抑制效果。所述双特异性抗体，包括具有轻链和重链IgG结构域以及可变区部分，所述IgG重链的N端或C端串联一个或者两个纳米抗体的可变区；所述纳米抗体结构域对第一抗原OX40具有结合特异性，IgG结构域对第二抗原PD-L1具有结合特异性。

Description

抗PD-L1与OX40双特异性抗体及其用途 技术领域
本发明属于生物技术领域，具体涉及特异性结合OX40和PD-L1的双特异性抗体和抗体片段以及含有所述抗体或抗体片段的组合物，还涉及编码所述抗体或其抗体片段的核酸及包含其的宿主细胞，以及相关用途，尤其是其在治疗与OX40或PD-L1相关疾病中的用途，例如癌症等。
背景技术
OX40，也称为CD134，属于肿瘤坏死因子(TNF)受体家族的成员，是T细胞反应的重要共同刺激因子。三分子OX40与三聚体OX40L蛋白结合，激活下游NF-κB、PI3K和AKT等途径，增加效应T细胞和记忆T细胞的存活和扩增，增加细胞因子分泌。理论上，在高度免疫抑制的肿瘤微环境中，利用激动剂刺激这些共刺激受体应该能够增强抗肿瘤免疫力。大量的小鼠数据也证实了这类药物的治疗潜力。例如，基因泰克将OX40激动剂抗体和PD-L1抗体联合起来，在一种小鼠模型中，90％的结直肠癌肿瘤获得了完全缓解。
OX40是一种I型跨膜糖蛋白，主要由T细胞表达(结构性地由调节性T细胞表达，激活后由效应T细胞表达)。OX40的配体为OX40L，最初发现于HTLV-1转换的T细胞，被称为pg34，主要表达于APC，在NK细胞、肥大细胞和激活的T细胞上也有表达。OX40表达于活化的T细胞表面，且主要是CD4+T细胞，CD8+T细胞表面有少量表达，在癌症中，在肿瘤浸润淋巴细胞中发现了表达OX40的活化T细胞，而OX40及其配体OX40-L在诱导及维持T细胞反应中起关键作用，因此OX40成为肿瘤免疫治疗的一个重要靶点。
机体的免疫应答过程需要多种免疫细胞和免疫分子共同参与，其中T细胞的活化则是免疫应答的核心。通常，T细胞活化需要至少两个信 号，除了第一信号T细胞识别MHC-肽化合物，识别过程中各种信号刺激也是必须的。这就是所谓的双信号假说。OX40的配体OX40L(又称CD252)以三聚体的形式和3个分子的OX40蛋白结合形成六聚体复合物，从而激活下游NF-κB、PI3K以及AKT等信号通路，这些下游信号的持续激活可以刺激细胞因子的产生，延长T细胞的存活时间，抑制调节性T细胞(Treg)的分化和活性，增强效应T细胞的杀伤能力。另外，OX40使T淋巴细胞在进行大量分裂时，将记忆T细胞的数量也进行分裂，从而利于第二次免疫应答的顺利进行。OX40的这种生物学特性，意味着基于这一靶点开发的抗体，有望对多种免疫系统疾病带来治疗效果，包括肿瘤、器官移植、哮喘、类风湿关节炎、系统性红斑狼疮等。
在免疫原性差的肿瘤中，单一的抗OX40治疗不能提供足够的抗肿瘤免疫原性。由于TNFR的激动作用需要受体聚集，本领域仍然需要关于TNF家族(尤其是OX40分子)的具有多价抗原结合功能的、具有改善的聚集和受体激动作用的单分子实体，以满足日益增加的健康和医疗需求。本发明满足了这些和其它需求。
PD-1/PD-L1信号通路在调节免疫耐受、微生物感染及肿瘤免疫逃逸中发挥着重要作用。PD1(programmed cell death 1，程序性细胞死亡因子1)主要表达在T细胞等免疫细胞，而PD-1的配体PD-L1主要在许多人类肿瘤组织呈高表达。靶向免疫检查点分子例如PD-1或者配体PD-L1能够增加现有的肿瘤治疗方式，阻断PD-1/PD-L1信号通路可使被抑制的T细胞激活，进而攻击癌细胞。目前，已先后在乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌等数十种人类肿瘤组织中检测到PD-L1蛋白的高表达，且PD-L1的表达水平与患者的临床及预后紧密相关。
虽然PD-L1在多种肿瘤中都有表达，但是却只有不到50％的肿瘤患者对针对PD-1/PD-L1的单克隆抗体的治疗有响应，因此瞄准其他免疫 检查点，例如OX40，一种能理论上有较普遍治疗相应的靶点，能够增加并改进单克隆抗体在肿瘤治疗中的应用。
发明内容
本发明的目的在于提供可以增强T效应细胞的免疫刺激、促进细胞因子分泌的抗OX40和PD-L1抗体，所述抗体在小鼠肿瘤模型中表现出了显著的肿瘤抑制效果。
为实现上述目的，本发明采用的技术方案为：一种双特异性抗体，包括具有轻链和重链IgG结构域以及纳米抗体可变区部分，所述纳米抗体可变区部分连接到所述重链的N端或C端并串联一个或者两个纳米抗体的可变区；所述纳米抗体结构域对第一抗原OX40具有结合特异性，IgG结构域对第二抗原PD-L1具有结合特异性。
本发明通过现有的人源化抗PD-L1单克隆抗体与全人源抗OX40单克隆抗体并以图1所示的5种结构组成形式组成了多种能够同时特异性结合PD-L1与OX40两种抗原的双特异性抗体以及OX40纳米抗体的四聚体形式。该双特异性抗体或纳米抗体四聚体(本申请包含5种，具体如下：L52-D7H232、L52-2D7H232、L52H232-D7、L52H232-2D7和2D7-Fc)都具有对PD1/PD-L1或OX40/OX40L的阻断活性。
本发明的双特异性抗体中，纳米抗体可变区(VHH)部分可以连接在抗PD-L1抗体重链的N端或者C端；还可以串联1个或者多个抗OX40纳米抗体VHH；所述双特异性抗体具有对OX40的第一结合特异性和对PD-L1的第二结合特异性，或，具有对PD-L1的第一结合特异性和对OX40的第二结合特异性。所获得双特异性抗体均能在ELISA水平结合人源OX40，且能结合食蟹猴子的OX40，但是不能结合鼠源OX40；能在ELISA水平结合PD-L1；能结合OX40后激活下游信号，且具有PD-L1 依赖性。
所述双特异性抗体选自全人源抗体、人源化抗体、嵌合抗体或重组抗体的一种或多种。
所述纳米抗体为全人源抗人OX40单克隆纳米抗体，包括重链可变区，所述重链可变区包含CDR1区、CDR2区和CDR3区，其中该CDR1区、CDR2区和CDR3区分别包含SEQ ID NO:34、35、36，所示氨基酸序列的任意一种或与其任意一种具有至少80％序列同一性的同源序列。
所述纳米抗体包括具有SEQ ID No:13、14、15的氨基酸序列或者具有与SEQ ID No:13、14、15至少90％、95％、96％、97％、98％相似性的氨基酸序列。
前述的以图1所示的5种结构组成形式组成了OX40纳米抗体的四聚体形式，所述四聚体纳米抗体融合蛋白，连接在IgG1Fc的N端，如SEQ ID NO:15所示；也可以是二聚体纳米抗体融合蛋白，连接在IgG1Fc的N端，如SEQ ID NO:14所示。
所述重链为PD-L1抗体的IgG1重链，所述重链包括具有SEQ ID No:10、12、17、20、23、26的氨基酸序列或者具有与SEQ ID No:10、12、17、20、23、26至少90％、95％、96％、97％、98％相似性的氨基酸序列。
所述轻链为PD-L1抗体的κ轻链，所述κ轻链包括具有SEQ ID No:9、11、16、18、19、21、22、24、25、27的氨基酸序列或者具有与SEQ ID No:9、11、16、18、19、21、22、24、25、27至少90％、95％、96％、97％、98％相似性的氨基酸序列。
所述双特异性抗体还包括人IgG1框架区域的CH1、CH2、CH3。
所述IgG结构域包括IgG1恒定区，所述IgG1恒定区具有SEQ ID No:7的氨基酸序列或者具有与SEQ ID No:7至少90％、95％、96％、97％、98％相似性的氨基酸序列。
连接可变区和重链IgG结构之间的linker部分为一段氨基酸序列，其接头为(GGGGS)n，其中n＝0-4。
所述抗体是IgG1形式的抗体或IgG2形式或IgG4形式的抗体或抗原结合片段，其Fc结构域分别IgG1、IgG2、IgG3、IgG4或相应工程化亚型。
所述轻链和重链的氨基酸序列分别为：SEQ ID No:9和SEQ ID No:10(L52H232)，或SEQ ID No:16和SEQ ID No:17(L52-D7H232)，或SEQ ID No:19和SEQ ID No:20(L52-2D7H232)，或SEQ ID No:22和SEQ ID No:23(L52H232-D7)，或SEQ ID No:25和SEQ ID No:27(L52H232-2D7)。
本发明所述双特异性抗体具有较好的加速稳定性，在反复冻融以及40℃保存时能保持较高的稳定性。
在一些实施方案中，抗OX40抗体的激动剂活性由OX40信号传导(例如检测NFκB下游信号传导)来评估。因此，本发明提供了与用IgG1对照抗体相比，提高NFκB介导的转录活性水平的抗OX40抗体或其片段。与相应的对照IgG1相比，本发明的抗OX40和PD-L1双特异性抗体或其片段能够将NFκB介导的转录活性水平提高数倍。
本发明的双特异性抗体在人FcγIIb细胞的存在下，展现了显著的激动活性。
在一些实施方案中，本发明的抗OX40和PD-L1双特异性抗体相比已知的抗OX40抗体和抗PD-L1抗体具有更好的抗肿瘤活性，例如相比于已知的抗OX40抗体或PD-L1抗体，本发明的双特异性抗体能够显著降低受试者中的肿瘤体积，优选地同时不影响受试者的体重。
本发明的优选抗体在对小鼠肿瘤治疗给药时，产生的转氨酶含量变化相对较低。
本发明中OX40抗体部分是通过淘筛获得的纳米抗体VHH形式的， 这种形式可以通过本领域的技术人员熟知的基因工程技术方便地进一步转换成Fc融合蛋白。
本发明中抗PD-L1抗体部分是通过兔源抗体人源化获得的，兔源抗体具有极高的亲和活性，人源化后可降低体内免疫原性的产生。
本发明的双特异性抗体在各项评价中均为IgG1形式，且Fc具有强ADCC活性。
本发明中优选地抗人OX40和PD-L1双特异性抗体L52-2D7H232，在小鼠的MC38肿瘤模型中展现出了强有力的肿瘤抑制活性。
本发明涉及的Fc区，以及人OX40序列是直接或间接从人体得到的。所述的直接方法包括但不限于基因组DNA克隆或cDNA文库。所述的间接方法包括但不限于Genbank或其他出版物或网站提供的生物信息为基础部分，合成或完全从头合成完整的DNA。DNA合成技术包括但不限于以PCR为基础的DNA合成方法。
全人源抗人huOX40和PD-L1双特异性抗体，所述抗体能特异性地与人huOX40和PD-L1抗原结合；所述双抗有IgG部分和VHH部分组成；IgG部分包括SEQ ID NO:16,SEQ ID NO:17,SEQ ID NO:19,SEQ ID NO:20,SEQ ID NO:22,SEQ ID NO:23,SEQ ID NO:25,SEQ ID NO:26所示氨基酸序列的任意一种或与其任意一种具有至少80％序列同一性的同源序列；即上述的4个优选的双特异性抗体：L52-D7H232、L52-2D7H232、L52H232-D7和L52H232-2D7。VHH部分包括即包括SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15所示氨基酸序列的任意一种或与其任意一种具有至少90％序列同一性的同源序列；即上述的3个形式的纳米抗体：D7、D7-Fc和2D7-Fc。
制备上述双特异性抗体的方法，包括培养上述宿主细胞或其他表达载体，从所述表达载体或宿主细胞中表达所述双特异性抗体。
所述双特异性抗体可以用于制备药物组合物或用于制成试剂盒或 制品。制备的药物可以用于治疗癌症，治疗方法包括向受试者施用有效量的所述双特异性抗体或其抗原结合片段或上述药物组合物；所述癌症是肺癌、结肠癌、胃癌、肾癌或肝癌。
为了表达本发明所述的抗体或抗体的片段，可以把其相应的重链或轻链编码序列插入到表达载体的转录和翻译控制序列之间。本发明所述的表达载体包含调控序列如启动子、增强子等。所述的表达载体及其控制序列应当与受体细胞兼容。
本发明中双特异性抗体的表达还可以通过瞬时表达来实现。该表达策略包括用一种或多种带有编码抗体轻链和重链DNA片段的表达载体转染哺乳动物细胞，从而使双特异性抗体在受体细胞中表达并组装,优选的表达方式是分泌到培养基中,可以用本领域技术人员熟知的层析等方法从中回收抗体。
附图说明
图1为所述双特异性抗体和纳米抗体四聚体结构示意图；
图2为ELISA法测定抗OX40和PD-L1双特异性抗体与OX40抗原的结合活性；
图3为ELISA法测定抗OX40和PD-L1双特异性抗体与PD-L1抗原的结合活性；
图4为OX40优选抗体阻断OX40L与OX40结合的活性检测；
图5为优选双特异性抗体阻断PD-L1与PD1的结合活性检测；
图6-a和图6-b为OX40和PD-L1双特异性抗体的激动活性检测；
图7显示了优选的OX40和PD-L1双特异性抗体的激动活性具有PD-L1依赖性；
图8-a至图8-e显示了OX40和PD-L1双特异性抗体的SEC纯度检测；
图8-a为2D7-Fc检测图；
图8-b为L52-2D7H232检测图；
图8-c为L52H232-2D7检测图；
图8-d为L52-D7H232检测图；
图8-e为L52H232-D7检测图；
图9显示了优选OX40和PD-L1双特异性抗体的交叉结合人源、鼠源和猴子OX40抗原的活性；
图10-a展示了本发明优选的双特异性抗体L52-2D7H232在MC38肿瘤小鼠模型中具有较好的抑瘤效果；
图10-b至图10-f显示了各组小鼠肿瘤体积变化情况；
图11记录了本发明抗体在对肿瘤小鼠进行治疗的过程中，体重变化情况。
具体实施方案
以下实施例可以进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。实施例不包括对传统方法的详细描述。实施例中未注明的技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用仪器未注明生产厂商者，均为可以通过市场购买获得的常规产品。
本发明主要涉及特异性结合PD-L1和OX40的双特异性抗体，在一些实施案例中，双特异性抗体包括结合PD-L1的第一臂和结合OX40的第二臂，在一些实施案例中，双特异性抗体包括结合OX40的第一臂和结合PD-L1的第二臂；含有臂的结构域包括但不限于Fab和VHH结构，Fc结构域可以是IgG1、IgG2、IgG3、IgG4或相应工程化亚型。
实施例1：双特异性抗体分子构建
将OX40的纳米抗体D7分别以二聚体或四聚体的方式连接在 PD-L1重链的N端或C端，并构建了纳米抗体D7的四聚体Fc融合形式；所形成的双特异性抗体形式均为对称型结构，Fc段为IgG1的野生型，各分子形式见图1。
实施例2：PD-L1和OX40双特异性抗体ELISA水平结合OX40检测
将表达纯化的rhOX40-Avi-His蛋白稀释至1.0μg/ml铺96孔ELISA板并4℃过夜；第二天TPBS(PBS+0.1％Tween 20)洗涤三遍，3％脱脂奶粉溶于TPBS，37℃封闭1小时；然后TPBS洗三遍，加入待测抗体(首孔0.1μM，连续5倍梯度稀释)，室温震荡温育2小时；TPBS洗三遍，加入1:5000稀释的goat-anti human Fc，每孔100μl，室温震荡温育30min；TPBS洗三遍，每孔加入混有0.1％过氧化氢的OPD邻苯二胺(购自Sigma公司，货号78412)底物工作液100μl进行显色，约3-7分钟后加入100μl 1M硫酸终止，酶标仪(购自Biotek公司，货号ELX800)测定OD490值；结果分析见图2。
实施例3：PD-L1和OX40双特异性抗体ELISA水平结合PD-L1检测
用ELISA法检测双特异性抗体与抗原蛋白PD-L1的结合情况。将表达纯化的抗原蛋白PD-L1-avi-his 1.0μg/ml浓度铺ELISA板并4℃放置过夜，ELISA板用PBS+0.1％Tween20溶液冲洗3次，用PBS-3％脱脂奶粉溶液37℃封闭1h。用PBS+0.1％Tween20溶液冲洗3次，用浓度梯度的双特异性抗体(首孔1μg/ml，5倍梯度稀释)与抗原结合，再用羊抗人Fc-HRP的第二抗体孵育，OPD显色，1M H 2SO 4终止。显色ELISA板用酶标仪在OD490下读数，将读数与浓度制图(图3)。
实施例4：双特异性抗体阻断OX40与OX40L结合检测
在ELISA水平检测双特异性抗体是否可以阻断OX40L与OX40的结合。将OX40抗原以1μg/ml浓度铺板于96孔ELISA板，4℃过夜，第二天ELISA板用PBS+0.1％Tween20溶液冲洗3次，用PBS-3％脱脂 奶粉溶液37℃封闭1h。用PBS+0.1％Tween20溶液冲洗3次，以20μg/ml的OX40L-mouse Fc为稀释buffer将待测抗体进行梯度稀释(首孔10μg/ml，5倍梯度稀释)，分别加入对应ELISA孔中室温2h；再用羊抗鼠Fc-HRP的第二抗体孵育1h，OPD显色，1M H 2SO 4终止。显色ELISA板用酶标仪在OD490下读数，利用读数与浓度制图(图4)。数据显示，纳米抗体D7-Fc和优选双特异性抗体L52-2D7H232可以在ELISA水平阻断OX40L与OX40的结合。
实施例5：双特异性抗体阻断PD1/PD-L1通路信号传导
复苏CHO-PDL1-TCR和Jurkat-PD1-NFAT细胞，用培养基使细胞混悬后，在黑色康宁96孔板中加入100μl细胞混悬液(使孔中细胞数达到50000cells/well)，并在黑色康宁96孔板四周加入80μl的PBS缓冲液，随后放入CO2培养箱培养16-24h。用稀释buffer稀释抗体浓度至最高浓度100μmol，再做3倍梯度稀释，共9个梯度备用；吸取掉CHO-PDL1-TCR细胞上清，将配制好的PDL1单抗稀释液(40μl每个孔)与铺好板的CHO-PDL1-TCR细胞室温孵育20分钟后，再用移液枪加入40μl Jurkat-PD1-NFAT(6.25x 105cells/ml)混悬液到黑色康宁96孔板中，随后放入CO2培养箱中培养6h。最后将CO2培养箱中的黑色康宁96孔板每孔中加入80ul已经达到室温的Promega Bio-GloTM Luciferase Assary System，并放置在室温用振荡器振荡10分钟后上机检测，读取RLU值并作图分析(5)。
结果显示优选抗体L52-D7H232和L52-2D7H232与对照抗体L52H232和Durvalumab能不同程度的阻断PD1/PD-L1通路信号传导。
实施例6：荧光素酶报告基因T细胞活化测定法：
可以通过在荧光素酶报告基因测定法中测量NFκB介导的转录活化的促进来评估本发明的双特异性抗体的激动剂活性。各收集一定数量的 CHO-DG44-FcγIIB(44F10)和Jurkat-OX40-NFκB-Luc(21C6)细胞置于离心管中，用理论buffer(含指定终浓度的CD3或CD28抗体)重悬细胞；将混合细胞加入白底96孔板中，每孔50μL；配制抗体稀释液：用理论buffer稀释抗体浓度至首孔浓度0.48μM，再做2倍梯度稀释，共10个梯度备用，加入上述铺有细胞的96孔板中，50μL/well；将上述细胞-抗体混合悬液置于混匀仪上充分混合2min，随后放入CO2培养箱培养18h，提前1-2h取出Promega Bio-GloTM Luciferase Assary System在避光条件下平衡至室温，取出96孔板室温平衡10min后加入80μl Luciferase Assary，轻轻加入后室温避光反应3-5min，上机检测RLU信号(图6-a，6-b)。
在如上实验方法中，测得优选抗体L52-D7H232、L52-2D7H232和2D7-Fc与对照IgG1抗体相比，表现出了显著激动剂活性；但L52H232-2D7并未表现出明显的激动剂活性，而L52-2D7H232却表现出了最强的激动剂活性。
实施例7：优选双特异性抗体激动剂活性为PD-L1依赖性
CHO-DG44-PDL1、CHO-K1细胞分别胰酶消化后，分别用理论Buffer:1640+10％FBS重悬至密度1.6x10 6cells/ml，各25μl/孔即40000cells/孔铺于白色康宁96孔板。用1640+10％FBS培养基稀释抗体，初浓度150nM，4倍梯度稀释至10孔。用理论Buffer混悬Jurkat-OX40-NFκB-21C6细胞浓度达到8ⅹ10 5cells/ml，白色康宁96孔板中加入25μl Jurkat-OX40-NFκB-21C6，细胞浓度20000cells/孔，25μl/孔，分别向铺板的两种细胞中加入L52H232、2D7-FC、L52-2D7H232、L52H232+2D7-FC抗体稀释液50μl每孔后于振荡器上混匀，随后放入CO 2培养箱中培养15h后每孔加入100μl已经达到室温的Promega Bio-GloTM luciferase Assary System，并放置在室温10min后上机检测。 实验结果见图7，结果表明优选抗体L52-2D7H232在DG44-PDL1-FL-4B6和Jurkat-OX40-NFκB-21C6检测体系中有明显的PDL1依赖的活性。
实施例8：本发明的抗OX40和PD-L1双特异性抗体的纯度检测
用分子排阻的方式检测双特异性抗体的纯度，结果表明OX40和PD-L1双特异性抗体L52-D7H232、L52-2D7H232、L52H232-D7和L52H232-2D7纯度较高，OX40纳米抗体四聚体2D7-Fc的纯度也较高；这与对称型双特异性抗体的结果稳定性相符。
实施例9：本发明的抗PD-L1和OX40双特异性抗体与鼠源和食蟹猴OX40结合检测
在ELISA水平检测本发明的抗PD-L1和OX40双特异性抗体是否能够与鼠源和食蟹猴OX40结合。分别将人鼠猴三种来源的OX40以适当浓度铺板后，先后加入待测抗体和检测抗体，根据OD490读数来分析实验结果，见图9；实验结果表明，本发明的抗PD-L1和OX40双特异性抗体除了与人源OX40能较好结合之外，对食蟹猴OX40也具有较好的结合活性，但不特异性识别鼠源OX40。另本实验室还鉴定了优选的PD-L1和OX40双特异性抗体也不与鼠源PD-L1结合。
实施例10：本发明抗体的抗肿瘤活性
执行以下实验以确定本发明的抗PD-L1和OX40双特异性抗体是否展现强有力的抗肿瘤活性。为此，从百奥赛图基因生物技术有限公司购买PD-L1/OX40双人源化C57小鼠，获得阻断OX40/OX40L或PD1/PD-L1相互作用抗体L52-D7H232、L52-2D7H232、L52H232和Durvalumab并在小鼠皮下注射MC38-PD-L1细胞，待肿瘤体积均值100-120mm 3后分组并开始IP给药，用双特异性抗体和对照抗体进行一周2次的给药处理，并一周2次测量肿瘤体积和称量动物体重，共给药四周。实验结果表明 测试的双特异性抗体能够比对照单药抗体更好地抑制肿瘤的生长，见图10(a-f)；优选抗体L52-2D7H232组6只小鼠的肿瘤在给药后不同时间肿瘤完全消除，见图10-e。且双特异性抗体实验组相比对照组也没有显著影响小鼠的体重；即优选抗PD-L1和OX40双特异性抗体在小鼠体内具有强有力的抗肿瘤活性。
本发明还涵盖本文所述的任何实施方案的任意组合。本文所述的任何实施方案或其任何组合适用于本文所述发明的任何PD-L1和OX40双特异性抗体或其片段、方法和用途。

Claims (16)

  1. 一种双特异性抗体，其特征在于：所述双特异性抗体包括IgG重链、轻链、连接到重链的C端或N端的纳米抗体部分，所述纳米抗体结构域对第一抗原OX40具有结合特异性，IgG结构域对第二抗原PD-L1具有结合特异性。
  2. 根据权利要求1所述的双特异性抗体，其特征在于：所述双特异性抗体选自全人源抗体、人源化抗体、嵌合抗体或重组抗体的一种或多种。
  3. 根据权利要求1所述的双特异性抗体，其特征在于：所述纳米抗体为全人源抗人OX40单克隆纳米抗体，包括重链可变区，所述重链可变区包含CDR1区、CDR2区和CDR3区，其中该CDR1区、CDR2区和CDR3区分别包含SEQ ID NO:34、35、36，所示氨基酸序列的任意一种或与其任意一种具有至少80％序列同一性的同源序列。
  4. 根据权利要求1或3所述的双特异性抗体，其特征在于：所述纳米抗体包括具有SEQ ID No:13、14、15的氨基酸序列或者具有与SEQ ID No:13、14、15至少90％、95％、96％、97％、98％相似性的氨基酸序列。
  5. 根据权利要求1所述的双特异性抗体，其特征在于：所述纳米抗体为：四聚体纳米抗体融合蛋白，连接在IgG1 Fc的N端，如SEQ ID NO:15所示；和/或，二聚体纳米抗体融合蛋白，连接在IgG1 Fc的N端，如SEQ ID NO:14所示。
  6. 根据权利要求1所述的双特异性抗体，其特征在于：所述重链为PD-L1抗体的IgG1重链，所述重链包括具有SEQ ID No:10、12、17、20、23、26的氨基酸序列或者具有与SEQ ID No:10、12、17、20、23、26至少90％、95％、96％、97％、98％相似性的氨基酸序列。
  7. 根据权利要求1所述的双特异性抗体，其特征在于：所述轻链为PD-L1抗体的κ轻链，所述κ轻链包括具有SEQ ID No:9、11、16、18、19、21、22、24、25、27的氨基酸序列或者具有与SEQ ID No:9、 11、16、18、19、21、22、24、25、27至少90％、95％、96％、97％、98％相似性的氨基酸序列。
  8. 根据权利要求1所述的双特异性抗体，其特征在于：连接可变区和重链IgG结构之间的linker部分为一段氨基酸序列，其接头为(GGGGS)n，其中n＝0-4。
  9. 根据权利要求1-8任一项所述的双特异性抗体，其特征在于：所述抗体是IgG1形式的抗体或IgG2形式或IgG4形式的抗体或抗原结合片段，其Fc结构域分别IgG1、IgG2、IgG3、IgG4或相应工程化亚型。
  10. 根据权利要求1所述的双特异性抗体，其特征在于：所述轻链和重链的氨基酸序列分别为：SEQ ID No:9和SEQ ID No:10，或SEQ ID No:16和SEQ ID No:17，或SEQ ID No:19和SEQ ID No:20，或SEQ ID No:22和SEQ ID No:23，或SEQ ID No:25和SEQ ID No:27。
  11. 一种核酸，编码权利要求1-10任一项所述的双特异性抗体或其抗原结合部分。
  12. 一种表达载体，其特征在于：利用所述表达载体表达权利要求11所述核酸。
  13. 一种宿主细胞，其特征在于：所述宿主细胞包括权利要求12所述的表达载体。
  14. 一种药物组合物，其包含权利要求1-10任一项所述的双特异性抗体或其抗原结合片段。
  15. 一种治疗癌症的方法，其特征在于：所述方法包括向受试者施用有效量的权利要求1-10任一项所述的双特异性抗体或其抗原结合片段或权利要求14的药物组合物；所述癌症是肺癌、结肠癌、胃癌、肾癌或肝癌。
  16. 一种试剂盒或制品，其包含权利要求1-10任一项所述的双特异性抗体。
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