WO2023186061A1 - 抗pd-1纳米抗体、其应用及其治疗疾病的方法 - Google Patents
抗pd-1纳米抗体、其应用及其治疗疾病的方法 Download PDFInfo
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- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70521—CD28, CD152
Definitions
- the invention belongs to the field of biopharmaceutical technology, and specifically relates to nanobodies targeting programmed death receptor 1 (PD-1) and their applications.
- PD-1 programmed death receptor 1
- PD-1 belongs to the immunoglobulin superfamily CD28/B7. It is a type I transmembrane glycoprotein composed of 288 amino acids and is an immunosuppressive receptor.
- PD-1 is structurally composed of an extracellular domain, a hydrophobic span region and a cytoplasmic region.
- the extracellular domain contains an IgV (immunoglobulin variable region) domain, which is related to cytotoxic T lymphocyte antigen 4. (CTLA-4) and other costimulatory molecules have 22-33% homology and contain 4 sites that can be glycosylated; the cytoplasmic tail contains two tyrosine residues at the amino terminus and carboxyl terminus. They constitute an immunoreceptor tyrosine inhibition motif (ITIM) and an immunoreceptor tyrosine switching motif (ITSM) respectively.
- ITIM immunoreceptor tyrosine inhibition motif
- ITDM immunoreceptor tyrosine switching motif
- PD-1 When PD-1 binds to PD-L1, it causes phosphorylation of phosphatidylinositol-3-kinase, further activation of protein kinase B, activation of stimulatory T cell signaling pathways, glucose metabolism, and secretion of interferon, etc.
- the downstream signals of T cell activation are blocked, thereby effectively inhibiting the transcription of T cells, and ultimately inhibiting the immune function of T cells, playing an important role in the negative regulation of immune responses. Therefore, blocking the PD-1/PD-L1 signaling pathway can upregulate T cell activation and activate endogenous anti-tumor immune responses, thereby exerting a therapeutic effect on tumors.
- Pembrolizumab is a world-leading anti-PD-1 monoclonal antibody developed by Merck & Co., which was launched in the United States, Europe and China in September 2014, July 2015 and July 2018 respectively.
- This blockbuster tumor immunotherapy has shown promising results in a variety of cancers and can be used to treat melanoma, colorectal cancer, head and neck cancer, non-small cell lung cancer, and classic Hodgkin There are currently 13 approved indications for lymphoma, bladder cancer, gastric cancer, etc. In 2021, Keytruda's global sales reached US$17.18 billion, making it the absolute TOP1 product among the anti-tumor monoclonal antibody drugs.
- Keytruda has achieved great commercial success, it is a traditional monoclonal antibody and has certain disadvantages compared to nanobodies in terms of stability and production cost.
- Nanobody (Nb) that is, heavy chain single domain antibody VHH (variable domain of heavy chain of heavy-chain antibody), with a diameter of 2.5nm and a length of 4nm, is the smallest naturally occurring fragment that can bind to an antigen.
- Nanobodies can be expressed in both mammals and E. coli expression systems. The expressed antibodies have lower immunogenicity, are easier to produce cheaply in batches, and have higher stability over a wider temperature and pH range. , which has certain advantages over traditional antibodies.
- CN107814845A is the applicant's prior patent, which discloses an anti-PD-1 nanobody.
- the document describes: using human PD-1 antigenic protein to immunize camels to obtain an immune nanobody gene library; coupling PD-1 protein molecules Connect to the enzyme plate to display the spatial structure of the PD-1 protein, and then use phage display technology to screen the immune nanobody gene library obtained with this form of antigen to obtain the PD-1 specific nanobody gene; then use this form of antigen to screen the immune nanobody gene library obtained The gene was transferred into E. coli and a nanobody strain with high specificity that could be expressed efficiently in E. coli was obtained.
- CN107814845A also discloses humanized anti-PD-1 Nanobodies obtained by humanizing camel-derived Nanobodies.
- the Nanobody VHH sequence was synthesized into the pFUSE-hIgG1-Fc2 vector, and after extracting the pFUSE-Nb-hIgG1-Fc2 plasmid, it was transfected into eukaryotic cells HEK293 for expression and purification, and the result was VHH-IgG1-Fc antibody.
- the appearance of the product sample was found to have obvious floc; the CE-SDS test showed obvious split peaks, indicating that the homogeneity of the antibody was relatively poor, and the applicant believed that it was difficult to meet the requirements for development as a drug.
- the standards of candidate molecules must be improved according to the thinking and methods of drug development to meet the requirements of drug development and commercial production.
- the purpose of the present invention is to provide improved anti-PD-1 Nanobodies based on the Nanobody sequence disclosed in CN107814845A.
- the Nanobodies retain high affinity for the antigen PD-1 but have significantly improved uniformity, and have good drug efficacy.
- the efficacy of the drug has been significantly improved, which meets the requirements of being used as a candidate molecule for drug development, is suitable for industrial production, is suitable for subcutaneous administration, and has clinical application potential.
- the invention provides an anti-PD-1 Nanobody, which is composed of a VHH chain and an IgG4-Fc containing a modified hinge region, wherein:
- the anti-PD-1 Nanobody VHH chain includes CDR1 shown in SEQ ID NO.2, CDR2 shown in SEQ ID NO.3, CDR3 shown in SEQ ID NO.4 and SEQ ID NO.28
- the framework region FR2 between CDR1 and CDR2 is shown;
- the modified hinge region in the anti-PD-1 Nanobody includes the amino acid sequence shown in SEQ ID NO:9, or consists of a connecting peptide and the hinge region sequence in IgG4-Fc, and the connecting peptide includes SEQ ID The amino acid sequence shown in NO.12, SEQ ID NO.21 or SEQ ID NO.24.
- anti-PD-1 Nanobody In the present invention, the terms "anti-PD-1 Nanobody”, “anti-PD-1 antibody” and “fusion protein” are used interchangeably depending on the context.
- the CDR1, CDR2 and CDR3 contained in the anti-PD-1 Nanobody VHH chain of the present invention are derived from SEQ ID NO.1.
- CN107814845A discloses a reference Nanobody (VHH-IgG1-Fc) containing the VHH chain of the amino acid sequence shown in SEQ ID NO: 1.
- VHH-IgG1-Fc a reference Nanobody containing the VHH chain of the amino acid sequence shown in SEQ ID NO: 1.
- Those skilled in the art can routinely determine the VHH chain CDR1, CDR2 and CDR3 segments it contains.
- Combinations of VHH chain CDR1, CDR2 and CDR3 divided by methods known in the art are all encompassed by the scope of the present invention.
- the amino acid sequences of the VHH chain CDR1, CDR2 and CDR3 and the amino acid sequence of the framework region FR2 are as follows:
- amino acid sequence of the modified hinge region or the amino acid sequence of the connecting peptide is as follows:
- amino acid sequence of the modified hinge region is shown in SEQ ID NO.9:
- the connecting peptide amino acid sequence is shown in SEQ ID NO.12, SEQ ID NO.21 or SEQ ID NO.24: SEQ ID NO.12:
- the connecting peptide is connected to the N-terminus of the pre-modified IgG4-Fc hinge region shown in SEQ ID NO. 6.
- VHH (SEQ ID NO.1) (CDR1/CDR2/CDR3: SEQ ID NO.2/SEQ ID NO.3/SEQ ID NO.4, underlined and bolded):
- VHH+hinge region (SEQ ID NO.10):
- IgG4-Fc (SEQ ID NO. 5) (hinge region: SEQ ID NO. 6, shown in italics and bold):
- IgG4-Fc (SEQ ID NO. 5) (hinge region: SEQ ID NO. 6, shown in bold):
- IgG4-Fc (SEQ ID NO. 5) (hinge region: SEQ ID NO. 6, shown in bold):
- VHH chain + hinge region (SEQ ID NO.19):
- Antibody full length (SEQ ID NO.20):
- IgG4-Fc (SEQ ID NO. 5) (hinge region: SEQ ID NO. 6, shown in bold):
- Antigen coating Take 100 ⁇ L/well of human PD-1 protein (Acro PD1-HB2F2) with a concentration of 20 ng/mL in a 96-well plate, incubate at room temperature for 2 hours, and wash the plate 5 times;
- step d Add detection antibody: transfer the diluted detection antibody to the sample in step c at a rate of 100 ⁇ L/well. Cover the plate, incubate at room temperature for 1 hour, and then wash the plate 5 times;
- chromogenic substrate Add TMB chromogenic substrate to the sample plate, cover it, and incubate at room temperature for 10-15 minutes;
- CHO-PD-L1- CD3L cell complete culture medium Add 100mL of FBS, 10mL of NEAA, 10mL of penicillin and streptomycin solution, 4mL of Hygromycin B (50mg/L) and 800 ⁇ L of Puromycin (10mg/L) to 1L of DMEM/F12 basic medium to prepare CHO-PD-L1- CD3L cell complete culture medium; inoculate CHO-PD-L1-CD3L cells into the culture medium, and subculture the cells every 2 days to the logarithmic growth phase;
- step d Take out the cell culture plate from step b, aspirate and discard the supernatant, and add the diluted test product, reference product and quality control product, 50 ⁇ L per well;
- Chip Protein A (manufacturer: Pall Fortebio, article number: 18-5010)
- Buffer pH 7.4 PBST (pH 7.4 PBS, Tween20 0.05% v/v)
- the antibodies to be tested are specifically captured through the Protein A chip.
- the signal reaches 3nm and binds to gradient dilutions of PD-1 protein at different concentrations.
- the sample protein to be tested was diluted with PBST buffer to a final concentration of 5ug/mL.
- the lyophilized PD-1 protein was diluted to 250ug/mL with ddH 2 O, and then diluted with PBST, with the highest concentration being 50nM, and 2-fold concentration gradient dilution, for a total of 7 concentrations.
- MC38-hPD-L1 tumor cells colon cancer cells
- mice were isolated and adaptively raised for one week before experimental treatment;
- mice were divided into 5 groups using random block method, namely control group (group 1), Keytruda (group 2), PR antibody group (group 3), optimized nanobody N7 group (group 4), optimized nanobody N15 Group (Group 5), 8 animals in each group.
- the drug was administered according to the schedule in Table 9. Each group was administered intraperitoneally. The administration volume in each group was 10 ml/kg. The drug was administered continuously for 3 weeks, twice a week, for a total of 6 times.
- the antibodies in each group were prepared at a concentration of 1 mg/ml, and the control group used physiological saline, and administration began on the day of grouping.
- the tumor diameter was measured twice a week, the weight of the mice was weighed, the living conditions of the mice were observed, and abnormal conditions were recorded. Use the following calculation formula:
- a represents the long diameter of the tumor
- b represents the short diameter of the tumor.
- V initial is the tumor volume measured during group administration (i.e. d initial )
- V t is the tumor volume at each measurement.
- T/C (%) (T RTV /C RTV ) ⁇ 100%
- T RTV represents the relative tumor volume of the treatment group
- C RTV represents the relative tumor volume of the solvent group. product.
- TGI Tumor volume inhibition rate
- the anti-PD-1 Nanobody of the present invention also has broad clinical treatment prospects as an alternative Nanobody drug that is not inferior to its efficacy.
- the anti-PD-1 Nanobody of the present invention provides drug candidate molecules that are more in line with drug development requirements than PR.
- s.c. means subcutaneous injection
- i.v. means intravenous injection
- BIW*7 means twice a week, a total of 7 times
- P values are all compared with the G1 group, the same below.
- TGI Relative tumor inhibition rate
- TGI relative tumor inhibition rate
- the relative tumor inhibition rates of each group at each time point are shown in Table 15. On D28 after administration, the relative tumor inhibition rates of the G2-G6 group were 45.26%, 55.03%, 51.50%, 46.83% and 46.39% respectively. It can be seen that the same Under dosage conditions, subcutaneous injection of the anti-PD-1 Nanobody of the present invention has comparable or even superior tumor inhibitory effects compared to its intravenous injection, intravenous injection of Keytruda and sintilimab.
- the tumor weights of groups G2 to G6 were 2.25 ⁇ 0.08g, 1.81 ⁇ 0.10g, 2.00 ⁇ 0.11g, 2.28 ⁇ 0.14g and 2.29 ⁇ 0.09g respectively.
- the tumor weights of mice in each experimental group are shown in Table 16. Compared with the G1PBS group, the tumor weights in each group were significantly reduced (p ⁇ 0.01). It can be seen from the tumor weight of each treatment group that compared with the anti-PD-1 Nanobody intravenous injection G6 group, Keytruda G4 group and sintilimab G5 group of the present invention, the anti-PD-1 Nanobody subcutaneous injection G3 group of the present invention showed Equivalent or even better anti-tumor effect.
- the anti-PD-1 Nanobody of the present invention is suitable for subcutaneous and intravenous administration.
- the subcutaneous injection group of the anti-PD-1 Nanobody of the present invention showed better performance than the intravenous injection of Keytruda and sintilimab. It has equivalent or even better anti-tumor effect, is safe and controllable, and has good drug potential.
- subcutaneous administration is more convenient, the infusion time is shorter, the infusion reaction is smaller, the infusion risk is lower, and the administration cost is lower. Therefore, the anti-PD-1 Nanobody of the present invention has good drug potential. and clinical application potential.
Abstract
一种新型的抗PD-1纳米抗体、该纳米抗体的应用及其治疗疾病的方法。该抗体为纳米抗体VHH链与包含经修饰铰链区的IgG4-Fc的融合蛋白。该纳米抗体在表达纯化后无絮状物、均一性好,对抗原PD-1的结合亲和力高,体内抑瘤效果好,适合皮下给药,适用于工业化生产和商品化应用。
Description
相关申请的交叉引用
本专利申请要求于2022年3月31日提交的申请号为CN202210337061.1的中国发明专利申请的优先权权益,在此将其全部内容引入作为参考。
本发明属于生物制药技术领域,具体涉及针对程序性死亡受体1(PD-1)的纳米抗体及其应用。
程序性死亡受体1(PD-1)属于免疫球蛋白超家族CD28/B7,是由288个氨基酸组成的Ⅰ型跨膜糖蛋白,为免疫抑制性受体。PD-1在结构上由胞外结构域、疏水性跨区和胞质区组成,其中,胞外结构域包含一个IgV(免疫球蛋白可变区)结构域,与细胞毒性T淋巴细胞抗原4(CTLA-4)等其他共刺激分子具有22-33%的同源性,同时含有4个能被糖基化的位点;胞质尾部含有氨基端和羧基端两个酪氨酸残基,分别构成一个免疫受体酪氨酸抑制基序(ITIM)和一个免疫受体酪氨酸转换基序(ITSM)。
PD-1的主要配体为PD-L1,其又称B7-H1,是一种由人CD274基因编码的Ⅰ型跨膜糖蛋白。PD-L1含IgV样区、IgC(免疫球蛋白恒定区)样区、跨膜区和细胞质尾区,其中细胞质尾区与细胞内的信号转导相关;IgV区和IgC区则参与细胞间的信号转导。
PD-1与PD-L1结合时,通过发生磷脂酰肌醇-3-激酶的磷酸化、蛋白激酶B的进一步激活、刺激性T细胞信号通路的活化、葡萄糖代谢以及干扰素的分泌等,导致T细胞活化的下游信号受阻,进而有效抑制T细胞的转录,最终抑制T细胞的免疫功能,在免疫应答的负性调控方面发挥着重要作用。因此,阻断PD-1/PD-L1信号通路可使T细胞活化上调,激活内源性抗肿瘤免疫反应,从而发挥对肿瘤的治疗作用。
(帕博利珠单抗)为默沙东公司研制的一款全球领先的抗PD-1单抗,分别于2014年9月、2015年7月及2018年7月在美国、欧洲和中国上市。这款重磅肿瘤免疫疗法在多种癌症中展现出了喜人的疗效,可用于治疗黑色素瘤、结直肠癌、头颈癌、非小细胞肺癌、经典霍奇金
淋巴瘤、膀胱癌、胃癌等,目前获批的适应症达13个。2021年Keytruda全球销售额高达171.8亿美元,在一众抗肿瘤单抗药物中成为绝对的TOP1产品。但是,Keytruda虽然取得了商业上的巨大成功,然而其为传统的单抗,相对纳米抗体在稳定性、生产成本等方面存在一定的劣势。
纳米抗体(nanobody,Nb),即重链单域抗体VHH(variable domain of heavy chain of heavy-chain antibody),直径2.5nm,长4nm,是自然存在的可以和抗原结合的最小片段。纳米抗体既能在哺乳动物中表达,又可在大肠杆菌表达系统中表达,表达的抗体具有较低的免疫原性,更容易廉价批量生产,在更宽的温度和pH范围内稳定性更高,相比传统抗体具有一定的优势。
CN107814845A为申请人在先专利,公开了一种抗PD-1纳米抗体,该文件描述了:利用人源的PD-1抗原蛋白免疫骆驼,获得免疫纳米抗体基因库;将PD-1蛋白分子偶联在酶标板上,展示PD-1蛋白的空间结构,然后以此形式的抗原利用噬菌体展示技术筛选获得的免疫纳米抗体基因库,从而获得PD-1特异性的纳米抗体基因;再将此基因转至大肠杆菌中,获得了能在大肠杆菌中高效表达的、且特异性高的纳米抗体株。此外,CN107814845A还公开了通过对骆驼源的纳米抗体进行人源化改造而获得的人源化的抗PD-1纳米抗体。
但是,CN107814845A中在载体构建时,将纳米抗体VHH序列合成到pFUSE-hIgG1-Fc2载体上,提取pFUSE-Nb-hIgG1-Fc2质粒后,转染至真核细胞HEK293中进行表达及纯化,得到了VHH-IgG1-Fc抗体。但是,该抗体进行纯化后,产物样品经外观检测发现有明显絮状物;CE-SDS检测显示出明显的分裂峰,表明该抗体均一性相对较差,申请人认为其难以达到作为药物开发的候选分子的标准,须按照药物开发的思维和方法对其进行改进,以满足药物开发及商业化生产的要求。
发明内容
本发明的目的在于,基于CN107814845A中公开的纳米抗体序列,提供改进的抗PD-1纳米抗体,该纳米抗体保留对抗原PD-1的高亲和力但均一性得到明显改善,并具有良好药物疗效,药物疗效得到显著提升,符合将其作为药物开发候选分子的要求,并适于工业化生产,适合皮下给药,且具有临床应用潜力。
本发明的技术方案如下:
一方面,本发明提供一种抗PD-1纳米抗体,其由VHH链与包含经修饰铰链区的IgG4-Fc组成,其中:
(i)所述抗PD-1纳米抗体VHH链包含SEQ ID NO.2所示的CDR1、SEQ ID NO.3所示的CDR2、SEQ ID NO.4所示的CDR3及SEQ ID NO.28所示的CDR1和CDR2之间的框架区FR2;并且
(ii)所述抗PD-1纳米抗体中的经修饰铰链区包含SEQ ID NO:9所示氨基酸序列,或者由连接肽与IgG4-Fc中的铰链区序列组成,所述连接肽包含SEQ ID NO.12、SEQ ID NO.21或SEQ ID NO.24所示氨基酸序列。
在本发明中,术语“抗PD-1纳米抗体”、“抗PD-1抗体”和“融合蛋白”根据上下文可互换使用。
本发明的抗PD-1纳米抗体VHH链包含的CDR1、CDR2和CDR3源自SEQ ID NO.1。
SEQ ID NO.1:
CN107814845A公开了包含SEQ ID NO:1所示氨基酸序列的VHH链的参照纳米抗体(VHH-IgG1-Fc),本领域技术人员可以常规地确定其包含的VHH链CDR1、CDR2和CDR3区段。以本领域已知方法划分得到的VHH链CDR1、CDR2和CDR3组合均被涵盖在本发明的范围内。
根据本发明的具体实施方式,在本发明提供的抗PD-1纳米抗体中,所述VHH链CDR1、CDR2和CDR3的氨基酸序列以及框架区FR2的氨基酸序列分别如下所示:
SEQ ID NO.2:
SEQ ID NO.3:
SEQ ID NO.4:
SEQ ID NO.28:
根据本发明的具体实施方式,在本发明提供的抗PD-1纳米抗体中,所
述经修饰铰链区的氨基酸序列或者所述连接肽的氨基酸序列分别如下所示:
经修饰铰链区的氨基酸序列如SEQ ID NO.9所示:
修饰前IgG4-Fc中的铰链区序列如SEQ ID NO.6所示:
连接肽氨基酸序列如SEQ ID NO.12、SEQ ID NO.21或SEQ ID NO.24所示:SEQ ID NO.12:
SEQ ID NO.21:
SEQ ID NO.24:
所述连接肽连接至SEQ ID NO.6所示的修饰前IgG4-Fc铰链区的N端。
优选地,本发明提供的抗PD-1纳米抗体为抗人PD-1纳米抗体。
进一步地,在本发明提供的抗PD-1纳米抗体中,所述VHH链包含上述CDR1、CDR2和CDR3以及其间的4个框架区(framework region,FR),各个区的排列方式为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。优选地,在本发明提供的抗PD-1纳米抗体中,所述VHH链包含SEQ ID NO.27所示的FR1、SEQ ID NO.29所示的FR3和SEQ ID NO.30所示的FR4。
SEQ ID NO.27:
SEQ ID NO.29:
SEQ ID NO.30:
根据本发明的具体实施方式,在本发明提供的抗PD-1纳米抗体中,所述VHH链包含SEQ ID NO.7所示氨基酸序列。
SEQ ID NO.7:
优选地,本发明提供的抗PD-1纳米抗体包含SEQ ID NO.10、SEQ ID NO.
13、SEQ ID NO.22或SEQ ID NO.25所示氨基酸序列。
SEQ ID NO.10:
SEQ ID NO.13:
SEQ ID NO.22:
SEQ ID NO.25:
进一步地,根据本发明的具体实施方式,本发明提供的抗PD-1纳米抗体由VHH链与包含经修饰铰链区的IgG4-Fc组成,所述纳米抗体氨基酸全长序列如SEQ ID NO.11、SEQ ID NO.14、SEQ ID NO.23或SEQ ID NO.26所示。
SEQ ID NO.11:
SEQ ID NO.14:
SEQ ID NO.23:
SEQ ID NO.26:
另一方面,本发明提供一种核酸分子,所述核酸分子包含核苷酸序列,所述核苷酸序列编码本发明提供的抗PD-1纳米抗体中包含的氨基酸序列。所述氨基酸序列可以是上文提供的任一序列。本发明所述的核苷酸序列可以为DNA序列或RNA序列。所述核酸分子可以是DNA分子或RNA分子,
可以是单链或双链的。
再一方面,本发明提供一种载体,所述载体含有本发明提供的核酸分子。优选地,所述载体可以为表达载体。根据本发明的具体实施方式,所述载体为真核细胞表达载体或原核细胞表达载体。
还一方面,本发明提供一种宿主细胞,所述宿主细胞含有本发明提供的载体,例如被所述载体转化或转染,或所述宿主细胞在基因组中整合有本发明提供的核酸分子。根据本发明的具体实施方式,所述宿主细胞为真核细胞,例如真菌、昆虫、植物或动物细胞,或为原核细胞,例如细菌细胞。
另一方面,本发明提供一种组合物,所述组合物包含本发明提供的抗PD-1纳米抗体、核酸分子、载体和/或宿主细胞。
再一方面,本发明提供所述抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物在制备用于治疗PD-1或PD-L1相关疾病的药物中的用途。优选地,所述疾病为肿瘤或癌症,或感染性疾病。进一步优选地,所述疾病为肺癌(例如非小细胞肺癌)、胃癌、肝癌、黑色素瘤、宫颈癌、结直肠癌、膀胱癌、乳腺癌、白血病、淋巴瘤、肾细胞癌、盲肠癌、胰腺癌、胆管癌、头颈癌、梅克尔细胞癌、卵巢癌、鼻咽癌、胶质瘤、食管癌、骨癌或前列腺癌。优选地,所述疾病为结直肠癌或肺癌。
因此相应地,本发明还提供一种治疗PD-1或PD-L1相关疾病的方法,所述方法包括给有此需要的受试者施用本发明提供的抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物。优选地,所述疾病为肿瘤或癌症。进一步优选地,所述疾病为肺癌(例如非小细胞肺癌)、胃癌、肝癌、黑色素瘤、宫颈癌、结直肠癌、膀胱癌、乳腺癌、白血病、淋巴瘤、肾细胞癌、盲肠癌、胰腺癌、胆管癌、头颈癌、梅克尔细胞癌、卵巢癌、鼻咽癌、胶质瘤、食管癌、骨癌或前列腺癌。优选地,所述疾病为结直肠癌或肺癌。所述受试者是哺乳动物,例如人、家畜和农用动物以及动物园、运动或宠物动物,比如绵羊、狗、马、猫、牛、大鼠、猪、猕猴。根据本发明的具体实施方式,所述受试者是人。
优选地,本发明抗PD-1纳米抗体施用方式可以是静脉给药或皮下给药,更优选地,选择皮下给药,例如皮下注射。
还一方面,本发明提供所述抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物在制备PD-1检测试剂中的用途。优选地,所述检测试剂为基于流式细胞术或酶联免疫吸附法的检测试剂。
因此相应地,本发明还提供一种PD-1检测试剂,所述PD-1检测试剂包含本发明提供的抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物。优选地,所述检测试剂为基于流式细胞术或酶联免疫吸附法的检测试剂。
本发明还提供一种检测来自受试者的样品中PD-1的方法,所述方法包括使本发明提供的抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物与所述样品相接触。
又一方面,本发明提供一种试剂盒,所述试剂盒包含本发明提供的抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物。优选地,所述试剂盒为检测试剂盒,例如基于流式细胞术或酶联免疫吸附法的检测试剂盒。
另一方面,本发明提供一种产生所述抗PD-1纳米抗体的方法,所述方法包括以下步骤:
(a)培养本发明提供的宿主细胞,从而获得含所述抗PD-1纳米抗体的培养物;和,(b)从所述培养物中回收所述抗PD-1纳米抗体。
与现有技术相比,本发明提供了一种抗PD-1纳米抗体,所述纳米抗体为纳米抗体VHH链与包含经修饰铰链区的IgG4-Fc的融合蛋白,其是基于CN107814845A中公开的纳米抗体的改进纳米抗体。
考虑到CN107814845A中表达抗体时采用的真核细胞HEK293并非抗体工业化生产用菌株,采用的IgG1也非针对PD-1的常规抗体亚型(抗PD-1单抗治疗药物均为IgG4型,从而仅保留阻断功能,弱化ADCC类效应子功能),因此本发明的发明人将CN107814845A中纳米抗体VHH链序列合成到IgG4-Fc上并采用工业化使用的CHO细胞进行表达及纯化,结果发现,纯化后的VHH-IgG4-Fc(以下简称PR抗体)同样存在明显絮状物,经CE-SDS检测,也出现了明显的分裂峰,说明经IgG4-Fc优化后的抗体絮状物及均一性未得到明显改善,难以实现基于该VHH序列的抗体成药性研究、工业化生产及临床治疗应用。
鉴于此,本发明的发明人进一步从成药性研发角度对序列进行了优化,特别是对VHH链、铰链区和Fc区域中的多个氨基酸位点进行特定氨基酸置换,通过对所得蛋白的理化、生化性质(包括均一性、结合亲和力、抗原活性阻断作用以及体内抑瘤实验)检测,筛选得到了具有更优活性、更具应用价值的新型抗PD-1纳米抗体,特别是该纳米抗体在表达纯化后无絮状物、均一性好,对抗原PD-1的结合亲和力高,体内抑瘤效果显著提升,特别是疗效可与(帕博利珠单抗)匹敌,符合将其作为药物开
发候选分子的要求,并适于工业化生产,适合皮下给药,且具有临床应用潜力。
以下,结合附图来详细说明本发明的实施方案,其中:
图1:VHH-IgG1-Fc和VHH-IgG4-Fc(PR抗体)的CE-SDS检测结果。
图2:PR抗体与优化纳米抗体的序列比对结果。
图3:PR抗体与优化纳米抗体的CE-SDS检测结果。
图4:优化纳米抗体N7和N15的SEC检测结果。
图5:给药后肿瘤体积增长趋势图。
图6:试验结束后小鼠肿瘤重。
实施发明的最佳方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解,这些实施例仅用于说明本发明,其不以任何方式限制本发明的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的原料、试剂材料等,如无特殊说明,均为市售购买产品。
下述实施例中,采用以下程序:
(一)纳米抗体样品的制备与纯化:
a.合成编码纳米抗体序列的基因序列;
b.将合成的基因序列构建到pcDNA3.1载体上,转化至大肠杆菌DH5α菌株中,大摇并用Omega质粒大提试剂盒提取pcDNA3.1质粒,并过滤除菌;
c.将CHO-S细胞培养至5×106个/ml;
d.将步骤b得到的pcDNA3.1质粒与转染试剂PEI以1:3的比例在转染培养基ExpiCHOTM Expression Medium中混合均匀并静置20min,然后加入到步骤c的CHO-S细胞中,于37℃、6%CO2、115rpm培养5天;
e.培养产物离心,0.2μm滤膜过滤,得到离心上清;
f.采用ProA亲和层析法纯化离心上清,使用pH7.0的Tris体系缓冲液清洗掉杂蛋白和其他杂质;
g.使用24mM醋酸洗脱;
h.将得到的洗脱液用Tris Base回调pH至5.0,再用0.2μm滤膜过滤,得到待测样品。
(二)纳米抗体样品的外观及可见异物检测
样品纯化后收集于西林瓶,观测纳米抗体样品外观及可见异物情况。
(三)纳米抗体样品的CE-SDS检测:
a.溶液制备:分别配制稀释缓冲液,DTT,NEM,染色凝胶和脱色液,分子量标准品和变性剂;
b.样品处理:在样品中加入超纯水和还原试剂(蛋白230试剂盒提供的样品缓冲液,与1M DTT混合)稀释后变性;非还原条件下,样品加入120mM N乙基马来酰亚胺(NEM)溶液和非还原试剂(样品溶液与超纯水混合);将还原和非还原条件下的ladder(阶梯式标记物)和样品加热后加到芯片上;样品在充满聚合物溶液的微通道中进行电泳分离;
c.检测:基于一种荧光染料的激光诱导荧光,样品添加到聚合物溶液中,并以非共价结合到蛋白质-SDS胶束上;
d.结果分析:生物分析仪软件根据ladder(阶梯式标记物)自动确定大小,并计算电泳图中每个分离峰的百分比。
(四)纳米抗体样品的SEC检测
a.试剂制备:制备溶液A(100mM磷酸盐缓冲液、100mM硫酸钠,pH=2.8)、溶液B(100mM磷酸盐缓冲液、100mM硫酸钠,pH=7.0);
b.检测系统设置:运行“U3000SEC”项目和“3000”色谱系统,使用A溶液清洗A管路,B溶液清洗B管路,柱温设置为25℃,样品室温度设置为8℃,关闭清洗阀,用超纯水清洗系统后,平衡系统压;
c.分析样品:在Empower3上设置样品序列,分析6针标准品后,每个样品分析1针2mg/ml,所有样品分析完后再分析1针标准品,进样体积为5微升;然后加载并运行序列;
d.运行结束后,分析数据。
实施例1已知抗PD-1纳米抗体的检测
CN107814845A中公开了一种新的抗PD-1纳米抗体及其应用,该抗体的VHH序列如下:
SEQ ID NO.1(CDR1/CDR2/CDR3:SEQ ID NO.2/SEQ ID NO.3/SEQ ID NO.4,下划线并加粗示出):
按照上文“(一)纳米抗体样品的制备”所述程序制备VHH-IgG4-Fc(PR抗体),其中IgG4-Fc的氨基酸序列示于SEQ ID NO.5。
对CN107814845A中公开的在HEK293中表达的VHH-IgG1-Fc(参见CN107814845A实施例8)和VHH-IgG4-Fc纯化后,按照上文“(二)纳米抗体样品的外观及可见异物检测”所述程序进行观测,发现,纯化后的抗体样品均出现明显的絮状物。按照上文“(三)纳米抗体样品的CE-SDS检测”程序进行检测,发现均检测到分裂峰,结果分别见图1中的1A(
VHH-IgG1-Fc)和1B(VHH-IgG4-Fc)。
不受限于任何理论,认为可能由于VHH、铰链区和Fc区域的特定氨基酸造成纯化后絮状物产生,且均一性很差,须进一步从成药性研发角度对现有序列进行优化。
实施例2已知抗PD-1纳米抗体的序列优化设计及初步筛选
为解决PR抗体纯化后出现絮状物及均一性较差的缺陷,对PR抗体进行优化,优化设计方案见表1。
表1.PR抗体的优化方案
注:表1中CDR,优化数2,是指针对CDR区设计了2种序列优化方案。同理,针对FR区设计了8种序列优化方案;同时针对FR和CDR区两者设计了8种序列优化方案;同时针对FR和Hinge(铰链区)区两者设计了1种序列优化方案;对FR序列优化的同时增加连接polypeptide(连接肽)的方案设计了3种;构型变化即VHH由原来的连接至Fc的N端改为连接至C端的方案。
按照上文“(一)纳米抗体样品的制备”所述程序制备优化纳米抗体样品,并按照上文“(二)纳米抗体样品的外观及可见异物检测”和“(三)纳米抗体样品的CE-SDS检测”程序进行样品检测。结果见表2。
表2.优化纳米抗体样品的检测结果
从CE-SDS检测结果及纯化后样品外观情况可以看出,N7、N10、N12、N13、N14、N15均一性好,且无明显絮状物形成。
实施例3优化纳米抗体的理化、生化分析
对初步筛选出的N7、N10、N12、N13、N14、N15进行理化、生化分析及进一步筛选。PR抗体与优化纳米抗体的序列见下(加粗、斜体并带下划线示出的为优化位点;按照IMGT定义CDR区域),序列比对见图2。PR:
VHH(SEQ ID NO.1)(CDR1/CDR2/CDR3:SEQ ID NO.2/SEQ ID NO.3/SEQ ID NO.4,以下划线并加粗示出):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以加粗示出):
N7:
VHH(SEQ ID NO.7)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.27/SEQ ID NO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR2突变氨基酸以下划线、斜体并加粗示出):
IgG4-Fc(SEQ ID NO.8)(铰链区:SEQ ID NO.9,以加粗示出,铰链区突变氨基酸以下划线示出):
VHH+铰链区(SEQ ID NO.10):
抗体全长(SEQ ID NO.11):
N10:
VHH(SEQ ID NO.7)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.27/SEQ ID NO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR2突变氨基酸以下划线、斜体并加粗示出):
连接肽(SEQ ID NO.12):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以斜体并加粗示出):
VHH链+连接肽+铰链区(SEQ ID NO.13):
抗体全长(SEQ ID NO.14):
N12:
VHH(SEQ ID NO.15)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.31/SEQ ID NO.2/SEQ ID NO.32/SEQ ID NO.3/SEQ ID NO.33/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR1和FR3突变氨基酸以下划线、斜体并加粗示出):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以加粗示出):
VHH链+铰链区(SEQ ID NO.16):
抗体全长(SEQ ID NO.17):
N13:
VHH(SEQ ID NO.18)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.34/SEQ ID NO.2/SEQ ID NO.32/SEQ ID NO.3/SEQ ID NO.35/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR1和FR3突变氨基酸以下划线、斜体并加粗示出):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以加粗示出):
VHH链+铰链区(SEQ ID NO.19):
抗体全长(SEQ ID NO.20):
N14:
VHH(SEQ ID NO.7)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.27/SEQ ID NO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR2突变氨基酸以下划线、斜体并加粗示出)
连接肽(SEQ ID NO.21):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以加粗示出):
VHH链+连接肽+铰链区(SEQ ID NO.22):
抗体全长(SEQ ID NO.23):
N15:
VHH(SEQ ID NO.7)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.27/SEQ ID NO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR2突变氨基酸以下划线、斜体并加粗示出)
连接肽(SEQ ID NO.24):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以加粗示出):
VHH链+连接肽+铰链区(SEQ ID NO.25):
抗体全长(SEQ ID NO.26):
3.1CE-SDS检测
按照上文“(二)纳米抗体样品的CE-SDS检测”程序进行样品检测。结果见表3和图3中的3A(PR)、3B(N7)、3C(N10)、3D(N12)、3E(N13)、3F(N14)和3G(N15)。
表3.优化纳米抗体样品的CE-SDS检测结果
3.2 SEC检测
按照上文“(四)纳米抗体样品的SEC检测”程序进行样品检测。结果见表4和图4中的4A(N7)和4B(N15)。
表4.优化纳米抗体样品的SEC检测结果
3.3抗原结合(ELISA)
通过ELISA检测优化纳米抗体对抗原PD-1蛋白的结合能力。方法如下:
a.抗原包被:取100μL/孔浓度为20ng/mL的人PD-1蛋白(Acro PD1-HB2F2)于96孔板中,室温孵育2小时后,洗板5次;
b.参比品及供试品准备:参比品和供试品稀释至1000ng/mL后,进行梯度稀释;
c.反应:将梯度稀释后的样品按100μL/孔加入到步骤a中预先包被的96孔板中,盖上板盖,室温孵育2小时后,洗板5次;
d.加入检测抗体:按100μL/孔将稀释后的检测抗体转移至步骤c中样品
板中,盖上盖板,室温孵育1小时后,洗板5次;
e.加入显色底物:在样品板中加入TMB显色底物,盖上盖板,室温孵育10-15分钟;
f.终止显色反应:在样品板中加入2M硫酸,终止显色反应;
g.读板:将样品板放置于酶标仪中,进行读板。
结果见表5。
表5.优化纳米抗体的抗原结合检测结果
3.4抗原活性的阻断检测
检测优化纳米抗体对抗原PD-1蛋白活性的阻断作用。方法如下:
a.在1L的DMEM/F12基础培养基中加入FBS 100mL、NEAA 10mL、青链霉素溶液10mL、Hygromycin B(50mg/L)4mL和Puromycin(10mg/L)800μL,配制CHO-PD-L1-CD3L细胞完全培养基;将CHO-PD-L1-CD3L细胞接种到培养基中,每2天进行细胞传代培养至对数生长期;
在1640基础培养基1L中加入FBS 100mL、NEAA 10mL、青链霉素溶液10mL、Hygromycin B(50mg/L)8mL和Puromycin(10mg/L)400μL,配制Jurkat-PD-1-NFAT细胞完全培养基;将Jurkat-PD-1-NFAT细胞接种到培养基中,每2天进行细胞传代培养至对数生长期;
b.消化并离心CHO-PD-L1-CD3L细胞,用DMEM/F12完全培养基重悬细胞至5×105个/mL,每孔100μL加入到白底96孔板中,于37±2℃、(5±1)%CO2培养箱中孵育16±2h;
c.将供试品、参比品及质控品预稀释至100μg/mL,再用分析培养基(RPMI1640基础培养基+2%FBS)进行梯度稀释;
d.将步骤b的细胞培养板取出,吸取上清液弃掉,加入稀释后的供试品、参比品及质控品,每孔50μL;
e.取出Jurkat-PD-1-NFAT细胞,用分析培养基重悬细胞至2×106个/mL;以每孔50μL将该Jurkat细胞悬液加入到步骤d的培养板中,于培养箱中孵育6h;
f.提前1h取出Bio-Glo Luciferase Assay System试剂,室温放置,每孔100μL加入到细胞板;室温避光孵育2~3min后用酶标仪进行读数。
结果见表6。
表6.优化纳米抗体对抗原活性的阻断作用的检测结果
3.5结合亲和力(fortebio)
检测优化纳米抗体对抗原PD-1蛋白的结合亲和力(fortebio)。方法如下:
检测仪器:Octet K2,pall-fortebio
芯片:Protein A(厂家:Pall Fortebio,货号:18-5010)
缓冲液:pH 7.4 PBST(pH 7.4 PBS,Tween20 0.05%v/v)
软件版本:fortebio data analysis 10.0
通过Protein A芯片特异性捕获待测抗体(PR,N7,N10,N12,N13,N14,N15),信号达到3nm,与梯度稀释的不同浓度PD-1蛋白进行结合。
其中,待测样品蛋白用PBST缓冲液稀释至终浓度为5ug/mL。将冻干PD-1蛋白用ddH2O稀释为250ug/mL,然后用PBST稀释,最高浓度为50nM,2倍浓度梯度稀释,共7个浓度。
结果见表7。
表7.优化纳米抗体对抗原的结合亲和力
将3.1至3.5项检测数据结果汇总于表8。
表8.优化纳米抗体的理化、生化分析结果
实施例4优化纳米抗体的抑瘤作用检测
在C57BL/6hPD1人源化小鼠中检测本发明的优化纳米抗体对皮下肿瘤的治疗作用。
在C57BL/6J-hPD1人源化小鼠皮下移植MC38-hPD-L1肿瘤细胞(结肠癌细胞),建立MC38-hPD-L1移植瘤模型。方法如下:
1.试验处理前小鼠进行隔离与适应性饲养一周;
2.将MC38-hPD-L1细胞在37℃、5%CO2的培养箱中培养扩增,收取对数生长期细胞重悬于PBS中,加入基质胶,调整细胞浓度到1×107个细胞/mL;
3.用1mL注射器将细胞悬液注入C57BL/6J-hPD1人源化小鼠右侧皮下,每只小鼠注射100μL;
4.待平均肿瘤体积约为112mm3时,淘汰体积过大、过小或者肿瘤形状不规则的小鼠。
采用随机区组法将小鼠分为5组,分别为对照组(组1)、Keytruda(组2)、PR抗体组(组3)、优化纳米抗体N7组(组4)、优化纳米抗体N15组(组5),每组8只。按照表9中的方案给药,各组均采用腹腔给药,各组给药体积均为10ml/kg,连续给药3周,每周给药2次,共给药6次。其中各组抗体配制成1mg/ml的使用浓度,对照组采用生理盐水,分组当天开始给药。
表9.给药方案
每周测量2次瘤径、称量小鼠体重,观察小鼠生活状态,对异常情况进行记录。采用以下计算公式:
1.肿瘤体积(Tumor volume,TV)
TV=1/2×a×b2
TV=1/2×a×b2
其中:a表示肿瘤长径;b表示肿瘤短径。
2.相对肿瘤体积(Relative tumor volume,RTV)
RTV=Vt/Vinitial×100(%)
RTV=Vt/Vinitial×100(%)
其中,Vinitial为分组给药时(即dinitial)测量所得肿瘤体积,Vt为每次测量时的肿瘤体积。
3.相对肿瘤增殖率T/C(%)
T/C(%)=(TRTV/CRTV)×100%
T/C(%)=(TRTV/CRTV)×100%
其中,TRTV表示治疗组的相对肿瘤体积,CRTV表示溶剂组的相对肿瘤体
积。
4.肿瘤体积抑瘤率(TGI)
本实验中,肿瘤体积抑瘤率(TGI)如下计算:
TGI=[1-(TVt-TVinitial)/(CVt-CVinitial)]×100%
TGI=[1-(TVt-TVinitial)/(CVt-CVinitial)]×100%
其中,TVt表示治疗组每次测量时的肿瘤体积;TVinitial表示分组给药时治疗组的肿瘤体积;CVt表示对照组每次测量时的肿瘤体积;CVinitial表示分组给药时对照组的肿瘤体积。
结果用平均数和标准误(Mean±SEM)表示,使用T-test分析对照组和给药组的肿瘤体积差异,p<0.05表示存在统计学差异。
一、小鼠肿瘤体积变化见表10和图5。
表10.小鼠肿瘤体积(mm3)
注:数据用Mean±SEM表示
“-”:动物安乐死
给药开始后第14天,对照组平均肿瘤体积为4057.43±288.73mm3。组2、组3、组4和组5肿瘤体积分别为884.86±194.57mm3、1584.55±186.82mm3、672.45±101.01mm3和815.51±131.88mm3,肿瘤抑制率分别为80.42%、62.69%、85.79%和82.16%。组2、组3、组4和组5与对照组肿瘤体积比较,有显著性差异(P<0.01)。
以第14天测得的肿瘤体积为基础,计算抗体对移植hPD-L1肿瘤的雌性人源化小鼠的肿瘤生长抑制能力。结果见表11和表12。
表11.肿瘤生长抑瘤率分析
表12.各组之间肿瘤体积比较
注:数据用Mean±SEM表示
采用T-test分析比较vehicle组和治疗组之间的肿瘤体积,****表示p<0.0001,*表示p<0.05。
二、小鼠肿瘤重
试验结束时,对动物实施安乐死,剥取瘤块称重,小鼠瘤重情况见图6,具体的,对照组平均肿瘤重为7.0907±0.8349g,受试样品Keytruda、PR、N7和N15平均肿瘤重分别为3.0556±0.9384g、5.1787±0.3078g、2.4394±0.7359g和3.0210±0.8778g。
表10至表12及图5和图6的实验结果表明,除受试物PR外,受试物N7、N15和Keytruda抗体单独治疗在hPD-L1-MC38肿瘤模型上均相对对照
组取得了优异的抗肿瘤效果。N7、N15、Keytruda在抗肿瘤效果上显著优于PR抗体,N7、N15相较Keytruda在hPD-L1-MC38肿瘤模型上显示相当的抗肿瘤效果。鉴于Keytruda作为PD-1/PD-L1单抗类药物已被国内外药监机构批准用于多达13种肿瘤或癌症的治疗,给患者带来了巨大的临床获益,也取得了巨大商业成功,本发明抗PD-1纳米抗体作为药效不逊于其的可替代的纳米抗体药物也具有广阔的临床治疗前景。另外本发明抗PD-1纳米抗体提供了相较PR而言,更符合药物开发要求的药物候选分子。
实施例5优化纳米抗体在HCC827(非小细胞肺癌细胞)皮下移植瘤动物模型中的抗肿瘤作用
将含5×106个人非小细胞肺癌HCC827细胞/100μL的PBS和Matrigel等体积混匀后,通过皮下注射接种于裸鼠背部右边靠近腋下,接种体积为200μL左右(5×106个细胞/鼠)。HCC827细胞接种后7天,根据小鼠当日体重和肿瘤体积随机分成6组,每组6只动物。分组后按照表13给药方案进行给药,分组给药当天定义为Day 0。
给药期间每周2次测量并记录小鼠体重与肿瘤体积,Day 28小鼠安乐死,收集肿瘤,拍照。
表13.给药方案
(一)抑瘤效果
1.肿瘤体积
结果见表14。实验期间,G1PBS组小鼠的肿瘤体积持续增长,说明人非小细胞肺癌HCC827在人源化小鼠皮下移植瘤模型在本实验中成功建立。相较G1组,G2-G6组肿瘤体积均显著减小。
表14.各组各时间点动物肿瘤体积统计表(mm3)
注:s.c.表示皮下注射;i.v.表示静脉注射;BIW*7表示一周2次,共7次;P值均为与G1组比较结果,下同。
(2)相对肿瘤抑制率(TGI)
本实验中,相对肿瘤抑制率(TGI)如下计算:
TGI(%)=[1-治疗组(T)相对肿瘤体积平均值/溶媒对照组(C)相对
肿瘤体积平均值]×100%。
TGI(%)=[1-治疗组(T)相对肿瘤体积平均值/溶媒对照组(C)相对
肿瘤体积平均值]×100%。
各组各时间点相对肿瘤抑制率见表15,给药后D28,G2-G6组相对肿瘤抑制率在Day28分别为45.26%、55.03%、51.50%、46.83%和46.39%,可以看出,同剂量条件下,本发明抗PD-1纳米抗体皮下注射相较其静脉注射、Keytruda和信迪利单抗静脉注射具有相当甚至更优的抑瘤效果。
表15.相对肿瘤抑制率(%)
(3)瘤重
实验结束时(Day 28),G2~G6组瘤重分别为2.25±0.08g、1.81±0.10g、2.00±0.11g、2.28±0.14g和2.29±0.09g。各实验组小鼠肿瘤重量见表16。与G1PBS组相比,各组肿瘤重量均显著降低(p<0.01)。从各处理组瘤重可以看出,相较本发明抗PD-1纳米抗体静脉注射G6组、Keytruda G4组和信迪利单抗G5组,本发明抗PD-1纳米抗体皮下注射G3组显示出相当甚至更优的抑瘤效果。
表16.各组小鼠Day 28平均瘤重(g)
(二)动物体重
在实验过程中,各组动物体重相对平稳。给药期间,各组动物身体状况一切正常,活动自如,没有出现不良反应和动物死亡的现象。与同时期的G1PBS组相比,各实验组动物体重在Day 0~Day 28期间均没有显著性差异,上述结果表明本发明抗PD-1纳米抗体安全可控。
综上可知,本发明抗PD-1纳米抗体适合皮下及静脉给药,在HCC827皮下移植瘤动物模型中,本发明抗PD-1纳米抗体皮下注射组相较Keytruda和信迪利单抗静脉注射展现出了相当甚至更优的抑瘤效果,且安全可控,具有良好的成药潜力。另外,相较静脉给药,皮下给药更方便,输液时间更短,输液反应更小,输注风险更低,给药成本更低,因此本发明抗PD-1纳米抗体具有良好的成药潜力及临床应用潜能。
以上对本发明具体实施方式的描述并不限制本发明,本领域技术人员可以根据本发明作出各种改变或变形,只要不脱离本发明的精神,均应属于本发明所附权利要求的范围。
Claims (22)
- 一种抗PD-1纳米抗体,其特征在于,所述抗PD-1纳米抗体由VHH链与包含经修饰铰链区的IgG4-Fc组成,其中:(i)所述抗PD-1纳米抗体VHH链包含SEQ ID NO.2所示的CDR1、SEQ ID NO.3所示的CDR2、SEQ ID NO.4所示的CDR3及SEQ ID NO.28所示的CDR1和CDR2之间的框架区FR2;并且(ii)所述抗PD-1纳米抗体中的经修饰铰链区包含SEQ ID NO:9所示氨基酸序列,或者由连接肽与SEQ ID NO:6所示的IgG4-Fc铰链区序列组成,所述连接肽包含SEQ ID NO.12、SEQ ID NO.21或SEQ ID NO.24所示氨基酸序列。
- 根据权利要求1所述的抗PD-1纳米抗体,其特征在于,所述纳米抗体VHH链包含所述CDR1、CDR2和CDR3以及其间的4个框架区(FR),各个区的排列方式为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4,所述FR1、FR3、FR4分别包含SEQ ID NO.27、SEQ ID NO.29、SEQ ID NO.30所示的氨基酸序列。
- 根据权利要求2所述的抗PD-1纳米抗体,其特征在于,所述纳米抗体VHH链包含SEQ ID NO.7所示氨基酸序列。
- 根据权利要求3所述的抗PD-1纳米抗体,其特征在于,所述纳米抗体包含SEQ ID NO.10、SEQ ID NO.13、SEQ ID NO.22或SEQ ID NO.25所示氨基酸序列。
- 根据权利要求4所述的抗PD-1纳米抗体,其特征在于,所述纳米抗体氨基酸全长序列如SEQ ID NO.11、SEQ ID NO.14、SEQ ID NO.23或SEQ ID NO.26所示。
- 一种核酸分子,所述核酸分子包含核苷酸序列,所述核苷酸序列编码权利要求1至5中任一项所述的抗PD-1纳米抗体中包含的氨基酸序列。
- 一种载体,所述载体含有权利要求6所述的核酸分子。
- 根据权利要求7所述的载体,其特征在于,所述载体为表达载体。
- 一种宿主细胞,所述宿主细胞含有权利要求7或8所述的载体,或所述宿主细胞在基因组中整合有权利要求6所述的核酸分子。
- 一种组合物,所述组合物包含权利要求1至5中任一项所述的抗 PD-1纳米抗体。
- 权利要求1至5中任一项所述的纳米抗体、权利要求6所述的核酸分子、权利要求7或8所述的载体、权利要求9所述的宿主细胞或权利要求10所述的组合物在制备用于治疗PD-1或PD-L1相关疾病的药物中的用途。
- 根据权利要求11所述的用途,其特征在于,所述疾病为肿瘤或癌症。
- 根据权利要求12所述的用途,其特征在于,所述肿瘤或癌症为肺癌、胃癌、肝癌、黑色素瘤、宫颈癌、结直肠癌、膀胱癌、乳腺癌、白血病、淋巴瘤、肾细胞癌、盲肠癌、胰腺癌、胆管癌、头颈癌、梅克尔细胞癌、卵巢癌、鼻咽癌、胶质瘤、食管癌、骨癌或前列腺癌。
- 根据权利要求13所述的用途,其特征在于,所述疾病为结直肠癌或肺癌。
- 根据权利要求14所述的用途,其特征在于,所述肺癌为非小细胞肺癌。
- 一种治疗PD-1或PD-L1相关疾病的方法,所述方法包括给有此需要的受试者施用权利要求1至5中任一项所述的纳米抗体或权利要求10所述的组合物。
- 根据权利要求16所述的方法,其特征在于,所述施用采用皮下给药或静脉给药。
- 根据权利要求16或17所述的方法,其特征在于,所述疾病为肿瘤或癌症。
- 根据权利要求18所述的方法,其特征在于,所述肿瘤或癌症为肺癌、胃癌、肝癌、黑色素瘤、宫颈癌、结直肠癌、膀胱癌、乳腺癌、白血病、淋巴瘤、肾细胞癌、盲肠癌、胰腺癌、胆管癌、头颈癌、梅克尔细胞癌、卵巢癌、鼻咽癌、胶质瘤、食管癌、骨癌或前列腺癌;所述受试者是哺乳动物。
- 一种PD-1检测试剂,其特征在于,所述PD-1检测试剂包含权利要求1至5中任一项所述的抗PD-1纳米抗体。
- 根据权利要求20所述的PD-1检测试剂,其特征在于,所述PD-1检测试剂为基于流式细胞术或酶联免疫吸附法的检测试剂。
- 一种试剂盒,其特征在于,所述试剂盒包含权利要求20或21所述的PD-1检测试剂。
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