WO2022061720A1 - 与SARS-CoV-2 RBD结合的羊驼源纳米抗体 - Google Patents

与SARS-CoV-2 RBD结合的羊驼源纳米抗体 Download PDF

Info

Publication number
WO2022061720A1
WO2022061720A1 PCT/CN2020/117771 CN2020117771W WO2022061720A1 WO 2022061720 A1 WO2022061720 A1 WO 2022061720A1 CN 2020117771 W CN2020117771 W CN 2020117771W WO 2022061720 A1 WO2022061720 A1 WO 2022061720A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
arbd
antigen
binding fragment
cov
Prior art date
Application number
PCT/CN2020/117771
Other languages
English (en)
French (fr)
Inventor
金腾川
马欢
曾威红
Original Assignee
中国科学技术大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学技术大学 filed Critical 中国科学技术大学
Priority to PCT/CN2020/117771 priority Critical patent/WO2022061720A1/zh
Publication of WO2022061720A1 publication Critical patent/WO2022061720A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to nanobody sequences against SARS-CoV-2 RBD used for treatment and diagnosis.
  • SARS-CoV-2 is a coronavirus and the pneumonia it causes is called COVID-19. SARS-CoV-2 enters cells through the receptor binding region (RBD) of its surface spike protein (spike) and binds to angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells to complete the infection.
  • RBD receptor binding region
  • ACE2 angiotensin-converting enzyme 2
  • Fully human antibodies isolated from recovered patients have been shown to have good antiviral effects, but these are traditional monoclonal antibodies consisting of 2 heavy and 2 light chains. It has the limitations of large molecular weight, complex production process and difficult processing and transformation.
  • variable region is only composed of heavy chains.
  • the variable region is abbreviated as VHH.
  • the diameter of the variable region protein is less than 10 nanometers, so it is also known as nanobodies. Nanobodies have the advantages of small molecular weight, strong penetrability, easy expression, easy genetic modification, and easy binding of multiple epitopes.
  • the present disclosure provides an alpaca-derived heavy chain antibody variable region sequence (VHH) that can bind to the receptor binding region (RBD) of the novel coronavirus (SARS-CoV-2) with high affinity, the variable region sequence is also referred to as a nanobody , which can be used to prevent, treat and/or diagnose SARS-CoV-2 infection.
  • VHH alpaca-derived heavy chain antibody variable region sequence
  • RBD receptor binding region
  • SARS-CoV-2 novel coronavirus
  • the inventors used the SARS-CoV-2 RBD protein recombinantly expressed in vitro to immunize two llamas three times, then isolated peripheral blood lymphocytes and extracted total RNA from the cells, which were then reverse transcribed into cDNA. Using this cDNA as a template, the nanobody sequence was amplified with specific primers. We isolated and obtained 7 nanobodies. They are named aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54, respectively, and their amino acid sequences are as follows:
  • CDR1, CDR2 and CDR3 The three antigenic complementarity determining regions (CDR1, CDR2 and CDR3) of the 7-strain Nanobodies are shown in the underlined part, specifically:
  • CDR2 EFVAAMRWSDTD (SEQ ID NO: 2)
  • CDR3 AGEAWLARSTHHYDY (SEQ ID NO: 3)
  • CDR2 EGVSCISHPGGSTN (SEQ ID NO: 5)
  • CDR3 ASPLALFRLCVLPSPLPYDY (SEQ ID NO: 6)
  • CDR1 GFTLDYYAI (SEQ ID NO: 7)
  • CDR2 EGVSCISGSGGITN (SEQ ID NO: 8)
  • CDR3 PVSHTVVAGCAFEAWTDFGS (SEQ ID NO: 9)
  • CDR1 ERTFSGGVM (SEQ ID NO: 10)
  • CDR2 EFVAAIRWNGASTF (SEQ ID NO: 11)
  • CDR3 RAVRTYASSDYYFQERTYDY (SEQ ID NO: 12)
  • CDR1 GFTSGHYAI (SEQ ID NO: 13)
  • CDR2 EFVAAISWSGLSRY (SEQ ID NO: 17)
  • CDR3 ARISSAYYTRSSSYAY (SEQ ID NO: 21).
  • Nanobodies aRBD-2 and aRBD-5 bind different epitopes, and aRBD-2 and aRBD-7 bind different epitopes, so they were combined to construct two corresponding bi-epitope-specific antibodies. aRBD-2-5 and aRBD-2-7.
  • bi-epitope-specific antibody refers to connecting two nanobodies that can respectively bind to two independent epitopes on SARS-CoV-2 RBD with flexible polypeptide chains, so as to construct an antibody that can bind to the RBD. Antibodies to two epitopes.
  • the present invention provides the following technical solutions:
  • VHH comprises:
  • the antibody or antigen-binding fragment thereof of item 1 or 2 which is a bi-epitope-specific antibody, the sequence of the bi-epitope-specific antibody (for example in the order of N-terminal to C-terminal) comprising SEQ ID NO : 22 and SEQ ID NO: 24, or SEQ ID NO: 22 and SEQ ID NO: 25, preferably, wherein SEQ ID NO: 22 and SEQ ID NO: 24 or SEQ ID NO: 22 and SEQ ID NO: 25
  • Linkers eg, flexible polypeptide chains such as GS linkers
  • the antibody or antigen-binding fragment thereof according to any one of items 1 to 3, which further has an Fc domain, preferably an IgG1 Fc domain, more preferably a human IgG1 Fc domain, the The sequence is shown, for example, as SEQ ID NO: 30, and the nucleotide sequence of the gene encoding the sequence of the human IgG1 Fc domain is shown, for example, as SEQ ID NO: 31.
  • An expression vector eg using an expression vector based on one or more promoters and a host cell, comprising the polynucleotide of item 5.
  • a host cell comprising the expression vector of item 6, the host cell being a host cell for expressing a foreign protein, eg bacteria, yeast, insect cells, mammalian cells.
  • a foreign protein eg bacteria, yeast, insect cells, mammalian cells.
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of items 1 to 4 and a pharmaceutically acceptable carrier.
  • VHH nanobody
  • the circular dichroism experiment showed that the half-dissolution temperature (Tm value) of the above seven nanobodies were all above 70°C.
  • nanobodies were fused with human IgG1 Fc segment, they were cloned into pTT5 vector, and expressed in mammalian cells 293F for secretory expression.
  • the yields of the seven antibody strains were all above 90 mg/L.
  • All seven antibodies can bind SARS-CoV-2 RBD with high affinity.
  • ELISA assay showed that, except for aRBD-42, the Fc fusion proteins of other antibodies of the present disclosure had higher affinity for binding the extracellular segment of the SARS-CoV-2 spike protein (S1+S2) than ACE2.
  • SPR surface plasmon resonance
  • aRBD-42 the other six nanobodies of the present disclosure can well inhibit the binding of human ACE2 to SARS-CoV-2 RBD after fusion with human IgG1 Fc.
  • Competitive ELISA experiments showed that the Fc fusion proteins of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41 and aRBD-54 could compete with 10nM ACE2-Fc for SARS-CoV-2 RBD, IC50s were 2.68, 2.59, 1.89, 1.42, 5.76 and 2.07 nM, respectively.
  • Nanobodies aRBD-2 and aRBD-5 of the present disclosure bind different epitopes, and aRBD-2 and aRBD-7 bind different epitopes, so two bi-epitope-specific antibodies aRBD-2-5 and aRBD- 2-7, SPR showed greatly enhanced affinity to the SARS-CoV-2 RBD with KD values of 59.2 pM (picomoles per liter) and 0.25 nM, respectively.
  • the Fc fusion proteins of the Nanobodies aRBD-2, aRBD-5 and aRBD-7 of the present disclosure can all neutralize the infection of Vero E6 cells by SARS-CoV-2 in vitro.
  • the ND50 of the Fc fusion proteins of aRBD-2, aRBD- 5 and aRBD-7 to neutralize 200 PFU of SARS-CoV-2 infecting Vero E6 in a 100 ⁇ L system were 0.092, 0.413 and 0.591 ⁇ g/mL, respectively.
  • the Fc fusion proteins of bi-epitope-specific antibodies aRBD-2-5 and aRBD-2-7 neutralized 200 PFU of SARS-CoV-2 infecting Vero E6 in a 100 ⁇ L system with a ND 50 of 0.0104 and 0.0067 ⁇ g/mL, respectively .
  • FIG. 1 Results of phage display screening of seven Nanobodies of the present disclosure.
  • A is the phage count result of two rounds of panning;
  • B is the result of monoclonal phage ELISA.
  • FIG. 1 SDS-PAGE gel electrophoresis results of the Nanobody Fc fusion protein (A) and Nanobody (B).
  • Lane M is the marker
  • lanes 1 to 7 are aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54 fusion proteins (A) and their respective Fc-cutting nanoparticles Antibody protein (B).
  • FIG. 1 Results of circular dichroism (CD) experiments to detect the denaturation temperature of seven Nanobodies of the present disclosure.
  • (A)-(G) are the detection results of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54 in sequence.
  • Figure 4 The result of detecting the binding between the Fc fusion protein of the Nanobody and the extracellular segment protein of the SARS-CoV-2 spike protein (S1+S2) by ELISA.
  • FIG. 5 The affinity between the Nanobodies and the SARS-CoV-2 RBD was detected by SPR.
  • (A) to (I) are the sequential detection of Nanobodies aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42, aRBD-54, aRBD-2-5 and Kinetic curve of binding between aRBD-2-7 and SARS-CoV-2 RBD protein.
  • the solid line is the kinetic curve of real-time monitoring, and the dashed line is the curve fitted by biacore evaluation software.
  • the kinetic curves of different antibody concentration gradients correspond from top to bottom with the concentrations marked on the right from top to bottom.
  • Figure 7 In vitro virus neutralization experiments verify the function of the disclosed antibodies. Fc fusion proteins of nanobodies aRBD-2, aRBD-5 and aRBD-7 and bi-epitope specific antibodies aRBD-2-5 and aRBD-2-7 and their Fc fusion proteins neutralize SARS-CoV-2 virus in vitro Results of experimental data analysis of infected Vero E6 cells.
  • SARS-CoV-2 RBD QKV42562.1, aa 321-591 expressed and purified by HEK293F cells (ATCC, CBP60437) was mixed with Freund's adjuvant, and the alpacas were immunized by subcutaneous injection at a dose of 500 ⁇ g/time for three times, A total of 2 6-month-old alpacas were immunized at 2-week intervals.
  • TG1 Electroporation of TG1 to amplify the nanobody phagemid library: Escherichia coli TG1 competent cells were prepared by washing with 10% glycerol, and then the above Gibson assembly products were electroporated into TG1 competent cells, and 5 pieces of 150 mm containing 2 were coated. % glucose and 100 ⁇ g/mL ampicillin in LB (LB/2% G/Amp) plates to amplify the phagemid library.
  • Amplify the nanobody phage library after scraping, take an appropriate amount of bacterial liquid to inoculate 200 mL of 2TY (containing 2% glucose and 100 ⁇ g/mL ampicillin) and cultivate it to the logarithmic growth phase, and add 10 12 pfu of KM13 helper phage (purchased from MRC). Laboratory of Molecular Biology), infect 45min at 37°C, centrifuge 100mL of bacterial liquid, resuspend the cells with 200mL of 2TY (containing 0.1% glucose, 100 ⁇ g/mL and 50 ⁇ g/mL kanamycin), and culture at 25°C for 20h to Amplification of phage displaying Nanobodies. The phage was concentrated by PEG precipitation, and finally resuspended in PBS and stored on ice.
  • the first round Dilute the SARS-CoV-2 RBD expressed and purified in Example 1 to 0.1 mg/mL with PBS, add 100 ⁇ L to one well of a 96-well immune plate (Nunc maxsorp plate), and coat overnight at 4°C , and set up a well without antigen control at the same time.
  • the cells were washed three times with PBS, and 300 ⁇ L of MPBS (PBS containing 5% nonfat milk) was added to each well for blocking at room temperature for 2 h. Washed 3 times with PBS, added 1 ⁇ 10 11 pfu or more to each well to prepare a phage library phage (dissolved in 100 ⁇ L MPBS), and incubated at room temperature at 80 rpm for 1 h.
  • the remaining phage solution was all infected with 3 mL of logarithmic growth phase TG1 bacteria, water bathed at 37 °C for 45 min, coated with a 150 mm LB/2% G/Amp plate, and cultured at 37 °C overnight.
  • Second round add 4mL 2TY to the above 150mm plate, scrape off the colonies, mix the bacterial liquid and inoculate 100 ⁇ L to 100mL 2TY/2%G/Amp medium, culture to logarithmic growth phase and add KM13 Infection to prepare Nanobody-displayed phage.
  • SARS-CoV-2 RBD was diluted with PBS to 0.02 mg/mL, 100 ⁇ L was added to one well of a 96-well immunoplate, and coated overnight at 4°C, while a well-free antigen control was set.
  • the cells were washed three times with PBS, and 300 ⁇ L of MPBS (PBS containing 5% skim milk) was added to each well for blocking at room temperature for 2 h.
  • MPBS MPBS
  • Phage counts for two rounds of panning elution are shown in Figure 1A. Compared with the control wells, the number of phage eluted from the RBD-coated wells was significantly higher. The number of phage eluted from the RBD-coated wells in the first round was more than 70 times that of the control wells, and the ratio was higher in the second round. This indicated that phages specific for RBD were successfully isolated and enriched.
  • A. Preparation of monoclonal phage Pick 31 single clones from the plates counted after the above 2 rounds of screening and inoculation into 96 wells containing 100 ⁇ L 2TY medium (containing 2% glucose and 100 ⁇ g/mL ampicillin) per well In the cell culture plate, one well of one clone was cultured at 37° C. and 250 rpm with shaking for 12 h.
  • Phage ELISA detection Dilute the SARS-CoV-2 RBD protein with PBS to 1 ⁇ g/mL, take 100 ⁇ L/well to coat the 96-well immune plate, and set a blank control (PBS well, 4 °C overnight coating) Wash 3 times with PBS, add 300 ⁇ L MPBS to each well, and block for 2 h at room temperature. Add 100 ⁇ L of the above-prepared phage MPBS mixture to each well, and incubate for 1 h at room temperature. Wash the plate 4 times with PBST. Use MPBS to moderately dilute HRP-anti M13 antibody (sense). Add 100 ⁇ L to each well of the immune plate above, and incubate for 1 h at room temperature.
  • Non-competitive ELISA was used to preliminarily characterize the binding of the Nanobody Fc fusion protein to the extracellular segment of the SARS-CoV-2 spike protein (S1+S2): (S1+S2) Extracellular segment (Val 16-Pro 1213, Beijing Yiqiao Shenzhou) was diluted with PBS to 2 ⁇ g/mL, and 100 ⁇ L was added to each well for coating. After routine washing and blocking, 1:1 was added in turn.
  • Nanobody Fc fusion protein and ACE2-Fc protein the aa 19-615 segment of human ACE2 was fused to human IgG1 Fc for secretory expression using HEK293F cells, followed by protein A purification) solution, incubated at room temperature for 1 Hour. After washing, HRP-conjugated anti-IgG1 Fc antibody (Beijing Yiqiao Shenzhou) was added to detect the bound VHH-Fc and ACE2-Fc. The results are shown in Figure 4.
  • fusion proteins namely aRBD-2-Fc, aRBD-3-Fc, aRBD-5-Fc, aRBD-7-Fc, aRBD-41-Fc and aRBD-54-Fc, all had higher affinity than ACE2-Fc, and their EC50s were 0.256, 0.098, 0.077, 0.105, 0.226 , 0.164 nM, respectively.
  • the binding affinity of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD -54 to RBD was KD
  • the values were 2.60, 3.33, 16.3, 3.31, 21.9, 113 and 5.49 nM, respectively.
  • the blocking function of the screened nanobodies was characterized by competitive ELISA.
  • SARS-CoV-2 RBD was diluted to 1 ⁇ g/mL in PBS, 100 ⁇ L was added to each well for coating, washed and blocked as usual.
  • Dilute the biotinylated ACE2-Fc to 10nM then use the ACE2-Fc solution to sequentially dilute the nanobody Fc fusion protein in a 1:3 gradient, add 100 ⁇ L of each gradient mixture to the antigen-coated wells, and incubate at room temperature 1 hour. After washing 4 times with PBST, HRP-conjugated Streptavidin (Biyuntian) was added to detect the bound biotinylated ACE2-Fc.
  • Vero E6 cells ATCC CBP60972
  • the Fc fusion protein of Nanobody aRBD-2 was diluted in a 1:3 gradient from 10 ⁇ g/mL to 0.041 ⁇ g/mL
  • the Fc fusion proteins of aRBD-5 and aRBD-7 were diluted in a 1:3 gradient from 30 ⁇ g/mL
  • the bi-epitope-specific antibodies aRBD-2-5 and aRBD-2-7 and their Fc fusion proteins were diluted from 1 ⁇ g/mL to 0.0041 ⁇ g/mL according to a 1:3 gradient, and the dilutions were both DMEM+1% FBS, and then 50 ⁇ L were added to 96-well plates.
  • a control without antibody was set at the same time, mixed well, and incubated at 37°C for half an hour.
  • Aspirate the medium of Vero E6 cells transfer the above 100 ⁇ L of antibody and virus incubations to the wells inoculated with Vero E6 cells, and incubate at 37° C. and 5% CO 2 for 1 h.
  • the fitting shows that aRBD-2-Fc, aRBD-5-Fc, aRBD-7-Fc, aRBD-2-5-Fc and aRBD-2-7-Fc
  • the ND 50 (half neutralizing dose concentration) of Vero E6 cells infected with SARS-CoV-2 were 0.092, 0.413, 0.591, 0.0104 and 0.0067 ⁇ g/mL, respectively, while the ND of aRBD-2-5 and aRBD-2-7 50 was less than 0.004 ⁇ g/mL. It can be seen that the virus neutralization ability of the bi-epitope-specific antibody was significantly better than that of a single nanobody. .

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Pulmonology (AREA)
  • Mycology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

涉及与SARS-CoV-2 RBD结合的羊驼源抗体或其抗原结合片段，具体涉及能以高亲和力结合新冠病毒(SARS-CoV-2)受体结合区域(RBD)的羊驼源纳米抗体或由其组成的双表位特异性抗体或其抗原结合片段，其能够用于预防、治疗和/或诊断SARS-CoV-2感染。

Description

与SARS-CoV-2 RBD结合的羊驼源纳米抗体 技术领域
本发明属于生物技术领域，具体涉及用于治疗和诊断用的抗SARS-CoV-2 RBD的纳米抗体序列。
背景技术
SARS-CoV-2属于冠状病毒，其导致的肺炎称为COVID-19。SARS-CoV-2通过其表面突刺蛋白(spike)的受体结合区域(RBD)与上皮细胞表面的血管紧张素转换酶2(ACE2)结合后进入细胞，完成侵染。
从康复患者体内分离的全人源抗体被证实具有很好的抗病毒作用，但这些都是传统的单克隆抗体，由2条重链和2条轻链组成。具有分子量大，生产工艺复杂，不易加工改造等局限性。
在骆驼科动物体内存在一种天然缺失轻链的抗体，即重链抗体，其可变区仅由重链组成，该可变区简写为VHH，该可变区蛋白直径小于10纳米，因此又被称为纳米抗体。纳米抗体具有分子量小、穿透性强、易于表达、易于基因改造以及易于结合多个表位等优点。
目前尚无抗SARS-CoV-2 RBD的羊驼源天然纳米抗体获批用于治疗COVID19。
发明内容
本公开提供了能以高亲和力结合新冠病毒(SARS-CoV-2)受体结合区域(RBD)的羊驼源重链抗体可变区序列(VHH)，该可变区序列又称为纳米抗体，其能够用于预防、治疗和/或诊断SARS-CoV-2感染。
发明人采用体外重组表达的SARS-CoV-2 RBD蛋白对2头小羊驼进行3次免疫，然后分离出外周血淋巴细胞并抽提细胞的总RNA，随后反转录为cDNA。再以此cDNA为模板，用特异引物扩增出纳米抗体序列。我们分离获得7株纳米抗体。分别命名为aRBD-2、aRBD-3、aRBD-5、aRBD-7、aRBD-41、aRBD-42和aRBD-54，其氨基酸序列分别如下：
aRBD-2的氨基酸序列：
Figure PCTCN2020117771-appb-000001
aRBD-3的氨基酸序列：
Figure PCTCN2020117771-appb-000002
aRBD-5的氨基酸序列：
Figure PCTCN2020117771-appb-000003
aRBD-7的氨基酸序列：
Figure PCTCN2020117771-appb-000004
aRBD-41的氨基酸序列：
Figure PCTCN2020117771-appb-000005
aRBD-42的氨基酸序列：
Figure PCTCN2020117771-appb-000006
aRBD-54的氨基酸序列：
Figure PCTCN2020117771-appb-000007
所述7株纳米抗体的3个抗原互补决定区(CDR1、CDR2和CDR3)如划线部分所示，具体地：
aRBD-2：
CDR1：GRTYTM(SEQ ID NO：1)
CDR2：EFVAAMRWSDTD(SEQ ID NO：2)
CDR3：AGEAWLARSTHHYDY(SEQ ID NO：3)
aRBD-3：
CDR1：GLTLDYYAI(SEQ ID NO：4)
CDR2：EGVSCISHPGGSTN(SEQ ID NO：5)
CDR3：ASPLALFRLCVLPSPLPYDY(SEQ ID NO：6)
aRBD-5：
CDR1：GFTLDYYAI(SEQ ID NO：7)
CDR2：EGVSCISGSGGITN(SEQ ID NO：8)
CDR3：PVSHTVVAGCAFEAWTDFGS(SEQ ID NO：9)
aRBD-7：
CDR1：ERTFSGGVM(SEQ ID NO：10)
CDR2：EFVAAIRWNGASTF(SEQ ID NO：11)
CDR3：RAVRTYASSDYYFQERTYDY(SEQ ID NO：12)
aRBD-41：
CDR1：GFTSGHYAI(SEQ ID NO：13)
CDR2：EGVSCIGSSDGSTY(SEQ ID NO：14)
CDR3：AGLWYGRSLNSFDYDY(SEQ ID NO：15)
aRBD-42：
CDR1：GRTFSSATM(SEQ ID NO：16)
CDR2：EFVAAISWSGLSRY(SEQ ID NO：17)
CDR3：DSWGCSGLGC(SEQ ID NO：18)
aRBD-54：
CDR1：GRTFGSFM(SEQ ID NO：19)
CDR2：DFVAAITWSGGSTY(SEQ ID NO：20)
CDR3：ARISSAYYTRSSSYAY(SEQ ID NO：21)。
而后，发明人发现纳米抗体aRBD-2和aRBD-5结合不同的表位，aRBD-2和aRBD-7结合不同的表位，因此用他们分别组合构建了对应的两个双表位特异性抗体aRBD-2-5和aRBD-2-7。
如本所用的双表位特异性抗体，是指将能够分别结合如SARS-CoV-2 RBD上两个独立表位的两个纳米抗体用柔性多肽链连接，从而构建的能够结合所述RBD的两个表位的抗体。
具体地，本发明提供了以下各项技术方案：
1.与SARS-CoV-2 RBD结合的羊驼源抗体或其抗原结合片段，其具有VHH，所述VHH具有选自以下各项组成的组：
如SEQ ID NO：1所示的CDR1，
如SEQ ID NO：2所示的CDR2和
如SEQ ID NO：3所示的CDR3；
如SEQ ID NO：4所示的CDR1，
如SEQ ID NO：5所示的CDR2和
如SEQ ID NO：6所示的CDR3；
如SEQ ID NO：7所示的CDR1，
如SEQ ID NO：8所示的CDR2和
如SEQ ID NO：9所示的CDR3；
如SEQ ID NO：10所示的CDR1，
如SEQ ID NO：11所示的CDR2和
如SEQ ID NO：12所示的CDR3；
如SEQ ID NO：13所示的CDR1，
如SEQ ID NO：14所示的CDR2和
如SEQ ID NO：15所示的CDR3；
如SEQ ID NO：16所示的CDR1，
如SEQ ID NO：17所示的CDR2和
如SEQ ID NO：18所示的CDR3；和/或
如SEQ ID NO：19所示的CDR1，
如SEQ ID NO：20所示的CDR2和
如SEQ ID NO：21所示的CDR3。
2.如项1所述的抗体或其抗原结合片段，其中所述VHH包含：
如SEQ ID NO：22所示的氨基酸序列，
如SEQ ID NO：23所示的氨基酸序列
如SEQ ID NO：24所示的氨基酸序列。
如SEQ ID NO：25所示的氨基酸序列，
如SEQ ID NO：26所示的氨基酸序列，
如SEQ ID NO：27所示的氨基酸序列，和/或
如SEQ ID NO：28所示的氨基酸序列。
3.如项1或2所述的抗体或其抗原结合片段，其是双表位特异性抗体，所述双表位特异性抗体(例如以N端到C端的顺序)的顺序包含SEQ ID NO：22和SEQ ID NO：24，或SEQ ID NO：22和SEQ ID NO：25，优选地，其中SEQ ID NO：22和SEQ ID NO：24或SEQ ID NO：22和SEQ ID NO：25之间用接头(例如柔性多肽链，例如GS接头)连接。
4.如项1-3中任一项所述的抗体或其抗原结合片段，其进一步具有Fc结构域，优选IgG1 Fc结构域，更优选人IgG1 Fc结构域，所述人IgG1 Fc结构域的序列例如如SEQ ID NO：30所示，所述人IgG1 Fc结构域的序列的编码基因的核苷酸序列例如如SEQ ID NO：31所示。
5.多核苷酸，其编码项1-4中任一项所述的抗体或其抗原结合片段。
6.表达载体，例如采用基于一种或更多种启动子和宿主细胞的表达载体，其包含项5所述的多核苷酸。
7.宿主细胞，其包含项6所述的表达载体，所述宿主细胞是用于表达外源蛋白的宿主细胞，例如细菌、酵母、昆虫细胞、哺乳动物细胞。
8.药物组合物，其含有项1-4中任一项所述的抗体或其抗原结合片段和药用载体。
9.项1-4中任一项所述的抗体或其抗原结合片段在制备预防、治疗和/或诊断SARS-CoV-2感染的试剂盒或药物中的用途。
本公开的优点和积极效果
由于本公开所述的纳米抗体(VHH)来源于天然的羊驼重链抗体，因此其具备稳定性高、表达量高以及亲和力高的特点。
采用圆二色谱实验显示以上7株纳米抗体的半溶解温度(Tm值)均在70℃以上。
将以上7株纳米抗体与人IgG1 Fc段融合后，克隆至pTT5载体，采用哺乳动物细胞293F进行分泌性表达，表达3天后，采用Protein A柱子对培养基上清中的融合蛋白进行纯化发现，所述7株抗体的产量均在90mg/L以上。
7株抗体均能高亲和力的结合SARS-CoV-2 RBD。采用ELISA实验检测显示，除aRBD-42外，本公开的其它抗体的Fc融合蛋白结合SARS-CoV-2刺突蛋白(S1+S2)胞外段的亲和力均高于ACE2。采用表面等离子共振(SPR)实验表明，aRBD-2、aRBD-3、aRBD-5、aRBD-7、aRBD-41、aRBD-42、aRBD-54与SARS-CoV-2 RBD的亲和力解离常数(K D)值分别为2.60、3.33、16.3、3.31、21.9、113和5.49nM(纳摩尔每升)。
除aRBD-42外，本公开的另外6株纳米抗体在融合人IgG1 Fc后均能很好地抑制人ACE2与SARS-CoV-2 RBD的结合。采用竞争性ELISA实验显示aRBD-2、aRBD-3、aRBD-5、aRBD-7、aRBD-41和aRBD-54的Fc融合蛋白均能与10nM的ACE2-Fc竞争SARS-CoV-2 RBD，其IC 50分别为2.68、2.59、1.89、1.42、5.76和2.07nM。
本公开的纳米抗体aRBD-2和aRBD-5结合不同的表位，aRBD-2和aRBD-7结合不同的表位，因此构建了两个双表位特异性抗体aRBD-2-5和aRBD-2-7，SPR显示其与SARS-CoV-2 RBD亲和力大大增强，K D值分别为59.2pM(皮摩尔每升)和0.25nM。
本公开的纳米抗体aRBD-2、aRBD-5和aRBD-7的Fc融合蛋白均能在体外中和SARS-CoV-2侵染Vero E6细胞。aRBD-2、aRBD-5和aRBD-7的Fc融合蛋白在100μL体系下中和200 PFU SARS-CoV-2侵染Vero E6 的浓度ND 50分别是0.092、0.413和0.591μg/mL。双表位特异性抗体aRBD-2-5和aRBD-2-7的Fc融合蛋白在100μL体系下中和200 PFU SARS-CoV-2侵染Vero E6的浓度ND 50分别是0.0104和0.0067μg/mL。
附图说明
图1.噬菌体展示筛选本公开的7个纳米抗体的结果。(A)为两轮淘选的phage计数结果；(B)为单克隆噬菌体ELISA结果。
图2.所述纳米抗体Fc融合蛋白(A)及纳米抗体(B)的SDS-PAGE凝胶电泳结果。泳道M为marker，泳道1到7依次为aRBD-2、aRBD-3、aRBD-5、aRBD-7、aRBD-41、aRBD-42和aRBD-54融合蛋白(A)及其各自切除Fc的纳米抗体蛋白(B)。
图3.圆二色谱(CD)实验检测本公开的7个纳米抗体的变性温度的结果图。(A)-(G)依次是aRBD-2、aRBD-3、aRBD-5、aRBD-7、aRBD-41、aRBD-42和aRBD-54的检测结果。
图4.采用ELISA检测所述纳米抗体的Fc融合蛋白与SARS-CoV-2刺突蛋白(S1+S2)胞外段蛋白之间的结合的结果图。
图5.采用SPR检测所述纳米抗体与SARS-CoV-2 RBD之间的亲和力。(A)到(I)依次是采用SPR的方法检测纳米抗体aRBD-2、aRBD-3、aRBD-5、aRBD-7、aRBD-41、aRBD-42、aRBD-54、aRBD-2-5和aRBD-2-7与SARS-CoV-2 RBD蛋白之间结合的动力学曲线。其中实线是实时监测的动力学曲线，虚线是采用biacore evaluation软件拟合的曲线。不同抗体浓度梯度的动力学曲线从上到下与右侧标识的从上到下的浓度依次对应。
图6.采用竞争性ELISA检测所述纳米抗体的Fc融合蛋白阻断ACE2与SARS-CoV-2 RBD结合的结果图。
图7.体外病毒中和实验验证本公开抗体的功能。纳米抗体aRBD-2、aRBD-5和aRBD-7的Fc融合蛋白以及双表位特异性抗体aRBD-2-5和aRBD-2-7及其Fc融合蛋白在体外中和SARS-CoV-2病毒侵染Vero E6细胞的实验数据分析结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。
实施例1采用SARS-CoV-2 RBD免疫羊驼并筛选纳米抗体
1)采用HEK293F细胞(ATCC，CBP60437)表达纯化的SARS-CoV-2 RBD(QKV42562.1，aa 321-591)与弗氏佐剂混匀，以500μg/次的剂量皮下注射免疫羊驼三次，每次间隔2星期，共免疫2头6个月大小的母羊驼。
2)第三次免疫2周后，静脉取血并分离血液中的白细胞。采用omegabiotek公司的RNA抽提试剂盒提取总RNA，同时采用DNA酶除去基因组DNA。采用TAKARA公司的PrimeScript TM II 1st Strand cDNA Synthesis Kit对RNA进行反转录，将RNA反转录为cDNA。
3)制备纳米抗体噬菌粒文库：采用我们设计的羊驼VHH特异性引物，以以上cDNA为模板扩增获得VHH的编码基因片段，采用Gibson assembly的方法将扩增的VHH序列克隆至噬菌粒pR2的NcoI和NotI位点中，得到的Gibson assembly产物即为初始纳米抗体噬菌粒文库。
4)电转化TG1扩增纳米抗体噬菌粒文库：采用10％甘油洗涤法制备大肠杆菌TG1感受态细胞，然后将以上Gibson assembly产物电转化至TG1感受态细胞中，涂布5块150mm含有2％葡萄糖和100μg/mL氨苄青霉素的LB(LB/2％G/Amp)平板中以扩增噬菌粒文库。
5)扩增纳米抗体噬菌体文库：刮板后取适量菌液接种200mL 2TY(含2％葡萄糖和100μg/mL氨苄青霉素)培养至对数生长期，加入10 12pfu的KM13辅助噬菌体(购自MRC Laboratory of Molecular Biology)，37℃侵染45min，取100mL菌液离心，菌体用200mL的2TY(含0.1％葡萄糖、100μg/mL和50μg/mL卡那霉素)重悬，25℃培养20h以扩增展示纳米抗体的phage。采用PEG沉淀的方法浓缩phage，最终用PBS重悬，冰面保存。
6)淘选(Panning)
A.第一轮：将实施例1中表达纯化的SARS-CoV-2 RBD用PBS稀释至0.1mg/mL，取100μL加入96孔免疫板(Nunc maxsorp plate)的一孔，4℃包被过夜，同时设置一孔无抗原对照。采用PBS洗3次，每孔加入300 μL MPBS(含5％脱脂牛奶的PBS)室温封闭2h。采用PBS洗3次，每孔加入1x 10 11pfu以上制备噬菌体文库phage(溶于100μL MPBS)，80rpm室温孵育1h。采用PBST(0.1％Tween 20)洗30次。每孔加入100μL浓度为0.5mg/mL的胰蛋白酶，室温消化1h，结合在孔中的phage被洗脱。取10μL洗脱的phage侵染1mL对数生长期TG1细菌，37℃水浴45min。分别取100μL、10μL和1μL涂布LB/2％G/Amp平板计数。剩余phage溶液全部侵染3mL对数生长期TG1细菌，37℃水浴45min，涂布1块150mm LB/2％G/Amp平板，37℃过夜培养。
B.第二轮：加入4mL 2TY至以上150mm平板中，将菌落刮下，将菌液混匀后接种100μL至100mL 2TY/2％G/Amp培养基中，培养至对数生长期后加入KM13侵染以制备纳米抗体展示的phage。随后将SARS-CoV-2 RBD用PBS稀释至0.02mg/mL，取100μL加入96孔免疫板的一孔，4℃包被过夜，同时设置一孔无抗原对照。采用PBS洗3次，每孔加入300μL MPBS(含5％脱脂牛奶的PBS)室温封闭2h。采用PBS洗3次，每孔加入1x 10 8pfu以上扩增的第一轮洗脱phage(溶于100μL MPBS)，80rpm室温孵育1h。采用PBST(0.2％Tween 20)洗30次。每孔加入100μL浓度为0.5mg/mL的胰蛋白酶，室温消化1h，结合在孔中的phage被洗脱。取10μL洗脱的phage侵染1mL对数生长期TG1细菌，37℃水浴45min。分别取100μL、10μL和1μL涂布LB/2％G/Amp平板计数。
C.两轮panning洗脱的phage计数见图1A。与对照孔相比，包被RBD的孔洗脱的phage数明显多很多，第一轮包被RBD孔洗脱的phage数量是对照孔的70倍以上，第二轮这个比值更高。说明特异性针对RBD的噬菌体被成功分离并富集。
7)噬菌体ELISA筛选抗SARS-CoV-2 RBD的纳米抗体单克隆。
A.制备单克隆phage：分别从以上2轮筛选洗脱后计数的平板上挑取31个单克隆接种至每孔含有100μL 2TY培养基(含2％葡萄糖和100μg/mL氨苄青霉素)的96孔细胞培养板中，1个克隆1个孔，37℃、 250rpm震荡培养12h。转移5μL以上菌液至新的每孔含有200μl 2TY培养基(含2％葡萄糖和100μg/mL Ampicillin)的96孔板中进行培养(剩余的菌液加入终浓度为15％甘油，-80℃储存)，37℃、250rpm震荡培养1.5h至OD600为约0.5，每孔吸除100μL菌液。每孔加入50μL含有4×10 8pfu KM13噬菌体的2TY，混匀，37℃孵育45min。3500g离心10min，弃上清，沉淀用200μL含有0.1％葡萄糖、100μg/mL Ampicillin和50μg/mL Kanamycin的2TY重悬，25℃、250rpm震荡培养20h。3500g离心10min，取75μL上清转移至每孔含有225μL MPBS的96孔板的孔中，混匀，4℃暂存备用，至此单克隆噬菌体制备完成。
B.噬菌体phage ELISA检测：将SARS-CoV-2 RBD蛋白用PBS稀释至1μg/mL，分别取100μL/孔对96孔免疫板进行包被，另外设置空白对照(PBS孔，4℃包被过夜。采用PBS洗3次，每孔加入300μL MPBS，室温封闭2h。每孔加入以上制备phage MPBS混合液100μL，室温孵育1h。采用PBST洗板4次。采用MPBS适度稀释HRP-anti M13抗体(义翘神州)，分别加100μL至以上免疫板的各孔中，室温孵育1h。采用PBST洗板4次。每孔加入100μL TMB显色底物(碧云天)，用铝箔纸包好避光，室温反应5min。每孔加入50μL的1M H 2SO 4终止反应，测量OD 450nm值。结果如图1B所示。
C.将所有OD 450nm值大于1的阳性克隆送公司进行测序，分析比对测序结果，最终确定7个阳性单克隆，分别命名如上所述的aRBD-2、aRBD-3、aRBD-5、aRBD-7、aRBD-41、aRBD-42和aRBD-54。
实施例2表达纯化所得纳米抗体及其Fc融合蛋白
1)设计引物，将所述纳米抗体的基因序列N端融合IFNα蛋白信号肽以引导分泌表达，将所述纳米抗体的基因序列C末端融合人IgG1 Fc，同时在它们之间引入一个TEV酶切位点，随后克隆至哺乳动物表达载体pTT5中。将构建载体用PEI瞬时转染哺乳动物细胞HEK293F中，培养3天后收集上清，采用Protein A柱子对上清中的融合蛋白进行纯化，进行SDS-PAGE电泳，结果如图2A所示，从上清中，我们获得了高 纯度的纳米抗体Fc融合蛋白。
2)采用TEV酶切融合蛋白，随后将酶切产物分别流过Protein G柱子和镍柱，从而分别除去未酶切完全的蛋白、Fc和TEV酶，收集流穿，浓缩后进行SDS-PAGE电泳，结果如图2B所示，从流穿中，我们获得了高纯度的纳米抗体蛋白。
实施例3表征所述纳米抗体
1)采用圆二色谱(CD)表征纳米抗体的稳定性：将实施例纳米抗体溶液分别置换为PBS，稀释到OD 280nm为0.6左右，随后采用圆二色谱仪检测，检测波长范围为280nm-180nm，温度从20-95℃。每个检测重复两次。采用Prism软件处理数据，选取205nm处的光谱值随温度的变化情况，并进一步拟合出Tm值。结果如图3所示，aRBD-2-Fc、aRBD-3-Fc、aRBD-5-Fc、aRBD-7-Fc、aRBD-41-Fc、aRBD-42-Fc和aRBD-54-Fc的Tm值分别为72.33、75.44、73.37、78.98、71.26、98.23和71.07℃。
2)采用非竞争性ELISA初步表征所述纳米抗体Fc融合蛋白与SARS-CoV-2刺突蛋白(S1+S2)胞外段的结合情况：将SARS-CoV-2SARS-CoV-2刺突蛋白(S1+S2)胞外段(Val 16-Pro 1213，北京义翘神州)用PBS稀释至2μg/mL，每个孔分别加100μL用于包被，经过常规洗涤和封闭后，依次添加1∶2.5梯度稀释的纳米抗体Fc融合蛋白和ACE2-Fc蛋白(将人ACE2的aa 19-615段融合人IgG1 Fc后采用HEK293F细胞进行分泌性表达，随后采用Protein A纯化)溶液，在室温下孵育1小时。洗涤后，加入HRP偶联的抗IgG1 Fc抗体(北京义翘神州)检测结合的VHH-Fc和ACE2-Fc，结果如图4所示，除aRBD-42-Fc外，其它6个纳米抗体Fc融合蛋白，即aRBD-2-Fc、aRBD-3-Fc、aRBD-5-Fc、aRBD-7-Fc、aRBD-41-Fc和aRBD-54-Fc的亲和力均高于ACE2-Fc，它们的EC 50分别是0.256、0.098、0.077、0.105、0.226、0.164nM。
3)采用SPR表征所述纳米抗体与SARS-CoV-2 RBD之间的亲和力：将RBD蛋白溶于pH 4.5的醋酸钠，偶联至CM5芯片的一个通道上，同 时设置一个不偶联蛋白的对照通道，采用乙醇胺封闭。将所述7个纳米抗体按1∶1用PBS稀释5个梯度，随后分别以30μL/min的速度流过以上2个通道，同时检测信号值(RU)。在一个循环完成后，采用50mM的NaOH吸掉结合的抗体以再生芯片。所有操作均在Biacore T200系统上完成。结果如图5所示，采用Biacore evaluation程序对结果进行分析，aRBD-2、aRBD-3、aRBD-5、aRBD-7、aRBD-41、aRBD-42和aRBD-54与RBD结合的亲和力K D值分别为2.60、3.33、16.3、3.31、21.9、113和5.49nM。同时我们根据抗体间的竞争实验，设计出2个双表位特异性抗体aRBD-2-5(用序列如SEQ ID NO：29(GGGGSGGGGSGGGGS)所示的GS接头将aRBD-2和aRBD-5首尾相连)和aRBD-2-7(用序列如SEQ ID NO：29(GGGGSGGGGSGGGGS)所示的GS接头将aRBD-2和aRBD-7首尾相连)，相比单体，双表位特异性抗体的亲和力大大提高，aRBD-2-5和aRBD-2-7的亲和力K D值分别为59.2pM和0.25nM。
实施例4表征所述纳米抗体抑制ACE2与RBD的结合功能
采用竞争性ELISA的方法对筛选所得的纳米抗体阻断功能进行表征。将SARS-CoV-2 RBD用PBS稀释至1μg/mL，每个孔分别加100μL用于包被，经过常规洗涤和封闭。将生物素化的ACE2-Fc稀释至10nM，然后用该ACE2-Fc溶液去依次1∶3梯度稀释纳米抗体Fc融合蛋白，每个梯度的混合物取100μL分别加入包被抗原的孔中，室温孵育1小时。用PBST洗涤4次后，加入HRP偶联的Streptavidin(碧云天)检测结合的生物素化ACE2-Fc，结果如图6所示，除aRBD-42外，其它筛选到的6个纳米抗体Fc融合蛋白aRBD-2-Fc、aRBD-3-Fc、aRBD-5-Fc、aRBD-7-Fc、aRBD-41-Fc和aRBD-54-Fc均具有抑制ACE2-Fc与SARS-CoV-2 RBD结合的功能，抑制10nM的ACE2-Fc与SARS-CoV-2 RBD结合的IC 50分别是2.68、2.59、1.89、1.42、5.76和2.07nM。
实施例5表征所述纳米抗体体外中和SARS-CoV-2侵入细胞实验
1)在96孔板中接种Vero E6细胞(ATCC CBP60972)，培养基为DMEM+ 10％FBS，37℃、5％CO 2下培养过夜。将纳米抗体aRBD-2的Fc融合蛋白按照1∶3的梯度从10μg/mL稀释到0.041μg/mL，将aRBD-5和aRBD-7的Fc融合蛋白按照1∶3的梯度从30μg/mL稀释到0.123μg/mL，将双表位特异性抗体aRBD-2-5和aRBD-2-7及其Fc融合蛋白按照1∶3的梯度从1μg/mL稀释到0.0041μg/mL，稀释液均为DMEM+1％FBS，随后分别取50μL加入96孔板中。将SARS-CoV-2(USA-WA1/2020分离株)稀释至4000PFU/mL，稀释液也为DMEM+1％FBS，随后分别取50μL SARS-CoV-2稀释液加入装有梯度稀释的抗体的孔中，同时设置不加抗体的对照，混匀，37℃孵育半小时。吸掉Vero E6细胞的培养基，将以上100μL抗体和病毒的孵育物分别转移到接种Vero E6细胞的孔中，37℃、5％CO 2下孵育1h。吸出孵育物，换PBS洗2次，每孔加入100μL DMEM(含10％FBS+0.5％甲基纤维素)，37℃、5％CO2下培养48h。每个抗体浓度均包含2个重复孔。
2)吸掉培养基上清，PBS洗2次，每孔加入50μL含4％多聚甲醛的PBS，固定15分钟，PBS洗两次。用含有0.1％Triton X-100的PBS孵育样品10分钟，使细胞膜穿孔，PBS洗3次。加入含10％FBS的DMEM封闭非特异性结合位点，室温放置30min。PBS洗2次，用稀释的抗SARS-CoV-2 N蛋白抗体(GeneTex，GTX635679)至合适浓度，每孔加入50μL，室温下孵育1小时。PBST洗3次。加入稀释的Alexa Fluor 488-conjugated二抗(Thermo)至合适浓度，每孔加入50μL，室温下孵育1小时。用Hoechst 33342染色细胞核。用细胞成像仪Cytation 5(BioTek)中的4倍物镜获取整个孔的荧光图像，用Gen5软件(BioTek)的细胞分析模块定量细胞总数(如核染色所示)和感染细胞的总数(如N蛋白染色所示)，从而计算出感染细胞的百分数。中和率＝100×(1-抗体孔感染细胞百分数/无抗体孔感染细胞百分数)。采用Prism软件分析数据结果，如图7所示，拟合显示aRBD-2-Fc、aRBD-5-Fc、aRBD-7-Fc、aRBD-2-5-Fc和aRBD-2-7-Fc中和SARS-CoV-2侵染Vero E6细胞的ND 50(半中和剂量浓度)分别是0.092、0.413、0.591、0.0104和0.0067μg/mL，而aRBD-2-5和aRBD-2-7的ND 50则小于0.004μg/mL可见，双表位特异性抗体的病毒中和能力明显好于单个纳米抗体。。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (11)

  1. 与SARS-CoV-2 RBD结合的羊驼源双表位特异性抗体或其抗原结合片段，其具有VHH1和VHH2，其中所述VHH1具有
    如SEQ ID NO：1所示的CDR1，
    如SEQ ID NO：2所示的CDR2，和
    如SEQ ID NO：3所示的CDR3；
    所述VHH2具有
    如SEQ ID NO：7所示的CDR1，
    如SEQ ID NO：8所示的CDR2，和
    如SEQ ID NO：9所示的CDR3。
  2. 如权利要求1所述的双表位特异性抗体或其抗原结合片段，其中所述VHH1包含如SEQ ID NO：22所示的氨基酸序列，所述VHH2包含如SEQ ID NO：24所示的氨基酸序列。
  3. 如权利要求1或2所述的双表位特异性抗体或其抗原结合片段，其(例如以N端到C端的顺序)包含SEQ ID NO：22和SEQ ID NO：24，优选地，其中SEQ ID NO：22和SEQ ID NO：24之间用接头(例如柔性多肽链，例如GS接头)连接。
  4. 如与SARS-CoV-2 RBD结合的羊驼源抗体或其抗原结合片段，其具有VHH，其中所述VHH具有
    如SEQ ID NO：1所示的CDR1，
    如SEQ ID NO：2所示的CDR2，和
    如SEQ ID NO：3所示的CDR3，
    优选地，所述VHH具有如SEQ ID NO：22所示的氨基酸序列。
  5. 如与SARS-CoV-2 RBD结合的羊驼源抗体或其抗原结合片段，其具有VHH，其中所述VHH具有
    如SEQ ID NO：7所示的CDR1，
    如SEQ ID NO：8所示的CDR2，和
    如SEQ ID NO：9所示的CDR3，
    优选地，所述VHH具有如SEQ ID NO：24所示的氨基酸序列。
  6. 如权利要求1-3中任一项所述的双表位特异性抗体或其抗原结合片段或如权利要求4或5所述的抗体或其抗原结合片段，其进一步具有Fc结构域，优选IgG1 Fc结构域，更优选人IgG1 Fc结构域，所述人IgG1 Fc结构域的氨基酸序列例如如SEQ ID NO：30所示。
  7. 多核苷酸，其编码如权利要求1-3和6中任一项所述的双表位特异性抗体或其抗原结合片段或如权利要求4、5或6所述的抗体或其抗原结合片段。
  8. 表达载体，其包含权利要求7所述的多核苷酸。
  9. 宿主细胞，其包含权利要求8所述的表达载体，所述宿主细胞是用于表达外源蛋白的宿主细胞，例如细菌、酵母、昆虫细胞、哺乳动物细胞。
  10. 药物组合物，其含有如权利要求1-3和6中任一项所述的双表位特异性抗体或其抗原结合片段或如权利要求4、5或6所述的抗体或其抗原结合片段和药用载体。
  11. 如权利要求1-3和6中任一项所述的双表位特异性抗体或其抗原结合片段或如权利要求4、5或6所述的抗体或其抗原结合片段在制备预防、治疗和/或诊断SARS-CoV-2感染的试剂盒或药物中的用途。
PCT/CN2020/117771 2020-09-25 2020-09-25 与SARS-CoV-2 RBD结合的羊驼源纳米抗体 WO2022061720A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/117771 WO2022061720A1 (zh) 2020-09-25 2020-09-25 与SARS-CoV-2 RBD结合的羊驼源纳米抗体

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/117771 WO2022061720A1 (zh) 2020-09-25 2020-09-25 与SARS-CoV-2 RBD结合的羊驼源纳米抗体

Publications (1)

Publication Number Publication Date
WO2022061720A1 true WO2022061720A1 (zh) 2022-03-31

Family

ID=80846025

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/117771 WO2022061720A1 (zh) 2020-09-25 2020-09-25 与SARS-CoV-2 RBD结合的羊驼源纳米抗体

Country Status (1)

Country Link
WO (1) WO2022061720A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111303279A (zh) * 2020-03-17 2020-06-19 中国医学科学院病原生物学研究所 一种针对新型冠状病毒的单域抗体及其应用
CN111647077A (zh) * 2020-06-02 2020-09-11 深圳市因诺赛生物科技有限公司 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用
CN111647076A (zh) * 2020-04-27 2020-09-11 南京医科大学 抗新型冠状病毒SARS-Cov-2的中和性单域抗体及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111303279A (zh) * 2020-03-17 2020-06-19 中国医学科学院病原生物学研究所 一种针对新型冠状病毒的单域抗体及其应用
CN111647076A (zh) * 2020-04-27 2020-09-11 南京医科大学 抗新型冠状病毒SARS-Cov-2的中和性单域抗体及其应用
CN111647077A (zh) * 2020-06-02 2020-09-11 深圳市因诺赛生物科技有限公司 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DU, JIAN-HUI ET AL.: "Development Strategies and Research Progress of SARS-CoV-2 Neutralizing Antibody", CHINESE JOURNAL OF BIOLOGICALS, vol. 33, no. 9, 20 September 2020 (2020-09-20), pages 1070 - 1075, XP055913978 *
HANKE LEO, VIDAKOVICS PEREZ LAURA, SHEWARD DANIEL J., DAS HRISHIKESH, SCHULTE TIM, MOLINER-MORRO AINHOA, CORCORAN MARTIN, ACHOUR A: "An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction", NATURE COMMUNICATIONS, vol. 11, no. 1, 1 December 2020 (2020-12-01), pages 1 - 9, XP055788435, DOI: 10.1038/s41467-020-18174-5 *
HE, LEI ET AL.: "Construction and Identification of Nanobody Phage Display Library Targeting Middle East Respiratory Syndrome Coronavirus", CHINESE JOURNAL OF CELLULAR AND MOLECULAR IMMUNOLOGY, vol. 33, no. 12, 31 December 2017 (2017-12-31), pages 1662 - 1668, XP055913982 *
HUO JIANGDONG; LE BAS AUDREY; RUZA REINIS R.; DUYVESTEYN HELEN M. E.; MIKOLAJEK HALINA; MALINAUSKAS TOMAS; TAN TIONG KIT; RIJAL PR: "Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2", NATURE STRUCTURAL & MOLECULAR BIOLOGY, vol. 27, no. 9, 13 July 2020 (2020-07-13), New York , pages 846 - 854, XP037271196, ISSN: 1545-9993, DOI: 10.1038/s41594-020-0469-6 *
JIANBO DONG, HUANG BETTY, JIA ZHEJUN, WANG BO, GALLOLU KANKANAMALAGE SACHITH, TITONG ALLISON, LIU YUE: "Development of multi-specific humanized llama antibodies blocking SARS-CoV-2/ACE2 interaction with high affinity and avidity", EMERGING MICROBES & INFECTIONS, vol. 9, no. 1, 1 January 2020 (2020-01-01), pages 1034 - 1036, XP055735314, ISSN: 2222-1751, DOI: 10.1080/22221751.2020.1768806 *

Similar Documents

Publication Publication Date Title
CN112094342B (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
CN112094343B (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
CN109096395B (zh) 阻断型cd47纳米抗体及其用途
WO2022061706A1 (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
WO2021244089A1 (zh) 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用
CN114262377B (zh) 一种阻断cd70与其配体cd27结合的抗人cd70纳米抗体的制备方法及其编码序列
CN109232739B (zh) 一种抗cd38纳米抗体、编码基因及应用
WO2022061594A1 (zh) 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用
CN114106187A (zh) 一种靶向ogt的特异性鲨鱼单域抗体及其制备方法和应用
US11136387B2 (en) IL-13 antibody and preparation method and use thereof
CN117777292B (zh) 抗整合素α4β7纳米抗体及其用途
CN108640993B (zh) 一种抗重组人碱性成纤维细胞生长因子纳米抗体及其应用
CN109021103B (zh) 抗人血管内皮生长因子的抗体及其制备方法和应用
CN114249820B (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
CN112961250A (zh) 抗体融合蛋白及其应用
WO2022061720A1 (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
CN114249822B (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
CN114249821B (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
CN115975015A (zh) 一种小反刍兽疫病毒（pprv）f蛋白纳米抗体和纳米抗体的制备、纯化及中和试验方法
CN114478761A (zh) 绿色荧光蛋白鲨源纳米抗体、制备方法及其应用
CN113583119A (zh) 抗金黄色葡萄球菌的纳米抗体Nb56、应用及试剂盒
CN114957469B (zh) 一种抗NKp30抗体及其应用
CN114605555B (zh) 一种抗新型冠状病毒SARS-CoV-2的双特异中和抗体及其应用
CN116874600B (zh) 来自纳米抗体可靶向cd163受体的短肽的制备方法及其应用
CN114380905B (zh) 靶向SARS-CoV-2RBD蛋白的鲨源纳米抗体、制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20954572

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20954572

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 20954572

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC

122 Ep: pct application non-entry in european phase

Ref document number: 20954572

Country of ref document: EP

Kind code of ref document: A1