CN114456260A - Novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof - Google Patents
Novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof Download PDFInfo
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- CN114456260A CN114456260A CN202111554248.9A CN202111554248A CN114456260A CN 114456260 A CN114456260 A CN 114456260A CN 202111554248 A CN202111554248 A CN 202111554248A CN 114456260 A CN114456260 A CN 114456260A
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Abstract
The invention relates to the technical field of biomedicine, and particularly discloses a novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof. The binding molecules are capable of specifically binding to the spike protein of SARS-COV-2 and comprise at least one immunoglobulin single variable domain. The SARS-COV-2-Spike protein binding molecule provided by the invention can specifically bind to SARS-COV-2-Spike protein, and effectively block the binding of SARS-COV-2-Spike protein and human body cell ACE2 receptor, thereby blocking the infection process of SARS-COV-2 to cells, inhibiting the infection and amplification of SARS-COV-2, playing the role of long-acting inhibition of SARS-COV-2 in vivo, and effectively avoiding the repeated occurrence of SARS-COV-2 in vivo.
Description
The application is a divisional application of Chinese patent application CN202080002271.4 filed on 23/09/2020. The 14 th scheme is separated from 21 parallel schemes in the parent scheme and is the application.
Technical Field
The invention relates to the technical field of biological medicines, in particular to a novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof.
Background
The COVID-19 currently lacks specific and effective treatment means clinically. In addition, more and more studies have shown that infection with the novel coronavirus (SARS-COV-2) may present a chronic carrier state; partial discharge of the patient with regaining yang also suggests that the virus may be present in the human body for a long time. At present, the key factors of long-term carrying, such as mechanism, time and the like are not clear, and the prevention of SARS-COV-2 soil rolling is important in the future.
At present, no specific medicine exists in COVID-19, and rapid development of effective medicines is urgently needed. Many research and development organizations both at home and abroad have a second conflict in the research of the treatment strategy aiming at the COVID-19. Although the developed broad-spectrum small-molecule antiviral drugs such as Reidcisvir, Favipiravir and the like have certain curative effect on COVID-19, the drug has no specificity to SARS-COV-2 and has limited curative effect, so the drug is difficult to become a specific drug of COVID-19.
Disclosure of Invention
Aiming at the problems that the prior antiviral drug has no specificity to the novel coronavirus of the novel coronavirus, has poor treatment effect and is difficult to become a specific drug for SARS-COV-2, the invention provides a novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a SARS-COV-2 spike protein binding molecule capable of specifically binding to SARS-COV-2 spike protein and comprising an immunoglobulin single variable domain in which CDR1, CDR2 and CDR3 are:
CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3.
Compared with the prior art, the SARS-COV-2 Spike protein (SARS-COV-2-Spike protein) binding molecule provided by the invention can specifically bind to SARS-COV-2-Spike protein, effectively block the binding of SARS-COV-2-Spike protein and human body cell ACE2 receptor, further block the infection process of SARS-COV-2 to cells, and inhibit the infection and amplification of SARS-COV-2. The SARS-COV-2-Spike protein binding molecule provided by the invention also has the characteristics of good specificity of binding with SARS-COV-2-Spike protein, high biological activity and stability and no toxic or side effect. Meanwhile, the SARS-COV-2-Spike protein binding molecule provided by the invention can play a role of long-acting inhibition on SARS-COV-2 in vivo, and effectively avoids the repeated infection or positive infection of SARS-COV-2 in vivo.
Preferably, the immunoglobulin single variable domain is a single domain antibody.
Preferably, the single domain antibody comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO. 4.
Preferably, the single domain antibody comprises an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO. 4.
Preferably, the single domain antibody comprises an amino acid sequence having at least 99% sequence identity to the sequence of SEQ ID NO. 4.
Preferably, the single domain antibody comprises the amino acid sequence of SEQ ID NO 4.
Preferably, the SARS-COV-2 spike protein binding molecule further comprises an immunoglobulin Fc region.
The inclusion of an immunoglobulin Fc region in the SARS-COV-2 spike protein binding molecule allows the binding molecule to form a dimer while further extending the in vivo half-life of the molecule. The Fc region used in the present invention may be from different subtypes of immunoglobulin, for example, IgG (IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM.
Preferably, the immunoglobulin Fc region is the Fc region of human IgG 1.
Preferably, the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO 5.
The stability and biological activity of the combined molecule fused with the Fc region are further improved, and the KD value of the combined molecule combined with SARS-COV-2 spike protein is further reduced.
Preferably, the SARS-COV-2 spike protein binding molecule comprises the amino acid sequence of SEQ ID NO 6.
The invention also provides a nucleic acid molecule for coding the SARS-COV-2 spike protein binding molecule, wherein the nucleic acid molecule is RNA, DNA or cDNA, which can be obtained by artificial synthesis or separated from proper natural sources.
The invention also provides an expression vector containing the nucleic acid molecule and an expression control element thereof. The expression vector typically comprises at least one nucleic acid molecule provided herein operably linked to one or more suitable expression regulatory elements (promoters, enhancers, terminators, integration factors, selection markers, leaders, reporters, and the like). The selection of such elements and their sequences for expression in a particular host cell is within the knowledge of one skilled in the art.
The invention also provides host cells comprising and expressing the nucleic acid molecules. The host cell is a cell for expressing a heterologous protein, including a bacterial cell, a fungal cell, or a mammalian cell.
The invention also provides a method for obtaining the SARS-COV-2 spike protein binding molecule, which comprises the following steps:
a. culturing the above host cell under conditions that allow expression of the SARS-COV-2 spike protein binding molecule;
b. collecting the SARS-COV-2 spike protein binding molecule expressed by the host cell from the culture of step a.
The recombination of specific nucleic acid molecules into expression vectors and expression into host cells by transformation or transfection methods, selection of markers, methods of inducing protein expression, culture conditions, and the like are known in the art. Techniques for the isolation and purification of protein binding molecules are well known to those skilled in the art.
In addition, the SARS-COV-2 spike protein binding molecule of the invention can also be obtained by other methods known in the art for producing proteins of known sequence, such as chemical synthesis.
The invention also provides an immunoconjugate comprising the SARS-COV-2 spike protein binding molecule described above conjugated to a therapeutic moiety.
The invention also provides a pharmaceutical composition, which comprises the SARS-COV-2 spike protein binding molecule and/or the immunoconjugate, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the invention may further comprise other adjuvants and auxiliary materials, etc. according to the needs.
The "pharmaceutically acceptable carrier" according to the present invention includes any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent, and the like which are physiologically compatible. The carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the binding molecule, immunoconjugate, may be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound, as is well known to those skilled in the art.
The invention also provides application of the pharmaceutical composition in preparing a medicine for treating or preventing novel pneumonia caused by the coronavirus disease.
The invention also provides a kit for detecting SARS-COV-2, which comprises the SARS-COV-2 spike protein binding molecule.
The application method of the kit for detecting SARS-COV-2 comprises the following steps: contacting the SARS-COV-2 spike protein binding molecule with the test sample and the control sample under the condition that a complex can be formed between the SARS-COV-2 spike protein binding molecule and the SARS-COV-2 spike protein, and detecting the formation of the complex; the presence of SARS-COV-2 in the sample is determined by the difference in complex formation between the test sample and the control sample.
Drawings
FIG. 1 is an agarose gel electrophoresis of total RNA extracted in example 1 of the present invention, wherein M: DNA marker 2000, lane 1: total RNA;
FIG. 2 is an agarose gel electrophoresis image of the PCR amplification product in Step1 of nested PCR amplification single domain antibody gene in example 1 of the present invention, wherein M: DNA marker 2000, lane 1: (ii) amplification products;
FIG. 3 is an agarose gel electrophoresis of PCR amplification products in Step2 of nested PCR amplification of single domain antibody gene in example 1 of the present invention, wherein DNA marker 2000, lane 1: (ii) amplification products;
FIG. 4 is an agarose gel electrophoresis of colony PCR amplification products for calculating library insertion rates in example 1 of the present invention, wherein M: DNA marker 2000; lanes 1-8: 8 colonies were picked;
FIG. 5 is a graph showing the change in viral load in the respiratory tracts of rhesus monkeys in the treatment group and the control group according to the change in days in example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Definition of
Unless otherwise indicated or defined, all terms used have the ordinary meaning in the art that will be understood by those skilled in the art. Moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically recited may be and have been performed in a manner known per se to those of skill in the art.
The term "immunoglobulin single variable domain" as used herein refers to an immunoglobulin domain consisting essentially of four "framework regions" referred to in the art as "framework region 1" or "FR 1", "framework region 2" or "FR 2", "framework region 3" or "FR 3", and "framework region 4" or "FR 4", wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art as "complementarity determining region 1" or "CDR 1", "complementarity determining region 2" or "CDR 2", and "complementarity determining region 3" or "CDR 3". Thus, the general structure or sequence of an immunoglobulin single variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR 3-FR 4. Immunoglobulin single variable domains confer antigen specificity to antibodies by virtue of having an antigen binding site.
Conventional IgG antibody molecules generally consist of a light chain comprising 1 variable region (VL) and 1 constant region (CL) and a heavy chain comprising 1 variable region (VH) and 3 constant regions (CH1, CH2, CH 3). Single domain antibodies (sdabs), which are antibodies lacking the light chain of the antibody and having only the variable region of the heavy chain, are also called nanobodies (nanobodies) because of their small molecular weight. Single domain antibodies specifically bind epitopes without the need for additional antigen binding domains. Single domain antibodies are small, stable and efficient antigen recognition units formed from immunoglobulin single variable domains.
It is well known in the art that the total number of amino acid residues in each CDR may differ for single domain antibodies.
The total number of amino acid residues in a single domain antibody will generally range from 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.
Methods for obtaining single domain antibodies that bind to a particular antigen or epitope have been previously disclosed in the following references: van der linden et al, Journal of Immunological Methods,240(2000) 185-195; li et al, JBiol chem.,287(2012) 13713-13721; deffar et al, African Journal of Biotech biology Vol.8(12), pp.2645-2652,17June,2009 and WO 94/04678.
In addition, those skilled in the art will also appreciate that it is possible to "graft" one or more of the above CDRs onto other "scaffolds," including but not limited to human scaffolds or non-immunoglobulin scaffolds. Scaffolds and techniques suitable for such CDR grafting are known in the art.
In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., an immunoglobulin single variable domain of the invention) molecule can bind. Specificity of an antigen-binding molecule can be determined based on its affinity and/or avidity. The affinity, expressed by the dissociation equilibrium constant (KD) of an antigen to an antigen binding protein, is a measure of the strength of binding between an epitope and the antigen binding site on the antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen-binding molecule (alternatively, affinity can also be expressed as the association constant (KA), which is 1/KD). As will be appreciated by those skilled in the art, affinity can be determined in a known manner depending on the particular antigen of interest. Avidity is a measure of the strength of binding between an antigen binding molecule (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and an associated antigen. Affinity is related to both: affinity to its antigen binding site on the antigen binding molecule, and the number of relevant binding sites present on the antigen binding molecule.
The term "SARS-COV-2 Spike protein binding molecule (SARS-COV-2-Spike protein binding molecule)" as used herein means any molecule capable of specifically binding to SARS-COV-2 Spike protein. The SARS-COV-2 spike protein binding molecule may comprise a single domain antibody or conjugate thereof as defined herein directed against the SARS-COV-2 spike protein. SARS-COV-2 spike protein binding molecules also encompass so-called "SM IP" ("small modular immunopharmaceuticals"), or immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.
The "SARS-COV-2 spike protein binding molecule" of the invention may comprise at least one immunoglobulin single variable domain, such as a single domain antibody, that binds to the SARS-COV-2 spike protein. In some embodiments, a "SARS-COV-2 spike protein binding molecule" of the invention can comprise two immunoglobulin single variable domains, such as single domain antibodies, that bind to the SARS-COV-2 spike protein. SARS-COV-2 spike protein binding molecules containing more than one immunoglobulin single variable domain are also known as "formatted" SAR S-COV-2 spike protein binding molecules. The formatted SARS-COV-2 spike protein binding molecule may also comprise, in addition to the immunoglobulin single variable domain that binds to SARS-COV-2 spike protein, a linker and/or a moiety with effector function, for example a half-life extending moiety (such as an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as mpeg) and/or an Fc region. The "SARS-COV-2 spike protein binding molecule" of the invention also encompasses bispecific antibodies that contain immunoglobulin single variable domains that bind different antigens.
Generally, the SARS-COV-2 spike protein binding molecules of the invention will be preferably 10 as measured in a Biacore or Kin ExA assay-8To 10-12Mole/liter (M), more preferably 10-9To 10-11Mole/liter, even more preferably 10-10To 10-12Even more preferably 10-11To 10-12Or a lower dissociation constant (KD). Any greater than 10-4The KD value of M is generally considered to indicate non-specific binding. Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, Surface Plasmon Resonance (SPR) assays, as described herein, and/or competitive binding assays (e.g., Enzyme Immunoassay (EIA) and sandwich competitive assays).
Amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is well known and agreed upon in the art. Such conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably made where one amino acid within the following groups (1) - (5) is replaced with another amino acid residue within the same group: (1) a smaller aliphatic non-polar or weakly polar residue: ala, Ser, Thr, Pro, and Gly; (2) polar negatively charged residues and their (uncharged) amides: asp, Asn, Glu and Gln; (3) polar positively charged residues: his, Ar g and Lys; (4) larger aliphatic non-polar residues: met, Leu, Ile, Val and Cys; and (5) aromatic residues: phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: ala substituted by Gly or Ser; arg is replaced by Lys; asn is replaced by Gln or His; asp substituted by Glu; cys is substituted with Ser; gln is substituted by Asn; glu is substituted with Asp; gly by Ala or Pro; his is substituted with Asn or Gln; ile is substituted by Leu or Val; leu is substituted by Ile or Val; lys is substituted with Arg, Gln, or Glu; met is substituted by Leu, Tyr or Ile; phe is substituted by Met, Leu or Tyr; ser substituted by T hr; thr is substituted by Ser; trp is substituted by Tyr; tyr is substituted with Trp or Phe; val is substituted by Ile or Leu.
"sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity. For the determination of sequence identity see, for example: sequence Analysis in Molecular Biology, von Heinje, G., Academic Press,1987 and Se Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, Newyo rk, 1991.
A polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been separated from at least one other component with which it is normally associated in the source or medium (culture medium), such as another protein/polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or minor component, as compared to the reaction medium or culture medium from which it is naturally derived and/or from which it is obtained. In particular, a polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more than 1000-fold. The "substantially isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous, as determined by suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).
Example 1
Screening of Single Domain antibodies against SARS-COV-2-Spike protein
1.1 construction of the library
1.1.1 immunization
The alpaca is immunized by Spike-RBD protein of the novel coronavirus, and is immunized 4 times in 1, 2, 4 and 6 weeks respectively, and the dosage of each immunization is 300 ug.
1.1.2 extraction of Total RNA
Taking 50ml of peripheral blood of alpaca immunized in the 6 th week, separating lymphocytes, extracting total RNA of the lymphocytes by using Trizol, and detecting the extracted RNA by using an ultraviolet spectrophotometer to obtain a result: OD260/280 is 1.97, OD260/230 is 2.14, which shows that the extracted RNA is not obviously degraded and has better purity; the total RNA concentration was 937.5 ng/. mu.L. Agarose gel electrophoresis was performed using the extracted total RNA, and as a result, two bands, 28S and 18S, were observed as shown in FIG. 1.
1.1.3 reverse transcription of RNA
The RNA reverse transcription system is as follows:
Step 1：
mixing, keeping at 65 deg.C for 5min, and rapidly ice-cooling;
after mixing evenly, carrying out reverse transcription to obtain cDNA, wherein the reverse transcription conditions are as follows: 30min at 42 ℃; 15min at 50 ℃; 70 ℃ for 15 min.
1.1.4 Single Domain antibody (VHH) Gene amplification
The VHH gene is amplified by adopting nested PCR, and the method comprises the following steps:
Step1
after mixing, carrying out PCR reaction under the following reaction conditions: 10s at 98 ℃, 30s at 50 ℃ and 1min at 72 ℃ for 20 cycles. The sequence of the amplification primers was: primer For-1: 5'-GTCCTGGCTGCTCTTCTAC AAGG-3' (SEQ ID NO: 8); primer Rev-1: 5'-GGTACGTGCTGTTGAACTGTT CC-3' (SEQ ID NO: 9).
Purifying and concentrating the PCR product by using a DNA purification kit, performing agarose gel electrophoresis to obtain an agarose gel electrophoresis image shown in figure 2, recovering a 750bp strip by using a DNA product gel recovery kit, and quantifying by using an ultraviolet spectrophotometer to serve as a DNA template of Step 2;
after mixing, carrying out PCR reaction under the following reaction conditions: 10s at 98 ℃, 30s at 55 ℃ and 30s at 72 ℃ for 20 cycles. The sequence of the amplification primers was: primer For-2: 5'-CTAGTGCGGCCGCTGGAGA CGGTGACCTGGGT-3' (SEQ ID NO: 10); primer Rev-2:5 '-GATGTGCAGCT GCAGGAGTCTGGRGGAGG-3' (SEQ ID NO: 11).
The obtained PCR product was subjected to agarose gel electrophoresis, and the agarose gel electrophoresis pattern is shown in FIG. 3, which was recovered using a DNA product gel recovery kit and quantified using an ultraviolet spectrophotometer. Finally, 200. mu.L of the target gene (VHH) of about 500 bp was obtained at a concentration of 458 ng/. mu.L.
1.1.5 library transformation
The obtained target gene and the vector pHEN1 are subjected to double enzyme digestion by SfiI and Not1, the target gene after enzyme digestion and the pHEN1 fragment are connected by T4 DNA ligase and then are transformed into an escherichia coli electroporation competent cell TG1, and a single domain antibody gene library aiming at SARS-COV-2-Spike protein is constructed and named as S2-Lib. Co-transforming for 15 times, mixing and then uniformly spreading in 6 pieces of culture dish (LB solid culture medium containing ampicillin) with phi 150 mm.
The mixed solutions of 0.1. mu.L, 0.01. mu.L, 0.001. mu.L and 0.0001. mu.L were applied to a Phi 90mm petri dish (LB solid medium containing ampicillin) and used for calculation of library volume (counting on plates with a colony count of 30-300), as shown in Table 1, the library volume was calculated to be 1.425X 109cfu。
TABLE 1
Randomly selecting 8 colonies from the culture dish for calculating the library volume, performing colony PCR, performing agarose gel electrophoresis on the PCR product, and calculating the target gene insertion rate of the library, wherein the agarose gel electrophoresis is shown in FIG. 4The library insertion rate was 100%, and the actual library volume was 1.425X 109cfu。
The colony PCR system was as follows:
the colony PCR reaction conditions are as follows: 31 cycles of 98 ℃ for 10s, 50 ℃ for 30s and 72 ℃ for 1 min.
1.1.6 library rescue
Inoculating 10-100 times of live cells from the S2-Lib gene library, culturing to logarithmic phase, rescuing with M13K07 phage, centrifugally collecting phage, purifying with PEG-NaCl to obtain phage display library named S2-PDL with titer of 3.5 × 1013cfu/mL. Can be directly used for affinity screening of subsequent specific phage.
1.2 screening against the Single Domain antibody against SARS-COV-2-Spike protein
Coating the plate with 3. mu.g/well of Spike-RBD protein (Spike protein receptor binding domain protein), and standing at 4 ℃ overnight; blocking with 1 wt% skimmed milk powder at room temperature for 2h, adding 100. mu.l phage (8X 10)11tfu from the phage display library S2-PDL constructed at 1.1.6) at room temperature for 1 h. Followed by elution 5 times with PBST (0.05 vt% tween 20 in PBS) to wash away unbound phage; phages specifically bound to Spike-RBD protein were dissociated with triethylamine (100mM) and infected with E.coli TG1, which was grown in log phase, and phages were generated and purified for the next round of screening. The same screening process was repeated for 3 rounds. Thus, positive clones are enriched, and the aim of screening the Spike-RBD protein specific antibody in the antibody library by using the phage display library is fulfilled. Sequencing the obtained positive phage to obtain an antibody gene sequence.
The obtained antibody gene sequences are respectively constructed on pcDNA3.4 vectors, HEK-293 cells are used for expressing the antibodies, and the proteinA medium is used for purifying and collecting the antibodies in the culture medium supernatant. The purified antibody was incubated with a Spike-RBD coated plate for ELISA assay. Obtaining the antibody which can specifically bind to the Spike-RBD protein.
The obtained antibody sequences were analyzed according to the sequence alignment software Vector NTI. Obtaining a single domain antibody strain which can specifically bind to the Spike-RBD protein, wherein the sequence of the single domain antibody is shown as SEQ ID NO. 4, and the single domain antibody strain carries the CDR1-3 sequences in SEQ ID NO. 1-3 respectively, and is specifically shown as table 2.
TABLE 2
| Antibodies | CDR1 | CDR2 | CDR3 |
| Single domain antibodies | SEQ ID NO:1 | SEQ ID NO:2 | SEQ ID NO:3 |
The single domain antibody strains were incubated with a plate coated with Spike-RBD and measured by ELISA, and the values of OD450 in the wells after the reaction of the single domain antibodies with Spike-RBD were shown in Table 3.
TABLE 3
Wherein, blank is the OD450 value in the duplicate wells without the antibody.
From the data in Table 3, it is clear that the single domain antibody was bound to the Spike-RBD protein.
1.3 evaluation and identification of Single-Domain antibodies against SARS-COV-2-Spike protein
1.3.1 expression and purification of Single Domain antibodies in host bacteria Escherichia coli
The gene coding sequences of the obtained single domain antibodies were recombined into expression vectors PET32b (Novagen, product No. 69016-3), and recombinant plasmids identified to be correct by sequencing were transformed into expression host bacteria BL1(DE3) (Tiangen Biochemical technology, product No. CB105-02), which were spread on LB plates containing 100. mu.g/mL ampicillin overnight at 37 ℃. Selecting single colony, inoculating, culturing overnight, transferring overnight strain for amplification next day, shake culturing at 37 deg.C until OD value reaches 0.5-1, adding 0.5mM IPTG for induction, shake culturing at 28 deg.C overnight. The next day, the cells were collected by centrifugation, and the collected cells were disrupted to obtain a crude antibody extract. Then purifying the single domain antibody protein to ensure that the purity of the single domain antibody protein reaches more than 90 percent.
1.3.2 Competition ELISA to investigate the effect of the above-mentioned SARS-COV-2-Spike protein single-domain antibody on the binding of Spike-RBD protein and the receptor ACE2
The Spike-RBD protein and ACE2 protein were obtained by expression from HEK293 cells (pCDNA4, Invitrogen, CatV 86220). Then biotinylated ACE2 protein was obtained using the biotinylation kit from Thermo.
The plate was coated with 0.5. mu.g/well of Spike-RBD protein overnight at 4 ℃ after which 100ng of 1.3.1 purified single domain antibody and 5. mu.g of biotinylated ACE2 protein were added per well, and a control was set, in which no single domain antibody was added to the wells of control 1 and no biotinylated ACE2 protein was added to the wells of control 2 and reacted for 2h at room temperature. Then, SA-HRP (purchased from Sigma) was added, and after 1 hour of reaction at room temperature, a color developing solution was added, and the absorbance was read at a wavelength of 450 nm. When the OD value of the sample is less than 0.8 than that of the control, the single-domain antibody is considered to have a blocking effect.
As a result, as shown in Table 4, the single domain antibody strain showed a blocking effect on the Spike protein/ACE 2 protein interaction.
TABLE 4
| Sample (I) | |
| Control group | |
| 1 | 2.271 |
| |
0.022 |
| Single domain antibody strains | 0.016 |
In the laboratory with biological safety grade P3, the purified single domain antibody is added into a culture system by infecting a VERO cell model with virus, and the specific operation is as follows: will 104The/well VERO cells were added to a 96 well plate and after 24 hours the cells were washed 2 times with PBS, single domain antibody was mixed with virus and added to the 96 well plate at an initial concentration of 10 μ g/mL, diluted 2-fold with 10 gradients, 5 wells, incubated for 2 hours at 37 ℃ and on day 5 to check whether the VERO cells were infected with virus (if the cells were not diseased indicating that the single domain antibody had neutralized the virus and blocked the process of virus infection of the VERO cells).
As shown in Table 5, the single domain antibody was effective in blocking the process of viral infection of cells at a concentration of 0.078125. mu.g/ml or more. The IC50(μ g/ml) data obtained in Table 5 indicate that the obtained single domain antibody is capable of blocking the process of virus infection of cells and is an effective neutralizing antibody.
TABLE 5
Note: "+" indicates that viral infection of cells can be blocked, and "-" indicates that viral infection of cells cannot be blocked.
Example 2
1.1 Fc fusion protein for preparing single domain antibody of SARS-COV-2-Spike protein
The amino acid sequence of human IgG1-Fc region (SEQ ID NO:5) was obtained from the amino acid sequence of the constant region of human immunoglobulin (IgG1) on Uniprot, a protein database. A nucleic acid fragment (nucleic acid sequence is shown as SEQ ID NO:7) encoding human IgG1-Fc was obtained from human PBMC total RNA by reverse transcription PCR, and a nucleic acid fragment encoding a fusion protein of a single domain antibody of SARS-COV-2-Spike protein and Fc was obtained by overlapping PCR and recombined into vector pCDNA4(Invitrogen, Cat V86220).
The constructed pCDNA4 plasmid containing the nucleic acid fragment of the fusion protein of the SARS-COV-2-Spike protein single domain antibody and Fc is transfected into HEK293 cells for expression. Specifically, the recombinant expression plasmid is diluted by Freestyle293 culture medium and added with PEI (polyethylenimine) solution required for transformation, and the plasmid/PEI mixture is respectively added into HEK293 cell suspension and placed at 37 ℃ with 10% CO2Culturing in a shaker at 100 rpm; adding 50 μ g/LIGF-1. After 4h, it was supplemented with EX293 medium, 2mM glutamine and 50. mu.g/LIGF-1, and shaken at 120 rpm. After 24h, 3.8mM VPA was added. After culturing for 5 days, collecting the expression culture supernatant, and purifying by a ProteinA affinity chromatography to obtain the fusion protein of the SARS-COV-2-Spike protein single domain antibody and Fc. The sequence of the fusion protein of the obtained SARS-COV-2-Spike protein single-domain antibody and Fc is shown in SEQ ID NO 6.
1.2 characterization of the function of the fusion protein of the SARS-COV-2-Spike protein single domain antibody with Fc (SEQ ID NO:6)
The binding capacity of the SARS-COV-2-Spike protein single-domain antibody and the Fc fusion protein to the SARS-COV-2-Spike protein is identified by the SPR method. The specific operation is as follows: the binding kinetics of the obtained fusion protein of SARS-COV-2-Spike protein single domain antibody and Fc against Spike-RBD was measured by the surface plasmon resonance (SRP) method using a BIAcoreX100 instrument, and the Spike-RBD protein was directly coated on a CM5 biosensor chip to obtain approximately 1000 Response Units (RU). For kinetic measurements, the fusion protein of the single domain antibody of SARS-COV-2-Spike protein and Fc was serially diluted three times (1.37nm to 1000nm) with HBS-EP +1 Xbuffer (GE, cat # BR-1006-69), injected at 25 ℃ for 120s with a dissociation time of 30min, and regenerated for 120s by addition of 10mM glycine-HCl (pH 2.0). The binding rate (kon), dissociation rate (koff) and equilibrium dissociation constant (kD) (calculated as the ratio koff/kon) of the fusion protein to SARS-COV-2-Spike protein were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2). The calculation results are shown in table 6.
TABLE 6
| Binding Rate (kon) | Off rate (koff) | Equilibrium dissociation constant (kD; koff/kon) | |
| Fusion proteins | 3.26E+06 | 6.51E-05 | 1.97E-11 |
As can be seen from Table 6, the binding rate of the SARS-COV-2-Spike protein single domain antibody and the Fc fusion protein to the SARS-COV-2-Spike protein is high, the dissociation rate is low, and the equilibrium dissociation constant KD is 1.97E-11, which indicates that the fusion protein can more rapidly bind to the SARS-COV-2-Spike protein and is difficult to dissociate, and further indicates that the SARS-COV-2-Spike protein single domain antibody and the Fc fusion protein are used as a blocking antibody and have excellent blocking effect.
1.3 identification of the blocking Capacity of the SARS-COV-2-Spike protein Single Domain antibody and the Fc fusion protein for the interaction of Spike protein/ACE 2 by competitive ELISA
The ACE2 protein is obtained by expression of HEK293 cells. The biotinylated protein ACE2-Biotin was obtained using the Biotinysis kit from Thermo.
The plate was coated with 0.5. mu.g of Spike-RBD protein overnight at 4 ℃ and then 200ng of the obtained SARS-COV-2-Spike protein single domain antibody and Fc fusion protein and ACE2-Biotin 5ug were added to each well, and the reaction was carried out at room temperature for 2 hours without adding the fusion protein to control 1 and without adding ACE2-Biotin to control 2. After washing, SA-HRP (purchased from Sigma) was added, the reaction was carried out at room temperature for 1 hour, and after washing, a color developing solution was added and absorbance was read at a wavelength of 450 nm. The results are shown in Table 7.
TABLE 7
| Sample (I) | |
| Control group | |
| 1 | 2.414 |
| |
0.019 |
| SARS-COV-2-Spike protein single domain antibody | 0.016 |
The result shows that the SARS-COV-2-Spike protein single-domain antibody and Fc fusion protein can effectively block the interaction between Spike protein and ACE 2.
1.4 analysis of the specificity of the binding of the SARS-COV-2-Spike protein single domain antibody and Fc fusion protein to Spike protein
Plasmids (pCDNA4, Invitrogen, Cat V86220) carrying the full-length genes of the currently known 7 coronavirus (SARS-COV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV) Spike proteins were obtained by transient transfection using human HEK293 cells, and the Spike proteins were transiently expressed on the membrane. The plasmid enables the C end of the Spike protein to be fused with the EGFP protein, so that the expression level of the Spike protein on the membrane can be investigated through the green fluorescence intensity. The constructed cells were resuspended in 0.5% PBS-BSA Buffer, and SARS-COV-2-Spike protein single domain antibody and Fc fusion protein were added, while negative control was set, and incubated on ice for 20 min. After washing, the eBioscience secondary antibody anti-hIg-PE was added and the mixture was frozen for 20 min. After washing, the cells were resuspended in 500. mu.l of 0.5% PBS-BSABuffer and examined by flow cytometry. The results showed that the SARS-COV-2-Spike protein single domain antibody-Fc fusion protein specifically binds only to the SARS-COV-2-Spike protein, but not to the Spike proteins of other coronaviruses.
1.5SARS-COV-2-Spike protein single domain antibody and Fc fusion protein block SARS-COV-2 infection of rhesus monkey
6 of 12 rhesus monkeys infected with SARS-COV-2 virus and showing symptoms (treatment group) were treated by administration of the SARS-COV-2-Spike protein single domain antibody and Fc fusion protein (SARS-COV-2-Spike protein single domain antibody and Fc fusion protein 100. mu.g/rhesus monkey) provided by the present invention, and 6 (control group) were not treated by administration. The respiratory viral load was measured once daily within 6 days after treatment. The mean load of the new coronavirus in the respiratory tracts of 6 rhesus monkeys in the treatment group was significantly reduced compared to the control group, as shown in fig. 5. The treatment group was continued with 6 rhesus monkeys and their symptoms and respiratory viral load were measured every other week. After 2 weeks of continuous observation, the viral load in the respiratory tract was not detected and no corresponding symptoms of the disease were observed. The continuous observation for 3 months shows that the virus in 6 rhesus monkeys of the treatment group has no recurrence, which shows that the SARS-COV-2-Spike protein single domain antibody and Fc fusion protein can play a long-acting role in vivo, and can avoid the relapse of positive after the living body infected with the novel coronavirus is cured. Wherein the detection process of the load of the new coronavirus is as follows: the throat swabs of the rhesus monkeys (treatment group) that were subjected to the administration treatment and the rhesus monkeys (control group) that were not subjected to the administration treatment were respectively collected, and nucleic acids of the viruses in the throat swabs were extracted and detected, and the detection process was: SARS-COV-2 RNA was extracted using an RNA extraction kit (Qiagen) according to the instructions, and the obtained RNA was dissolved in 50. mu.L of elution buffer and used as a template for RT-PCR amplification. The viral S region genes were amplified using primers RBD-qF1 (5'-CAATGGTTTAACAGGCACAGG-3', SEQ ID NO:12) and RBD-qR1 (5'-CTCAAGTGTCTGTGGATCACG-3', SEQ ID NO: 13). Adopting a HiScriptR II One Step qRT-PCR SYBRRGreen Kit (Vazyme Biotech Co., Ltd.) Kit, operating according to the Kit instruction, and setting the PCR amplification condition as follows; 50 ℃ for 3min, 95 ℃ for 10s, 60 ℃ for 30s, 40 cycles, and the PCR amplification instrument is an ABI quantitative PCR instrument.
The results of the in vivo experiments show that the SARS-COV-2-Spike protein single domain antibody and Fc fusion protein of the invention show significant long-acting SARS-COV-2 infected cell inhibition and amplification effects in rhesus monkeys infected with SARS-COV-2, and the sun recovery rate of the treated rhesus monkeys is 0.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen Nennuosai Biotech Co., Ltd
<120> novel coronavirus (SARS-COV-2) spike protein binding molecule and use thereof
<130> 2020
<160> 13
<170> PatentIn version 3.5
<210> 1
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20 25 30
Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
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Ala Gly Ile Met Pro Gly Gly Gly Phe Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
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Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
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Ala Ala Ala Arg Ser Glu Thr Gly Glu Tyr Asp Asn Arg Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 5
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Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
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Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
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Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
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Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
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Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
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tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420
tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540
acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600
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ggtacgtgct gttgaactgt tcc 23
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Claims (11)
1. A SARS-COV-2 spike protein binding molecule, characterized by: is capable of specifically binding to the spike protein of SARS-COV-2 and comprises an immunoglobulin single variable domain in which CDR1, CDR2 and CDR3 are:
CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3.
2. The SARS-COV-2 spike protein binding molecule of claim 1, wherein: the immunoglobulin single variable domain is a single domain antibody.
3. The SARS-COV-2 spike protein binding molecule of claim 2, wherein: the single domain antibody comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO. 4.
4. The SARS-COV-2 spike protein binding molecule of claim 2, wherein: the single domain antibody comprises an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO. 4.
5. The SARS-COV-2 spike protein binding molecule of claim 2, wherein: the single domain antibody comprises an amino acid sequence having at least 99% sequence identity to the sequence of SEQ ID NO. 4.
6. The SARS-COV-2 spike protein binding molecule of claim 2, wherein: the single domain antibody comprises the amino acid sequence of SEQ ID NO 4.
7. The SARS-COV-2 spike protein binding molecule of any one of claims 1 to 6, wherein: further comprises an immunoglobulin Fc region, and the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO. 5.
8. The SARS-COV-2 spike protein binding molecule of claim 7, wherein: comprises the amino acid sequence of SEQ ID NO 6.
9. A pharmaceutical composition comprising a SARS-COV-2 spike protein binding molecule according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.
10. Use of the pharmaceutical composition of claim 9 for the manufacture of a medicament for the treatment or prevention of pneumonia as a novel coronavirus disease.
11. A kit for detecting SARS-COV-2, comprising the SARS-COV-2 spike protein binding molecule of any one of claims 1 to 8.
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| MX2022014852A (en) | 2020-06-03 | 2023-02-01 | Regeneron Pharma | METHODS FOR TREATING OR PREVENTING SARS-CoV-2 INFECTIONS AND COVID-19 WITH ANTI-SARS-CoV-2 SPIKE GLYCOPROTEIN ANTIBODIES. |
| CN113388031B (en) * | 2021-08-17 | 2021-11-09 | 上海浙江大学高等研究院 | Monoclonal antibody 35B5, and preparation method and application thereof |
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| CN115057938B (en) * | 2022-06-24 | 2023-01-06 | 广东菲鹏制药股份有限公司 | Novel coronavirus resistant humanized multivalent binding protein and application thereof |
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