CN117327174A - Humanized multivalent binding protein against novel coronaviruses and application thereof - Google Patents
Humanized multivalent binding protein against novel coronaviruses and application thereof Download PDFInfo
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- CN117327174A CN117327174A CN202211668151.5A CN202211668151A CN117327174A CN 117327174 A CN117327174 A CN 117327174A CN 202211668151 A CN202211668151 A CN 202211668151A CN 117327174 A CN117327174 A CN 117327174A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- 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
-
- 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
- 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
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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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]
-
- 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®
-
- 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
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of antibodies, in particular to an anti-novel coronavirus humanized multivalent binding protein and application thereof. The multivalent binding protein provided by the invention comprises at least two antigen epitope binding domains, wherein the antigen epitope binding domains are VHH; the amino acid sequence of the VHH is shown as SEQ ID NO. 1 or SEQ ID NO. 2, or as an amino acid sequence with at least 95% similarity with SEQ ID NO. 1 or SEQ ID NO. 2. The multivalent binding protein can effectively block the binding of wild strain, delta variant strain and Omacron variant strain SARS-COV-2RBD protein and human ACE2 receptor protein, has more remarkable neutralizing activity of novel coronavirus than monovalent binding protein, and can be widely used for preventing and treating novel coronavirus.
Description
The application is a divisional application of Chinese patent application 202210724686.3 (application number: 202210724686.3, application date: 2022-06-24, invention name: anti-novel coronavirus humanized multivalent binding protein and application thereof).
Technical Field
The invention belongs to the technical field of antibodies. More particularly, to humanized multivalent binding proteins against novel coronaviruses and uses thereof.
Background
The traditional monoclonal antibody has over-large molecular mass (150 kD), is difficult to penetrate tissues, and has lower effective concentration in tumor areas and insufficient treatment effect; in addition, traditional antibodies have high immunogenicity, and engineered antibodies are difficult to achieve the original affinity, which limits their wide clinical application.
Since the 2019 outbreak, the new coronavirus (SARS-COV-2) has spread widely around the world; in addition, various types of novel coronavirus variants were discovered successively, such as the first discovery of novel coronavirus Delta variants in india in month 10 of 2020, and the first discovery of novel coronavirus omacron variants from south africa in month 11 of 2021 on day 24. A variant refers to a mutation of a certain gene base or deletion of a certain base based on the genome of an original virus, the mutation or deletion of the base can cause the property of the virus to change, and the virus can have such changes as infectivity, host range, transmission strength, virulence, pathogenicity, severity of illness, prognosis and immunogenicity. This results in a clinical overall course of disease and epidemiology change, and even in the case of immunogenicity and prophylaxis against immunity, serious economic losses, social burden and other negative effects. Therefore, there is a need to develop drugs with specific therapeutic effects on novel coronavirus wild-type strains and mutant strains.
Disclosure of Invention
The object of the present invention is to provide humanized multivalent binding proteins against novel coronaviruses and uses thereof. The multivalent binding protein provided by the invention can effectively block the binding of wild strain, delta variant strain and Omicron variant strain SARS-COV-2RBD protein and human ACE2 receptor protein, and compared with monovalent binding protein, the humanized multivalent binding protein has more remarkable novel coronavirus neutralization activity.
The above object of the present invention is achieved by the following technical scheme:
in a first aspect, the present invention provides an anti-novel coronavirus humanized multivalent binding protein comprising at least two epitope binding domains, which are VHHs; the amino acid sequence of the VHH is shown as SEQ ID NO. 1 or SEQ ID NO. 2, or as an amino acid sequence with at least 95% similarity with SEQ ID NO. 1 or SEQ ID NO. 2.
In a second aspect, the invention provides a fusion protein comprising the multivalent binding protein.
In a third aspect, the invention provides a conjugate comprising the multivalent binding protein.
In a fourth aspect, the invention provides a nucleic acid encoding the multivalent binding protein, or encoding the fusion protein, or encoding the conjugate.
In a fifth aspect, the invention provides a recombinant vector carrying the nucleic acid.
In a sixth aspect, the invention provides a host cell carrying the nucleic acid, or comprising the recombinant vector.
In a seventh aspect, the invention provides a pharmaceutical composition comprising the multivalent binding protein, the fusion protein, the conjugate, the nucleic acid, the recombinant vector, or the host cell.
In an eighth aspect, the invention provides the use of said multivalent binding protein, said fusion protein, said conjugate, said nucleic acid, said recombinant vector, or said host cell for the preparation of a medicament for the treatment and/or prevention of a novel coronavirus.
In a ninth aspect, the present invention provides a method of preparing the multivalent binding protein, comprising: culturing the host cell, and separating and purifying the culture product to obtain the multivalent binding protein.
Drawings
FIG. 1 is a block diagram of multivalent binding proteins R1406, R1407, R1475, R1464 of the invention.
FIG. 2 is a graph showing the result of neutralizing activity of R1382, R1406 and R1407 against a novel coronavirus of a wild strain.
FIG. 3 is a graph showing the result of neutralizing activity of R1463, R1475 and R1464 against a wild-type novel coronavirus.
FIG. 4 is a graph showing the results of neutralization activity of R1382, R1406 and R1407 on novel coronaviruses of Delta variants.
FIG. 5 is a graph showing the results of neutralization activity of R1463, R1475, and R1464 on novel coronaviruses of the Delta variant.
FIG. 6 is a graph showing the results of neutralization activities of R1382, R1406 and R1407 against novel coronaviruses of Omacron variants.
FIG. 7 is a graph showing the results of neutralization activities of R1463, R1475 and R1464 on novel coronaviruses of Omacron variants.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
In the present invention, the term "amino acid" means a naturally occurring or non-naturally occurring carboxy alpha-amino acid. The term "amino acid" as used herein may include naturally occurring amino acids and non-naturally occurring amino acids. Naturally occurring amino acids include alanine (three letter code: ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Non-naturally occurring amino acids include, but are not limited to, alpha-aminoadipic acid, aminobutyric acid, citrulline, homocysteine, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.
In the present invention, the term "amino acid sequence" refers to the order in which amino acids are linked to each other to form a peptide chain (or polypeptide), and the amino acid sequence can be read in only one direction. There are 100 different types of amino acids, 20 of which are commonly used, and the present invention does not exclude other substances on the amino acid chain, such as sugar, lipid, etc., and the present invention is not limited to the commonly used amino acids in 20.
In the present invention, the term "Fc region" refers to the C-terminal region of an immunoglobulin, which is a functional building block consisting of only CH2 and CH3 in the constant domain of the heavy chain. The Fc region has no ability to bind antigen, however it has the property of having an extended half-life and has a constant amino acid sequence.
The invention provides an anti-novel coronavirus humanized multivalent binding protein, which is characterized in that it comprises at least two epitope binding domains, said epitope binding domains being VHH.
In some embodiments, the amino acid sequence of the VHH is as shown in SEQ ID NO. 1 or SEQ ID NO. 2, or as shown in an amino acid sequence having at least 95% similarity to SEQ ID NO. 1 or SEQ ID NO. 2.
In some embodiments, the number of epitope binding domains is 2-6.
In some embodiments, the epitope binding domains are linked by a linking peptide. Connecting peptides commonly used in the art can be used in the present invention.
In a preferred embodiment, the amino acid sequence of the connecting peptide is: GGGGSGGGGSGGGGS (SEQ ID NO: 11).
In a preferred embodiment, the connections are in series.
In some embodiments, the multivalent binding protein further comprises a half-life extending domain.
In a preferred embodiment, the half-life extending domain is selected from an immunoglobulin Fc region or a serum albumin binding domain.
In a preferred embodiment, the immunoglobulin is selected from IgA, igD, igE, igG and IgM
In some embodiments, the half-life extending domain is an immunoglobulin Fc region, and the epitope binding domain is linked to the N-terminus or the C-terminus of the Fc region.
In some embodiments, the multivalent binding protein forms a dimer structure through interchain disulfide bonds of the Fc region.
In a preferred embodiment, the amino acid sequence of the Fc region is shown in SEQ ID NO. 3.
In some embodiments, the multivalent binding protein has an amino acid sequence as shown in any one of SEQ ID NOs 6 to 9.
In some embodiments, the amino acid sequence of the multivalent binding protein may not contain a His tag, but may also contain a His tag.
The invention also provides fusion proteins comprising the multivalent binding proteins.
In the present invention, the term "fusion protein" refers to a fusion protein obtained by fusing the multivalent binding protein of the present invention with other functional protein fragments and the like.
The invention also provides conjugates comprising the multivalent binding proteins.
In the present invention, the term "conjugate" refers to a conjugate obtained by coupling the multivalent binding protein of the present invention with one or more of an enzyme phase (e.g., horseradish peroxidase, alkaline phosphatase, etc.), a radioisotope, a fluorescent compound or a chemiluminescent compound, a therapeutic agent, etc., which can be used for detecting a novel coronavirus, for preparing a medicament for treating and/or preventing a novel coronavirus, or for treating a disease infected with a novel coronavirus.
The invention also provides nucleic acids encoding the multivalent binding proteins, or encoding the fusion proteins, or encoding the conjugates.
In the present invention, the nucleic acid is usually RNA or DNA, and the nucleic acid molecule may be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acids are used when they are incorporated into vectors.
At present, the nucleic acid molecule sequences encoding the proteins of the invention are already available entirely by chemical synthesis.
The invention also provides a recombinant vector carrying the nucleic acid.
In the present invention, the term "vector" includes plasmids, expression vectors, cloning vectors, viral vectors and the like. Various vectors known in the art may be used. For example, a recombinant vector can be formed by selecting a commercially available vector and then operably linking a nucleic acid sequence encoding a multivalent binding protein of the invention to an expression control sequence.
The invention also provides a host cell carrying the nucleic acid, or comprising the recombinant vector.
In the present invention, the term "host cell" includes both prokaryotic and eukaryotic cells. Examples of commonly used prokaryotic host cells include E.coli, bacillus subtilis, and the like. Host cells for expressing the multivalent binding protein include E.coli, yeast cells, insect cells, COS cells, CHO cells, and the like. After obtaining the transformed host cell, the cell may be cultured under conditions suitable for expression of the multivalent binding protein of the invention, thereby expressing the multivalent binding protein; the expressed multivalent binding protein is then isolated.
The invention also provides a pharmaceutical composition comprising the multivalent binding protein, the fusion protein, the conjugate, the nucleic acid, the recombinant vector, or the host cell.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. As pharmaceutically acceptable carriers, binders, glidants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavoring agents and the like can be used for oral administration; buffers, preservatives, pain reducing agents, solubilizers, isotonic agents, stabilizers and the like can be used in the injectable mixture; as well as substrates, excipients, lubricants, preservatives, and the like, may be used for topical application.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.
The invention also provides the use of the multivalent binding protein, the fusion protein, the conjugate, the nucleic acid, the recombinant vector, or the host cell in the preparation of a medicament for the treatment and/or prevention of novel coronaviruses.
The invention also provides a method of preparing the multivalent binding protein, comprising: culturing the host cell, and separating and purifying the culture product to obtain the multivalent binding protein.
EXAMPLE 1 design and construction of humanized multivalent binding proteins against novel coronaviruses
Humanized multivalent binding proteins of different structures are designed in tandem using an Fc region or His tag, binding VHH domains. The structure of the modified humanized multivalent binding protein is shown in figure 1, and the specific amino acid sequence is shown in table 1:
wherein, the female parent sequences of R1406 and R1407 are RX011 (the amino acid sequence is shown as SEQ ID NO: 1), and the sequence structure is as follows: r1406 is a VHH-Linker-VHH-Fc region, R1407 is a Fc region-VHH-Linker-VHH; the female parent sequences of R1475 and R1464 are RX017 (the amino acid sequence is shown as SEQ ID NO: 2), and the sequence structure is as follows: r1475 is VHH-Linker-VHH-His tag, R1464 is His tag-VHH-Linker-VHH; the amino acid sequence of the Fc region is shown as SEQ ID NO. 3, and the amino acid sequence of the His tag is HHHHH (SEQ ID NO. 10).
And R1382 (the amino acid sequence is shown as SEQ ID NO: 4) and R1463 (the amino acid sequence is shown as SEQ ID NO: 5) are used as controls, and the sequence structure is as follows: r1382: RX011 VHH-Fc region, R1463: RX017 VHH-His tag.
SEQ ID NO:1:
QVQLVESGGGPVQAGGSLRLSCTCSRCTFNWDGMGWFRQAPGKEREFVATISWSGQEPAYADSVKGRFTISRDKPKNTVYLQMTSLKSEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTRVTVSA
SEQ ID NO:2:
QVQLVESGGGVVQPGGSLRLSCTCSRCTFNWDGMGWFRQAPGKGLEFVATISWSGQEPAYADSVKGRFTISRDNSKNTLYLQMTSLRAEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTLVTVSS
SEQ ID NO:3:
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:4:
QVQLVESGGGPVQAGGSLRLSCTCSRCTFNWDGMGWFRQAPGKEREFVATISWSGQEPAYADSVKGRFTISRDKPKNTVYLQMTSLKSEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTRVTVSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:5:
QVQLVESGGGVVQPGGSLRLSCTCSRCTFNWDGMGWFRQAPGKGLEFVATISWSGQEPAYADSVKGRFTISRDNSKNTLYLQMTSLRAEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTLVTVSSHHHHHH
TABLE 1 amino acid sequence of humanized multivalent binding proteins
EXAMPLE 2 preparation of humanized multivalent binding proteins against novel coronaviruses
Transient protein expression:
the plasmid containing the target gene is introduced into host cell Expi293 after forming a cationic complex with transfection reagent PEI, and the exogenous gene on the plasmid is transcribed and translated in the cell during the period of the plasmid in the cell, thereby obtaining the target protein.
The Expi293 was incubated at 37℃with 8% carbon dioxide at 130rpm and 2E6 cells were inoculated into 1L shake flasks by cell counting prior to transfection, the culture system being approximately 300ml. Preparing transfection complex for transfection: firstly, 750 mug of target plasmid is added into a 50ml centrifuge tube containing 15ml of Opti-MEM reagent, and the mixture is gently mixed and marked as a tube A; 1.5mg of the transfection reagent PEI was added to a 50ml centrifuge tube containing 15ml of Opti-MEM reagent, gently mixed, incubated at room temperature for 5min, labeled as tube B; and (3) dropwise adding the PEI diluent of the B tube into the DNA diluent of the A tube, slightly mixing, incubating for 15min at room temperature, adding the PEI-target plasmid complex into the Expi293 cells after incubation, and placing the mixture in a shaking table at 37 ℃ for continuous culture. Until D7-D10, the sample is collected.
Purification of complex samples:
the transient cell expression liquid is centrifuged at 9000rpm/20min, and the supernatant is collected and sterilized and filtered by a 0.22 mu m filter membrane. ProA affinity chromatography is adopted for purification. The procedure is as follows, using an AKTA avant 150 chromatography apparatus, a chromatography column (e.g., mabselectsurex LX, GE) is equilibrated with at least 5CV equilibration buffer (10 mM PBS), and a sample is loaded onto the column to allow the target protein to adsorb onto the column while other impurities penetrate the column. After loading was completed, the column was again rinsed with at least 5CV of equilibration buffer (10 mM PBS), followed by elution of the target protein with elution buffer (20 mM naac, ph=3.4), and the collection tube was pre-loaded with neutralization buffer (1 m tris, ph 8.0) at a volume of 10% of the elution volume depending on the estimated amount of eluted sample.
The binding proteins were prepared by conventional methods and the expression supernatants were purified by ProA affinity chromatography. The procedure is as follows, using an AKTA avant 150 chromatography apparatus, a chromatography column (e.g., mabselectsurex LX, GE) is equilibrated with at least 5CV equilibration buffer (10 mM PBS), and a sample is loaded onto the column to allow the target protein to adsorb onto the column while other impurities penetrate the column. After loading was completed, the column was again rinsed with at least 5CV of equilibration buffer (10 mM PBS), followed by elution of the target protein with elution buffer (20 mM naac, ph=3.4), and the collection tube was pre-loaded with neutralization buffer (1 m tris, ph 8.0) at a volume of 10% of the elution volume depending on the estimated amount of eluted sample.
The concentration of the binding protein was measured using a Biotek-Epoch-Take-3 using the sample, using the a280 method, i.e. with an extinction coefficient e.c. =1.37 (predicted from amino acid sequence), optical path=0.05 mm (slight differences in the optical path lengths of the different wells of the Take-3 plate, which will be automatically corrected), the absorbance of the sample was measured by the apparatus, and the concentration of the binding protein to be measured was calculated according to Lambert-Beer law. If the concentration of the sample is too low, ultrafiltration concentration is needed, and ultrafiltration concentration tube is usedUltra-15 Centrifugal Filter Devices,30kD) concentrating the sample to a concentration of > 0.5mg/ml according to the general procedure provided in the specification; collecting concentrated end sample, sterilizing and filtering with 0.22um sterile needle filter (Kebaite, PES,0.22um, diameter 13 mm), and packaging for freezing storage.
The results of titers and purities of humanized multivalent binding proteins against novel coronaviruses are shown in Table 2, which shows that the titers and purities of humanized multivalent binding proteins are all ideal.
TABLE 2 titre and purity results of humanized multivalent binding proteins against novel coronaviruses
EXAMPLE 3 neutralization Activity of humanized multivalent binding proteins against wild-type novel coronaviruses
The neutralization activity of the humanized multivalent binding protein prepared in example 2 on wild strain SARS-COV-2RBD protein is detected by using a competition method, and specific experimental steps are as follows:
coating conditions:
viral proteins: wild strain SARS-COV-2RBD protein (Ag 27), 2ug/ml;
coating liquid: 50mm pH 9.51CB;
coating volume: 100 ul/well;
coating temperature: 2-8 ℃;
coating time: 18 hours;
sealing liquid: 1% BSA+1×PBS;
sealing temperature: 37 ℃;
closing time: 3 hours;
sample adding: 50ul of the binding protein to be detected (initial concentration of all binding proteins is 5ug/mL, serial gradient dilution is carried out according to 5 times), incubation is carried out for 30min, washing is carried out for 5 times by using plate washing liquid (1 XPBS), 50ul of ACE2 protein is added, washing is carried out for 3 times by using plate washing liquid (1 XPBS), and color development detection is carried out.
The neutralization activity results of the humanized multivalent binding protein on the novel coronaviruses of the wild strain are shown in table 3, fig. 2 and fig. 3, and the results of table 3, fig. 2 and fig. 3 show that the humanized multivalent binding protein prepared in example 2 has the neutralization activity of the novel coronaviruses and can effectively block the combination of the SARS-COV-2RBD protein of the wild strain and the human ACE2 receptor protein; wherein, compared with the control group, R1406, R1407, R1475 and R1464 show remarkable neutralizing activity of the novel coronavirus of the wild strain.
TABLE 3 neutralization Activity results of humanized multivalent binding proteins on wild strain novel coronaviruses
EXAMPLE 4 neutralization Activity of humanized multivalent binding proteins on novel coronaviruses of Delta variant
The neutralization activity of the humanized multivalent binding protein prepared in example 2 on the Delta variant SARS-COV-2RBD protein was detected by competition method, and the virus proteins coated in the experiment were: the Delta variant SARS-COV-2RBD protein (Ag 101), 2ug/ml, was prepared as described in example 3.
The neutralization activity results of the humanized multivalent binding protein on the novel coronaviruses of the Delta variant strains are shown in Table 4, FIG. 4 and FIG. 5, and the results of Table 4, FIG. 4 and FIG. 5 show that the humanized multivalent binding protein prepared in example 2 can effectively block the binding between the SARS-COV-2RBD protein of the Delta variant strain and the human ACE2 receptor protein, and has the neutralization activity of the novel coronaviruses; wherein, compared with the control group, R1406, R1407, R1475 and R1464 show remarkable neutralizing activity of the novel coronavirus of the Delta variant strain.
TABLE 4 neutralization Activity results of humanized multivalent binding proteins on novel coronaviruses of Delta variant
Numbering device | Delta variant SARS-COV-2 IC50 (nM) |
R1382 | 0.01369 |
R1406 | 0.01476 |
R1407 | 0.01582 |
R1463 | 0.01805 |
R1475 | 0.003341 |
R1464 | 0.004316 |
EXAMPLE 5 neutralization Activity of humanized multivalent binding proteins against novel coronaviruses of Omacron variants
The neutralization activity of the humanized multivalent binding protein prepared in example 2 on the omacron variant SARS-COV-2RBD protein was examined by competition method, and the virus proteins coated in the experiment were: the Omicron variant SARS-COV-2RBD protein (covsK 20), 2ug/ml, was prepared as described in example 3.
The neutralization activity results of the humanized multivalent binding protein on the novel coronaviruses of the Omicron variant strain are shown in table 5, fig. 6 and fig. 7, and the results show that the humanized multivalent binding protein prepared in example 2 can effectively block the binding between SARS-COV-2RBD protein of the Omicron variant strain and human ACE2 receptor protein, and has the neutralization activity of the novel coronaviruses; wherein, compared with the control group, R1406, R1407, R1475 and R1464 show remarkable novel Omicron variant coronavirus neutralization activity.
TABLE 5 neutralization Activity results of humanized multivalent binding proteins on novel coronaviruses of Omacron variants
Numbering device | Omicron variant SARS-COV-2 IC50 (nM) |
R1382 | 0.17 |
R1406 | 0.1277 |
R1407 | 0.1591 |
R1463 | 11.1 |
R1475 | 0.07167 |
R1464 | 0.06166 |
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. An anti-novel coronavirus humanized multivalent binding protein comprising at least two epitope binding domains, said epitope binding domains being VHHs; the amino acid sequence of the VHH is shown as SEQ ID NO. 1 or SEQ ID NO. 2, or as an amino acid sequence with at least 95% similarity with SEQ ID NO. 1 or SEQ ID NO. 2;
alternatively, the process may be carried out in a single-stage,
the amino acid sequence of the VHH has at least 95% sequence similarity to SEQ ID NO. 1 and the VHH comprises complementarity determining regions CDR1-3 of SEQ ID NO. 1; or (b)
The amino acid sequence of the VHH has at least 95% sequence similarity to SEQ ID NO. 2, and the VHH comprises complementarity determining regions CDR1-3 of SEQ ID NO. 2.
2. The multivalent binding protein of claim 1, wherein the number of epitope binding domains is 2-6;
optionally, the epitope binding domains are linked by a linker peptide;
optionally, in the multivalent binding protein, the epitope binding domains are connected in series;
alternatively, the multivalent binding protein comprises 2 epitope binding domains, the 2 epitope binding domains being connected in series by a connecting peptide, or the multivalent binding protein comprises 3 epitope binding domains, the 3 epitope binding domains being connected in series by a connecting peptide:
optionally, the amino acid sequence of the connecting peptide is shown as SEQ ID NO. 11;
optionally, the C-terminus or N-terminus of the polypeptide chain of the multivalent binding protein is linked to a His tag; optionally, the amino acid sequence of the His tag is shown as SEQ ID NO. 10;
alternatively, the multivalent binding protein has an amino acid sequence as shown in SEQ ID NO. 8 or 9.
3. The multivalent binding protein of claim 1 or 2, wherein the multivalent binding protein further comprises a half-life extending domain; alternatively, the half-life extending domain is selected from an immunoglobulin Fc region or a serum albumin binding domain; alternatively, the immunoglobulin is selected from IgA, igD, igE, igG and IgM; optionally, the amino acid sequence of the Fc region is shown as SEQ ID NO. 3;
alternatively, the half-life extending domain is an immunoglobulin Fc region, the epitope binding domain being linked to the N-terminus or C-terminus of the Fc region;
alternatively, the multivalent binding protein forms a dimer structure through interchain disulfide bonds of the Fc region;
alternatively, the multivalent binding protein comprises 3 epitope binding domains, the 3 epitope binding domains are connected in series by a connecting peptide, and the N-terminus of the Fc region is connected to the C-terminus of the 3 rd epitope binding domain by a connecting peptide; alternatively, the multivalent binding protein comprises 3 epitope binding domains, the 3 epitope binding domains are connected in series by a connecting peptide, and the C-terminus of the Fc region is connected to the N-terminus of the 1 st epitope binding domain by a connecting peptide;
optionally, the amino acid sequence of the connecting peptide is shown as SEQ ID NO. 11;
alternatively, the multivalent binding protein has an amino acid sequence as shown in any one of SEQ ID NO. 6 or 7.
4. Fusion protein, characterized in that it comprises a multivalent binding protein according to any one of claims 1 to 3.
5. Conjugate, characterized in that it comprises a multivalent binding protein according to any one of claims 1 to 3.
6. Nucleic acid encoding a multivalent binding protein according to any one of claims 1 to 3, or encoding a fusion protein according to claim 4, or encoding a conjugate according to claim 5.
7. A recombinant vector carrying the nucleic acid of claim 6.
8. A host cell carrying the nucleic acid of claim 6 or comprising the recombinant vector of claim 7.
9. A pharmaceutical composition comprising the multivalent binding protein of any one of claims 1-3, the fusion protein of claim 4, the conjugate of claim 5, the nucleic acid of claim 6, the recombinant vector of claim 7, or the host cell of claim 8.
10. Use of a multivalent binding protein according to any one of claims 1 to 3, a fusion protein according to claim 4, a conjugate according to claim 5, a nucleic acid according to claim 6, a recombinant vector according to claim 7, a host cell according to claim 8 or a pharmaceutical composition according to claim 9 for the preparation of a medicament for the treatment and/or prophylaxis of novel coronaviruses.
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EP3944877A1 (en) * | 2020-07-29 | 2022-02-02 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Nanobodies sars-cov2 |
WO2022032660A1 (en) * | 2020-08-14 | 2022-02-17 | 武汉友微生物技术有限公司 | Novel coronavirus rbd fusion protein |
CN114106162A (en) * | 2020-08-28 | 2022-03-01 | 安源医药科技(上海)有限公司 | Novel coronavirus Spike protein antibody and application thereof |
CN114276443B (en) * | 2020-09-23 | 2023-05-16 | 深圳市因诺赛生物科技有限公司 | Novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof |
WO2022105772A1 (en) * | 2020-11-18 | 2022-05-27 | 三优生物医药(上海)有限公司 | Bispecific antibody having neutralizing activity against coronavirus, and use thereof |
CN112707968B (en) * | 2020-12-17 | 2023-10-20 | 苏州科锐迈德生物医药科技有限公司 | Recombinant receptor binding proteins, recombinant receptor proteins for detection of novel coronavirus neutralizing antibodies |
WO2022184027A1 (en) * | 2021-03-01 | 2022-09-09 | 中国科学院微生物研究所 | Novel coronavirus multivalent antigen, and preparation method therefor and application thereof |
CN113018427B (en) * | 2021-03-10 | 2023-09-19 | 江苏健安生物科技有限公司 | Multivalent fusion protein vaccine based on neutralizing epitope of novel coronavirus |
CN114349851B (en) * | 2021-12-22 | 2023-08-15 | 上海纳为生物技术有限公司 | New coronavirus neutralizing antibody and preparation method and application thereof |
CN114634556B (en) * | 2022-04-01 | 2023-12-19 | 中国科学院微生物研究所 | New coronavirus Delta and Omicron variant chimeric antigen, preparation method and application thereof |
CN117327174A (en) * | 2022-06-24 | 2024-01-02 | 广东菲鹏制药股份有限公司 | Humanized multivalent binding protein against novel coronaviruses and application thereof |
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2022
- 2022-06-24 CN CN202211668151.5A patent/CN117327174A/en active Pending
- 2022-06-24 CN CN202210724686.3A patent/CN115057938B/en active Active
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2023
- 2023-06-21 WO PCT/CN2023/101644 patent/WO2023246853A1/en unknown
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CN115057938B (en) | 2023-01-06 |
WO2023246853A1 (en) | 2023-12-28 |
CN115057938A (en) | 2022-09-16 |
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