CN113769080B

CN113769080B - Polypeptide immunoconjugates and uses thereof

Info

Publication number: CN113769080B
Application number: CN202111092938.7A
Authority: CN
Inventors: 刘刚; 李雪媛; 高军; 杨俊伟; 张伟
Original assignee: Liaoning Chengda Biotechnology Co ltd; Tsinghua University
Current assignee: Liaoning Chengda Biotechnology Co ltd; Tsinghua University
Priority date: 2021-09-17
Filing date: 2021-09-17
Publication date: 2023-04-07
Anticipated expiration: 2041-09-17
Also published as: CN113769080A

Abstract

The invention provides a polypeptide immunoconjugate and an application thereof, and the polypeptide immunoconjugate comprises: RBD of polypeptides and novel coronavirus S proteins; wherein the molar ratio of the polypeptide to the RBD of the novel coronavirus S protein is 15:1 to 1:1. the vaccine prepared by the polypeptide immunoconjugate has high neutralizing antibody to various novel coronaviruses, and can effectively prevent or treat the novel coronavirus pneumonia.

Description

Polypeptide immunoconjugates and uses thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a polypeptide-RBD (radial basis function) immunoconjugate and application thereof, and more particularly relates to the polypeptide immunoconjugate, application of the immunoconjugate, a pharmaceutical composition, a vaccine, a method for preparing the vaccine, a method for preparing an antibody and a method for stimulating generation of an anti-peptide antibody or an anti-RBD antibody in a subject.

Background

Coronavirus is a forward enveloped virus with RNA and has a genome size of about 26-32 kb. The genomic RNA and phosphorylated nucleocapsid (N) proteins are buried in a phospholipid bilayer and covered by a spike glycoprotein trimer (S), with membrane (M) proteins (type III transmembrane glycoproteins) and envelope (E) proteins located between the S proteins of the viral envelope. Coronaviruses are a class of viruses that are widely found in nature and cause multiple system diseases including respiratory, digestive and nervous systems.

The S protein of SARS-CoV-2 is located at the outermost layer of virus, regularly arranged in corona structure on the membrane, and is involved in the process of binding virus with virus receptor on the surface of host cell and mediating virus to enter cell through membrane fusion, and plays an important role in inducing host to produce neutralizing antibody. The development of the COVID-19 vaccine currently uses the full S protein or partial S protein (receptor binding domain (RBD) in S protein) as the target antigen. Research reports that the S protein of SARS-CoV of coronaviruses of the same genus may induce antibody dependent infection enhancement reaction (ADE), the molecular weight of RBD is low, the immunogenicity using RBD monomer as antigen is poor, and part of SARS-CoV-2 mutant strains have RBD amino acid single site and multi site mutation, therefore, how to improve the immunogenicity of RBD needs further exploration.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

research reports that the molecular weight of the novel coronavirus RBD is low, and the immunogenicity taking the RBD monomer as an antigen is poor, and through a large number of experimental researches, the inventor unexpectedly obtains a polypeptide immunoconjugate for preventing the novel coronavirus pneumonia COVID-19; the polypeptide immunoconjugate is added with an adjuvant to prepare a vaccine, so that the immunogenicity of the polypeptide can be improved, the immunogenicity of the RBD protein can be improved, and the broad spectrum of the immune effect of the immunoconjugate can be improved.

To this end, in a first aspect of the invention, the invention provides a polypeptide immunoconjugate. According to an embodiment of the invention, comprising: RBD of polypeptides and novel coronavirus S proteins; wherein the molar ratio of the polypeptide to the RBD of the novel coronavirus S protein is 1:1 to 15:1. the inventor creatively discovers that the polypeptide immunoconjugate obtained by combining the polypeptide and the RBD of the novel coronavirus S protein can remarkably improve the content of the anti-peptide antibody or the anti-RBD antibody when the molar ratio of the polypeptide to the RBD of the novel coronavirus S protein is 1:1 to 1:30 hours, the polypeptide immunoconjugate produced a higher level of neutralizing antibodies.

According to embodiments of the invention, the polypeptide immunoconjugate may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the polypeptide is a novel coronavirus S protein epitope.

According to an embodiment of the invention, the polypeptide has the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75% identity to SEQ ID No. 1.

CFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLN-NH2(SEQ ID NO:1)。

According to an embodiment of the invention, the RBD of the novel coronavirus S protein has the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75% identity to SEQ ID No. 2.

MLRGLCCVLL LCGAVFVSPS QEIHARFRRG ARGRVQPTES IVRFPNITNL CPFGEVFNAT RFASVYAWNR KRISNCVADY SVLYNSASFS TFKCYGVSPT KLNDLCFTNV YADSFVIRGD EVRQIAPGQT GKIADYNYKL PDDFTGCVIA WNSNNLDSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPCNGVEG FNCYFPLQSY GFQPTNGVGY QPYRVVVLSF ELLHAPATVC GPKKSTNLVK NKCVNFWSHP QFEKDYKDDD DK(SEQ ID NO：2)。

According to an embodiment of the invention, said polypeptide is linked to said RBD by a linker.

According to an embodiment of the invention, the linker is sulfosuccinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate.

In a second aspect, the invention provides the use of a polypeptide immunoconjugate of the first aspect in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the prevention or treatment of a novel coronavirus pneumonia.

In a third aspect of the invention, the invention provides the use of a polypeptide immunoconjugate of the first aspect in the preparation of a vaccine. According to an embodiment of the invention, the vaccine is for the prevention or treatment of novel coronavirus pneumonia. The vaccine provided by the invention has high neutralizing antibody to various novel coronaviruses, and can effectively prevent or treat the novel coronavirus pneumonia.

In a fourth aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, there is included a polypeptide immunoconjugate according to the first aspect. The pharmaceutical composition provided by the embodiment of the invention can obviously improve the content of the anti-peptide antibody or the anti-RBD antibody, and effectively prevent or treat the novel coronavirus pneumonia.

In a fifth aspect of the invention, a vaccine is presented. According to an embodiment of the invention, there is provided a polypeptide immunoconjugate according to the first aspect; and a pharmaceutically acceptable adjuvant. The vaccine provided by the embodiment of the invention has the polypeptide immunoconjugate, and can remarkably improve the content of anti-peptide antibodies or anti-RBD antibodies, so that the vaccine obtained by combining the polypeptide immunoconjugate and the adjuvant has the characteristic of being capable of generating higher anti-peptide and anti-RBD antibodies in a human body stimulated by various novel coronaviruses, and meanwhile, due to the addition of the adjuvant, the immunogenicity of the polypeptide and the RBD to the body is further improved, so that the titer of the antibodies is improved, and the effect of effectively preventing or treating the novel coronaviruses is achieved.

According to an embodiment of the invention, the vaccine may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the adjuvant comprises at least one of aluminium hydroxide, aluminium phosphate, monophosphoryl lipid, oligonucleotide CpG. The vaccine obtained by combining the adjuvant and the polypeptide immunoconjugate according to the embodiment of the invention has significantly improved titer of anti-peptide antibody and anti-RBD antibody compared with vaccines prepared by using other adjuvants, and has better effect of preventing or treating novel coronaviruses.

According to an embodiment of the invention, the adjuvant is aluminium hydroxide.

According to an embodiment of the invention, the adjuvant comprises 3D-mLA at a final concentration of 100. Mu.g/L, and aluminium hydroxide at a final concentration of 1 mg/L. The vaccine obtained by combining the adjuvant and the polypeptide immunoconjugate provided by the embodiment of the invention has significantly improved titer of anti-peptide antibody and anti-RBD antibody compared with vaccines prepared by using other adjuvants, and has better effect of preventing or treating novel coronaviruses.

According to an embodiment of the invention the amount of adjuvant in said vaccine is 1 μ g to 1000 μ g per dose, preferably 10 μ g to 500 μ g, more preferably 10 to 100 μ g, most preferably 10 to 50 μ g per dose. According to the adjuvant additive amount of the embodiment of the invention, the prepared vaccine has better effect of preventing or treating the novel coronavirus.

According to an embodiment of the invention, the vaccine is in any pharmaceutically acceptable dosage form.

According to an embodiment of the present invention, the vaccine immunization mode is at least one of intramuscular injection, subcutaneous injection, intradermal injection and microneedle injection.

In a sixth aspect of the invention, there is provided a method of making a vaccine according to the fifth aspect. According to an embodiment of the invention, the method comprises the following steps: the polypeptide immunoconjugate of the first aspect is mixed with an adjuvant. The vaccine provided by the embodiment of the invention has the polypeptide immunoconjugate, and can remarkably improve the content of anti-peptide antibodies or anti-RBD antibodies, so that the vaccine obtained by combining the polypeptide immunoconjugate and the adjuvant has the characteristic of generating higher anti-peptide and anti-RBD antibodies in a human body stimulated by various novel coronaviruses, and meanwhile, due to the addition of the adjuvant, the immunogenicity of the polypeptide and the RBD to the body is further improved, so that the titer of the antibodies is improved, and the effect of effectively preventing or treating the novel coronaviruses is achieved.

According to an embodiment of the invention, the adjuvant comprises at least one of aluminium hydroxide, aluminium phosphate, monophosphoryl lipid, oligonucleotide CpG.

According to an embodiment of the present invention, 3D-mLA is included in the adjuvant at a final concentration of 100. Mu.g/L, and aluminum hydroxide is included at a final concentration of 1 mg/L.

In a seventh aspect of the invention, the invention features a method of making an antibody. According to an embodiment of the invention, comprising: immunizing an animal with a polypeptide immunoconjugate of the first aspect; collecting serum of the immunized animal; and purifying the antibody of interest from the serum. The method for preparing the antibody provided by the embodiment of the invention is simple and convenient to operate, and the antibody can specifically recognize the polypeptide immunoconjugate.

In an eighth aspect of the invention, a method of stimulating the production of anti-peptide antibodies or anti-RBD antibodies in a subject is provided. According to the embodiment of the invention, the method is realized by at least one of the following ways: 1) Administering to a subject an immunoconjugate comprising a polypeptide of the first aspect; 2) Administering to the subject the pharmaceutical composition of the fourth aspect; and 3) administering the vaccine of the fifth aspect to the subject.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a SDS-PAGE gel of polypeptides (7 #, S2#, 45#, and 46 #) according to an embodiment of the present invention coupled to RBD domains of novel coronavirus S proteins, respectively, wherein RBD represents: only the set of RBD domains in the S protein of the novel coronavirus; RBD-SMCC denotes: reacting RBD protein with sulfosuccinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate to obtain a product group; RBD-7#, RBD-S2#, RBD-45#, and RBD-46# represent RBD-polypeptide group;

FIG. 2 is a photograph of SDS-PAGE electrophoresis gel obtained after polypeptides (7 # and S2 #) according to an embodiment of the present invention are coupled to the RBD domain of the S protein of the novel coronavirus, respectively, wherein the molar ratio of the 7# polypeptide to the RBD domain of the S protein of the novel coronavirus is 10: the molar ratio of 1,S2 # polypeptide to the RBD structural domain of the novel coronavirus S protein is 10:1;

FIG. 3 is a SDS-PAGE gel of polypeptides (45 # and 46 #) coupled to the RBD domain of the novel coronavirus S protein, respectively, according to an embodiment of the present invention, wherein the molar ratio of the 45# polypeptide to the RBD domain of the novel coronavirus S protein is 10: the molar ratio of the 1,46 # polypeptide to the RBD domain of the novel coronavirus S protein was 10:1; and

FIG. 4 is a graph showing the results of detecting the level of neutralizing antibodies of polypeptide immunoconjugates of the RBD-HA204 adjuvant group and the RBD-45# + HA204 adjuvant group against the novel coronavirus of the south Africa strain according to the embodiment of the invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Interpretation of terms

As used herein, the terms "novel coronavirus", "COVID-19", and the like, all refer to the causative agent of the novel coronavirus pneumonia.

Identity, the present invention, in order to compare two or more nucleotide sequences, the percentage of "sequence identity" between a first sequence and a second sequence can be calculated by dividing [ the number of nucleotides in the first sequence that are identical to the nucleotide at the corresponding position ]. Nucleotide in the second sequence ] minus [ the total number of nucleotides in the first sequence ], and then multiplied by [100% ], wherein the deletion, insertion, substitution, or addition of each nucleotide in the second nucleotide sequence-relative to the first nucleotide sequence-is considered to be a difference in a single nucleotide (position).

Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings using known computer algorithms for sequence alignment, such as NCBI Blast v 2.0.

Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768-A.

Identity, the present invention, for comparing two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence can be determined by dividing the number of amino acid residues in the first amino acid sequence by the same as the [ amino acid residue at the corresponding position in the second amino acid sequence ], to be [ total number of nucleotides in the first amino acid sequence ], and then multiplying by [100% ], wherein each deletion, insertion, substitution or addition of a "residue" of an amino acid residue in the second amino acid sequence is considered a difference in a single amino acid residue (position), i.e., an "amino acid difference" as defined herein, as compared to the first amino acid sequence.

Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as the algorithms described above for determining the degree of sequence identity of nucleotide sequences, again using standard settings.

Typically, to determine the percentage of "sequence identity" between two amino acid sequences according to the calculation methods outlined above, the amino acid sequence with the largest number of amino acid residues will be the "first" amino acid sequence and the other amino acid sequence will be the "second" amino acid sequence.

Likewise, in determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called "conservative" amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced. Another amino acid residue having a similar chemical structure, which has little or no effect on the function, activity or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example, from WO 04/037999, GB-A-2357768, WO 98/49185, WO 00/46383, and WO01/09300; and WO 01/09300. The type and/or combination of such substitutions may be selected and/or (preferably) selected based on the relevant teachings from WO 04/037999 and WO 98/49185, as well as from other references cited therein.

The invention provides a polypeptide immunoconjugate, comprising: RBD of polypeptides and novel coronavirus S proteins; wherein the molar ratio of the polypeptide to the RBD of the novel coronavirus S protein is 1:1 to 15:1. the inventor creatively discovers that the polypeptide immunoconjugate obtained by combining the polypeptide and the RBD of the novel coronavirus S protein can remarkably improve the content of the anti-peptide antibody or the anti-RBD antibody when the molar ratio of the polypeptide to the RBD of the novel coronavirus S protein is 1:1 to 15:1, the polypeptide immunoconjugate produces a higher level of neutralizing antibodies.

According to a particular embodiment of the invention, the polypeptide is a novel coronavirus S protein epitope. The polypeptide is not particularly limited, and any novel coronavirus S protein epitope can be used.

According to a particular embodiment of the invention, the polypeptide has the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75% identity to SEQ ID No. 1.

CFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLN-NH2(SEQ ID NO:1)。

According to a particular embodiment of the invention, the RBD of the novel coronavirus S protein has the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75% identity to SEQ ID No. 2.

According to a particular embodiment of the invention, said polypeptide is linked to said RBD by a linker.

According to a particular embodiment of the invention, the linker is sulfosuccinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate.

The invention provides the application of the polypeptide immunoconjugate in the preparation of medicines. According to a particular embodiment of the invention, the medicament is for the prevention or treatment of novel coronavirus pneumonia.

The invention provides the application of the polypeptide immunoconjugate in the preparation of vaccines. According to a particular embodiment of the invention, the vaccine is for the prevention or treatment of novel coronavirus pneumonia.

The invention provides a pharmaceutical composition comprising the polypeptide immunoconjugate described above. The pharmaceutical composition may comprise: pharmaceutically acceptable adjuvants including at least one of stabilizers, wetting agents, emulsifiers, binders, isotonizing agents; the pharmaceutical composition is at least one of tablets, granules, powder, capsules, solutions, suspensions and freeze-dried preparations. The pharmaceutical composition is effective in preventing or treating novel coronavirus pneumonia.

The invention provides a vaccine, which comprises the polypeptide immunoconjugate; and a pharmaceutically acceptable adjuvant. The vaccine according to the specific embodiment of the invention comprises the polypeptide immunoconjugate, and the content of an anti-peptide antibody or an anti-RBD antibody can be remarkably improved, so that the vaccine obtained by combining the polypeptide immunoconjugate and the adjuvant has the characteristic of being capable of generating higher anti-peptide and anti-RBD antibodies in a human body stimulated by various novel coronaviruses, and meanwhile, due to the addition of the adjuvant, the immunogenicity of the polypeptide and the RBD to the body is further improved, so that the titer of the antibodies is improved, and the effect of effectively preventing or treating the novel coronavirus pneumonia is achieved.

According to a particular embodiment of the invention, the adjuvant comprises at least one of aluminium hydroxide, aluminium phosphate, monophosphoryl lipids, oligonucleotide CpG.

According to a particular embodiment of the invention, the adjuvant is aluminium hydroxide.

According to a specific embodiment of the invention, the adjuvant comprises 3D-mLA at a final concentration of 100. Mu.g/L and aluminum hydroxide at a final concentration of 1 mg/L.

According to a particular embodiment of the invention, the adjuvant is present in the vaccine in an amount of 1. Mu.g to 1000. Mu.g, preferably 10. Mu.g to 500. Mu.g, more preferably 10 to 100. Mu.g, most preferably 10 to 50. Mu.g per dose.

According to a particular embodiment of the invention, the vaccine is in any pharmaceutically acceptable dosage form.

According to a specific embodiment of the present invention, the vaccine immunization mode is at least one of intramuscular injection, subcutaneous injection, intradermal injection and microneedle injection.

The invention provides a method for preparing the vaccine, which comprises the following steps: the polypeptide immunoconjugate described above is mixed with an adjuvant.

According to a specific embodiment of the invention, the adjuvant comprises at least one of aluminium hydroxide, aluminium phosphate, monophosphoryl lipid, oligonucleotide CpG.

According to a specific embodiment of the invention, the adjuvant comprises 3D-mLA in a final concentration of 100. Mu.g/L and aluminum hydroxide in a final concentration of 1 mg/L.

The invention provides a method for preparing an antibody, which comprises the following steps: immunizing an animal with a polypeptide immunoconjugate as described above; collecting serum of the immunized animal; and purifying the antibody of interest from the serum.

The present invention provides a method of stimulating the production of anti-peptide antibodies or anti-RBD antibodies in a subject by at least one of: 1) Administering to the subject an immunoconjugate as described above; 2) Administering to the subject the aforementioned pharmaceutical composition; and 3) administering the aforementioned vaccine to the subject.

The term "administering" as used herein refers to introducing a predetermined amount of a substance into a patient by some suitable means. The polypeptide immunoconjugate, pharmaceutical composition or vaccine of the present embodiment may be administered by any common route, as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal, but the invention is not limited to these exemplified modes of administration. However, because of oral administration, the active ingredients of orally administered compositions should be coated or formulated to prevent degradation in the stomach. Preferably, the composition of the present invention can be administered in an injectable formulation. In addition, the pharmaceutical compositions of the present invention may be administered using a specific device that delivers the active ingredient to the target cells.

The administration frequency and dose of the polypeptide immunoconjugate, pharmaceutical composition or vaccine of the present embodiment may be determined by a number of relevant factors, including the type of disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient, and the type of drug as an active ingredient. According to some embodiments of the invention, the daily dose may be divided into 1, 2 or more doses in a suitable form, to be administered 1, 2 or more times over the entire period, as long as a therapeutically effective amount is achieved.

The term "therapeutically effective amount" refers to an amount sufficient to significantly ameliorate some of the symptoms associated with a disease or condition, i.e., to provide a therapeutic effect for a given condition and dosing regimen. The term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. As used herein, "treatment" encompasses administration of the polypeptide immunoconjugates, pharmaceutical compositions or vaccines of the embodiments of the invention to an individual for treatment, including but not limited to administration to an individual in need thereof containing the teachings herein.

It should be noted that the polypeptide immunoconjugates, the pharmaceutical compositions, the vaccines, the methods for preparing antibodies, and the methods for stimulating the production of anti-peptide antibodies or anti-RBD antibodies in a subject according to the embodiments of the present invention are discovered and completed by the inventors of the present application through hard inventive work and optimization work.

The embodiments will be described in detail below. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 Synthesis of polypeptide of interest

In this example, polypeptides 7#, 45#, 46# and S2 were prepared by conventional solid phase synthesis of polypeptides, the amino acid sequences of which are shown in Table 1.

Table 1:

note: the C end of the amino acid sequences of the synthesized No. 7 peptide, the synthesized No. 45 peptide and the synthesized No. 46 peptide is-CONH ₂ Form or COOH form; n terminal is NH ₂ Or CH ₃ CONH-form.

The specific experimental procedures for preparing the polypeptide are as follows:

(1) DeFmoc protection

A commercially available Rink Amide AM resin was charged into a reaction tube having a filter and resistant to an organic solvent, and was closed with an organic solvent-resistant cap. After washing with DMF (dimethylformamide) for 1 min, an excess of 20% piperidine/DMF (volumetric ratio) solution was added, the reaction tube was gently shaken after the cover was closed, mixed well and kept free of Fmoc protection for 15 min, and then washed three times with DMF after draining.

(2) Peptide bond condensation

Adding 3 times of resin amino molar weight of Fmoc protected amino acid, 3 times of resin amino molar weight of activating agent HBTU and 6 times of resin amino molar weight of DIPEA (N, N-diisopropylethylamine) which are subjected to preactivation (after 2-3 minutes of preactivation) into the reaction tube obtained in the step (1), using DMF as a solvent, ensuring that the reagents are completely dissolved, and completely covering the resin. The resin was mixed by shaking every 2-3 minutes to allow reaction for 20-30 minutes.

After the reaction was completed, a 50-fold molar excess of acetic anhydride in DMF was added to the reaction tube at room temperature, then the mixture was drained after 10 minutes, and an excess of 20% piperidine/DMF (volume ratio) solution was added thereto at room temperature to react for 30 minutes. The solution was filtered off and the resin was washed 5 times with DMF.

The above steps are repeated in cycles until all the Fmoc-protected amino acids fitted to the polypeptide are attached to the resin in a linear fashion.

(3) Polypeptide cleavage

The polypeptide lysate is treated with a strong acid, such as TFA (trifluoroacetic acid). The resin from step (2) was treated with TFA, water, EDT dimercaptoethanol, phenol (volume ratio: 92.5. Then collecting the lysate carefully into a glass collector, adding ether precooled with ice, collecting the precipitated polypeptide, and continuing to wash with cold ether for 5-6 times to obtain crude peptide.

(4) And (5) polypeptide purification.

The crude peptide is purified by HPLC (high performance liquid chromatography), collected, lyophilized, and finally tested by HPLC for purity (214 nm wavelength) of greater than 85% and mass spectrometry for correct molecular weight.

After the above steps, polypeptides 7#, 45#, 46# and S2 can be obtained, wherein the amino acid sequence shown in SEQ ID NO: the purity of the synthetic peptide shown in 1 (Table 1) was 95%.

EXAMPLE 2 preparation of immunogenic conjugates

In this example, the synthetic peptide prepared in example 1 was conjugated to RBD protein via a linker, and the following experimental procedures were performed:

preparation of RBD protein: the RBD plasmid was transfected into HEK293F cells, which were cultured to a logarithmic growth phase and cell supernatants collected. After concentration, the protein was purified by Anti-flag G1 Affinity resin. And purifying the crude protein by using a molecular sieve to obtain the RBD protein.

The RBD protein was formulated in PBS to a concentration of 1 mg/mL. Taking a required amount of RBD solution, and mixing the RBD solution according to a molar ratio of 1:2-1:40 sulfol-SMCC (10 mg/mL) was added thereto, the reaction mixture was reacted at room temperature for 1 hour, and the reaction mixture was concentrated by centrifugation at 3500rpm at 4 ℃ using an ultrafiltration concentration tube. After the volume is concentrated to 1/10 of the reaction solution, PBS is added to the volume of the original reaction solution, centrifugal concentration is continued, and centrifugal concentration is continuously carried out for 5 times to remove free Sulfo-SMCC. The final RBD-SMCC stock solution concentration was 10mg/mL. The desired amount of the synthetic peptide prepared in example 1 was weighed, dissolved in PBS or a PBS solution containing 10% dmso, and the inventors optimized the addition ratio of the polypeptide to RBD protein, in a molar ratio of 1:1-15:1 adding an RBD-SMCC stock solution, wherein the ratio of polypeptide to RBD addition is as shown in table 1; PBS is then added to the appropriate reaction volume, and the resulting mixture is reacted at room temperature for 1h, and checked for conjugation by SDS-PAGE, wherein the loading of protein is 2 μ g, the electrophoretogram of SDS-PAGE is shown in FIG. 1, the molar ratio of 7# peptide to protein is 10, the molar ratio of 45# peptide to protein is 7.5, the molar ratio of 46# peptide to protein is 7, and the molar ratio of S2# peptide to protein is 6. In fig. 2, the molar ratio of peptide # 7 to protein is 10, and the molar ratio of peptide # S2 to protein is 10. In fig. 3, the molar ratio of 45# peptide to protein is 10, and the molar ratio of 46# peptide to protein is 10. According to the experimental results shown in fig. 1-3, when the molar ratio of 7# peptide to protein is 10, the molar ratio of 45# peptide to protein is 7.5.

Table 2:

item	Molar ratio of 1	Molar ratio of 2
			7#-RBD	-	10：1
45#-RBD	7.5：1	10：1
			46#-RBD	7：1	10：1
S2#-RBD	6：1	10：1

Example 3 preparation of vaccine and detection of immune Effect

1. And (4) preparing the vaccine.

(1) Experimental materials

An immunoconjugate: a conjugate of RBD protein and polypeptide shown in SEQ ID NO. 1;

adjuvant: HA204[ 100. Mu.g of 3D-MLA, 1mg of aluminum hydroxide (calculated as aluminum ion) per mL ];

(2) Experiment grouping

The experimental group of this example is shown in table 3, wherein the molar ratio of 7# peptide to RBD is 10, the molar ratio of 45# peptide to RBD is 7.5, the molar ratio of 46# peptide to RBD is 7, the molar ratio of S2# peptide to RBD is 6.

Table 3:

(3) Procedure for the preparation of the

The immunogenic conjugate prepared in example 2 was mixed with the adjuvant HA204 and PBS solution, and shaken at room temperature and 30RPM for 1 hour to obtain final samples with a final concentration of polypeptide-containing RBD protein antigen of 100 μ g/mL and adjuvant HA 204.5% (v/v).

2. Experiment of immune Effect

Grouping of immune effect experiments is shown in table 3, the immunogenicity of mice was studied using the samples to be tested prepared in table 3, healthy BALB/c mice weighing 16-18g were randomly grouped into 6 mice each; using samples to be tested of each experimental group (RBD, RBD + HA204, RBD-7# + HA204, RBD-45# + HA204, RBD-46# + HA204, RBD-S2+ HA 204) in the table 3 to carry out intraperitoneal injection on mice, wherein the intraperitoneal injection is carried out 1 time on each day 0, 14 and 21, and the administration volume is 200 mu L/mouse; injecting normal saline into the abdominal cavity of mice in Control group, injecting 1 time into the abdominal cavity on the 0 th day, the 14 th day and the 21 st day, wherein the administration volume is 100 mu L/mouse, collecting blood of all mice 1 time respectively before injection on the 0 th day, the 21 st day and the 28 th day, separating serum, diluting the serum 20 times, and measuring the serum antibody level of each group of mice on the 21 st day and the 28 th day by adopting an enzyme linked immunosorbent assay (ELISA) method, wherein the specific operation is as follows:

(1) And (3) diluting the RBD solution into working solution with the final concentration of 1 mu g/mL by using coating solution (0.05M carbonate-bicarbonate buffer solution), fully and uniformly mixing, adding an enzyme label plate according to 100 mu L/hole, attaching sealing paper, and coating at 2-8 ℃ overnight.

(2) The supernatant from the wells was aspirated, washed 5 times with PBST, and then blocked for 1h at room temperature by adding 100. Mu.L of 1% BSA-containing PBS.

(3) After blocking, wash 5 times with PBST.

(4) Sample adding: after washing in step (3), 100. Mu.L of diluted serum sample was added to each well, incubated at room temperature for 2h, and washed 5 times with PBST.

(5) Adding an enzyme-labeled antibody: and (4) adding 100 mu L of diluted goat anti-mouse IgG HRP enzyme-labeled secondary antibody into each well washed in the step (4), and incubating for 1h at room temperature.

(6) After incubation was complete, washed 5 times with PBST.

(7) Adding a substrate solution for color development: after washing in step (6), 100. Mu.L of TMB was added to each well to develop color.

(8) And (3) terminating the reaction: and (4) adding 50 mu L of 1N sulfuric acid into each hole after the substrate color developing solution is added in the step (7) to terminate the reaction.

Determination of OD of serum antibodies using microplate reader ₄₅₀ Values, anti-RBD antibody levels results after combination of the polypeptide conjugate with different adjuvants are shown in table 4, and the results show that peptide No. 45 conjugated with RBD significantly increased anti-RBD antibody titers.

Table 4:

3. pseudovirus neutralization assay

Based on the above results, the inventors compared the level of neutralizing antibodies against south African pseudovirus strain produced by the serum of mice D21 and D28 in RBD-HA204 group and RBD-45# + HA204 group, and the following experimental procedures were performed:

(1) Mouse serum obtained in experiment 2 of this example was inactivated in 56 ℃ water bath for 30min, diluted in 96-well cell culture plates in duplicate with DMEM complete medium;

(2) Adding pseudovirus (650 TCID) to serum ₅₀ Perwell), cell culture incubator (37 ℃,5% ₂ ) Incubating for 1 hour;

(3) Add 100. Mu.L of Huh7 cells per well in 96-well plates to make cells per well 2X 10 ⁴ A plurality of;

(4) Placing the 96-well plate in a cell incubator, incubating at 37 ℃ for 20 to 28 hours with 5% of CO2;

(5) Taking out the 96-well plate from the cell incubator, absorbing 150 mu L of supernatant from each well, then adding 100 mu L of luciferase detection reagent, and reacting for 2min at room temperature in a dark place;

(6) After the reaction is finished, repeatedly blowing and sucking the liquid in the reaction holes for 6-8 times to fully crack the cells, sucking 150 mu L of liquid from each hole, adding the liquid into a corresponding 96-hole chemiluminescence detection plate, and placing the plate in a chemiluminescence detector to read a luminescence value;

the neutralizing antibody level of the pseudoviruses generated by the RBD-HA204 group and the RBD-45# + HA204 group is shown in figure 4, wherein after the peptide No. 45 is conjugated with the RBD, the neutralizing antibody level of the RBD group at 21 days is obviously higher than that of the RBD group, and the neutralizing antibody level of the RBD-45# + HA204 group at 28 days is higher than that of the RBD-HA204 group, but the difference is not obvious, and the two groups both have good immune effect.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Liaoning Dabie shares GmbH of Qinghua university

<120> polypeptide immunoconjugate and use thereof

<130> BI3211207

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<223> 1

<400> 1

Cys Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1 5 10 15

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

20 25 30

Asn Glu Val Ala Lys Asn Leu Asn

35 40

<210> 2

<211> 272

<212> PRT

<213> Artificial Sequence

<220>

<223> 2

<400> 2

Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val Phe

1 5 10 15

Val Ser Pro Ser Gln Glu Ile His Ala Arg Phe Arg Arg Gly Ala Arg

20 25 30

Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr

35 40 45

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser

50 55 60

Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr

65 70 75 80

Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly

85 90 95

Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala

100 105 110

Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly

115 120 125

Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

130 135 140

Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val

145 150 155 160

Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu

165 170 175

Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser

180 185 190

Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln

195 200 205

Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg

210 215 220

Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys

225 230 235 240

Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

245 250 255

Trp Ser His Pro Gln Phe Glu Lys Asp Tyr Lys Asp Asp Asp Asp Lys

260 265 270

<210> 3

<211> 29

<212> PRT

<213> Artificial Sequence

<220>

<223> 3

<400> 3

Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala

1 5 10 15

Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly

20 25

<210> 4

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> 4

<400> 4

Cys Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu

1 5 10 15

Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile

20 25 30

Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu

35 40

<210> 5

<211> 47

<212> PRT

<213> Artificial Sequence

<220>

<223> 5

<400> 5

Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp

1 5 10 15

Ile Ala Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr Ala Ile

20 25 30

His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr Gly

35 40 45

Claims

1. A polypeptide immunoconjugate, consisting of a polypeptide and an RBD of a novel coronavirus S protein;

wherein the molar ratio of the polypeptide to the RBD of the novel coronavirus S protein is 7.5:1;

the amino acid sequence of the polypeptide is shown as SEQ ID NO:1 is shown in the specification;

the RBD of the novel coronavirus S protein has the sequence shown in SEQ ID NO: 2;

the polypeptide is connected with the RBD through sulfosuccinimide 4- (N-maleimide methyl) cyclohexane-1-carboxylate.

2. The polypeptide immunoconjugate of claim 1, wherein the polypeptide is a novel coronavirus S protein epitope.

3. Use of the polypeptide immunoconjugate of any one of claims 1 to 2 for the preparation of a medicament for the prevention or treatment of novel coronavirus pneumonia.

4. Use of the polypeptide immunoconjugate of any one of claims 1 to 2 for the preparation of a vaccine for the prevention or treatment of novel coronavirus pneumonia.

5. A pharmaceutical composition comprising the polypeptide immunoconjugate of any one of claims 1 to 2.

6. A vaccine comprising the polypeptide immunoconjugate of any one of claims 1 to 2; and

a pharmaceutically acceptable adjuvant.

7. The vaccine of claim 6, wherein the adjuvant comprises at least one of aluminum hydroxide, aluminum phosphate, monophosphoryl lipid, oligonucleotide CpG.

8. The vaccine of claim 7, wherein the adjuvant is aluminum hydroxide.

9. The vaccine according to claim 7, characterized in that 3D-MLA is included in the adjuvant at a final concentration of 100 μ g/L, and aluminium hydroxide is included at a final concentration of 1 mg/L.

10. The vaccine according to any one of claims 6 to 9, wherein the adjuvant is contained in an amount of 1 μ g to 1000 μ g per dose of the vaccine.

11. The vaccine according to claim 10, wherein the adjuvant is present in an amount of 10 μ g to 500 μ g per dose of the vaccine.

12. The vaccine according to claim 10, wherein the adjuvant is present in an amount of 10 to 100 μ g per dose of the vaccine.

13. The vaccine according to claim 10, wherein the adjuvant is present in an amount of 10 to 50 μ g per dose of the vaccine.

14. The vaccine of claim 6, wherein the vaccine is in any pharmaceutically acceptable dosage form.

15. The vaccine of claim 6, wherein the vaccine immunization is at least one of intramuscular injection, subcutaneous injection, intradermal injection, and microneedle injection.

16. A method for preparing the vaccine of claims 6-15, comprising the steps of: mixing the polypeptide immunoconjugate of any one of claims 1 to 2 with an adjuvant.

17. The method of claim 16, wherein the adjuvant comprises at least one of aluminum hydroxide, aluminum phosphate, monophosphoryl lipid, oligonucleotide CpG.

18. The method of claim 17, wherein the adjuvant is aluminum hydroxide.

19. The method of claim 16, wherein the adjuvant comprises 3D-MLA at a final concentration of 100 μ g/L, and aluminum hydroxide at a final concentration of 1 mg/L.

20. A method of producing an antibody, comprising:

immunizing an animal with the polypeptide immunoconjugate of any one of claims 1-2;

collecting serum of the immunized animal; and

the antibody of interest is purified from the serum.