WO2023184280A1 - Anti-sars-cov-2 nanobody and use thereof - Google Patents

Abstract

Provided are an anti-SARS-Cov-2 nanobody and use thereof. The nanobody comprises CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 2, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 3. Provided is a fusion protein, which is formed by the conjugation of the nanobody and an Fc, or by the conjugation of two or more nanobodies. The use refers to use of the nanobody or the fusion protein in the preparation of a drug for treating SARS-Cov-2. The nanobody has the potential to neutralize the Omicron strain, and has the advantages of small molecular weight, high binding activity, stable physical and chemical properties, ease of mass production, transport, storage, and the like. The nanobody can bind to RBDO and almost completely inhibits the binding of RBDO to ACE2, providing the foundations for the design and assembly of bispecific or even multispecific antibodies and CAR molecules.

Description

An anti-coronavirus nanobody and its application Technical field

The invention belongs to the field of biomedicine and relates to an anti-coronavirus nanobody and its application.

Background technique

The novel coronavirus (SARS-Cov-2) is a new beta-coronavirus that mainly uses angiotensin-converting enzyme 2 (ACE2) as a receptor to invade the human body and cause lung damage, which may then cause severe systemic inflammatory reactions. , acute respiratory syndrome and shock may occur, and even multiple organ failure may occur, leading to death. As of February 22, 2022, WHO reported that more than 400 million people had been infected with the new coronavirus (SARS-Cov-2), and the death toll was as high as 5.8 million, posing a huge threat to global economic development and public safety. Through the joint efforts of the whole society, several vaccines and therapeutic antibodies have been successfully developed, achieving effective response to a certain extent. However, given the uncertainty of the spread of the new coronavirus and the emergence of mutant strains, it still poses a serious threat to human health. Especially the recently emerged Omicron mutant strains, although their toxicity has been reduced, they have serious immune evasion, spread more quickly, and cause serious sequelae. As the epidemic develops, further substrains of Omicron (eg. BA.2) have emerged. Studies have shown that it has higher infectivity and virulence, and because it is difficult to detect with conventional PCR technology, it is called "stealth". "Stealth variant strain" of the new coronavirus. Therefore, there is an urgent need to develop neutralizing antibodies against Omicron mutant strains and their substrains.

Neutralizing antibodies are an important weapon in dealing with the new coronavirus at this stage. It has important applications at all stages of COVID-19 infection, including prevention and treatment. Traditional antibodies have large molecular weights, insufficient stability, and high production and transportation costs, all of which limit their application. Nanobodies are small molecule antibodies developed in recent years. While retaining the high affinity of traditional antibodies, they have the advantages of high stability, small molecular weight, easy production and storage, and low cost. They are another important option to deal with the new coronavirus. .

Contents of the invention

In order to solve the problem of lack of antibodies against the new coronavirus in the prior art, the present invention provides an anti-new coronavirus nanobody and its application. The present invention provides a variety of nanobodies with high affinity to the receptor binding domain (RBDO) of the Spike protein of the new coronavirus Omicron mutant strain (including BA.1 and BA.2). They have the ability to compete with RBDO for binding to ACE2, indicating that they prevent The potential for COVID-19 to invade the human body.

The present invention relates to a plurality of Nanobodies with neutralizing potential against Omicron strains. The nanobody that can bind to RBDO in the present invention has the advantages of small molecular weight (only 1/10 the size of a monoclonal antibody), high binding activity (high affinity with Omicron strain RBD), and easy mass production. In addition, the Nanobodies involved in the present invention have the advantages of safety research foundation and rapid entry into clinical research.

The present invention relates to a screening method for Nanobodies. The RBD domain (RBDO) of the Spike protein of the Omicron mutant strain was used as the antigen and recombinantly expressed so that it has a unique biotinylation tag, increasing the possibility of screening more epitopes. Based on the artificially synthesized humanized Nanobody library, Panning solution is used, combined with magnetic bead sorting and antigen concentration gradient reduction scheme, to obtain Nanobodies with high affinity to the antigen. Poly-ELISA was used to test the degree of enrichment in each round of screening to evaluate the screening effect of each round. After the enrichment effect reaches a certain level, continue to conduct phage ELISA experiments at the monoclonal level to identify Nanobodies with affinity for the antigen. The identified Nanobody nucleic acid sequence is transferred to a new expression vector, and the periplasmic expression method of E. coli is used to maintain the disulfide bonds within the Nanobody and maintain its stability. Afterwards, affinity chromatography is used for purification to obtain nanobodies with good purity, with a yield of 10 to 60 mg/L. The biochemical identification uses the Bio-Layer Interferometry (BLI) method. First, a single concentration binding test is performed. After the bound nanobody is found, a concentration gradient experiment is performed to determine its binding kinetic parameters. After screening the Nanobodies that bind to RBDO, competitive binding experiments were used to identify whether the binding of different Nanobodies affects the binding of RBDO to ACE2, and further identify the binding epitopes of Nanobodies that affect the binding of ACE2 to clarify the different nanobodies. Whether there is synergy between antibodies will lay the foundation for subsequent cocktail therapy.

In order to solve the above problems, the first aspect of the present invention provides a Nanobody, which includes CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is as shown in SEQ ID NO: 1, and the amino acid sequence of the CDR2 is as SEQ ID As shown in NO:2, the amino acid sequence of the CDR3 is as shown in SEQ ID NO:3. That is Nb4 antibody.

Or, the amino acid sequence of the CDR1 is shown in SEQ ID NO:10, the amino acid sequence of the CDR2 is shown in SEQ ID NO:11, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:12. That is Nb24 antibody.

Or, the amino acid sequence of the CDR1 is shown in SEQ ID NO:14, the amino acid sequence of the CDR2 is shown in SEQ ID NO:15, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:16. That is Nb30 antibody.

Or, the amino acid sequence of the CDR1 is shown in SEQ ID NO:18, the amino acid sequence of the CDR2 is shown in SEQ ID NO:19, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:20. That is Nb38 antibody.

In some specific embodiments, the Nanobody also includes FR1, FR2, FR3 and FR4; the amino acid sequence of FR1 is shown in SEQ ID NO:4, and the amino acid sequence of FR2 is shown in SEQ ID NO:5 , the amino acid sequence of the FR3 is shown in SEQ ID NO:6, and the amino acid sequence of the FR4 is shown in SEQ ID NO:7.

In some preferred embodiments, the amino acid sequence of the Nanobody is as shown in SEQ ID NO: 8, 13, 17 or SEQ ID NO: 21, or is the same as SEQ ID NO: 8, 13, 17 or SEQ ID The amino acid sequence shown in NO: 21 has at least 85%, 90%, 95%, 98%, and 99% identity.

Biochemical analysis of the Nanobody involved in the present invention shows that Nb4 almost completely inhibits the binding of RBDO to ACE2, indicating that Nb4 is likely to bind to the ACE2 binding region of RBD, and the crystal structure also confirms this. Some other Nanobodies that partially compete with ACE2 for binding to the RBD are likely to have different binding epitopes, laying the foundation for the design and assembly of bispecific or even multispecific antibodies, which also take advantage of the small size of Nanobodies.

In order to solve the above problems, the second aspect of the present invention provides a fusion protein, which is conjugated by the Nanobody and Fc according to any one of the first aspect of the present invention.

Preferably, the Fc is selected from human IgGl, IgG2, IgG3 and IgG4.

More preferably, the nucleotide sequence encoding said Fc is shown in SEQ ID NO: 22.

In some specific embodiments, the fusion protein has a monovalent, bivalent or multivalent Nanobody, that is, it is formed by conjugating two or more Nanobodies. Monovalent Nanobodies are also called monomers. Bivalent Nanobodies are fusion proteins formed by connecting two Nb4s in series, also called duplexes or trimers. Multivalent Nanobodies such as trivalent Nanobodies, such as fusion proteins obtained by connecting three Nb4s in series, are also called triplets or trimers. The bivalent or multivalent Nanobodies are directly connected or connected with a linker, and the linker includes G and S, preferably ( _GmS ) _n ; where m and n are, for example, natural numbers from 0 to 10, and The total number of amino acids is preferably 30 (also called 30GS), such as (G ₄ S) ₅ and (G ₂ S) ₁₀ . In the present invention, after the nanobodies such as Nb4 are transformed in series or triplet, their binding ability to RBDO is greatly improved, and the affinity reaches the pM level.

In order to solve the above problems, the third aspect of the present invention provides a CAR or TCR molecule, which includes a Nanobody as described in any one of the first aspects of the present invention, or a fusion as described in the second aspect of the present invention. protein.

In order to solve the above problems, the fourth aspect of the present invention provides an isolated nucleic acid, wherein the nucleic acid encodes a Nanobody as described in any one of the third aspect of the present invention, or as described in the third aspect of the present invention fusion protein, or a CAR or TCR molecule as described in the third aspect of the invention.

Preferably, the nucleotide sequence encoding the Nanobody is shown in SEQ ID NO: 9.

In order to solve the above problems, the fifth aspect of the present invention provides a recombinant expression vector, which includes the nucleic acid as described in the fourth aspect of the present invention.

In order to solve the above problems, the sixth aspect of the present invention provides a transformant, which includes the nucleic acid as described in the fourth aspect of the present invention, or the recombinant expression vector as described in the fifth aspect of the present invention.

In order to solve the above problems, the seventh aspect of the present invention provides a pharmaceutical composition, which includes the Nanobody as described in any one of the first aspects of the present invention, or the fusion protein as described in the second aspect of the present invention. ;

Preferably, the pharmaceutical composition also includes other anti-COVID-19 antibodies, small molecule drugs, nucleic acid drugs that treat COVID-19, or antibodies targeting other viruses.

In order to solve the above problems, the eighth aspect of the present invention provides a medicine box combination, which includes a medicine box A and a medicine box B, wherein the medicine box A includes any one of the medicines according to the first aspect of the present invention. Nanobodies, or fusion proteins as described in the second aspect of the present invention, or pharmaceutical compositions as described in the seventh aspect of the present invention; the kit B includes other antibodies, small molecules or other antibodies targeting the new coronavirus Nucleic acid drugs, or antibodies, small molecules or nucleic acid drugs that target other viruses.

In order to solve the above problems, the ninth aspect of the present invention provides the Nanobody as described in any one of the first aspect of the present invention, the fusion protein as described in the second aspect of the present invention, and the third aspect of the present invention. CAR or TCR molecule, the nucleic acid described in the fourth aspect of the present invention, the recombinant expression vector described in the fifth aspect of the present invention, the transformant described in the sixth aspect of the present invention or the seventh aspect of the present invention Use of pharmaceutical compositions in the preparation of drugs for treating new coronavirus.

Preferably, the new coronavirus is a new coronavirus Omicron mutant strain such as BA.1, BA.1+R346K and BA.2.

In order to solve the above problems, the tenth aspect of the present invention provides a Nanobody as described in any one of the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention or a third aspect of the present invention. The CAR or TCR molecule described in this aspect is used to prevent (e.g., block new coronavirus infection caused by the respiratory system) and diagnose (diagnostic kit) new coronavirus-related infections, and to treat related diseases caused by new coronavirus infection (such as Respiratory syndrome, pneumonia, etc.).

In order to solve the above problems, the eleventh aspect of the present invention provides a method for treating novel coronavirus-related diseases, which is to administer the Nanobody as described in any one of the first aspect of the present invention to a subject in need. , the fusion protein as described in the second aspect of the present invention or the CAR or TCR molecule as described in the third aspect of the present invention.

In order to solve the above problems, a twelfth aspect of the present invention provides a diagnostic kit, which includes a Nanobody as described in any one of the first aspects of the present invention and a fusion protein as described in the second aspect of the present invention. Or a CAR or TCR molecule as described in the third aspect of the present invention, which can be used for in vitro diagnosis of the presence of the new coronavirus, for example, by diagnosing the new coronavirus Omicron mutant strains such as BA.1, BA.1+R346K and BA.2.

On the basis of common sense in the field, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progressive effects of the present invention are:

The nanobody with neutralizing potential against Omicron strains of the present invention has the advantages of small molecular weight, high binding activity, stable physical and chemical properties, and easy large-scale production and transportation. It can bind to RBDO and almost completely inhibit the binding of RBDO to ACE2, laying the foundation for the design and assembly of bispecific or even multispecific antibodies and CAR molecules. In addition, the Nanobodies involved in the present invention have the advantages of safety research foundation and rapid entry into clinical research.

Description of drawings

Figure 1 shows the screening strategy and the enrichment detection using Poly-ELISA after each round of screening. Five rounds of screening are used (A in Figure 1) to continuously reduce the concentration of the antigen and increase the harshness of the screening conditions to obtain Nanobodies with higher affinity. Poly-ELISA takes an equal amount of phage produced in each round and incubates it with 2 μg/mL RBDO coated on a 96-well plate. After a series of color development operations, the enrichment of each round is observed (Figure 1 B). It can be seen that the degree of enrichment is increasing, and screening at the single clone level can be performed.

Figure 2 shows the results of a representative Mono-ELISA to identify monoclones that bind to RBDO after screening. A and B in Figure 2 are both monoclonal screening ELISA results. The ordinate is the ratio of the RBDO experimental group to the BSA control group. The gray dotted line is an indicator line with a ratio of 0.25. If it is higher than this value, single clones are selected for sequencing to obtain the nucleic acid sequence of the antibody with binding ability.

Figure 3 shows the typical purification results and molecular sieve diagram of Nanobodies. Figure 3A shows the purification results of several Nanobodies, demonstrating their purity. B in Figure 3 is a representative molecular sieve peak pattern (Superdex 75 increase 10/300 GL).

Figure 4 shows the determination of binding kinetic parameters. The figure shows the binding kinetic curves of RBDO to ACE2 and four nanobodies under the same buffer conditions.

Figure 5 shows the competitive binding experiment between Nanobodies and ACE2. A, B, C, and D in Figure 5 are four more competitive nanobodies, namely Nb4, Nb24, Nb30, and Nb38.

Figures 6 and 7 are competitive binding experiments between Nanobodies and ACE2, showing the competition binding curves of Nanobodies with partial competitive binding.

Figure 8 shows the crystal and thermal stability detection of Nb4 and RBDO complex. Figure 8A shows the molecular sieve peak pattern of the complex formed by RBDO and Nb4. Figure 8 B is the SDS-PAGE analysis gel image of the component corresponding to Figure A. It can be seen that a stable complex is formed. C in Figure 8 shows the crystallization experiment performed after the complex was concentrated, and the crystal photo. D of Figure 8 shows the thermal stability curve of Nb4 obtained using nanoDSF.

Figure 9 shows the detection of the redissolution properties of Nb4 after it was frozen into dry powder. In order to demonstrate the superiority of nanobodies, Nb4 was taken and lyophilized into dry powder and then reconstituted. The measured physical and chemical properties of Nb4 were consistent with those before freeze-drying. Figure 9 A) Molecular sieve peak shape diagram. It shows that freeze-dried powder does not increase the aggregation of Nb4. B in Figure 9 shows that the binding kinetics results are consistent with those before freeze-drying the powder (B in Figure 4). C in Figure 9 shows the competitive binding experiment showing that Nb4 maintains its good competitive binding ability. D in Figure 9 shows that the stability of Nb4 remains unchanged before and after freeze-drying.

Figure 10 shows the typical purification results and molecular sieve diagrams of nanobody Nb4 monomers, doublets and triplets. Figure 10, A shows the purification results of the Nanobody, demonstrating its purity. B in Figure 10 is a representative molecular sieve peak pattern (Superdex 200 increase 10/300 GL).

Figure 11 shows the measurement of the binding kinetic parameters of nanobody Nb4 monomer, doublet and triplet to RBDO. The figure shows the binding kinetic curves of RBDO and Nb4 monomer, doublet and triplet under the same buffer conditions.

Figure 12 shows the measurement of the binding kinetic parameters of nanobody Nb4 monomers, doublets and triplets to the new coronavirus Omicron BA.2 spike protein. The figure shows the binding kinetic curves of Nb4 monomer, doublet and triplet to Omicron BA.2 spike protein under the same buffer conditions.

Figure 13 shows the determination of the ability of nanobody Nb4 monomers, doublets and triplets to competitively bind to ACE2 and Omicron BA.2 spike protein.

Detailed ways

The present invention is further described below by means of examples, but the present invention is not limited to the scope of the described examples. Experimental methods that do not indicate specific conditions in the following examples should be selected according to conventional methods and conditions, or according to product specifications.

Example 1 Construction, expression and purification of human ACE2 protein

Construction of human ACE2 expression plasmid: from N-terminus to C-terminus are Kozak sequence (nucleotide GCCACC), wild-type ACE2 signal peptide (nucleotide sequence: SEQ ID NO:23), human ACE2 protein truncation ( 20-615 amino acids) (SEQ ID NO:26), Gly-Thr linker sequence (nucleotide sequence: GGTACC), Avi tag (nucleotide sequence: SEQ ID NO:24), Gly-Ser linker sequence (nucleoside Acid sequence: GGAAGT), His8 tag (nucleotide sequence: SEQ ID NO: 25). pCAG plasmid was used as expression vector.

The nucleotide sequence of wild-type ACE2 signal peptide is (SEQ ID NO:23):

The nucleotide sequence of the Avi tag is (SEQ ID NO:24):

The nucleotide sequence of the His8 tag is (SEQ ID NO:25):

The nucleotide sequence of the human ACE2 protein truncate (20-615) is (SEQ ID NO:26):

Expression process of human ACE2: Transient transfection method is used to transfect the constructed ACE2 expression plasmid into mammalian cells HEK293F. PEI 25K is used as a transfection reagent. Mix the in vitro purified plasmid and PEI 25K at a mass ratio of 1:3 and incubate for 15 minutes. Then add it dropwise into the HEK293F cells prepared in advance. Centrifuge at 37°C for 3 days after transfection to collect the post-expression supernatant. After incubating the supernatant with a certain amount of Ni-NTA agarose gel beads at 4°C for 2 hours, collect the Ni-NTA beads and wash them with a buffer containing 20mM imidazole (150mM NaCl, 20mM Tris HCl pH8.0) Mix, and then use a buffer containing 300mM imidazole to elute the target protein. The collected target protein was concentrated and further purified using size exclusion chromatography (Superdex Increase 200 10/300 GL). Finally, ACE2 protein with uniform molecular weight and purity greater than 95% was obtained.

Example 2 Expression and purification of SARS-CoV-2 Spike protein RBD domain (RBDO)

Expression construction of RBDO: from N-terminus to C-terminus are GP64 signal peptide (nucleotide sequence: SEQ ID NO:27), RBDO (amino acids 319-531; nucleotide sequence: SEQ ID NO:29), Gly -Thr linker sequence (nucleotide sequence: GGTACC), Avi tag (SEQ ID NO:24), Gly-Ser linker sequence (nucleotide sequence: GGAAGT), His6 tag (nucleotide sequence: SEQ ID NO:28 ). The vector is regular pFastBac. The expression of RBDO is secreted in Trichoplusia ni High Five insect suspension cells. Collect the supernatant after expression and add 20mM imidazole, 1mM nickel sulfate, 2mM calcium chloride, 150mM sodium chloride, 20mM Tris HCl (pH8.0 ), stir at 4°C for 30 minutes. Mix the supernatant collected by centrifugation with Ni-NTA beads. After incubation at 4°C for 2 hours, add the supernatant and Ni-NTA mixture to the gravity column. Collect the beads. Use 10 column volumes of buffer A (150mM) with 20mM imidazole added. NaCl, 20mM Tris HCl pH 8.0), and then the protein was eluted with buffer A containing 300mM imidazole.

The nucleotide sequence of GP64 signal peptide is (SEQ ID NO:27):

The nucleotide sequence of the His6 tag is (SEQ ID NO:28):

The nucleotide sequence of the human RBDO(319-531) protein truncate is (SEQ ID NO:29):

Example 3 Biotinylation

Biotinylation labeling of RBDO: Add purified biotin ligase (fusion protein of BirA (11582-E10E) and MBP (maltose binding protein)) to 1 mg/mL RBDO, 1/25 of the molar concentration of RBDO , add 5mM ATP, 10mM magnesium acetate and 3 times the RBDO molar concentration of biotin, mix well, place at 4°C for 16h, and then use size exclusion chromatography (Superdex Increase 75 10/300 GL) to further separate and purify the protein. The protein-containing fractions were collected, aliquoted, quick-frozen in nitrogen, and stored in a -80°C refrigerator for phage display screening.

Example 4 Phage display of Nanobodies and screening

A total of five rounds of phage display were performed in the present invention (see A in Figure 1). The first and second rounds used 700 nM of antigen. The purified phages are first incubated with magnetic beads to remove phages that have the ability to bind to the magnetic beads. The phages are then incubated with 700nM biotinylated RBDO, and then transferred to another set of magnetic beads. After binding at room temperature, the impurities are washed away. Unless the specifically bound phage is present, use 0.2M glycine solution (pH 3) to release the bound phage, and add Tris-HCl in time to adjust the pH to neutral. The screened phages were amplified in vivo, purified in vitro, and then subjected to the third and fourth rounds of phage display. At this time, everything was the same as before except that the antigen concentration was reduced to 100 nM. Then the fifth round of screening was carried out, the antigen concentration was reduced to 10 nM, and after washing, 5 μM non-biotinylated RBDO was added for competitive binding, and some phages with low affinity or fast dissociation were removed, and finally elution was performed. The screened phages were then subjected to Poly-ELISA experiments to obtain the enrichment status after each round of screening (see Figure 1, B). The final two rounds of screening results were used to select single clones for ELISA experiments and sequencing to obtain the nanobody nucleic acid sequence (Nb, see A and B in Figure 2) that can bind to RBDO. The screened Nb gene was cloned into the expression vector pSb and transformed into E. coli MC1061 for expression.

The sequences of Nb4, Nb24, Nb30 and Nb38 are as follows:

Nb4 amino acid sequence (SEQ ID NO:8):

CDR1 sequence of Nb4 (SEQ ID NO:1): WAETFGH;

CDR2 sequence of Nb4 (SEQ ID NO:2): DWWDTVH;

CDR3 sequence of Nb4 (SEQ ID NO:3): YWDMDYLQNSIPVD.

Nb24 amino acid sequence (SEQ ID NO:13):

CDR1 sequence of Nb24 (SEQ ID NO:10): HWPWTGF;

CDR2 sequence of Nb24 (SEQ ID NO:11): VEYGWPT;

CDR3 sequence of Nb24 (SEQ ID NO:12): IAGLIAPEFEQMPV.

Nb30 amino acid sequence (SEQ ID NO:17):

CDR1 sequence of Nb30 (SEQ ID NO:14): MNWDTDW;

CDR2 sequence of Nb30 (SEQ ID NO:15): EAKTFSP;

CDR3 sequence of Nb30 (SEQ ID NO:16): MQMHLKNSMYEGYT.

Nb38 amino acid sequence (SEQ ID NO:21):

CDR1 sequence of Nb38 (SEQ ID NO:18): FTVQLDW;

CDR2 sequence of Nb38 (SEQ ID NO:19): HQMDWYR;

CDR3 sequence of Nb38 (SEQ ID NO:20): SMIHAPKYGYEEWF.

The nucleotide sequence of Nb4 (SEQ ID NO:9) is:

Table 1 Sequence numbers of antibodies of the present invention

Example 5 Expression and Purification of Nanobodies

After the Nanobody expression plasmid was transferred into E.coli MC1061, single clones were picked and cultured overnight in 1 mL TB medium containing chloramphenicol (37°C, 220 rpm), and then transferred into fresh TB medium (containing chloramphenicol) at 1:100. Chloramphenicol), when the OD600 reaches 0.5, lower the temperature to 22°C and continue culturing for 1.5 to 2 hours, then add 0.02% (w/v) arabinose for induction and culture for 16 hours. Centrifuge at 5000g for 15 minutes to collect the cultured cells, resuspend in 5mL TES (0.5M sucrose, 0.5mM EDTA, 0.2M Tris-HCl pH 8.0), rotate for 30 minutes, add 10mL milliQ H ₂ O, rotate for 1 hour, and centrifuge to collect. Supernatant, and add 20mM imidazole, 2mM MgCl2, 150mM NaCl, 20mM Tris-HCl pH 8.0 to the supernatant, and add 200μL Ni-NTA affinity column. After incubating for 1.5 hours, flow through the gravity column, add 30mM imidazole to the buffer (150mM NaCl, 20mM Tris-HCl pH 8.0) to wash the impurities, and use a buffer containing 300mM imidazole to elute the protein. The purified Nanobodies were subjected to SDS-PAGE gel and SEC analysis (see Figure 10, A and B). Expression and purification of tandems and triplets of Nanobody Nb4 were the same as above (see Figure 3, A and B).

Example 6 BLI method to determine the affinity of the antigen RBDO with ACE2 and Nanobodies

Use the Octet RED96 instrument to detect the interaction of ACE2 or different nanobodies with RBDO using biofilm layer optical interference (BLI) technology. Dilute the ACE2 or Nanobody to be tested to different concentration gradients with buffer (20mM HEPES (8.0), 150mM NaCl, 10mg/mL BSA). Biotinylated RBDO is fixed on a streptavidin probe, and then the probe is inserted into a gradient dilution solution containing ACE2 or Nanobodies to be tested, and the affinity between RBDO and ACE2 or different Nanobodies is determined.

Experimental results show that the RBDO used in this patent binds strongly to ACE2 (K _D =9.24nM) (see Figure 4, A). The binding of Nb4 to RBDO (K _D =7.65 nM) is comparable to the binding of ACE2 to RBDO (see Figure 4, B), and it is one of the strongest Nanobodies that binds to RBDO among all the Nanobodies to be tested. Other Nanobodies also bind strongly to RBDO (see Figure 4, C, D, and E).

Experimental results show that after Nb4 is transformed into a tandem or triplet, the binding ability of Nb4 to RBDO is greatly improved, and the affinity reaches the pM level (A-E in Figure 11). Among them, k _on refers to the binding rate constant, k _off refers to the dissociation rate constant, and K _D (K _D = k _off /k _on ) refers to the equilibrium dissociation constant, which is used to characterize the affinity between the antibody and the antigen. The larger the _KD value, the higher the concentration of drug required to cause the maximum effect, and the lower the affinity.

Example 7 BLI method to determine the ability of different Nanobodies to competitively bind to RBDO with ACE2

Immobilize the biotinylated RBDO protein on the streptavidin probe, first saturate the RBDO with a buffer containing 500nM of the Nanobody to be detected, and then extend the probe into a mixture containing 500nM ACE2 and 500nM of the Nanobody to be detected. solution, or 500nM Nanobody buffer without ACE2, observe whether ACE2 can continue to bind after the RBDO binding site is saturated. If the binding level is equivalent to the control group, it indicates that the Nanobody to be tested has no competitive ability with ACE2. If If the binding level decreases compared with the control group, it indicates that the Nanobody to be tested partially or completely competes with ACE2. As a control, first extend the streptavidin probe with biotinylated RBDO protein fixed into the buffer, and then into the buffer containing 500nM ACE2 to obtain the binding of RBDO and ACE2 without the nanobody to be detected. Signal.

Experimental results show that among all the Nanobodies to be tested, Nanobody Nb4 can almost completely compete for the binding of ACE2 and RBDO (see Figure 5, A), and is the Nanobody with the strongest competitive ability among the Nanobodies to be tested. Nanobodies Nb24, Nb30 and Nb38 can mostly compete for the binding of ACE2 to RBDO (see Figure 5, B, C and D). Nanobodies Nb1, Nb8, Nb9, Nb10, Nb15, Nb18, Nb20, Nb23 and Nb26 can partially compete for the binding of ACE2 to RBDO (see A to E of Figure 6 and A to D of Figure 7). In summary, Nb4 is the Nanobody with the most neutralizing potential. The black solid line ACE2 refers to the binding signal of RBDO and ACE2 measured as a control without the nanobody to be detected. The black dotted line Nb+ACE2 refers to the binding signal measured when a probe that has been saturated with Nb is inserted into a mixture containing Nb and ACE2. The black dotted line Nb+Nb refers to the binding signal measured when a probe that has been saturated with Nb is inserted into a pool solution containing only Nb.

Example 8 Co-migration experiment of Nanobody Nb4 and RBDO

The purified nanobody Nb4 and RBDO were mixed at a molar ratio of 2:1. After incubation on ice for 1 hour, size exclusion chromatography was used to analyze whether Nb4 could co-migrate with RBDO on the molecular sieve to obtain stable Nb4/ RBDO complex.

Experimental results show that Nb4 and RBDO can form a stable complex on molecular sieves (see Figure 8, A), and are detected using SDS-PAGE (see Figure 8, B) and crystal screening using the hanging drop method (see Figure 8 C).

Example 9 Determination of thermal stability of Nanobodies

Use the nanoDSF instrument to perform thermal stability analysis of the nanobodies to be detected. Dilute the nanobody to be detected with buffer (1×PBS pH7.4) to 0.05mg/mL~0.1mg/mL, use a capillary tube to absorb about 10μL protein sample, place it in the center of the sample stage of the instrument, and set the variable temperature The range is 20~90℃, 1℃/min, and the stability analysis of the sample to be tested is carried out. The protein stability parameters of the Nanobody to be detected were obtained through the denaturation curve. The melting temperature (Tm) of Nanobody Nb4, that is, the temperature when half of the protein is unfolded, is 64.5°C, and has high thermal stability (see D in Figure 8).

Example 10 Lyophilization of Nanobody Nb4

After quick-freezing the Nb4 protein in liquid nitrogen for 20 minutes, a vacuum freeze dryer was used for further freeze-drying overnight. Store dry protein powder in a -80°C refrigerator. After 7 days, use buffer (1×PBS pH7.4) to dissolve the Nb4 protein lyophilized powder. After high-speed centrifugation, there will be no visible precipitation. For the reconstituted Nb4 sample, size exclusion chromatography was used to analyze its protein molecule aggregation state, BLI technology was used to analyze the kinetic parameters of its binding to RBDO and its competition with ACE2 for binding to RBDO, and nanoDSF was used to analyze its thermal stability. sex.

The experimental results show that the reconstituted Nb4 protein dry powder is in a molecular aggregation state (see Figure 9 A), has the ability to bind to RBDO (see Figure 9 B), and competes with ACE2 for binding to RBDO (see Figure 9 C). It performs very well in terms of its thermal stability (see D in Figure 9), and has the same physical and chemical properties as freshly purified Nb4.

Example 11 Construction, expression and purification of antibody Nb-Fc protein

Construction of Nb4-Fc expression plasmid: from N-terminus to C-terminus are Kozak sequence (nucleotide GCCACC), immunoglobulin Kappa signal peptide (nucleotide sequence: SEQ ID NO:30), His6 tag (nucleotide Sequence: SEQ ID NO:28), Nb4 nucleotide sequence (nucleotide sequence: SEQ ID NO:X), linker sequence (nucleotide sequence: SEQ ID NO:31), Fc nucleotide sequence (nucleoside Acid sequence: SEQ ID NO: 22). PcDNA3.1 plasmid was used as expression vector.

The nucleotide sequence of Fc (SEQ ID NO:22) is:

The nucleotide sequence of immunoglobulin Kappa signal peptide (SEQ ID NO:30) is:

The nucleotide sequence of the connecting sequence (SEQ ID NO:31) is: ATGGTGCGCTCT

Expression process of Nb4-Fc: Transfect the constructed Nb4-Fc expression plasmid into mammalian cells HEK293F using transient transfection method. PEI 25K is used as a transfection reagent. Mix the in vitro purified plasmid and PEI 25K at a mass ratio of 1:3 and incubate for 15 minutes. Then add it dropwise into the prepared HEK293F cells. Centrifuge at 37°C for 3 days after transfection to collect the post-expression supernatant. After incubating the supernatant with a certain amount of Ni-NTA agarose gel beads at 4°C for 2 hours, collect the Ni-NTA beads and wash them with a buffer containing 20mM imidazole (150mM NaCl, 20mM Tris HCl pH 8.0). , and then use a buffer containing 300mM imidazole to elute the target protein. The collected target protein was concentrated and further purified using size exclusion chromatography (Superdex Increase 200 10/300 GL). Finally, Nb4-Fc protein with uniform molecular weight and purity greater than 95% was obtained.

Example 12 BLI method to determine the affinity of Nb4 monomer, Nb4 bivalent Nanobody and Nb4 trivalent Nanobody to the new coronavirus Omicron BA.2 Spike protein

The Octet RED96 instrument is used to detect the interaction between Nb4 monomer, Nb4 dimer and Nb4 trimer and the new coronavirus Omicron BA.2 Spike protein using biofilm layer optical interference (BLI) technology. The Omicron BA.2 Spike protein to be tested was diluted to different concentration gradients with buffer (20mM HEPES (8.0), 150mM NaCl, 10mg/mL BSA). The biotinylated Nb4 monomer, Nb4 dimer or Nb4 trimer was fixed on the streptavidin probe, and then the probe was inserted into the gradient dilution solution containing the Omicron BA.2 Spike protein to be tested. In this study, the affinity of Nb4 monomer, Nb4 dimer and Nb4 trimer to the new coronavirus Omicron BA.2 Spike protein was determined.

Experimental results show that Nb4 monomer, Nb4 dimer and Nb4 trimer have very strong affinity with Omicron BA.2 Spike protein, and the binding force is higher than nM level. Among them, the Nb4 trimer constructed with 30GS as a linker has strong affinity with Omicron BA. 2 Spike protein has the strongest affinity, higher than pM (A-E in Figure 12).

Example 13 BLI method was used to determine the ability of Nb4 monomer, Nb4 dimer and Nb4 trimer to competitively bind to Omicron BA.2 Spike protein with ACE2.

Immobilize the biotinylated Nb4 monomer, Nb4 dimer or Nb4 trimer on the streptavidin probe respectively. First, use a buffer containing 100nM of the Omicron BA.2 Spike protein to be detected to saturated and fix it on the streptavidin probe. Nanobody of avidin probe, and then insert the probe into a mixture containing 100nM ACE2 and 100nM Omicron BA.2 Spike protein, or a 100nM Omicron BA.2 Spike protein buffer without ACE2, and observe the nanometer After the antibody binding site is saturated, whether ACE2 can continue to bind to the Omicron BA.2 Spike protein that has been bound to the stationary phase (nanobody). If it can continue to bind, it indicates that the nanobody to be detected has no competitive ability with ACE2. If it binds If the level is equivalent to or lower than that of the control group, it indicates that the Nanobody to be tested competes with ACE2.

Experimental results show that Nb4 monomer, Nb4 dimer and Nb4 trimer can almost completely compete for the binding of ACE2 and Omicron BA.2 Spike (see Figure 13 A to E). Compared with Nb4 monomer, Nb4 dimer and Nb4 trimer have the strongest ability to compete with ACE2 for binding to Omicron BA.2 Spike protein (see Figure 13). The black solid line BA.2+ACE2 refers to the binding signal measured when a probe that has been saturated with BA.2 is inserted into a mixture containing BA.2 and ACE2. The black dotted line BA.2+BA.2 refers to the binding signal measured when a probe that has been saturated with BA.2 is inserted into the pool solution containing only BA.2.

Claims (12)

1. A kind of Nanobody, characterized in that, the Nanobody includes CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID NO: 1, and the amino acid sequence of the CDR2 is shown in SEQ ID NO: 2, The amino acid sequence of the CDR3 is shown in SEQ ID NO: 3; or,

   The amino acid sequence of the CDR1 is shown in SEQ ID NO:10, the amino acid sequence of the CDR2 is shown in SEQ ID NO:11, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:12; or,

   The amino acid sequence of the CDR1 is shown in SEQ ID NO: 14, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 15, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 16; or,

   The amino acid sequence of the CDR1 is shown in SEQ ID NO: 18, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 19, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 20.
2. The Nanobody of claim 1, wherein the Nanobody further includes FR1, FR2, FR3 and FR4; the amino acid sequence of FR1 is as shown in SEQ ID NO: 4, and the amino acid sequence of FR2 is as SEQ ID NO:5 is shown, the amino acid sequence of FR3 is shown in SEQ ID NO:6, and the amino acid sequence of FR4 is shown in SEQ ID NO:7.
3. The Nanobody according to claim 1 or 2, wherein the amino acid sequence of the Nanobody is as shown in SEQ ID NO: 8, 13, 17 or SEQ ID NO: 21.
4. A fusion protein, characterized in that the fusion protein is formed by conjugating the Nanobody and Fc according to any one of claims 1 to 3; and/or the fusion protein has a monovalent, bivalent or Multivalent Nanobodies; preferably, the Fc is selected from human IgG1, IgG2, IgG3 and IgG4; and/or, the bivalent or multivalent Nanobodies are directly connected or connected with a linker, and the linker Preferably, it is (G _m S) _n ; wherein m and n are, for example, natural numbers from 0 to 10, such as (G ₄ S) ₅ and (G ₂ S) ₁₀ ;

   More preferably, the nucleotide sequence of the Fc is shown in SEQ ID NO: 22.
5. A CAR or TCR molecule, characterized in that it includes the Nanobody according to any one of claims 1 to 3, or the fusion protein according to claim 4.
6. An isolated nucleic acid, characterized in that the nucleic acid encodes the Nanobody according to any one of claims 1 to 3, or the fusion protein according to claim 4, or the CAR according to claim 5 or TCR molecules.
7. The nucleic acid of claim 6, wherein the nucleotide sequence encoding the Nanobody is shown in SEQ ID NO: 9.
8. A recombinant expression vector, characterized in that it includes the nucleic acid according to claim 6 or 7.
9. A transformant, characterized in that it includes the nucleic acid according to claim 6 or 7, or the recombinant expression vector according to claim 8.
10. A pharmaceutical composition, characterized in that it includes the Nanobody according to any one of claims 1 to 3, or the fusion protein according to claim 4;

    Preferably, the pharmaceutical composition also includes other anti-COVID-19 antibodies, small molecule drugs, nucleic acid drugs that treat COVID-19, or antibodies targeting other viruses.
11. A medicine kit combination, which includes medicine box A and medicine box B, characterized in that, the medicine box A includes the Nanobody as described in any one of claims 1 to 3, or the nanobody as claimed in claim 4 Fusion protein, or the pharmaceutical composition according to claim 10; the kit B includes other antibodies, small molecules or nucleic acid drugs targeting the new coronavirus, or antibodies, small molecules or nucleic acid drugs targeting other viruses.
12. The Nanobody according to any one of claims 1 to 3, the fusion protein according to claim 4, the CAR or TCR molecule according to claim 5, the nucleic acid according to claim 6 or 7, the nucleic acid according to claim 8 The use of the recombinant expression vector, the transformant according to claim 9 or the pharmaceutical composition according to claim 10 in the preparation of drugs for the treatment of new coronavirus;

    Preferably, the new coronavirus is a new coronavirus Omicron mutant strain such as BA.1, BA.1+R346K and BA.2.

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