CN116675766A - Humanized high-neutralization-activity anti-novel coronavirus monoclonal antibody and application thereof - Google Patents
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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Abstract
The invention discloses a humanized high neutralizing activity anti-novel coronavirus monoclonal antibody, which is obtained by screening a single B cell flow separation-antibody gene amplification pairing expression technology, has a unique CDR region, can be specifically combined with SARS-COV-2 and can effectively neutralize the current multi-strain international epidemic virus (Prototype strain and Omicron B.1.1.529, BF.7 and XBB.1.5 variant strain). The invention also relates to application of the antibody in preparing a novel coronavirus disease treatment or prevention medicament and application in preparing a novel coronavirus detection reagent. The antibody can be used for diagnosis, emergency prevention and/or treatment of COVID-19, has the characteristics of full humanization, high expression and good stability, and is suitable for industrial preparation.
Description
Technical Field
The invention discloses a polypeptide, and more particularly, an antibody.
Background
SARS-CoV-2 has evolved into a variety of variants, including Alpha, beta, gamma, delta, lambda, and Omicron, among others. At present, omicron is a main epidemic strain and has the characteristics of high transmission speed, strong infectivity, short incubation period, strong immune escape capability and the like. In order to control the incidence and severity of SARS-CoV-2, there is a need to develop effective antiviral drugs, vaccines and neutralizing antibodies.
In the new diagnosis and treatment scheme for coronavirus infection, the patient with rapid, heavy and critical disease progress can be treated by using the convalescent blood plasma. One of the main reasons for the effectiveness of plasma therapy is that patients in convalescence may contain polyclonal antiviral antibodies that help kill or eliminate the virus. However, the main drawbacks of this method are limited sources, difficult quality control, low neutralizing antibody titers. The training drawn from SARS suggests that some non-neutralizing antibodies (nAbs) against non-RBD regions in S protein may cause antibody dependent enhancement effects (ADE). The development of monoclonal antibodies with broad-spectrum neutralizing activity is therefore essential for the prevention or treatment of covd-19.
Studies have shown that many fragments of the novel coronavirus S protein (S1-NTD, RBD, S2) can be targeted by broad-spectrum neutralizing antibodies. They target the S1-RBD, S1-NTD or S2 region, block the binding of SARS-CoV-2 to cellular receptors, interfere with S2-mediated membrane fusion or entry into host cells, thereby inhibiting viral infection. It was found that although NTD antibodies have a strong neutralizing activity, NTD has the characteristic of high glycosylation, and the action epitopes are generally aggregated, so that the neutralizing ability on variant strains is poor. The S2 subunit is a key protein for fusion of virus and cell membrane, is the most conserved structure in the S protein, and has 63-98% sequence identity with human pathogenic coronavirus. However, the neutralizing ability of the isolated S2 antibody is limited, and there is no report on in vivo protection data, whether the S2 antibody can prevent infection or not has yet been determined. Compared with the prior art, the RBD antibody has strong neutralizing capacity and broad spectrum, and is more suitable for the research and development of the current anti-SARS-CoV-2 medicine.
At present, new crown neutralizing antibody separation researches are reported at home and abroad, and a batch of RBD-targeted humanized monoclonal antibodies are separated by adopting methods such as single cell sorting, antibody genome deep sequencing, phage display technology and the like, such as 1F11, 2F6, CA1, CB6, BD-368-2 and the like, and the antibodies show stronger in-vitro neutralizing activity (IC 50 is less than 1 mug/mL). However, most of the monoclonal antibodies which enter clinical use are found to be ineffective in neutralizing Omicron variants, and therefore, previously approved monoclonal antibodies such as Bamlanivimab, etesevelimab have been limited to use in individuals who may be infected with Omicron variants. Under the background that the number of SARS-CoV-2 infection people is continuously increased and the mutant strains are immune escaped, more strong neutralizing antibodies are separated to be very necessary as alternatives, meanwhile, the neutralizing antibodies aiming at different epitopes are subjected to various compatibility, and cocktail therapy is explored, so that the immune escaped of viruses can be more effectively avoided, and similar broad-spectrum antibodies and antibody compositions which are not reported in the prior art are available. The invention aims to provide a group of anti-novel coronavirus monoclonal antibodies with high neutralization activity, and provides application of the anti-novel coronavirus monoclonal antibodies with high neutralization activity in preparation of novel coronavirus disease treatment medicines.
Disclosure of Invention
Based on the above objects, the present invention provides a humanized broad-spectrum high neutralizing activity anti-novel coronavirus monoclonal antibody, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region of which are respectively as follows:
(1) Amino acids 26-33, 51-58 and 97-115 of SEQ ID NO.1, amino acids 27-32, 50-52, 89-97 of SEQ ID NO.3; or alternatively
(2) Amino acids 26-33, 51-57 and 96-111 of SEQ ID NO.5, amino acids 26-34, 52-54 and 91-102 of SEQ ID NO.7; or alternatively
(3) Amino acids 26-33, 51-58 and 97-115 of SEQ ID NO.9 and amino acids 26-31, 49-51 and 88-97 of SEQ ID NO.11.
In a preferred embodiment, the amino acid sequences of the variable region of the heavy chain and the variable region of the light chain of the monoclonal antibody are shown below, respectively:
(1) SEQ ID NO.1 and SEQ ID NO.3; in the present invention, an antibody of a specific embodiment having the variable region of the heavy chain and the variable region amino acid sequence of the light chain is designated "LY-F6HK"; or alternatively
(2) SEQ ID NO.5 and SEQ ID NO.7; in the present invention, an antibody of a specific embodiment having the variable region of the heavy chain and the variable region of the light chain is designated as "LQL-B6HL"; or alternatively
(3) SEQ ID NO.9 and SEQ ID NO.11; in the present invention, an antibody of a specific embodiment having the variable region of the heavy chain and the variable region of the light chain is designated as "LQL-D6HL".
In a more preferred embodiment, the amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown in SEQ ID NO.13, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.15 or SEQ ID NO.17, wherein SEQ ID NO.15 is the amino acid sequence of the constant region of a Kappa-type light chain and SEQ ID NO.17 is the amino acid sequence of the constant region of a Lamda-type light chain.
Secondly, the invention provides a polynucleotide for coding the humanized broad-spectrum high-neutralization activity anti-novel coronavirus monoclonal antibody, wherein the polynucleotide for coding the variable region of the heavy chain of the monoclonal antibody and the polynucleotide combination for coding the variable region of the light chain of the monoclonal antibody are respectively shown as follows:
SEQ ID NO.2 and SEQ ID NO.4; an antibody having one specific embodiment of the heavy chain variable region encoding polynucleotide and the light chain variable region encoding polynucleotide is designated "LY-F6HK"; or alternatively
SEQ ID NO.6 and SEQ ID NO.8; an antibody having one specific embodiment of the heavy chain variable region encoding polynucleotide and the light chain variable region encoding polynucleotide is designated "LQL-B6HL"; or alternatively
SEQ ID NO.10 and SEQ ID NO.12, the antibody having one specific embodiment of the heavy chain variable region encoding polynucleotide and the light chain variable region encoding polynucleotide is designated as "LQL-D6HL".
In a preferred embodiment, the polynucleotide sequence encoding the heavy chain constant region of the antibody is shown in SEQ ID NO.14, and the polynucleotide sequence for the light chain constant region is shown in SEQ ID NO.16 or SEQ ID NO.18, wherein SEQ ID NO.16 is the constant region polynucleotide sequence for a Kappa-type light chain and SEQ ID NO.18 is the constant region polynucleotide sequence for a Lamda-type light chain.
Third, the present invention also provides a vector for expressing the humanized high neutralizing activity anti-novel coronavirus monoclonal antibody described above, which contains the polynucleotide encoding the variable region of the antibody heavy chain and the polynucleotide encoding the variable region of the antibody light chain described above, which may be eukaryotic expression vectors conventionally used in genetic engineering, and in one specific embodiment of the present invention, the vector is IgH (heavy chain expression vector), igkappa (kappa light chain expression vector), iglambda (lambda light chain expression vector) (see in particular Tiller et al Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCRand expression vector cloning, J Immunol methods 2008 January 1; 329 (1-2): 112-124), which is incorporated herein by reference.
Fourth, the present invention provides a host cell expressing the humanized high neutralizing activity anti-novel coronavirus monoclonal antibody described above, which contains the vector described above. The host cell may be a eukaryotic host cell conventionally used in genetic engineering, and in one embodiment of the invention, the host cell is a 293F cell.
Fifth, the invention provides the application of the humanized high neutralizing activity anti-novel coronavirus monoclonal antibody in preparing the novel coronavirus disease treatment and/or prevention drugs, wherein the antibody can be prepared into clinical treatment drugs, and can also be prepared into targeting drugs such as fusion proteins, antibody conjugates and the like of targeting novel coronaviruses for treating and/or preventing novel coronavirus diseases.
Sixth, the present invention provides the use of the humanized high neutralizing activity anti-novel coronavirus monoclonal antibody in preparing novel coronavirus detection reagents, said antibody can be used in various laboratory detection applications of novel coronaviruses, such as various immunological methods for detecting novel coronaviruses based on antigen-antibody specific binding reaction, including but not limited to enzyme-linked immunosorbent assay, radioimmunoassay, chemiluminescent immunoassay, westernblot, etc.
Seventh, the present invention provides an antibody composition comprising a first antibody and a second antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region of the first antibody are shown as amino acids 26-33, 51-58 and 97-115 of SEQ ID NO.1 and amino acids 27-32, 50-52, 89-97 of SEQ ID NO.3, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region of the second antibody are shown as amino acids 26-33, 51-57 and 96-111 of SEQ ID NO.5 and amino acids 26-34, 52-54 and 91-102 of SEQ ID NO.7, respectively, or the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region are shown as amino acids 26-34, 52-54 and 91-102 of SEQ ID NO.7, respectively, and amino acids 26-33-51-33, 52-54 and amino acids 26-35 and 35-33 of the light chain variable region of the second antibody. In the present invention, the "first" and "second" antibodies are not prioritized, and are used only to distinguish between two different antibodies. In a specific definition of the invention, the first antibody is "LY-F6HK" which has a different antigen recognition epitope than the other two antibodies "LQL-B6HL" and "LQL-D6HL" provided by the invention. The 'LQL-B6 HL' and the 'LQL-D6 HL' have the same antigen recognition epitope, and the two antibodies have an epitope competition relationship. Thus, in the present invention, the composition comprises an "LY-F6HK" antibody and at least one of the two antibodies of "LQL-B6HL" or "LQL-D6HL" to exert sufficient binding to the novel coronavirus epitope to block viral invasion of body cells.
In a preferred embodiment, the amino acid sequences of the heavy chain variable region and the light chain variable region of the first antibody are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the second antibody are shown in SEQ ID NO.5 and SEQ ID NO.7, respectively; alternatively, as shown in SEQ ID NO.9 and SEQ ID NO.11.
Finally, the invention provides application of the antibody composition in preparing novel medicaments for treating and/or preventing coronavirus diseases. The 3 antibodies provided by the invention not only have high neutralization activity, but also have broad spectrum, and have strong neutralization capability on various new coronavirus variants in the world at present, so that the application of the combination of at least two antibody compositions containing the invention, such as LQL-D6HL and LY-F6HK or LQL-B6HL and LY-F6HK, can more effectively improve the broad spectrum neutralization capability on the multiple variants of the new coronavirus, and therefore, the antibody composition form containing the invention can be used for cocktail therapy for treating the new coronavirus diseases and/or preventing the new coronavirus infection.
The humanized high neutralizing activity anti-novel coronavirus monoclonal antibody provided by the invention is obtained by screening a single B cell flow sorting-antibody gene amplification pairing expression technology, has a unique CDR region, can be specifically combined with SARS-COV-2, can effectively neutralize the current multi-strain international epidemic virus (Prototype and Omicron variant B.1.1.529, BF.7 and XBB.1.5), and has remarkable broad-spectrum neutralization capability on a plurality of novel coronavirus epidemic strains in the world. Therefore, the antibody and the composition containing the two antibodies can be used for preparing the COVID-19 emergency preventive and/or therapeutic drug, have the characteristics of full humanization, high expression and good stability, and are suitable for industrialization. In addition, the monoclonal antibody provided by the invention can also be used for preparing SARS-COV-2 virus detection reagent, for detecting virus antigen and for finding effective neutralization antigen epitope.
Drawings
FIG. 1 shows a graph of the potency detection of biotinylated RBD protein, wherein A. The magnetic bead method detects the RBD biotinylation efficiency; B. ELISA method for detecting biotinylation efficiency;
FIG. 2 is a schematic representation of flow-sorted RBD-specific B cells;
FIG. 3 is a graph of ELISA results for antibodies against RBD binding capacity; wherein A, 3 monoclonal antibodies and Prototype-RBD binding capacity curves; B. 3 mab and Omicron b.1.1.529-RBD binding capacity curves;
FIG. 4 is a graph of LY-F6HK affinity assay using BLI technique; wherein a. Mab and Prototype-RBD interaction profile; B. mab and Omicron b.1.1.529-RBD interaction curves;
FIG. 5 shows a graph of LQL-B6HL affinity detection results using BLI technique; wherein a. Mab and Prototype-RBD interaction profile; B. mab and Omicron b.1.1.529-RBD interaction curves;
FIG. 6 shows a graph of LQL-D6HL affinity detection results using BLI technique; wherein a. Mab and Prototype-RBD interaction profile; B. mab and Omicron b.1.1.529-RBD interaction curves.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are only exemplary and do not limit the scope of the invention in any way, which is defined by the claims.
Example 1: synthesis, expression and biotinylation and staining of novel coronavirus RBD probes
(1) According to the data published by Genbank (NC_ 045512), the RBD full-length gene sequence carrying a 6 XHis-Avi (His-His-His-His-His-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-Asp-Ile-Thr-Asn-Trp-Leu-Trp-Tyr-ILe-Lys-Lys-Lys) tag was synthesized.
(2) The eukaryotic expression vector pDRI1.0 (constructed and stored by the inventor) is re-connected after double digestion by EcoRI and EcoRV, and the cloning is selected and sequenced without error.
(3) Respectively transfecting 293F cells with the two probe plasmids for expression, centrifuging the culture solution after 5-6 days to obtain cell supernatant, and purifying antigen proteins by a nickel column.
(4) The probe protein was biotinylated using the BirA500 biotin protein ligase kit (BirA 500, avidity). Dissolving 1 mg molecule of probe protein in 0.7 mL of PBS buffer solution, respectively adding 0.1 mL of 10 Xbuffer solution A and 0.1 mL of 10 Xbuffer solution B, adding 4 mu L of BirA500 enzyme, uniformly mixing, and incubating at 30 ℃ for 30 minutes; the mixture was transferred to a 10 KD concentrate tube, 10 mL PBS was added, 4000 g 15 min centrifuged to a residual volume of 0.5 mL, and this operation was repeated 5 times; the concentrated protein was collected and the concentration was determined and stored in a-80 ℃ freezer. Detecting biotinylation activity of molecular probes, namely shaking 60 mu L of streptomycin-labeled agarose+10 mu g of protein+500 mu L of PBS in a room temperature oscillator for 30 min, briefly centrifuging, washing 3 times by using 1 mL PBS, absorbing liquid as clean as possible for the last time, and finally leaving about 30 mu L of agarose, simultaneously preparing 30 mu L of 2 XSDS gel loading buffer (100 mM pH6.8 Tris-HCl, 4% SDS, 0.2% bromophenol blue, 20% glycerol and 200 mM beta-mercaptoethanol) loading buffer, mixing agarose and mixed buffer 1:1, absorbing 20 mu L of mixed buffer at 100 ℃, performing SDS-PAGE electrophoresis for 5-10 min; after electrophoresis, adding a proper amount of coomassie brilliant blue for dyeing for 1 hour, placing the mixture in an oscillator for decolorization, observing the depth of an electrophoresis band, and judging the efficiency of biotin labeling, wherein fluorescent labeling can be considered at least over 50 percent.
(5) The biotinylated activity of both probes was detected using an Enzyme-linked immunosorbent assay (Enzyme-linked Immunosorbent Assay, ELISA). The new crown antigen is diluted to 2 mug/mL by PBS, added into a 96-well ELISA plate, 100 mug/well is placed at 4 ℃ overnight, and washed 3 times by PBST (PBS containing 0.05% Tween 20) for the next day; each well was blocked with 370 μl of blocking solution (PBS containing 2% milk and 5% FBS) at room temperature for 1 hour, and washed 3 times with PBST (PBS containing 0.05% tween 20); adding 100 μl of blocking solution to each well, adding 25 μl of diluted biotin-labeled probe protein (the storage concentration of biotinylated probe protein is 50 μg/mL, 5 times diluted before use) to each well of the first row, sucking 25 μl from each well of the first row into the second row after mixing, sucking 25 μl into the third row after mixing, sequentially until 25 μl from the last row is sucked out and discarded (five-fold dilution gradient is formed between each row), incubating at 37deg.C for 1 hr, and washing 5 times with PBST (PBS containing 0.05% Tween 20); horseradish peroxidase-labeled streptavidin (Sigma KPL HRP-SA) was diluted 1000-fold in a blocking solution containing 0.05% Tween 20, 100. Mu.L was added to each well, incubated for 1 hour at 37℃and plates were washed 5 times with PBST; 100. Mu.L of substrate was added to each well, and after incubation at room temperature for 20 minutes, 50. Mu.L of sulfuric acid stop solution was added to each well to stop the reaction immediately, and the reaction was read and stored on an microplate reader. FIG. 1 shows the results of a titer assay for biotinylated RBD protein, wherein A is the magnetic bead assay for RBD biotinylation efficiency, lanes 1-2 are the RBDs successfully biotinylated captured by streptavidin magnetic beads from 10 μg of biotinylated RBD protein, lane 3 is 10 μg of biotinylated RBD, and lane 4 is the molecular weight marker; as can be seen from FIG. 1A, RBD biotinylation efficiency exceeds 50%; b is ELISA method to detect biotinylation efficiency, and the final concentration (end-titer) of the probe is 0.0032 mug/mL. From the results, it can be seen that the biotinylation of RBD was successful.
(6) Biotinylated probe protein was fluorescently labeled RBD-Avi probe protein with PE (phycoerythrin) for single cell flow sorting.
Example 2: screening of humanized monoclonal antibody against SARS-COV-2
2.1 Preparing cell lysate: mu.L of cell lysate per well, containing 0.5. Mu.L of denrnase, 5. Mu.L of 5 XFirst Strand buffer, 1.25. Mu.L of 0.1M DTT, 0.0625. Mu.L of Igepal, 13.25. Mu.L of water, covered with sealing plate membrane, and placed in a 4℃refrigerator for use.
2.2 Sample preparation:
recovery of PBMCs cells following new crown infection: after the frozen cell tube is taken out of liquid nitrogen, the frozen cell tube is quickly placed in a water bath at 37 ℃ and taken out when the frozen cell tube is melted to have ice cores, the frozen cell tube is opened in a biosafety cabinet, and R10+Benzonase culture medium (5 mL R10+Benzonase culture medium is used for 1 cell) is slowly dripped. 1500 Centrifuging at rpm for 10 min, discarding supernatant, suspending cells with residual liquid, adding 10 mL R10, mixing, taking 50 μL for cell counting, centrifuging at 1500 rpm for 10 min; cell concentration was adjusted: the supernatant was discarded, cells were suspended with the residual liquid, and the concentration was adjusted using R10 medium, and the cells were plated into 96-well U-plates at 2.5X10 6 A/hole;
an EDTA/PBS solution of 2 mM was prepared, hereinafter denoted by E-PBS;
placing 96 Kong Xibao plate in a centrifuge, centrifuging at 4deg.C and 2000 rpm for 3 min, discarding supernatant (operating in biosafety cabinet, placing paper towel in high pressure table);
50 mu L of Vivid working solution (prepared by diluting Vivid (UV) working solution with PBS at a ratio of 1:1000, mixing well) is added into each hole, and the mixture is incubated on light-proof ice for 20 minutes;
150 mu L E-PBS was added to each well and centrifuged at 2000 rpm at 4℃for 3 minutes;
the supernatant was discarded, 50. Mu.L of extracellular antibody mix (2. Mu.L of Anti-CD 3-Pacific Blue, 2. Mu.L of Anti-CD8-Pacific Blue, 1.5. Mu.L of Anti-CD14-Pacific Blue,1. Mu.L of Anti-CD19-BV510, 2. Mu.L of Anti-CD20-ECD, 2.5. Mu.L of Anti-CD27-APC-cy7, 5. Mu.L of Anti-IgG-FITC, 2.5. Mu.L of Anti-IgM-PercpCs 5.5, 2.5. Mu.L of Anti-PD-1-PECy7, 1. Mu.L of Anti-CXCR5-APC-R700, 5. Mu.L of Anti-CXCR3-PECy5, 1. Mu.L of Anti-CD45RA-BV, 1. Mu.L of Anti-CD4-BV605, 5. Mu.L of Anti-IgG-PE, and insufficient E) was added, and the mixture was incubated for 60 minutes in the dark place;
150 mu L E-PBS was added to each well, centrifuged at 2000 rpm for 3 minutes at 4℃and the supernatant was discarded;
200 mu L E-PBS was added to each well, centrifuged at 2000 rpm for 3 minutes at 4℃and the supernatant was discarded;
200 mu L E-PBS is added into each hole, the mixture is uniformly mixed, the same sample cell suspension is filtered into the same branch-flow tube, and the branch-flow tube is stored at 4 ℃ in a dark place for sorting.
Single-dye tube control: 1 undyed control (1 drop of compensation microspheres, 200. Mu. L E-PBS) and 15 individually stained tubes (1 drop of compensation microspheres per tube, 2. Mu.L of Anti-CD 3-Pacific Blue,1. Mu.L of Anti-CD19-BV510, 2. Mu.L of Anti-CD20-ECD, 2.5. Mu.L of Anti-CD27-APC-cy7, 5. Mu.L of Anti-IgG-FITC, 2.5. Mu.L of Anti-IgM-PercpCy5.5, 2.5. Mu.L of Anti-PD-1-PECy7, 1. Mu.L of Anti-CXCR5-APC-R700, 5. Mu.L of Anti-CXCR3-PECy5, 1. Mu.L of Anti-CD45 RA-650, 1. Mu.L of Anti-CD4-BV605, 5. Mu.L of Anti-RBD-PE, 50. Mu.L of UV working solution) were set, incubated for 20 minutes at room temperature, and after centrifugation was performed at 35 min at 150. Mu.L of PBS for 200. Mu.3-PBS, and centrifugation was performed at 35 min.
2.3 Single B cell flow sorting: selection of CD3 - CD8 - CD14 - CD19 + CD20 + CD27 + IgG + IgM - RBD + Is selected from the group consisting of 157 cells. The first circle circumscribes single lymphocyte population and then circumscribes CD3 - CD8 - CD14 - To exclude T cells and macrophages and to re-circle CD19 + CD20 + B cells of (C) and then circling to define CD27 + Memory B cells of (C) and circling IgG + IgM - Finally, memory B cells combined with the probe RBD are circled. 1 of these B cells were sorted per well into 96-well plates containing the following lysate system (Table 1). After the sorting was completed, the 96-well plate was immediately sealed with a sealing film, coagulated on dry ice, transferred to a-80 ℃ refrigerator overnight, and subjected to PCR operation the next day. Results: the results of the flow sorting are shown in fig. 2.
TABLE 1B cell lysate System
2.4 The variable region gene of the whole human antibody is amplified by using the RT-PCR technology, an RT-PCR reaction system shown in Table 2 is configured, and 6 mu L of the variable region gene is added into each hole to perform the RT-PCR reaction.
TABLE 2 RT-PCR reaction System
RT-PCR reaction program setting: 42 ℃ for 10 min,25 ℃ for 10 min,50 min,94 ℃ for 5 min, and 4 ℃ for preservation.
(1) Two rounds of PCR reactions were performed, and the reaction system was as follows
The first round PCR reaction (Table 3) complements the primer sequences (Table 4)
TABLE 3 first round PCR reaction System
TABLE 4 primer sequences for first round PCR amplification
The target fragments amplified by the primers for the first round of PCR amplification are shown in Table 5.
TABLE 5 amplification of fragments of interest by primers for the first round of PCR amplification
The first round PCR procedure is shown in Table 6.
TABLE 6 first round PCR procedure
After the completion of the first round of PCR, a second round of PCR was performed, and the second round of PCR was performed according to the system shown in Table 7.
TABLE 7 second round PCR reaction System
The second round PCR primer sequences are shown in Table 8.
TABLE 8 second round PCR primer sequences
The target fragments amplified by the primers used for the second round of PCR amplification are shown in Table 9.
TABLE 9 amplification of fragments of interest by primers for the second round of PCR amplification
The second round PCR procedure is shown in Table 10.
TABLE 10 second round PCR procedure
(2) Electrophoresis, sequencing, and family analysis
Detecting the two rounds of PCR products by electrophoresis, and directly sequencing heavy chain products and light chain products; sequencing results were analyzed using an antibody family gene database (http:// www.imgt.org/IMGT_vquest/vquest), and antibody variable region sequences with cleavage sites (heavies chain: 5'-Age I,3' -Sal I; kappa chain: 5'-Age I,3' -BsiW I; lambda chain: 5'-Age I,3' -Xho I) were designed and synthesized to obtain Heavy and light chain paired clones.
2.5 Monoclonal antibody expression vector construction and plasmid transformation
The synthesized genes were digested with the corresponding enzymes and then recovered by gel electrophoresis again, and the variable region genes were transformed with the corresponding vectors IgH (heavy chain expression vector), igkappa (kappa light chain expression vector), iglambda (lambda light chain expression vector) using T4 DNA ligase (see in particular Tiller et al efficiency generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning, J Immunol methods 2008 January 1; 329 (1-2): 112-124. The present invention incorporates this prior art document by reference into the present specification) on a linker overnight in a 16℃water bath. mu.L of the ligation product was added to 50. Mu.L of DH 5. Alpha. Competent cells, shaken well, ice-bath for 30 min, and heat-shocked in a 42℃water bath for 45 s. After the centrifuge tube was placed in an ice bath for 2 min, 1 mL non-resistant LB medium was added, shaking culture was performed at 37℃and 200 rpm for 1 hour, centrifugation was performed at 4000 rpm for 4 min, and the remaining bacterial liquid was spread on a resistant LB plate. Inverted culturing at 37 ℃ for 14-16 hours. The monoclonal colonies were picked up and inoculated into a resistant LB liquid medium, and subjected to shaking culture at 37℃and 200 rpm for 14-16 hours, followed by Plasmid extraction (Plasmid Midi Kit from Omega).
2.6 Antibody expression and purification
293F cell concentration was adjusted to 1.2X10 6 Each ml was cultured for 2 hours. Preparing solution A: 25 500. Mu.g of antibody heavy chain DNA and 500. Mu.g of antibody light chain DNA were added to mL opti-MEM, solution B: 25 PEI transfection reagent 5 mL was added to the mL opti-MEM and allowed to stand for 5 minutes. Mixing the solution A and the solution B, standing for 20 minutes, dropwise adding the mixture into 1L of 293F cells, shaking the mixture while dripping the mixture, and culturing the mixture at the temperature of 8% CO2 and the temperature of 37 ℃ for 5 days by shaking. The antibody was purified by using Protein A affinity column (product of GEhealth Co.). The antibody concentration was determined using a NanoDrop2000 ultra-micro spectrophotometer (Thermo company product) and placed at 4 ℃ to be detected. 133 antibodies (antibody amount) were expressed in the light/heavy chain pairing of this step>Concentration of antibody 10. Mu.g>10μg/mL）。
EXAMPLE 3 identification of humanized monoclonal antibodies against SARS-CoV-2
3.1 Antigen-antibody binding Capacity detection
The monoclonal antigen binding capacity of SARS-CoV-2 was determined by ELISA. RBD proteins from Prototype and Omicron B.1.1.529 variants were diluted to 0.5. Mu.g/mL with PBS and 100. Mu.L per well coated 96-well ELISA plates (Corning Costar Co.) overnight at 4 ℃. Wash plate 5 times with PBS-T solution (0.05% tween-20); mu.L of blocking solution (PBS, 2% BSA+5% skimmed milk powder) was added to each well and blocked at 37℃for 2 hours. PBS-T was washed 5 times. The antibodies were serially diluted 5-fold with blocking solution at an initial concentration of 10. Mu.g/mL, 100. Mu.L of each sample was added to ELISA plates and incubated at 37℃for 1 hour. PBS-T was washed 5 times. 100 μl of blocking solution 1 was added per well: 5000. the diluted horseradish enzyme-labeled goat anti-human IgG (h+l) (sequoyis gold-bridge biotechnology limited in beijing) was incubated at 37 ℃ for 1 hour. PBS-T was washed 5 times. 100 μl of TMB developing solution (Beijing Jinhao pharmaceutical Co., ltd.) was added, and developed at room temperature in a dark place for 15 minutes. The reaction was stopped by adding 50. Mu.L of stop solution (Beijing Jinhao pharmaceutical Co., ltd.) to each well, and the absorbance (OD) values at two wavelengths of 450nm to 630nm were read by an ELISA reader.
From 133 test antibodies, 3 monoclonal antibodies with strong binding capacity against SARS-CoV-2 prototype,Omicron B.1.1.529,BF.7 and XBB.1.5 variants were selected, and the numbers of the 3 monoclonal antibodies were LY-F6HK, LQL-B6HL and LQL-D6HL, respectively. 3. The results of the binding capacity of the antibodies against the different variants are shown in Table 11 and FIG. 3. In fig. 3 VRC01 is an anti-HIV neutralizing antibody as a negative control.
TABLE 11 End-titer values of neutralizing antibodies
3.2 Antigen-antibody affinity kinetic assay
LY-F6HK, LQL-B6HL and LQL-D6HL were tested for affinity to Prototype and Omicron B.1.1.529 variant RBD Protein using the Octet Red 96 system (Fortebio, USA) and streptavidin sensor using BLI technique, diluted to a concentration of 2. Mu.g/mL with buffer (PBS solution containing 0.1% BSA+0.02% Tween 20) and then fixed to Protein sensor (Sartorius AG, germany) for 120 seconds. After 120 seconds of washing step with PBST, the biosensor probe was immersed in a well containing serial dilutions of RBD (25 nM, 12.5 nM, 6.25 nM, 3.13 nM and 1.56 nM) and combined for 120 seconds, followed by 300 seconds of dissociation step. The KD values were calculated using the 1:1 binding model in data analysis software 9.0 as shown in Table 12 below, and the results are shown in FIGS. 4-6. These 3 antibodies were shown to have very strong affinity for RBD.
TABLE 12 affinity values for antigen-antibodies
3.3 Neutralization activity detection of SARS-CoV-2 pseudovirus by antibody
(1) Packaging of pseudoviruses: the total length of S protein gene of artificial synthesized SARS-CoV-2 (GenBank: MN 908947) is inserted into pcDNA3.1 expression plasmid to construct pcDNA-SARS-CoV2-S. The pseudoviral mutation sites with mutations are introduced on the S gene as shown in Table 13.
TABLE 13 pseudoviral mutation sites
Will be 3X 10 6 (300 ten thousand) 293T cells were seeded in T75 cell culture flasks at 5% CO 2 Culturing at 37 deg.C for 20-24 hr. Transfection was performed using Fugene 6 Transfection Reagent (Promega, cat#E2691): mu.g of plasmid pcDNA-SARS-CoV-2-S was transfected into 293T cells with 1.05X10 6 G.DELTA.G-VSV virus infection with TCID50 was performed for 293T and medium was changed 8 hours later. 24 hours after transfection, the culture supernatant was collected and filtered to obtain the pseudovirus of SARS-CoV-2S protein, which was stored frozen at-80 ℃.
(2) Neutralization experiments of pseudoviruses by antibodies:
add each well to a 96 well plate100. mu.L of gradient diluted antibody dilutions. Subsequently, pseudoviruses were diluted to 1.3X10 with DMEM complete medium 4 TCID50/mL was added at 50. Mu.L per well in columns 3-11 to give a pseudovirus addition of 650 TCID 50/well. The 96-well plate was placed in a cell incubator (37 ℃ C., 5% CO) 2 ) Incubate for 1 hour. When the incubation time reaches half an hour, taking out the prepared Vero cells, sucking and removing the culture medium, and adding PBS buffer solution to clean the cells; PBS was discarded, and after centrifugation by digestion with pancreatin-EDTA, cells were resuspended in complete medium and counted. Dilution of the cell suspension to 2X 10 5 And each mL. Incubation to 1 hour, 100. Mu.L of cells were added to each well of a 96-well plate to give 2X 10 cells per well 4 And each. Slightly shaking the 96-well plate back and forth and left and right to uniformly disperse cells in the well, placing the 96-well plate into a cell incubator at 37 ℃ and 5% CO 2 Culturing for 48 hours. The 96-well plate was removed from the cell incubator, 150. Mu.L of the supernatant was pipetted from each well using a multi-channel pipette, and 100. Mu.L of the luciferase assay reagent was added thereto, and the reaction was carried out at room temperature for 2 minutes in a dark place. After the reaction is finished, the mixture is evenly mixed by vibrating in a flat-plate oscillator, and the mixture is put into a multifunctional plate reader to read the luminous value. And (3) calculating the neutralization inhibition rate: based on the neutralization inhibition results, IC50 of the antibody was calculated.
The neutralizing capacity results of LY-F6HK, LQL-B6HL and LQL-D6HL 3 strain antibodies against pseudoviruses of the different variant strains are shown in Table 14.
TABLE 14 neutralizing Capacity of antibodies against pseudoviruses of different variants (IC 50, μg/mL)
Neutralization activity detection of SARS-CoV-2 live virus by antibody
Vero-E6 cells were plated at 2.5X10 s1 day in advance 4 Cell number per well was inoculated in a 48-well plate and cultured in MEM medium containing 10% FBS and a double antibody. Anti-cancer agentAnd (3) body configuration: the antibodies were diluted with MEM medium containing 5% FBS and diabody. The amount of antibody was 750. Mu.L (250. Mu.L/well) per concentration calculated as 500. Mu.L/well of the final volume of the 48 well plate and 3 wells per concentration, and 2 times the actual concentration. From the highest concentration, the dilution is performed at a multiple (e.g., 3-fold). At the same time, corresponding positive and negative controls were set. The prepared antibodies were added to a 48-well plate and brought into the P3 laboratory. The new coronavirus stock was diluted with MEM medium containing 5% FBS according to 1: 1. to the prepared antibody was added virus solution (250. Mu.L/well) and incubated at 37℃for 3 hours. The supernatant of the logarithmic phase of Vero-E6 cell plate was blotted, and the incubated antibody virus mixture (500. Mu.L/well) was added thereto, followed by culturing at 37℃for 7 days. And (3) subsequent detection: cytopathy is observed, and the concentration of neutralizing antibody with the neutralization percentage of 50% is calculated as IC 50 . The results are shown in Table 15. All three monoclonal antibodies can effectively neutralize the viable viruses of Prototype, omicron B.1.1.529 and BA.5 variants, and LY-F6HK has the strongest ability to neutralize the Prototype and Omicron BA.5 variants, and the IC50 can reach 0.055 mug/mL and 0.081 mug/mL respectively.
TABLE 15 neutralizing Capacity of antibodies against live Virus (IC 50, μg/mL)
By sequencing the clones encoding the above antibody strains, the amino acid and nucleotide sequences of the 3-strain antibodies were as follows:
the nucleotide sequence of the heavy chain variable region of the antibody LY-F6HK is shown as SEQ ID NO.2, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as amino acids 26-33, 51-58 and 97-115 of SEQ ID NO.1 respectively; the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as 27-32, 50-52 and 89-97 of SEQ ID NO.3 respectively.
The nucleotide sequence of the heavy chain variable region of the antibody LQL-B6HL is shown as SEQ ID NO.6, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.5, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as amino acids 26-33, 51-57 and 96-111 of SEQ ID NO.5 respectively; the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.8, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.7, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as amino acids 26-34, 52-54 and 91-102 of SEQ ID NO.7 respectively.
The nucleotide sequence of the heavy chain variable region of the antibody LQL-D6HL is shown as SEQ ID NO.10, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.9, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as amino acids 26-33, 51-58 and 97-115 of SEQ ID NO.9 respectively; the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.12, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.11, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as amino acids 26-31, 49-51 and 88-97 of SEQ ID NO.11 respectively.
The polynucleotide sequence of the heavy chain constant region of the antibody is shown as SEQ ID NO.14, the polynucleotide sequence of the light chain constant region is shown as SEQ ID NO.16 (kappa chain) or SEQ ID NO.18 (lambda chain). The amino acid sequence of the heavy chain constant region of the antibody is shown as SEQ ID NO.13, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO.15 or SEQ ID NO. 17. In one embodiment of the invention, the light chain constant region of antibody LY-F6HK employs a Kappa chain; the light chain constant regions of antibodies LQL-B6HL and LQL-D6HL both employ Lambda chains.
EXAMPLE 4 determination of anti-SARS-CoV-2 humanized monoclonal antibody combination
4.1 Epitope pairing detection of monoclonal antibody combinations
The epitope of the combination of two monoclonal antibodies was detected using the Octet in-tandem method using the Octet Red 96 system (Fortebio, USA) and a streptavidin sensor.
Diluting the biotinylated RBD to 1 mug/mL to serve as a fixture;
diluting the human monoclonal antibody to a concentration of 30 mug/mL by using a buffer solution (PBS solution containing 0.1% BSA and 0.02% Tween 20);
selecting an SA biosensor probe, and prewetting in advance by using a buffer solution;
the molecular interaction procedure was set as follows: baseline 30 s,loading 120 s,baseline 30 s,1st antibody 180s,baseline 30 s,2nd antibody 180s,regeneration 30 s;
performing on-machine detection on the 96-well plate added with the sample;
after detection, the reaction signal between antibodies was obtained by analysis using ForteBio Data Analysis and 9.0 software.
The LY-F6HK, the LQL-B6HL and the LQL-D6HL monoclonal antibodies are combined pairwise, and the results show that the reaction signals of the LY-F6HK and the LQL-B6HL and the LQL-D6HL are both more than 70%, the binding epitope of the LY-F6HK is different from the epitope of the LQL-B6HL and the epitope of the LQL-D6HL, and the antibodies are not in competition; while LQL-B6HL and LQL-D6HL reacted with 7% signal, the two antibodies were epitope-competing (Table 16).
TABLE 16 monoclonal antibody competitive epitope detection values
4.2 Neutralization activity detection of SARS-CoV-2 pseudovirus by combined antibody
The neutralizing ability of the combined antibodies against SARS-CoV-2 Prototype, omicron B.1.1.529, omicron BF.7 and Omicron XBB.1.5 variants was examined by combining 3 monoclonal antibodies (LY-F6 HK, LQL-B6HL and LQL-D6 HL) in equal proportions in a pseudo-virus neutralization method (see section 3.3 for specific experimental procedures), and the results are shown in Table 17. The LY-F6 HK+LQL-D6 HL and the LQL-D6 HL+LQL-B6 HL combined antibody have strong capability of neutralizing Prototype and Omicron variant strains. And the epitope detection LY-F6HK and LQL-D6HL antibodies have no competitive epitope, further showing that the combination of LY-F6HK and LQL-D6HL antibodies can be used as candidate neutralizing antibodies for SARS-CoV-2 prevention and treatment.
TABLE 17 neutralizing Capacity of antibodies against pseudoviruses of different variants (IC 50, μg/mL)
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Claims (12)
1. A humanized high neutralizing activity anti-novel coronavirus monoclonal antibody characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region of said monoclonal antibody are as follows:
(1) Amino acids 26-33, 51-58 and 97-115 of SEQ ID NO.1, amino acids 27-32, 50-52, 89-97 of SEQ ID NO.3; or alternatively
(2) Amino acids 26-33, 51-57 and 96-111 of SEQ ID NO.5, amino acids 26-34, 52-54 and 91-102 of SEQ ID NO.7; or alternatively
(3) Amino acids 26-33, 51-58 and 97-115 of SEQ ID NO.9 and amino acids 26-31, 49-51 and 88-97 of SEQ ID NO.11.
2. The monoclonal antibody according to claim 1, wherein the amino acid sequences of the variable region of the heavy chain and the variable region of the light chain of the monoclonal antibody are as follows:
(1) SEQ ID NO.1 and SEQ ID NO.3; or alternatively
(2) SEQ ID NO.5 and SEQ ID NO.7; or alternatively
(3) SEQ ID NO.9 and SEQ ID NO.11.
3. The monoclonal antibody according to claim 2, wherein the amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID No.13 and the amino acid sequence of the light chain constant region is shown in SEQ ID No.15 or SEQ ID No. 17.
4. A polynucleotide encoding the humanized broad spectrum high neutralizing activity anti-novel coronavirus monoclonal antibody of claim 2 or 3, wherein the polynucleotide encoding the variable region of the heavy chain of said antibody and the polynucleotide encoding the variable region of the light chain of said antibody are each as follows:
(1) SEQ ID NO.2 and SEQ ID NO.4; or alternatively
(2) SEQ ID NO.6 and SEQ ID NO.8; or alternatively
(3) SEQ ID NO.10 and SEQ ID NO.12.
5. The polynucleotide encoding a monoclonal antibody according to claim 4, wherein the sequence of the polynucleotide encoding the heavy chain constant region of the antibody is shown in SEQ ID NO.14 and the sequence of the polynucleotide encoding the light chain constant region is shown in SEQ ID NO.16 or SEQ ID NO. 18.
6. A vector expressing the humanized high neutralizing activity anti-novel coronavirus monoclonal antibody of claim 2 or 3, said vector comprising the polynucleotide encoding the variable region of said antibody heavy chain of claim 4 and the polynucleotide encoding the variable region of said antibody light chain.
7. A host cell expressing the humanized high neutralizing activity anti-novel coronavirus monoclonal antibody of claim 2 or 3, said host cell comprising the vector of claim 6.
8. Use of a humanized high neutralizing activity anti-novel coronavirus monoclonal antibody according to any one of claims 1-3 for the preparation of a novel coronavirus therapeutic and/or prophylactic medicament.
9. Use of a humanized high neutralizing activity anti-novel coronavirus monoclonal antibody according to any one of claims 1-3 for the preparation of a novel coronavirus detection reagent.
10. An antibody composition comprising a first antibody and a second antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region of the first antibody are as shown in amino acids 26-33, 51-58 and 97-115 of SEQ ID No.1 and amino acids 27-32, 50-52, 89-97 of SEQ ID No.3, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region of the second antibody are as shown in amino acids 26-33, 51-57 and 96-111 of SEQ ID No.5 and amino acids 26-34, 52-54 and 91-102 of SEQ ID No.7, respectively, or the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region of the second antibody are as shown in amino acids 26-34, 52-54 and amino acids 26-102 of SEQ ID No.7 and amino acids 26-33, 52-54 and amino acids 9-97 of the light chain variable region of the second antibody are as shown in amino acids 26-33-35 and 35-35 and 88-35.
11. The antibody composition of claim 11, wherein the amino acid sequences of the heavy and light chain variable regions of the first antibody are set forth in SEQ ID No.1 and SEQ ID No.3, respectively, and the amino acid sequences of the heavy and light chain variable regions of the second antibody are set forth in SEQ ID No.5 and SEQ ID No.7, respectively; alternatively, as shown in SEQ ID NO.9 and SEQ ID NO.11.
12. Use of an antibody composition according to claim 10 or 11 for the preparation of a novel medicament for the treatment and/or prophylaxis of coronavirus diseases.
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