CN114805556A - Neutralizing antibody for SARS-CoV-2 virus - Google Patents

Neutralizing antibody for SARS-CoV-2 virus Download PDF

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Publication number
CN114805556A
CN114805556A CN202110125913.6A CN202110125913A CN114805556A CN 114805556 A CN114805556 A CN 114805556A CN 202110125913 A CN202110125913 A CN 202110125913A CN 114805556 A CN114805556 A CN 114805556A
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antibody
variable region
light chain
heavy chain
chain variable
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唐宏
江陆斌
张晓明
孟璐
曹龙
李腾
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Nanjing Institute Of Life And Health Sciences
Institut Pasteur of Shanghai of CAS
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Nanjing Institute Of Life And Health Sciences
Institut Pasteur of Shanghai of CAS
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Priority to CN202110125913.6A priority Critical patent/CN114805556A/en
Priority to PCT/CN2022/074998 priority patent/WO2022161491A1/en
Publication of CN114805556A publication Critical patent/CN114805556A/en
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    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
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Abstract

The invention provides a monoclonal antibody with a neutralizing effect against SARS-CoV-2 virus. Meanwhile, the invention provides a method for detecting the neutralizing capacity and the binding capacity of the antibody. The preparation process of the antibody is simple, the product purity is high, the antibody is a fully human IgG antibody, and the antibody has a strong neutralizing effect with SARS-CoV-2 virus.

Description

Neutralizing antibody for SARS-CoV-2 virus
Technical Field
The invention belongs to the field of biotechnology or medicine, and particularly relates to preparation and application of a neutralizing monoclonal antibody for resisting SARS-CoV-2 virus.
Background
In acute infectious diseases, most of the infectious diseases are viral infectious diseases, the incidence rate of the viral infectious diseases is high, and the death rate is high. Because the detection and diagnosis means are limited, the outbreak of new epidemic caused by new viruses often has the characteristics of paroxysmal, random, unpredictable and the like, once the outbreak occurs, if no effective prevention and treatment means exists, the large-scale epidemic is very easy to cause, and the health and life safety of people is seriously threatened.
One of the key steps of SARS-CoV-2 virus invading host cell is that the RBD region of its structural protein Spike is combined with angiotensin converting enzyme 2(ACE2) as host cell membrane surface receptor to form ACE2-RBD complex. As early as 2 months in 2020, the protein structure of Spike protein has been resolved by the Zhouqiang team by cryoelectron microscopy. The Wrapp team has further shown that the affinity of the SARS-CoV-2S protein for binding to the host cell ACE2 receptor is more than 10 times higher than that of SARS-CoV. Based on these studies, monoclonal antibodies targeting the S protein were designed to block fusion of the virus to the host by competitively inhibiting binding of the S protein to the receptor, thereby inhibiting infection of the host cells by the virus, thereby preventing neocoronary infection or treating already infected patients. Antibody drugs will likely become a new therapeutic strategy.
Throughout the world, antibody drug development has long been a research hotspot, and most of the antibody drugs take S protein as a drug target. Currently, over 200 institutions are involved in antibody drug development in at least 26 countries. By day 6/11 of 2020, a total of 105 (mostly monoclonal antibodies) Antibody, polypeptide or recombinant protein development strategies for S protein design could be searched globally on the COVID-19Antibody Therapeutics Tracker, with first clinical 10 (JS016, TY027, CT-P59, BRII-196, BRII-198, SCTA01, MW33, STI-1499/COVI-SHIELD, SAB-185, IgY-110/anti-SARS-CoV-2IgY), second 2 (DXP-593, APN01), and third 4 (REGN-COV2, LY3819253, VIR-7831, AZD 7442).
However, currently there are only a few neutralizing antibodies to S protein approved for marketing, and there are only two in the global antibody therapy study: levilimab (BCD-089, Ilsira trade name) approved by the Russian Federal health agency in this year 6 and Itolizumab (EQ001, H-T1, T1-H) approved by India in 7 months are both used to control cytokine storms to reduce mortality in neonatal patients
At present, all domestic approved antiviral drugs have toxic and side effects to a certain extent and need to be used under the supervision of doctors. The convalescent plasma therapy, as one of the new coronary therapies approved at present, can be used for resisting virus infection to a certain extent, but has different plasma sources, relatively complex components, more adverse reactions and incapability of ensuring safety and effectiveness. The successful use of restorative plasma still suggests that neutralizing monoclonal antibodies may be used to treat new coronavirus infections.
In view of the foregoing, there is an urgent need in the art to develop effective diagnostic and therapeutic methods against SARS-CoV-2 coronavirus for the diagnosis and treatment of pneumonia caused by infection with the novel coronavirus.
Disclosure of Invention
The invention aims to provide an effective diagnosis and treatment method aiming at SARS-CoV-2 coronavirus, which is used for diagnosing and treating pneumonia caused by novel coronavirus infection.
In a first aspect of the invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region having complementarity determining regions CDRs selected from the group consisting of:
VH-CDR1 shown in SEQ ID NO. 3, VH-CDR2 shown in SEQ ID NO. 4, and VH-CDR3 shown in SEQ ID NO. 5;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO 2.
In a second aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region as described in the first aspect of the invention.
In another preferred embodiment, the heavy chain further comprises a heavy chain constant region.
In another preferred embodiment, the heavy chain constant region is of human or murine origin.
In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region.
In a third aspect of the present invention, there is provided a light chain variable region of an antibody, said light chain variable region having complementarity determining regions CDRs selected from the group consisting of:
VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO.9, and VL-CDR3 shown in SEQ ID NO. 10;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
In another preferred embodiment, the variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 7.
In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region as described in the third aspect of the invention.
In another preferred embodiment, the light chain further comprises a light chain constant region.
In another preferred embodiment, the light chain constant region is of human or murine origin.
In another preferred embodiment, the light chain constant region is a human antibody light chain lambda constant region.
In another preferred embodiment, the amino acid sequence of the light chain is shown in SEQ ID NO 7.
In a fifth aspect of the invention, there is provided an antibody having a heavy chain variable region as described in the first aspect of the invention, and/or a light chain variable region as described in the third aspect of the invention;
alternatively, the antibody has a heavy chain as described in the second aspect of the invention, and/or a light chain as described in the fourth aspect of the invention;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
In another preferred embodiment, the number of the amino acids to be added, deleted, modified and/or substituted is 1 to 5 (e.g., 1 to 3, preferably 1 to 2, and more preferably 1).
In another preferred embodiment, the derivative sequence which is added, deleted, modified and/or substituted with at least one amino acid and which retains the binding affinity of the RBD domain of the SARS-CoV-2S protein is an amino acid sequence having a homology or sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
In another preferred embodiment, the antibody further comprises a heavy chain constant region and/or a light chain constant region.
In another preferred embodiment, said heavy chain constant region is of human origin and/or said light chain constant region is of human origin.
In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region and the light chain constant region is a human antibody light chain λ constant region.
In another preferred embodiment, the heavy chain variable region of the antibody further comprises a framework region of human origin, and/or the light chain variable region of the antibody further comprises a framework region of human origin.
In another preferred embodiment, the heavy chain variable region of the antibody further comprises a murine framework region, and/or the light chain variable region of the antibody further comprises a murine framework region.
In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.
In another preferred embodiment, the antibody is a partially or fully humanized, or fully human monoclonal antibody.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the antibody is a full-length protein, or an antigen-binding fragment of an antibody.
In another preferred embodiment, the antibody is a bispecific antibody, or a multispecific antibody.
In another preferred embodiment, the antibody is in the form of a drug conjugate.
In another preferred embodiment, the antibody has one or more properties selected from the group consisting of:
(a) specifically binds to the RBD domain of SARS-CoV-2S protein;
(b) blocking the combination of SARS-CoV-2 virus and human angiotensin converting enzyme 2(ACE 2); and
(c) effectively neutralize SARS-CoV-2 virus infection.
In another preferred embodiment, the antibody has a heavy chain variable region according to the first aspect of the invention and a light chain variable region according to the third aspect of the invention;
wherein said heavy chain variable region and said light chain variable region comprise CDRs selected from the group consisting of:
(1) VH-CDR1 shown in SEQ ID NO. 3, VH-CDR2 shown in SEQ ID NO. 4, VH-CDR3 shown in SEQ ID NO. 5, VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO.9, and VL-CDR3 shown in SEQ ID NO. 10.
In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 7.
In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence set forth in SEQ ID NO. 2.
In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence set forth in SEQ ID NO. 7.
In a sixth aspect of the present invention, there is provided a recombinant protein comprising:
(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a seventh aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of: the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, the antibody according to the fifth aspect of the invention, or the recombinant protein according to the sixth aspect of the invention.
In another preferred embodiment, the polynucleotide encoding the heavy chain variable region is represented by SEQ ID NO. 1; and/or, the polynucleotide for encoding the light chain variable region is shown as SEQ ID NO. 6.
In an eighth aspect of the invention, there is provided a vector comprising a polynucleotide according to the seventh aspect of the invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a ninth aspect of the invention there is provided a genetically engineered host cell comprising a vector according to the eighth aspect of the invention or having integrated into its genome a polynucleotide according to the seventh aspect of the invention.
In a tenth aspect of the present invention, there is provided an antibody conjugate comprising:
(a) an antibody moiety selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or a combination thereof; and
(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnet, a viral coat protein, or a VLP, or a combination thereof.
In another preferred embodiment, said antibody moiety is coupled to said coupling moiety by a chemical bond or a linker.
In another preferred embodiment, the radionuclide includes:
(i) a diagnostic isotope selected from the group consisting of: tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
(ii) A therapeutic isotope selected from the group consisting of: lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, or a combination thereof.
In another preferred embodiment, the coupling moiety is a drug or toxin.
In another preferred embodiment, the drug is a cytotoxic drug.
In another preferred embodiment, the cytotoxic agent is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, an anti-metabolite drug, a chemotherapeutic sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycins/duocarmycins (duocarmycins), etoposides (etoposides), maytansinoids (maytansinoids) and maytansinoids (e.g., DM1 and DM4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indobenzodiazepines (indoxazepines) and benzodiazepines (oxyphenoxazepines)), or combinations thereof.
In another preferred embodiment, the toxin is selected from the group consisting of:
auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytansinoid, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthrax toxin dione, actinomycin, diphtheria toxin, Pseudomonas Exotoxin (PE) A, PE40, abrin a chain, modeccin a chain, alpha-sarcina, gelonin, mitogelonin (mitogellin), restrictocin (rettstricon), phenomycin, enomycin, curcin (curcin), crotin, calicheamicin, soapwort (Sapaonaria officinalis) inhibitor, glucocorticoid, or a combination thereof.
In another preferred embodiment, the conjugated moiety is a detectable label.
In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin).
In another preferred embodiment, the immunoconjugate comprises: multivalent (e.g., divalent) of (a).
In another preferred embodiment, the multivalent is (a) comprising multiple repeats in the amino acid sequence of the immunoconjugate.
In an eleventh aspect of the invention there is provided an immune cell which expresses or has exposed outside the cell membrane an antibody according to the fifth aspect of the invention.
In another preferred embodiment, the immune cells comprise NK cells and T cells.
In another preferred embodiment, the immune cell is from a human or non-human mammal (e.g., a mouse).
In a twelfth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) an active ingredient selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an antibody conjugate according to the tenth aspect of the invention, an immune cell according to the eleventh aspect of the invention, or a combination thereof; and
(ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a liquid preparation.
In another preferred embodiment, the pharmaceutical composition is an injection.
In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the antibody conjugate according to the tenth aspect of the present invention, the immune cell according to the eleventh aspect of the present invention, or the combination thereof, and 0.01 to 99.99% of the pharmaceutically acceptable carrier, wherein the percentages are mass percentages of the pharmaceutical composition.
In a thirteenth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an antibody conjugate according to the tenth aspect of the invention, an immune cell according to the eleventh aspect of the invention, or a combination thereof, wherein the active ingredients are used (a) in the preparation of a diagnostic reagent or kit for SARS-CoV-2 viral infection; and/or (b) preparing a medicament for preventing and/or treating SARS-CoV-2 virus infection.
In another preferred embodiment, the diagnostic reagent is a test strip or test plate.
In another preferred embodiment, the diagnostic reagent or kit is used for: detecting SARS-CoV-2S protein or a fragment thereof in the sample.
In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).
In a fourteenth aspect of the present invention, there is provided a method for in vitro detection of SARS-CoV-2 virus or SARS-CoV-2S protein or a fragment thereof in a sample, the method comprising the steps of:
(1) contacting said sample in vitro with an antibody according to the fifth aspect of the invention;
(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex indicates the presence of SARS-CoV-2 virus or SARS-CoV-2S protein or a fragment thereof in the sample.
In another preferred embodiment, said detection comprises diagnostic or non-diagnostic.
In a fifteenth aspect of the present invention, there is provided a kit comprising:
(1) a first container comprising an antibody according to the fifth aspect of the invention; and/or
(2) A second container comprising a secondary antibody directed against the antibody of the fifth aspect of the invention;
alternatively, the first and second electrodes may be,
the kit contains a detection plate, and the detection plate comprises: a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an antibody conjugate according to the tenth aspect of the invention, an immune cell according to the eleventh aspect of the invention, or a combination thereof.
In a sixteenth aspect of the present invention, there is provided a method of producing a recombinant polypeptide which is an antibody according to the fifth aspect of the present invention or a recombinant protein according to the sixth aspect of the present invention, the method comprising:
(a) culturing a host cell according to the ninth aspect of the invention under conditions suitable for expression; and
(b) isolating said recombinant polypeptide from the culture.
In a seventeenth aspect of the present invention, there is provided a pharmaceutical combination comprising:
(i) a first active ingredient comprising an antibody according to the fifth aspect of the invention, or a recombinant protein according to the sixth aspect of the invention, or an antibody conjugate according to the tenth aspect of the invention, or an immune cell according to the eleventh aspect of the invention, or a pharmaceutical composition according to the twelfth aspect of the invention, or a combination thereof;
(ii) a second active ingredient comprising an additional drug for the treatment of SAR-CoV-2 viral infection.
In another preferred example, the other drugs for treating SAR-CoV-2 virus infection include: other protective monoclonal antibodies or small molecular chemical drugs such as Reidesvir or other Chinese patent drugs.
In an eighteenth aspect of the present invention, there is provided a method for diagnosing SAR-CoV-2 virus infection, comprising the steps of:
(i) obtaining a sample from a subject, contacting said sample with an antibody according to the fifth aspect of the invention; and
(ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is a confirmed patient of SAR-CoV-2 virus.
In another preferred embodiment, the sample is a blood sample or a pharyngeal swab sample, or a sample from another tissue or organ.
In a nineteenth aspect of the present invention, there is provided a method for treating a disease infected with SARS-CoV-2 virus, comprising the steps of: administering to a subject in need thereof an effective amount of an antibody according to the fifth aspect of the invention, or a recombinant protein according to the sixth aspect of the invention, or an antibody conjugate according to the tenth aspect of the invention, or an immune cell according to the eleventh aspect of the invention, a pharmaceutical composition according to the twelfth aspect of the invention, or a combination thereof.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows an SDS-PAGE electrophoresis of neutralizing antibody expression.
Wherein lane 1 is protein marker; lane 2 is control antibody IgG; lane 3 is monoclonal antibody XG-Ab 008.
FIG. 2 shows the results of the antibody neutralization test and the antigen-antibody affinity test.
FIGS. 2A and 2B show the results of neutralization assay of candidate antibody Ab001-Ab010 against SARS-CoV-2 (FIG. 2A) and SARS-CoV lentivirus infection system (FIG. 2B). FIG. 2C shows the neutralizing effect of XG-Ab008 in the SARS-CoV-2 pseudovirus system, at about 1 nM. FIG. 2D shows the neutralizing effect of XG-Ab008 in SARS-Cov-2 euvirus system, approximately 4.5 nM.
FIG. 3 shows B cells sorted by flow cytometry to CD20-/CD38+/IgD-/IgM-/RBD +.
FIG. 4 shows the light and heavy chains amplified using reverse transcribed cDNA from B cells as a template: the successful amplification of the double-stranded template was: 3. 4, 6, 7 and 8.
FIG. 5 shows the affinity assay results for the XG-Ab008 neutralizing antibody with a KD of about 1.39 nM.
Detailed Description
The inventor of the invention develops an antibody which is highly effective and specific to 2019 novel coronavirus for the first time through extensive and intensive research and a large amount of screening. Specifically, the inventor screens out a monoclonal antibody which is extracted from a blood sample of a new coronavirus healer and has high binding force and neutralization effect on the surface Spike protein of SARS-Cov-2 virus. The experimental results show that the neutralizing antibody of the invention can effectively inhibit infection of pseudoviruses at a cellular level, and can play a role in neutralization by inhibiting the binding of ACE2 receptor and RBD protein.
Specifically, the inventor firstly separates PBMC from blood samples of COVID-19 healers, and obtains CD19+ B cells through magnetic bead enrichment. After adding SARS-CoV-2 target antigen marked by fluorescein for incubation, single IgG + B cell is flow sorted into 96-well plate lysate. Respectively carrying out PCR amplification on the heavy chain and the light chain of each single cell, and recombining and inserting the monoclonal antibody expression plasmid; after transfection of the recombinant plasmid into CHO cells, cell culture supernatants were collected and purified using protein G to obtain monoclonal antibodies. Through Vero-E6 cells expressing ACE2 in vitro and a SARS-Cov-2 pseudovirus expression system, the monoclonal antibody with high neutralization effect and high binding capacity on virus S protein is screened out.
On the basis of this, the present invention has been completed.
Antibodies
As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.
In the present invention, "VH-CDR 1" and "CDR-H1" are used interchangeably and refer to CDR1 of the heavy chain variable region; "VH-CDR 2" and "CDR-H2" are used interchangeably and refer to CDR2 of the heavy chain variable region; "VH-CDR 3" and "CDR-H3" are used interchangeably and refer to CDR3 of the heavy chain variable region. "VL-CDR 1" and "CDR-L1" are used interchangeably and refer to CDR1 of the light chain variable region; "VL-CDR 2" and "CDR-L2" are used interchangeably and refer to CDR2 of the light chain variable region; "VL-CDR 3" and "CDR-L3" are used interchangeably and refer to CDR3 of the light chain variable region.
The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, and are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as chimeric antibodies having a variable region derived from a murine monoclonal antibody, and a constant region derived from a human immunoglobulin (see, e.g., U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, which are hereby incorporated by reference in their entirety). Humanized antibodies refer to antibody molecules derived from non-human species having one or more Complementarity Determining Regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, herein incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.
In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.
TABLE A
Initial residue(s) Representative substitutions Preferred substitutions
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
In the present invention, the antibody is an antibody that specifically binds to the RBD domain of SARS-CoV-2S protein. The invention provides a high specificity and high affinity antibody directed against the RBD domain of SARS-CoV-2S protein, comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.
Preferably, the first and second electrodes are formed of a metal,
the heavy chain variable region (VH) has complementarity determining regions CDRs selected from the group consisting of:
VH-CDR1 shown in SEQ ID NO. 3,
VH-CDR2 shown in SEQ ID NO:4, and
VH-CDR3 shown in SEQ ID NO. 5;
the light chain variable region (VL) has Complementarity Determining Regions (CDRs) selected from the group consisting of:
VL-CDR1 shown in SEQ ID NO. 8,
VL-CDR2 shown in SEQ ID NO.9, and
VL-CDR3 shown in SEQ ID NO. 10;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence with homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: computer Molecular Biology (computerized Molecular Biology), Lesk, a.m. ed, oxford university press, new york, 1988; biological calculation: informatics and genomic Projects (Biocomputing: information and Genome Projects), Smith, d.w. eds, academic press, new york, 1993; computer Analysis of Sequence Data (Computer Analysis of Sequence Data), first part, Griffin, a.m. and Griffin, h.g. eds, Humana Press, new jersey, 1994; sequence Analysis in Molecular Biology (Sequence Analysis in Molecular Biology), von Heinje, g., academic Press, 1987 and Sequence Analysis primers (Sequence Analysis Primer), Gribskov, m. and Devereux, j. eds M Stockton Press, New York, 1991 and Carllo, h. and Lipman, d.s., SIAM j.applied Math., 48:1073 (1988). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG program package (Devereux, J. et al, 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al, 1990). BLASTX programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al, NCBI NLM NIH Bethesda, Md.20894; Altschul, S. et al, 1990). The well-known Smith Waterman algorithm can also be used to determine identity.
Preferably, the antibody described herein is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv), a single domain antibody (sdAb), and a single domain antibody (sign-domain antibody), and a monoclonal antibody or a polyclonal antibody produced from the above antibodies. The monoclonal antibody can be developed by various means and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the monoclonal antibody is prepared from wild-type or transgenic mice by the hybridoma technology in the mainstream.
The antibody full-length protein is conventional in the field, and comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and the light chain variable region of the protein, the humanized heavy chain constant region and the humanized light chain constant region form a fully humanized antibody full-length protein. Preferably, the antibody full-length protein is IgG1, IgG2, IgG3 or IgG 4; more preferably IgG 1.
The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and still more preferably a fully humanized antibody.
The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab') 2 or other antibody derivatives known in the art, and the like, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subtypes.
The single-chain antibody is a conventional single-chain antibody in the field and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
Among them, the animal is preferably a mammal such as a mouse.
The antibodies of the invention may be chimeric, humanized, CDR grafted and/or modified antibodies targeting the SARS-CoV-2Spike protein.
In the above-mentioned aspect of the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.
In the above-mentioned aspect of the present invention, the number of the amino acids to be added, deleted, modified and/or substituted may be 1 to 7, more preferably 1 to 5, still more preferably 1 to 3, and still more preferably 1 to 2.
In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 2.
In another preferred embodiment, the variable region of the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO. 7.
In an embodiment of the invention, the antibody targeting the RBD domain of the SARS-CoV-2S protein is XG-Ab 008.
In a more preferred embodiment, each antibody of the invention specifically comprises each of the following VL and VH sequences, as well as Fc, CL and CH1 sequences.
TABLE B XG-Ab008 antibody sequences
Figure BDA0002924009190000161
Encoding polynucleotides
The present invention also provides a polynucleotide encoding the above antibody or a recombinant protein comprising the same or a heavy chain variable region or a light chain variable region thereof.
Preferably, the nucleotide sequence of the nucleic acid for encoding the heavy chain variable region is shown as a sequence table SEQ ID NO 1; and/or the nucleotide sequence of the nucleic acid for encoding the light chain variable region is shown as the sequence table SEQ ID NO. 6.
More preferably, the nucleotide sequence of the nucleic acid for encoding the heavy chain variable region is shown as the sequence table SEQ ID NO 1; and the nucleotide sequence of the nucleic acid for coding the light chain variable region is shown as a sequence table SEQ ID NO. 6.
The preparation method of the nucleic acid is a preparation method which is conventional in the field, and preferably comprises the following steps: obtaining the nucleic acid molecule coding the protein by gene cloning technology, or obtaining the nucleic acid molecule coding the protein by artificial complete sequence synthesis method.
Those skilled in the art know that the base sequence of the amino acid sequence encoding the above protein may be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a polynucleotide homolog. The homologue of the polynucleotide of the present invention may be prepared by substituting, deleting or adding one or more bases of a gene encoding the protein sequence within a range in which the activity of the antibody is maintained.
Carrier
The invention also provides a recombinant expression vector comprising the nucleic acid.
Wherein said recombinant expression vector is obtainable by methods conventional in the art, i.e.: the nucleic acid molecule is connected to various expression vectors to construct the nucleic acid molecule. The expression vector is any vector conventionally used in the art so long as it can carry the aforementioned nucleic acid molecule. The carrier preferably comprises: various plasmids, cosmids, bacteriophages or viral vectors, etc.
The invention also provides a recombinant expression transformant containing the recombinant expression vector.
Wherein, the preparation method of the recombinant expression transformant is a preparation method which is conventional in the field, and preferably comprises the following steps: transforming the recombinant expression vector into a host cell. The host cell is any host cell conventionally used in the art, so long as it is sufficient that the recombinant expression vector is stably self-replicating and the nucleic acid carried thereby can be efficiently expressed. Preferably, the host cell is an e.coli TG1 or e.coli BL21 cell (expressing a single chain antibody or Fab antibody), or an HEK293 or CHO cell (expressing a full length IgG antibody). The recombinant expression plasmid is transformed into a host cell to obtain a recombinant expression transformant preferred in the present invention. Wherein the transformation method is a transformation method conventional in the art, preferably a chemical transformation method, a thermal shock method or an electric transformation method.
Preparation of antibodies
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, the DNA sequence encoding the antibody of the invention (or a fragment thereof, or a derivative thereof) has been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal. biochem.,107:220 (1980).
The antibody of the present invention may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
Antibody-drug conjugates (ADC)
The invention also provides an antibody-conjugated drug (ADC) based on the antibody of the invention.
Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.
The antibody of the invention may be conjugated to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent is a compound which enables effector molecules and antibodies to form covalent bonds, such as glutaraldehyde and the like. The coupling agent using carboxyl group may be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (coupling site is acylhydrazone).
Certain residues on the antibody (e.g., Cys or Lys, etc.) are used to attach to a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers) and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be attached to the antibody directly, or indirectly through a linker.
Antibodies may be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, the ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., the linker degrades at the site of interest, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may comprise, for example, a dipeptide such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.
Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and attachment is achieved by the reactive group. Thiol-specific reactive groups are preferred and include: for example maleimide compounds, haloamides (for example iodine, bromine or chlorine); halogenated esters (e.g., iodo, bromo, or chloro); halomethyl ketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodo, bromo, or chloro); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-bis- (mercuric methyl) dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiolsulfonate. The linker may comprise, for example, a maleimide linked to the antibody via a thiosuccinimide.
The drug may be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker links the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or keto group that may form a bond with the linker. In the case of a drug directly attached to a linker, the drug has a reactive group prior to attachment to the antibody.
In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, bifunctional linker compounds may be used to form ADCs in a two-step or multi-step process. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.
Generally, the functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, azide-based moieties may be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as those described in bioconjugation technology, second edition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of a drug moiety and a linker, each member of a complementary pair may be used for both the linker and the drug when the reactive functional group of the complementary pair is selected.
The present invention also provides a method of preparing an ADC, which may further comprise: the antibody is conjugated to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
In certain embodiments, the methods of the invention comprise: the antibody is conjugated to the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: the antibody linker conjugate is bound to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.
In some embodiments, the antibody drug conjugate ADC has the formula:
Figure BDA0002924009190000211
wherein:
ab is an antibody, and Ab is an antibody,
LU is a joint;
d is a drug;
and subscript p is a value selected from 1 to 8.
Applications of
The invention also provides the use of the antibodies, antibody conjugates ADC, recombinant proteins, and/or immune cells of the invention, for example for the preparation of a diagnostic formulation or for the preparation of a medicament.
Preferably, the medicament is a medicament for preventing and/or treating SARS-CoV-2 virus infection.
Detection use and kit
The antibodies of the invention or ADCs thereof may be used in detection applications, for example for the detection of samples, to provide diagnostic information.
In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens.
Preferably, the sample is a blood sample or a pharyngeal swab sample from a subject.
The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus biopsies as used in the present invention may comprise e.g. resection samples of tumours, tissue samples prepared by endoscopic methods or needle biopsy of organs.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit containing the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding immune cell, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, typically having a pH of from about 5 to about 8, preferably a pH of from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated.
The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection administration or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a granule, an injection or an infusion, etc.
The antibody of the present invention may also be used for cell therapy by intracellular expression of a nucleotide sequence, for example, for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
The pharmaceutical composition of the invention is a pharmaceutical composition for preventing and/or treating SARS-CoV-2 virus infection diseases.
The pharmaceutical composition of the present invention can be directly used for binding SARS-CoV-2S protein molecules or fragments thereof, and thus can be used for preventing and treating diseases caused by viral infection.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers. The medicinal carrier is a conventional medicinal carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable medicinal auxiliary material. The pharmaceutical adjuvant is conventional in the field, and preferably comprises pharmaceutically acceptable excipient, filler or diluent and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the protein and 0.01-99.99% of a pharmaceutical carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.
In the present invention, preferably, the pharmaceutical composition is administered in an effective amount, which is an amount that alleviates or delays the progression of the disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the result sought. One skilled in the art can determine an effective amount by using such factors as an individual basis and using no more than routine experimentation.
In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The invention provides the application of the pharmaceutical composition in preparing a medicament for preventing and/or treating SARS-CoV-2 virus infection diseases. Preferably, the SARS-CoV-2 virus infection is pneumonia.
Coronavirus (coronavirus)
As used herein, the terms "novel coronavirus", "2019-nCov" or "SARS-CoV-2" are used interchangeably, the 2019 novel coronavirus being the 7 th coronavirus known to infect humans and causing new coronary pneumonia (COVID-19), one of the serious infectious diseases threatening global human health.
Coronaviruses (CoV) belong to the family of the Nidovirales (Nidovirales) Coronaviridae (Coronaviridae), a enveloped positive-strand RNA virus, a subfamily of which contains four genera, alpha, beta, delta and gamma.
Among the coronaviruses known to infect humans, HCoV-229E and HCoV-NL63 belong to the genus alpha coronavirus, and HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all the genus beta coronavirus. SARS-CoV-2 is also known as 2019-nCov.
Highly pathogenic coronavirus "SARS-CoV" and "middle east respiratory syndrome" MERS-CoV both belong to the genus beta coronavirus. The novel coronavirus (SARS-CoV-2) has about 80% similarity to SARS-CoV and 40% similarity to MERS-CoV, and also belongs to the genus beta coronavirus.
The genome of the virus is a single-strand positive-strand RNA, is one of RNA viruses with the largest genome, and codes comprise replicase, spike protein, envelope protein, nucleocapsid protein and the like. In the initial stage of viral replication, the genome is translated into two peptide chains of up to several thousand amino acids, the precursor Polyprotein (Polyprotein), which is subsequently cleaved by proteases to yield nonstructural proteins (e.g., RNA polymerase and helicase) and structural proteins (e.g., spike protein) and accessory proteins.
The main advantages of the invention include:
(1) the preparation process of the antibody is simple. The invention uses the CHO expression system, has simple operation, can continuously express a large amount of antibodies and meets the requirements of downstream experiments and detection.
(2) The monoclonal antibody provided by the invention is a fully human antibody, and has no antigenicity of other heterologous proteins.
(3) The in vitro S protein lentivirus infection system verifies that the antibody has stronger neutralization effect (4.5nM) on SARS-CoV-2 virus.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The following basic experimental protocol is required to obtain the monoclonal antibodies described for the purposes of the invention.
1. Collecting 12-15ml of blood collected from the vein of a patient with new coronavirus infection and carrying out heparin anticoagulation.
2. Peripheral heparin anticoagulation mononuclear cells (PBMC) were obtained using Lymphoprep density gradient centrifugation
3. Enrichment of B cells in Peripheral Blood Mononuclear Cells (PBMC) using magnetic bead negative selection
4. Adding fluorescent antibody and SARS-CoV-2 target antigen marked by fluorescein for incubation, sorting single IgG + B cell by flow method into 96-well plate, theoretically collecting one cell per well, adding cell lysis solution containing RNase inhibitor into each well
5. Then extracting single B cell RNA, and reverse transcribing to cDNA
6. Taking cDNA as a template, carrying out First PCR, then taking the First PCR product as a template, amplifying light and heavy chains by a seq-PCR method, screening and only amplifying a single band template with correct size and sequencing.
7. Amplification of total light and heavy chain genes using the selected template
8. Recombining light and heavy chain gene to obtain expression vector
9. The expression vector was transfected into DH 5. alpha. competent cells and single clones were selected by screening with agarose plates containing ampicillin.
10. Selected monoclonals are cultured in small volume and the expression vector is purified
11. And (3) amplifying light and heavy chains by using a PCR method, sequencing, comparing the light and heavy chain sequences obtained in the step (6) and the step (11), and screening out light and heavy chain vectors with consistent results.
12. Transfecting an expression vector into a CHO cell, expressing and purifying to obtain the fully human antibody.
13. In a BSL-2 laboratory, 10 candidate antibody strains with a neutralizing effect are screened and obtained in an established S protein lentivirus infection system: ab001, 002, 003 to 010. By comparing the neutralization effect, the target-specific neutralizing antibody in 3.1 was obtained: XG-Ab 008. (Ab 008 for short)
14. In addition, the binding coefficient of XG-Ab008 to the S protein RBD was obtained using Octet RED 96 protein interaction workstation fitting.
The monoclonal neutralizing antibody XG Ab-008 can be obtained by collecting CHO cell expression supernatant solution and purifying through the steps 1-12, and the purity of the purified candidate antibody can meet the requirement of subsequent experiments as shown by SDS-PAGE results (figure 1).
The inhibitory effect of the candidate antibody against SARS-CoV-2 lentivirus (FIG. 2A) and SARS-CoV lentivirus (FIG. 2B) was obtained by using Vero-E6 cells overexpressing ACE2 as responder cells in the steps 13 to 14 as described above and using pseudovirus systems expressing SARS-CoV-2 and SARS-CoV S proteins, respectively, and it was confirmed that Ab008, a candidate antibody, has a specific neutralizing ability against SARS-CoV-2 virus. Among them, Ab008 antibody specifically had a good neutralizing effect against SARS-CoV-2 of 1nM (FIG. 2C). After the neutralization effect on SARS-CoV-2 in a euvirus infection system can reach 4.5nM (figure 2D) and the S-RBD structure domain is solidified by a streptavidin sensor (the RBD detection gradient concentration range is 1.56-50nM), an Octet instrument detects the affinity of a candidate antibody Ab008, and the antibody has very strong S-RBD binding capacity (KD-1.39 nM) (figure 5)
Example 1: obtaining mononuclear cells (PBMC) from blood of patients with new crown recovery
Collecting 12-15ml of heparin anticoagulated blood of a patient with a new crown recovery, centrifuging for 10 minutes at 500g, and sucking upper plasma. Then, according to the following steps of 1: adding physiological saline into 1 volume of the mixture to dilute the lower layer blood cells, and uniformly mixing. The mixture was slowly added to a centrifuge tube containing an equal volume of Lymphoprep solution, and mononuclear cells (PBMC) were obtained by density gradient centrifugation.
The formulations of the solutions required in the above examples are as follows:
physiological saline: 0.9 percent of sodium chloride, ultrapure water and a 0.22um filter membrane are used after filtration.
Lymphoprep solution: LymphopreptM, 1 XD-PBS, 0.9% sodium chloride, 100 Xstreptomycin double antibody, 0.5M EDTA, adjusted to pH 8.0.
Example 2: b cell enrichment
LS column was prepared according to the method of the commercial description.
"antibody dilution mixture" was prepared by diluting 0.6ul each of biotin-labeled anti-CD 3, anti-CD 11b, anti-CD 14, anti-CD 16, anti-CD 56, and anti-CD 235a antibodies in 150ul of antibody dilution.
"an avidin magnetic bead suspension" was prepared, and 20ul of avidin magnetic beads were added to 200ul of the antibody dilution.
The PBMC suspension prepared in example 1 was incubated with the antibody dilution mixture according to the commercial instructions, after washing off excess antibody, the treated PBMC suspension was incubated with the anti-biotin magnetic bead suspension and the enriched B cells were collected with LS gum.
The formulations of the solutions required in the above examples are as follows:
MACS solution: 10ml Fetal Bovine Serum (FBS), 5mM EDTA, 500ml 1 XDPBS, adjusted to pH 8.0, 0.22um filter, using 4 ℃ storage.
Example 3: b cell sorting
Prepare Mouse FC dilution: to 100ul of the antibody dilution, 4ul mouse FC was added
Preparing flow-type fluorescent antibody dilution and mixing: the following antibodies were added to 100ul of the antibody dilution
Antibody/protein name Fluorescein Dosage of
S-RBD FITC 5ug/ml
S-RBD PE 5ug/ml
IgA VioBlue(PB channel) 1/25
IgM A647 1/100
IgD BV510 1/100
CD3 PerCPCy5.5 1/250
CD20 BV785 1/100
CD38 PECy7 1/250
The enriched B cells from example 2 were used, 100ul Mouse FC dilution was added and incubated for 15 min at room temperature in the dark. Excess antibody was washed away with MACS, 100ul of flow-through fluorescent antibody dilution was added, and incubated for 15 min at room temperature in the dark. After washing away excess antibody with MACS, individual B cells that specifically bound S-RBD were sorted using flow cytometer Melody (FIG. 3). Sorted B cells were collected on a 96-well PCR plate containing 4ul of cell lysate, one cell per well, immediately after sorting, membrane-sealed and snap frozen with dry ice.
The formulations of the solutions required in the above examples are as follows:
lysis solution: 300ul RNase, 40U/ul RNase inhibitor, 200ul 1 DPBS, 400ul DL-DTT (100mM), 3100ul nuclease free water.
Example 4: screening candidate antibody expression vector templates
The individual B cells sorted in example 3 were inverted with SuperScript III RT reverse transcriptase (Invitrogen,18080-044) to obtain cDNA.
Thereafter, 1st PCR, sequencing PCR and cloning PCR were carried out using TransTaq High Fidelity (HiFi) PCR Supermix II (Transgen, AS131-22) using cDNA AS a template according to commercial instructions, and light and heavy chains were identified by running agarose gel. The light and heavy chain pairs that could be matched were chosen (fig. 4).
Example 5: construction of expression vector, antibody expression and purification
The light chain expression plasmid and the heavy chain expression plasmid were constructed from the paired light and heavy chain sequences obtained in example 4, using AbVec hIgG antibody expression plasmid as a backbone, and the method described in the ExpicHO expression System (ExpicCHO) commercial Specification TM Expression System Kit, a29133), transfected and expressing CHO cells. The cell suspension was collected by centrifugation at 500G for 10min and submitted to the commercial instructions of Protein G Sepharose (Protein G)
Figure BDA0002924009190000271
4Fast Flow, 17-0618-01), purification of IgG antibodies
Example 6: antibody neutralization effect detection
Preparing SARS-Cov-2 pseudovirus, constructing expression vector containing: mouse Leukemia Virus (MLV) gag/pol, retrovirus EGFP, SARS-CoV-2S protein full-length sequence (Wuhan-Hu-1, GenBank: QHD43419.1) or SARS-CoV S protein full-length sequence (AY 569693). HEK293T was transfected with PEI transfection reagent as described in commercial instructions and after 48 hours the pseudovirus was collected in the supernatant. Subsequently, VeroE6 cells highly expressing hACE2 were infected with the premixed pseudovirus/antibody suspension as an experimental group (SD, n ═ 3) (fig. 2A), and the neutralizing effect of the antibody by flow analysis was about-10 nM (fig. 2C) with the pseudovirus suspension alone as a control group (SD, n ═ 3) (fig. 2B).
Example 7: antibody affinity detection
Biotin solution (10mM), ZB desalting column 130ul, was prepared as described in the Octet RED 96 protein interaction workstation commercial instructions. Labeled S protein was incubated with Biotin solution 1:1 and excess unbound Biotin was removed using a desalting column. S protein samples, XG-Ab008 gradient dilutions 50mM, 25mM, 12.5mM, 6.25mM, 3.125mM, 1.563mM were added sequentially to 96-well plates. The binding coefficient of XG-Ab008 to S protein was fitted to the instrumental values (FIG. 2D).
Discussion of the related Art
The existing common SARS-CoV-2 antibody screening technology includes hybridoma, bacteriophage, transgenic mouse, single B cell cloning technology, etc. The immunogenicity of antibodies can be greatly reduced by humanizing conventional murine antibodies to produce fully human antibodies (REGN-COV 2). With the development of flow cytometry, single B cell separation techniques have been widely used. Through marking the surface antigen of the B cell, single B cells with specific antigen are screened, and then the single B cells sorted are subjected to antibody gene amplification. Then, antigen-specific neutralizing antibodies (BRII-198 and BRII-196, university of Qinghua) are obtained by methods such as vector construction and antibody expression. Screening for effective neutralizing antibodies by high throughput single cell sequencing is also available (DXP-593, Baiji Shenzhou)
Although the technology of monoclonal antibodies targeting SARS-CoV-2 virus S protein is becoming more and more sophisticated, different sorting strategies are used, and the amplification conditions still show a very different screening result. In this regard, the present invention provides a strategy based on single B cell expansion and expression of monoclonal antibodies, and the selected high neutralizing monoclonal antibodies targeting S protein.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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Qilin Institute of innovation, Shanghai Pasteur Institute, Chinese Academy of Sciences
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Claims (10)

1. An antibody heavy chain variable region having Complementarity Determining Regions (CDRs) selected from the group consisting of:
VH-CDR1 shown in SEQ ID NO. 3, VH-CDR2 shown in SEQ ID NO. 4, and VH-CDR3 shown in SEQ ID NO. 5;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
2. An antibody heavy chain having the heavy chain variable region of claim 1.
3. An antibody light chain variable region having Complementarity Determining Regions (CDRs) selected from the group consisting of:
VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO.9, and VL-CDR3 shown in SEQ ID NO. 10;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
4. A light chain of an antibody, wherein said light chain has the variable region of the light chain of claim 3.
5. An antibody having the heavy chain variable region of claim 1, and/or the light chain variable region of claim 3;
alternatively, the antibody has a heavy chain according to claim 2, and/or a light chain according to claim 4;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
6. A recombinant protein, said recombinant protein comprising:
(i) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
7. A polynucleotide encoding a polypeptide selected from the group consisting of: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, the antibody of claim 5, or the recombinant protein of claim 6.
8. A vector comprising the polynucleotide of claim 7.
9. A genetically engineered host cell comprising the vector or genome of claim 8 having the polynucleotide of claim 7 integrated therein.
10. An antibody conjugate, comprising:
(a) an antibody moiety selected from the group consisting of: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, the antibody of claim 5, the recombinant protein of claim 6, or a combination thereof; and
(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnet, a viral coat protein, or a VLP, or a combination thereof.
CN202110125913.6A 2021-01-29 2021-01-29 Neutralizing antibody for SARS-CoV-2 virus Pending CN114805556A (en)

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