CN117736314A - Neutralizing antibodies against SARS-CoV-2 and variants thereof and uses thereof - Google Patents

Neutralizing antibodies against SARS-CoV-2 and variants thereof and uses thereof Download PDF

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CN117736314A
CN117736314A CN202311088752.3A CN202311088752A CN117736314A CN 117736314 A CN117736314 A CN 117736314A CN 202311088752 A CN202311088752 A CN 202311088752A CN 117736314 A CN117736314 A CN 117736314A
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antibody
seq
antigen
binding fragment
heavy chain
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王乔
张璐楠
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Advaccine Suzhou Biopharmaceutical Co ltd
Fudan University
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Advaccine Suzhou Biopharmaceutical Co ltd
Fudan University
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Abstract

The present invention relates to highly neutralizing antibodies and antigen binding fragments thereof directed against SARS-CoV-2 and variants thereof, and methods of making and using the neutralizing antibodies and antigen binding fragments thereof. The isolated antibody or antigen binding fragment thereof specifically binds to the S protein of SARS-CoV-2, and is capable of efficiently neutralizing SARS-CoV-2 and variants thereof, including but not limited to Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and Omicron variants, including but not limited to B.1.1.529, BA.1, BA.2, BA.2.12.1, BA.3, BA.4, and BA.5.

Description

Neutralizing antibodies against SARS-CoV-2 and variants thereof and uses thereof
Technical Field
The present invention relates to highly neutralizing antibodies and antigen binding fragments thereof directed against SARS-CoV-2 and variants thereof, and methods of making and using the neutralizing antibodies and antigen binding fragments thereof.
Background
Variants of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to occur and spread worldwide. To date, 5 high-interest Variants (VOCs) have been identified, including Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and newly identified Omicron (B.1.1.529) variants (Karim, S.S.A.et al. (2021). Omicron SARS-CoV-2 variant:a new chapter in the COVID-19pandemic.Lancet 398,2126-2128;Mannar,D.et al. (2022). SARS-CoV-2 Omicron variant:Antibody evasion and cryo-EM structure of spike protein-ACE2 complex.science 375,760-764; viana, R. (2022). Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa.Nature 603,679-686). These VOCs harbor mutations in the viral spike protein (S protein), not only increasing viral infectivity or virulence, but also facilitating immune escape (Altmann, D.M., et al (2021) Immunity to SARS-CoV-2 variants of concern.Science 371,1103-1104; mlcochova, P., et al (2021) SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion.Nature 599,114-119; planas, D., et al (2021) Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization.Nature 596,276-280; wang, G.L., et al (2021) Susceptibility of Circulating SARS-CoV-2 Variants to Neutralization.N Engl J Med 384,2354-2356; wang, P., et al (2021) Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7.Nature 593,130-135).
Many monoclonal antibodies (mabs) identified from convalescent or vaccinated individuals showed reduced or abolished neutralizing activity against various VOCs (Schmidt, f., et al (2021). High genetic barrierto SARS-CoV-2 p olyclonal neutralizing antibody escape.Nature 600,512-516; wang, k., et al (2022). MemoryB cell repertoire from triple vaccinees against diverse SARS-CoV-2 variants.Nature). In particular, the emerging omacron variant encodes 37 amino acid substitutions in the viral S protein, 15 of which are located in the Receptor Binding Domain (RBD) and which cause significant humoral immune escape, which presents significant challenges for the effectiveness of vaccine and mAb therapies (Cameroni, e., et al (2022) Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift.Nature 602,664-670; cao, y., et al (2022) Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies.Nature 602,657-663; carreno, j.m., et al (2022) Activity of convalescent and vaccine serum against SARS-CoV-2 Omicron.Nature602,682-688; cele, S., et al (2022) Omicron extensively but incompletely escapes Pfizer BNT b2 linkage. Nature602,654-656; iketani, S., et al (2022) Antibody evasion properties of SARS-CoV-2 Omicron sublineages.Nature;Liu,L, et al (2) Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2.202602, 676-681, plas, et al (2022) and 676-681, p.v. 20292-675, et al (2022).
These emerging SARS-CoV-2 variants with strong immune escape capability have prompted researchers to identify broad-spectrum neutralizing antibodies (bNAb) that may have potential clinical benefits. The combination of two mabs recognizing two different epitopes is a popular strategy to increase the broad spectrum of neutralization and avoid escape mutations in viruses (Baum, a., et al (2020). Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies.Science 369,1014-1018; dong, j., et al (2021). Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-anti-cocktail. Biorxiv; li, c., et al (2022). Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain anti-body. Cell 185,1389-1401.e 1318). For example, a combination of two RBD-binding mAbs, bamlanivimab (LY-CoV 555) and etesevelimab (Eli Lilly) has been approved for emergency use after exposure to SARS-CoV-2 virus (Dougan, M., et al (2021), bamlanivimab plus Etesevimab in Mild or Moderate Covid-19.N Engl J Med 385,1382-1392). The combination of Tixagevimab (AZD 8895) and cilgavimab (AZD 1061) showed prophylactic and therapeutic efficacy in non-human primate models of SARS-CoV-2 infection (lo, y.m., et al (2022). The SARS-CoV-2 monoclonal antibody combination,AZD7442,is protective in nonhuman primates and has an extended half-life in human sci trans Med 14, eabl 8124). Bispecific antibodies formed by linking two single domain antibodies n3113v and n3130v have also been shown to produce excellent broad spectrum and high efficacy of neutralization by inhaled administration (Li, c., et al (2022). Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody. Cell 185,1389-1401.e 1318).
The use of only a single monoclonal bNAb with broad spectrum and higher neutralizing potency may also have significant efficacy for clinical prophylaxis or treatment. For example, LY-CoV1404 (also known as bebtelovimab) showed excellent neutralizing activity against various SARS-CoV-2 variants, independent of the mutation of most of these variants (Iketani, S., et al (2022). Antibody evasion properties of SARS-CoV-2 Omicron sublineages.Nature;Westendorf,K, et al (2022). LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.bioRxiv;Zhou,T, et al (2022). Structural basis for potent antibody neutralization of SARS-CoV-2 variants including B.1.1.529.Science,eabn8897).
However, the number of superantibodies with extremely broad spectrum of activity and ultra-high potency is still very limited. Many RBD neutralizing antibodies have been shown to have a varying degree of reduction in neutralization potency for Omicron (Yunlong Cao, et al (2021): omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies.Nature.https:// doi. Org/10.1038/d 41586-021-03796-6). Omicron is able to escape most therapeutic monoclonal antibodies and vaccine raised antibodies (Delphine Planas, et al (2021): considerable escaple of SARS-CoV-2 Omicron to antibody neutralization.Nature.https:// doi. Org/10.1038/d 41586-021-03827-2). It has been found that new Omicron mutants, such as ba.2.12.1, ba.4and ba.5, may evolve to mutate, thereby evading humoral immunity caused by ba.1 infection, suggesting that ba.1-derived vaccine boosters may not be able to achieve broad spectrum protection against the new Omicron mutant (Yunlong Cao, et al (2022). BA.2.12.1, BA.4andBA.5escape antibodies elicitedby Omicron in section. Nature. Https:// doi.org/10.1038/s 41586-022-04980-y).
In the present invention, the inventors isolated mAbs from the recovery of infection with excellent serum neutralization activity and identified a strain of fully human IgG mAb targeting SARS-CoV-2S protein, designated "antibody 09C7" or "09C7". The antibodies have extremely high neutralizing efficacy against known VOCs with severe immune escape capacity and the newly occurring Omicron variants BA.1, BA.2, BA.2.12.1, BA.3, BA.4 and BA.5.
Disclosure of Invention
The present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to the S protein of SARS-CoV-2, characterized in that said isolated antibody or antigen-binding fragment thereof comprises the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3 of the amino acid sequences shown in SEQ ID NOs 1, 2 and 3, and the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 of the amino acid sequences shown in SEQ ID NOs 4, 5 and 6.
In one embodiment, an antibody or antigen binding fragment thereof of the invention comprises the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3 of the amino acid sequences shown in SEQ ID nos. 1, 2 and 11, and the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 of the amino acid sequences shown in SEQ ID nos. 4, 5 and 6. In one embodiment, an antibody or antigen binding fragment thereof of the invention comprises the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3 of the amino acid sequences shown in SEQ ID nos. 1, 2 and 14, and the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 of the amino acid sequences shown in SEQ ID nos. 4, 5 and 6.
In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH of the amino acid sequence shown in SEQ ID No. 7 and a light chain variable region VL of the amino acid sequence shown in SEQ ID No. 8.
In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH of the amino acid sequence shown in SEQ ID No. 12 and a light chain variable region VL of the amino acid sequence shown in SEQ ID No. 8. In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH of the amino acid sequence shown in SEQ ID No. 15 and a light chain variable region VL of the amino acid sequence shown in SEQ ID No. 8.
In one embodiment, an isolated antibody or antigen binding fragment thereof described herein comprises a mutation in the heavy chain constant region. In one embodiment, the mutation is selected from LS, kdel, GRLR, YTE, LALA and combinations thereof.
In one embodiment, the isolated antibody or antigen binding fragment thereof described herein is of the IgG1, igG2, igG3 or IgG4 type.
In one embodiment, the antibody or antigen binding fragment thereof of the invention comprises a heavy chain and a light chain of an amino acid sequence selected from the group consisting of seq id nos:
A heavy chain shown in SEQ ID NO. 9 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 13 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 16 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 17 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 18 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 19 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 20 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 21 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 22 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 23 and a light chain shown in SEQ ID NO. 10; and
a heavy chain shown in SEQ ID NO. 24 and a light chain shown in SEQ ID NO. 10.
In one embodiment, the isolated antibody or antigen-binding fragment thereof described herein is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment thereof.
In one embodiment, the antigen binding fragment is a Fab fragment, a Fab 'fragment, F (ab') 2 Fragments, fv fragments, diabodies or single chain antibody molecules.
The invention also provides an isolated polynucleotide encoding an isolated antibody or antigen binding fragment thereof as described herein.
The invention also provides an expression vector comprising a polynucleotide as described herein.
The invention also provides a host cell comprising an expression vector as described herein.
The invention also provides a method of producing an isolated antibody or antigen-binding fragment thereof as described herein, characterized in that the method comprises culturing a host cell as described herein under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof produced by the host cell.
The invention also provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier.
The invention also provides the use of the isolated antibody or antigen binding fragment thereof in the manufacture of a medicament for treating or preventing SARS-CoV-2 and mutant infections thereof in a subject in need thereof.
In one embodiment, the mutants include, but are not limited to Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) and Omicron variants. In further embodiments, the omacron variant includes, but is not limited to, b.1.1.529, ba.1, ba.2, ba.2.12.1, ba.3, ba.4 and ba.5.
In certain aspects, the invention provides methods for treating or preventing SARS-CoV-2 and mutant infections thereof in a subject in need thereof, comprising administering to the subject an effective amount of at least one isolated antibody or antigen-binding fragment thereof disclosed herein.
Drawings
The present invention will be better understood when considered in conjunction with the accompanying drawings.
Fig. 1.09C7 neutralization potency. Luciferase-based pseudoviruses of SARS-CoV-1 and five SARS-CoV-2 VOCs (A), SARS-CoV-1 and four SARS-CoV-2 variants (B), and six Omicron variants (C) were used for cell infection, luciferase signals after infection were measured as infection substitutes, and normalized to no antibody control (dotted line). In vitro neutralization assays of antibodies were performed at least twice, expressed as mean ± SEM. IC (integrated circuit) 50 Values, average of two independent experiments.
FIG. 2 (A) 09C7 and control antibody LY-CoV1404 (bebtelovimab) induced in vitro ADE effects. In vitro ADE assays were performed in Raji cells by using SARS-CoV-2 pseudovirus that expresses luciferase. The presence of various dilutions of antibody induced different levels of luciferase signal. (B) The 09C7 variants of GRLR and LALA forms did not induce ADE effects.
The various variants of figure 3.09C7 maintained in vitro neutralising activity against b.1.1.529 (Omicron) pseudovirus. Kdel: mAb mutants lacking heavy chain C-terminal lysine in order to maintain antibody homogeneity (A, B, C and E). And YTE: mAb mutants with triple mutations M255Y, S257T and T259E in the Fc domain (B, C and E). LS: mAb mutant (D) with M431L and N437S mutations in the Fc domain. Both YTE and LS substitutions resulted in increased binding to human FcRn and an extended serum half-life of the antibody. GRLR: mAb mutants (C and D) with G239R and L331R mutations in the Fc domain. LALA: mAb mutants (C and E) with L237A and L238A mutations in the Fc domain. Both GRLR and LALA substitutions abrogated antibody binding to fcγr and abrogated ADE effect. C109Y: mAb mutant (E) with a C109Y mutation in the heavy chain complementarity determining region CDRH 3. C109S: mAb mutant (E) with a C109S mutation in the heavy chain complementarity determining region CDRH 3.
FIG. 4 efficacy of antibodies 09C7, 09C7-C109S and 09C7-C109Y in neutralizing SARS-CoV-2 and variants thereof in vitro. A: wild type; b: alpha (B.1.1.7); c: beta (B.1.351); d: gamma (P.1); e: delta (B.1.617.2); f: omicron (b.1.1.529); g: b.1.351-L242H; h: b.1.617.1 (Kappa); i: c.37 (Lambda); j: b.1.621 (Mu); k: omicron (ba.2); l: omicron (ba.2.12.1); m: omicron (ba.3); n: omicron (BA.4/5).
FIG. 5 efficacy of antibodies 09C7, 09C7-C109S-YTE-LALA-Kdel and Bebtelovimab in neutralizing SARS-CoV-2 and its Omicron variant in vitro. A: wild type; b: omicron (ba.4/5); c: omicron (ba.2); d: omicron (ba.2.75).
FIG. 6 neutralizing effect of antibodies 09C7-C109S-YTE-LALA-Kdel and Bebtelovimab on SARS-Cov-2 OmicronBA.2 in Vero cells.
FIG. 7 neutralizing effect of antibodies 09C7-C109S-YTE-LALA-Kdel and Bebtelovimab on SARS-Cov-2 OmicronBA.5 in Vero cells.
FIG. 8 effect of antibody 09C7-C109S-YTE-LALA-Kdel on live viral load in the lungs of BA.2-challenged mice.
FIG. 9 effect of antibody 09C7-C109S-YTE-LALA-Kdel on live viral load in the lungs of BA.5-challenged mice.
FIG. 10 SEC-HPLC purity chromatograms of antibodies 09C7-C109S-YTE-LALA-Kdel, 09C7-YTE-LALA-Kdel and 09C 7.
FIG. 11 non-reducing SDS-PAGE blue of antibodies 09C7-C109S-YTE-LALA-Kdel, 09C7-YTE-LALA-Kdel and 09C 7.
FIG. 12 reduction SDS-PAGE blue gel of antibodies 09C7-C109S-YTE-LALA-Kdel, 09C7-YTE-LALA-Kdel and 09C 7.
FIG. 13 non-reducing immunoblots of antibodies 09C7-C109S-YTE-LALA-Kdel, 09C7-YTE-LALA-Kdel and 09C 7.
Detailed Description
For convenience, the following sections outline the various meanings of the terms used herein, followed by discussion of various aspects regarding the antibodies or antigen-binding fragments thereof, followed by a demonstration of various embodiments of the antibodies or antigen-binding fragments thereof by specific examples.
1. Definition of the definition
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms such as "comprising" is not limiting and encompasses the term "consisting of … …".
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents or portions of documents (including but not limited to patents, patent applications, articles, books, and treatises) cited in this application are hereby incorporated by reference in their entirety for any purpose.
The term "S-protein", also known as spike protein, is one of the structural proteins of a virus that mediates the entry of the virus into a host cell. The S-protein contains 1273 amino acids, including a large extracellular domain (S-ECD), one transmembrane helix, and a small intracellular C-terminus. Two major domains within the S-ECD have been identified as the S1 head region and the S2 stem region, and the critical receptor-binding domain (RBD) is located in the S1 portion.
The term "polynucleotide" or "nucleic acid" includes both single-and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides or any type of nucleotide in modified form. The modification includes: base modifications such as bromouridine and inosine derivatives; ribose modifications, such as 2',3' -dideoxyribose; and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenate, phosphorodiselenate, phosphorophosphoramidate, and the like. The term "oligonucleotide" means a polynucleotide comprising 200 nucleotides or less. In certain embodiments, the oligonucleotide is 10-60 bases long. In other embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. The oligonucleotide may be single-stranded or double-stranded, for example, for constructing a mutant gene. The oligonucleotide may be a sense or antisense oligonucleotide. The oligonucleotides may include labels for detection assays, including radiolabels, fluorescent labels, haptens or antigenic labels. The oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.
The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) used to transfer protein-encoding information to a host cell.
The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and/or modulates the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to: sequences that affect or regulate transcription and translation; and sequences that, if present, affect RNA splicing of the coding region to which they are operably linked.
As used herein, "operably linked" means that the components in which the term is employed are in a relationship that permits the components to perform their inherent functions under the appropriate conditions. For example, a regulatory sequence in a vector "operably linked" to a protein coding sequence is linked to the protein coding sequence such that expression of the protein coding sequence is accomplished under conditions compatible with the transcriptional activity of the regulatory sequence.
The term "host cell" means a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a gene of interest. Whether the progeny are identical in morphology or in genetic constitution to the original parent cell, the term encompasses the progeny of the parent cell as long as the progeny have the gene of interest.
The term "transfection" means the uptake of foreign or exogenous DNA by a cell, which is "transfected" when the exogenous DNA is introduced into the cell membrane. A variety of transfection techniques are well known in the art and are disclosed herein. See, e.g., graham et al, 1973,Virology 52:456; sambrook et al, 2001,Molecular Cloning:A Laboratory Manual; davis et al, 1986,Basic Methods in Molecular Biology,Elsevier; chu et al, 1981, gene 13:197. The techniques may be used to introduce one or more exogenous DNA portions into a suitable host cell.
The term "transformation" refers to the alteration of a genetic trait in a cell that has been transformed when the cell has been modified to contain new DNA or RNA. For example, a cell is transformed when the natural state of the cell is genetically modified by introducing new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transformed DNA may be recombined with cellular DNA by physically integrating into the cell chromosome, or may be transiently retained as an extrachromosomal element that is not replicated, or may be replicated independently as a plasmid. When the transformed DNA replicates as the cell divides, the cell is considered to have been "stably transformed".
The term "amino acid" includes its ordinary meaning in the art. Conventional single letter amino acid codes and three letter amino acid codes are used herein, as shown in table 1.
TABLE 1
Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids such as alpha-, alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components of the antibodies of the invention. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamic acid, epsilon-N, N, N-trimethyllysine, epsilon-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma-N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left hand direction is the amino terminal direction and the right hand direction is the carboxy terminal direction, consistent with standard usage and habits.
A "variant" of a protein (e.g., an antibody) comprises an amino acid sequence in which one or more amino acid residues are inserted, deleted, and/or substituted in the amino acid sequence relative to another protein sequence. Variants include fusion proteins.
The term "identity" refers to the relationship between two or more polypeptide molecules or two or more nucleic acid molecule sequences as determined by aligning and comparing the sequences. "percent identity" means the percentage of identical residues between amino acids or nucleotides within a molecule being compared, calculated based on the smallest molecule being compared. For these calculations, the gaps (if any) in the alignment are preferably accounted for by a specific mathematical model or computer program (i.e., an "algorithm"). Methods useful for calculating the identity of aligned nucleic acids or polypeptides include those described in the following documents: computational Molecular Biology, (Lesk, a.m. edit), 1988,New York:Oxford University Press; biocomputing Informatics and Genome Projects, (Smith, d.w. edit), 1993,New York:Academic Press; computer Analysis of Sequence Data, partI, (Griffin, A.M. and Griffin, H.G. editions), 1994,New Jersey:Humana Press; von Heinje, g.,1987,Sequence Analysis in Molecular Biology,New York:Academic Press; sequence Analysis Primer, (Gribskov, m. And deveerux, j. Edit), 1991,New York:M.Stockton Press; and Carilo et al, 1988,SIAM J.Applied Math.48:1073.
In calculating the percent identity, sequences to be compared are typically aligned in such a way as to obtain the greatest match between the sequences. One example of a computer program that may be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al, 1984,Nucl.Acid Res.12:387;Genetics Computer Group,University of Wisconsin,Madison,WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which the percentage of sequence identity is to be determined. The optimally matched sequences are aligned according to their respective amino acids or nucleotides (the "match span" is determined by an algorithm). Gap opening penalties (calculated as a 3x average diagonal, where the "average diagonal" is the average of the diagonals of the comparison matrix used; the "diagonal" is the score or value assigned to each perfect amino acid match by a particular comparison matrix) and gap extension penalties (which are typically 1/10 times the gap opening penalty) are used in conjunction with the method, as well as comparison matrices such as PAM250 or BLOSUM62. In certain embodiments, the algorithm also uses a standard comparison matrix (see Dayhoff et al, 1978,Atlas of Protein Sequence and Structure,5:345-352 for a PAM250 comparison matrix; see Henikoff et al, 1992, proc. Natl. Acad. Sci. U.S. A.89:10915-10919 for a BLOSUM62 comparison matrix).
Examples of parameters that may be employed in determining the percent identity of a polypeptide or nucleotide sequence using the GAP program are as follows: algorithm: needleman et al, 1970, J.mol. Biol.48:443-453; comparison matrix: BLOSUM62, described above, from Henikoff et al, 1992; gap penalty: 12 (but no penalty for terminal gaps); gap length penalty: 4, a step of; similarity threshold: 0.
some alignment designs for aligning two amino acid sequences can result in a short region match of only two sequences, and such small alignment regions can have extremely high sequence identity even though there is no significant relationship between the two full-length sequences. Thus, if it is desired to generate an alignment of at least 50 or other numbers of contiguous amino acids across the target polypeptide, the selected alignment method (GAP program) may be adjusted.
The term "derivative" refers to a molecule that includes chemical modifications other than insertions, deletions, or substitutions of amino acids (or nucleic acids). In certain embodiments, the derivatives include covalent modifications, including, but not limited to, chemical bonding to polymers, lipids, or other organic or inorganic moieties. In certain embodiments, the chemically modified antigen binding protein may have a longer circulating half-life than an antigen binding protein that has not been chemically modified. In certain embodiments, the chemically modified antigen binding proteins may improve the ability to target a desired cell, tissue, and/or organ. In certain embodiments, the derivatized antigen binding protein is covalently modified to include one or more water-soluble polymeric linkers, including, but not limited to, polyethylene glycol, polyoxyethyleneglycol, or polypropylene glycol. In certain embodiments, the derivatized antigen binding protein comprises one or more polymers including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose or other saccharide-based polymers, poly- (N-vinylpyrrolidone) -polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., ethylene glycol) and polyvinyl alcohol, and mixtures of such polymers.
In certain embodiments, the derivatives are covalently modified with polyethylene glycol (PEG) subunits. In certain embodiments, one or more water-soluble polymers are bonded at one or more specific positions (e.g., at the amino terminus of the derivative). In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivative. In certain embodiments, PEG is used to improve the therapeutic ability of an antibody or antigen binding fragment thereof. In certain embodiments, PEG is used to improve the therapeutic ability of the humanized antibodies. Some of these methods are discussed, for example, in U.S. patent No. 6,133,426, which is incorporated herein by reference for any purpose.
The term "antibody" refers to an intact immunoglobulin of any isotype or fragment thereof that competes with the intact antibody for specific binding to a target antigen, and includes, for example, chimeric antibodies, humanized antibodies, human whole antibodies, and bispecific antibodies. An "antibody" is a class of antigen binding proteins. An intact antibody will typically comprise at least two full length heavy chains and two full length light chains, but in some cases may comprise fewer chains, e.g. an antibody naturally occurring in a camel may comprise only heavy chains. Antibodies may be derived from only a single source, or may be "chimeric", i.e., different portions of an antibody may be derived from two different antibodies. Can be in hybridomas; the antigen binding proteins, antibodies or binding fragments are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact antibody. Unless otherwise indicated, the term "antibody" includes derivatives, variants and fragments thereof in addition to antibodies comprising two full length heavy chains and two full length light chains. Further, unless expressly excluded, antibodies include monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively.
Naturally occurring antibody building blocks typically comprise tetramers. Each tetramer typically consists of two identical pairs of polypeptide chains, each pair having one full length "light chain" (in certain embodiments, about 25 kDa) and one full length "heavy chain" (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically comprises a variable region of about 100-110 amino acids or more, which is typically responsible for antigen recognition. The carboxy-terminal portion of each chain is generally defined as a constant region that may be responsible for effector function. Human light chains are generally classified into kappa and lambda light chains. Heavy chains are generally classified as mu, delta, gamma, alpha or epsilon chains, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. IgG has several subclasses including, but not limited to, igG1, igG2, igG3, and IgG4.IgM has subclasses including, but not limited to, igM1 and IgM2.IgA is also subdivided into subclasses, including but not limited to IgA1 and IgA2. Within full length light and heavy chains, typically the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, e.g., fundamental Immunology, ch.7 (Paul, W.edit, second edition, raven Press, N.Y. (1989)) (which is incorporated herein by reference in its entirety for all purposes). The variable region of each light chain/heavy chain pair typically forms an antigen binding site.
In certain embodiments, the variable regions of different antibodies vary widely in amino acid sequence, even between antibodies of the same species. The antibody variable region generally determines the specificity of a particular antibody for its target.
The variable regions typically exhibit the same general structure of relatively conserved Framework Regions (FR) joined by three hypervariable regions, also known as complementarity determining regions or CDRs. CDRs from each pair of two chains are typically located by framework regions, which allow binding to specific epitopes. The two light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus. Amino acid assignment to each domain is generally consistent with the definition: kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)) or Chothia & Lesk, J.mol.biol.,196:901-917 (1987); chothia et al, nature,342:878-883 (1989). The International Immunogenetics (IMGT) database (http:// www.imgt.org) provides standardized numbering and definition of antigen binding sites. Correspondence between CDRs and IMGT partitions is described in Lefranc et al, dev Comparat Immunol27:55-77,2003. The amino acid numbering rules for antibody CDRs described herein are divided by IMGT, unless explicitly indicated.
Conventionally, CDR regions in the heavy chain are commonly referred to as H1, H2, and H3, and are numbered sequentially from the amino terminus to the carboxy terminus. CDR regions in the light chain are commonly referred to as L1, L2 and L3, and are numbered sequentially from the amino terminus to the carboxy terminus.
The term "light chain" includes full length light chains and fragments thereof having variable region sequences sufficient to confer binding specificity. Full length light chain comprises variable region domain V L And constant region domain C L . The variable region domain of the light chain is located at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.
The term "heavy chain" includes full-length heavy chains and fragments thereof having variable region sequences sufficient to confer binding specificity. Full length heavy chain comprises variable region domain V H And three constant region domains C H 1、C H 2 and C H 3。V H The domain is at the amino terminus of the polypeptide, while C H The domain is at the carboxy terminus, and C H 3 nearest the carboxy terminus of the polypeptide. The heavy chain may be of any isotype including IgG (including IgG1, igG2, igG3 and IgG4 subtypes), igA (including IgA1 and IgA2 subtypes), igM and IgE.
The term "antigen binding fragment" includes, but is not limited to, fab fragments, fab 'fragments, F (ab') 2 Fragments, fv fragments, diabodies, and single chain antibody molecules.
The "Fc" region comprises two antibody-containing C H 1 and C H 2 domain. The two heavy chain fragments are linked by two or more disulfide bonds and by C H The hydrophobic effect of the 3 domains remains together.
"Fab fragment" comprises the C of a light chain and a heavy chain H 1 and a variable region. The heavy chain of a Fab molecule is unable to form disulfide bonds with another heavy chain molecule.
“Fab’Fragments "comprise a light chain and contain a VH domain and C H 1 domain also has C H 1 and C H 2, so that an interchain disulfide bond can be formed between the two heavy chains of two Fab 'fragments to form F (ab') 2 A molecule.
“F(ab’) 2 Fragments "comprise two light chains and C H 1 and C H 2, thereby forming an interchain disulfide bond between the two heavy chains. F (ab') 2 Fragments consist of two Fab' fragments held together by disulfide bonds between the two heavy chains.
An "Fv fragment" comprises variable regions from both the heavy and light chains, but lacks constant regions.
A "single chain antibody" is an Fv molecule in which the heavy and light chain variable regions are joined together by an elastic linker to form a single polypeptide chain, thereby forming an antigen-binding region. Single chain antibodies are described in detail in International patent application publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the contents of which are incorporated herein by reference.
"monoclonal antibody" refers to a homogeneous population of antibodies having a single molecular composition. Monoclonal antibodies may be non-specific or multispecific.
A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the heavy chain variable region or the light chain variable region. In some cases, two or more V H The regions are covalently linked to a peptide linker to form a bivalent domain antibody. These two V of bivalent domain antibody H The regions may target the same or different antigens.
A "multispecific antibody" is an antibody that targets more than one antigen or epitope. "bispecific", "dual specificity" or "bifunctional" antibodies are hybrid antibodies having two different antigen binding sites, respectively. The two binding sites of a bispecific antibody will bind two different epitopes, which may be located on the same or different protein targets.
An antibody is a "selective" antibody when it binds to one target more tightly than it binds to a second target.
A "recombinant antibody" is an antibody produced by recombinant techniques. Methods and techniques for preparing recombinant antibodies are well known in the art.
Bispecific or bifunctional antibodies are typically artificial hybrid antibodies having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be prepared by a variety of methods, including but not limited to fusion hybridomas or linked Fab' fragments. See, e.g., songsivilai et al, clin. Exp. Immunol.,79:315-321 (1990); kostelny et al, J.Immunol.,148:1547-1553 (1992).
Each individual immunoglobulin chain is typically composed of several "immunoglobulin domains," each consisting of about 90-110 amino acids and having a characteristic folding pattern. These domains are the basic units that make up the antibody polypeptide. In humans, igA and IgD isoforms contain four heavy and four light chains; igG and IgE isotypes contain two heavy chains and two light chains; while IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically comprises one or more domains that may be responsible for effector functions. The number of heavy chain constant region domains will depend on the isotype. For example, an IgG heavy chain contains three chains called C H 1、C H 2 and C H 3. Antibodies may be provided having any of these isoforms and subtypes.
The term "neutralising" refers to binding of a ligand and preventing or reducing the biological effect of the ligand. This can be achieved, for example, by directly blocking the binding site on the ligand or by binding the ligand and changing the binding capacity of the ligand by indirect means (e.g. structural or energy change of the ligand). In assessing the binding and/or specificity of an antibody or antigen binding fragment thereof, an antibody or fragment may substantially inhibit the binding of a ligand to its binding partner when an excess of antibody reduces the amount of binding partner bound to the ligand by at least about 1-20, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-98%, 98-99% or more (as measured by an in vitro competitive binding assay). In some embodiments In the case of the present invention, the neutralizing antibodies reduce the binding of RBD to its receptor ACE 2. In certain embodiments, the neutralization capacity is characterized and/or described via a competition assay. In certain embodiments, the neutralization capacity is measured at a 50% inhibition concentration (IC 50 ) Values are described.
The term "antibody-dependent enhanced (ADE) effect" refers to the fact that after certain virus-specific antibodies bind to a virus, the antibodies that bind to the virus can bind to FcR-expressing cells on their surface via their Fc region, thereby mediating the entry of the virus into these cells, resulting in an enhancement of the infectious process of the virus.
The term "target" refers to a molecule or portion of a molecule capable of being bound by an antibody or antigen binding fragment thereof. In certain embodiments, the target may have one or more epitopes. In certain embodiments, the target is an antigen. The use of "antigen" in the phrase "antigen binding fragment" merely refers to a protein sequence that comprises an antigen that can be bound by an antibody. In this case, the protein is not necessarily exogenous or is not necessarily capable of inducing an immune response.
When the term "competition" is used in the context of neutralizing antibodies competing for the same epitope, it is meant competition between antibodies, as determined by the following assay: in the assay, the antibody or antigen-binding fragment thereof to be detected prevents or inhibits (e.g., reduces) specific binding of the reference antibody to a common antigen (e.g., S protein or domain thereof). Numerous types of competitive binding assays can be used to determine whether an antibody competes with another, such as: solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assay (see, e.g., stahli et al, 1983,Methods in Enzymology 9242-253); solid phase direct biotin-avidin EIA (see, e.g., kirkland et al, 1986, J.Immunol).1373614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., harlow and Lane,1988,Antibodies,A Laboratory Manual (antibodies, laboratory Manual), cold Spring Harbor Press); RIA is directly labeled with the solid phase of the I-125 label (see, e.g., morel et al, 1988, molecular immunol.).257-15); solid phase direct biotin-philicAnd element EIA (see, e.g., cheung, et al, 1990, virology176546-552); and directly labeled RIA (Moldenhauer et al, 1990, scand. J. Immunol).32:77-82). Typically the assay involves the use of a purified antigen that binds to a solid surface or cell bearing either an unlabeled detection antibody or a labeled reference antibody. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antibody being measured. Typically the antibody to be detected is present in excess. Typically, when competing antibodies are present in excess, they will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more of the specific binding of the reference antibody to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
The term "antigen" refers to a molecule or portion of a molecule that is capable of being bound by a selective binding agent, such as an antibody or immunologically functional fragment thereof. In certain embodiments, an antigen can be used in an animal to produce an antibody capable of binding the antigen. An antigen may possess one or more epitopes that are capable of interacting with different antibodies.
The term "epitope" includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets that antigen. When the antigen is a protein, it comprises specific amino acids that are in direct contact with the antibody. In most cases, the epitope is located on a protein, but in some cases may be located on other types of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three dimensional structural characteristics and/or specific charge characteristics.
The term "therapeutically effective amount" refers to an amount of an antibody or antigen-binding fragment thereof of the invention that is determined to produce a therapeutic response in a mammal. The therapeutically effective amount is readily determined by one of ordinary skill in the art.
The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, subjects with formally established disease, subjects with unidentified disease, subjects receiving medical treatment, subjects at risk of developing disease, and the like.
The term "treatment" includes therapeutic treatment, prophylactic treatment, and the use of reducing the risk of a subject developing a disease or other risk factor. Treatment need not cure the disease entirely, but includes embodiments in which symptoms are alleviated or potential risk factors are alleviated.
The term "prevention" does not require 100% elimination of the possibility of an event. More precisely, it means that the probability of an event occurring in the presence of said antibody or method is reduced.
Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). The enzymatic reactions and purification techniques may be carried out according to manufacturer's instructions, or may be accomplished according to methods common in the art or as described herein. The foregoing techniques and methods may be generally implemented according to conventional methods well known in the art, as well as various general and more specific references cited and discussed throughout the present specification. See, e.g., sambrook et al Molecular Cloning: A Laboratory Manual (2 nd edition, cold Spring Harbor Laboratory Press, coldSpring Harbor, n.y. (1989)), which is incorporated herein by reference for any purpose. Unless clearly defined otherwise, the terms and experimental methods and techniques used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry described herein are well known in the art and are commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, drug preparation, formulation, delivery and treatment of patients.
2. Antibodies and methods of making the same
The present disclosure provides antibodies or antigen-binding fragments thereof (hereinafter collectively referred to as "antibodies") capable of binding SARS-CoV-2 and effectively neutralizing its activity. The inventors have unexpectedly found that the antibodies are capable of neutralizing SARS-CoV-2 and variants thereof, including but not limited to Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) and Omicron variants. In one embodiment, the omacron variant includes, but is not limited to, b.1.1.529, ba.1, ba.2, ba.2.12.1, ba.3, ba.4 and ba.5.
In some embodiments, the antibodies described herein are administered at a 50% Inhibitory Concentration (IC) of less than 0.010 μg/ml 50 ) The SARS-CoV-2 and its variant are neutralized in vitro. IC of antibodies described herein is measured by luciferase activity as described in the cell-based in vitro neutralization assay in the examples 50 Values.
In one aspect, the invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2 and variants thereof, comprising: heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3 shown in SEQ ID nos. 1, 2 and 3, and light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 shown in SEQ ID nos. 4, 5 and 6, wherein NO more than 2 amino acid substitutions, deletions or insertions are contained at one or more of the sequences.
In one embodiment, an antibody or antigen binding fragment thereof of the invention comprises the heavy chain complementarity determining region CDRH3 shown in SEQ ID NO. 11 or 14.
In one aspect, the invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2 and variants thereof, comprising: a heavy chain variable region VH shown in SEQ ID NO. 7 and a light chain variable region VL shown in SEQ ID NO. 8.
In certain embodiments, the amino acid sequence of the heavy chain variable region VH has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 7. In certain embodiments, the amino acid sequence of the light chain variable region VL has 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 8.
In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH shown in SEQ ID NO. 12 or 15 and a light chain variable region VL shown in SEQ ID NO. 8.
In one aspect, the invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2 and variants thereof, comprising a heavy chain and a light chain selected from the group consisting of:
A heavy chain shown in SEQ ID NO. 9 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 13 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 16 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 17 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 18 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 19 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 20 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 21 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 22 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 23 and a light chain shown in SEQ ID NO. 10; and
a heavy chain shown in SEQ ID NO. 24 and a light chain shown in SEQ ID NO. 10.
In certain embodiments, the amino acid sequence of the heavy chain has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 9, 13, 16, 17, 18, 19, 20, 21, 22, 23 or 24. In certain embodiments, the amino acid sequence of the light chain has 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 10.
Variants of the antibodies of the invention comprising the amino acid sequences described above are within the scope of the invention. For example, a variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions in VH and/or VL that do not adversely affect antibody properties. In some embodiments, the sequence identity relative to a VH or VL amino acid sequence of the invention may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Exemplary modifications are conservative amino acid substitutions, e.g., in the antigen binding site or in the framework, which do not adversely alter the properties of the antibody. Conservative substitutions may also be made to improve antibody properties, such as stability or affinity. Conservative substitutions are those that occur within the family of related amino acids in their side chains. The amino acids encoded by genes can be divided into four classes: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) Nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) non-charge polarity (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. Alternatively, the amino acid repertoires may be grouped into: (1) acidic (aspartic acid, glutamic acid); (2) Basic (lysine, arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), wherein serine and threonine are optionally grouped separately as hydroxyl-containing aliphatic; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amides (asparagine, glutamine); and (6) sulfur (cysteine and methionine) (strer (edit), biochemistry, 2 nd edition, WH Freeman and co., 1981). In addition, any natural residue in the polypeptide can be replaced with alanine as described previously for alanine scanning mutagenesis (MacLennan et al (1998) Acta Physiol. Scan. Suppl.643:55-67; sasaki et al (1998) adv. Biophys. 35:1-24). The desired amino acid substitutions may be determined by one skilled in the art when such substitutions are desired. The characteristics of the resulting antibody variants can be tested using the assays described herein.
In one embodiment, the isolated antibody or antigen binding fragment thereof described herein comprises a mutation selected from LS, kdel, GRLR, YTE, LALA, and combinations thereof.
Amino acid substitutions may be made, for example, by PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, a library of variants may be generated using known methods, for example using random (NNK) codons or non-random codons (e.g., DVK codons, which encode 11 amino acids (Ala, cys, asp, glu, gly, lys, asn, arg, ser, tyr, trp)), and then screening the library for variants having the desired properties.
Although the antibodies of the invention include paired variable regions, one from the heavy chain and one from the light chain, one skilled in the art will recognize that alternative embodiments may include a single heavy chain variable region or a single light chain variable region. A single variable region can be used to screen for variable domains capable of forming a two-domain specific antigen binding fragment. The screening can be accomplished by phage display screening methods using, for example, the hierarchical double combination method disclosed in International patent publication No. WO 92/01047. In this combinatorial approach, a single colony comprising either an H-chain clone or an L-chain clone is used to infect a complete library of clones encoding the other chain (L or H), and the resulting double-stranded specific antigen binding domain is then selected according to the phage display technique described. Thus, individual VH and VL polypeptide chains can be used to identify additional antibodies that specifically bind to the S protein domain using the methods disclosed in international patent publication No. WO 92/01047.
A variety of techniques for producing antibodies can be used to prepare antibodies of the invention. For example, monoclonal antibodies can be produced using the hybridoma method as set forth in Nature 256:495,1975 by Kohler and Milstein. In the hybridoma method, a mouse or other host animal (such as hamster, rat, or monkey) is immunized with S protein or a fragment thereof, and spleen cells from the immunized animal are then fused with myeloma cells to form hybridoma cells using standard methods (Goding, monoclonal Antibodies: principles and Practice, pp.59-103 (Academic Press, 1986)). Colonies generated by individual immortalized hybridoma cells are screened for the production of antibodies having desired properties, such as binding specificity, cross-reactivity or lack thereof, lack of cross-reactivity, and affinity for antigen.
Antibodies of the invention can be prepared using a variety of host animals. For example, balb/c mice can be used to make antibodies. Antibodies prepared in Balb/c mice and other non-human animals can be humanized using a variety of techniques to produce more human-like sequences. Exemplary humanization techniques including selection of human acceptor frameworks are known to those skilled in the art and include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), surface remodeling (Padlan, mol Immunol28:489-499, 1991), specificity determining residue surface remodeling (U.S. Pat. Pub. No. 20100261620), human modification (or human framework modification) (U.S. Pat. No. 2009/018127), superhumanization (U.S. Pat. No. 7,709,226), and targeting selection (Osbourn et al (2005) Methods 36:61-68,2005; U.S. Pat. No. 5,565,332).
The humanized antibodies can be further optimized to improve their selectivity or affinity for the desired antigen by introducing modified framework support residues to maintain binding affinity (back-mutations) using techniques such as those described in International patent publication No. WO90/007861 and International patent publication No. WO 92/22653.
Transgenic mice bearing human immunoglobulin (Ig) loci in their genomes can be used to produce human antibodies against proteins of interest, as described, for example, in international patent publication No. WO90/04036, U.S. patent No. 6150584, international patent publication No. WO99/45962, international patent publication No. WO02/066630, international patent publication No. WO02/43478; lonberg et al (1994) Nature 368:856-9; green et al (1994) Nature Genet.7:13-21; green & Jakobovits (1998) exp. Med.188:483-95; lonberg and Huszar (1995) int.rev.immunol.13:65-93; bruggemann et al (1991) Eur.J.Immunol.21:1323-1326; fishwild et al (1996) Nat. Biotechnol.14:845-851; mendez et al (1997) Nat. Genet.15:146-156; green (1999) J.immunol.methods 231:11-23; yang et al (1999) Cancer Res.59:1236-1243; brU ggemann and Taussig (1997) Curr.Opin.Biotechnol.8:455-458; international patent publication No. WO 02/043478). Endogenous immunoglobulin loci in such mice may be disrupted or deleted and at least one full or partial human immunoglobulin locus may be inserted into the mouse genome by homologous or nonhomologous recombination using a transchromosome or minigene.
The human antibody may be selected from phage display libraries, wherein phage are engineered to express human immunoglobulins or portions thereof, such as Fab, single chain antibodies (scFv) or unpaired or paired antibody variable regions (Knappiket al (2000) J.mol. Biol.296:57-86; krebs et al (2001) J.Immunol. Meth.254:67-84; vaughan et al (1996) Nature Biotechnology 14:309-314; sheets et al (1998) PITAS (USA) 95:6157-6162;Hoogenboom and Winter, (1991) J.mol. Biol.227:381; marks et al (1991) J.mol. Biol. 222:581). Antibodies of the invention can be isolated, for example, from phage display libraries that express the heavy and light chain variable regions of the antibodies as fusion proteins with phage pIX coat proteins, as described in Shi et al (2010) J.mol.biol.397:385-96 and International patent publication No. WO 09/085462. The library may be screened for phage binding to protein S and the positive clones obtained may be further characterized, and Fab isolated from clone lysates and expressed as full length IgG. Such phage display methods for isolating human antibodies are described, for example, in: U.S. Pat. Nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner et al; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al; U.S. patent nos. 5,969,108 and 6,172,197 to McCafferty et al; U.S. Pat. nos. 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915 and 6,593,081 to Griffiths et al.
The preparation of the immunogenic antigen and the generation of monoclonal antibodies can be performed using any suitable technique, such as recombinant protein production. The immunogenic antigen may be administered to the animal as a purified protein or mixture of proteins (including whole cells, cell extracts or tissue extracts), or the antigen may be formed de novo in the animal from nucleic acids encoding the antigen or portions thereof.
Another embodiment of the invention is an isolated polynucleotide encoding any one of the antibody heavy chain variable regions and/or antibody light chain variable regions of the invention. It is also within the scope of the invention for multiple polynucleotides encoding the same antibody of the invention to be encoded in view of the degeneracy of the genetic code or codon-preference in a given expression system. The polynucleotide sequence encoding the VH or VL of the antibodies of the invention or fragments thereof may be operably linked to one or more regulatory elements, such as promoters or enhancers, which allow expression of the nucleotide sequence in the intended host cell. The polynucleotide may be a cDNA.
Another embodiment of the invention is a vector comprising a polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors or any other suitable vector for introducing the synthetic polynucleotide of the invention into a given organism or genetic background by any means. For example, a polynucleotide that is optionally linked to a constant region and encodes a light chain variable region and/or a heavy chain variable region of an antibody of the invention is inserted into an expression vector. The light chain and/or heavy chain may be cloned into the same or different expression vectors. The DNA fragments encoding the immunoglobulin chains may be operably linked to control sequences in one or more expression vectors that ensure expression of the immunoglobulin polypeptides. Such control sequences include signal sequences, promoters (e.g., naturally associated or heterologous promoters), enhancer elements, and transcription terminator sequences, which are selected to be compatible with the host cell selected for expression of the antibody. Once the vector is incorporated into an appropriate host, the vector maintains the host under conditions suitable for high levels of expression of the protein encoded by the bound polynucleotide.
Suitable expression vectors can typically replicate in a host organism as an episome or as part of the host chromosomal DNA. Typically, the expression vector comprises a selectable marker, such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance or neomycin resistance, to allow for detection of those cells transformed with the desired DNA sequence.
Suitable promoter and enhancer elements are known in the art. For expression in bacterial cells, exemplary promoters include lacl, lacZ, T, T7, gpt, λp and trc. For expression in eukaryotic cells, exemplary promoters include light and/or heavy chain immunoglobulin gene promoters and enhancer elements; a cytomegalovirus very early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in the long terminal repeat of the retrovirus; a mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. For expression in yeast cells, exemplary promoters are constitutive promoters such as the ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, and the like; or regulatory promoters such as GAL1 promoter, GAL10 promoter, ADH2 promoter, PH05 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter and AOX1 (e.g., for Pichia (Pichia)). The selection of suitable vectors and promoters is within the ability of one of ordinary skill in the art.
Numerous suitable vectors and promoters are known to those skilled in the art; many are commercially available for generating subject recombinant constructs. The following vectors are provided by way of example. Bacterial vector: pBs, phagescript, psiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, la Jolla, calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, uppsala, sweden). Eukaryotic vectors: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia).
Another embodiment of the invention is a host cell comprising one or more vectors of the invention. The term "host cell" refers to a cell into which a vector has been introduced. It should be understood that the term "host cell" is intended to refer not only to a particular subject cell, but also to the progeny of such a cell, but also to stable cell lines produced from a particular subject cell. Such progeny may not be identical to the parent cell, but are still encompassed within the scope of the term "host cell" as used herein, because of mutations or because of environmental effects, certain modifications may occur in the progeny. Such host cells may be eukaryotic, prokaryotic, plant or archaeal.
Coli (Escherichia coli), bacillus (such as bacillus subtilis (Bacillus subtilis)) and other enterobacteriaceae (such as Salmonella (Salmonella), serratia (Serratia)) and various Pseudomonas (Pseudomonas) species are examples of prokaryotic host cells. Other microorganisms such as yeast may also be used for expression. Saccharomyces(s) (e.g., saccharomyces cerevisiae) and Pichia (Pichia) are examples of suitable yeast host cells. Exemplary eukaryotic cells may be from mammalian, insect, avian, or other animal sources. Mammalian eukaryotic cells include immortalized cell lines such as hybridoma or myeloma cell lines, such as SP2/0 (American type culture Collection (ATCC), manassas, VA, CRL-1581), NS0 (European Collection of cell cultures (ECACC), salisbury, wiltshire, UK, ECACC No. 8510503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTCCRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, walkersville, MD), CHO-K1 (ATCC CRL-61), or DG44.
Another embodiment of the invention is a method of producing an antibody of the invention, comprising culturing a host cell of the invention under conditions such that the antibody is expressed, and recovering the antibody produced by the host cell. Methods for preparing antibodies and purifying them are well known in the art. Once synthesized (chemically or recombinantly), the whole antibody, its dimer, each light and/or heavy chain, or other antibody fragment(s), such as VH and/or VL, may be purified according to standard procedures including ammonium sulfate precipitation, affinity chromatography columns, column chromatography, high Performance Liquid Chromatography (HPLC) purification, gel electrophoresis, and the like (see general protocols, protein Purification (Springer-Verlag, n.y., (1982)). Antibodies may be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99% pure, or more pure, e.g., free of contaminants (such as cell debris, macromolecules other than the subject antibody, etc.).
Another embodiment of the invention is a method for preparing an antibody of the invention, comprising:
Incorporating a first polynucleotide encoding an antibody heavy chain and a second polynucleotide encoding an antibody light chain into an expression vector;
transforming a host cell with the expression vector;
culturing the host cell in a medium under conditions that allow expression of the heavy and light chains and formation of the antibody; antibodies are then recovered from the host cells or culture medium.
Polynucleotides encoding antibodies of the invention are incorporated into vectors using standard molecular biological methods. Host cell transformation, culture, antibody expression and purification are accomplished using well known methods.
3. Compositions and methods of administration
The invention provides pharmaceutical compositions comprising an antibody as described herein, and a pharmaceutically acceptable carrier. For therapeutic use, the antibodies of the invention may be formulated into pharmaceutical compositions comprising an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle from which the active compound is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% brine and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by well known conventional sterilization techniques (e.g., filtration). The composition may contain pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, colorants, and the like as needed to approximate physiological conditions. The concentration of the antibodies of the invention in such pharmaceutical formulations can vary widely, i.e., from less than about 0.5% by weight, typically up to at least about 1% by weight up to 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight, and will be selected based primarily on the desired dosage, fluid volume, viscosity, etc., depending on the particular mode of administration selected. Suitable vehicles and formulations (including other human proteins such as human serum albumin) are described, for example, in Remington, the Science and Practice of Pharmacy, 21 st edition, troy, d.b. editions, lipincott Williams and Wilkins, philiadelphia, PA 2006, section 5, pharmaceutical Manufacturing, pages 691-1092, see in particular pages 958-989.
The manner of administering the antibodies of the invention in the methods of the invention described herein may be by any suitable route, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, mucosal (buccal, intranasal, intravaginal, rectal) or other means known to the skilled artisan.
The antibodies in the methods of the invention described herein can be administered to a patient by any suitable route, such as parenterally by intravenous (i.v.) infusion or bolus injection, intramuscularly, subcutaneously or intraperitoneally. Intravenous infusion may be administered within, for example, 15, 30, 60, 90, 120, 180, or 240 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.
In one embodiment, a therapeutically effective amount of an antibody of the invention is administered to a subject in a method of the invention described herein. The "therapeutically effective amount" of an antibody can be determined by standard research techniques. For example, in vitro assays may be employed to help identify optimal dosage ranges. Optionally, a therapeutically effective amount of an antibody of the invention can be determined by administering the antibody to a related animal model well known in the art. The selection of a particular effective dose can be determined by one of skill in the art based on consideration of several factors (e.g., via clinical trials). Such factors include the disease to be treated or prevented, the symptoms involved, the patient's weight, the patient's immune status, and other factors known to the skilled artisan. The precise dosage to be used in the formulation will also depend on the route of administration and the severity of the disease and should be determined according to the judgment of the practitioner and each patient's circumstances. The effective dose can be deduced from dose response curves from in vitro or animal model test systems. The efficacy and effective dose of the antibodies of the invention can be tested using any of the models described herein.
The antibodies in the methods of the invention may be repeatedly administered after one, two, three, four, five, six, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer. The treatment course may also be repeated, as is chronic administration. Repeated administration may be the same dose or different doses.
The antibodies in the methods of the invention can also be administered prophylactically to reduce the risk of a subject to infection with SARS-CoV-2 and mutants thereof, delay the onset of infection with SARS-CoV-2 and mutants thereof, and/or reduce the risk of recurrence of infection with SARS-CoV-2 and mutants thereof in remission.
The antibodies in the methods of the invention described herein can be stored lyophilized and reconstituted in a suitable carrier prior to use. This technique has proven effective for conventional protein formulations and can employ well known lyophilization and reconstitution techniques.
The invention will now be described with reference to the following specific non-limiting examples which are intended to illustrate the invention and are not intended to limit the scope of the invention.
Example 1 Experimental methods
ELISA
For ELISA, 96-well plates were coated (50. Mu.l/well) overnight at 4℃with antigen protein (10. Mu.g/ml) dissolved in Phosphate Buffered Saline (PBS). The antigenic proteins are the S protein extracellular domains of SARS-CoV-2 wild-type and its related variants, including Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) and Omicron (B.1.1.529). These coated plates were then blocked (200 μl/well) with PBS containing 2% Bovine Serum Albumin (BSA). After blocking, the plates were incubated (8 dilutions, maximum concentration 10 μg/ml, 3-fold serial dilutions) (50 μl/well) with primary antibody dissolved in PBS at room temperature for 1 hour. After washing, secondary antibody (goat anti-human IgG conjugated with HRP, thermo Fisher Scientific) (50 μl/well) dissolved in PBS was added to each well, incubated for 1 hour, and then detected. To evaluate antigen binding capacity, the area under the curve (AUC) of each purified recombinant IgG1 monoclonal antibody was calculated using PRISM software.
Production of SARS-CoV-1/2 pseudovirus
Pseudoviruses of SARS-CoV-1, SARS-CoV-2 and SARS-CoV-2 related variants were generated as previously described (Xia, S.et al (2020). Inhibition ofSARS-CoV-2 (previous 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion.cell Res 30,343-355; zhou, Y., et al (2021). Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on the RBD.Cell Rep 34,108699). Firstly, constructing S-protein expression plasmid pcDNA3.1-SARS-CoV-2-S or pcDNA3.1-SARS-CoV-1-S according to related nucleotide and amino acid sequences of S protein of wild type and variant strain. The constructed pcDNA3.1 plasmid was then co-transfected with the backbone plasmid of pNL4-3.Luc.R-E into HEK293T cells. After two days, the cell supernatant containing pseudoviruses was collected and stored at-80 ℃ for in vitro neutralization assay.
Pseudo-virus based in vitro neutralization assay
In vitro pseudovirus neutralization assays (Xia, S., et al (2020) Inhibition of SARS-CoV-2 (previous 2019-nCoV) infection by ahighly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion.Cell Res 30,343-355; zhou, Y., et al (2021) Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on the RBD.Cell Rep 34,108699) were performed as described previously. The resulting pseudoviruses were first checked by infecting Huh-7 cells and measuring luciferase activity to determine the virus concentration. The concentrated virus stock was then aliquoted and stored at-80. For in vitro neutralization experiments, the sample was taken at 10 4 Individual cells/well Huh-7 cells were seeded into 96-well plates and serial dilutions (1:3) of overexpressed monoclonal antibody (maximum concentration 10 μg/ml) were made for a total of 9 dilutions. The antibodies were mixed with concentrated pseudovirus at 37 ℃ and incubated for 30 minutes, after which they were added to Huh-7 cells and incubated for 24 hours. ThenCell supernatants were replaced with fresh DMEM containing 10% fbs and cells were collected after 36 hours of cell culture. Finally, the cultured cells were lysed and luciferase activity was measured using a firefly luciferase assay kit (Promega, USA) and a microplate reader (Infinite M200PRO, switzerland) according to the manufacturer's instructions. In view of the apparent variation in absolute luciferase values, relative luminescence values were calculated by normalizing to control wells of pseudovirus only. IC in neutralization assay 50 Values were calculated by nonlinear regression analysis using PRISM software.
Pseudo-virus based in vitro ADE assay
An in vitro SARS-CoV-2 pseudovirus Antibody Dependence Enhancement (ADE) assay (Zhou, Y., et al (2021). Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on the RBD.Cell Rep 34,108699) was performed as described previously. For in vitro ADE assay experiments, 3×10 4 Individual cells/well Raji cells were seeded into 96-well plates coated with 0.01% polylysine. The overexpressed monoclonal antibodies were serially diluted (1:2) for a total of 9 dilutions (maximum concentration 100. Mu.g/ml). After that, the antibodies were mixed with the concentrated pseudoviruses at 37℃and incubated for 30 minutes, and then they were added to Raji cells and incubated for 60 hours. When the cultured cells were collected, the cell culture broth was discarded, the cultured cells were lysed, and luciferase activity was measured using a firefly luciferase assay kit (Promega, USA) and a microplate reader (Infinite M200PRO, switzerland) according to the manufacturer's instructions. Only the Raji cell well, to which no monoclonal antibody was added, was used as a negative control, confirming that the pseudovirus itself did not infect Raji cells and produced a luciferase signal.
Antibody cloning and production
Single B cell-based antibody amplification and sequence analysis was performed as described previously (Wang, Q., et al (2020) A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape modifications.cell Host micro, zhou, Y., et al (2020)) Single-Cell Sorting of HBsAg-Binding Memory B Cells from Human Peripheral Blood Mononuclear Cells and Antibody cloning.STAR Protoc 1,100129. Briefly, for single B cells sorted, reverse transcription and nested PCR amplification were performed (Zhou, Y., et al (2021). Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on the RBD.Cell Rep 34,108699). All Sanger sequencing results of heavy and light chains were analyzed and V (D) J genes and BLASR sequences were identified using IMGT/V-QUEST (Brochet, X., lefranc, M.P., and Giudicelli, V. (2008): IMGT/V-QUEST: the highly customized and integrated system for IG andTR standardized V-J and V-D-J sequence analysis.nucleic Acids Res 36, W503-508) and/or IgBLAST (Ye, J., ma, N., madden, T.L., and Ostell, J.M. (3): igST: an immunoglobulin variable domain sequence analysis tool.201nucleic Acids Res 41, W34-40). For antibody expression, HEK293F cells were transiently transfected with heavy/light chain plasmids and supernatants were harvested after 7 days for antibody purification.
Example 2: broad spectrum neutralizing Activity of human monoclonal antibody 09C7
Antibody 09C7, a fully human IgGmAb targeting the SARS-CoV-2S protein, was isolated from a person recovering infection with excellent serum neutralization activity as described in example 1. 09C7 has the sequence of SEQ ID NO:9 and the heavy chain of the amino acid sequence shown in SEQ ID NO:10, and a light chain of the amino acid sequence shown in seq id no. Control antibodies 05H9 and 02F6 were also obtained for comparison. The antigen binding capacity was assessed by ELISA and AUC values for the S protein of the various SARS-CoV-2 VOC mutants are provided in Table 1.
Table 1: AUC value of antibody against S protein of various SARS-CoV-2 VOC mutant strains
Wild type Alpha Beta Gamma Delta Omicron
09C7 30 31 31 31 31 26
05H9 29 29 26 29 30 4
02F6 7 6 6 2 4 3
Various SARS-CoV-2 pseudoviruses expressing luciferase were constructed, including SARS-CoV-2 wild-type Alpha (B.1.1.7)Beta (B.1.351), gamma (P.1), delta (B.1.617.2) and Omicron (B.1.1.529) variants, and in vitro neutralization assays of antibodies were performed (see FIG. 1A for results) and IC calculations were performed 50 Values. The resulting IC 50 The results of the values are shown in Table 2.
Table 2:09C7 IC against various SARS-CoV-2 pseudoviruses 50 Value (μg/ml)
Wild type Alpha Beta Gamma Delta Omicron
0.004 0.005 0.003 0.006 0.003 0.005
The neutralization profile of 09C7 was further evaluated in view of the outstanding neutralization activity of 09C 7. In addition, several types of pseudoviruses were constructed, including SARS-CoV-1, SARS-CoV-2 variant [ B.1.351-L242H, B.1.617.1 (Kappa), C.37 (Lambda), B.1.621 (Mu) ]And SARS-CoV-2 Omicron variant [ B.1.1.529, BA.1, BA.2, BA.2.12.1, BA.3, BA.4 and BA.5 ]]And pseudo-virus neutralization assays were performed. 09C7 neutralizes all of these variantsRemain effective in aspects, including omacron sub-lineages, IC 50 Values were below 0.01. Mu.g/ml, but there was no neutralization activity against SARS-CoV-1 (see FIGS. 1B and 1C for results).
Example 3: variants of human monoclonal antibody 09C7 and characterization thereof
09C7 induces antibody-mediated viral entry and S-protein mediated membrane fusion through its interaction with Fc receptor (FcR) (Liu, Z., et al (2021). An ultrapotent pan-beta-coronavirus lineage B (beta-CoV-B) neutralizing antibody locks the receptor-binding domain in closed conformation by targeting its conserved epitope.protein Cell; zhou, Y., et al (2021). Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on the RBD.Cell Rep 34, 108699). Like 09C7, LY-CoV1404 (bebtelovimab) is also a broad-spectrum and highly potent monoclonal neutralizing antibody encoded by IGHV2-5/IGHV2-14 (Westendorf, k., et al (2022). LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.Cell Rep 39, 110812). LY-CoV1404 (bebtelovimab) was able to induce antibody-dependent viral entry, thereby generating an ADE effect in vitro (FIG. 2A).
The 09C7 Fc amino acid was engineered to reduce its FcR interactions (GRLR modification-G239R/L331R; or LALA modification-L237A/L238A) and/or to extend the antibody half-life (YTE modification-M255Y/S257T/T259E; or LS modification-M431L/N437S) and/or to maintain antibody homogeneity (Kdel-mAb mutant lacking heavy chain C-terminal lysine).
As expected, the in vitro SARS-CoV-2 pseudovirus antibody-dependent enhancement (ADE) assay (Zhou, y., et al (2021). Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on theRBD.Cell Rep 34, 108699) showed that 09C7 engineered Fc variants with GRLR or LALA substitutions did not induce ADE effects in cultured Raji cells, whereas more pronounced ADE effects were observed in wild-type 09C7 as well as 09C7 treated Raji cells with YTE substitutions (fig. 2B).
In addition, a series of in vitro neutralization assays of 09C7 variants were performed as described in example 1 to confirm that these variants retained their neutralizing efficacy against SARS-CoV-2 Omicron pseudovirus as compared to the wild-type 09C7 monoclonal antibody (FIGS. 3A-D).
Example 4: artificial engineering of HCDR3
In the same volunteer from which the antibody 09C7 was cloned, two antibodies of 09C7 family were isolated, the C109 site of which was not identical to that of the 09C7 antibody. Based on the amino acid residues at position 109, the cloning construct yielded antibodies 09C7-C109S and 09C7-C109Y.09C7-C109S has sEQ ID NO:13 and the light chain of the amino acid sequence shown in SEQ ID NO: 10. 09C7-C109Y has the amino acid sequence of SEQ ID NO:16 and the heavy chain of the amino acid sequence shown in SEQ ID NO:10, and a light chain of the amino acid sequence shown in seq id no.
As described in example 1, various luciferase-expressing SARS-CoV-2 pseudoviruses were constructed, including SARS-CoV-2 wild-type (WT), alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), omicron (B.1.1.529), B.1.351-L242H, B.1.617.1 (Kappa), C.37 (Lambda), B.1.621 (Mu), omicron (BA.2), omicron (BA.2.12.1), omicron (BA.3), omicron (BA.4/5), and pseudovirus neutralization assays were performed, and IC was calculated 50 Values.
Antibodies 09C7, 09C7-C109S and 09C7-C109Y remained equally neutralizing in neutralizing all of these variants (see FIGS. 4A-N for results). Fc variants of antibodies 09C7-C109S and 09C7-C109Y were also constructed. Like 09C7, these variants also retained the neutralizing potency against SARS-CoV-2 Omicron pseudovirus (fig. 3E).
Example 5: comparison with LY-CoV1404 antibody
LY-CoV1404 (bebtelovimab) antibody is a novel monoclonal antibody directed against the Omicron variant that has been authorized for urgent use in the United states. The in vitro neutralization activity of the antibodies 09C7, 09C7-C109S-YTE-LALA-Kdel and Bebtelovimab of the invention was compared using a pseudovirus neutralization method according to the following procedure.
1. Sample dilution: the 09C7, 09C7-C109S-YTE-LALA-Kdel, bebtelovimab antibodies were moderately diluted, respectively, followed by a 3-fold gradient dilution. Taking a 96-well plate, adding 90 mu L of serum-free DMEM culture medium into the 3 rd-9 th column, adding 135 mu L of diluted initial antibody (diluted by serum-free culture medium) into the second column, transferring 45 mu L from the second column to the third column for 3-time gradient dilution, diluting to the ninth column, taking 8 gradients of each antibody, discarding the last 45 mu L, and repeatedly blowing and beating 5-10 times by a pipette for uniformly mixing every dilution.
2. Virus dilution: the pseudovirus used was purchased from Nanjinopran and diluted to 2×10 with DMEM medium containing 10% fbs according to COA recommendation 4 TCID 50 /mL。
3. Neutralization reaction: the sample wells and virus control wells were each filled with 90. Mu.L of virus dilution (final dilution of the sample was 1/2 of the initial concentration), the cell control wells were filled with 90. Mu.L of DMEM medium containing 10% FBS, and repeatedly blown up and down 5 times using a lance at 37℃with 5% CO 2 Neutralization is carried out for 1 hour.
4. Rotating plate: from a 96-well transparent plate, 50 μl/well was transferred to a 96 Kong Quanbai plate, 3 multiple wells.
5. Adding cells: HEK293-ACE2 cells were purchased from nankingdom and diluted to a viable cell density of 4 x 10 according to COA recommendations 5 (cells need to be passaged one day in advance, the cells are ensured to be in an exponential phase, the activation rate is more than 90%, the cells which are just recovered need to be passaged twice and used), and the sample holes, the virus control holes and the cell control holes are added according to 50 mu L/hole, the temperature is 37 ℃, and the CO is 5 percent 2 Culturing for 48 hours.
6. And (3) adding a detection reagent: the cultured white plate was left at room temperature for 1 hour, and at the same time, the detection reagent (purchased from novzan) was left at room temperature, and after all equilibrated to room temperature, the detection reagent was added at 100. Mu.L/well for detection, and data were recorded.
7. And (3) data processing: IC using Graphpad 8.0 50 Analyzing, namely carrying out nonlinear fitting of four parameter equations by taking antibody dilution as an abscissa and RLU value as an ordinate to determine the IC 50 And non-linear fitting R 2 。
The pseudovirus neutralization results are shown in Table 3 below and in FIGS. 5A-D. It can be seen that the 09C7 and 09C7-C109S-YTE-LALA-Kdel antibodies had excellent neutralizing activity against both SARS-CoV-2 wild-type (WT) and Omacron mutant strains. The 09C7-C109S-YTE-LALA-Kdel antibody has a superior effect to that of the Bebtelovimab antibody, particularly against the Omicron BA.2 variant.
Table 3: pseudo-virus neutralization results of 09C7, 09C7-C109S-YTE-LALA-Kdel and Bebtelovimab
Example 6: in vitro neutralization assay based on true viruses
The neutralizing capacity of antibody 09C7-C109S-YTE-LALA-Kdel against SARS-CoV-2 OmicronBA.2 and BA.5 was measured in vitro and compared to the control antibody Bebtelovimab. The eukaryotic strains Omicron BA.2 and BA.5 were obtained from the Guangzhou customs technical center. Control antibody Bebtelovimab was obtained from genescript profio. The virus infected cells were stained using plaque formation assay (FFA), and the stained spots on the plates were counted and analyzed by ELispot analyzer (ImmunoSpot S6 Ultra, CTL) and the half-maximal Inhibition (IC) 50 ) To assess the neutralizing capacity of the antibodies.
The method comprises the following specific steps:
vero cell resuscitation and culture
1.1 Resuscitates cells in a water bath at 37 ℃ and shakes the freezing tube rapidly to accelerate the thawing process, and after the cells are completely thawed, the outer side of the freezing tube is wiped by 75% ethanol;
1.2 immediately transferring the entire contents of the frozen stock tube to a flask preheated in advance in the first step using a sterile pasteur tube;
1.3 cells were placed at 37℃with 5% CO 2 Culturing in an incubator;
1.4 cells were resuscitated and passaged 3 times before the experiment.
Vero cell plating
2.1, after the cell density of the T75 cell culture flask is about 90%, 10mL of DPBS is cleaned for 1 time, and the DPBS is discarded;
2.2T75 the flask was then filled with 2ml of 0.25% Trypsin-EDTA (Gibco) and digested at 37℃for 4-5min;
2.3 preparation of cell suspensions, counting, dilution of cells to 2X 10 with medium 5 Individual/ml;
2.496 well plates with 100. Mu.L concentration 2X 10 per well 5 Cell suspension per ml;
2.5 Culturing in a 37 ℃ incubator for 12-14h until the cells grow to about 90-95% for experiment.
3. Antibody dilution
All drug dilution processes were performed in a biosafety cabinet. In a microcentrifuge tube, the antibody 09C7-C109S-YTE-LALA-Kdel was diluted to 2.54mg/mL and 254. Mu.g/mL in sequence with PBS, and then diluted to 9.41. Mu.g/mL with DMEM medium (Gibco) without FBS. Antibody Bebtelovimab was diluted to 0.778mg/mL and 77.8. Mu.g/mL in PBS, followed by dilution to 2.88. Mu.g/mL in DMEM medium without FBS.
Antibody dilution was further performed in 96 well sterile cell culture plates. The antibody 09C7-C109S-YTE-LALA-Kdel was diluted 3-fold sequentially from 9.41. Mu.g/mL to 0.00078. Mu.g/mL with DMEM medium without FBS for a total of 10 gradients. Bebtelovimab was serially diluted 3-fold from 2.88 μg/mL to 0.000222 μg/mL with DMEM medium without FBS for a total of 10 gradients. Each well was run in triplicate at 25 μl.
4. Virus dilution
The true virus was transferred from-80℃to ice (complete immersion on ice was required) for thawing, and the virus was diluted to 4000-12000FFU/mL with DMEM without FBS. FFU: plaque forming units.
5. Neutralization reaction
In the antibody dilution plate, 25. Mu.L of Omicron BA.2 or BA.5 virus dilutions (containing 100-300FFU virus) were added per well and mixed well, 37℃at 5% CO 2 Incubate in incubator for 1h. Virus control wells CC (virus-free, antibody-free) and virus control wells VC (virus-containing, antibody-free) were set.
6. Rotating plate and infection
6.1 after incubation for 1h, the antibody-virus incubation mixtures were transferred in one-to-one correspondence to Vero E6 cell plates (2X 10) 4 Each well) (CORNING) was 50. Mu.L per well at 37℃with 5% CO 2 Incubating for 1h under the condition;
6.2 incubation for 1h the culture supernatant was discarded and 100. Mu.L of 1.6% CMC broth (Sigma), 37℃and 5% CO were added 2 Culturing for 24h.
7. Detection of
7.1 37℃、5％CO 2 After culturing for 24 hours, directly adding 300 mu L of 4% paraformaldehyde (Biosharp) to inactivate and fix the cells for more than 1 hour;
7.2 removing the fixing solution, and washing the cells 3 times with 200 mu L of PBS buffer solution per well;
7.3 then 50. Mu.L 1% BSA diluted 0.2% Triton X-100 (Sigma) per well was added and membrane rupture was blocked at room temperature for 30min;
7.4, discarding the sealing membrane rupture liquid, and cleaning the cells with PBS buffer solution for 3 times;
7.5 dilution of primary antibody with 1% BSA (1:4000 diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody) (Beijing SinoBiological Company), followed by addition of 50. Mu.L of diluted primary antibody per well and incubation at 37℃for 1h;
7.6 primary antibody was discarded and cells were washed 3 times with PBST (0.15% Tween 20) buffer;
7.7 dilution of secondary antibody with 1% BSA (1:6000 diluted HRP-labeled goat anti-rabbit secondary antibody) (Abcam) followed by addition of 50 μl of diluted secondary antibody per well and incubation at 37deg.C for 1h;
7.8 discarding the secondary antibody, washing the cells 3 times with PBST (0.15% Tween 20) buffer;
7.9 adding 50 mu L of True-blue color development liquid (KPL) into each hole, and incubating for 10min at room temperature in a dark place;
7.10, flushing with tap water to stop color development, and airing;
7.11 plate spot counts were scanned with an ELispot analyzer.
8. Data processing
8.1 calculation of inhibition ratio: 100% > (1-number of sample spots/number of positive well spots).
8.2IC 50 Is calculated by (1): IC using Graphpad 8.0 50 And (5) analyzing. Taking the dilution as the abscissa and the inhibition ratio as the ordinate, performing nonlinear fitting of four parameter equations, and determining the IC 50 And a non-wireR2 is fit sexually.
9. Experimental results:
the antibody 09C7-C109S-YTE-LALA-Kdel and Bebtelovimab can well protect Vero cells from being infected by Omicron BA.2, and IC 50 The method comprises the following steps of: 33.04ng/mL,11.2ng/mL. The results are shown in FIG. 6 and Table 4.
Table 4: antibody 09C7-C109S-YTE-LALA-Kdel and Bebtelovimab semi-Inhibitory Concentration (IC) on SARS-Cov-2 Omicron BA.2 in Vero cells 50 )
Antibodies to IC 50 (ng/mL)
09C7-C109S-YTE-LALA-Kdel 33.04
Bebtelovimab 11.2
The antibody 09C7-C109S-YTE-LALA-Kdel and Bebtelovimab can well protect Vero cells from being infected by Omicron BA.5, and IC 50 The method comprises the following steps of: 22.85ng/mL,7.99ng/mL. The results are shown in FIG. 7 and Table 5.
Table 5: antibody 09C7-C109S-YTE-LALA-Kdel and Bebtelovimab semi-Inhibitory Concentration (IC) on SARS-Cov-2 Omicron BA.5 in Vero cells 50 )
Antibodies to IC 50 (ng/mL)
09C7-C109S-YTE-LALA-Kdel 22.85
Bebtelovimab 7.99
Example 7: in vivo animal experiment-BA.2 toxicity counteracting
The protective effect of antibody 09C7-C109S-YTE-LALA-Kdel on SARS-COV-2 BA.2 challenge K18-hACE2 transgenic mice was evaluated.
SPF grade K18-hACe2_C57BL/6J mice, female, 6-8 weeks old, weighing 21.14-25.23 g, were purchased from Guangdong medical biotechnology Co., ltd (animal batch 44824700015438). Mice are kept in cages, and at most 5 mice are fed with drinking water and complete pellet feed freely. The raising temperature is 20-25 ℃, the humidity is 40-70%, and the light and shade alternate every day and every night for 12 hours. The eukaryotic strain Omicron BA.2 was obtained from Guangzhou customs technology center.
The method comprises the following specific steps:
1. grouping animals
On the day of challenge, all animals were weighed and randomly grouped according to body weight. The animals were grouped and dosed as follows:
2. Reagent preparation
The preparation of the compound solution is carried out in a biosafety cabinet or an ultra clean bench. Dosing volume = 10 μl/g. mu.L, 63 mu L and 187 mu L of 25.4mg/mL 09C7-C109S-YTE-LALA-Kdel are added to a sterile centrifuge tube, respectively, 2.307mL, 2.217mL and 2.073mL of sterile PBS are added, and the mixture is mixed to prepare 2.33mL of 0.25mg/mL, 2.28mL of 0.7mg/mL and 2.26mL of 2.1mg/mL antibody solution. PBS solution was used directly as vehicle control.
3. Attack toxin
Day of challenge (day 0), nasal drop infection with Omicron ba.2 virus, 10 5 FFU/dose, infectious volume 50. Mu.L/dose.
4. Administration of drugs
Mice were given intraperitoneal administration according to the above regimen 4h after challenge.
5. Experimental endpoint and sample collection
Mice were observed daily for status after dosing and weighed. Two days later, animals were euthanized and subjected to cervical scission and the lung tissue was collected as follows: multilobal lung was weighed and after tissue placement into 1mL DPBS mill homogenate, lung tissue live virus titer was measured by FFA method (see example 6 for detailed procedure except lung tissue homogenate was used instead of antibody-virus incubation mixture).
6. Data processing
FFA calculation mode: titer FFU/g = number of spots x dilution x 20/gram of tissue weight; data are described using Graphpad 8.0.
7. Experimental results
After the mice were detoxified, no significant weight change was seen. The effect of antibody 09C7-C109S-YTE-LALA-Kdel on the number of live viruses in lung tissue of BA.2-challenged mice is shown in FIG. 8. Live virus was detected in lung tissues of animals in PBS group, and no live virus was detected in lung tissues of animals in all of 09C7-C109S-YTE-LALA-Kdel treatment groups.
Example 8: in vivo animal experiment-BA.5 toxicity attack
The protective effect of antibody 09C7-C109S-YTE-LALA-Kdel on SARS-COV-2 BA.5 challenge K18-hACE2 transgenic mice was evaluated.
Male, 6-8 week old, SPF grade K18-hACe2_C57BL/6J mice weighing 21.78-27.75 g were purchased from Guangdong medical biotechnology Co., ltd (animal lot 44824700017522). Mice are kept in cages, and at most 5 mice are fed with drinking water and complete pellet feed freely. The raising temperature is 20-25 ℃, the humidity is 40-70%, and the light and shade alternate every day and every night for 12 hours. The eukaryotic strain Omicron BA.5 was obtained from Guangzhou customs technology center. Control antibody Bebtelovimab was obtained from Genescript ProBio.
The method comprises the following specific steps:
1. grouping animals
On the day of challenge, all animals were weighed and randomly grouped according to body weight. The animals were grouped and dosed as follows:
2. reagent preparation
The preparation of the compound solution is carried out in a biosafety cabinet or an ultra clean bench. Dosing volume = 10 μl/g. The corresponding volumes of antibody 09C7-C109S-YTE-LALA-Kdel were taken into a sterile centrifuge tube, and stoichiometric amounts of sterile PBS were added and mixed well to prepare 0.5mg/mL, 0.1mg/mL, 0.05mg/mL, 0.02mg/mL and 0.004mg/mL antibody solutions. The corresponding volumes of antibody Bebtelovimab were taken into a sterile centrifuge tube, added with stoichiometric amounts of sterile PBS, and mixed well to make 0.5mg/mL and 0.05mg/mL antibody solutions. PBS solution was used directly as vehicle control.
3. Attack toxin
Day of challenge (day 0), nasal drip infection with Omicron ba.5 virus, 10 5 FFU/dose, infectious volume 50. Mu.L/dose.
4. Administration of drugs
Mice were given intraperitoneal administration according to the above regimen 4h after challenge.
5. Experimental endpoint and sample collection
Mice were observed daily for status after dosing and weighed. Two days later, animals were euthanized and subjected to cervical scission, and right lung was collected, and after tissue was homogenized by grinding with 1ml pbs, lung tissue live virus titers were detected by FFA method (see example 6 for detailed procedures except that lung tissue homogenates were used instead of antibody-virus incubation mixtures).
6. Data processing
FFA calculation mode: titer FFU/g = number of spots x dilution x 20/gram of tissue weight; data are described using Graphpad 8.0.
7. Experimental results
After the mice were detoxified, no significant weight change was seen. The effect of antibody 09C7-C109S-YTE-LALA-Kdel on the number of live viruses in lung tissue of BA.5-challenged mice is shown in FIG. 9. Live virus was detected in lung tissue of animals in PBS group, but not in all animals in antibody 09C7-C109S-YTE-LALA-Kdel 5 and 1mg/kg dose group. The dose of antibody 09C7-C109S-YTE-LALA-Kdel was 0.5mg/kg, and live virus was detected in only one animal lung tissue, and when the dose was as low as 0.2mg/kg, the live virus amount in most animal lung tissues was still lower than in PBS group.
Example 9: antibody aggregation studies
1. Analysis of antibody purity Using SEC-HPLC
Exclusion Chromatography (SEC) is a chromatographic technique that separates sample molecules according to their size, with small molecules having long retention times, large elution volumes, and large molecules having short retention times and small elution volumes. The molecular weight size purity of the antibodies 09C7-C109S-YTE-LALA-Kdel, 09C7-YTE-LALA-Kdel and 09C7 were analyzed using this assay. The SEC-HPLC chromatographic conditions are shown in Table 4.
Table 6: SEC-HPLC chromatographic conditions
Table 7: SEC-HPLC purity results
As shown in fig. 10 and table 7, the ratio of pre-main peak High Molecular Weight (HMW) impurities of antibodies 09C7-C109S-YTE-LALA-Kdel was lower compared to 09C7 and 09C7-YTE-LALA-Kdel, indicating that the C109S mutation helped reduce the formation of HMW impurities.
2. Analysis of antibody aggregation using polyacrylamide gel electrophoresis (SDS-PAGE)
Reduced and non-reduced polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze whether the high molecular weight impurities were aggregates formed by intermolecular disulfide bonds. The experimental conditions were as follows: the prefabricated glue produced by gold was used in the specification SurePAGE, bis-Tris, 10X 8,USE MOPS,NO TRIS-GLYCINE,10%,10 wells, 10/pk, voltage 200V for 35 minutes. The antibodies 09C7-C109S-YTE-LALA-Kdel, 09C7-YTE-LALA-Kdel and 09C7 were subjected to sample preparation, gel running, staining and decoloring, and after completion, were photographed using a Bio-Rad gel imager (model: universal Hood II).
The non-reducing SDS-PAGE results are shown in FIG. 11, and the molecular weight of the major band (five-pointed star) under non-reducing conditions is practically consistent. Antibodies 09C7-YTE-LALA-Kdel and 09C7 had distinct bands at the arrowed positions (HMW impurity) under non-reducing conditions, possibly as aggregates of the master bands or related heteroproteins of the non-master bands. The significantly lower proportion of HMW (relative to the master band) in the antibodies 09C7-C109S-YTE-LALA-Kdel compared to 09C7-YTE-LALA-Kdel and 09C7, indicates that 09C7-C109S-YTE-LALA-Kdel is capable of reducing the formation of HMW impurities. The results of SDS-PAGE under reducing conditions are shown in FIG. 12, where the molecular weights of the heavy and light chains are practically identical.
3. Analysis of antibody aggregation using non-reducing immunoblotting (WB)
The WB method was used to examine whether HMW impurities under non-reducing SDS-PAGE conditions were aggregates of antibodies.
BD Pharmingen HRP Anti-Human IgG is used as a secondary antibody, and experimental conditions are as follows: the prefabricated glue produced by gold is used, and the specification is SurePAGE, bis-Tris, 10X 8,USE MOPS,NO TRIS-GLYCINE,4-12%,10 holes, 10/pk, voltage 200V, time 35 minutes.
According to the results shown in FIG. 13, antibodies 09C7-YTE-LALA-Kdel and 09C7 had distinct bands at the arrowed positions, which were HMW aggregates of antibodies. With consistent master band gray scale, the proportion of HMW aggregates for antibodies 09C7-C109S-YTE-LALA-Kdel was lower compared to 09C7-YTE-LALA-Kdel and 09C7, indicating that 09C7-C109S-YTE-LALA-Kdel reduced aggregate formation.
From the above analysis, it is expected that the antibodies 09C7-C109S-YTE-LALA-Kdel are more stable during product storage and shelf stability period longer than 09C7-YTE-LALA-Kdel and 09C 7.
Sequence listing
SEQ ID NO. 1: amino acid sequence of CDRH1 (IMGT) as heavy chain Complementarity Determining Region (CDRH) of antibody 09C7
GFSLSTPGGG
SEQ ID NO. 2: amino acid sequence of CDRH2 (IMGT) as heavy chain complementarity determining region of antibody 09C7
IYWDDDK
SEQ ID NO. 3: amino acid sequence of CDRH3 (IMGT) as heavy chain Complementarity Determining Region (CDRH) of antibody 09C7
ARLTAADTIFDC
SEQ ID NO. 4: amino acid sequence of CDRL1 (IMGT) of light chain Complementarity Determining Region (CDRL) of antibody 09C7
SSDVGAYNY
SEQ ID NO. 5: amino acid sequence of CDRL2 (IMGT) of light chain complementarity determining region (CDL) of antibody 09C7
DVS
SEQ ID NO. 6: amino acid sequence of CDRL3 (IMGT) of light chain Complementarity Determining Region (CDR) of antibody 09C7
SSYTTTSVV
SEQ ID NO. 7: amino acid sequence of heavy chain variable region VH of antibody 09C7
QVTLRESGPTLVKPKQTLTLTCTFSGFSLSTPGGGVGWIRQPPGKALEWLALIYWDDDKRYSPSLKSSLTITKDTSKNQVVLTMTNMDPVDTATYYCARLTAADTIFDCWGQGTLVTV
SEQ ID NO. 8: amino acid sequence of light chain variable region VL of antibody 09C7
QSALTQPASVSGSPGLSITISCTATSSDVGAYNYVSWYQQHPGQAPKLMIYDVSKRPSGVSNRFSGSKSANTASLTISGLQAEDEADYYCSSYTTTSVVFGGGTKLTVL
SEQ ID NO. 9: amino acid sequence of heavy chain of antibody 09C7 (wild type), bold-Fab, italic-constant region
SEQ ID NO. 10: amino acid sequence of light chain of antibody 09C7 (wild type), bold-Fab, italic-constant region
SEQ ID NO. 11: amino acid sequence of CDRH3 (IMGT) as heavy chain complementarity determining region of antibody 09C7C 109S variant
ARLTAADTIFDS
SEQ ID NO. 12: amino acid sequence of heavy chain variable region VH of antibody 09C7 c109s variant
QVTLRESGPTLVKPKQTLTLTCTFSGFSLSTPGGGVGWIRQPPGKALEWLALIYWDDDKRYSPSLKSSLTITKDTSKNQVVLTMTNMDPVDTATYYCARLTAADTIFDSWGQGTLVTV
SEQ ID NO. 13: amino acid sequence of heavy chain of antibody 09C7 c109s variant, bold-Fab, italic-constant region
SEQ ID NO. 14: amino acid sequence of CDRH3 (IMGT) as heavy chain complementarity determining region of antibody 09C7C 109Y variant
ARLTAADTIFDY
SEQ ID NO. 15: amino acid sequence of heavy chain variable region VH of antibody 09C7 c109y variant
QVTLRESGPTLVKPKQTLTLTCTFSGFSLSTPGGGVGWIRQPPGKALEWLALIYWDDDKRYSPSLKSSLTITKDTSKNQVVLTMTNMDPVDTATYYCARLTAADTIFDYWGQGTLVTV
SEQ ID NO. 16: amino acid sequence of heavy chain of antibody 09C7 c109y variant, bold-Fab, italic-constant region
SEQ ID NO. 17: the amino acid sequence of the heavy chain of the antibody 09C7 LS variant, bold-Fab, italic-constant region, M431L and N437S mutations are underlined
SEQ ID NO. 18: amino acid sequence of heavy chain of antibody 09C7 Kdel variant, bold-Fab, italic-constant region, C-terminal lysine deletion
SEQ ID NO. 19: the amino acid sequence of the heavy chain of the antibody 09C7 GRLR variant, bold-Fab, italic-constant region, G239R and L331R mutations are underlined
SEQ ID NO. 20: the amino acid sequence of the heavy chain of the antibody 09C7YTE-Kdel variant, bold-Fab, italic-constant region, M255Y, S257T and T259E mutations are underlined and the C-terminal lysine is deleted
SEQ ID NO. 21: the amino acid sequence of the heavy chain of the antibody 09C7YTE-GRLR-Kdel variant, bold-Fab, italic-constant region, M255Y, S257T and T259E and G239R and L331R mutations are underlined, C-terminal lysine deleted
SEQ ID NO. 22: the amino acid sequence of the heavy chain of the antibody 09C7YTE-LALA-Kdel variant, bold-Fab, italic-constant region, M255Y, S257T and T259E and L237A and L238A mutations are underlined and C-terminal lysine deleted
SEQ ID NO. 23: the amino acid sequence of the heavy chain of the variant of antibody 09C7C109S-YTE-LALA-Kdel, bold-Fab, italic-constant region, C109S, M255Y, S T and T259E and L237A and L238A mutations are underlined and the C-terminal lysine deletion
SEQ ID NO. 24: the amino acid sequence of the heavy chain of the antibody 09C7C109Y-YTE-LALA-Kdel variant, bold-Fab, italic-constant region, C109Y, M255Y, S T and T259E and L237A and L238A mutations are underlined and the C-terminal lysine deletion
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Claims (15)

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to the S protein of SARS-CoV-2, characterized in that the isolated antibody or antigen-binding fragment thereof comprises:
heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3 of the amino acid sequences shown in SEQ ID NOs 1, 2 and 3, and light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 of the amino acid sequences shown in SEQ ID NOs 4, 5 and 6;
heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3 of the amino acid sequences shown in SEQ ID nos. 1, 2 and 11, and light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 of the amino acid sequences shown in SEQ ID nos. 4, 5 and 6; or alternatively
Heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3 of the amino acid sequences shown in SEQ ID NOs 1, 2 and 14, and light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 of the amino acid sequences shown in SEQ ID NOs 4, 5 and 6.
2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH as set forth in SEQ ID No. 7, 12 or 15 and a light chain variable region VL as set forth in SEQ ID No. 8.
3. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that the antibody or antigen-binding fragment thereof comprises a mutation selected from LS, kdel, GRLR, YTE, LALA and combinations thereof.
4. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that the antibody or antigen-binding fragment thereof comprises the heavy and light chains of the amino acid sequences:
a heavy chain shown in SEQ ID NO. 9 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 13 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 16 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 17 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 18 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 19 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 20 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 21 and a light chain shown in SEQ ID NO. 10;
A heavy chain shown in SEQ ID NO. 22 and a light chain shown in SEQ ID NO. 10;
a heavy chain shown in SEQ ID NO. 23 and a light chain shown in SEQ ID NO. 10; or alternatively
A heavy chain shown in SEQ ID NO. 24 and a light chain shown in SEQ ID NO. 10.
5. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that the antibody or antigen-binding fragment thereof is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment thereof.
6. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that the antigen-binding fragment is a Fab fragment, a Fab 'fragment, a F (ab') 2 Fragments, fv fragments, diabodies or single chain antibody molecules.
7. The isolated antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is of the IgG1, igG2, igG3 or IgG4 type.
8. An isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof of any one of claims 1-7.
9. An expression vector comprising the polynucleotide of claim 8.
10. A host cell comprising the polynucleotide of claim 8 or the expression vector of claim 9.
11. A method of producing an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-7, characterized in that the method comprises culturing the host cell according to claim 10 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof produced by the host cell.
12. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1-7 and a pharmaceutically acceptable carrier.
13. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-7 in the manufacture of a medicament for treating or preventing SARS-CoV-2 and mutant infections thereof in a subject in need thereof.
14. The use of claim 13, wherein the mutants include, but are not limited to Alpha (b.1.1.7), beta (b.1.351), gamma (p.1), delta (b.1.617.2) and omacron variants.
15. The use according to claim 14, wherein the Omicron variant comprises, but is not limited to, b.1.1.529, ba.1, ba.2, ba.2.12.1, ba.3, ba.4 and ba.5.
CN202311088752.3A 2022-09-20 2023-08-28 Neutralizing antibodies against SARS-CoV-2 and variants thereof and uses thereof Pending CN117736314A (en)

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CN2022119868 2022-09-20

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