Patent Application
20210395346
This research was funded by the National Research Foundation of Korea [NRF-2016M3A9B6918973] and the Ministry of Science and ICT(MSIT) of the Republic of Korea and the National Research Foundation of Korea [NRF-2020R1A3B3079653]. This research was supported by the Global Research Development Center Program, through the NRF, funded by the MSIT [2015K1A4A3047345]. This work was supported by the Brain Korea 21 Plus Project in 2020.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 18, 2021, is named 106094-1255889-USNP101_SL.txt and is 218,628 bytes in size.
The coronavirus SARS-CoV-2 is responsible for the disease Covid-19. SARS-CoV-2 uses the spike (S) protein for receptor binding and membrane fusion. The S protein interacts with the cellular receptor angiotensin-converting enzyme II (ACE2) to gain entry into the host cell.
Stereotypic neutralizing antibodies (nAbs) that are identified in convalescent patients can be valuable, providing critical information regarding the epitopes that should be targeted during the development of a vaccine. Those antibodies with naïve sequences, little to no somatic mutations, and IgM or IgD isotypes are especially precious (1, 2) because these characteristics effectively exclude the possibility that these nAbs evolved from pre-existing clonotypes that are reactive to similar viruses. This critical phenomenon is referred to as original antigenic sin (OAS), and predisposed antibody-dependent enhancement (ADE) enhancing the severity of viral infections, which can sometimes be fatal, as in the case of the dengue virus vaccine (3-6). Several groups have identified nAbs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (7-11), and one report suggested the possibility that stereotypic nAbs utilizing germline immunoglobulin heavy variable (IGHV)3-53 and IGHV3-66 segments may exist among convalescent patients (7). Furthermore, the structural basis of the stereotypic nAb reaction to SARS-CoV-2 was clarified using the co-crystal structure of two IGHV3-53 nAbs in complex with SARS-CoV-2 receptor-binding domain (RBD) by defining critical germline-encoded residues in the binding site of angiotensin-converting enzyme II (ACE2) (12). However, the prevalence of these stereotypic nAb clonotypes among SARS-CoV-2 patients and their characteristics, such as frequency in immunoglobulin (IG) repertoires, somatic mutations, isotypes, and chronological changes remain to be elucidated. Described herein are neutralizing antibodies that bind SARS-CoV-2 and methods of using same.
Described herein are neutralizing antibodies that bind to a coronavirus, pharmaceutical compositions comprising the antibodies, methods for producing and using the antibodies to induce an immune response in a subject infected with a coronavirus or recovering from a coronavirus infection, and methods for treating a subject infected with a coronavirus. In some embodiments, the coronavirus is SARS-CoV-2, and the subject is suffering from Covid-19.
Thus, in one aspect, an isolated neutralizing antibody that binds SARS-CoV-2 is provided. In some embodiments, the antibody is an IgG, IgA, IgA or IgM class antibody. In some embodiments, the antibody is an IgG1, IgA1, or IgA2 subclass antibody.
In some embodiments, the antibody binds to the S1, S2, RBD and/or N proteins of SARS-CoV-2.
In some embodiments, the antibody comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to one or more sequences shown in
In some embodiments, the antibody comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a light chain and/or a heavy chain variable region amino acid sequence shown in Table 10. In some embodiments, the antibody comprises a light chain variable region (VL) having an amino acid sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25. In some embodiments, the antibody comprises a heavy chain variable region (VH) having an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26. In some embodiments, the antibody comprises:
i) a VL amino acid sequence of SEQ ID NO:1 and a VH amino acid sequence of SEQ ID NO:2;
ii) a VL amino acid sequence of SEQ ID NO:3 and a VH amino acid sequence of SEQ ID NO:4;
iii) a VL amino acid sequence of SEQ ID NO:5 and a VH amino acid sequence of SEQ ID NO:6;
iv) a VL amino acid sequence of SEQ ID NO:7 and a VH amino acid sequence of SEQ ID NO:8;
v) a VL amino acid sequence of SEQ ID NO:9 and a VH amino acid sequence of SEQ ID NO:10;
vi) a VL amino acid sequence of SEQ ID NO:11 and a VH amino acid sequence of SEQ ID NO:12;
vii) a VL amino acid sequence of SEQ ID NO:13 and a VH amino acid sequence of SEQ ID NO:14;
viii) a VL amino acid sequence of SEQ ID NO:15 and a VH amino acid sequence of SEQ ID NO:16;
ix) a VL amino acid sequence of SEQ ID NO:17 and a VH amino acid sequence of SEQ ID NO:18;
x) a VL amino acid sequence of SEQ ID NO:19 and a VH amino acid sequence of SEQ ID NO:20;
xi) a VL amino acid sequence of SEQ ID NO:21 and a VH amino acid sequence of SEQ ID NO:22,
xii) a VL amino acid sequence of SEQ ID NO:23 and a VH amino acid sequence of SEQ ID NO:24; or
xiii) a VL amino acid sequence of SEQ ID NO:25 and a VH amino acid sequence of SEQ ID NO:26;
or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%. or 95% sequence identity thereto.
In some embodiments, the antibody comprises a V gene and/or a J gene in
In some embodiments, the antibody comprises a HCDR3 amino acid sequence in
In some embodiments, the antibody inhibits binding of SARS-CoV-2 S glycoprotein to ACE2.
In some embodiments, the antibody binds to a mutant RBD comprising one or more amino acid substitutions selected from V341I; F342L; N354D; D364Y; N354D and D364Y; V367F; A435S; W436R; G476S; V483A; G476S and V483A; N501Y; N439K; K417V; K417V and N439K; K417N; E484K; K417N, E484K, and N501Y; K417T; K417T, E484K, and N501Y; L452R; S477N; E484K; E484Q; or E484Q and L452R, or combinations thereof.
In some embodiments, the clonotype is IGHV3-53/IGHV3-66 and IGHJ6. In some embodiments, the antibody is a naïve stereotypic IGHV3-53/IGHV3-66 and IGHJ6 clone.
In some embodiments, the antibody is an scFv, Fab, or other antigen binding fragment or format thereof.
In another aspect, a pharmaceutical composition comprising an antibody described herein is provided.
In another aspect, a nucleic acid encoding a heavy chain variable region and/or a light chain variable region of an antibody described herein is provided.
In another aspect, a vector comprising a nucleic acid encoding a heavy chain variable region and/or a light chain variable region of an antibody described herein is provided. In some embodiments, the vector further comprises a nucleic acid encoding a hIgG1 Fc region (hFc) or hCκ region operably linked to the nucleic acid encoding the heavy chain variable region or the nucleic acid encoding the light chain variable region.
In another aspect, a host cell comprising a vector described herein is provided.
In another aspect, a method for producing an antibody is described. In some embodiments, the method comprises culturing a host cell described herein under conditions in which the nucleic acids encoding the heavy and light chain variable regions are expressed.
In another aspect, an in vitro method for detecting binding of an antibody to SARS-CoV-2 antigens is described. In some embodiments, the method comprises contacting a cell infected with SARS-CoV-2 with an antibody described herein, and detecting binding of the antibody to the cell. In some embodiments, the method comprises contacting a recombinant SARS-CoV-2 antigen with an antibody described herein, and detecting binding of the antibody to the antigen. In some embodiments, the recombinant SARS-CoV-2 antigen comprises the SARS-CoV-2 spike, S1, S2, or N protein, or a recomdinant RBD domain of the S protein. In some embodiments, the recombinant SARS-CoV-2 antigen is fused to a molecular tag, such as a HIS tag, or fused to an antibody domain, such as a human Cκ domain.
In another aspect, a method of inducing an immune response in a subject is described. In some embodiments, the method comprises administering an antibody or pharmaceutical composition described herein to a subject.
In another aspect, a method of treating a patient infected with SARS-CoV-2 or suffering from COVID-19 is described. In some embodiments, the method comprises administering a therapeutically effective amount of an antibody or pharmaceutical composition described herein to the patient.
In another aspect, provided is an antibody or pharmaceutical composition described herein for use in the treatment of one or more symptoms of SARS-CoV-2 infection or COVID-19 disease in a subject. In some embodiments, provided is a pharmaceutical composition comprising an antibody described herein for the treatment of one or more symptoms of SARS-CoV-2 infection or COVID-19 disease in a subject.
The term “stereotypic” refers to a characteristic shared between many or most individuals, or a non-heterogeneous characteristic.
The term “clonotype” refers to a collection of B cell receptor sequences sharing identical or similar functions expected to be derived from the same progenitor cells, and includes stereotypic antibodies comprising a VH clonotype encoding the same VH and JH genes and perfectly matched HCDR3 sequences, at the amino acid level.
The term “antibody” refers to an immunoglobulin (Ig) molecule or fragment or format thereof that specifically binds to a target antigen. The term includes monoclonal antibodies and the IgA, IgD, IgE, IgG, and IgM isotypes and subtypes. The term also includes antigen-binding fragments or formats thereof, such as Fab (fragment, antigen binding), Fv (variable domain), scFv (single chain fragment variable), disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), dimeric and multimeric antibody formats like dia-, tria- and tetra-bodies, minibodies (miniAbs) comprising scFvs linked to oligomerization domains, VHH/VH of camelid heavy chain Abs and single domain Abs (sdAb). The term also includes fusion proteins of that antibodies or antigen-binding fragments thereof, such as scFv-light chain fusion proteins, or scFv-Fc fusion proteins. The term also includes antibodies or antigen-binding fragments thereof that include an Fc domain to provide effector functions such as Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC).
The term “neutralizing antibody” refers to an antibody or fragment thereof that prevents infection of a host cell by a virus, or blocks attachment to the cell and/or entry of the virus into the cell.
The term “subject” refers to an animal, for example a mammal, including but not limited to a human, a rodent such as a mouse or rat, a companion animal such as a dog or cat, and livestock such as cows, horses, and sheep. The term subject can also be used interchangeably with the term “patient.”
The term “sequence identity” refers to two or more amino acid or nucleic acid sequences, or subsequences thereof, that are the same. Sequences can also have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection. Two or more amino acid or nucleic acid sequences can also have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, thereby excluding sequences that are 100% identical (for example, a variant sequence is less than 100% identical to a wild-type or reference sequence). Two amino acid sequences can also be similar, i.e., they have a specified percentage of amino acid residues that are either the same or similar as defined by a conservative amino acid substitutions (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm described herein or by manual alignment and visual inspection. The above definitions also refer to the complement of a nucleotice sequence. For sequence comparison, one sequence typically acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are commonly used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities or similarities for the test sequences relative to the reference sequence, based on the program parameters.
The “percentage of sequence identity” can determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)). Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (see the internet at www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4.
The term “host cell” refers to both single-cell prokaryote and eukaryote organisms (e.g., bacteria, yeast, and actinomycetes) and single cells derived from multicellular plants or animals. Host cells are typically isolated and grown in cell culture.
The term “vector” refers to a nucleic acid sequence, typically double-stranded DNA, which can comprise a fragment of heterologous nucleic acid sequence (e.g., a heterologous DNA sequence) inserted into the vector sequence. The vector can be derived from a bacterial plasmid. Vectors can contain polynucleotide sequences that facilitate the autonomous replication of the vector in a host cell. The term “heterologous” refers to nucleic acid sequences not naturally found in the host cell, for example, sequences that function to replicate the vector molecule, or sequences that encode a selectable or screenable marker, or encode a transgene. A vector can used to transport the heterologous nucleic acid sequence into a suitable host cell. Once in the host cell, the vector can replicate independently of or coincidental with the host chromosomal DNA, and multiple copies of the vector and its inserted DNA can be generated. In addition, the vector can also contain the necessary elements that permit transcription of the heterologous DNA into an mRNA molecule or otherwise cause replication of the heterologous DNA into multiple copies of RNA. Expression vectors can contain additional sequence elements adjacent to the inserted DNA that increase the half-life of the expressed mRNA and/or allow translation of the mRNA into a protein molecule.
Neutralizing Antibodies
Described herein are stereotypic neutralizing antibodies (nAbs) that bind SARS-CoV-2 antigens. The antibodies can comprise naïve immunoglobulin (Ig) sequences having few or no somatic mutations. For example, described herein are stereotypic-naïve SARS-CoV-2 neutralizing antibody clonotypes that are present in the majority of patients with few somatic mutations and class-switched isotypes, and also pre-exist in the majority of individuals in the healthy human population, predominantly as an IgM isotype.
The inventors have unexpectedly found that the stereotypic-naïve nAbs described herein can rapidly initiate virus neutralization upon SARS-CoV-2 infection. The stereotypic-naïve SARS-CoV-2 nAbs described herein also provide the unexpected advantage of allowing a naïve heavy chain variable region sequence to pair with multiple light chain variable region sequences (referred to herein as light chain plasticity), and the resulting antibodies can bind the RBD and neutralize virus infection of host cells. The naïve heavy chain clonotypes described herein further provide the advantage of potentially providing near-immediate protection to subjects exposed to SARS-CoV-2 and thereby improve clinical outcomes. The nAbs described herein also provide the unexpected advantage of binding to known mutations within the RBD, therefore potentially providing protection against many SARS-CoV-2 mutants. Thus, the nAbs described herein may prevent “escape” of viral mutants in patients administered an antibody described herein, or prevent a reduction in the secondary immune response due to subsequent exposure to variant strains of SARS-CoV-2 (referred to as original antigenic sin).
In some embodiments, the nAbs described herein do not activate effector functions in response to closely related viruses. In some embodiments, the nAbs described herein do not trigger antibody-dependent enhancement (ADE) when administered to a subject.
In some embodiments, the stereotypic nAb is perfectly naïve and comprises a variable region encoded by a germline variable region gene (i.e., a genomic nucleic acid sequence). In some embodiments, the stereotypic nAb comprises a germline heavy chain variable region sequence joined to a germline J region sequence. In some embodiments, the stereotypic nAb has a low frequency of somatic mutations, for example, less than 2.695%+/−0.700%.
In some embodiments, the heavy chain of the stereotypic nAb is encoded by immunoglobulin heavy variable gene IGHV3-53. In some embodiments, the heavy chain of the stereotypic nAb is encoded by immunoglobulin heavy variable gene IGHV3-66. In some embodiments, the stereotypic nAb comprises a heavy chain variable region (VH) amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to one or more sequences shown in Table 10. In some embodiments, the VH sequence comprises an HCDR3 having the amino acid sequence shown in
In some embodiments, the joining region of the stereotypic nAb is encoded by the immunoglobulin heavy joining 6 gene IGHJ6.
In some embodiments, the stereotypic nAb is an IgM isotype. In some embodiments, the stereotypic nAb is an IgG (e.g., IgG1, IgG2, IgG3) isotype, IgA (e.g. IgA1, IgA2) isotype, or IgD isotype.
In some aspects, the stereotypic nAb comprises a common heavy chain paired with different light chains (referred to as “light chain plasticity”). For example, In some embodiments, the stereotypic nAb comprises a heavy chain encoded by IGHV3-53 or IGHV3-66, and a light chain encoded by one of five different Vκ/Vλ genes. Representative examples of a common heavy chain paired with different light chains are shown in
In some embodiments, the stereotypic nAb comprises a heavy chain variable region (VH) paired with a light chain variable region (VL) clone shown in
In some embodiments, the stereotypic nAb comprises a heavy chain variable region comprising a HCDR3 amino acid sequence shown in
In some embodiments, the stereotypic nAb comprises a light chain variable region (VL) amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to one or more sequences shown in Table 10. In some embodiments, the stereotypic nAb comprises a light chain variable region LCDR3 amino acid sequence shown in
In some aspects, the clonotypes described herein comprise substantially identical heavy chain variable region (VH) amino acid sequences, for example the VH amino acid sequences are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, the clonotypes comprise VH sequences having a low frequency of somatic mutations, for example, a frequency of less than 5%, 4%, 3%, 2%, or 1% somatic mutations.
In some embodiments, the nAbs inhibit the binding of the coronavirus spike (S) protein to angiotensin-converting enzyme II (ACE2). ACE2 is the cellular receptor for SARS-CoV-2 in humans, which allows the virus to gain entry into a host cell. In some embodiments, the nAbs bind recombinant S protein. In some embodiments, the nAbs bind recombinant receptor-binding domain (RBD) protein. The RBD is located within the S1 region of the S protein. In some embodiments, the nAbs bind recombinant SARS-CoV-2 nucleocapsid (NP), S, S1 subunit, S2 subunit, and/or RBD proteins. The S1 subunit of the spike protein contains the receptor binding domain and is responsible for recognition and binding to the host cell receptor. The S2 domain is thought to be responsible for fusion between the viral envelope and the host cell membrane.
In some embodiments, the nAb binds to a mutant RBD comprising an amino acid substitution selected from one or more of the following: V341I, F342L, N354D, D364Y; N354D and D364Y, V367F, A435S, W436R, G476S, V483A; G476S and V483A; N501Y; N439K; K417V; K417V and N439K; K417N; E484K; K417N, E484K, and N501Y; K417T; K417T, E484K, and N501Y; L452R; S477N; E484K; E484Q; or E484Q and L452R, or combinations thereof.
Neutralization Assays
Neutralizing antibodies can be identified using a suitable neutralization assay. In some embodiments, the neutralization assay comprises inoculating or infecting cells or a cell line with SARS-CoV-2 virus, culturing the cells under conditions whereby the cells produce the virus, isolating the virus from the cells, mixing an amount (e.g., a predetermined amount) of the isolated virus with the antibody, contacting the mixture of virus and antibodies with non-infected cells or a non-infected cell line, and culturing the cells or cell line for an amount of time (for example, 24, 48 or 72 hours, or 1 to 5 days). In some embodiments, culture supernatant is collected, the viral titer is determined, for example by using a TCID50 assay, and the amount of viral RNA in the supernatant is quantified, for example, based on a standard curve using in vitro transcribed RNA. In some embodiments, the cell line is a Vero cells (ATCC CCL-81). In some embodiments, the cells or cell line are incubated with 100 to 2,500 TCID50 of SARS-CoV-2 virus.
Another example of a neutralization assay comprises determining the cytopathic effect (CPE) of cells infected with SARS-CoV-2 virus in the presence and absence of an antibody described herein. In some embodiments, a cell or cell line is incubated with a mixture of SARS-CoV-2 virus and the antibody, cultured for an amount of time (for example, 24, 48 or 72 hours, or 1 to 5 days), and the CPE determined, for example by calculating an IC50. In some embodiments, the cell line is a Vero cells (ATCC CCL-81). In some embodiments, the cells or cell line are incubated with 100 to 2,500 TCID50 of SARS-CoV-2 virus.
In some embodiments, the antibodies described herein inhibit binding of the SARS-CoV-2 virus to a target cell. Thus, antibodies that inhibit binding of the SARS-CoV-2 virus to a target cell can be identified using an assay that measures inhibition of binding between a SARS-CoV-2 virus and a cells. In some embodiments, the assay detects inhibition of binding between recombinant SARS-CoV-2 S protein and cells expressing the ACE2 receptor. In some embodiments, the assay comprises mixing recombinant SARS-CoV-2 S protein with an antibody described herein, and determining the binding of the S protein to a cell or cell line expressing the ACE2 receptor. The binding can be measured by flow cytometry using an labeled antibody that binds to the recombinant SARS-CoV-2 S protein, where a decrease in signal from the label compared to a positive control indicates that the antibody inhibited binding of the S protein to the ACE2 receptor. In some embodiments, the recombinant SARS-CoV-2 S protein is fused with a polyhistidine (HIS)-tag, and the relative amount of bound, recombinant SARS-875 CoV-2 S glycoprotein is measured using a fluorescein isothiocyanate (FITC)-conjugated anti-HIS antibody. In some embodiments, the antibodies described herein inhibited binding between recombinant S protein and cells expressing the ACE2 receptor at an equimolar (1:1) ratio of recombinant S protein to antibody concentration, or up to a molar ration of 1:3 recombinant S protein to antibody concentration. In some embodiments, the antibodies described herein exhibit a half-maximal inhibitory concentration (IC50) from 0.1 to 0.8 μg/mL. In some embodiments, the cell expressing the ACE2 receptor is a Vero E6 cell.
Antibody Formats
In some embodiments, the nAbs described herein are monoclonal antibodies comprising two Fab arms and one Fc region. In some embodiments, the two Fab arms bind to the same epitope of SARS-CoV-2. In some embodiments, the antibody comprises a single-chain variable fragment (scFv). In some embodiments, the antibody comprises a scFv fusion protein. In some embodiments, the scFv fusion protein comprises a scFv-light chain fusion protein. In some embodiments, the scFv fusion protein comprises a scFv-human kappa light chain fragment (hCκ) fusion protein (scFv-hCκ fusion proteins). In some embodiments, the scFv fusion protein comprises a scFv-human Fc region fusion protein (scFv-hFc fusion proteins).
Variants
Also described herein are variants of the neutralizing antibodies described herein. In some embodiments, the variant antibodies comprise one or more amino acid substitutions in the heavy or light chain sequence of an antibody described herein. In some embodiments, the variant antibodies comprise one or more amino acid substitutions in the heavy chain variable region (VH) or light chain variable region (VL) sequence of an antibody described herein. In some embodiments, the variant antibodies comprise one or more amino acid substitutions in the complementarity-determining regions (CDRs) of an antibody described herein, for example one or more amino acid substitutions in the heavy chain CDR1 (HCDR1), HCDR2 or HCDR3 sequence, or one or more amino acid substitutions in the light chain CDR1 (LCDR1), LCDR2 or LCDR3 sequence.
Antibody Libraries
Also provided are libraries comprising the antibodies described herein. The libraries can be prepared from biological samples from subjects infected by SARS-CoV-2. In some embodiments, the biological sample is a blood sample. Peripheral blood mononuclear cells (PBMCs) present in the blood sample are then isolated, and total RNA is prepared from the PBMCs. cDNA is synthesized from the RNA using primers that bind to the poly A tail of mRNA, or using gene specific primers. In some embodiments, the gene specific primers bind to sequences in the constant region (CH1 domain) of each isotype (IgM, IgD, IgG, IgA, and IgE). Following second strand cDNA synthesis, the double stranded DNA is purified, and the IgG genes are amplified, for example by PCR. For example, the VH and VL (VK and Vλ) encoding genes can be amplified by PCR. In some embodiments, overlap extension PCR is used to link the amplified VH and VK/Vλ encoding fragments. In some embodiments, the VH and VK/Vλ encoding fragments are linked to produce scFv fusion constructs, that are then cloned into a phagemid vector. The synthesized VH and VL (VK and Vλ) encoding genes can be amplified to produce scFv libraries, for example by PCR. The amplified scFV fragments can be cloned into phagemid vectors to produce a phage library. In some embodiments, the library can contain VK/Vλ shuffled libraries.
Methods for Identifying Antibodies that Bind SARS-Cov-2 Antigens
The antibody libraries described herein can be used to identify antibodies that bind recombinant SARS-CoV-2 antigens, for example recombinant SARS-CoV-2 S and RBD proteins. In some embodiments, the recombinant SARS-CoV-2 antigenic proteins are fused to an Fc region or an antibody constant region, as described in the Examples. Methods for identifying antibodies that bind recombinant SARS-CoV-2 antigens include phage display followed by contacting the expressed antibodies to recombinant SARS-CoV-2 antigens, and eluting the bound antibodies. The recombinant SARS-CoV-2 antigens can be bound or conjugated to beads or magnetic beads. The bind and elute steps can be repeated multiple time, e.g., by biopanning, to identify high affinity antibodies.
In some embodiments, antibodies that bind SARS-CoV-2 antigens can be identified using an enzyme-linked immunosorbent assay (ELISA).
In some embodiments, neutralizing antibodies that bind SARS-CoV-2 antigens can be identified using a neutralization assay described herein. In some embodiments, neutralizing antibodies that bind SARS-CoV-2 antigens can be identified using an inhibition assay described herein.
In some embodiments, neutralizing antibodies that bind SARS-CoV-2 antigens can be identified using a phage ELISA. For example, antibodies can be selected that bind to SARS-CoV-2 S protein using recombinant S and RBD protein-coated microtiter plates, as described previously (45). In some embodiments, the antibody is an scFv. Antibodies can be sequenced to determine their nucleotide sequences.
Also provided are methods for identifying antibodies that have neutralizing activity against the SARS-CoV-2 virus. The method can comprise mutagenizing a polynucleotide encoding a heavy chain variable region or a light chain variable region of an antibody; expressing the antibody comprising the mutagenized heavy chain and/or light chain variable region; and selecting an antibody with neutralizing activity. The antibody with neutralizing activity can be selected using an assay described herein.
Representative antibody libraries and methods for producing same are described in the Examples.
Samples
To obtain antibodies against SARS-CoV-2, biological samples are typically obtained from the subject or patient. Samples include blood samples, plasma samples, and/or serum samples. In some embodiments, the sample comprises PBMCs. In some embodiments, the subject or patient is or has been infected by SARS-CoV-2. In some embodiments, the subject or patient is a human.
Pharmaceutical Compositions
Also described herein are pharmaceutical compositions comprising an antibody described herein. The pharmaceutical compositions can include additives such as a filler, bulking agent, buffer, stabilizer, or excipient. Standard pharmaceutical formulation techniques are well known to persons skilled in the art (see, e.g., 2005 Physicians' Desk Reference©, Thomson Healthcare: Montvale, N.J., 2004; Remington: The Science and Practice of Pharmacy, 20th ed., Gennado et al., Eds. Lippincott Williams & Wilkins: Philadelphia, Pa., 2000). In some embodiments, the pharmaceutical compositions contain pH buffering reagents, wetting or emulsifying agents, preservatives or stabilizers.
The pharmaceutical composition can also be formulated based on the intended route of administrations and other parameters (see, e.g., Rowe et al., Handbook of Pharmaceutical Excipients, 4th ed., APhA Publications, 2003). For example, the pharmaceutical composition be formulated for parental administration by intravenous, subcutaneous, intramuscular, or intra-articular administration. In some embodiments, the pharmaceutical composition is provided as a liquid or lyophilized form. In some embodiments, the pharmaceutical composition is a sterile, non-pyrogenic solution.
Nucleic Acids
Also provided are nucleic acid molecules such as polynucleotides that comprise a sequence encoding the amino acid sequence of an antibody described herein. In some embodiments, the nucleic acid molecule encodes a heavy chain and/or a light chain of an antibody described herein. In some embodiments, the nucleic acid molecule encodes a heavy chain variable region or a light chain variable region of an antibody described herein. In some embodiments, the nucleic acid molecule comprises sequences encoding both a heavy chain, or heavy chain variable region, and a light chain, or light chain variable region, of an antibody described herein. In some embodiments, the heavy and light chain variable regions are linked together. Methods for linking together the heavy and light chain variable regions, include but are not limited to ligation and overlap extension PCR. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is an RNA molecule.
In some embodiments, the nucleic acid molecule encodes an amino acid sequence shown in
Vectors
Also described herein are vectors comprising one or more nucleic acid sequences, for example, one or more nucleic acid sequences encoding an antibody described herein. In some embodiments, the vector comprises one or more nucleic acid sequences encoding a light chain variable region and/or a heavy chain variable region described herein. In some embodiments, the vector comprises one or more nucleic acid sequences encoding an amino acid sequence shown in
In some embodiments, the vector comprises one or more nucleic acid sequences encoding a light chain variable region (VL) having an amino acid sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25. In some embodiments, the vector comprises one or more nucleic acid sequences encoding a heavy chain variable region (VH) having an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26. In some embodiments, the vector comprises one or more nucleic acid sequences encoding a light chain variable region (VL) having an amino acid sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, and one or more nucleic acid sequences encoding a heavy chain variable region (VH) having an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26. In some embodiments, the vector comprises one or more nucleic acid sequences encoding a light chain variable region (VL) comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, and one or more nucleic acid sequences encoding a heavy chain variable region (VH) comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26.
In some embodiments, vector is an expression vector, such as a mammalian expression vector. In some embodiments, the vector is a phagemid vector. The expression vector can further comprise a constitutive or inducible promoter sequence for regulating transcription of the one or more nucleic acids, and a terminator sequence for terminating transcription. The one or more nucleic acids can be separated by internal ribosome entry sites (IRESes) that allow expression of different proteins from the same transcription unit.
In some embodiments, the vector comprises a nucleotide sequence encoding an Fc region or an antibody constant region at the 3′ end. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the constant region is a human kappa constant region (hCκ). In some embodiments, the vector comprises the CH1 and hinge regions of an antibody. In some embodiments, the vector comprises the CH1 and hinge regions of a human or humanized antibody. In some embodiments, the vector comprises the the CH2 and CH3 regions of an antibody. In some embodiments, the vector comprises the the CH2 and CH3 regions of a human or humanized antibody. In some embodiments, the vector comprises the CH1 and hinge regions of human IgG2 fused to the CH2 and CH3 regions of human IgG4.
Host Cells
Also described herein are host cells. The host cells can comprise a vector described herein, and/or can comprise a nucleic acid sequence encoding an antibody described herein. Examples of host cells include single celled prokaryotes and eukaryotes, such as bacteria or yeast, or cells derived from multicellular organisms such as plants or animals. In some embodiments, the host cell is from a mammalian cell line. In some embodiments, the host cell is an Expi293F cell (Invitrogen).
In some embodiments, the host cell is capable of being infected by SARS-CoV-2. In some embodiments, the host cell expresses the ACE2 receptor. In some embodiments, the host cell expressing the ACE2 receptor is a Vero cell, or aVero E6 cell.
Methods of Producing Antibodies
In some embodiments, the neutralizing antibodies to SARS-CoV-2 described herein can be produced by transfecting a host cell with a nucleic acid encoding a heavy chain variable region and/or a light chain variable region of the antibody, and culturing the host cell under conditions suitable for expressing the heavy and/or light chain variable region protein. In some embodiments, the host cell comprises one or more nucleic acids encoding both a heavy chain variable region and a light chain variable region of the antibody, and the heavy and light chains self-assemble to form a functional antibody that specifically binds a SARS-CoV-2 antigen.
In some embodiments, the host cell comprises an expression vector comprising one or more nucleic acid sequences encoding a heavy chain variable region and/or a light chain variable region of a neutralizing antibody to SARS-CoV-2 described herein.
In some embodiments, the method for producing an antibody comprises synthesizing the amino acid sequence of the heavy chain variable region and/or light chain variable region of an antibody described herein.
Neutralizing antibodies to SARS-CoV-2 can also be obtained from biological samples from subjects infected with SARS-CoV-2. In some embodiments, the biological sample is a blood sample. In some embodiments, the antibodies so obtained can be used to generate antibody libraries.
In Vitro Methods for Detecting Binding of an Antibody to SARS-Cov-2 Antigens
In another aspect, an in vitro method for detecting binding of an antibody to SARS-CoV-2 antigens is described. In some embodiments, the method comprises contacting a cell infected with SARS-CoV-2 with an antibody described herein in vitro, and detecting binding of the antibody to the cell.
In some embodiments, the method comprises contacting a SARS-CoV-2 antigen with an antibody described herein in vitro, and detecting binding of the antibody to the antigen. The binding of the antibody to the SARS-CoV-2 antigen can be detected using an enzyme-linked immunosorbent assay (ELISA), or by detecting the signal from a labeled antibody such as a fluorescein labeled antibody.
In some embodiments, binding of an antibody described herein to SARS-CoV-2 antigens is detected by inhibiting binding between the SARS-CoV-2 S protein and a cell that expresses the ACE2 receptor. In some embodiments, the SARS-CoV-2 S protein is recombinantly labeled with a poly-HIS tag, and the relative amount of bound, recombinant SARSCoV-2 S glycoprotein is measured using a FITC-conjugated anti-HIS antibody. A decrease in fluorescent signal indicates that the antibody inhibits binding between the S glycoprotein and the ACE2 receptor.
Method of Inducing an Immune Response in a Subject
In another aspect, a method of inducing an immune response in a subject is provided. In some embodiments, the method comprises administering an antibody described herein to a subject. Following administration of the antibody, induction of the immune response can be detected in a biological sample from the subject, such as a blood or serum sample. Induction of an immune response includes induction of cytokines such an Type I IFNs (IFN-α and IFN-β) and IFN-γ, and changes to the TCR and BCR repertoire.
Methods of Treatment
Also described are methods for treating a subject infected with SARS-CoV-2, or displaying the symptoms of Covid-19. In some embodiments, the method comprises administering a therapeutically effective amount of an antibody described herein to the subject or patient. In some embodiments, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody described herein to the subject or patient.
The antibodies described herein can be administered to a subject using an route of administration, such as parenterally, intravenously, subcutaneously, or intramuscularly. The antibody can be administered daily, weekly, or monthly. The antibody can be administered in a body—size-based, for example in a range from 1 milligram/square meter to 500 mg/square meter of body surface, or from 1 mg/kg to 10 mg/kg of body weight. The total single dose can range from 400 to 10,000 milligrams (0.4 to 10 grams) for a human subject. The dose can be a single dose, or multiple doses, such as two or more weekly doses.
The treatment method may further comprise administering one or more additional treatments, such as therapeutic agents or medical procedures, to the subject. In some embodiments, the one or more additional treatments include antivirals, immune-based therapies, other neutralizing antibodies, and administering oxygen and/or mechanical ventilators for patients with respiratory conditions or failure. In some embodiments, the antiviral is selected from Remdesivir, Lopinavir/Ritonavir (Kaletra®), Favipiravir, azithromycin or Arbidol. In some embodiments, the additional treatment comprises administering hydroxychloroquine or chloroquine to the subject.
In some embodiments, the additional treatment comprises administering an immune-based therapy, such as convalescent plasma and/or SARS-CoV-2-specific immune globulins, to the subject. In some embodiments, the additional treatment comprises immune suppressant drugs to treat the so-called “cytokine storm” associated with Covid-19 infection in patients that develop acute respiratory distress syndrome (ARDS). The immunosuppressant drug can be selected from those currently being tested in clinical trials, including baricitinib, a drug for rheumatoid arthritis; CM4620-IE, a drug for pancreatic cancer; and Interleukin inhibitors such as IL-6 inhibitors (e.g., sarilumab, siltuximab, or tocilizumab). In some embodiments, the additional treatment comprises administering immunomodulators, such as alpha and beta interferons and kinase inhibitors, to the patient.
In some embodiments, the additional treatment comprises administering corticosteroids to the patient. In some embodiments, the additional treatment comprises administering antithrombotic therapy to the patient. Antithrombotic therapy can include anticoagulants and antiplatelet therapy. Thus, in some embodiments, the additional treatment comprises administering venous thromboembolism (VTE) prophylaxis per the standard of care.
In some embodiments, the additional treatment comprises filtering cytokines out of the blood of Covid-19 patients. Suitable filters include those granted emergency use authorization by the FDA, including the Spectra Optia Apheresis System (Terumo BCT Inc.) and Depuro D2000 Adsorption Cartridge (Marker Therapeutics AG) devices.
In some embodiments, the additional treatment comprises administering oxygen therapy to the patient. In some embodiments, the additional treatment comprises placing the patient on ventilator support if the patient presents acute hypoxemic respiratory failure despite conventional oxygen therapy. In one embodiment, the treatment comprises administering high-flow nasal cannula (HFNC) oxygen to the patient.
This example describes the identification, cloning and expression of neutralizing antibodies that bind SARS-CoV-2.
Isolation and Characterization of Human nAbs
To obtain monoclonal nAbs against SARS-CoV-2, blood samples were collected from 17 SARS-CoV-2-infected patients (Patients A-Q) and used them to generate human antibody libraries. Similar to severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 also uses a spike (S) protein for receptor binding and membrane fusion (13). This protein interacts with the cellular receptor ACE2 to gain entry into the host cell (14, 15). A previous report suggested that a human monoclonal antibody (mAb), which reacted with the RBD, within the S1 region of the S protein, could hinder the initial interaction between the virus and the cell, effectively neutralizing SARS-CoV-2 (11). The reactivity of the sera derived from patients against recombinant SARS-CoV-2 S and RBD proteins was confirmed. Patients A and E, who presented with extensive pneumonic infiltrates, also showed high plasma IgG titers against all recombinant SARS-CoV-2 nucleocapsid (NP), S, S1, S2, and RBD proteins, which could be detected 11, 17, and 45 days after symptom onset in Patient A and 23, 44, and 99 days after symptom onset in Patient E (Table 2 and
Identification of Stereotypic Clonotypes from IGH Repertoire of SARS-CoV-2-Infected Patients
Deep profiling of the IG repertoire in three chronological blood samples each from Patients A and E, two chronological samples from each of Patients B, C, D, F, and G, and a single timepoint sample from each of the other ten patients (H-Q) was performed. nAb clonotypes that possessed identical variable (V) and joining (J) gene combinations and perfectly matched heavy chain complementarity-determining region 3 (HCDR3) amino acid sequences among the immunoglobulin heavy chain (IGH) repertoires of Patients A and E was determined. One and five nAb clonotypes were successfully identified in Patients A and E, respectively (
To test the reactivity of clonotypes homologous to E-3B1 against the SARS-CoV-2 S protein, 12 IGH clonotypes (
Among these IGH clonotypes, A,B,G-42 was quite unique, presenting little to no (0.6%+/−0.8%) somatic mutations and containing an HCDR3 (DLYYYGMDV (SEQ ID NO: 27)) formed by the simple joining of IGHV3-53 and IGHJ6. This naïve VH sequence existed in the IGH repertoire of five patients (Patients A, B, G, I, and K), as IgM and IgG1, IgM and IgG1, IgG1 and IgA1, IgM, or IgG1 subtypes, respectively (Table 1). More interestingly, the IGH clonotypes encoded by IGHV3-53/IGHV3-66 and IGHJ6 that possessed an HCDR3 (DLYYYGMDV (SEQ ID NO: 27)) with zero to one somatic mutation could be identified within the IGH repertoire of six of 10 healthy individuals, predominantly as an IgM isotype (16), based on publicly available IGH repertoires (Table 1). The A,B,G-42 clonotype showed light chain plasticity and paired with five Vκ/Vλ genes to achieve RBD binding. In particular, the Vκ gene (2J6H) accumulated only five somatic mutations (1.4% divergence). None of the 12 clones, including A,B,G-42, reacted against the recombinant RBD proteins from either SARS-CoV or MERS-CoV (
To further elucidate the preferential use of IGHV3-53/IGHV3-66 and IGHJ6 genes during the generation of SARS-CoV-2 RBD-binding antibodies, 252 predicted RBD-binding clones were extracted from the biopanning data (See Methods). It was previously shown that antibody clones with binding properties can be predicted by employing next-generation sequencing (NGS) technology and analyzing the enrichment patterns of biopanned clones (18, 19). Although the IGHJ4 gene was more prominent in the IGH repertoires of 17 patients, similar to healthy human samples (16, 20), the predicted RBD-binding clones primarily used the IGHJ6 gene (
Chronological Follow-Up of IGH Repertoire and the SARS-CoV-2 RBD-Binding Antibodies from Patients
Naïve B cells typically undergo somatic hypermutations, clonal selection, and class-switching following antigen exposure. Thus, the chronological events that occurred in all IGH clonotypes identified in Patients A-G and those that were reactive against the SARS-CoV-2 RBD were examined. In the entire patient IGH repertoire, naïve-derived IGH clonotypes with minimal somatic mutations (<2.695%+/−0.700%) showed increased IgG3 and IgG1 subtypes, and the proportion of the IgG1 subtype was dramatically increased for a period (
Selected nAbs Retained the Ability to Bind to Most Current SARS-CoV-2 Mutants
Because several mutations within the S1 have been identified along the course of the SARS-CoV-2 pandemic, worldwide (21), the probability of emerging escape mutants from the IGH repertoire induced by the wild-type virus infection was examined. The E-3B1, A-1H4, A-2F1, A-2H4, and E-3G9 nAbs successfully bound to recombinant mutant S1 proteins (V341I, F342L, N354D, V367F, R408I, A435S, G476S, V483A, and D614G) in a dose-dependent manner, with compatible reactivity against recombinant wild-type S1 and RBD protein (
In addition, the ability of nABs to bind to receptor binding domain variants of the SARS-CoV-2 spike protein was also determined. The A-1H4, A-2F1, A-2H4, E-3B1, and E-3G9 antibodies bound to wild-type (WT) and 9 different variants of the SARS-CoV-2 spike protein RBD shown in the Table below in a dose dependent manner. See
In response to SARS-CoV-2 infection, most human IGH repertoires efficiently generate clonotypes encoded by IGHV3-53/IGHV3-66 and IGHJ6, which can pair with diverse light chains, for both RBD binding and virus neutralization, with few to no somatic mutations. These clonotypes undergo swift class-switching to IgG1, IgA1, and even IgA2 subtypes. The expeditious development of these IGH clonotypes is possible because the naïve-stereotypic IGHV3-53/IGHV3-66 and IGHJ6 clonotypes pre-exist in the majority of the healthy population, predominantly as an IgM isotype. The data above show that IGHV3-53/IGHV3-66 and IGHJ6 are able to pair with diverse light chains to obtain reactivity to the RBD. It is expected that the extent of light chain plasticity is broad enough for virus-exposed people to successfully evolve nAbs because class-switched IGHV3-53/IGHV3-66 and IGHJ6 clonotypes were present in 13 of 17 patients from the current study.
Currently, it is not known whether the stereotypic nAbs are polyreactive or autoreactive. Rather, the selected stereotypic nAbs including A, B, G-42 do not cross-react with the recombinant RBD proteins of either SARS-CoV or MERS-CoV.
A possible correlation between clinical features and antibody response of 17 individuals who were infected with SARS-CoV-2 was analyzed. Of 17 laboratory-confirmed patients, two patients (Patients M and O) had a severe respiratory illness that required mechanical ventilation and six patients (Patients A, H, I, K, L, and P) with moderate illness required supplemental oxygenation. Together, these eight patients with relatively severe clinical courses had high titers of IgG antibody against SARS-CoV-2. However, some patients (Patients E, J, and Q) with mild/moderate symptoms also showed elevated titers of IgG antibody. Therefore, it is not clear whether antibody titer correlates with the clinical course of the patients.
In the humoral response to SAR-CoV-2, which elicits severe respiratory infection, it is beneficial for patients to produce both systemic and mucosal nAbs. The results presented herein showed that IGHV3-53/IGHV3-66 and IGHJ6 successfully class-switched to IgA1 in Patients G, I, and J, whereas they were class-switched to IgA1 and IgA2 in Patient E (Table 4). Furthermore, it deserves mention that after 99 days from the onset of symptoms, no RBD-reactive IGH clonotypes in the peripheral blood of Patient E were detected; however, the antibody titer to the RBD protein still remained high (
Meanwhile, in Patient A, only one of six nAbs was mapped to the IGH repertoire. It has been reported that the frequency of RBD-reactive B-cell clones is extremely low (0.07% to 0.005%) among circulating B cells (24). The frequency of isolated nAb clonotypes in the IGH repertoire was also extremely low (0.0004%-0.0064%) (
In summary, it was found that stereotypic nAb clonotypes pre-existed in the majority of the naïve population, were prevalent among the patients who displayed rapid class-switching to IgG and IgA isotypes, and exhibited light chain plasticity among the SARS-CoV-2 RBD-binding antibodies. These results strongly suggest that stereotypic nAb clonotypes could contribute to the milder clinical course and lower mortality rate seen in patients with SARS-CoV-2 compared to patients with SARS-CoV (9.5%) or MERS-CoV (34.4%) (33) in which similar stereotypic nAb clonotypes have not been reported.
To investigate stereotypic nAb clonotypes of SARS-CoV-2, 26 blood samples collected from 17 patients were subjected to NGS analysis of IG sequences. Human antibody libraries were prepared and subjected to biopanning against recombinant SARS-CoV-2 RBD proteins. RBD-binders were selected using ELISA and their neutralizing activity was tested using flow cytometry with ACE2-expressing cells and recombinant SARS-CoV-2 S protein and microneutralization assay. NGS analysis of the enrichment patterns of clones through biopanning was performed for in silico selection of RBD-binding clones. IG repertoire analyses were conducted to identify and characterize nAb clonotypes, including their prevalence among patients, frequency in IG repertoires, somatic mutations, isotypes, chronological changes, and existence in the naïve un-infected population.
Three chronological blood samples were drawn from Patients A and E. From Patients B, C, D, F, and G, two chronological samples were obtained. Blood samples were collected once from Patients H-Q. All patients were confirmed to be infected by SARS-CoV-2 by a positive reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) result, and sample collection was performed at Seoul National University Hospital. PBMCs and plasma were isolated using Lymphoprep (Stemcell Technologies, Vancouver, BC, Canada), according to the manufacturer's protocol. The PBMCs were subjected to total RNA isolation, using the TRI Reagent (Invitrogen, Carlsbad, Calif., USA), according to the manufacturer's protocol. The study involving human sample collection was approved by the Institutional Ethics Review Board of Seoul National University Hospital (IRB approval number: 2004-230-1119).
Genes encoding VH and part of the CH1 domain were amplified, using specific primers, as described previously (16, 34). All primers used are listed in Table 9. Briefly, total RNA was used as a template to synthesize cDNA, using the Superscript IV First-Strand Synthesis System (Invitrogen), with specific primers targeting the constant region (CH1 domain) of each isotype (IgM, IgD, IgG, IgA, and IgE) (34), according to the manufacturer's protocol. Following cDNA synthesis, 1.8 volumes of SPRI beads (AmpureXP, Beckman Coulter, Brea, Calif., USA) were used to purify cDNA, which was eluted in 40 μL water. The purified cDNA (18 μL) was subjected to second-strand synthesis in a 25-4 reaction volume, using V gene-specific primers (16) and KAPA Biosystems (KAPA HiFi HotStart, Roche, Basel, Switzerland). The PCR conditions were as follows: 95° C. for 3 min, 98° C. for 1 min, 55° C. for 1 min, and 72° C. for 5 min. Following the second-strand synthesis, double-strand DNA (dsDNA) was purified, using SPRI beads, as described above. VH genes were amplified using 15 μL eluted dsDNA and 2.5 pmol of the primers listed in Table 9, in a 50-μL total reaction volume (KAPA Biosystems), using the following thermal cycling program: 95° C. for 3 min; 17 cycles of 98° C. for 30 sec, 65° C. for 30 sec, and 72° C. for 1 min 10 sec; and 72° C. for 5 min. The number of PCR cycles was increased, from 17 to 19, for samples from Patients B (d10 and 19), C (d6), E (d23), and G (d9 and 22). PCR products were purified using SPRI beads and eluted in 30 μL water. Genes encoding Vκ and Vλ were amplified using specific primers, as described previously (20, 35). Briefly, total RNA was used as a template to synthesize cDNA, using the Superscript IV First-Strand Synthesis System (Invitrogen), with specific primers targeting the constant region, which are listed in Table 9, according to the manufacturer's protocol. Following cDNA synthesis, SPRI beads were used to purify cDNA, which was eluted in 40 μL water. Purified cDNA (18 μL) was used for the first amplification, in a 25-4 reaction volume, using VJ gene-specific primers, which are listed in Table 9, and KAPA Biosystems. The PCR conditions were as follows: 95° C. for 3 min, 4 cycles of 98° C. for 1 min, 55° C. for 1 min, and 72° C. for 1 min; and 72° C. for 10 min. Subsequently, DNA was purified using SPRI beads, and the Vκ and Vλ genes were amplified using 15 μL eluted dsDNA and 2.5 pmol of the primers listed in Table 9, in a 50-μL total reaction volume (KAPA Biosystems). The PCR conditions were as follows: 95° C. for 3 min; 17 cycles of 98° C. for 30 sec, 65° C. for 30 sec, and 72° C. for 1 min 10 sec; and 72° C. for 5 min. PCR products were purified using SPRI beads, as described above. For the amplification of VH from each round of biopanning (rounds 0-4), gene fragments were amplified from phagemid DNA, using the primers listed in Table 9. SPRI-purified sequencing libraries were quantified with a 4200 TapeStation System (Agilent Technologies), using a D1000 ScreenTape Assay, before performing sequencing on an Illumina MiSeq Platform.
The raw NGS forward (R1) and reverse (R2) reads were merged by PEAR, v0.9.10, in default setting (36). The merged reads were q-filtered using the condition q20p95, which results in 95% of the base-pairs in a read having Phread scores higher than 20. The location of the primers was recognized from the q-filtered reads while allowing one substitution or deletion (Table 9). Then, primer regions that specifically bind to the molecules were trimmed in the reads, to eliminate the effects of primer synthesis errors. Based on the primer recognition results, unique molecular identifier (UMI) sequences were extracted, and the reads were clustered according to the UMI sequences. To eliminate the possibility that the same UMI sequences might be used for different read amplifications, the clustered reads were sub-clustered, according to the similarity of the reads (Five mismatches were allowed in each sub-cluster). The sub-clustered reads were aligned, using a multiple sequence alignment tool, Clustal Omega, v1.2.4, in default setting (37, 38). From the aligned reads, the frequency of each nucleotide was calculated, and a consensus sequence of each sub-cluster was defined using the frequency information. Then, the read count of the consensus sequence was re-defined as the number of UMI sub-clusters that belong to the consensus sequences.
Sequence annotation consisted of two parts, isotype annotation and VDJ annotation. For annotation, the consensus sequence was divided into two sections, a VDJ region and a constant region, in a location-based manner. For isotype annotation, the extracted constant region was aligned with the IMGT (international immunogenetics information system) constant gene database (39). Based on the alignment results, the isotypes of the consensus sequences were annotated. Then, the VDJ regions of the consensus sequences were annotated, using IgBLAST, v1.8.0 (40). Among the annotation results, V/D/J genes (V/J genes for VL), CDR1/2/3 sequences, and the number of mutations from the corresponding V genes were extracted, for further analysis. Divergence values were defined as the number of mutations identified in the aligned V gene, divided by the aligned length. Then, the non-functional consensus reads were defined using the following criteria and filtered-out: 1. sequence length shorter than 250 bp; 2. existence of stop-codon or frame-shift in the full amino acid sequence; 3. annotation failure in one or more of the CDR1/2/3 regions; and 4. isotype annotation failure. Then, the functional consensus reads were random-sampled, to adjust the throughput of the VH data (Table 6). Throughput adjustment was not conducted for VL data (Table 7).
Pre-processing of the biopanning NGS data was performed as previously reported, except for the application of the q-filtering condition q20p95 instead of q20p100 (41).
To investigate the shared IGH sequences among the patients, the overlapping IGH repertoire of the patients was defined. First, histograms for the nearest-neighbor distances of the HCDR3 amino acid sequences were calculated for the repertoire data. A hierarchical, distance-based analysis, which was reported previously (42), was applied to the HCDR3 amino acid sequences, to cluster functionally similar IGH sequences. The IGH sequences for all repertoire data could be approximated into a bimodal distribution, allowing the functionally similar IGH sequences to be extracted by capturing the first peak of the distribution (
From each round of biopanning (rounds 0, 2, 3, and 4), the VH genes were amplified and subjected to NGS analysis, using the MiSeq platform, as described previously (19). Binding-predicted clones from biopanning were defined by employing frequency the values of the NGS data from four libraries, A_d17, A_d45, E_d23, and E_d44, at each round of biopanning. The enrichment of clones primarily occurred during the second round of biopanning, based on the input/output virus titer values for each round of biopanning and the frequencies of the clones in the NGS data (
Construction of a Human scFv Phage-Display Library and VL Shuffled Libraries
For the VH gene, the cDNA prepared for the NGS analysis was used. For the Vκ and Vλ genes, total RNA was used to synthesize cDNA, using the Superscript IV First-Strand Synthesis System (Invitrogen), with oligo(dT) primers, according to the manufacturer's instructions. Then, the genes encoding VK/Vλ and VH were amplified, from the oligo(dT)-synthesized cDNA and the cDNA prepared for NGS analysis, respectively, using the primers listed in Table 9 and KAPA Biosystems. The PCR conditions were as follows: preliminary denaturation at 95° C. for 3 min; 4 cycles of 98° C. for 1 min, 55° C. for 1 min, and 72° C. for 1 min; and 72° C. for 10 min. Subsequently, DNA was purified using SPRI beads, as described above. The purified DNA was amplified using the primers listed in Table 9 and KAPA Biosystems. The PCR conditions were as follows: preliminary denaturation, at 95° C. for 3 min; 25 cycles of 98° C. for 30 sec, 58° C. for 30 sec, and 72° C. for 90 sec; and 72° C. for 10 min. Then, the VH and VK/Vλ fragments were subjected to electrophoresis, on a 1% agarose gel, and purified, using a QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, Calif., USA), according to the manufacturer's instructions. The purified VH and VK/Vλ fragments were mixed, at equal ratios at 50 ng, and subjected to overlap extension, to generate scFv genes, using the primers listed in Table 9 and KAPA Biosystems. The PCR conditions were as follows: preliminary denaturation, at 94° C. for 5 min; 25 cycles of 98° C. for 15 sec, 56° C. for 15 sec, and 72° C. for 2 min; and 72° C. for 10 min. The amplified scFv fragment was purified and cloned into a phagemid vector, as described previously (43).
For the construction of VK/Vλ shuffled libraries, gBlocks Gene Fragments (Integrated DNA Technologies, Coralville, Iowa, USA), encoding A-11, E-12, A-31, A-32, B-33, E-34, A,B,G-42, G-44, D-51, F-53, E-52, and A-54, were synthesized. Synthesized VH and the VK/Vλ genes from Patients A, E, and G were used to synthesize the scFv libraries using PCR, as described previously (43). Then, the amplified scFv fragments were purified and cloned into the phagemid vector, as described above.
Phage display of the human scFv libraries exceeded complexity of 3.8×108, 6.7×108, 2.0×108, and 7.2×108 colony-forming units for A_d17, A_d45, E_d23, and E_d44, respectively. These libraries were subjected to four rounds of biopanning against the recombinant SARS-CoV-2 RBD protein (Sino Biological Inc., Beijing, China), fused to mFc or hCκ, as described previously (44). Briefly, 3 μg of the recombinant SARS-CoV-2 RBD protein was conjugated to 1.0×107 magnetic beads (Dynabeads M-270 epoxy, Invitrogen) and incubated with the scFv phage-display libraries (approximately 1012 phages), for 2 h at 37° C. During the first round of biopanning, the beads were washed once with 500 μL of 0.05% (v/v) Tween-20 (Sigma-Aldrich, St. Louis, Mo., USA) in phosphate-buffered saline (PBST). For the other rounds of biopanning, 1.5 μg of recombinant SARS-CoV-2 RBD protein was conjugated to 5.0×106 magnetic beads, and the number of washes was increased to three. After each round of biopanning, the bound phages were eluted and rescued, as described previously (44).
To select SARS-CoV-2 S reactive clones, phage ELISA was performed, using recombinant S and RBD protein-coated microtiter plates, as described previously (45). Reactive scFv clones were subjected to Sanger sequencing (Cosmogenetech, Seoul, Republic of Korea), to determine their nucleotide sequences.
A human, codon-optimized, SARS-CoV-2 RBD (YP 009724390.1, amino acids 306-543) gene was synthesized (Integrated DNA Technologies). Using a synthesized wild-type RBD gene as a template, RBD mutants (V341I, F342L, N354D, N354D/D364Y, V367F, R408I, A435S, W436R, G476S, and V483A) were generated through two-step PCR, using the primers listed in Table 9. The genes encoding wild-type or mutant SARS-CoV-2 RBD were cloned into a modified mammalian expression vector, containing the hCκ gene (44), and transfected into Expi293F (Invitrogen) cells. The fusion proteins were purified by affinity chromatography, using KappaSelect Columns (GE Healthcare, Chicago, Ill., USA), as described previously (46). Due to low expression yields, two RBD mutants (N354D/D364Y, W436R) were excluded from further studies.
The genes encoding the selected scFv clones were cloned into a modified mammalian expression vector, containing the hIgG1 Fc regions (hFc) or hCκ at the C-terminus (44, 47), before being transfected and purified by affinity chromatography, as described above.
Genes encoding VH and VL were amplified, cloned into a mammalian expression vector containing the CH1 and hinge regions of human IgG2 fused to the CH2 and CH3 regions of human IgG4 (48, 49), and transfected into Expi293F cells (Invitrogen) as described previously (50). Then, IgG2/4 was purified by affinity chromatography using MabSelect columns with the AKTA Pure chromatography system (GE Healthcare) following the manufacturer's protocol.
First, 100 ng of each recombinant SARS-CoV-2 S (Sino Biological Inc.), S1 (Sino Biological Inc.), S1 D614G (Sino Biological Inc.), S2 (Sino Biological Inc.), NP (Sino Biological Inc.), RBD, RBD mutants, SARS-CoV RBD (Sino Biological Inc.), MERS-CoV S (Sino Biological Inc.), RBD (Sino Biological Inc.), S2 (Sino Biological Inc.) proteins were added to microtiter plates (Costar), in coating buffer (0.1 M sodium bicarbonate, pH 8.6). After incubation at 4° C., overnight, and blocking with 3% bovine serum albumin (BSA) in PBS, for 1 h at 37° C., serially diluted plasma (5-fold, 6 dilutions, starting from 1:100) or scFv-hFc (5-fold, 12 dilutions, starting from 1,000 or 500 nM) in blocking buffer was added to individual wells and incubated for 1, h at 37° C. Then, the plates were washed three times with 0.05% PBST. Horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG antibody (Invitrogen) or anti-human Ig kappa light chain antibody (Millipore, Temecula, Calif., USA), in blocking buffer (1:5,000), was added into wells and incubated for 1 h at 37° C. After washing three times with PBST, 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic (ThermoFisher Scientific Inc., Waltham, Mass., USA) or 3,3′,5,5′-Tetramethylbenzidine liquid substrate system (ThermoFisher Scientific Inc.) was added to the wells. Absorbance was measured at 405 nm or 650 nm, using a microplate spectrophotometer (Multiskan GO; Thermo Scientific).
The recombinant SARS-CoV-2 S protein (200 nM), fused with a HIS-tag at the C-terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (molar ratio of 1:3), in 50 μL of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide (FACS buffer), at 37° C. for 1 h. Irrelevant scFv-hFc or scFv-hCκ fusion proteins were used as negative controls. Vero E6 cells (ACE2+) were seeded into v-bottom 96-well plates (Corning, Corning, N.Y., USA), at a density of 1.5×105 cells per well. Then, the mixture was added to each well and incubated, at 37° C. for 1 h. After washing three times with FACS buffer, FITC-labeled rabbit anti-HIS Ab (Abcam, Cambridge, UK) was incubated, at 37° C. for 1 h. Then, the cells were washed three times with FACS buffer, resuspended in 150 μL of PBS, and subjected to analysis by flow cytometry, using a FACS Canto II instrument (BD Bioscience, San Jose, Calif., USA). For each sample, 10,000 cells were assessed.
The virus (BetaCoV/Korea/SNU01/2020, accession number MT039890) was isolated at the Seoul National University Hospital and propagated in Vero cells (ATCC CCL-81), using Dulbecco's Modified Eagle's Medium (DMEM, Welgene, Gyeongsan, Republic of Korea) supplemented with 2% fetal bovine serum (Gibco) (51). The cells were grown in T-25 flasks, (ThermoFisher Scientific Inc.), inoculated with SARS-CoV-2, and incubated at 37° C., in a 5% CO2 environment. Then, 3 days after inoculation, the viruses were harvested and stored at −80° C. The virus titer was determined via a TCID50 assay (52).
Vero cells were seeded in T-25 flasks and grown for 24 h, at 37° C., in a 5% CO2 environment, to ensure 80% confluency on the day of inoculation. The recombinant scFv-hCκ fusion proteins (0.5, 5, or 50 μg/mL) were mixed with 2,500 TCID50 of SARS-CoV-2, and the mixture was incubated for 2 h, at 37° C. Then, the mixture (1 mL) was added to the Vero cells and incubated for 1 h, at 37° C., in a 5% CO2 environment. After incubation for 1 h, 6 mL of complete media was added to the flasks and incubated, at 37° C., in a 5% CO2 environment. After 0, 24, 48, and 72 h of infection, the culture supernatant was collected, to measure the virus titers. RNA was extracted, using the MagNA Pure 96 DNA and Viral NA small volume kit (Roche, Germany), according to the manufacturer's instructions. Viral RNA was detected using the PowerChek 2019-nCoV Real-time PCR Kit (Kogene Biotech, Seoul, Republic of Korea), for the amplification of the E gene, and quantified according to a standard curve, which was constructed using in vitro transcribed RNA, provided by the European Virus Archive (https://www.european-virus-archive.com). Another neutralization assay was performed as described previously (53). Briefly, Vero cells seeded in 96-well plates in DMEM medium were grown for 24 h at 37° C. in a 5% CO2 environment. 50 μl of two-fold serially diluted IgG2/4 were mixed with an equal volume of SARS-CoV-2 containing 100 TCID50 and the IgG2/4-virus mixture was incubated at 37° C. for 1 h. The mixture was then transferred into a 96-well microtiter plate containing Vero cells with 8 repeats and incubated for 5 days at 37° C. in a 5% CO2 environment. Cells infected with 100 TCID50 of SARS-CoV-2, isotype IgG2/4 control, or without the virus, were applied as positive, negative, and uninfected controls, respectively. The cytopathic effect (CPE) in each well was observed 5 days post-infection. The IC50 was calculated using GraphPad Prism 8 (GraphPad Software, San Diego, Calif., USA). All experiments using authentic SARS-CoV-2 were conducted in Biosafety Level 3 laboratory.
Data are represented as mean±standard deviation. Statistical analyses were performed using R software v.3.4.3. For the flow cytometry analysis using ACE2-expressing cells and recombinant SARS-CoV-2 S protein, results were analyzed by independent t-tests.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
The healthy samples based on publicly available IGH repertoires or patient identification can be found in the sample column. Clonotypes were mapped according to identical VJ gene usage of IGHV3-53/IGHV3-66 and IGHJ6 and perfectly matched HCDR3 amino acid sequence. Read counts of the mapped sequences in the repertoires of each sample were annotated in the occurrence column. For clonotypes with multiple occurrences, mean and standard deviation of divergence were represented. The proportion of each isotype is indicated for all samples.
This application claims priority to and benefit of U.S. Provisional Patent Application Nos. 63/042,396, filed Jun. 22, 2020, 63/042,901, filed Jun. 23, 2020, 63/044,707, filed Jun. 26, 2020, and 63/119,207, filed Nov. 23, 2020, which are all incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63042396 | Jun 2020 | US | |
| 63044707 | Jun 2020 | US | |
| 63042901 | Jun 2020 | US | |
| 63119207 | Nov 2020 | US |
