Patent Application
20230227539
The Coronavirus Disease 2019 (COVID-19), caused by the novel SARS-CoV-2 coronavirus, has quickly grown into a global pandemic and a major public health crisis. Since the virus has only recently emerged from bats and crossed over into humans, little is known about the immune response generated against it by infected patients.
Thus, a better understanding of the pathological mechanisms caused by the virus, and development of new therapeutic agents against it are urgently needed.
One aspect of the invention provides an isolated or recombinantly produced monoclonal antibody, or an antigen-binding fragment thereof, wherein said monoclonal antibody or antigen-binding fragment thereof is specific for the Spike protein or S protein of SARS-CoV-2, and wherein said monoclonal antibody specifically binds to and/or has a residue within 4 Å of residues T415, G416, K417, D420, Y421, Y453, L455, F456, R457, K458, N460, Y473, Q474, A475, G476, S477, F486, N487, Y489, Q493, S494, Y495, G496, Q498, T500, N501, G502, and Y505 of the S protein, optionally, said monoclonal antibody does not bind to and/or has no residue within 4 Å of residues G446 and Y449 of the S protein.
Another aspect of the invention provides an isolated or recombinantly produced monoclonal antibody, or an antigen-binding fragment thereof, wherein said monoclonal antibody or antigen-binding fragment thereof is specific for an antigen (e.g., the Spike protein or S protein responsible for ACE2 binding) of SARS-CoV-2, and wherein said monoclonal antibody comprises: (1a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence of SEQ ID NO: 1, a HCVR CDR2 sequence of SEQ ID NO: 2, and a HCVR CDR3 sequence of SEQ ID NO: 3; and, (1b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence of SEQ ID NO: 4, a LCVR CDR2 sequence of SEQ ID NO: 5, and a LCVR CDR3 sequence of SEQ ID NO: 6; or (2a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence of SEQ ID NO: 11, a HCVR CDR2 sequence of SEQ ID NO: 12, and a HCVR CDR3 sequence of SEQ ID NO: 13; and, (2b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence of SEQ ID NO: 14, a LCVR CDR2 sequence of SEQ ID NO: 15, and a LCVR CDR3 sequence of SEQ ID NO: 16; or (3a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence of SEQ ID NO: 21, a HCVR CDR2 sequence of SEQ ID NO: 22, and a HCVR CDR3 sequence of SEQ ID NO: 23; and, (3b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence of SEQ ID NO: 24, a LCVR CDR2 sequence of SEQ ID NO: 25, and a LCVR CDR3 sequence of SEQ ID NO: 26; or (4a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence of SEQ ID NO: 31, a HCVR CDR2 sequence of SEQ ID NO: 32, and a HCVR CDR3 sequence of SEQ ID NO: 33; and, (4b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence of SEQ ID NO: 34 or SEQ ID NO: 115, a LCVR CDR2 sequence of SEQ ID NO: 35, and a LCVR CDR3 sequence of SEQ ID NO: 36; (5a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence of SEQ ID NO: 51, a HCVR CDR2 sequence of SEQ ID NO: 52, and a HCVR CDR3 sequence of SEQ ID NO: 53; and, (5b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence of SEQ ID NO: 54, a LCVR CDR2 sequence of SEQ ID NO: 55, and a LCVR CDR3 sequence of SEQ ID NO: 56; or (6a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence of SEQ ID NO: 61, a HCVR CDR2 sequence of SEQ ID NO: 62, and a HCVR CDR3 sequence of SEQ ID NO: 63; and, (6b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence of SEQ ID NO: 64, a LCVR CDR2 sequence of SEQ ID NO: 65, and a LCVR CDR3 sequence of SEQ ID NO: 66; or (7a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence of SEQ ID NO: 71, a HCVR CDR2 sequence of SEQ ID NO: 72, and a HCVR CDR3 sequence of SEQ ID NO: 73; and, (7b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence of SEQ ID NO: 74, a LCVR CDR2 sequence of SEQ ID NO: 75, and a LCVR CDR3 sequence of SEQ ID NO: 76; or (8a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence of SEQ ID NO: 81, a HCVR CDR2 sequence of SEQ ID NO: 82, and a HCVR CDR3 sequence of SEQ ID NO: 83; and, (8b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence of SEQ ID NO: 84, a LCVR CDR2 sequence of SEQ ID NO: 85, and a LCVR CDR3 sequence of SEQ ID NO: 86; or (9a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence of SEQ ID NO: 91, a HCVR CDR2 sequence of SEQ ID NO: 92, and a HCVR CDR3 sequence of SEQ ID NO: 93; and, (9b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence of SEQ ID NO: 94, a LCVR CDR2 sequence of SEQ ID NO: 95, and a LCVR CDR3 sequence of SEQ ID NO: 96; optionally, said isolated monoclonal antibody is not naturally occurring; and/or, optionally further comprising a signal peptide sequence of SEQ ID NO: 41 at the N-terminus of said HCVR and/or LCVR.
In any of the proceding embodiments, in the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof: (1A) the HCVR sequence is SEQ ID NO: 7; and/or, (1B) the LCVR sequence is SEQ ID NO: 8, or, (2A) the HCVR sequence is SEQ ID NO: 17; and/or, (2B) the LCVR sequence is SEQ ID NO: 18, or, (3A) the HCVR sequence is SEQ ID NO: 27; and/or, (3B) the LCVR sequence is SEQ ID NO: 28, or, (4A) the HCVR sequence is SEQ ID NO: 37; and/or, (4B) the LCVR sequence is SEQ ID NO: 38 or SEQ ID NO: 114; (5A) the HCVR sequence is SEQ ID NO: 57; and/or, (5B) the LCVR sequence is SEQ ID NO: 58, or, (6A) the HCVR sequence is SEQ ID NO: 67; and/or, (6B) the LCVR sequence is SEQ ID NO: 68, or, (7A) the HCVR sequence is SEQ ID NO: 77; and/or, (7B) the LCVR sequence is SEQ ID NO: 78, or, (8A) the HCVR sequence is SEQ ID NO: 87; and/or, (8B) the LCVR sequence is SEQ ID NO: 88, or, (9A) the HCVR sequence is SEQ ID NO: 97; and/or, (9B) the LCVR sequence is SEQ ID NO: 98.
In any of the proceding embodiments, the monoclonal antibody has: (1a) a heavy chain sequence of SEQ ID NO: 9; and/or, (1b) a light chain sequence of SEQ ID NO: 10, or, (2a) a heavy chain sequence of SEQ ID NO: 19; and/or, (2b) a light chain sequence of SEQ ID NO: 20, or, (3a) a heavy chain sequence of SEQ ID NO: 29; and/or, (3b) a light chain sequence of SEQ ID NO: 30, or, (4a) a heavy chain sequence of SEQ ID NO: 39; and/or, (4b) a light chain sequence of SEQ ID NO: 40; (5a) a heavy chain sequence of SEQ ID NO: 59; and/or, (5b) a light chain sequence of SEQ ID NO: 60, or, (6a) a heavy chain sequence of SEQ ID NO: 69; and/or, (6b) a light chain sequence of SEQ ID NO: 70, or, (7a) a heavy chain sequence of SEQ ID NO: 79; and/or, (7b) a light chain sequence of SEQ ID NO: 80, or, (8a) a heavy chain sequence of SEQ ID NO: 89; and/or, (8b) a light chain sequence of SEQ ID NO: 90, or, (9a) a heavy chain sequence of SEQ ID NO: 99; and/or, (9b) a light chain sequence of SEQ ID NO: 100.
In any of the proceding embodiments, the monoclonal antibody has: (1a) a heavy chain sequence of SEQ ID NO: 101; and/or, (1b) a light chain sequence of SEQ ID NO: 10, or, (2a) a heavy chain sequence of SEQ ID NO: 102; and/or, (2b) a light chain sequence of SEQ ID NO: 20, or, (3a) a heavy chain sequence of SEQ ID NO: 103; and/or, (3b) a light chain sequence of SEQ ID NO: 30, or, (4a) a heavy chain sequence of SEQ ID NO: 104; and/or, (4b) a light chain sequence of SEQ ID NO: 40 or SEQ ID NO: 113.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof is a human antibody, a CDR-grafted antibody, or a resurfaced antibody.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof is a human IgG4 antibody, or an FcγR null monoclonal antibody engineered to prevent FcγR engagement.
In any of the proceding embodiments, the antigen-binding fragment thereof is an Fab, Fab′, F(ab′)2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab′)3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.
In any of the proceding embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to the S1 glycoprotein of SARS-CoV-2.
In any of the proceding embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to the S2 glycoprotein of SARS-CoV-2.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof binds to SARS-CoV-2 wild-type S protein and/or RBD/S1 variants selected from the group consisting of S477N, S494P, F490S, Y453F, N439K, N501Y, E484K, Q493R, and A222V/D614G.
In any of the proceding embodiments, the monoclonal antibody or antigen-binding fragment thereof binds the SARS-CoV-2 antigen with a Kd of less than about 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, or 0.05 nM.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 immobilized on a solid support (such as in ELISA assay), or inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 expressed on the surface of a cell (such as Vero E6 cell).
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 with an EC50 value of less than 2 nM, 1 nM or 0.1 nM.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof exhibits neutralizing activity against a pseudovirus of SARS-CoV-2 or a live SARS-CoV-2 virus with an IC50 value of less than 10 nM, 8 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 0.6 nM or less than 0.5 nM.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) at less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.1 nM, less than 0.08 nM, less than 0.06 nM, less than 0.02 nM, or less than 0.01 nM.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) with an IC50 of less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 500 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 80 pM, less than 50 pM, less than 30 pM, less than 10 pM, or less than 5 pM.
In any of the proceding embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof inhibits entry of wild-type SARS-CoV-2 and/or SARS-CoV-2 variants, e.g. WuhanD614, BavPat D614G, UK B.1.1.7, or South Africa B.1.351 lineage, into a target cell.
In any of the proceding embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof inhibits entry of a SARS-CoV-2 variant sharing one or more S protein mutations with the WuhanD614, BavPat D614G, UK B.1.1.7, and/or South Africa B.1.351 strain(s), into a target cell.
In any of the proceding embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof does not cause antibody-dependent enhancement (ADE).
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region is human IgG1, human IgG2, human IgG3 or human IgG4.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region is human IgG4.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence of SEQ ID NO: 11, a HCVR CDR2 sequence of SEQ ID NO: 12, and a HCVR CDR3 sequence of SEQ ID NO: 13, and a light chain variable region (LCVR), comprising a LCVR CDR1 sequence of SEQ ID NO: 14, a LCVR CDR2 sequence of SEQ ID NO: 15, and a LCVR CDR3 sequence of SEQ ID NO: 16.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof comprises HCVR sequence of SEQ ID NO: 17 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 17, and an LCVR sequence of SEQ ID NO: 18 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 18.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region is human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the heavy chain constant region is human IgG4.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof comprises an HC sequence of SEQ ID NO: 19 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 19.
In any of the proceding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof comprises an HC sequence of SEQ ID NO: 102 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 102.
Another aspect of the invention provides an isolated or recombinantly produced monoclonal antibody or an antigen-binding fragment thereof, which competes with the isolated monoclonal antibody or antigen-binding fragment thereof of for binding to the same epitope.
In a related aspect, the invention provides a non-naturally occurring or existing therapeutic antibody based on the antigen-binding sequences of an antibody isolated from the patient using the method of the invention. Such therapeutic antibody may share one or more CDRs, such as CDR1, CDR2, and/or CDR3 of heavy chain and/or light chain sequences, with the antibody isolated from the patient using the method of the invention.
For example, heavy chain CDR3 (HC-CDR3) sequences of certain isolated antibodies are listed in Example 1.
Another aspect of the invention provides a mixture of two or more isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of the invention.
In certain embodiments, the proportion of each of said two or more isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof is substantially the same, or is different.
Another aspect of the invention provides a method of treating or preventing a disease or condition arising from SARS-CoV-2 infection, the method comprising administering to a patient in need thereof an effective amount of the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of the invention, or an effective amount of the mixture of the invention.
In certain embodiments, the method is for treating COVID-19, wherein the method further comprises administering a second therapeutic agent, which may be effective to treat infection by SARS-CoV-2.
In certain embodiments, the second therapeutic agent comprises chloroquine or hydroxychloroquine, remdesivir, lopinavir and ritonavir, azithromycin, an immune system inhibitor to inhibits cytokine storm (such as an anti-IL-6 neutralizing antibody such as tocilizumab or sarilumab), CD24Fc, IFX-1, an anti-CCR5 antibody such as Leronlimab, DAS181, CM4620, an anti-IFNγ monoclonal antibody such as emapalumab, an IL-1R antagonist such as Anakinra, Danoprevir+Ritonavir, Calquence (acalabrutinib), Xeljanz (tofacitinib), Jakafi (ruxolitinib), Olumiant (baricitinib), Ilaris (canakinumab), Otezla (apremilast), Mavrilimumab, or combination thereof.
In certain embodiments, the second therapeutic agent comprises one or more of: an anti-viral agent, an antibiotic, an anti-inflammatory agent or DMARD (disease-modifying anti-rheumatic drug).
Another aspect of the invention provides a polynucleotide encoding the heavy chain or the light chain or the antigen-binding portion thereof of the invention.
In certain embodiments, the polynucleotide is codon optimized for expression in a human cell.
Another aspect of the invention provides a vector comprising the polynucleotide of the invention.
In certain embodiments, the vector is an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector).
Another aspect of the invention provides a host cell comprising the vector of the invention, which expresses said isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof.
Another aspect of the invention provides a pharmaceutical composition comprising the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of the invention, or the mixture of the invention. The pharmaceutical composition further comprises a pharmaceutically acceptable excipient or diluent.
In certain embodiments, the pharmaceutical composition is formulated for intravenous administration.
It should be understood that any one embodiment of the invention, including those only described under one aspect or section of the invention, and those only described in the examples or claims, can be combined with any other embodiment(s) of the invention unless improper or expressly disclaimed.
One aspect of the invention provides an antibody isolated from a convalescent COVID-19 patient using the method of the invention. Specifically, sera from convalescent COVID-19 (i.e., SARS-CoV-2) patients, a source of antiviral antibodies capable of conferring protective immunity on recipients, were obtained to identify effective antibodies against antigens of COVID-19 for therapeutic purposes. Antibodies identified from patients infected with the Ebola virus have been used as therapeutic antibodies (Bornholdt et al., 2016; Casadevall & Pirofski, 2020).
In a related aspect, the invention provides a non-naturally occurring or existing therapeutic antibody based on the antigen-binding sequences of an antibody isolated from the patient using the method of the invention. Such therapeutic antibody may share one or more CDRs, such as CDR1, CDR2, and/or CDR3 of heavy chain and/or light chain sequences, with the antibody isolated from the patient using the method of the invention. For example, heavy chain CDR3 (HC-CDR3) sequences of certain isolated antibodies are listed in
Another aspect of the invention provides a mixture of the antibodies of the invention. Such a mixture may provide better therapeutic efficacy compared to the individual component antibodies of the mixture.
Indeed, a convalescent patient's sera is a natural mixture of different antibodies against the same or different viral antigen or epitope. Further, Applicant has identified multiple antibodies from the serum of convalescent patients, including 10 antibodies capable of binding to the full length SARS-CoV-2 S protein, among which 8 antibodies recognizes S1 only, and 2 antibodies interacts with S2 only but not with S1. The data indicates that diverse epitopes on SARS-CoV-2 virus spike protein can be targeted by different antibodies, suggesting that the antibodies of the invention, either alone or in combination with antibodies that target different epitopes via different mechanisms, may serve as potent therapeutic agents to treat COVID-19 patients.
Another aspect of the invention provides a polynucleotide encoding the heavy or light chain of the antibodies of the invention. Such polynucleotide sequences may be codon optimized for expression in a host cell, such as a mammalian cell line (e.g., CHO cell line) for large scale production of antibody.
Another aspect of the invention provides a vector comprising the polynucleotide of the invention. Such vector may be used for expression of antibody in a suitable host cell.
A further aspect of the invention provides a host cell comprising the vector of the invention, or producing the antibody of the invention.
Yet another aspect of the invention provides a method of treating or preventing a disease or condition arising from SARS-CoV-2 infection, such as COVID-19, the method comprising administering to a patient in need thereof a therapeutically effective amount of the antibody of the invention, or a mixture thereof.
With the general aspects of the inventions described, the following sections provide more detailed aspects of the invention. It should be understood that any one embodiment of the invention, including those only described in one section or one example, can be combined with any one or more additional embodiment of the invention whenever proper.
The term “antibody,” in the broadest sense, encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). The term “antibody” may also broadly refers to a molecule comprising complementarity determining region (CDR) 1, CDR2, and CDR3 of a heavy chain and CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to an antigen. The term “antibody” also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc.
In a narrower sense, however, “antibody” refers to the various monoclonal antibodies, including chimeric monoclonal antibodies, humanized monoclonal antibodies, and human monoclonal antibodies, particularly humanized or human monoclonal antibodies of the invention.
In some embodiments, an antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR). In some embodiments, an antibody comprises at least one heavy chain (HC) comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain (LC) comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, an antibody comprises two heavy chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region.
As used herein, a single-chain Fv (scFv), or any other antibody that comprises, for example, a single polypeptide chain comprising all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have a heavy chain and a light chain. In some such embodiments, the heavy chain is the region of the antibody that comprises the three heavy chain CDRs and the light chain in the region of the antibody that comprises the three light chain CDRs.
The term “heavy chain variable region (HCVR)” as used herein refers to, at a minimum, a region comprising heavy chain CDR1 (CDR-H1), framework 2 (HFR2), CDR2 (CDR-H2), FR3 (HFR3), and CDR3 (CDR-H3). In some embodiments, a heavy chain variable region also comprises at least a portion (e.g., the whole) of an FR1 (HFR1), which is N-terminal to CDR-H1, and/or at least a portion (e.g., the whole) of an FR4 (HFR4), which is C-terminal to CDR-H3.
The term “heavy chain constant region” as used herein refers to a region comprising at least three heavy chain constant domains, CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, an antibody comprising an α constant region is an IgA antibody, an antibody comprising an F constant region is an IgE antibody, and an antibody comprising an μ constant region is an IgM antibody.
Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a γ1 constant region), IgG2 (comprising a γ2 constant region), IgG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 (comprising an 1 constant region) and IgM2 (comprising μ2 constant region).
The term “heavy chain” as used herein refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term “full-length heavy chain” as used herein refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence, and with or without a C-terminal lysine.
The term “light chain variable region (LCVR)” as used herein refers to a region comprising light chain CDR1 (CDR-L1), framework (FR) 2 (LFR2), CDR2 (CDR-L2), FR3 (LFR3), and CDR3 (CDR-L3). In some embodiments, a light chain variable region also comprises at least a portion (e.g., the whole) of an FR1 (LFR1) and/or at least a portion (e.g., the whole) of an FR4 (LFR4).
The term “light chain constant region” as used herein refers to a region comprising a light chain constant domain, CL. Non-limiting exemplary light chain constant regions include λ and κ.
The term “light chain” as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term “full-length light chain” as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.
The term “antibody fragment” or “antigen binding portion” (of antibody) includes, but is not limited to, fragments that are capable of binding antigen, such as Fv, single-chain Fv (scFv), Fab, Fab′, and (Fab′)2. In certain embodiments, an antibody fragment includes Fab, Fab′, F(ab′)2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab′)3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.
The term “Fab” refers to an antibody fragment with a molecular mass of approximately 50,000 Daltons, and has an activity of binding to the antigen. It comprises approximately half of the N-terminal side of the heavy chain and the whole of the light chain connected by a disulfide bridge. The Fab can be obtained in particular by treatment of immunoglobulin by a protease, papain.
The term “F(ab′)2” designates a fragment of approximately 100,000 Daltons and an activity of binding to the antigen. This fragment is slightly larger than two Fab fragments connected via a disulfide bridge in the hinge region. These fragments are obtained by treating an immunoglobulin with a protease, pepsin. The Fab fragment can be obtained from the F(ab′)2 fragment by cleaving of the disulfide bridge of the hinge region.
A single Fv chain “scFv” corresponds to a VH: VL polypeptide synthesized using the genes coding for the VL and VH domains and a sequence coding for a peptide intended to bind these domains. An scFv according to the invention includes the CDRs maintained in an appropriate conformation, for example using genetic recombination techniques.
The dimers of “scFv” correspond to two scFv molecules connected together by a peptide bond. This Fv chain is frequently the result of the expression of a fusion gene including the genes coding for VH and VL connected by a linker sequence coding a peptide. The human scFv fragment may include CDR regions that are maintained in an appropriate conformation, preferably by means of the use of genetic recombination techniques.
The “dsFv” fragment is a VH-VL heterodimer stabilized by a disulfide bridge; it may be divalent (dsFV2). Fragments of divalent Sc(Fv)2 or multivalent antibodies may form spontaneously by the association of monovalent scFvs or be produced by connecting scFvs fragments by peptide binding sequences.
The Fc fragment is the support for the biological properties of the antibody, in particular its ability to be recognized by immunity effectors or to activate the complement. It consists of constant fragments of the heavy chains beyond the hinge region.
The term “diabodies” signifies small antibody fragments having two antigen fixing sites. These fragments comprise, in the same VH-VL polypeptide chain, a variable heavy chain domain VH connected to a variable light chain domain VL. Using a binding sequence that is too short to allow the matching of two domains of the same chain, the matching with two complementary domains of another chain necessarily occurs and thus two antigen fixing sites are created.
An “antibody that binds to the same epitope” as a reference antibody can be determined by an antibody competition assay. It refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. The term “compete” when used in the context of an antibody that compete for the same epitope means competition between antibodies is determined by an assay in which an antibody being tested prevents or inhibits specific binding of a reference antibody to a common antigen.
Numerous types of competitive binding assays can be used, for example: 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 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid phase direct labeled assay; solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using I125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol.).
Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen binding protein and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Usually the test antibody is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibodies and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. In some embodiments, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.
The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody or immunologically functional fragment thereof, and additionally capable of being used in a mammal to produce antibodies capable of binding to that antigen. An antigen may possess one or more epitopes that are capable of interacting with antibodies.
The term “epitope” is the portion of an antigen molecule that is bound by a selective binding agent, such as an antibody or a fragment thereof. The term includes any determinant capable of specifically binding to an antibody. An epitope can be contiguous or non-contiguous (e.g., in a polypeptide, amino acid residues that are not contiguous to one another in the polypeptide sequence but that within in context of the molecule are bound by the antigen binding protein). In some embodiments, epitopes may be mimetic in that they comprise a three dimensional structure that is similar to an epitope used to generate the antibody, yet comprise none or only some of the amino acid residues found in that epitope used to generate the antibody. 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.
In some embodiments, an “epitope” is defined by the method used to determine it. For example, in some embodiments, an antibody binds to the same epitope as a reference antibody, if they bind to the same region of the antigen, as determined by hydrogen-deuterium exchange (HDX).
In certain embodiments, an antibody binds to the same epitope as a reference antibody if they bind to the same region of the antigen, as determined by X-ray crystallography.
A “human antibody” as used herein refers to antibodies of human origin or antibodies produced in humans, antibodies produced in non-human animals that comprise human immunoglobulin genes, such as XENOMOUSE®, and antibodies selected using in vitro methods, such as phage display, wherein the antibody repertoire is based on a human immunoglobulin sequences.
A “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively.
The term “isolated” as used herein refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or has been separated from at least some of the components with which it is typically produced. For example, a polypeptide is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be “isolating” the polypeptide.
In certain embodiments, an isolated antibody of the invention may have natural human antibody sequence, but is so purified that it consists essentially of the antibody, such as a monoclonal antibody recombinantly produced and isolated/purified from the cells which produce such antibody.
In certain embodiments, the isolated antibody is at least 90% pure, 95% pure, 97% pure, 99% pure, 99.5% pure, 99.9% pure or purer.
Similarly, a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, e.g., in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated” so long as that polynucleotide is not found in that vector in nature.
The terms “subject” and “patient” are used interchangeably herein to refer to a mammal such as human. In some embodiments, methods of treating other non-human mammals, including, but not limited to, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are also provided. In some instances, a “subject” or “patient” refers to a (human) subject or patient in need of treatment for a disease or disorder.
The term “sample” or “patient sample” as used herein, refers to material that is obtained or derived from a subject of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics. For example, the phrase “disease sample” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized.
By “tissue or cell sample” is meant a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as sputum, cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a disease tissue/organ. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
A “reference sample,” “reference cell,” or “reference tissue,” as used herein, refers to a sample, cell or tissue obtained from a source known, or believed, not to be afflicted with the disease or condition for which a method or composition of the invention is being used to identify. In one embodiment, a reference sample, reference cell or reference tissue is obtained from a healthy part of the body of the same subject or patient in whom a disease or condition is being identified using a composition or method of the invention. In one embodiment, a reference sample, reference cell or reference tissue is obtained from a healthy part of the body of at least one individual who is not the subject or patient in whom a disease or condition is being identified using a composition or method of the invention. In some embodiments, a reference sample, reference cell or reference tissue was previously obtained from a patient prior to developing a disease or condition or at an earlier stage of the disease or condition.
A “disorder” or “disease” is any condition that would benefit from treatment with one or more antibodies of the invention. This includes COVID-19 or any secondary infection by other bacteria or virus, in which the antibody of the invention is used in a combination therapy.
The term “antibody-dependent enhancement” (ADE) refers to a phenomenon in which binding of a virus to suboptimal antibodies enhances its entry into host cells. While not wishing to be bound by any particular theory and not implying that any actual mechanisms of ADE may have been operating in connection with any antibodies tested herein, ADE has been documented to occur through two distinct mechanisms in viral infections: by enhanced antibody-mediated virus uptake into Fc gamma receptor IIa (FcγRIIa)-expressing phagocytic cells leading to increased viral infection and replication, or by excessive antibody Fc-mediated effector functions or immune complex formation causing enhanced inflammation and immunopathology. Both ADE pathways can occur when non-neutralizing antibodies or antibodies at sub-neutralizing levels bind to viral antigens without blocking or clearing infection.
“Treatment” refers to therapeutic treatment, for example, wherein the object is to slow down (lessen) the targeted pathologic condition or disorder as well as, for example, wherein the object is to inhibit recurrence of the condition or disorder. “Treatment” covers any administration or application of a therapeutic for a disease (also referred to herein as a “disorder” or a “condition”) in a mammal, including a human, and includes inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, partially or fully relieving the disease, partially or fully relieving one or more symptoms of a disease, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process. The term “treatment” also includes reducing the severity of any phenotypic characteristic and/or reducing the incidence, degree, or likelihood of that characteristic. Those in need of treatment include those already with the disorder as well as those at risk of recurrence of the disorder or those in whom a recurrence of the disorder is to be prevented or slowed down.
The term “effective amount” or “therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a subject. In some embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of antibody of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antagonist to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of subject antibody are outweighed by the therapeutically beneficial effects.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.
A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritable to the skin and does not cause injection site reaction.
An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder, or a probe for specifically detecting a biomarker described herein. In some embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.
In various embodiments, antibodies of the invention may be administered subcutaneously or intravenously.
In some embodiments, the subject antibody may be administered in vivo by various routes, including, but not limited to, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, by inhalation, intradermal, topical, transdermal, and intrathecal, or otherwise, e.g., by implantation.
In some embodiments, the subject antibody or antigen-binding fragment thereof is administered intraveneously (i.v.) or subcutaneously (s.c.).
The subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.
In various embodiments, compositions comprising the subject antibody are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers, which include vehicles, adjuvants, and diluents, are available. Moreover, various pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are also available. Nonlimiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
In various embodiments, the subject antibody may be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
In various embodiments, the compositions may be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifiuoromethane, propane, nitrogen, and the like.
The compositions may also be formulated, in various embodiments, into sustained release microcapsules, such as with biodegradable or non-biodegradable polymers. A non-limiting exemplary biodegradable formulation includes poly lactic acid-glycolic acid (PLGA) polymer. A non-limiting exemplary non-biodegradable formulation includes a polyglycerin fatty acid ester. Certain methods of making such formulations are described, for example, in EP 1125584 A1.
Pharmaceutical dosage packs comprising one or more containers, each containing one or more types or doses of the subject antibody, are also provided. In some embodiments, a unit dosage is provided wherein the unit dosage contains a predetermined amount of a composition comprising the subject antibody, with or without one or more additional agents. In some embodiments, such a unit dosage is supplied in single-use prefilled syringe for injection. In various embodiments, the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water. In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, a composition of the invention comprises heparin and/or a proteoglycan.
Pharmaceutical compositions are administered in an amount effective for treatment or prophylaxis of the specific indication. The therapeutically effective amount is typically dependent on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated.
In some embodiments, the subject antibody may be administered in an amount in the range of about 50 μg/kg body weight to about 50 mg/kg body weight per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 100 μg/kg body weight to about 50 mg/kg body weight per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.
In some embodiments, the subject antibody may be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 20 mg to about 200 mg per dose.
The subject antibody compositions may be administered as needed to subjects. In some embodiments, an effective dose of the subject antibody is administered to a subject one or more times. In various embodiments, an effective dose of the subject antibody is administered to the subject once a day, less than once a week, such as, for example, every two days, every three days, or every six days. In other embodiments, an effective dose of the subject antibody is administered more than once a day, such as, for example, once or multiple times per day. An effective dose of the subject antibody is administered to the subject at least once. In some embodiments, the effective dose of the subject antibody may be administered multiple times, including for periods of at least a month, at least six months, or at least a year. In some embodiments, the subject antibody is administered to a subject as-needed to alleviate one or more symptoms of a condition.
The antibodies and functional fragments thereof of the invention may be administered to a subject in need thereof in combination with other biologically active substances or other treatment procedures for the treatment of diseases, e.g., COVID-19 and associated symptoms and/or complications. For example, the antibodies of the invention may be administered alone, together as a mixture or combination, or with other modes of treatment such as a second therapeutic agent effective to treat COVID-19 or symptoms/complications thereof. They may be provided before, substantially contemporaneous with, or after other modes of treatment.
In certain embodiments, the second therapeutic agent comprises one or more of: chloroquine or hydroxychloroquine, remdesivir, lopinavir and ritonavir, azithromycin, an immune system inhibitor to inhibits cytokine storm (such as an anti-IL-6 neutralizing antibody such as tocilizumab or sarilumab), CD24Fc, IFX-1, an anti-CCR5 antibody such as Leronlimab, DAS181, CM4620, an anti-IFNγ monoclonal antibody such as emapalumab, an IL-1R antagonist such as Anakinra, Danoprevir+Ritonavir, Calquence (acalabrutinib), Xeljanz (tofacitinib), Jakafi (ruxolitinib), Olumiant (baricitinib), Ilaris (canakinumab), Otezla (apremilast), Mavrilimumab, or combination thereof.
The administration of any two or more agents may start at times that are, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks apart, or administration of the second agent may start, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks after the first agent has been administered.
In certain aspects, the agents are administered simultaneously, e.g., are infused simultaneously, e.g., over a period of 30 or 60 minutes, to a patient.
One aspect of the invention provides human antibodies that block binding of SARS-CoV-2 virus to a human cell receptor to gain viral entry of the human cell, such as inhibiting binding of the S1 glycoprotein to the ACE2 receptor.
In some embodiments, the antibody of the invention has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤5 nM, ≤2 nM, ≤1 nM, ≤0.5 nM, ≤0.2 nM, ≤0.1 nM, ≤0.05 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10−8 M or less, e.g. from 10−8 M to 10−13 M, e.g., from 10−9 M to 10−13 M) for the SARS-CoV-2, such as the S1 glycoprotein.
In some embodiments, the antibody of the invention inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2. Such binding can be assessed in vitro using, for example, an ELISA assay using immobilized SARS-CoV-2 antigen on a solid support, or binding to a cell expressing ACE2 receptor on the surface.
In some embodiments, the antibody of the invention inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 with an EC50 value of less than 1 nM or 0.1 nM.
In some embodiments, the antibody of the invention exhibits neutralizing activity against a pseudovirus of SARS-CoV-2 or a live SARS-CoV-2 virus with an IC50 value of less than 10 nM, 6 nM, 3 nM, 2 nM, 1 nM, 0.6 nM or less than 0.5 nM.
In some embodiments, an antibody having any the characteristics provided herein inhibits at least 25%, 50%, 75%, 80%, 90% or 100% of the entry of SARS-CoV-2 into a host cell, such as according to the in vitro assay conditions used in the examples for entry into Vero E6 cells. Inhibition of live virus entry can be assayed based on the concentration of antibodies needed to protect about 50% SARS-CoV-2 susceptible cells, such as Vero E6 cells growing on monolayer, from exhibiting CPE (cytopathic effect) 3-5 days post infection (dpi).
In some embodiments, the antibody of the invention inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) at less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.1 nM, less than 0.08 nM, less than 0.06 nM, less than 0.02 nM, or less than 0.01 nM.
In some embodiments, the antibody of the invention inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) with an IC50 of less than 10 nM, 5 nM, 3 nM, 2 nM, 1 nM, 500 pM, 300 pM, 200 pM, 100 pM, 80 pM, 50 pM, 30 pM, 10 pM, or less than 5 pM.
In some embodiments, multispecific antibodies are provided. In some embodiments, bispecific antibodies are provided. Non-limiting exemplary bispecific antibodies include antibodies comprising a first arm comprising a heavy chain/light chain combination that binds a first epitope of SARS-CoV-2 and a second arm comprising a heavy chain/light chain combination that binds a second epitope of SARS-CoV-2. A further non-limiting exemplary multispecific antibody is a dual variable domain antibody.
In certain embodiments, the monoclonal antibodies of the invention or antigen-binding fragments thereof, including human monoclonal antibodies or antigen-binding fragments thereof, include one or more point mutations of in amino acid sequences that are designed to improve developability of the antibody. For example, Raybould et al. (Five computational developability guidelines for therapeutic antibody profiling, PNAS 116(10): 4025-4030, 2019) described Therapeutic Antibody Profiler (TAP), a computational tool that builds downloadable homology models of variable domain sequences, tests them against five developability guidelines, and reports potential sequence liabilities and canonical forms. The authors further provide TAP as freely available at opig.stats.ox.ac.uk/webapps/sabdab-sabpred/TAP.php.
There are many barriers to therapeutic mAb development, besides achieving the desired affinity to the antigen. These include intrinsic immunogenicity, chemical and conformational instability, self-association, high viscosity, polyspecificity, and poor expression. For example, high levels of hydrophobicity, particularly in the highly variable complementarity-determining regions (CDRs), have repeatedly been implicated in aggregation, viscosity, and polyspecificity. Asymmetry in the net charge of the heavy- and light-chain variable domains is also correlated with self-association and viscosity at high concentrations. Patches of positive and negative charge in the CDRs are linked to high rates of clearance and poor expression levels. Product heterogeneity (e.g., through oxidation, isomerization, or glycosylation) often results from specific sequence motifs liable to post- or co-translational modification. Computational tools are available to facilitate the identification of sequence liabilities. Warszawski et al. (Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces. PLoS Comput Biol 15(8): e1007207. https://doi.org/10.1371/journal.pcbi.1007207) also described methods of optimizing antibody affinity and stability by an automated design of the variable light-heave chain interfaces. Additional methods are available to identify potential developability issues of a candidate antibody, and in preferred embodiments of this invention, one or more point mutations can be introduced, via conventional methods, to the candidate antibody to address such issues to lead to an optimized therapeutic antibody of the invention.
The sequences of certain representative antibodies, including the light chain (LC) and heavy chain (HC) variable regions, the CDR regions, and the framework regions (FR), are listed below.
For all the antibody heavy chain sequences, the framework region sequences HFR1-HFR4 are defined by the VH-CDR sequences. For example, HFR1 is the sequence of HCVR 30 that is N-terminal to VH-CDR1. HFR2 is the sequence of HCVR that is between VH-CDR1 and VH-CDR2. HFR3 is the sequence of HCVR that is between VH-CDR2 and VH-CDR3. HFR4 is the most C-terminal sequence of HCVR.
Likewise, for all the antibody light chain sequences, the framework region sequences LFR1-LFR4 are defined by the VL-CDR sequences. For example, LFR1 is the sequence of LCVR that is N-terminal to VL-CDR1. LFR2 is the sequence of LCVR that is between VL-CDR1 and VL-CDR2. LFR3 is the sequence of LCVR that is between VL-CDR2 and VL-CDR3. LFR4 is the most C-terminal sequence of LCVR.
In certain embodiments, the HC and/or LC further includes a signal peptide sequence: MGWSCIILFLVATATGAHS (SEQ ID NO: 41).
The invention described herein provides human antibodies or functional fragment thereof specific for an antigen of SARS-CoV-2, such as the S1 glycoprotein.
In certain embodiments, the human antibodies are isolated/purified from convalescent patients recovering from SARS-CoV-2 infection.
In certain embodiments, the human antibodies share one or more CDR sequences with the patient-isolated antibodies described herein, such as antibodies having the same HCVR and/or LCVR CDR1-3 sequences, or antibodies having the same HCVR and/or LCVR sequences but different constant region sequences, such as modified Fc region sequence, or mutations in the constant region that enhances antibody stability and/or confers additional therapeutic benefits.
Human antibodies can be made by any suitable method. Non-limiting exemplary methods include making human antibodies in transgenic mice that comprise human immunoglobulin loci. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993); Jakobovits et al, Nature 362: 255-8 (1993); onberg et al, Nature 368: 856-9 (1994); and U.S. Pat. Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.
Non-limiting exemplary methods also include making human antibodies using phage display libraries. See, e.g., Hoogenboom et al., J. Mol. Biol. 227: 381-8 (1992); Marks et al, J. Mol. Biol. 222: 581-97 (1991); and PCT Publication No. WO 99/10494.
Human Antibody Constant Regions
In some embodiments, a human antibody described herein comprises human constant region sequences. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from K and X. In some embodiments, an antibody described herein comprises a human IgG constant region, for example, human IgG1, IgG2, IgG3, or IgG4. In some embodiments, an antibody or Fc fusion partner comprises a C237S mutation, for example, in an IgG1 constant region. In some embodiments, an antibody described herein comprises a human IgG2 heavy chain constant region. In some such embodiments, the IgG2 constant region comprises a P331S mutation, as described in U.S. Pat. No. 6,900,292. In some embodiments, an antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, an antibody described herein comprises an S241P mutation in the human IgG4 constant region. See, e.g., Angal et al. Mol. Immunol. 30(1):105-108 (1993). In some embodiments, an antibody described herein comprises a human IgG4 constant region and a human κ light chain.
The choice of heavy chain constant region can determine whether or not an antibody will have effector function in vivo. Such effector function, in some embodiments, includes antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), and can result in killing of the cell to which the antibody is bound. Typically, antibodies comprising human IgG1 or IgG3 heavy chains have effector function.
In some embodiments, effector function is not desirable. For example, in some embodiments, effector function may not be desirable in treatments of inflammatory conditions and/or autoimmune disorders, such as SARS-CoV-2 induced cytokine storm. In some such embodiments, a human IgG4 or IgG2 heavy chain constant region is selected or engineered. In some embodiments, an IgG4 constant region comprises an S241P mutation.
Any of the antibodies described herein may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the antigen and/or epitope to which the antibody binds, and ligands that bind antibody constant regions. For example, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an antibody.
In some embodiments, hydrophobic interactive chromatography (HIC), for example, a butyl or phenyl column, is also used for purifying some polypeptides. Many methods of purifying polypeptides are known in the art.
Alternatively, in some embodiments, an antibody described herein is produced in a cell-free system. Nonlimiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al, Biotechnol. Adv. 21: 695-713 (2003).
The invention also provides nucleic acid molecules comprising polynucleotides that encode one or more chains of an antibody described herein. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an antibody described herein. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain, of an antibody described herein. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.
In some such embodiments, the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, such as when an antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.
In some embodiments, a polynucleotide encoding a heavy chain or light chain of an antibody described herein comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N-terminus of the heavy chain or light chain. As discussed above, the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.
Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell, such as a mammalian cell.
Vectors comprising polynucleotides that encode heavy chains and/or light chains of the antibodies described herein are provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.
In some embodiments, a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain. In some embodiments, the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1:1 and 1:5 for the vector encoding the heavy chain and the vector encoding the light chain is used. In some embodiments, a mass ratio of 1:2 for the vector encoding the heavy chain and the vector encoding the light chain is used.
In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004). In some embodiments, a vector is chosen for in vivo expression of the subject antibody in animals, including humans. In some such embodiments, expression of the polypeptide or polypeptides is under the control of a promoter or promoters that function in a tissue-specific manner. For example, liver-specific promoters are described, e.g., in PCT Publication No. WO 2006/076288.
In various embodiments, heavy chains and/or light chains of the antibodies described herein may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, heavy chains and/or light chains of the antibodies described herein may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains of the subject antibody. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
Introduction of one or more nucleic acids into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc., Nonlimiting exemplary methods are described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
In some embodiments, one or more polypeptides may be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method.
Many antibodies specific for SARS-CoV-2 antigens were identified and further characterized herein, including antibodies Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, and Ab-9. Among them, Ab-5 to Ab-7 are weaker binders compared to Ab-1 to Ab-4. Ab-8 and Ab-9 shares the same VL sequence with that of Ab-2, but with 1 amino acid difference in the VH region. The other Ab-2 alternatives, all with the same CDR but different framework region (FR) sequences are not listed.
The relevant sequences of these representative antibodies are listed below.
QLQLQESGPGLVKPSETLSLTCTVSGGSISSSIYYWGWIRQPPGK
NFMLTQPHSVSASPGKTVTVSCT
GLEWIGSIYYSGNAYYNPSLKSRVTISVDTSKNQFSLKLNSVTAA
RSSGSIASNYVLWYQQRPGSAPT
DTAVYYCATPHTRWGPDYWGQGTLVTVSSASTKGPSVFPLAPS
TVIYEDDQRPSGVPDRFSASIDSS
SNSASLTISGLKTEDEADYYCQSY
DGDNLVFGGGTKLTVLGQPKAA
EVQLVESGGGLIQPGGSLRLSCAASGFIVSSNYMSWVRQAPGK
DIQMTQSPSSVSASVGDRVTITC
GLEWVSIIYSGGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRVE
RASQGISSWLAWYQQKPGKAPK
DTAVYYCARDLQELGSLDYWGQGTLVTVSSASTKGPSVFPLAPS
LLIYAASSLQSGVPSRFSGSGSGT
DFTLTISSLQPEDFATYYCQEANS
FPYTFGQGTKLEIKRTVAAPSVFIF
QLQLQESGPGLVKPSETLSLTCTVSGGSISSTIYYWGWIRQPPGK
NFMLTQPHSVSASPGKTVTVSCT
GLEWIGSIYYSGNAYYNPSLKSRVTISVDTSKNQFSLMLNSVTAS
RSSGSIASNYVLWYQQRPGSAPT
DTAVYYCATPHTRWGPDYWGQGTLVTVSSASTKGPSVFPLAPS
TVIYEDDQRPSGVPDRFSASIDSS
SNSASLTISGLKTEDEADYYCQSY
DGDNLVFGGGTKLTVLGQPKAA
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGK
DIVMTQSPLSLPVTPGEPASISCR
ALEWLALIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMA
SSQSLLHSNGYNYLDWYLQKPG
PVDTATYYCAHRLSNFWSGYYTGWGQGTLVTVSSASTKGPSVF
QSPQLLIYLGSNRASGVPDRFSGS
GSGTDFTLKISRVEAEDVGVYYC
MQALQTPNTFGQGTKLEIKRTVA
QVQLQESGPGLVKPSETLSLTCGVSGGSISSYYWSWIRQPPGKG
NFMLTQPHSVSESPGKTITISCTG
LEWIGHIYDSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTASDT
SSGSIASNYVQWYQQRPGSAPT
AVYYCARQLWLRGAFDIWGQGTMVTVSSASTKGPSVFPLAPSS
TVIYEDQQRPSGVPDRFSGSIDSS
SNSASLTISGLKTEDEADYYCQSY
DSTNQVFGGGTKLTVLGQPKAAP
QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPG
QSALTQPPSASGSPGQSVTISCTG
KGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELS
TSSDVGGYNYVSWYQQHPGKA
SLRSEDTAVYYCATGHQLLFYNWFDPWGQGTLVTVSSASTKGP
PKLMIYEVSKRPSGVPDRFSGSKS
GNTASLTVSGLQAEDEADYYCSS
YAGSNNLVFGGGTKLTVLGQPKA
EVQLVESGGGLVKPGGSLRLSCAASGFAFSSYTMNWVRQAPGK
QSALTQPPSASGSPGQSVTISCTG
GLAWVSSISSSSDYIFYADSVKGRCTISRDNAKNSLSLQMNSLRA
TSSDVGRYNYVSWYQQHPGKAP
EDTAVYYCARGSNTAWGGVPDAFDFWGLGTVVTVSSASTKGP
KLMIYEVSKRPSGVPDRFSGSKS
GNTASLTVSGLQTEDEADYYCSS
YAGSNNLVFGGGTKLTVLGQPKA
EVQLVESGGGLIQPGGSLRLSCAAPGFIVSSNYMSWVRQAPGK
DIQMTQSPSSVSASVGDRVTITC
GLEWVSHYSGGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRVE
RASQGISSWLAWYQQKPGKAPK
DTAVYYCARDLQELGSLDYWGQGTLVTVSSASTKGPSVFPLAPS
LLIYAASSLQSGVPSRFSGSGSGT
DFTLTISSLQPEDFATYYCQEANS
FPYTFGQGTKLEIKRTVAAPSVFIF
EVQLVESGGGLIQPGGSLRLSCAASGFIVSSNYMSWVRQAPGK
DIQMTQSPSSVSASVGDRVTITC
GLEWVSHYSGGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRVE
RASQGISSWLAWYQQKPGKAPK
DTAVYYCARDLQELGSLDCWGQGTLVTVSSASTKGPSVFPLAPS
LLIYAASSLQSGVPSRFSGSGSGT
DFTLTISSLQPEDFATYYCQEANS
FPYTFGQGTKLEIKRTVAAPSVFIF
HC and LC signal peptide sequence: MGWSCILFLVATATGAHS (SEQ ID NO: 41)
IgG4 versions of Ab-1 to Ab-4 have also been generated. These antibodies have the same light chain sequences, and heavy chain variable regions (bold sequences) as the sequences above, but have distinct IgG4 heavy chain region sequences, as shown below. The corresponding LC and HC leader sequences are also provided. The only exception is IgG4 Ab-4, in which its 33rd light chain variable region residue N is substituted by Q.
QLQLQESGPGLVKPSETLSLTCTVSGGSISSSIYYWGWIRQ
PPGKGLEWIGSIYYSGNAYYNPSLKSRVTISVDTSKNQFSL
KLNSVTAADTAVYYCATPHTRWGPDYWGQGTLVTVSSASTK
EVQLVESGGGLIQPGGSLRLSCAASGFIVSSNYMSWVRQAP
GKGLEWVSIIYSGGSTFYADSVKGRFTISRDNSKNTLYLQM
NSLRVEDTAVYYCARDLQELGSLDYWGQGTLVTVSSASTKG
QLQLQESGPGLVKPSETLSLTCTVSGGSISSTIYYWGWIRQ
PPGKGLEWIGSIYYSGNAYYNPSLKSRVTISVDTSKNQFSL
MLNSVTASDTAVYYCATPHTRWGPDYWGQGTLVTVSSASTK
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQ
PPGKALEWLALIYWDDDKRYSPSLKSRLTITKDTSKNQVVL
TMTNMAPVDTATYYCAHRLSNFWSGYYTGWGQGTLVTVSSA
Ab-4 IgG4 LC a.a. sequence (without leader sequence):
Ab-4 IgG4 LCVR a.a. sequence:
The antibodies of the invention have been demonstrated to be able to bind to the spike protein of the SARS-CoV-2 virus, based on binding assays for S1 and S2 domains, as well as the full length of the S protein. ELISA results showed that at least 20 antibodies bound to the viral S protein with different affinity, among which Ab-2 was the strongest binder, and Ab-4 is another top candidate which also binds to S full length protein (see
Next, the blocking effects of the top four antibodies on the binding of S1 to Vero E6 cell line was analyzed by FACS analysis (
The blocking activities of these antibodies were also tested for blocking S1 binding to ACE2 using ELISA assay (data not shown). Again, Ab-1, Ab-2, & Ab-3 showed potent blocking activities with potency at 10−9-10−10 M, and Ab-4 did not show any blocking activity on S1 binding to ACE2.
Next, the neutralizing activities of these antibodies against HIV-vectored pseudovirus were examined. Antibodies Ab-2 exhibited potent neutralizing activity, with IC50 of 0.53 nM on average. Ab-3 and Ab-1 showed IC50s at 5.80 nM and 4.07 nM respectively, whereas Ab-4 displayed no neutralizing activities (
Sequence analysis showed that each cluster of antibodies shared at least 93% sequence identity. The difference within the same sequence cluster may result from somatic hypermutation in which point mutations accumulated in the antibody. Because the somatic hypermutation does not distinguish between favorable and unfavorable mutations, 12 closely similar sequences in the cluster comprising Ab-2 were chosen to compare their activity experimentally.
As shown in
indicates data missing or illegible when filed
The neutralizing activities of the lead antibodies Ab-1 to Ab-3 were further confirmed in live SARS-CoV-2 virus entry assay. Vero E6 cells were infected with SARS-CoV-2 virus at 100 TCID50 in the presence of the lead antibodies at different concentrations. Using fluorescent labeled nuclear protein of SAR-CoV-2, infected cells can be observed by florescence microscopy. Antibody Ab-2 demonstrated strong anti-virus activities—more than 50% inhibition at 6.5 nM, while Ab-3 demonstrated 74% inhibition at 62.7 nM, and Ab-1 showed 93.8% inhibition at 50.2 nM (
As the pandemics progressed, the mutations located in S protein accumulated and may confer selective advantages in transmission and resistance to the antibody intervention. Thus, the affinity of Ab-2 for 4 dominant spike RBD mutations, and its ability to competing with ACE2 for RBD binding were examined. SPR results showed that Ab-2 bound to mutations with similar affinity as to the wild-type S protein. Ab-2 also blocked the interaction of ACE2 with all RBD mutations (data not shown).
The three most potent antibodies binding to S1 protein has been further profiled pharmacologically. A snapshot PK study at 1 h, 24 h and 72 h separately in wild-type (WT) mice showed good PK profile. While no significant difference was observed among the 3 Abs, Ab-2 showed slightly better exposure comparing with Ab-1 and Ab-3 (
This example demonstrates that certain subject antibodies with hIgG4 constant region (which does not engage or minimally engages FcγR), compared to their counterpart with the hIgG1 constant region, have comparable binding affinity for the SARS-CoV-2 S1 antigen, pseudovirus neutralization activity, and live virus neutralization activity. Such hIgG4 antibodies also have comparable favorable developability characteristics.
For binding affinity assay, SPR (surface plasmon resonance) with anti-hIgG Fc immobilized on CM5 chip was used to capture several subject monoclonal antibodies with hIgG1 vs. hIgG4 constant region, respectively, in order to compare their respective binding affinity for the soluble S1 RBD domain. The results are summarized in the table below.
As indicated before, Ab-4 binds S2 but not S1 protein, which served as a negative control. The measured KD values for the different antibodies in IgG1 vs. IgG4 formats were comparable.
Comparable binding affinity was also verified using Ab-2, either in hIgG1 or hIgG4 format, in ELISA assay. The result in
Next, pseudo-virus neutralization activity of the IgG1 vs. IgG4 formats of Ab-2 were compared (data for Ab-1 and Ab-3 were also included). The results were shown in
Live-virus neutralization activity between IgG1 vs IgG4 formats of Ab-2 were also compared. In one experiment, a fixed amount of live SARS-CoV-2 virus was mixed with about equal volumes of a serial dilution of the IgG4 version of the Ab-2 antibody, before the neutralized or partially neutralized virus was used to infect single layer of Vero E6 cells (2 repeats for each Ab dilution). CPE (cytopathic effect) was observed at 3-5 days post infection in lower dilutions but not in higher dilutions. Experimental results were summarized below.
According to the Reed-Muench method, about 0.448 μg/mL (or about 3 nM) of IgG4 Ab-2 protected about 50% of Vero E6 cells from exhibiting SARS-CoV-2 infection-induced CPE under the experimental condition.
In another experiment similar to
Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Booth et al., “Extending human IgG half-life using structure-guided design,” MAbs, 2018 Oct. 10(7) 1098-1110. Recombinant antibodies' therapeutic potential may be enhanced by the introduction of defined mutations in the crystallizable fragment (Fc) domains, such as for example, YTE (M252Y/S254T/T256E) and LS (M428L/N434S), as a consequence of increased half-lives and prolonged duration of protection. For example, The prototypical example of an FcRn affinity-enhancing Fc mutant is the YTE mutation which, when incorporated into motavizumab IgG1, is able to extend serum half-life in humans by more than four-fold. Robbie et al., A novel investigational Fc-modified humanized monoclonal antibody, motavizumab-YTE, has an extended half-life in healthy adults. Antimicrob Agents Chemother. 2013; 57:6147-6153. Booth et al. and Robbie et al. are incorporated herein.
A series of developability assays were performed for the IgG4 version of Ab-2 and Ab-3, including accelerated stability (2-3 mg/mL of Ab at 25 and 40° C. in D-PBS, pH7.4, for up to 14 days); forced degradation (2-3 mg/mL of Ab at 25° C. in 100 mM acetic acid at pH3.5, for up to 6 hours); and up to 5 freeze-thaw cycles (2-3 mg/mL of Ab). The results showed that all samples were stable in the accelerated stability study; both antibodies showed aggregation formation under the low pH stress condition; and all samples remained stable after 5 cycles of freeze-thaw.
The data provided herein provides a deeper understanding of the immune response mounted by patients recovering from COVID-19, and may give insights into disease epidemiology and inform the development of novel therapies.
For example, despite the documented greater severity of the disease in males as compared to females, males seem to demonstrate higher antiviral titers as a group. This may be related to higher viral loads during the period of peak infection. Several mechanisms have been proposed to explain the apparently greater susceptibility of males to COVID-19, including 1) A high prevalence of smoking among males in regions where the SARS-CoV-2 virus has spread to humans, and 2) the ACE2 gene that acts as a receptor for the virus on human epithelial cells being located on the X chromosome, which may lead to sex-specific differences in ACE2 expression and susceptibility to infection or viral burden. Children have been observed to have less severe disease symptoms as compared to adults (Dong et al., 2020). Interestingly, an 8-year-old patient profiled as part of the cohort studied here showed relatively high titers to the SARS-CoV-2 S protein, suggesting that despite their less mature immune systems, children are capable of mounting a robust antiviral immune response.
COVID-19 patients tend to display low lymphocyte counts, and reduced levels and functional exhaustion of T cells has been described in patients with severe disease (Ni et al., 2020). It is therefore notable to find increased levels of circulating plasma cells as well as memory B cells in convalescent patients weeks after initial infection and days after recovery, suggesting that the humoral immune response is critical in limiting viral activity.
Use of convalescent patient sera as an immediate way to transfer protective immunity to newly diagnosed patients and at-risk populations is a therapeutic strategy being applied to treat COVID-19, and neutralizing antiviral monoclonal antibodies identified from these sera have the utility to be scalable treatments for the disease. Further mining of immune repertoires from patients recovering from COVID-19, using their methods of the invention described herein can be key to controlling the future spread of this and other similar viruses.
The methods described herein identified antibodies binding to SARS-CoV-2 coronavirus, thus permitting further characterization of the neutralizing activities of these antibodies, as well as mapping out binding epitopes of these antibodies. In one embodiment, these neutralizing antibodies can be formulated for use as therapeutic antibodies for patient treatment. In another embodiment, they can also be used prophylactically to prevent virus infection. In further embodiments, certain binding antibodies can be used in combination with the vaccine approaches, even if they do not have neutralization activities. In yet another embodiments, different antibodies with either S1 or S2 binding capacity are used to generate multi-valent antibodies, or be used together, for combination therapy.
Similar to live virus study described above, Vero E6 cells were infected by the WuhanD614, BavPat D614G and UK B.1.1.7 (also named 20I/501Y.V1) SARS-CoV-2 variants and incubated with the subject antibodies with a series of two-fold dilutions in the 0.97-1000 ng/ml range in triplicate. Viral RNA in the supernatant was determined, and % of inhibition was calculated based on infected but untreated control. The results are shown in the table below:
Additional tests were performed to compare the effectiveness of the subject antibody (i.e., Ab-2 IgG4) against the ACE2-Fc fusion, in various variant strains. The results are listed in the table below.
Next, a pseudotyped lentiviral vector with SARS-CoV-2 S proteins as part of the envelope is constructed to mimic SARS-CoV-2 virus, which can infect target cells expressing hACE2. The neutralizing potency was then deduced by detecting the expression levels of luciferase reporter gene packaged into the lentiviral vector. Three variants of SARS-CoV-2 were tested in this experiment: SARS-CoV-2/Wild-type (WT), SARS-CoV-2/United Kingdom (UK, B.1.1.7 lineage) variant and SARS-CoV-2/South Africa (SA, B.1.351 lineage) variant. Those variants represent commonly isolated SARS-CoV-2 clinical isolates.
Under the current experimental condition, Ab-2 IgG4 showed a significant neutralizing potency against two SARS-CoV-2 variants—WT and UK variant. However, little neutralizing activity was observed against SA variant, indicating that mutations in SARS-CoV-2 Spike from SA variant gained the ability to void the neutralizing antibody tested. On the other hand, Ab-5 showed a significant neutralizing potency against SARS-CoV-2/SA variant (data not shown).
SARS-CoV-2 strains (strain BetaCoV/Wuhan/WIV04/2019) at 100 TCID50 per 50 μL was mixed with equal volume of culture medium containing serially Ab-2 IgG1 and Ab-2 IgG4 diluted antibodies and incubated at 37° C. for 1 hour, and then added to Vero E6 cells seeded in 96-well plates. After 48 hrs of culture at 37° C., cells were fixed and processed for SARS-CoV-2 nucleocapsid protein (NP) and nuclei staining. % Inhibition was calculated by (total nuclei-infected cells)/total nuclei ×100%. Fifty percent neutralization dose (ND50) and ninety percent neutralization dose (ND90) were calculated using 4-parameter non-linear regression with GraphPad Prism 8.0. The experiment was carried out in triplicate.
Ab-2 IgG4 and Ab-2 IgG1 exhibited potent neutralization activities against SARS-CoV-2 live virus infections of Vero E6 cells with ND50 between 2.18-6.5 nM (0.320-0.956 μg/mL) and ND90 between 2.18-11.68 nM (0.320-1.681 μg/mL), respectively.
The binding of the Ab-2 IgG4 antibody to SARS-CoV-2 S protein variants (S477N, S494P, F490S, Y453F, N439K, N501Y, E484K, Q493R, and A222V/D614G) was determined by enzyme linked immunosorbent assay (ELISA). The 384-well plates were coated with 20 nM of the different SARS-CoV-2 S protein RBD variants. The binding of Ab-2 IgG4 (12 concentrations obtained by 3-fold serial dilutions starting from 300 nM, in triplicate) to the SARS-CoV-2 S protein RBD (receptor binding domain) variants was detected by goat F(ab′)2 anti-human IgG (H+L)-HRP. In this experiment, Ab-2 IgG4 exhibited potent binding to all tested SARS-CoV-2 S protein RBD/S1 variants with comparable EC50 values ranging from 0.10-0.37 nM.
The blocking activity of Ab-2 IgG4 was determined by enzyme linked immunosorbent assay (ELISA). The 384-well plates were coated with 20 nM of the hACE2-mFc protein. SARS-CoV-2 S protein RBD/S1 variants (with His tag) at fixed concentration (binding EC90 of corresponding variant to hACE2-mFc) was pre-incubated with Ab-2 IgG4 or isotype control at different concentrations (12 concentrations obtained by 3-fold serial dilutions starting from final concentration of 300 nM, in duplicates), before incubated with coated hACE2-mFc protein. The binding of SARS-CoV-2 S protein RBD/S1 variants (with His tag) to the hACE2-mFc protein was detected by HRP anti-6×His tag antibody. In this experiment, Ab-2 IgG4 blocked the binding of 9 tested SARS-CoV-2 S protein RBD/S1 variants (S477N, S494P, F490S, Y453F, N439K, N501Y, E484K, Q493R, and A222V/D614G) to hACE2-mFc protein with IC50 ranging from 0.6-13.15 nM.
Additionally, binding affinities of Ab-2 IgG4 to WT RBD or 6 mutant RBD variants (Y453F, S477N, S494P, F490S, N439K, N501Y) and a S1 variant (A222V/D614G) were evaluated using SPR (Biacore T200).
One potential hurdle for antibody-based therapeutics is the risk of exacerbating COVID-19 severity via antibody-dependent enhancement (ADE). ADE has been documented to occur through two distinct mechanisms in viral infections: by enhanced antibody-mediated virus uptake into Fc gamma receptor IIa (FcγRIIa)-expressing phagocytic cells leading to increased viral infection and replication, or by excessive antibody Fc-mediated effector functions or immune complex formation causing enhanced inflammation and immunopathology. Both ADE pathways can occur when non-neutralizing antibodies or antibodies at sub-neutralizing levels bind to viral antigens without blocking or clearing infection.
Raji cells, originally derived from a Burkitt's lymphoma patient, have been shown to facilitate SARS-CoV-1 infection in the presence of anti-S-protein immune serum. Thus, this FcγRII-bearing human B lymphoblast cell line was used to study the antibody-dependent viral entry of SARS-CoV-2 as an indicator of ADE. Briefly, Raji cells were seeded in 96-well plates. Antibodies at different concentrations were pre-incubated with the SARS-CoV-2 pseudo virus encoding wild-type spike protein and luciferase. The mixture of antibody and pseudo virus was then added to plated Raji cells. The plates were incubated and pseudovirus infection of Raji cells was quantified by measuring luciferase activity.
Antibody-dependent entry of SARS-CoV-2 pseudovirus in the presence of a positive control antibody was observed. On the other hand, Ab-2 IgG4 did not show any sign of antibody-dependent enhancement of SARS-CoV-2 pseudovirus infection of Raji cells.
The data suggests that Ab-2 IgG4 does not exacerbate a SARS-CoV-2 infection via ADE.
A total of 9 Rhesus monkeys were included for efficacy study of Ab-2 IgG4. Monkeys were allocated into 3 groups (three per group) receiving a single intravenous infusion of isotype control at 50 mg/kg (group 1), Ab-2 IgG4 at 10 mg/kg (group 2) and Ab-2 IgG4 at 50 mg/kg (group 3) one day after intratracheal inoculation of SARS-CoV-2 at 1×105 TCID50. Serum samples were collected once daily from 0-7 days post infection (d.p.i.). The concentration of Ab-2 IgG4 in plasma samples was determined using validated ELISA methods.
Briefly, plates were coated at 4° C. overnight with anti-human (h) IgG (quantification of total hIgG) or SARS-CoV-2 S protein S1 subunit recombinant protein (quantification of unbound Ab-2 IgG4), before incubated with rhesus monkey plasma collected during Ab-2 IgG4-012 Study. The concentration of plasma antibodies was detected by HRP conjugated anti-hIgG antibody.
Under these experimental conditions, the systemic exposure of Ab-2 IgG4 were achieved in all treated Rhesus monkeys. Ab-2 IgG4 exhibited linear clearance with approximate dose proportionality. The mean AUC0-6d of Ab-2 IgG4 in 10 and 50 mg/kg dosing groups were 222 and 1643 μg/mL*day, respectively, by total hIgG, and 419 and 2398 μg/mL*day, respectively, by unbound Ab-2 IgG4.
A study was conducted to evaluate serum pharmacokinetics (PK) and immunogenicity following a single IV infusion administration of Ab-2 IgG4 in naïve male and female cynomolgus monkeys.
On Day 1 of the study, 3 male and 3 female cynomolgus monkeys were administered a single 10 mg/kg dose of Ab-2 IgG4 by IV infusion (60 minutes; 4 mL/kg). Blood was collected and processed for PK, anti-drug antibody (ADA), hematology, and clinical chemistry evaluations (data not shown). The concentration of Ab-2 IgG4 and ADA, was monitored in serum for up to 56 days (1345 hours) after beginning of infusion and are provided below. Safety was also monitored based on clinical observations and hematology and clinical chemistry evaluations.
The quantification of serum Ab-2 IgG4 concentrations and ADA were conducted at WuXi AppTec Bioanalytical Services Department using a validated ELISA method or a validated electrochemiluminescent (ECL) method, respectively. Serum concentration versus time data was analyzed by non-compartmental model using the WinNonlin software (version 6.3, Pharsight, Mountain View, Calif.).
Individual animal Ab-2 IgG4 serum concentrations over time were measured (not shown) and a summary of the Ab-2 IgG4 PK parameters is provided in the table below. A single dose of 10 mg/kg resulted in a combined (males and females) mean area under the serum drug concentration-time curve up to the last quantifiable time-point 56 days post start of infusion (AUC0-1345 h) of 77,500,000 ng·h/mL and mean maximum observed serum concentration (Cmax) of 266,000 ng/mL. The mean time to maximum concentration (Tmax) was 1.0833 hours post beginning of infusion and the mean terminal half-life (T½) was 459 hours. There were no notable gender-related differences in Ab-2 IgG4 systemic exposures in cynomolgus monkeys (data not shown). From Day 1 through Day 56 of the study, there were no adverse clinical observations, body weight changes, or abnormal hematology and blood chemistry test results.
The efficacy of Ab-2 IgG4 to treat SARS-CoV-2 infection was evaluated in Rhesus monkeys. On Day 0, 3 groups of 3 Rhesus monkeys (2 females and 1 male per group) were infected by intratracheal (IT) inoculation with SARS-CoV-2 at 1×105 TCID50/animal. On Day 1, following confirmation of infection via oropharyngeal swab, a single treatment with isotype control antibody (50 mg/kg) or Ab-2 IgG4 at 10 or 50 mg/kg was administered by IV infusion. Health status and infection were monitored via body temperature, body weight, hematology, and blood chemistry analyses from samples collected prior to viral challenge (pi0d) and on Days 1 (blood, swabs, and feces collected before Ab-2 IgG4 administration), 2, 3, 4, 5, 6, and 7. SARS-CoV-2 viral load was evaluated in blood; oropharyngeal, nasal and rectal swabs; and feces using RT-qPCR method. The lungs were X-rayed on Days 0 (prior to infection), 3, and 6. One monkey from each group was euthanized 5, 6 and 7 days post-infection and selected organs (lungs [6 lobes, trachea, left and right bronchia], spleen, pulmonary hilar lymph node, liver, and kidney) were processed, stained with hematoxylin and eosin [H&E] and Masson's trichrome staining, and associated pathological changes were evaluated microscopically. Serum samples were collected prior to viral challenge (pi0d) and on Days 1 (before Ab-2 IgG4 administration), 2, 3, 4, 5, 6, and 7, for evaluation of Ab-2 IgG4 levels. The findings for animal health (body weight, body temperature), viral load, X-rays, and microscopic evaluations of lung tissues have been reported, as well as pharmacokinetics results.
No significant changes in body temperature or body weight were noted throughout the study. A single treatment with Ab-2 IgG4 at 10 or 50 mg/kg markedly decreased detectable SARS-CoV-2 viral load in oropharyngeal swabs by Day 4 (
Viral load was also evaluated in different tissues and showed consistently detectable viral RNA (1×104-1×107 viral RNA copies/gram) in all 3 isotype control group animals in the trachea, left and right bronchus, and 2 or 3 of the 6 lung lobes in the 2 animals euthanized on days 6 and 7 post infection (
In contrast, in Ab-2 IgG4 50 mg/kg treatment group, viral RNA (1×105-1×107 viral RNA copies/gram) was only detected in the trachea, left and right bronchus, and 1 lung lobe in the animal euthanized 5 days post infection, but not in any of the tissues tested from the 2 animals euthanized on day 6 and 7 post infection (
Microscopic evaluation of the lungs (picture not shown) of the animals in the isotype control group or Ab-2 IgG4 10 mg/kg group on Day 7 post infection (6 days after treatment) revealed severe and moderate to severe lung lesions, respectively, characterized by: (1) thickened alveolar walls with large numbers of monocyte and lymphocyte infiltrates, fibroblast hyperplasia, and fibrosis; (2) monocyte and lymphocyte infiltrates, edema, cellulose exudation and/or hyaline membrane formation, fibroblast proliferation, and fibrosis in the alveolar cavities, with compensating emphysema in some alveoli; and (3) pulmonary hemorrhage in the lungs. The lesions generally centered around small bronchial tubes with numerous epithelial cells observed in some small bronchial cavities. Emphysema under the pleural was observed with localized pleural wall thickening and fibrosis on part of the pleural. The lung lesions in the 50 mg/kg Ab-2 IgG4 treatment group were reduced to mild-moderate with most of the alveoli displaying normal structure. A summary comparison of lung lesions in different animals is shown in the following table.
The serum drug concentration was determined using 2 optimized ELISA methods measuring total hIgG or unbound Ab-2 IgG4. After intravenous administration of Ab-2 IgG4, serum exposure was observed in all animals. Ab-2 IgG4 exhibited linear clearance with approximate dose proportionality. The mean AUC0-6days of Ab-2 IgG4 in 10 and 50 mg/kg dosing groups were 222 and 1643 μg/mL*day, respectively, by total hIgG, and 419 and 2398 μg/mL*day, respectively, by unbound Ab-2 IgG4 (see Table below).
In conclusion, intra-tracheal inoculation of SARS-CoV-2 (1×105 TCID50) in Rhesus monkeys resulted in viral replication in the respiratory tract and lung lesions in isotype control (50 mg/kg) treated animals.
In contrast to the treatment with isotype control antibody, Ab-2 IgG4 effectively inhibited viral replication, as shown by a progressive declining of viral loads in the oropharyngeal swabs of animals treated with a single dose of 10 mg/kg or 50 mg/kg Ab-2 IgG4 one day after viral inoculation (2 of 3 animals at 10 mg/kg and 3 of 3 animals at 50 mg/kg) and decreased viral distribution in respiratory tract and lungs.
Based on lung imaging and microscopic analysis, isotype control treated animals exhibited pathological changes including moderate to severe lesions characterized by thickened alveoli walls, fibroblast hyperplasia, fibrosis, large numbers of monocyte and lymphocyte infiltrates, alveoli edema with small amounts of cellulose exudate and/or hyaline membrane formation in alveolar cavities, and pulmonary hemorrhage. The lung lesions generally centered around small bronchial tubes with numerous epithelial cells observed in some small bronchial cavities. Emphysema under the pleural was observed with localized pleural wall thickening and fibrosis on part of the pleural.
Although similar microscopic changes were noted in the lungs in all groups, the severity was reduced to moderate in the 10 mg/kg Ab-2 IgG4 treatment group, and mild-moderate in the 50 mg/kg Ab-2 IgG4 treatment group with most of the alveoli displaying normal structure. After intravenous administration of Ab-2 IgG4, serum exposure was observed in all animals. Ab-2 IgG4 exhibited linear clearance with approximate dose proportionality.
Together, these data demonstrate the antiviral activity of Ab-2 IgG4 for the treatment of SARS-CoV-2 in a Rhesus monkey infection model.
The purpose of this study was to determine the potential toxicity of Ab-2 IgG4 when administered by intravenous infusion at 0, 50 and 300 mg/kg/dose to cynomolgus monkey once weekly for 14 days (two doses in total) and to assess the reversibility, persistence, or delayed occurrence of potential toxic effects following a 56-day recovery phase. In addition, the toxicokinetics (TK) of Ab-2 IgG4 were determined.
Thirty (15 per sex) cynomolgus monkeys were randomly assigned to 3 groups of 5/sex/group and given Ab-2 IgG4 at 0, 50, or 300 mg/kg/dose once weekly for 2 doses by intravenous (IV) infusion at a dosing volume of 10 mL/kg and dosing rate of 6.7 mL/kg/hour. The infusion duration was 1.5 hours. At initiation of dosing, male and female cynomolgus monkeys were approximately 3 to 5 years of age, and their body weights ranged from 2.2 to 3.6 kg for females and 2.3 to 5.2 kg for males. The dosing phase animals were necropsied on Day 15 and recovery animals were necropsied on Day 71.
Parameters evaluated during the study included viability (morbidity and mortality), clinical observations including tolerance at injection sites, body weight, body weight changes, qualitative food consumption, ophthalmology, body temperature, safety pharmacology (electrocardiography, blood pressure, respiration and neurological examinations), clinical pathology (hematology, serum chemistry, coagulation and urinalysis), toxicokinetics (TK), immunogenicity analysis (anti-drug-antibody), cytokine analysis (IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, TNF-α and IFN-7), gross pathology, organ weights, and histopathology.
Median Tmax values for Ab-2 IgG4 were observed between 1.6 and 5.5 hours post start of infusion. Mean or median T1/2 values were estimated between 167.0 and 350.3 hours. No marked sex differences in systemic exposure were observed at any dose level. The systemic exposure increased dose-proportionally in males and females on Days 1 and 8 as the dosage increased from 50 to 300 mg/kg. No marked drug accumulation was observed at either dose level. Relatively higher titers of anti-Ab-2 IgG4 antibodies were detected in one male and female given 300 mg/kg/dose during the recovery phase, which resulted in a slight decrease of Ab-2 IgG4 concentrations in these two animals during the recovery phase and has no impact on overall TK evaluation of the study.
There were no animals showing moribund or found dead during the study. All animals survived to scheduled termination. There were no test article-related clinical signs, or effects on body weight, appetite, or findings in ophthalmologic examinations, body temperature, safety pharmacology parameters (quantitative and qualitative electrocardiography, cardiovascular, respiration and neurological changes), hematology, coagulation, urinalysis, serum cytokine (IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, TNF-α, and IFN-7) concentrations, organ weight changes, or macroscopic and microscopic findings. Toward the end of the dosing phase, increased serum globulin (GLB) was noted in males and females given 300 mg/kg/dose, which was likely related to the presence of the test article in the blood, as the test article itself is a monoclonal antibody. Increased serum GLB was not apparent toward the end of the recovery phase.
In conclusion, administration of Ab-2 IgG4 to cynomolgus monkeys once weekly for 2 doses by intravenous infusion at dosages of 0, 50, or 300 mg/kg/dose followed by a 56-day recovery period was well tolerated and did not result in any adverse changes. The no observed adverse effect level (NOAEL) for this study was considered to be 300 mg/kg/dose, the highest dose tested. The corresponding Cmax and AUC0-169.5h of Ab-2 IgG4 following the last dose at the NOAEL were 9,880,000 ng/mL and 949,000,000 h*ng/mL for males, and 9,470,000 ng/mL and 916,000,000 h*ng/mL for females, respectively.
Epitope mapping for Ab-2 was conducted to determine the specific residues on the SARS-CoV-2 spike (S) protein that may be involved in binding to Ab-2. Meanwhile, the specific residues on the SARS-CoV-2 spike (S) protein that may be responsible for ACE2 binding and host cell entry were also determined.
Specifically, based on the 3-D crystallography structure of the Ab-2/S protein complex, a first set of S protein residues within 4A of Ab-2 were labeled, and based on the 3-D crystallography structure of the ACE2/S protein complex, a second set of S protein residues within 4 Å of the ACE2 receptor were also labeled. Common residues between the first and the second sets were noted.
It is apparent that among the 17 S protein residues that are within 4 Å of the ACE2 receptor (which are likely involved in ACE2 receptor binding and host cell entry), 15 of these residues are also within 4 Å of Ab-2. This suggests that Ab-2 binding to the Spike protein S will almost completely block off S protein binding to ACE2 receptor, thus preventing SARS-CoV-2 infection of host cells.
The partial protein sequence of the SARS-CoV-2 Wuhan-Hu-1 strain sequence including residues for ACE2 receptor binding and/or Ab-2 binding, from N334 to P527, is shown below and in
Thus the S protein epitope required for Ab-2 binding includes residues T415, G416, K417, D420, Y421, Y453, L455, F456, R457, K458, N460, Y473, Q474, A475, G476, S477, F486, N487, Y489, Q493, S494, Y495, G496, Q498, T500, N501, G502, and Y505 (bold residues are also involved in ACE2 binding).
IADYNYKLPDDFTG
GN
NYL
R
SNLKPFERDISTEI
GS
C
FPL
F
P
VG
QPYRVVVLSFELLHAPAT
Antibody Binding and Competition with Receptor ACE2:
The binding affinity of antibodies to spike protein was analyzed by ELISA. 384 well plate (Corning #3700), was coated overnight at 4° C. with PBS containing 30 μL 20 nM of the SARS-CoV-2 Spike S1+S2 ECD, his Tag protein. The next day the plate was washed 5 times with washing buffer (PBS and 0.05% Tween) and then incubated 1 hour at room temperature in blocking buffer (PBS with 2% BSA). After 5 washes the plate was incubated with serial dilution of purified mAbs for 1 hour at room temperature. The plates were then washed 5 times and incubated for 1 hour in detection reagent (Mouse anti-Human IgG Fc HRP labeled (Thermo Fisher 05-4220) at 0.2 μg/ml in 1×PBS with 0.05% Tween and 1% BSA) for 1 hour at room temperature. Following this the plate was washed again 5 times and developed in TMB substrate for 5 min before stopping the reaction with the stop solution. The OD values were determined using Thermo MultiSkan or MD SpectraMax i3X at 450 nm wavelength.
The blocking with receptor ACE2 was performed using cell surface expressed ACE2. 10 nM SARS-CoV-2 Spike S1, mFc tag spike protein was incubated with serial dilution of purified mAbs at room temperature for 1 h and then added to Vero E6 cells (approximately 105 per well) in duplicate. Then detection reagent rabbit anti mouse IgG Fc-AF647 was used. Half-maximal inhibitory concentration (IC50) of the evaluated mAbs were determined with Beckman Cytoflex and FlowJo software analysis.
Murine leukemia virus-based SARS-CoV-2 S pseudotyped virus were prepared by GenScript as previous described. Neutralization assay were performed by incubating pseudo virus with serial dilution of purified antibodies at room temperature for 1 h. ACE2 overexpression Hela cells (approximately 8×104 per well) were cultured in DMEM containing 10% FBS, 1 μg/mL puromycin were added in triplicate into virus-antibody mixture. Following infection at 37° C., 5% CO2 for 48 h, half-maximal inhibitory concentration (IC50) were determined by luciferase activity using Promega Bio-Glo luciferase assay system with GraphPad Prism.
Vero E6 cells infected by SARS-CoV-2 at 100 50% tissue-culture infectious doses (TCID50) or infected with a mixture of viruses and diluted antibodies (incubated together for 1 hr) were cultured at 37° C. for 48 hours, and then fixed with 4% paraformaldehyde diluted in PBS (pH=7.2) for 15 minutes at room temperature and then penetrated with 0.25% triton-X 100 for 10-15 minutes. After three washes, cells were blocked at 37° C. for 1 hour using PBS containing 5% BSA, then incubated with in-house prepared anti-SARS-CoV-2 NP rabbit serum as primary antibody and FITC or Alexa Fluor® 488-conjugated goat anti-mouse IgG antibody as the secondary antibody. Cell nuclei were stained using Hoechst 33258 at room temperature for 10 minutes. Images were taken under an inverted fluorescence microscope (Nikon). In some experiments, numbers of nuclei and cells infected with viruses were counted using an Operetta CLSTM system. % inhibition was calculated by (total nuclei-infected cells)/(total nuclei)×100%. Fifty percent neutralization dose (ND50) and ninety percent neutralization dose (ND90) were calculated using 4-parameter non-linear regression with GraphPad Prism 8.0.
All references are incorporated herein by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/096360 | Jun 2020 | WO | international |
This International Patent Application claims priority to International Patent Application No. PCT/CN2020/096360, filed on Jun. 16, 2020, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/037615 | 6/16/2021 | WO |
