Patent Application
20240018217
The present invention relates in general to the field of full length, fully human monoclonal antibodies, and more particularly, to human monoclonal antibodies to SARS-CoV-2.
The present application includes a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on Jun. 28, 2023, is named OMRF1039.xml and is 8,487,350 bytes in size.
Without limiting the scope of the invention, its background is described in connection with broadly specific, neutralizing, full-length, fully human monoclonal antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a novel Betacoronavirus that emerged in Wuhan, China, in late 2019. The rapid spread of infection globally led to the WHO declaring it a pandemic by March 2020. This novel coronavirus was initially referred to as 2019-nCoV and later renamed SARS-CoV-2 and disease caused by this virus was termed Coronavirus Disease-2019 (COVID-19) by WHO.
As of Mar. 8 2022, infection with SARS-CoV-2 has resulted in approximately 425 million COVID-19 cases and approximately 6 million deaths worldwide. The global research effort to combat this pandemic has led to rapid advances in preventative and therapeutic modalities. However, the increased incidence of breakthrough infections due to the emergence of SARS-CoV-2 variants, with mutations in their spike (S) glycoprotein, have rendered some existing therapeutics ineffective. In late 2020, a SARS-CoV-2 variant classified as Delta (B.1.617.2 and AY lineages) was identified in India, followed by the Omicron variant (B.1.1.529 and BA lineages) identified in 2021 in South Africa. Both, the Delta and Omicron variants were classified as Variants of Concern (VOC) by the Centers for Disease Control and Prevention due to their increased transmissibility, ability to cause more severe disease, significant reduction in their neutralization by Abs generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, or diagnostic detection failures (https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-classifications.html). This rise in prevalence of SARS-CoV-2 variants underscores the need for effective diagnostic tools for early detection of SARS-CoV-2 infection and for interventions that can prevent and treat SARS-CoV-2 infections.
In one embodiment, this invention includes an isolated antibody (Ab), or antigen (Ag)-binding fragment thereof, capable of binding to a SARS-CoV-2 spike (S) glycoprotein comprising: a heavy chain variable domain (VH) that comprises complementarity determining region (CDR)H1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO.:1 or 11, SEQ ID NO.:2 or 12 and SEQ ID NO.:3 or 13, respectively, and a light chain variable domain (VL) that comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO.: 6 or 16, SEQ ID NO.: 7 or 17, and SEQ ID NO.: 8 or 18, respectively. In another aspect, the isolated Ab or Ag-binding fragment is a human Ab or a human Ag-binding fragment. In another aspect, the Ab or fragment comprises a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4 or 14 and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9 or 19.
In one aspect, the SARS-CoV-2 spike (S) glycoprotein is expressed on a cell surface of a host cell; on a SARS-CoV-2 virion; or both. In another aspect, the Ab or fragment thereof binds to the spike (S) glycoprotein of: (i) a SARS-CoV-2 Wuhan-Hu-1 (GenBank QHD43416.1); (ii) a SARS-CoV-2 B.1.1.7; (iii) a SARS-CoV-2 B.1.351; (iv) a SARS-CoV-2 B.1.1.529; (v) a SARS-CoV-2 comprising any one or more of the following substitution mutations relative to amino acid sequence provided in GenBank QHD43416.1: N501Y; S477N; N439K; L452R; E484K; Y453F; A520S; K417N; K417V; S494P; N501T; 5477R; V367F; P384L; A522S; A522V; V382L; P330S; T478I; S477I; P479S; or (vi) any combination of (i)-(v). In another aspect, the Ab or fragment thereof neutralizes a SARS-CoV-2 infection: (i) in an in vitro model of infection; (ii) in an in vivo animal model of infection; (iii) in a human; or (iv) any combination of (i)-(iii).
In another aspect, the Ab or fragment thereof comprises a monoclonal Ab, a single chain Ab, a Fab, a Fab′, a F(ab′)2, a Fv, a scFv, or a scFab. In another aspect, the Ab or fragment thereof is a bivalent or bispecific Ab and comprises (a) a first target binding site that specifically binds to an epitope within the SARS-CoV-2 spike (S) polypeptide, and (b) a second target binding site that binds to a different epitope on the SARS-CoV-2 spike (S) polypeptide or a different molecule. In yet another aspect, the Ab or fragment thereof is a bivalent or bispecific Ab and comprises (a) a first target binding site that specifically binds the spike (S) glycoprotein of one of (i) a SARS-CoV-2 Wuhan-Hu-1 (GenBank QHD43416.1); (ii) a SARS-CoV-2 B.1.1.7; (iii) a SARS-CoV-2 B.1.351; or (iv) a SARS-CoV-2 comprising any one or more of the following substitution mutations relative to amino acid sequence provided in GenBank QHD43416: 1N501Y; S477N; N439K; L452R; E484K; Y453F; A520S; K417N; K417V; S494P; N501T; 5477R; V367F; P384L; A522S; A522V; V382L; P330S; T478I; S477I; P479S, and (b) a second target binding site that specifically binds the spike (S) glycoprotein of one of (i) a SARS-CoV-2 Wuhan-Hu-1 (GenBank QHD43416.1); (ii) a SARS-CoV-2 B.1.1.7; (iii) a SARS-CoV-2 B.1.351; or (iv) a SARS-CoV-2 comprising any one or more of the following substitution mutations relative to amino acid sequence provided in GenBank QHD43416: N501Y; S477N; N439K; L452R; E484K; Y453F; A520S; K417N; K417V; S494P; N501T; 5477R; V367F; P384L; A522S; A522V; V382L; P330S; T478I; S477I; P479S, not bound by (a).
In one aspect, the Ab or fragment thereof in which (i) the VH comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID NO.: 4 or 14; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID NO.: 9 or 19. In another aspect, the Ab or fragment thereof is a IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgE, or IgD isotype. In yet another aspect, the Ab or fragment thereof, further comprising (a) heavy chain constant regions (CH)1-CH3 that comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 5 or 15 and (b) a light chain constant region (CL) that comprises or consists of the amino acid sequence set forth in SEQ ID NO.:10 or 20.
In one aspect, the Ab or fragment thereof binds to a receptor-binding domain (RBD) in the “up” conformation, wherein the Ab or fragment thereof is able to bind RBD in the “up” conformation and is not able to bind RBD in the “down” conformation. In another aspect, the Ab or fragment thereof binds to a RBD in the “down” conformation, wherein the Ab or fragment thereof is able to bind RBD in the “down” conformation and is not able to bind RBD in the “up” conformation.
In one aspect, the present invention comprises a hybridoma or engineered cell comprising a polynucleotide encoding the Ab or fragment thereof described above or herein.
In one aspect, the present disclosure comprises a nucleic acid molecule encoding the Ab or fragment thereof discussed above or herein. In another aspect, the present disclosure includes a vector comprising the nucleic acid molecule encoding the Ab or fragment thereof discussed above or herein.
In yet another aspect the present disclosure includes a host cell comprising the vector comprising the nucleic acid molecule encoding the Ab or fragment thereof discussed above or herein. In yet another aspect, the present invention describes a method of preparing an Ab, or Ag-binding fragment thereof, comprising: obtaining the host cell described above or herein; culturing the host cell in a medium under conditions permitting expression of the Ab or fragment thereof encoded by the vector described above or herein; and purifying the Ab or fragment thereof from the cultured cell or a medium thereof.
In one aspect, the present disclosure comprises a pharmaceutical composition comprising one or more of the Ab or fragment thereof discussed above or herein and a pharmaceutically acceptable carrier or excipient. In another aspect, the pharmaceutical composition discussed herein further comprises a second therapeutic agent. In yet another aspect, the second therapeutic agent is selected from the group consisting of: an anti-inflammatory agent, or an anti-viral agent.
In one embodiment, this invention includes a method of treating a subject infected with SARS-Cov-2 or preventing SARS-CoV-2 infection in a subject at risk of infection with SARS-CoV-2 comprising administering to a subject in need thereof a therapeutically effective amount of a SARS-CoV-2 neutralizing Ab or Ag-binding fragment. In one aspect, the Ab or Ag-binding fragment capable of binding to a SARS-CoV-2 spike (S) glycoprotein comprising: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4 or 14 or CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO.:1 or 11, SEQ ID NO.:2 or 12 and SEQ ID NO.:3 or 13, respectively; and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9 or 19 or CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO.: 6 or 16, SEQ ID NO.: 7 or 17, and SEQ ID NO.: 8 or 18, respectively. In one aspect, the infected subject is a mammal. In another aspect, the infected subject is human.
In one embodiment, the present disclosure comprises a method of detecting SARS-CoV-2 infection in a subject comprising the steps of: (a) contacting a sample from the subject suspected to be infected with SARS-CoV-2 with an Ab or Ag-binding fragment capable of binding to a SARS-CoV-2 spike (S) glycoprotein; and (b) detecting binding of the Ab or Ab fragment to a SARS-CoV-2 Ag in the sample. In one aspect, the Ab or Ag-binding fragment capable of binding to a SARS-CoV-2 spike (S) glycoprotein comprising: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4 or 14 or CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO.:1 or 11, SEQ ID NO.:2 or 12 and SEQ ID NO.:3 or 13, respectively; and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9 or 19 or CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO.: 6 or 16, SEQ ID NO.: 7 or 17, and SEQ ID NO.: 8 or 18, respectively. In one aspect, the infected subject is a mammal. In another aspect, the infected subject is human.
In one aspect, the sample in which SARS-CoV-2 infection is detected according to the present disclosure is selected from, a nasopharyngeal swab, a nares swab, saliva, urine, tears, cerebrospinal fluid, amniotic fluid, serum, plasma, whole blood, bronchopulmonary lavage, vaginal sampling and a rectal/stool sampling obtained from the subject. In yet another aspect, the SARS-CoV-2 Ag detected in the samples according to the present disclosure comprises a spike (S) glycoprotein of a human or an animal SARS-CoV-2.
In one aspect the Ab or Ab fragment contacting the sample for detection of SARS-CoV-2 infection according to the present disclosure is conjugated to at least one of: a nanoparticle, a liposome, or a detectable label. In yet another aspect, the detectable label conjugated to the Ab or Ab fragment for detection of SARS-CoV-2 infection according to the present disclosure comprises a radioactive tag, a fluorescent tag, a biological, or an enzymatic tag.
In one embodiment, the present disclosure comprises a kit for the detection of SARS-Cov-2 infection in a subject comprising: (a) an Ab or Ag-binding fragment capable of binding to a SARS-CoV-2 spike (S) glycoprotein, (b) a suitable container, and (c) an immunodetection reagent. In one aspect, the Ab or Ag-binding fragment capable of binding to a SARS-CoV-2 spike (S) glycoprotein comprising: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4 or 14 or CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO.:1 or 11, SEQ ID NO.:2 or 12 and SEQ ID NO.:3 or 13, respectively; and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9 or 19 or CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO.: 6 or 16, SEQ ID NO.: 7 or 17, and SEQ ID NO.: 8 or 18, respectively. In one aspect, the subject is a mammal. In another aspect, the subject is human.
In another aspect, the Ab or fragment thereof, comprised in the kit according to the present disclosure and as discussed above or herein, are affixed to a support selected from one or more beads, a dipstick, a filter, a membrane, a plate, a chip, or a column matrix. In yet another aspect, the immunodetection reagent, comprised in the kit according to the present disclosure, comprises at least a second Ab that binds an immunocomplex formed when the Ab or Ab fragment discussed above binds SARS-CoV-2 or SARS-CoV-2 Ag. In yet another aspect, the kit for the detection of SARS-CoV-2 infection in a subject, according to the present disclosure further comprises SARS-CoV-2 or SARS-CoV-2 Ag for use as a standard.
In one aspect, at least one of the Ab or Ab fragment, or the immunodetection reagent, comprised in the kit according to the present disclosure, are linked to a detectable label. In another aspect, the detectable label linked to the Ab or Ab fragment, or the immunodetection reagent, according to the present disclosure, comprises a radioactive tag, a fluorescent tag, a biological, or an enzymatic tag.
For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
The present invention relates to novel mAbs that bind to the SARS-CoV-2 spike (S) glycoprotein. The present invention provides such mAbs and Ab fragments thereof, which are useful for detection or prevention and/or treatment of SARS-CoV-2. The present invention also provides a pharmaceutical composition comprising the novel MAbs or Ab fragments thereof. In addition, the present invention provides a kit and method for detecting SARS-CoV-2 and a method for preventing or treating SARS-CoV-2, using the novel MAbs or Ab fragments thereof as described herein.
SARS-CoV-2 belongs to the Betacoronavirus genus, a group of related RNA viruses that can cause fatal respiratory tract infections. This genus includes the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS), and SARS-CoV-2 [1]. Only four common mildly pathogenic coronaviruses are endemic to humans, including HCoV-0C43, HCoV-HKU1, HCoV-NL63, and HCoV-229E [2]. Both MERS and SARS-CoV are zoonotic pathogens transmitted to humans from intermediate hosts such as dromedary camels and civet cats, respectively [3,4]. While the origin of SARS-CoV-2 is still unclear, bats are the likely to be the reservoir hosts with other mammalian species acting as intermediaries to promote the zoonotic transfer [5].
These coronaviruses are all enveloped viruses with a positive-sense single-stranded RNA genome surrounded by nucleocapsid phosphoproteins. The coronavirus virion is made up of the nucleocapsid (N), membrane (M), envelope (E) and spike (S) proteins, which are structural proteins. The coronavirus S protein consists of two subunits, S1 and S2. S1 subunit contains a receptor-binding domain (RBD) which mediates binding to the receptor on host cell, whereas S2 plays a role in membrane fusion. Angiotensin-converting enzyme 2 (ACE2) was previously shown to be a crucial receptor for SARS-CoV infection in vivo [6], and was rapidly identified as a receptor for SARS-CoV-2. Although several SARS-CoV-2 candidate receptors have been identified in addition to ACE2, whether these receptor interactions are biologically relevant remains to be established. Upon binding of SARS-CoV-2 particles to receptors on target cells, the S protein is cleaved by host proteases leading to fusion of viral and cellular membranes. In the absence of cellular proteases, coronaviruses enter host cells through the endocytic pathways where fusion is induced by low pH and S cleavage by endosomal/lysosomal proteases (cathepsins) [reviewed in refs. 7,8]. Following fusion, the nucleocapsid is released into the cytoplasm and the genomic RNA is translated after dissociation from N. Viral genomes, coated with N protein bud into the lumen of the ER-Golgi intermediate compartment to form enveloped particles containing M, E and S proteins and the progeny virions are finally trafficked to the cell surface for release.
SARS-CoV-2 results in a disease called COVID-19, which involves both pneumonia and acute respiratory distress syndrome (ARDS). COVID-19 can also lead to complications such as acute liver, cardiac and kidney injury, as well as secondary infection and inflammatory response. Upon infection with an RNA virus, the process of innate immune sensing begins with the engagement of pattern recognition receptors (PRRs) by viral RNA. Activation of PRR leads to secretion of anti-viral cytokines such as type I/III interferons (IFNs) and other cytokines such as proinflammatory tumor necrosis factor alpha (TNF-α), and interleukin-1 (IL-1), IL-6, and IL-18. These cytokines can then potentiate an adaptive immune response including activation of T cells and B cells. A cytotoxic CD8+ T cell response typically observed within 7 days of symptoms and peaking at 14 days, correlates with mild disease and while type 1 CD4+ phenotype is associated with an effective viral control, a type 2 profile is often seen in those with severe disease [9]. Activation of B cells lead to antibody (Ab) responses predominantly against S (particularly RBD) and N proteins. The median time to seroconversion for anti-RBD Abs is 11-13 days after the onset of symptoms with decline in anti-RBD IgG observed around 5-8 months [10]. However, the existence and duration of a memory response to SARS-CoV-2 is yet to be clearly established.
In severe COVID-19, the immune system over activated and causes a ‘cytokine storm’ characterized by the release of high levels of cytokines, especially IL-6 and TNF-α, into the circulation, causing a local and systemic inflammatory response [11, 12]. Additionally, there is release of pro-IL-1β, which is cleaved into the active mature IL-1β that mediates lung inflammation and ultimately, fibrosis [13]. Although the respiratory system is the principal target for SARS-CoV-2, it can affect the gastrointestinal tract (GI), hepatobiliary, cardiovascular, renal, and central nervous system. Either one or a combination of mechanisms explains SARS-CoV-2—induced organ dysfunction: direct viral toxicity, ischemic injury caused by vasculitis, thrombosis, or thrombo-inflammation, immune dysregulation, and renin-angiotensin-aldosterone system (RAAS) dysregulation [14]. The median incubation period for SARS-CoV-2 is around 5.1 days, and the majority of patients develop symptoms within 11.5 days of infection [15]. The clinical spectrum of COVID-19 varies from asymptomatic or mildly symptomatic forms to clinical illness characterized by acute respiratory failure requiring mechanical ventilation, septic shock, and multiple organ failure.
Diagnosis: The standard test for diagnosing a SARS-CoV-2 infection is testing a nasopharyngeal swab for SARS-CoV-2 nucleic acid using a real-time PCR assay. The US Food and Drug Administration (FDA) has provided emergency use authorizations (EUAs) for commercial PCR assays validated for the qualitative detection of SARS-CoV-2 nucleic acid in other specimen such as, oropharyngeal swabs, anterior/mid-turbinate nasal swabs, nasopharyngeal aspirates, bronchoalveolar lavage (BAL) and saliva. An antibody test can evaluate for the presence of antibodies that are generated as a result of infection. Antibody tests play an important role in broad-based surveillance of COVID-19, and many commercial manufactured antibody testing kits are available to evaluate the presence of antibodies against SARS-CoV-2 are available. Considering this viral illness commonly manifests itself as pneumonia, radiological imaging has a fundamental role in the diagnostic process, management, and follow-up. Imaging studies may include chest x-ray, lung ultrasound, or chest computed tomography (CT).
Treatment: Several therapeutic options to treat COVID-19 are currently available. These include antiviral drugs (e.g., molnupiravir, paxlovid, remdesivir), anti-SARS-CoV-2 mAbs (e.g., bamlanivimab/etesevimab, casirivimab/imdevimab), anti-inflammatory drugs (e.g., dexamethasone), and immunomodulator agents (e.g., baricitinib, tocilizumab) that are available under FDA issued EUA or are being evaluated in the management of COVID-19 [14]. Antiviral medications and Ab-based treatments are likely to be more effective during the early phase of infection when viral replication is at its peak while anti-inflammatory drugs, immunomodulating therapies, or a combination of these therapies may help combat the hyperinflammatory, prothrombotic state observed in the later phase.
The invention described herein is based, at least in part, on the disclosed anti-SARS-CoV-2 Abs or Ab fragments thereof and their unexpected activity to broadly bind and neutralize SARS-CoV-2. These Abs and Ab fragments constitute a novel strategy in treating and preventing SARS-CoV-2 infections.
As used herein, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally, occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
As used herein, “antibody” or “Antigen-binding fragment” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen (Ag) binding portion that immunospecifically binds a glycoprotein. As such, the term antibody (Ab) encompasses not only whole Ab molecules, but also Ab fragments as well as variants (including derivatives) of Abs and Ab fragments. In natural Abs, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (κ). Five main heavy chain classes (or isotypes) determine the functional activity of an Ab molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000.
The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the Ag. The light and heavy chains of an Ab each have three complementarity determining regions (CDRs), designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3, respectively. An Ag-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain variable region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.
Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.
An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V H domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V H or V L domain. In certain embodiments, an Ab or Ag-binding fragment of the present disclosure specifically binds to a SARS-CoV-2 Ag or to a SARS-CoV-2 spike (S) glycoprotein or polypeptide.
As used herein, the term “Antigen” (Ag), as used herein, refers to an immunogenic molecule that provokes an immune response. This immune response may involve Ab production, activation of specific immunologically-competent cells, activation of complement, Ab-dependent cytotoxicicity, or any combination thereof. An antigen (Ag-immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like. It is readily apparent that an Ag can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more Ags include tissue samples, stool samples, cells, biological fluids, or combinations thereof. Ags can be produced by cells that have been modified or genetically engineered to express an Ag. Ags can also be present in a SARS-CoV-2 (e.g., a surface or a spike glycoprotein or portion thereof), such as present in a virion, or expressed or presented on the surface of a cell infected by SARS-CoV-2.
As used herein, “anti-inflammatory agent” includes any therapeutic drug or substance that reduces inflammation (redness, swelling, and pain) in the body. The one or more anti-inflammatory agents for use in a combination therapy of the present disclosure comprises a corticosteroid such as, for example, dexamethasone, prednisone, or the like. In some embodiments, the one or more anti-inflammatory agents comprise a cytokine antagonist such as, for example, an antibody that binds to IL6 (such as siltuximab), or to IL-6R (such as tocilizumab), or to IL-1β, IL-7, IL-8, IL-9, IL-10, FGF, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1A, MIP1-B, PDGR, TNF-α, or VEGF. In some embodiments, anti-inflammatory agents such as ruxolitinib and/or anakinra are used. Anti-inflammatory agents for use in a combination therapy of the present disclosure also include non-steroidal anti-inflammatory drugs (NSAIDS).
As used herein, “anti-viral agent” includes any drug or substance to treat a viral infection. The one or more anti-viral agents for use in a combination therapy of the present disclosure comprises nucleotide analogs or nucelotide analog prodrugs such as, for example, remdesivir, sofosbuvir, acyclovir, and zidovudine. The anti-viral agent also includes lopinavir, ritonavir, favipiravir, or any combination thereof. In certain embodiments, the anti-viral agent administered with the Ab or Ag-binding fragment of the present disclosure comprises leronlimab.
As used herein, the phrase “bispecific antibody” or “bivalent antibody” includes an antibody (Ab) capable of selectively binding two or more epitopes. Bispecific Abs include fragments of two different monoclonal Abs and generally comprise two nonidentical heavy chains derived from the two different monoclonal Abs, with each heavy chain specifically binding a different epitope—either on two different molecules (e.g., different epitopes on two different immunogens) or on the same molecule. Bispecific Abs can be made, for example, by combining heavy chains that recognize different epitopes of the same or different immunogen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same or different immunogen can be fused to nucleic acid sequences encoding the same or different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain. A typical bispecific Ab has two heavy chains each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer epitope-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain epitope-binding regions, or that can associate with each heavy chain and enable binding or one or both of the heavy chains to one or both epitopes.
As used herein, “epitope” or “antigenic epitope” includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Where an Ag comprises a peptide or protein, the epitope can be comprised of consecutive amino acids (e.g., a linear epitope), or can be comprised of amino acids from different parts or regions of the protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope), or non-contiguous amino acids that are in close proximity irrespective of protein folding.
As used herein, the term “host” refers to a cell or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., an antibody of the present disclosure). A host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989).
As used herein, the term “Fab” (fragment antigen binding) refers to the part of an Ab that binds to Ag and includes the variable region and CH1 of the heavy chain linked to the light chain via an inter-chain disulfide bond. Each Fab fragment is monovalent with respect to Ag binding, i.e., it has a single Ag-binding site. Pepsin treatment of an Ab yields a single large F(ab′)2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent Ag-binding activity and is still capable of cross-linking Ag. Both the Fab and F(ab′)2 are examples of “Ag-binding fragments.” Fab′ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the Ab hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 Ab fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of Ab fragments are also known. Fab fragments may be joined, e.g., by a peptide linker, to form a single chain Fab, also referred to herein as “scFab.” In these embodiments, an inter-chain disulfide bond that is present in a native Fab may not be present, and the linker serves in full or in part to link or connect the Fab fragments in a single polypeptide chain.
As used herein, the term “Fv” is a small Ab fragment that contains a complete Ag-recognition and Ag-binding site. This fragment generally consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an Ag) has the ability to recognize and bind Ag, although typically at a lower affinity than the entire binding site.
As used herein, the term “Single-chain Fv” also abbreviated as “sFv” or “scFv”, are Ab fragments that comprise the VH and VL Ab domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide comprises a polypeptide linker disposed between and linking the VH and VL domains that enables the scFv to retain or form the desired structure for Ag binding. Such a peptide linker can be incorporated into a fusion polypeptide using standard techniques well known in the art. Additionally or alternatively, Fv can have a disulfide bond formed between and stabilizing the VH and the VL. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra. In certain embodiments, the Ab or Ag-binding fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain. In particular embodiments, a scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH-linker-VL orientation or in a VL-linker-VH orientation. Any scFv of the present disclosure may be engineered so that the C-terminal end of the VL domain is linked by a short peptide sequence to the N-terminal end of the VH domain, or vice versa (i.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C). Alternatively, in some embodiments, a linker may be linked to an N-terminal portion or end of the VH domain, the VL domain, or both. scFv can be constructed using any combination of the VH and VL sequences or any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences disclosed herein.
As used herein, a “monoclonal antibody” refers to an Ab obtained from a population of substantially homogeneous Abs, e.g., the individual Abs comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. In contrast to polyclonal Ab preparations, which typically include different Abs directed against different determinants (epitopes), each monoclonal Ab is directed against a single determinant on the Ag (epitope). The modifier “monoclonal” indicates the character of the Ab as being obtained from a substantially homogeneous population of Abs, and is not to be construed as requiring production of the Ab by any particular method. For example, the monoclonal Abs to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal Abs may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature, 348:552-554, for example. Other methods are known in the art and are contemplated for use herein.
As used herein, “neutralizing antibody” is an Ab that can neutralize, i.e., prevent, inhibit, reduce, impede, or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms “neutralizing Ab” and “an Ab that neutralizes” or “Abs that neutralize” are used interchangeably herein. In any of the presently disclosed embodiments, the Ab or Ag-binding fragment is capable of preventing and/or neutralizing a SARS-CoV-2 infection in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human.
As used herein, the term “Nucleic acid molecule” or “polynucleotide” or “polynucleic acid” refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), which includes mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense) strand. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.
Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68° C. or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42° C. Nucleic acid molecule variants retain the capacity to encode a binding domain thereof having a functionality described herein, such as binding a target molecule.
As used herein, the term “percent sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. “Default values” mean any set of values or parameters which originally load with the software when first initialized.
As used herein, the term “pharmaceutical composition” comprises the combination of an active agent, such as any one or more of the presently disclosed Abs, Ag-binding fragments, polynucleotides, peptides, vectors, or host cells, singly or in any combination, and can further comprise a pharmaceutically acceptable carrier, excipient, or diluent, inert or active, in a sterile composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. Carriers, excipients, and diluents are discussed in further detail herein. Such compositions comprising the disclosed Ab, Ag-binding fragment, polynucleotide, vector, host cell, peptides, or composition of the present disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents such as anti-viral or anti-inflammatory agents.
As used herein, the term “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. This further includes materials from such compounds that are appropriate for use in pharmaceutical contexts, i.e., materials which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).
As used herein, the term “protein” or “polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and non-naturally occurring amino acid polymers. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated. In certain embodiments, variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.
As used herein, the term “SARS-CoV-2”, also referred to herein as “Wuhan coronavirus”, or “Wuhan seafood market pneumonia virus”, or “Wuhan CoV”, or “novel CoV”, or “nCoV”, or “2019 nCoV”, or “Wuhan nCoV” is a betacoronavirus believed to be of lineage B (sarbecovirus). SARS-CoV-2 was first identified in Wuhan, Hubei province, China, in late 2019 and spread within China and to other parts of the world by early 2020. Symptoms of SARS-CoV-2 include fever, dry cough, and dyspnea. The genomic sequence of SARS-CoV-2 isolate Wuhan-Hu-1 is provided in GenBank MN908947.3 and the amino acid translation of the genome is provided in GenBank QHD43416.1. Like other coronaviruses (e.g., SARS CoV), SARS-CoV-2 comprises a “spike” or surface (“S”) type I transmembrane glycoprotein containing a receptor binding domain (RBD). RBD is believed to mediate entry of the lineage B SARS coronavirus to respiratory epithelial cells by binding to the cell surface receptor angiotensin-converting enzyme 2 (ACE2). In particular, a receptor binding motif (RBM) in the virus RBD is believed to interact with ACE2. Ab and Ag-binding fragments of the present disclosure are capable of binding to a SARS CoV-2 surface glycoprotein (S), such as that of Wuhan-Hu-1. For example, in certain embodiments, an Ab or Ag-binding fragment binds to an epitope in Wuhan-Hu-1 S protein RBD. SARS-CoV-2 Wuhan-Hu-1 S protein has approximately 73% amino acid sequence identity with SARS-CoV S protein. SARS-CoV-2 RBD has approximately 75% to 77% amino acid sequence similarity to SARS coronavirus RBD, and SARS-CoV-2 Wuhan Hu-1 RBM has approximately 50% amino acid sequence similarity to SARS coronavirus RBM. Unless otherwise indicated herein, SARS-CoV-2 Wuhan Hu-1 refers to a virus comprising the amino acid sequence set forth in GenBank QHD43416.1, optionally with the genomic sequence set forth in GenBank MN908947.3. There have been a number of emerging SARS-CoV-2 variants. Some SARS-CoV-2 variants contain an N439K mutation, which has enhanced binding affinity to the human ACE2 receptor [16]. Some SARS-CoV-2 variants contain an N501Y mutation, which is associated with increased transmissibility, including the lineages B.1.1.7 (also known as 20I/501Y.Vi1 and VOC 202012/01; (de169-70, de1144, N501Y, A570D, D614G, P681H, T7161, S982A, and D1118H mutations)) and B.1.351 (also known as 20H/501Y.V2; L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G, and A701V mutations), which were discovered in the United Kingdom and South Africa, respectively [17, 18]. B.1.351 also include two other mutations in the RBD domain of SARS-CoV2 spike protein, K417N and E484K [17]. Other SARS-CoV-2 variants include the Lineage B.1.1.28, which was first reported in Brazil; the Variant P.1, lineage B.1.1.28 (also known as 20J/501Y.V3), which was first reported in Japan; Variant L452R, which was first reported in California in the United States (Pan American Health Organization, Epidemiological update: Occurrence of variants of SARS-CoV-2 in the Americas, Jan. 20, 2021, available at reliefweb.int/sites/reliefweb.int/files/resources/2021-jan-20-phe-epi-update-SARS-CoV-2.pdf). Other SARS-CoV-2 variants include a SARS CoV-2 of Glade 19A; SARS CoV-2 of Glade 19B; a SARS CoV-2 of Glade 20A; a SARS CoV-2 of Glade 20B; a SARS CoV-2 of Glade 20C; a SARS CoV-2 of Glade 20D; a SARS CoV-2 of Glade 20E (EU1); a SARS CoV-2 of Glade 20F; a SARS CoV-2 of Glade 20G; and SARS CoV-2 B1.1.207; and other SARS CoV-2 lineages [19]. Another variant of SARS-CoV-2, B.1.1.529 (Omicron) was first reported to the World Health Organization (WHO) by South Africa on Nov. 24, 2021 [20]. The foregoing SARS-CoV-2 variants, and the amino acid and nucleotide sequences thereof, are incorporated herein by reference.
As used herein, the term “specifically binds” refers to an association or union of an antibody or antigen-binding fragment to an antigen with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 105 M−1 (which equals the ratio of the on-rate [Kon] to the off rate [Koff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Alternatively, affinity may be defined as an equilibrium dissociation constant (K d) of a particular binding interaction with units of M (e.g., 10−5 M to 10−13 M). Antibodies may be classified as “high-affinity” antibodies or as “low-affinity” antibodies. “High-affinity” antibodies refer to those antibodies having a Ka of at least 107 M−1, at least 108 M−1, at least 109 M−1, at least 1010 M−1, at least 1011 M−1, at least 1012 M−1, or at least 1013 M−1. “Low-affinity” antibodies refer to those antibodies having a Ka of up to 107 M−1, up to 106 M−1, up to 105 M−1. Alternatively, affinity may be defined as an equilibrium dissociation constant (K a) of a particular binding interaction with units of M (e.g., 10−5 M to 10−13 M).
In some contexts, Ab and Ag-binding fragments may be described with reference to affinity and/or to avidity for antigen. Unless otherwise indicated, avidity refers to the total binding strength of an Ab or Ag-binding fragment thereof to Ag, and reflects binding affinity, valency of the Ab or antigen-binding fragment (e.g., whether the antibody or antigen-binding fragment comprises one, two, three, four, five, six, seven, eight, nine, ten, or more binding sites), and, for example, whether another agent is present that can affect the binding (e.g., a non-competitive inhibitor of the antibody or antigen-binding fragment).
As used herein, the phrases “therapeutically effective amount” and “therapeutic level” mean that drug dosage or plasma concentration in a subject, respectively, that provides the specific pharmacological effect for which the drug is administered in a subject in need of such treatment, i.e., to reduce, ameliorate, or eliminate the symptoms or effects of SARS-CoV-2 infection. It is emphasized that a therapeutically effective amount or therapeutic amount of an antibody will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the subject's condition, including the SARS-CoV-2 variant and severity of the infection, among other factors. The human subject treated according to the present disclosure include an infant, a child, a young adult, an adult of middle age, or an elderly person. The human subject treated according to the present disclosure include those less than 1 year old, or those 1 to 5 years old, or those between 5 and 125 years old (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115, or 125 years old, including any and all ages therein or there between). The human subject treated includes male and female.
As used herein, the term “treat”, “treating”, and “treatment” refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration of an Ab to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate, partially or completely alleviate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition or to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
As used herein, the term “vector” or “expression vector” refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself or deliver the polynucleotide contained in the vector into the genome without the vector sequence. In the present specification, “plasmid,” “expression plasmid,” “virus,” and “vector” are often used interchangeably.
General Methods for monoclonal antibody production: It will be understood that monoclonal Abs binding to SARS-CoV-2 will have utility in several applications. These include the production of diagnostic kits for use in detecting and diagnosing disease. In these contexts, one may link such Abs to diagnostic or therapeutic agents, or use them as capture agents or competitors in competitive assays. Means for preparing and characterizing Abs are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Pat. No. 4,196,265).
The methods for generating monoclonal Abs (MAbs) generally begin along the same lines as those for preparing polyclonal Abs. The first step for both these methods is immunization of an appropriate host or identification of subjects who are immune due to prior natural infection. In the case of human monoclonal Abs, one may instead simply look for an individual already known to have generated an immune response, in this case, to have been exposed to SARS-CoV-2 or immunized with vaccines to prevent SARS-COV-2, such as COMIRNATY®, Spikevax™, or others.
Monoclonal Abs useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal Abs may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example. Monoclonal Abs may also be obtained using methods disclosed in PCT Publication No. WO 2004/076677A2.
It also is contemplated that a molecular cloning approach may be used to generate monoclonal Abs. For this, RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.
Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present invention include: U.S. Pat. No. 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 which describes recombinant immunoglobulin preparations; U.S. Pat. No. 4,867,973 which describes antibody-therapeutic agent conjugates; and U.S. Pat. No. 8,563,305 which describes methods for rapidly producing monoclonal antibodies.
Included in this disclosure is a method of antibody generation from peripheral blood samples obtained from healthy donors immunized with two doses of the Pfizer (COMIRNATY®) vaccine. Also included in this disclosure is a method of antibody generation from peripheral blood samples collected from donors with history of COVID-19 infection who were later immunized with two doses of the Pfizer (COMIRNATY®) vaccine. The disclosure further includes the method wherein, blood samples were collected 7 days after the second vaccine dose followed by purification of peripheral blood mononuclear cells (PBMCs) and IgG-positive antibody-secreting cell isolation. One example of the method for generating human monoclonal antibodies from such samples is described below.
Lymphoprep and B-Cell Enrichment
Staining and Flow Cytometry
Reverse Transcription, Nested and Cloning PCRs
PCR Purification
Digestion Purification
Second Digestion
Gel Purification
Ligation
Transformation of DH5a Cells
Miniprep
Maxiprep
Transfection of 293A Cells
Protein Purification
Protein Quantification
Gel Confirmation of Protein Quality
Reagents for Steps 1-80
Ig gamma, Ig kappa and Ig lambda expression vectors: The expression vectors contain a murine immunoglobulin signal peptide sequence and variable-gene cloning sites upstream of the appropriate human immunoglobulin constant regions followed by an SV40 polyadenylation sequence. Transcription is under the HCMV (human cytomegalovirus immediate—early) promoter and clones are selected based on ampicillin resistance. The antibody variable-heavy and variable-light rearranged genes from each single cell are cloned into the respective vectors in frame with the signal peptide and constant region genes. These vectors are then co-transfected into the 293A cell line for expression. The resultant antibodies are properly trafficked and secreted after cleavage of the signal peptide, resulting in fully human IgG/kappa or IgG/lambda amino acid sequences. The vector sequences are available through the NCBI GenBank (accession numbers: FJ475055, FJ475056 and FJ517647).
Basal media: An aliquot of 250 ml each of sterile RPMI and DMEM; 3.75 ml of antibiotic/antimycotic and 5 ml each of L-glutamine (200 mM), 100× Nutridoma and sodium pyruvate (100 mM) was used. Basal media must be made fresh every 7 d. L-Glutamine can be stored at −20° C. for up to 1 year, Nutridoma can be stored at room temperature (20-25° C.) for up to 1 year and sodium pyruvate can be stored for up to 6 months at 4° C.
0.1 M glycine-HCl: Here 0.1 M glycine solution is equilibrated to pH 2.7 with 12 M HCl and filter sterilized. Solution can be stored up to 60 d at room temperature.
1M Tris-HCl: Here 1M Tris solution is equilibrated to pH 9.0 with HCl and filter sterilized. Solution can be stored up to 60 d at 4° C.
ACK lysing buffer: Here 0.15 M NH4Cl, 10 mM KHCO3 and 0.1 mM Na2 EDTA. Adjust pH to 7.2-7.4 with 1M HCl and filter sterilized. Solution can be stored up to 1 year at room temperature (20-25° C.).
LB agar plates: LB agar dissolved in dH2O according to package directions and autoclaved. When cooled to 45° C., 50 μg/ml ampicillin is added. Dispense 20-25 ml agar solution into 100 mm×15 mm petri dishes. Cool and store at 4° C. for up to 6 months.
AEC substrate: Prepare AEC stock (20 mg/ml AEC in dimethylformamide). Dilute AEC from stock to 0.3 mg/ml in 0.1 M sodium acetate buffer (pH 5.0) just prior to use. Filter sterilized with a syringe filter. The stock solution may be made and stored for up to 2 months. The diluted solution must be made fresh each time used.
RNAse-inhibiting RT-PCR catch buffer: To 5 ml of RNAse-free water, add 50 μl of 1M Tris pH 8.0 and 125 μl of Rnasin. Keep on ice. This makes enough for 10 half plates. Catch buffer must be made fresh each time used.
PEI solution: prepare a 1 mg/ml PEI solution in 100 mL dH2O. Heat to 80° C. (do not boil) to allow all of the PEI to dissolve, then allow to cool. Adjust pH to 7.2 with HCl. Filter sterilize with a syringe filter. Store aliquots at −20° C. for up to 1 year.
To test the binding specificity and affinity of the antibodies p2C01 and p3C03, binding curves were generated as described herein. Wells were coated with 200 ng of Wuhan or variant 51 or RBD blocked with 0.1% BSA in PBS, and developed with anti-human IgG-HRP (Jackson ImmunoResearch, West Grove, PA) and Super Aqua Blue substrate (EBiosciences, San Diego CA). The absorbance was measured at 405 nm on a microplate reader (Molecular Devices, Sunnyvale, CA). Antibody affinities (Kd) were calculated by curve fitting analysis of individual ELISA curves plotted from a dilution series of 16 two-fold dilutions of antibody beginning at 10 μg/ml. Binding curves were generated with a saturation binding, non-linear curve fit using GraphPad Prism software. Equilibrium dissociation constants (Kd) values for each hmAb were calculated using the equation Y=Bmax*X/(Kd+X) where Bmax is the maximum number of binding sites, X is the concentration of the antibody and Y is the specific binding. Therefore, the reported dissociation constants are equal to the concentration of antibody where half the binding sites are occupied at equilibrium. Each antibody was run in duplicate in at least three unique experiments. The binding of Ab to various SARS-CoV-2 strains such as Wuhan, Epsilon (B.1.427 and B.1.429), Gamma (P.1), Alpha (B1.1.7), Beta (B.1.351), Delta (B.1.617.2.1) and Omicron (B.1.1.529) and SARS-CoV was examined. The results for each experiment were then averaged to obtain the reported Kd in Table 4.
To test the neutralization capacity of the antibodies generated, pseudovirus/lentiviral neutralization assays were conducted using commercial S-pseudotyped lentivirus (BPS Biosciences) in a 96-well format. The virus was incubated with the monoclonal antibodies samples of interest (15 two-fold dilutions) for an hour at 37° C. The virus/antibody mixture was then added to HEK-293T-hACE2 cells for 60-72 hours. Luminescence was measured using the One-Step Luciferase Assay System (BPS Biosciences) on a luminescence plate reader. Each sample was run in duplicate, the values for each dilution were averaged and IC50's determined using a 4-parameter/sigmoidal curve fit (
To further assess SARS-CoV-2 neutralization, each antibody was serially diluted 1:2 across a 96-well plate and mixed with enough virus (isolate USA-WA1/2020) to yield a final MOI of 0.01. After incubating the antibody virus mixture for 1 h at 37° C., the virus mixture was transferred to a 96-well plate containing VERO E6 cells seeded at 10,000 cells per well. SARS-CoV-2 activity was then determined 96 h after infection by visually observing cytopathic effects (CPE). The antibody dilution at which virus positive wells was observed was recorded as shown in
The 810p2C01 heavy and light chain nucleotide and protein sequences are as follows:
The 810p3C03 heavy and light chain protein and nucleotide sequences are as follows:
Additional oligonucleotides for use with the invention are listed as SEQ ID NOS: 73-100, which include the restriction enzyme cleavage site and the location on the antibody, as follows:
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the phrase “consisting essentially of” requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method/process steps or limitation(s)) only.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 12 or 15%.
Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.
For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.
This application claims priority to U.S. Provisional Application Ser. No. 63/356,682, filed Jun. 29, 2022, the entire contents of which are incorporated herein by reference.
This invention was made with government support under U19AI062629-1752 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63356682 | Jun 2022 | US |
