Patent Grant
12115219
All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
In particular U.S. patent application Ser. No. 14/438,511, filed Apr. 24, 2015, claiming priority under 35 U.S. § 371 to PCT/US2013/067328, filed Oct. 29, 2013, titled “Compositions and Methods for Inhibiting Pathogen Infection”, claiming priority to U.S. provisional patent application No. 61/719,689, filed on Oct. 29, 2012 is herein incorporated by reference in its entirety.
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 5470-843_ST25.txt, 13,552 bytes in size, generated on Sep. 21, 2020 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.
The presently-disclosed subject matter relates to antibodies, compositions, and methods for inhibiting and treating pathogen infection and providing contraception. In particular, the presently-disclosed subject matter relates to inhibiting and treating pathogen infection and providing contraception in a subject using compositions and antibodies that trap pathogens in mucus, thereby inhibiting transport of pathogens or sperm across or through mucus secretions. The subject matter further relates to methods for monitoring the effectiveness of vaccines by detecting antibodies capable of trapping pathogens in mucus. For example, the presently-disclosed subject matter relates to antibodies, compositions, and methods for inhibiting and treating virus infection in the respiratory tract and virus transmission through the respiratory tract, including using compositions and antibodies that trap viruses in mucus of the respiratory tract, thereby inhibiting transport of virus across or through mucus secretions.
Large quantities of IgG are transported into female genital tract mucus secretions by the MHC class I-related neonatal Fc receptor (Li et al., Proc. Natl. Acad. Sci. U.S.A. 108:4388 (2011), resulting in at least ten-fold more IgG than IgA (Usala et al., J. Reprod. Med. 34:292 (1989)). However, despite this predominance of IgG, the precise mechanism(s) by which secreted IgG can prevent vaginal infections are not well understood. Previous animal studies have shown that antibodies (Ab) can provide robust protection against vaginal challenge with pathogens, including human immunodeficiency virus (HIV) and herpes simplex virus-2 (HSV-2), when applied intravaginally (Burton et al., Proc. Natl. Acad. Sci. U.S.A. 108:11181 (2011); Sherwood et al., Nature Biotechnol. 14:468 (1996); Veazey et al., Nature Med. 9:343 (2003); Whaley et al., J. Infect. Dis, 169:647 (1994)) or even intravenously (Hessell et al., PLoS Pathogens 5:e1000433 (2009); Mascola et al., Nature Med. 6:207 (2000)). Many investigators have focused on neutralizing Ab, which, at sufficiently high doses, provided sterilizing immunity against simian-human immunodeficiency virus (SHIV) challenge in rhesus macaques (Burton et al., Proc. Natl. Acad. Sci. U.S.A. 108:11181 (2011)). In the same studies, non-neutralizing or poorly neutralizing Ab provided at best only partial protection, with some infected animals exhibiting reduced viral load. Studies by Hessell et al. and Mascola et al. showed complete protection in some animals even by weakly neutralizing antibodies, as well as reduced viremia in others (Hessell et al., PLoS Pathogens 5:e1000433 (2009); Mascola et al., Nature Med. 6:207 (2000)).
Few studies have explored the potential protective role of IgG within the mucus secretions overlaying the epithelial tissue, which sexually transmitted viruses invariably encounter and must penetrate in order to reach target cells. Well-known Ab effector functions in blood and lymph (e.g., complement activation, opsonization, and ADCC) are absent or limited in healthy female genital secretions, which typically have little complement activity and few if any active leukocytes (Cone, In Handbook of Mucosal Immunology. P. L. Ogra et al., editors. Academic Press, San Diego, Calif. 43-64 (1999); Hill et al., Am. J. Obstet. Gynecol. 166:720 (1992); Schumacher, Hum. Reprod. 3:289 (1988)). These classical mechanisms of systemic immune protection do not adequately account for the moderate but significant protection observed in the landmark Thai RV144 HIV vaccine trial (Kresge, IAVI Report Vol 13, Number 5 (2009); Rerks-Ngarm et al., N. Engl. J. Med. 361:2209 (2009)). The vaccination regimen modestly reduced the risk of HIV acquisition despite inducing primarily non-neutralizing Ab and otherwise offering little to no protection against systemic progression of infections once acquired, suggesting that protection likely occurred prior to initiation of infection. A better understanding of potential additional mechanisms of vaginal mucosal immunity will also likely be critical for developing effective vaccines against other sexually transmitted infections, including HSV, which has been shown to evade complement and other classical antibody-mediated protective mechanisms (Brockman et al., Vaccine 26 Suppl 8:194 (2008); Hook et al., J. Virol. 80:4038 (2006); Lubinski et al., J. Exp. Med. 190:1637 (1999)). To date, herpes vaccine candidates have achieved only transient and partial protection despite inducing high neutralizing serum antibody titers and cellular immunity (Chentoufi et al., Clin. Dev. Immunol. 2012:187585 (2012)). A Phase III clinical trial, using a subunit vaccine based on HSV-2 glycoprotein D, demonstrated some protection against HSV-1 infection but no efficacy against HSV-2 infection and no overall efficacy against genital disease (Belshe et al., New Eng. J. Med. 366:34 (2012)).
There is a need in the art for new compositions, and methods of using such compositions, to prevent and treat infectious diseases and provide contraception.
For example, respiratory infections, such as influenza virus and respiratory syncytial virus (RSV) infections, are a tremendous health and financial burden. Additionally, the mucosal lining of the respiratory tract is a point of entry for non-respiratory viruses such as Ebola virus. Ideally, viral infection in or through the lungs would be prevented by transporting virions that enter the respiratory tract out of the tract before they have a chance to contact cells.
Thus, there is a need for new compositions, and methods of using such compositions, to prevent and treat viral infectious diseases, including in particular diseases in the respiratory tract.
Viruses must penetrate mucus to reach and infect their target cells; indeed, HIV and human papillomavirus (HPV) are both capable of rapidly diffusing through human genital mucus secretions (Lai et al., J. Virol. 83:11196 (2009); Olmsted et al., Biophys. J. 81:1930 (2001)). It was previously found that the diffusion of IgG (11 nm) was slowed slightly in human cervical mucus compared to saline buffer, while much larger virus-like particles, including the capsids of norovirus (38 nm) and HPV (55 nm), were not slowed by this mucus (Olmsted et al., Biophys. J. 81:1930 (2001)). It was hypothesized that the slight retardation of the much smaller IgG molecules may be due to very transient (<1 s), low-affinity bonds with the mucin mesh (Olmsted et al., Biophys. J. 81:1930 (2001)). In other words, by making only transient low-affinity bonds with mucins, IgG is able to diffuse rapidly through mucus, and in theory is therefore free to quickly accumulate on a pathogen surface.
Surprisingly, as described herein, Ab bound to a pathogen surface can effectively trap the pathogen in mucus gel by ensuring at least some low-affinity bonds to the mucin mesh are present at any given time (illustrated in
Thus, one aspect of the methods and compositions described herein may relate to an isolated antibody comprising an oligosaccharide at a glycosylation site, the oligosaccharide comprising a glycosylation pattern that enhances trapping potency of the antibody in mucus, wherein the antibody specifically binds an epitope of a target pathogen. In general, these methods and compositions may include trapping antibodies directed against a target viron in mucus (e.g., within the lungs, vagina, etc.).
A further aspect may relate to compositions, e.g., pharmaceutical compositions, and kits comprising the antibodies of the invention.
An additional aspect of the invention relates to a method of inhibiting an infection by a pathogen or a disease or disorder caused by an infection by a pathogen in a subject in need thereof, comprising administering to a mucosa of the subject an antibody (e.g., an antibody that is adapted to have an enhanced trapping potency in mucus) in an amount effective to inhibit an infection.
Another aspect of the invention relates to a method of treating an infection by a pathogen or a disease or disorder caused by an infection by a pathogen in a subject in need thereof, comprising administering to a mucosa of the subject the antibody of the invention in an amount effective to treat the infection.
An additional aspect of the invention relates to a method of monitoring the effectiveness of a vaccination in a subject that has been vaccinated against a target pathogen, comprising determining in a mucus sample from said subject the amount of an antibody comprising an oligosaccharide at a glycosylation site, the oligosaccharide having a glycosylation pattern that enhances trapping potency of the antibody in mucus, wherein the antibody specifically binds an epitope of said target pathogen.
Typically, viruses must penetrate the mucus of the respiratory tract to reach and infect their target cells in the epithelium. The present invention is based in part on the finding that antibodies in the respiratory tract can bind to virions and effectively trap the virions in mucus gel by ensuring at least some low-affinity bonds to the mucin mesh are present at any given time. Virions trapped in the mucus cannot reach their target cells, and will instead be shed from the respiratory tract with the mucus. This provides novel methods for preventing and treating infection in the respiratory tract.
Thus, one aspect of the invention relates to method for treating or preventing a viral infection in a subject in need thereof, the viral infection characterized by a virion in the lung of the subject, the method comprising administering, via an inhaled route, an antibody, e.g., a recombinant monoclonal antibody molecule, comprising a human Fc portion and a set of CDRs with specific affinity for the virion, thereby treating or preventing the infection. The antibody may be delivered in an aerosol composition, e.g., by nebulizer.
A further aspect of the invention relates to compositions comprising an antibody, e.g., a recombinant monoclonal antibody molecule, comprising a human Fc portion and a set of CDRs with specific affinity for a virion present in the lung of a subject, wherein the composition is suitable for inhalation.
An additional aspect of the invention relates to a kit comprising the compositions of the invention.
For example, described herein are methods for treating or preventing an infection by a pathogen (e.g., treating and/or preventing a viral infection by a virion, treating and/or preventing a bacterial infection, treating and/or preventing a fungal infection) in a subject in need thereof. For example, a method for treating and/or preventing a viral infection by a virion in a subject in need thereof may include: administering to the subject, via an inhaled route, a recombinant antibody with a specific affinity for the virion, the recombinant antibody comprising a human or humanized Fc region, wherein the recombinant antibody comprises an oligosaccharide having a glycosylation pattern that enhances the trapping potency of the recombinant antibody in mucus, so that the recombinant antibody binds to the virion to form an antibody/virion complex that is trapped in the subject's mucus thereby treating or preventing the infection.
The administration of the mAb may refer to administering a population of mAbs that has been enriched for mAbs having a glycosylation pattern that enhances mucosal trapping.
The recombinant antibody may, when not bound to target (e.g., virion) be weakly interacting with mucus and may therefore freely diffuse through the mucus; however, once bound to the target (e.g., virion) to form an antibody/viron complex, it may trap the antibody/virion complex within the mucus, allowing it to be removed and/or destroyed by the body. The concentration of antibody in the mucus (and particularly the concentration of mAb having an enhanced trapping efficiency, such as having a G0/G0F (i.e. GnGn) glycosylation pattern) may be, e.g., 50 ng/mL or greater, (e.g., 100 ng/mL or greater, 200 ng/mL or greater, 500 ng/mL or greater, 1 μg/mL or greater, 10 μg/mL or greater, 12.5 μg/mL or greater, 15.0 μg/mL or greater, 20 μg/mL or greater, etc., e.g., between 0.1 μg/mL and 20 μg/mL).
In general, recombinant antibodies having an increased mucosal trapping potency may comprise an N-linked glycosylation site on the Fc region of the antibodies to which the oligosaccharide is attached. For example, the glycosylation pattern may comprise a biantennary core glycan structure of Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminal N-acetylglucosamine on each branch. This glycosylation pattern may be with or without fucose, and/or with or without a bisecting GlcNAc.
In some variations at least 20% (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, etc., and particularly 50% or more, e.g., 55% or greater, 60% or greater, 65% or greater, between 80-100%, etc.) of the recombinant antibodies in the population administered to the patient have a glycosylation pattern comprising the biantennary core glycan structure Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminal N-acetylglucosamine on each branch. For example, at least 50% of the recombinant antibodies in the population have a glycosylation pattern comprising the biantennary core glycan structure Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminal N-acetylglucosamine on each branch. As mentioned, this glycosylation pattern may be with or without fucose, and/or with or without a bisecting GlcNAc.
As mentioned, any of these methods may include forming an antibody/virion complex and trapping an antibody/virion complex in the mucus. The recombinant antibody may be configured to bind to a non-neutralizing epitope of the virion. Administering may comprise administering a dose at a sub-neutralization dose level.
Any of these methods may include reducing the mobility of the virion in the patient's mucus to no more than about 50% relative to its mobility in water. For example, any of these methods may include reducing the percentage of virion that can penetrate the patient's mucus by at least 10% (e.g., at least 15%, at least 20%, at least 25%, etc.).
The recombinant antibody may be a human or humanized IgG or IgM monoclonal antibody, or a fragment or derivative thereof.
The recombinant antibody may be formulated as an aerosol composition. For example, the recombinant antibody may be formulated to have a neutral pH (e.g., approximately neutral pH); in some variations the recombinant antibody may be formulated as a hypotonic solution.
Administering may comprise administering a nebulized composition of recombinant antibody having a Mass Median Aerodynamic Diameter (MMAD) in the 2-5 μm range, as measured using a Next Generation Impactor.
Appropriate dosing regimens may include dosing the subject with the recombinant antibody molecule via the inhaled route once every 3 to 24 hours. The dosing regimen may include dosing between about 5-100 mg/day (e.g., between about 15-50 mg/day, between about 5-200 mg/day, between about 15-200 mg/day, between about 5-150 mg/day, between about 15-150 mg/day, between about 5-100 mg/day, between about 15-100 mg/day, etc.). In some variations the dosing may be configured to maintain a sustained concentration in the mucus (e.g., for approximately at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, etc.) of between about 0.5-1000 μg/mL in the mucus (e.g., 1 μg/mL or greater, 10 μg/mL or greater, 15 μg/mL or greater, 20 μg/mL or greater, 30 μg/mL or greater, 40 μg/mL or greater, 50 μg/mL or greater, 60 μg/mL or greater, 70 μg/mL or greater, 80 μg/mL or greater, 19 μg/mL or greater, 100 μg/mL or greater, 120 μg/mL or greater, 150 μg/mL or greater, etc.). This dosing may be used for adult patients and/or infant patients. Alternatively, for children (including infants), the dosing may be different.
For example, in some variations dosing may be per body weight. For example, for children, the dosing may be in the range of between 0.1-25 mg/kg range per day (e.g., 0.5-20 mg/kg per day, 1-15 mg/kg per day, etc.), e.g., 5-75 mg/day for a 5 kg infant. For adults, higher dose levels may be used, e.g., >500 mg/day to even 1 g/day. The concentration in mucus may be between 0.05-1000 μg/mL (e.g., between about 0.5-1000 μg/mL), etc.
As described herein, antibodies of the presently-disclosed subject matter (having an enhanced trapping potency in mucus) are capable of diffusing through mucus when they are unbound, to allow the antibody to bind a target pathogen or sperm at a desirable rate. However, when antibodies are bound to the target (e.g., virion), the cumulative effect of the antibody-mucin interactions effectively traps the pathogen or sperm in the mucus. Because in the unbound state the antibodies may diffuse quickly through the mucus, the half-life of a typical dose is likely to be between 12-24 hours, allowing once or twice daily dosing and still remain above a certain minimal threshold despite the natural renewal and clearance of mucus. The unbound antibodies that are topically delivered (e.g., by inhalation) are not likely to be cleared quickly from the lungs, and the antibody may be at a steady state with the fluid environment even though the mucin mesh is being cleared quickly.
The patient may be an infant (e.g., a human infant), a child, an adult, or an elderly adult (e.g., 55 or older, 60 or older, 65 or older, 70 or older, 75 or older, etc.) In some variations the patient is immunocompromised (e.g., due to a pre-existing condition, due to chemotherapy, due to bone marrow transplant, etc.)
In some embodiments, the virion may be selected from respiratory viruses and/or viruses that are not typically considered respiratory viruses, including but not limited to: Ebola virus, respiratory syncytial virus, influenza virus, adenovirus, human rhinovirus, coronavirus, norovirus, human metapneumovirus, parainfluenza virus, Hantavirus, MERS, Bocavirus, Marburg virus, enterovirus, etc. In particular, the viron may be respiratory syncytial virus.
For example, described herein are methods of immobilizing a respiratory virion in mucus of a subject's lung, the method comprising administering to the subject, via an inhaled route, a recombinant antibody with a specific affinity for the virion so that the recombinant antibody is trapped in the subject's mucus, the recombinant antibody comprising a human or humanized Fc region, wherein the recombinant antibody comprises an oligosaccharide having a glycosylation pattern that enhances the trapping potency of the recombinant antibody in mucus, wherein the recombinant antibody is trapped in the subject's mucus at a concentration of 50 ng/mL or greater (e.g., between 0.1 μg/mL and 20 μg/mL).
Also described herein are methods for blocking, preventing or eliminating the proliferation, infiltration or spreading of a respiratory virion in mucus of a subject's lung, the method comprising immobilizing the virion by administering to the subject, via an inhaled route, a recombinant antibody with a specific affinity for the virion so that the recombinant antibody is trapped in the subject's mucus, the recombinant antibody comprising a human or humanized Fc region, wherein the recombinant antibody comprises an oligosaccharide having a glycosylation pattern that enhances the trapping potency of the recombinant antibody in mucus.
Also descried herein are nebulized solutions for treating viron, including, in particular, a viron causing a respiratory disorder. In general, these nebulized solutions may be aerosolized by a nebulizer (e.g., any appropriate nebulizer) to form particles having a mass median aerodynamic diameter (MMAD) that is less than about 5 μm diameter. The nebulized solution may generally comprise a recombinant antibody having a human or humanized Fc region and an oligosaccharide having a glycosylation pattern that enhances the trapping potency of the recombinant antibody in mucus, so that the recombinant antibody binds to a viron to form an antibody/viron complex that is trapped in the subject's mucus thereby treating or preventing the infection. The nebulized solution may have a concentration of antibody within the particles of between 10 mg/mL and 100 mg/mL, and a pH that is neutral or between neutral (pH of about 7) and mildly acidic (pH of about 4.5). In some variations, the nebulized solution may be hypotonic or hypertonic relative to the lung tissue. Since the compositions descried herein may be effective outside of the lung tissue (e.g., in the mucus), hypertonic (including mildly hypertonic) solutions may be preferred in some variations. For example, hypertonic saline may refer to any saline solution with a concentration of sodium chloride (NaCl) higher than physiologic (0.9%). For example, a hypertonic solution may include 2%, 3%, 5%, etc. NaCl. In some variations the nebulized solution may be mildly hypertonic (e.g., 2% or 3% NaCl). In some variations, the nebulized solution is isotonic.
In some variations it may be beneficial to include sugars, polyols and/or amino acids in the nebulized solution. For example in some variations, the formulation may include one or more (or all) of: histidine, glycine and/or mannitol (e.g., 47 mM Histidine, 4 mM glycine and 5.6% Mannitol). As mentioned, any of these formulations may be hypertonic. In some variations the formulation may include NaCl and/or citrate (e.g., 1.8% NaCl and 10 mM citrate).
Examples of virons may include: Ebola virus, respiratory syncytial virus (RSV), influenza virus, adenovirus, human rhinovirus, coronavirus, human metapneumovirus, parainfluenza virus, hantavirus, Middle Eastern Respiratory Syndrome (MERS) coronavirus, Bocavirus, and Marburg virus.
In particular, described herein are nebulized solutions for treating or preventing Respiratory syncytial virus (RSV) in a subject inhaling the nebulized solution, the nebulized solution comprising a recombinant antibody having a human or humanized Fc region and an oligosaccharide having a glycosylation pattern that enhances the trapping potency of the recombinant antibody in mucus, so that the recombinant antibody binds to the RSV to form an antibody/RSV complex that is trapped in the subject's mucus thereby treating or preventing the infection, wherein the nebulized solution comprises particles having a mass median aerodynamic diameter (MMAD) of between 2-6 μm (e.g., between 3-4.5 μm, less than 6 μm, less than 5 μm, etc.) diameter and a concentration of antibody within the particles of between 10 mg/mL and 100 mg/mL.
The pH of the nebulized solution may be neutral pH (e.g., pH approximately 7) or between neutral and mildly acidic (e.g., between about 4.5 and 7.4, between about 4.5 and 7, etc.).
As mentioned, the nebulized solutions described herein may be hypertonic (including mildly hypertonic) relative to the lungs, which may thin the mucus, slightly while keeping the antibody within the mucus. In some variations the nebulized solution may be hypotonic (including mildly hypotonic) relative to the lungs. In some variations the nebulized solution may be isotonic.
In any of these examples, the recombinant antibody may comprise an N-linked glycosylation site on the Fc region of the antibodies to which the oligosaccharide is attached. For example, the glycosylation pattern may comprise a biantennary core glycan structure of Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminal N-acetylglucosamine on each branch. In general, the recombinant antibody may comprise a human or humanized IgG or IgM monoclonal antibody, or a fragment or derivative thereof.
For example, recombinant antibody in the nebulized solution may comprises palivizumab, or a variant of palivizumab, including but not limited to motavizumab (or a variant of motavizumab).
Any of the nebulized solutions described herein may include a surfactant. The surfactant may be a nonionic surfactant (e.g., olyoxyethylene glycol octylphenol ethers: e.g., C8H17-(C6H4)-(O—C2H4)1-25-OH; Polyoxyethylene glycol alkylphenol ethers: e.g., C9H19-(C6H4)-(O—C2H4)1-25-OH; Polyoxyethylene glycol sorbitan alkyl esters; Sorbitan alkyl esters; block copolymers of polyethylene glycol and polypropylene glycol, etc.), anionic surfactants (e.g., dioctyl sodium sulfosuccinate (DOSS); Perfluorooctanesulfonate (PFOS); linear alkylbenzene sulfonates; sodium lauryl ether sulfate; lignosulfonate; sodium stearate, etc.), cationic surfactants (e.g., benzalkonium chloride (BAC), cetylpyridinium chloride (CPC), benzethonium chloride (BZT), etc.), and zwitterionic surfactants (e.g., sultaines such as CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), betaines, etc.).
The range of antibody concentration may be particularly critical. As described above, the concentration of antibody within the particles may be between 10 mg/mL and 100 mg/mL (e.g., between about 12 mg/mL and 85 mg/mL). Although the antibodies may be formed outside of this range of values, the efficacy falls of sharply. For example at concentrations between about 10 mg/mL (e.g., 5 mg/mL), the nebulized solution may have no appreciable effect, or significantly reduced effect. Concentrations of antibody greater than about 100 mg/mL were also ineffective, likely due to aggregation of the mAb, and an increase in the viscosity of the nebulized solution. This unexpected concentration dependence of the nebulized solution may explain, at least in part, the general belief that anti-RSV mAbs cannot be aerosolized.
For example, described herein are nebulized solutions for treating or preventing Respiratory syncytial virus (RSV) in a subject inhaling the nebulized solution, the nebulized solutions comprising a recombinant antibody against an epitope of the F protein of RSV, the recombinant antibody having a human or humanized Fc region and an oligosaccharide having a glycosylation pattern that enhances the trapping potency of the recombinant antibody in mucus, so that the recombinant antibody binds to the RSV to form an antibody/RSV complex that is trapped in the subject's mucus thereby treating or preventing the infection, wherein the nebulized solution comprises particles having a mass median aerodynamic diameter (MMAD) of between about 2-6 μm (e.g., between about 3-4.5 less than about 6 μm, less than about 5 μm diameter, etc.), a pH between 7.0 and 8.5, and a concentration of antibody within the particles of between 10 mg/mL and 100 mg/mL.
The epitope of the F protein of RSV may be in the A antigenic site, site II, and/or site Ø, and/or antigenic site VIII, which occupies an intermediate position between the previously defined major antigenic sites II and site Ø.
In general, the methods and compositions (including nebulized solutions) described herein may be directed to respiratory viruses. In addition to RSV, described above, the methods and compositions described herein may be used for treating one or more of: metapneumovirus, influenza virus, adenovirus, and parainfluenza. For example, the recombinant antibody may be directed against metapneumovirus, e.g., human mAb 54G10 (J Infect Dis. 2015 Jan. 15; 211 (2): 216-225, doi: 10.1093/infdis/jiu307), monoclonal antibody (MAb 338, MedImmune, Antiviral Research Volume 88, Issue 1, October 2010, Pages 31-37; doi.org/10.1016/j.antiviral.2010.07.001), etc. See also Journal of Virological Methods 254 (2018) 51-64 (doi.org/10.1016/j.jviromet.2018.01.011), J Virol. 2006 August; 80 (16): 7799-7806 (doi: 10.1128/JVI.00318-06), J Gen Virol. 2008 December; 89 (Pt 12): 3113-3118 (doi: 10.1099/vir.0.2008/005199-0), and Nat Microbiol. 2017 Jan. 30; 2:16272 (doi: 10.1038/nmicrobiol.2016.272) for a description of mAbs against metapneumovirus and/or RSV that may be used herein. Another example of two mAbs against metapneumovius are described in U.S. Pat. No. 9,498,531 (note that
For example, a nebulized solution for treating or preventing a respiratory virus in a subject inhaling the nebulized solution may include: a recombinant antibody having a human or humanized Fc region and an oligosaccharide having a glycosylation pattern that enhances the trapping potency of the recombinant antibody in mucus, so that the recombinant antibody binds to the respiratory to form an antibody/respiratory virus complex that is trapped in the subject's mucus thereby treating or preventing the infection, wherein the nebulized solution comprises particles having a mass median aerodynamic diameter (MMAD) of between about 2 and 6 μm diameter and a concentration of antibody within the particles of between 10 mg/mL and 100 mg/mL. As mentioned, the respiratory virus may be one of: Respiratory syncytial virus (RSV), metapneumovirus, influenza virus, adenovirus, and parainfluenza.
The novel features of the invention are set forth with particularity in the claims that follow. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
The present invention is based, in part, on the discovery and characterization of weak binding interactions between antibodies and mucins and the ability of such antibodies to stop the penetration of pathogens through mucus layers. The antibody-mucin interaction can be used advantageously in methods for preventing and treating infection, providing contraception, and monitoring the effectiveness of vaccines.
The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.
Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Except as otherwise indicated, standard methods known to those skilled in the art may be used for production of recombinant mid synthetic polypeptides, antibodies or antigen-binding fragments thereof, manipulation of nucleic acid sequences, and production of transformed cells. Such techniques are known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).
All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference in their entirety.
Definitions
As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.
Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
The transitional phrase “consisting essentially of” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03.
As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.
A “nucleic acid” or “nucleotide sequence” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotide), but is preferably either single or double stranded DNA sequences.
As used herein, an “isolated” antibody means an antibody separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell structural components or other polypeptides or nucleic acids commonly found associated with the antibody. The term also encompasses antibodies that have been prepared synthetically.
By the terms “treat,” “treating,” or “treatment of” (or grammatically equivalent terms) it is meant that the severity of the subject's condition is reduced or at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the condition.
As used herein, the terms “prevent,” “prevents,” or “prevention” and “inhibit,” “inhibits,” or “inhibition” (and grammatical equivalents thereof) are not meant to imply complete abolition of disease and encompasses any type of prophylactic treatment that reduces the incidence of the condition, delays the onset of the condition, and/or reduces the symptoms associated with the condition after onset.
An “effective,” “prophylactically effective,” or “therapeutically effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, an “effective,” “prophylactically effective,” or “therapeutically effective” amount is an amount that will provide some delay, alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the effects need not be complete or curative, as long as some benefit is provided to the subject.
As used herein, the term “trapping potency” refers to the ability of an antibody that specially binds to a target pathogen or sperm to inhibit movement of the pathogen or sperm through mucus. Trapping potency can be measured by methods known in the art and as disclosed herein. Trapping potency can be quantitated, e.g., as the amount of antibody (e.g., concentration of antibody in mucus) needed to reduce the mobility of at least 50% of the pathogen or sperm Within the mucus gel to at least one-tenth of its mobility in solution (e.g., saline). Mobility in mucus can be measured using techniques well known in the art and described herein. Alternatively, trapping potency can be quantitated as the reduction in percentage of pathogens or sperm that penetrate mucus.
The term “enhances trapping potency” refers to an antibody comprising an oligosaccharide that provides an increased trapping potency relative to the trapping potency of antibodies as found in nature and/or antibodies prior to any modification and/or selection.
As used herein, the term “bind specifically” or “specifically binds” in reference to an antibody of the presently-disclosed subject matter means that the antibody of the invention will bind with an epitope (including one or more epitopes) of a target pathogen or sperm, but does not substantially bind to other unrelated epitopes or molecules. In certain embodiments, the term refers to an antibody that exhibits at least about 60% binding, e.g., at least about 70%, 80%, 90%, or 95% binding, to the target epitope relative to binding to other unrelated epitopes or molecules.
Antibodies and Compositions
The presently-disclosed subject matter includes antibodies, compositions, and methods for inhibiting and/or treating pathogen infection, eliminating pathogen from a mucosal surface, providing contraception, and/or monitoring the effectiveness of vaccines. In particular, the presently-disclosed subject matter relates to antibodies and compositions capable of trapping pathogens and sperm in mucus, thereby inhibiting transport of pathogens or sperm across or through mucus secretions.
The prevailing view of how antibodies protect a subject at mucosal surfaces assumes that neutralization of the pathogen is the primary mechanism of protection. Surprisingly and unexpectedly, in light of this widespread view, the present inventors disclose herein that neutralization is not necessary to protect against infection at mucosal surfaces in a subject. Indeed, it is demonstrated herein that sub-neutralization doses of antibodies to neutralizing epitopes of pathogens can be quite effective at inhibiting infection. Furthermore, it is demonstrated herein that use of antibodies to non-neutralizing epitopes of pathogens can also be quite effective at inhibiting infection.
Antibodies are naturally found in mucus. The current thoughts on antibody-mediated mucosal protection are that secretory IgA (sIgA) antibodies are important for protection because very large amounts of this isotype are found in the gastrointestinal tract. It is further thought that IgG does not play a role in mucosal protection. However, IgG is the dominant isotype in genital secretions and there are substantial quantities of IgG found in respiratory mucus secretions. In contrast to the prevailing thought in the scientific community, it is shown herein that certain antibodies, e.g., IgG, found in mucus, e.g., CVM, can diffuse rapidly through the mucus, slowed only slightly by weak, transient adhesive interactions with mucins within the mucus. This rapid diffusion allows antibodies to accumulate rapidly on pathogen or sperm surfaces. When a plurality of antibodies have accumulated on the surface of a pathogen, the adhesive interactions between the plurality of antibodies and the mucus become sufficient to trap the bound pathogen or sperm in the mucus, thereby preventing infection/providing contraception. Pathogens or sperm trapped in CVM cannot reach their target cells in the mucosal surface, and will instead be shed with post-coital discharge and/or inactivated by spontaneous thermal degradation as well as additional protective factors in mucus, such as defensins (Cole, Curr. Top. Microbiol. Immunol. 306:199 (2006); Doss et al., J. Leukoc. Biol. 87:79 (2010). As disclosed herein, this pathogen trapping activity provides for protection without neutralization, and can effectively inhibit infection at sub-neutralization doses and/or using antibodies to non-neutralizing epitopes of a pathogen.
The present invention additionally provides the discovery that the low-affinity interactions that an antibody forms with mucins are not only Fe-dependent, but also influenced by antibody glycosylation.
Accordingly, the presently-disclosed subject matter includes an isolated antibody comprising an oligosaccharide at a glycosylation site, the oligosaccharide comprising, consisting essentially of, or consisting of a pattern correlating with (providing) enhanced trapping potency of the antibody in mucus, and wherein the antibody specifically binds an epitope of a target pathogen or sperm. The unique glycosylation pattern/unique oligosaccharide component of the antibody is designed to maximize trapping potency of the antibody once a plurality of antibodies are bound to the target pathogen or sperm, without unduly hindering the ability of the unbound antibody to diffuse readily through mucus to rapidly bind a target pathogen or sperm. In certain embodiments, the antibody is one that exhibits a mobility in mucus that is reduced no more than about 50%, e.g., no more than about 40%, 30, 20%, 10%, or 5%, relative to its mobility in solution (e.g., saline or water) and effectively traps a target pathogen or sperm in mucus based on a plurality of bound antibodies (e.g., at least 50% of pathogens or sperm slowed by at least 90%). In some embodiments, the antibody reduces the mobility of at least 50%, e.g., at least 60%, 70%, 80%, or 90% or more of the pathogen or sperm by at least 90%, e.g., at least 95%, 96%, 97%, 98%, or 99% or more. In other embodiments, the antibody reduces the percentage of pathogens or sperm that can penetrate mucus by at least 10%, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Based on the disclosure herein, one of skill in the art can readily identify/design oligosaccharide patterns that provide the desired trapping potency. In other embodiments, the antibody has a sufficient binding rate to an epitope of the target pathogen or sperm to accumulate on the surface of the target pathogen or sperm at sufficient levels to trap the target pathogen or sperm in mucus within one hour (e.g., within 30 minutes or 15 minutes) at an antibody concentration in the mucus of less than 5 μg/mL (e.g., less than 1 μg/mL or 0.1 μg/mL).
In some embodiments, the oligosaccharide component is bound to an N-linked glycosylation site in an Fc region of the antibody. The N-linked glycosylation site can be an asparagine residue on the Fc region of the antibody, for example, the Asn 297 asparagine residue. The amino acid numbering is with respect to the standard amino acid structure of a human IgG molecule.
The N-glycan structure on human IgG-Fc is typically dominated by a biantennary core structure that shares a common core sugar sequence (as shown in
In some embodiments, the oligosaccharide component, i.e., the glycan, attached to the antibody comprises, consists, essentially of, or consists of the core structure depicted in
In some embodiments, the antibody of the invention is a mixture of antibodies having different oligosaccharide components. In some embodiments, the mixture of antibodies comprises at least about 30% antibodies having the core glycan structure depicted in
In some embodiments, the mixture of antibodies is the mixture generated in a human cell line, e.g., a 293 cell line, e.g., a 293T cell line.
The antibody is useful for binding target pathogens to trap the pathogen in mucus to inhibit infection by the pathogen. Target pathogens of the antibody can include any pathogen that can infect a subject through a mucus membrane. Pathogens can be in the categories of algae, bacteria, fungi, parasites (helminths, protozoa), viruses, and subviral agents. Target pathogens further include synthetic systems comprising an antigen having an epitope, for example particles or particulates (e.g., polystyrene beads) comprising attached proteins, e.g., as might be used for bioterrorism.
Pathogens include those that cause sexually-transmitted diseases (listed with the diseases caused by such pathogens), including, without limitation, Neisseria gonorrhoeae (gonorrhea); Chlamydia trachomatis (chlamydia, lymphogranuloma venereum); Treponema pallidum (syphilis); Haemophilus ducreyi (chancroid); Klebsiella granulomatis or Calymmatobacterium granulomatis (donovanosis) Mycoplasma genitalium, Ureaplasma urealyticum (mycoplasmas); human immunodeficiency virus HIV-1 and HIV-2 (HIV, AIDS); HTLV-1 (T-lymphotrophic virus type 1); herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2); Epstein-Barr virus; cytomegalovirus; human herpesvirus 6; varicella-zoster virus; human papillomaviruses (genital warts); hepatitis A virus, hepatitis B virus, hepatitis C virus (viral hepatitis); molluscum contagiosum virus (MCV); Trichomona vaginalis (trichomoniasis); and yeasts, such as Candida albicans (vulvovaginal candidiasis). The antibodies and compositions may also be active against other diseases that are transmitted by contact with bodily fluids that may also be transmissible by sexual contact and are capable of being prevented by administration of the compositions according to this invention. Accordingly, the phrase, “sexually transmitted diseases (STDs),” is to be interpreted herein as including any disease that is capable of being transmitted in the course of sexual contact, whether or not the genital organs are the site of the resulting pathology.
Pathogens also include those that cause respiratory diseases, including, without limitation, influenza (including influenza A, B, and C); severe acute respiratory syndrome (SARS); respiratory syncytial virus (RSV); parainfluenza; adenovirus; human rhinovirus; coronavirus; and norovirus.
Other pathogens include, without limitation, Salmonella and Escherichia coli.
Pathogens include those that affect non-human animals, such as livestock, e.g., swine (e.g., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotavirus, classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), encephalomyocarditis virus (EMCV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), pseudorabies virus (PRV), Japanese encephalitis virus (JEV), Brucella, Leptospira, Salmonella, and Lawsonia intracellularis, Pasteurella multocida, Brachyspira hyodysenteriae, Mycoplasma hyopneumoniae), ruminants (e.g., bovine virus diarrhoea virus (BVDV), border disease virus (BDV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV), Pasteurella haemolytica, Pasteurella multocida, Haemophilus somnus, Haemophilus agnii, Moraxella bovis, Mycoplasma mycoides, Theileria annulata, Mycobacterium avium paratuberculosis), ungulates (e.g., Brucella abortus, Mycobacterium bovis, Theileria parva, Rift Valley fever virus, foot-and-mouth disease virus, lumpy skin disease virus), horses (e.g., Rhodococcus equi, Salmonella choleraesuis, Pasteurella multocida, equine herpesvirus-1, equine herpesvirus-4, equine influenza virus, Streptococcus equi), poultry (e.g., fowl pox virus, Newcastle disease virus, Marek's disease virus, avian influenza virus, infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV)), and the like.
The terms virus and viral pathogen are used interchangeably herein, and further refer to various strains of virus, e.g., influenza is inclusive of new strains of influenza, which would be readily identifiable to one of skill in the art. The terms bacterium, bacteria, and bacterial pathogen are used interchangeably herein, and further refer to antibiotic-resistant or multidrug resistant strains of bacterial pathogens. As used herein when referring to a bacterial pathogen, the term “antibiotic-resistant strain” or “multidrug resistant strain” refers to a bacterial pathogen that is capable of withstanding an effect of an antibiotic or drug used in the art to treat the bacterial pathogen (i.e., a non-resistant strain of the bacterial pathogen).
In some embodiments, it is contemplated that an antibody according to the presently-disclosed subject matter is capable of broadly binding to viruses containing lipid envelopes, which are not necessarily specific to one virus.
As noted above, it was surprisingly discovered that sub-neutralization doses of an antibody can be used to effectively trap a target pathogen or sperm in mucus. As such, in some embodiments, wherein the antibody specifically binds a neutralizing epitope of the target pathogen, a sub-neutralization dose can be used. A sub-neutralization doses is a dose below that which would be needed to achieve effective neutralization. For example, in the case of polyclonal anti-HSV gG antibodies targeting HSV, as described hereinbelow, an effective neutralization dose is approximately 5 μg/mL. However, effective trapping using the antibody can be achieved at a dose below 5 μg/mL, and even below a dose of 1 μg/mL.
As will be recognized by one of skill in the art, doses appropriate for trapping bacterial pathogens can be higher in some embodiments than the doses appropriate for trapping viral pathogens. It will further be recognized that appropriate doses may differ between pathogens, between mucosal surfaces, and also between individuals. It will also be recognized that different subjects and different mucosal surfaces may have different optimal glycan patterns and optimal antibody-mucin affinities, contributing to different optimal doses.
It is further proposed herein that antibodies that selectively bind non-neutralizing epitopes of a target pathogen can be used to effectively trap the target pathogen in mucus. As such, in some embodiments, the antibody specifically binds a non-neutralizing epitope, e.g., one or more non-neutralizing epitopes.
The presently-disclosed subject matter further includes an antibody that selectively binds a conserved epitope of a target pathogen, a benefit of targeting a conserved epitope would be to preserve efficacy of the antibody as against new strains of the pathogen. Targeting such epitopes has been avoided at times in the past because they were viewed as being ineffective targets; however, in view of the disclosure herein that non-neutralizing epitopes can serve as effective targets and/or that sub-neutralization doses can be effective for inhibiting infection, previously dismissed conserved epitopes of target pathogens can be seen as effective targets.
Antibodies of the invention are useful for binding sperm to trap the sperm in mucus to inhibit fertilization of an egg by the sperm. Sperm specific antigens that can be used as antibody targets are well known in the art. See, e.g., U.S. Pat. Nos. 8,211,666, 8,137,918, 8,110,668, 8,012,932, 7,339,029, 7,230,073, and 7,125,550, each incorporated by reference in its entirety.
As noted above, it was determined that the low-affinity binding interactions that an antibody forms with mucins are influenced by antibody glycosylation, and are also Fc-dependent. As such, the presently-disclosed subject matter includes antibodies having a preserved and/or engineered. Fc region. Such antibodies can be, for example, one or more of IgG, IgA, IgM, IgD, or IgE. In certain embodiments, the antibodies are IgG. In some embodiments, the antibodies are one or more subclasses of IgG, e.g., IgG1, IgG2, IgG3, IgG4, or any combination thereof.
In some embodiments, the antibody has a sufficient binding rate and/or binding affinity to an epitope of the target pathogen or sperm to accumulate on the surface of the pathogen or sperm at sufficient levels to trap the pathogen or sperm within one hour after administration of the antibody at an antibody concentration of less than about 5 μg/mL. The term “trap” in this instance refers to reduction of further movement through the mucus. In some embodiments, the target pathogen or sperm is trapped within about 30 minutes, e.g., about 25, 20, 15, or 10 minutes after administration of the antibody. In some embodiments, the antibody traps the target pathogen or sperm at an antibody concentration of less than about 4, 3, 2, or 1 μg/mL.
The following discussion is presented as a general overview of the techniques available for the production of antibodies; however, one of skill in the art will recognize that many variations upon the following methods are known.
The term “antibody” or “antibodies” as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibody can be monoclonal or polyclonal and can be of any species of origin, including (for example) mouse, rat, rabbit, horse, goat, sheep, camel, or human, or can be a chimeric or humanized antibody. See, e.g., Walker et al., Molec. Immunol. 26:403 (1989). The antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies can also be chemically constructed according to the method disclosed in U.S. Pat. No. 4,676,980.
Antibody fragments included within the scope of the present invention include, for example, Fab, Fab′, F(ab)2, and Fv fragments; domain antibodies, diabodies; vaccibodies, linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Such fragments can be produced by known techniques. For example, F(ab′)2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., Science 254:1275 (1989)). In some embodiments, the term “antibody fragment” as used herein may also include any protein construct that is capable of binding a target pathogen or sperm and associate with mucin to trap the target pathogen or sperm in mucus.
Antibodies of the invention may be altered or mutated for compatibility with species other than the species in which the antibody was produced. For example, antibodies may be humanized or camelized. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions (i.e., the sequences between the CDR regions) are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature, 332:323 (1988); and Presta, Curr. Op. Struct. Biol. 2:593 (1992)).
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain Humanization can essentially be performed following the method of Winter and co-workers (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues (e.g., all of the CDRs or a portion thereof) and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147:86 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10:779 (1992); Lonberg et al., Nature 368:856 (1994); Morrison, Nature 368:812 (1994); Fishwild et al., Nature Biotechnol. 14:845 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65 (1995).
Immunogens (antigens) are used to produce antibodies specifically reactive with target polypeptides. Recombinant or synthetic polypeptides and peptides, e.g., of at least 5 (e.g., at least 7 or 10) amino acids in length, or greater, are the preferred immunogens for the production of monoclonal or polyclonal antibodies. In one embodiment, an immunogenic polypeptide conjugate is also included as an immunogen. The peptides are used either in pure, partially pure or impure form. Suitable polypeptides and epitopes for target pathogens and sperm are well known in the art. Polynucleotide and polypeptide sequences are available in public sequence databases such as GENBANK®/GENPEPT®. Large numbers of neutralizing and non-neutralizing antibodies that specifically bind to target pathogens and sperm have been described in the art and can be used as starting material to prepare the antibodies of the present invention. Alternatively, new antibodies can be raised against target pathogens and sperm using the techniques described herein and well known in the art.
Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells and purified using standard techniques. The polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypeptide.
Methods of producing polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, e.g., a purified or synthetic peptide, a peptide coupled to an appropriate carrier (e.g., glutathione-S-transferase, keyhole limpet hemanocyanin, etc.), or a peptide incorporated into an immunization vector such as a recombinant vaccinia virus is optionally mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the peptide of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the peptide is performed where desired. Antibodies, including binding fragments and single chain recombinant versions thereof, against the polypeptides are raised by immunizing animals, e.g., using immunogenic conjugates comprising a polypeptide covalently attached (conjugated) to a carrier protein as described above. Typically, the immunogen of interest is a polypeptide of at least about 10 amino acids, in another embodiment the polypeptide is at least about 20 amino acids in length, and in another embodiment, the fragment is at least about 30 amino acids in length. For example, the polypeptide can comprise amino acids acid residues 1 through 200 from the N-terminal of the papillomavirus L2 protein. The immunogenic conjugates are typically prepared by coupling the polypeptide to a carrier protein (e.g., as a fusion protein) or, alternatively, they are recombinantly expressed in an immunization vector.
Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified peptides, or screened for agonistic or antagonistic activity. Specific monoclonal and polyclonal antibodies will usually bind with a KD of at least about 50 mM, e.g., at least about 1 mM, e.g., at least about 0.1 mM or better. In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in Kohler and Milstein 1975 Nature 256:495-497. Summarized briefly, this method proceeds by injecting an animal with an immunogen, e.g., an immunogenic peptide either alone or optionally linked to a carrier protein. The animal is then sacrificed and cells taken from its spleen, which are fused with myeloma cells. The result is a hybrid cell or “hybridoma” that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells is enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate (preferably mammalian) host. The polypeptides and antibodies of the present invention are used with or without modification, and include chimeric antibodies such as humanized murine antibodies. Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. 1989 Science 246:1275-1281; and Ward et al. 1989 Nature 341:544-546.
Antibodies specific to the target polypeptide can also be obtained by phage display techniques known in the art.
Antibodies can sometimes be labeled by joining, either covalently or noncovalently, a substance which provides a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Such antibodies are useful for detecting or diagnosing the presence of a microbe on which an antigen is found.
Method of making antibodies with a glycosylation pattern of interest can be achieved by any method known to those or skill in the art. For example, in some embodiments, mammalian cells can be used, such as, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and NS0- and SP2/0-mouse myeloma cells, to produce antibodies having the desired glycosylation pattern. In certain embodiments, human cell lines can be used, e.g., 293 cells. In some embodiments, non-mammalian cells can be used. The cell line can be genetically engineered to produce the antibodies with the desired oligosaccharide. Such cell lines can have altered expression, for example, of one or more enzymes affecting glycosylation patterns, e.g., glycosyltransferases. Glycosyltransferases include, without limitation, a galactosyltransferase, a fucosyltransferase, a glucosyltransferase, an N-acetylgalactosaminyltransferase, an N-acetylglucosaminyltransferase, a glucuronyltransferase, a sialyltransferase, a mannosyltransferase, a glucuronic acid transferase, a galacturonic acid transferase, an oligosaccharyltransferase, or any combination thereof. Specific examples include, without limitation, oligosaccharyltransferase, UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, GDP-fucose protein:O-fucosyltransferase 1, GDP-fucose protein:O-fucosyltransferase 2, protein:O-glucosyltransferase, UDP-N-acetylglucosamine:peptide N-acetylglucosaminyltransferase, protein:O-mannosyltransferase, β1,4 galactosyltransferase, and any combination thereof. Enzymes involved in glycosylation of proteins are well known in the art and can be manipulated using routine techniques. See, for example, U.S. Pat. Nos. 8,383,106, 8,367,374, 8,080,415, 8,025,879, 8,021,856, 7,906,329, and 7,846,434, each incorporated herein by reference in its entirety. In other embodiments, glycans can be synthesized in specific patterns and linked to antibodies. In further embodiments, antibodies with mixed glycosylation patterns can be separated to isolate antibodies with the desired glycosylation pattern.
As would be recognized by one skilled in the art, the antibodies of the presently-disclosed subject matter can also be formed into suitable compositions, e.g., pharmaceutical compositions for administration to a subject in order to treat or prevent an infection caused by a target pathogen or a disease or disorder caused by infection by a target pathogen or to provide contraception. In one embodiment, the compositions comprise, consist essentially of, or consist of an antibody of the invention in a prophylactically or therapeutically effective amount and a pharmaceutically-acceptable carrier.
Pharmaceutical compositions containing the antibodies as disclosed herein can be formulated in combination with any suitable pharmaceutical vehicle, excipient or carrier that would commonly be used in this art, including such conventional materials for this purpose, e.g., saline, dextrose, water, glycerol, ethanol and combinations thereof. As one skilled in this art would recognize, the particular vehicle, excipient or carrier used will vary depending on the subject and the subject's condition, and a variety of modes of administration would be suitable for the compositions of the invention. Suitable methods of administration of any pharmaceutical composition disclosed in this application include, but are not limited to, topical, oral, intranasal, buccal, inhalation, anal, and vaginal administration, wherein such administration achieves delivery of the antibody to a mucus membrane of interest.
The composition can be any type of composition suitable for delivering antibody to a mucosal surface and can be in various forms known in the art, including solid, semisolid, or liquid form or in lotion form, either oil-in-water or water-in-oil emulsions, in aqueous gel compositions. Compositions include, without limitation, gel, paste, suppository, douche, ovule, foam, film, spray, ointment, pessary, capsule, tablet, jelly, cream, milk, dispersion, liposomes, powder/talc or other solid, suspension, solution, emulsion, microemulsion, nanoemulsion, liquid, aerosol, microcapsules, time-release capsules, controlled release formulation, sustained release formulation or bioadhesive gel (e.g., a mucoadhesive thermogelling composition) or in other forms embedded in a matrix for the slow or controlled release of the antibody to the surface onto which it has been applied or in contact. Of particular interest herein are nebulized compositions, suitable for aerosol delivery.
The composition may contain conventional additives, such as preservatives, solvents to promote penetration, and emollients. Topical formulations may also contain conventional carriers such as cream or ointment bases, ethanol, or oleyl alcohol. Other formulations for administration, including intranasal administration, etc., are contemplated for use in connection with the presently-disclosed subject matter. All formulations, devices, and methods known to one of skill in the art which are appropriate for delivering the antibody or composition containing the antibody to one or more mucus membranes of a subject can be used in connection with the presently-disclosed subject matter.
The compositions used in the methods described herein may include other agents that do not negatively impact or otherwise affect the inhibitory and/or contraceptive effectiveness of the components of the composition, including antibodies, antimicrobial agents, and/or sperm-function inhibitors. For example, solid, liquid or a mixture of solid and liquid pharmaceutically acceptable carriers, diluents, vehicles, or excipients may be employed in the pharmaceutical compositions. Suitable physiologically acceptable, substantially inert carriers include water, a polyethylene glycol, mineral oil or petrolatum, propylene glycol, hydroxyethylcellulose, carboxymethyl cellulose, cellulosic derivatives, polycarboxylic acids, linked polyacrylic acids. such as carbopols; and other polymers such as poly(lysine), poly(glutamic acid), poly(maleic acid), polylactic acid), thermal polyaspartate, and aliphatic-aromatic resin; glycerin, starch, lactose, calcium sulphate dihydrate, terra alba, sucrose, talc, gelatin, pectin, acacia, magnesium stearate, stearic acid, syrup, peanut oil, olive oil, saline solution, and the like.
The pharmaceutical compositions described herein useful in the methods of the present invention may further include diluents, fillers, binding agents, colorants, stabilizers, perfumes, gelling agents, antioxidants, moisturizing agents, preservatives, acids, and other elements known to those skilled in the art. For example, suitable preservatives are well known in the art, and include, for example, methyl paraben, propyl paraben, butyl paraben, benzoic acid and benzyl alcohol.
Compositions can be powdered or liquid and may include one or more inactive ingredients and/or carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
The composition can comprise a powder or an aerosolized or atomized solution or suspension comprising the antibody. Such powdered, aerosolized, or atomized compositions, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers.
The antibody can be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the antibody, which the subject inhales. The respirable particles can be liquid or solid. The term “aerosol” includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Harwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth. 27:143 (1992). Aerosols of liquid particles comprising the antibody can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer, vibrating mesh nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles comprising the antibody can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
Alternatively, one can administer the antibody in a local rather than systemic manner, for example, in a depot or sustained-release formulation.
As noted herein, antibodies of the presently-disclosed subject matter are capable of diffusing through mucus when they are unbound, to allow the antibody to bind a target pathogen or sperm at a desirable rate. It is also desirable that, when antibodies are bound to the pathogen or sperm, the cumulative effect of the antibody-mucin interactions effectively traps the pathogen or sperm in the mucus. To facilitate this goal, in some embodiments, it can be desirable to provide a composition that includes more than one antibody, wherein each antibody specifically binds a different epitope of the target pathogen or sperm. Such a composition provides the ability for an increased number of antibodies to become bound to the pathogen or sperm, thereby strengthening the antibody-mucin interactions that serve to trap the pathogen or sperm in the mucus.
In some embodiments of the presently-disclosed subject matter, a composition includes a first antibody and a second antibody, as disclosed herein, wherein the first antibody specifically binds a first epitope of the target pathogen or sperm and the second antibody specifically binds a second epitope of the target pathogen or sperm, wherein said first epitope is distinct from the second epitope. In certain embodiments, the composition includes three or more different antibodies, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different antibodies, wherein each antibody specifically binds a different epitope of the target pathogen or sperm.
In some embodiments of the presently-disclosed subject matter, a composition includes a first antibody that specifically binds a gG surface glycoprotein of HSV, a second antibody that specifically binds a gD surface glycoprotein of HSV, and/or a third antibody that specifically binds a gB surface glycoprotein of HSV.
It is also desirable to provide a composition that can provide treatment or prevention of infection due to more than one target pathogen. In some embodiments of the presently-disclosed subject matter, a composition includes a first antibody and a second antibody, as disclosed herein, wherein the first antibody specifically binds an epitope of a first target pathogen and the second antibody specifically binds an epitope of second target pathogen. In certain embodiments, the composition includes three or more different antibodies, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different antibodies, wherein each antibody specifically binds an epitope of a different target pathogen.
In other embodiments, a composition provides treatment or prevention of infection by one or more target pathogens. In some embodiments of the presently-disclosed subject matter, a composition includes a first antibody and a second antibody, as disclosed herein, wherein the first antibody specifically binds an epitope of sperm and the second antibody specifically binds an epitope of a target pathogen. In certain embodiments, the composition includes three or more different antibodies, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different antibodies, wherein one or more antibodies bind different epitopes of sperm and one or more antibodies specifically binds an epitope of a target pathogen or multiple target pathogens.
In some embodiments, the pharmaceutical composition can further include an additional active agent, e.g., a prophylactic or therapeutic agent. For example, the additional active agent can be an antimicrobial agent, as would be known to one of skill in the art. The antimicrobial agent may be active against algae, bacteria, fungi, parasites (helminths, protozoa), viruses, and subviral agents. Accordingly, the antimicrobial agent may be an antibacterial, antifungal, antiviral, antiparasitic, or antiprotozoal agent. The antimicrobial agent is preferably active against infectious diseases.
Suitable antiviral agents include, for example, virus-inactivating agents such as nonionic, anionic and cationic surfactants, and C31 G (amine oxide and alkyl betaine), polybiguanides, docosanol, acylcarnitine analogs, octyl glycerol, and antimicrobial peptides such as magainins, gramicidins, protegrins, and retrocyclins. Mild surfactants, e.g., sorbitan monolaurate, may advantageously be used as antiviral agents in the compositions described herein. Other antiviral agents that may advantageously be utilized in the compositions described herein include nucleotide or nucleoside analogs, such as tenofovir, acyclovir, amantadine, didanosine, foscarnet, ganciclovir, ribavirin, vidarabine, zalcitabine, and zidovudine. Further antiviral agents that may be used include non-nucleoside reverse transcriptase inhibitors, such as UC-781 (thiocarboxanilide), pyridinones, TIBO, nevaripine, delavirdine, calanolide A, capravirine and efavirenz. From these reverse transcriptase inhibitors, agents and their analogs that have shown poor oral bioavailability are especially suitable for administration to mucosal tissue, in combination with antibodies and compositions of the invention, to prevent sexual transmission of HIV. Other antiviral agents that may be used are those in the category of HIV entry blockers, such as cyanovirin-N, cyclodextrins, carregeenans, sulfated or sulfonated polymers, mandelic acid condensation polymers, monoclonal antibodies, chemokine receptor antagonists such as TAK-779, SCH-C/D, and AMD-3100, and fusion inhibitors such as T-20 and 1249.
Suitable antibacterial agents include antibiotics, such as aminoglycosides, cephalosporins, including first, second and third generation cephalosporins; macrolides, including erythromycins, penicillins, including natural penicillins, penicillinase-resistant penicillins, aminopenicillins, extended spectrum penicillins; sulfonamides, tetracyclines, fluoroquinolones, metronidazole and urinary tract antiseptics.
Suitable antifungal agents include amphotericin B, nystatin, griseofulvin, flucytosine, fluconazole, potassium iodide, intraconazole, clortrimazole, miconazole, ketoconazole, and tolnaftate.
Suitable antiprotozoal agents include antimalarial agents, such as chloroquine, primaquine, pyrimethamine, quinine, fansidar, and mefloquine; amebicides, such as dioloxamide, emetine, iodoquinol, metronidazole, paromomycine and quinacrine; pentamidine isethionate, atovaquone, and eflornithine.
The presently-disclosed subject matter further includes a kit, including the antibody or composition comprising the antibody as described herein; and optionally a device for administering the antibody or composition. In some embodiments, the kit can include multiple antibodies and/or compositions containing such antibodies. In some embodiments, each of multiple antibodies provided in such a kit can specifically bind to a different epitope of the target pathogen or sperm. In other embodiments, each of multiple antibodies provided in such a kit can specifically bind to an epitope of a different target pathogen or to an epitope of sperm. In some embodiments, the kit can further include an additional active agent, e.g., an antimicrobial, such as an antibiotic, an antiviral, or other antimicrobial, or a sperm-function inhibitor as would be known to one of skill in the art.
Prevention and Treatment of Infection
The presently-disclosed subject matter further includes methods of inhibiting or treating an infection by a target pathogen in a subject, including administering to a mucosa of the subject an antibody and/or composition as disclosed herein. The mucosa can be selected from, for example, a respiratory tract mucosa (e.g., a nasal mucosa, a lung mucosa), a reproductive tract mucosa (e.g., a genital mucosa, a uterine mucosa, a vaginal mucosa), an ocular mucosa, and a gastrointestinal mucosa (e.g., an oral mucosa, an anal mucosa), and any combination thereof. In certain embodiments, the methods comprise additional steps such as one or more of isolating the antibodies, preparing a composition of the isolated antibodies, determining the level of antibodies in the mucus of the subject before administering the antibodies, and determining the level of antibodies in the mucus of the subject after administering the antibodies.
The antibodies and compositions of the present invention according to the methods described herein are administered or otherwise applied by delivering the composition, typically to a site of infection. The site of infection may be one where an infection is already present (an actual site of infection) or where an infection is likely to occur (a potential site of site of infection in or on an uninfected individual). In some embodiments, the antibodies and compositions may be topically delivered. In other embodiments, the antibodies and compositions may be systemically delivered such that the antibodies are secreted into the mucus of the subject. Accordingly, the compositions as described above may be delivered the respiratory tract, e.g., the nasal cavity and the lungs.
An effective amount of the antibody can be administered. As used herein, an “effective amount” of the antibody for inhibition of infection refers to a dosage sufficient to inhibit infection by the target pathogen. As used herein, an “effective amount” of the antibody for treatment of infection refers to a dosage sufficient to inhibit spread of the target pathogen from infected cells to non-infected cells in the subject and/or to inhibit spread of the target pathogen from the infected subject to another subject, e.g., a non-infected subject. The effective amount can be an amount sufficient to trap an amount of the target pathogen in mucus. As will be recognized by one of skill in the art, the amount can vary depending on the patient and the target pathogen. The exact amount that is required will vary from subject to subject, depending on the species, age, and general condition of the subject, the particular carrier or adjuvant being used, mode of administration, and the like. As such, the effective amount will vary based on the particular circumstances, and an appropriate effective amount can be determined in a particular case by one of skill in the art using only routine experimentation. In some instances, an effective amount of the antibody that specifically binds the target pathogen or sperm can be an amount that achieves a concentration of the antibody in the mucus of about 0.1 μg/mL to about 1000 μg/mL, e.g., about 0.5 μg/mL to about 100 μg/mL, e.g., about 1 μg/mL to about 50 μg/mL or any range therein. In some embodiments, the antibody may be administered in two or more stages with different doses in each stage. For example, higher doses can be administered initially in order to clear target pathogens that are present in the mucus of exposed or infected subjects and ensure that sufficient amounts of antibody remain in the mucus to provide protection, e.g., for about 24 hours. In later stages, lower doses can be administered to maintain protective levels of the antibody. In other embodiments, protective doses can be administered to subjects that are likely to be exposed to a pathogen and higher doses can be administered if infection occurs.
As will be recognized by one of skill in the art, the term “inhibiting” or “inhibition” does not refer to the ability to completely eliminate the possibility of infection in all cases. Rather, the skilled artisan will understand that the term “inhibiting” refers to reducing the chances of pathogens moving through mucus beyond the mucus membrane such that infection of a subject can occur, such as reducing chances of infection by a pathogen when such pathogen is bound to trapping antibodies in mucus. Such decrease in infection potential can be determined relative to a control that has not been administered the antibodies of the invention. In some embodiments, the decrease of inhibition potential relative to a control can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease.
In some embodiments inhibiting or treating an infection in a subject can comprise trapping a pathogen in mucus. As such, in some embodiments a method of trapping a target pathogen in mucus is provided, which method includes administering to a mucosa of the subject an antibody or composition as described herein.
In some embodiments, a method of inhibiting or treating an infection in a subject, and/or trapping a pathogen in the mucus of a subject, involves administering to a mucosa of the subject a composition comprising an isolated antibody that specifically binds a non-neutralizing epitope of a target pathogen. The antibody can be a non-neutralizing antibody. In some embodiments, the non-neutralizing antibody is provided at a concentration above a predetermined amount.
In some embodiments, a method of inhibiting or treating an infection in a subject, and/or trapping a pathogen in the mucus of a subject, involves administering to a mucosa of the subject a composition comprising an isolated antibody that specifically binds a neutralizing epitope of a target pathogen, wherein the antibody is provided at a sub-neutralization dose.
As used herein, the term “subject” refers to humans and other animals. Thus, veterinary treatment is provided in accordance with the presently-disclosed subject matter. As such, the presently-disclosed subject matter provides for the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Thus, also provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), poultry, and the like.
A subject in need of inhibiting an infection or a disease or disorder caused by an infection is a subject that has been identified as being at risk of infection. In some embodiments, the subject is identified as having been exposed to the target pathogen. In other embodiments, the subject is in contact with other subjects that are infected or are likely to become infected, e.g., a subject that is living with, working with, and/or attending school with infected subjects.
A subject in need of treating an infection or a disease or disorder caused by an infection is a subject that has been diagnosed as infected with a pathogen or is suspected of being infected with a pathogen, e.g., exhibiting symptoms of infection.
In particular embodiments of the invention, more than one administration (e.g., two, three, four, or more administrations) of the antibody, composition, or device comprising the composition can be employed over a variety of time intervals (e.g., hourly, daily, weekly, monthly, etc.) to achieve prophylactic and/or therapeutic effects.
In some embodiments, the method include administering an antibody, and further administering an additional active agent, e.g., prophylactic or therapeutic agent, e.g., an antimicrobial, such as an antibiotic, an antiviral, or other antimicrobial as would be known to one of skill in the art. The additional active agents can be delivered concurrently with the antibodies and compositions of the invention. The additional active agents can be delivered in the same composition as the antibody or in separate compositions. As used herein, the word “concurrently” means sufficiently close in time to produce a combined effect (that is, concurrently can be simultaneously, or it can be two or more events occurring within a short time period before or after each other).
Monitoring Vaccine Effectiveness
Having discovered the usefulness of the antibodies of the invention in preventing and treating infection by pathogens and/or diseases or disorders caused by infection by pathogens, another aspect of the invention relates to detecting the presence of such antibodies in a subject in order to determine how well the subject is protected against possible infection. For example, a subject that has been vaccinated against a particular pathogen can be monitored for the presence and level of antibodies that have trapping potency in mucus or other body fluids, e.g., saliva. If such antibodies do not appear in the mucus or appear at non-prophylactic or non-therapeutic levels, the subject can be administered a booster vaccination and/or a different vaccine in order to generate an effective amount of antibodies. For example, subjects can be monitored to ensure a level of antibodies in mucus that at least equal to the level of antibodies observed in a subject that has been exposed previously to a pathogen.
Thus, one embodiment relates to a method of monitoring the effectiveness of a vaccination in a subject that has been vaccinated against a target pathogen, comprising determining in a mucus sample from said subject the amount of an antibody comprising an oligosaccharide at a glycosylation site, the oligosaccharide having a glycosylation pattern that enhances trapping potency of the antibody in mucus, wherein the antibody specifically binds an epitope of said target pathogen, wherein the amount of said antibody is indicative of the effectiveness of the vaccination.
The mucus to be sampled can be from the appropriate mucosal surface where the antibody is expected to be found. Samples of mucus can be obtained from a subject by methods routinely used in the art. Similarly, samples of other body fluids such as saliva can be obtained from a subject by methods routinely used in the art.
A variety of assays can be employed for detection and/or quantitation of the antibody. For example, various immunoassays can be used to detect antibodies of this invention. Such immunoassays typically involve the measurement of antigen/antibody complex formation between a protein or peptide (i.e., an antigen) and its specific antibody.
The immunoassays of the invention can be either competitive or noncompetitive and both types of assays are well-known and well-developed in the art. In competitive binding assays, antigen or antibody competes with a detectably labeled antigen or antibody for specific binding to a capture site bound to a solid surface. The concentration of labeled antigen or antibody bound to the capture agent is inversely proportional to the amount of free antigen or antibody present in the sample.
Noncompetitive assays of this invention can be, for example, sandwich assays, in which, for example, the antigen is bound between two antibodies. One of the antibodies is used as a capture agent and is bound to a solid surface. The other antibody is labeled and is used to measure or detect the resultant antigen/antibody complex by e.g., visual or instrument means. A number of combinations of antibody and labeled antibody can be used, as are well known in the art. In some embodiments, the antigen/antibody complex can be detected by other proteins capable of specifically binding human immunoglobulin constant regions, such as protein A, protein L or protein G. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong nonimmunogenic reactivity with immunoglobulin constant regions from a variety of species. (See, e.g., Kronval et al., J. Immunol., 111:1401 (1973); Akerstrom et al., J. Immunol., 135:2589 (1985)).
In some embodiments, the non-competitive assays need not be sandwich assays. For instance, the antibodies or antigens in the sample can be bound directly to the solid surface. The presence of antibodies or antigens in the sample can then be detected using labeled antigen or antibody, respectively.
In some embodiments, antibodies and/or proteins can be conjugated or otherwise linked or connected (e.g., covalently or noncovalently) to a solid support (e.g., bead, plate, slide, dish, membrane or well) in accordance with known techniques. Antibodies can also be conjugated or otherwise linked or connected to detectable groups such as radiolabels (e.g., 35S, 125I, 32P, 13H, 14C, 131D, enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), gold beads, chemiluminescence labels, ligands (e.g., biotin) and/or fluorescence labels (e.g., fluorescein) in accordance with known techniques.
A variety of organic and inorganic polymers, both natural and synthetic can be used as the material for the solid surface. Nonlimiting examples of polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like. Other materials that can be used include, but are not limited to, include paper, glass, ceramic, metal, metalloids, semiconductive materials, cements and the like. In addition, substances that form gels, such as proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides can be used. Polymers that form several aqueous phases, such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like are also suitable. Where the solid surface is porous, various pore sizes can be employed depending upon the nature of the system.
A variety of immunoassay systems can be used, including but not limited to, radio-immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA) assays, enzyme immunoassays (EIA), “sandwich” assays, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, immunofluorescence assays, fluorescence activated cell sorting (FACS) assays, immunohistochemical assays, protein A immunoassays, protein G immunoassays, protein L immunoassays, biotin/avidin assays, biotin/streptavidin assays, immunoelectrophoresis assays, precipitation/flocculation reactions, immunoblots (Western blot; dot/slot blot); immunodiffusion assays; liposome immunoassay, chemiluminescence assays, library screens, expression arrays, immunoprecipitation, competitive binding assays and immunohistochemical staining. These and other assays are described, among other places, in Hampton et al. (Serological Methods, a Laboratory Manual, APS Press, St Paul, Mimi (1990)) and Maddox et al. (J. Exp. Med. 158:1211 (1993); the entire contents of which are incorporated herein by reference for teachings directed to immunoassays).
The methods of this invention can also be carried out using a variety of solid phase systems, such as described in U.S. Pat. No. 5,879,881, as well as in a dry strip lateral flow system (e.g., a “dipstick” system), such as described, for example, in U.S. Patent Publication No. 2003/0073147, the entire contents of each of which are incorporated by reference herein.
The detection and/or quantitation of the antibody in the mucus sample can further comprise characterization of the glycosylation pattern of the antibody. Determination of the glycosylation pattern can be carried out using methods well known in the art and described herein, including glycan sequencing and separation techniques such as gel electrophoresis, fluorescence-activated sorting, and mass spectrometry.
Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.
Culture and Purification of Fluorescent HSV-1: The HSV-1 mutant 166v (Elliott et al., J. Virol. 73:4110 (1999)), encoding a VP22-Green Fluorescent Protein (GFP) tegument protein packaged into HSV-1 at relatively high copy numbers (Heine et al., J Virol. 14:640 (1974)), was kindly provided by Richard Courtney and used in all microscopy and ELISA studies, besides those for in vivo experiments. The addition of GFP to the VP22 protein appears to have no deleterious effects on viral replication (Elliott et al., J. Virol. 73:4110 (1999)), and the fluorescence of 166v was consistently more intense than that of HSV-1 mutants encoding other GFP fusion proteins. 166v was expanded at an MOI of 3 on confluent monolayers of HaCat cells maintained in DMEM (Life Technologies, Grand Island, N.Y.) supplemented with 5% FBS, 1×L-glutamine and 1× Penicillin/Streptomycin. Culture medium was collected 16-18 hr post-infection and twice centrifuged at 1000×g for 5 min to remove cell debris. The resulting supernatant was split into 30 mL aliquots and precipitated overnight with a polyethylene glycol (PEG)/salt solution. Briefly, 10 mL of 1.55 M NaCl was added to 30 mL of crude virus supernatant, followed by 10 mL of 40% PEG 8000 (Sigma, St. Louis, Mo.). After an overnight incubation at 4° C. the virus/PEG solution was centrifuged at 2555×g and 4° C. for 1 hr. The virus pellet was then resuspended in 1×PBS and centrifuged through a continuous 20-50% (w/w) sucrose in PBS gradient for 1 hr at 74,119×g. The resulting virus band was collected, diluted 1:5 in PBS, layered over 30% (w/w) sucrose in PBS, and centrifuged for 1.5 hr at 83,472×g to pellet virus for further purification. Purified virus pellet was resuspended in PBS and stored as single use aliquots at −80° C.
Cervicovaginal Mucus (CVM) Collection and Characterization: CVM collection was performed as published previously (Lai et al., J. Virol. 83:11196 (2009); Lai et al., Proc. Natl. Acad. Sci. U.S.A. 107:598 (2010)). Briefly, undiluted CVM secretions, averaging 0.3 g per sample, were obtained from women of reproductive age, ranging from 20 to 32 years old (27.4±0.9 years, mean±SEM), by using a self-sampling menstrual collection device following protocols approved by the Institutional Review Board of the University of North Carolina—Chapel Hill. Informed consent of participants was obtained after the nature and possible consequences of the study were explained. Participants inserted the device into the vagina for at least 30 s, removed it, and placed it into a 50 mL centrifuge tube. Samples were centrifuged at 230×g for 2 min to collect the secretions. Aliquots of CVM for lactic acid and Ab measurements (diluted 1:5 with 1×PBS and stored at −80° C.) and slides for gram staining were prepared immediately, and the remainder of the sample was stored at 4° C. until microscopy, typically within a few hours. Samples were collected at random times throughout the menstrual cycle, and cycle phase was estimated based on the last menstrual period date normalized to a 28 day cycle. No samples were ovulatory based on visual observation (none exhibited spinnbarkeit). Samples that were non-uniform in color or consistency were discarded. Donors stated they had not used vaginal products nor participated in unprotected intercourse within 3 days prior to donating. All samples had pH<4.5; none had bacterial vaginosis (BY) based on Gram staining and Nugent scoring, following scoring criteria described previously (Nugent et al., J. Clin. Microbiol. 29:297 (1991)). For lactic acid and Ab measurements, CVM aliquots were thawed and centrifuged for 2 min at 21,130×g to obtain cell-free supernatant containing lactic acid and Ab. Lactic acid content was measured using a DA-lactic acid kit (R-Biopharm, Darmstadt, Germany) according to manufacturer protocol, but adapted to a 96-well format.
Total immunoglobulin levels in CVM were quantified using the Human Isotyping Kit (HGAMMAG-301K; Millipore, Billerica, Mass.) according to manufacturer protocol. Briefly, 20× stock isotyping beads were vortexed, sonicated, diluted to IX, and incubated with 50 μL of serially diluted CVM supernatant at 1:2 beads:CVM volume ratio. After 1 hr, the beads were separated from CVM supernatant using a magnetic plate, and washed twice with wash buffer. The beads were then incubated with 25 μL of 1× anti-Human Kappa and Lambda-PE for 1 hr, washed twice, and resuspended in Luminex Drive fluid. Fluorescence intensities indicative of immunoglobulin levels present in CVM were measured using the Luminex MAGPIX system, and data analysis was performed using Milliplex Analyst (v3.5.5.0; Vigene Tech Inc., Carlisle, Mass.). All incubations were carried out at room temperature in the dark with vigorous agitation.
Whole-virus ELISA was used to quantify HSV-1 specific IgG. Briefly, high-affinity 96-well half-area plates (Thermo Scientific, Rockford, Ill.) were coated overnight at 4° C. with 25 μL per well of affinity-purified intact HSV-1 at 20 μg/mL (measured using BCA assay). The plates were washed four times with 0.05% Tween in PBS (PBS-T), blocked with 5% milk for at least 1 hr followed by PBS-T washes, then incubated for at least 1 hr with serial dilutions of CVM supernatant. Following PBS-T washes, virion-bound IgG was quantified using F(ab′)2 anti-human IgG Fc (Goat)-HRP conjugate (709-1317; Rockland, Gilbertsville, Pa.) and 1-Step Ultra TMB substrate (Thermo Scientific, Rockford, Ill.), and compared to a standard generated on the same plate using twice-purified anti-HSV-1 IgG, which was assumed to be >90% pure. TMB conversion was terminated with 2 N sulfuric acid, and absorbance was measured at 450 nm using a BioTek Synergy 2 plate reader. HSV-1 specific IgA and IgM levels were too low to be detected by this assay.
Preparation and Characterization of Anti-HSV-1 IgG: Anti-HSV-1 IgG was purified from intravenous immunoglobulin (IVIg, Privigen®; ≥98% IgG; CSL Behring, King of Prussia, Pa.) by affinity column purification. Briefly HSV glycoproteins were extracted from purified HSV-1 by overnight incubation with Triton X-100 (final concentration 0.05%) at 4° C., followed by centrifugation at 21,130×g and 4° C. for 1.5 hr. The resulting supernatant containing HSV glycoproteins was coupled to AminoLink Plus Coupling Resins (Thermo Scientific, Rockford, Ill.) according to manufacturer protocol, and stored at 4° C. until use. To extract HSV-1 specific IgG, the column was first warmed to room temperature and washed twice with 3 mL of equilibrium buffer (150 mM NaCl, 0.05% Tween, 10 mM sodium phosphate at pH 6): The column was then loaded with 2 mL of IVIg buffer exchanged into equilibrium buffer using a 50 kDa MWCO concentrator (Corning, Tewksbury, Mass.), and incubated at room temperature for at least 45 min with end-over-end mixing. Unbound, non-specific Ab was removed by washing with 1 mL of equilibrium buffer followed by three additional rounds of wash buffer (4 mL each round; 500 mM NaCl, 0.05% Tween, 10 mM sodium phosphate at pH 6). Bound Ab was then eluted using IgG Elution Buffer (Thermo Scientific, Rockford, Ill.); each elution consisted of three 3 mL volumes of elution buffer, and was collected into tubes containing 100 μL of 10 mM sodium phosphate pH 6 to neutralize the elutions. Elutions from multiple runs were pooled together, concentrated and buffer exchanged into PBS using a 30 kDa MWCO concentrator tube, supplemented with sodium azide (final concentration 0.03%) and stored at 4° C. until use. A single purification round removed over 99% of non-specific IgG, tested by spiking IVIg with mouse IgG. Final concentration of purified IgG was measured via sandwich ELISA, with plates coated with 2 μg/mL of anti-human IgG Fc (Alpha Diagnostics, San Antonio, Tex.) followed by detection with F(ab′)2 anti-human IgG Fc (Goat)-HRP, and compared to a standard curve generated with serial dilutions of stock IVIg.
Preparation and Characterization of Anti-HSV-1 F(Ab′)2: HSV-1 specific IgG was fragmented using a F(ab′)2 Preparation Kit (Thermo Scientific, Rockford, Ill.). Briefly, IgG was desalted into IgG Digestion Buffer using 3 kDa MWCO concentrator tubes (Amicon 0.5 mL; Millipore, Billerica, Mass.) and incubated with pepsin at 37° C. for 3.5 hr with end-over-end mixing, followed by purification using a Protein A column (0.2 mL resin; Thermo Scientific, Rockford, Ill.) and PBS washes to remove undigested IgG. Fragmentation was confirmed using a non-reducing 4-12% Bis-Tris gel with MOPS running buffer. The gel was stained with Coomassie Blue for imaging; note that the gel image presented in
Preparation and Characterization of Deglycosylated Anti-HSV-1 IgG: N-linked oligosaccharides on purified anti-HSV-1 IgG were cleaved by incubating 200 μg of IgG with 10 μL of PNGase and 11 tit of 10×G7 reaction buffer (New England Biolabs, Ipswich, Mass.) for at least 24 hr at 37° C. IgG was then recovered using a Protein A column, eluted with IgG Elution Buffer into 100 μL of 10 mM sodium phosphate pH 6 and buffer exchanged into PBS using a 30 kDa MWCO concentrator tube. The deglycosylated IgG was further purified using a 0.8 mL spin column immobilized with Con A-agarose slurry (Vector Labs, Burlingame, Calif.) and washed thrice with equilibrium buffer (10 mM HEPES with 0.15 M NaCl at pH 7.5) prior to incubation with end-over-end mixing for 45 min at room temperature. Following the incubation, the column was spun at 5000×g for 1 min to collect the flow through, which contains the deglycosylated IgG, and washed thrice with equilibrium buffer to maximize recovery. The flow through and the washes were pooled, buffer exchanged into PBS using a 30 kDa MWCO concentrator tube, supplemented with sodium azide (final concentration 0.03%) and stored at 4° C. until use. Total amounts of deglycosylated IgG were measured as described above. Deglycosylation was confirmed using a lectin-ELISA assay. Briefly, high-affinity 96-well plates were coated overnight at 4° C. with 50 μL per well of 1 μg/mL purified deglycosylated HSV-1 IgG. The plates were washed 4× with PBS-T, blocked with 300 μL/well of 1× Carbo Free solution (Vector Labs, Burlingame, Calif.) for at least 1 hr followed by PBS-T washes, then incubated with 50 μL/well of 1 μg/mL biotinylated Con A lectin (Vector Labs, Burlingame, Calif.) for at least 2 hr. Following PBS-T washes, IgG-bound lectin was quantified using anti-biotin-HRP conjugate (Vector Labs, Burlingame, Calif.) and 1-Step Ultra TMB substrate, and compared to wells coated with affinity-purified HSV-1 IgG or IVIg. TMB conversion was terminated with 2 N sulfuric acid, and absorbance was measured at 460 nm using a BioTek Synergy 2plate reader and normalized to the amount of IgG bound to coated wells quantified using F(ab′)2 anti-human IgG Fc (Goat)-HRP conjugate.
Neutralization Assay: Purified HSV-1 (˜550 PFU; 5 μL) was incubated with 95 μL of HSV-1 specific IgG solution at different final concentrations for 1 hr with end-over-end mixing. The mixture was then diluted with 210 μL of media, of which duplicate 150 μL aliquots were transferred to confluent Vero cell monolayers in a 6-well plate. Plates were incubated at 37° C. for 1 hr with periodic rocking to ensure that the plates did not dry out, before the HSV-1/Ab mixture was aspirated off and wells were washed with 2 mL of PBS. The plates were then incubated for 3 days at 37° C. in 2% carboxymethyl cellulose in EMEM supplemented with 1×L-glutamine and 1× Penicillin/Streptomycin, before staining with 1% crystal violet solution, and the resulting plaques were manually counted and compared to control wells to determine the extent of neutralization.
Multiple Particle Tracking of HSV-1 in CVM: To mimic neutralization of CVM by alkaline seminal fluid, we titrated CVM to pH 6.8-7.1 using small volumes C3% v/v) of 3 N NaOH, and confirmed pH using a micro pH electrode (Microelectrodes, Inc., Bedford, N.H.) calibrated to pH 4, 7 and 10 buffers. Samples were either untreated or treated by addition of known amounts of purified anti-HSV-1 IgG or control (anti-biotin) IgG. Control beads consisted of red or green fluorescent 200 nm carboxyl-modified polystyrene particles (Molecular Probes, Eugene, Oreg.), either uncoated (PS; muco-adhesive) or covalently conjugated with low molecular weight (2 kDa), amine-functionalized polyethylene glycol (PEG; Rapp Polymere, Tuebingen, Germany) to produce coated particles (PS-PEG; muco-inert), as previously described (Lai et al., Proc. Natl. Acad. Sci. U.S.A. 104:1482 (2007)). Fluorescent virions or control beads (approximately 108-109 particles/mL) were added at 5% v/v to 20 μL of CVM placed in a custom-made glass chamber, and incubated for 1 hr at 37° C. prior to microscopy. The translational motions of the particles were recorded using an EMCCD camera (Evolve 512; Photometrics, Tucson, Ariz.) mounted on an inverted epifluorescence microscope (AxioObserver D1; Zeiss, Thornwood, N.Y.), equipped with an Alpha Plan-Apo 100×/1.46 NA objective, environmental (temperature and CO2) control chamber and an LED light source (Lumencor Light Engine DAPI/GFP/543/623/690). Videos (512×512, 16-bit image depth) were captured with MetaMorph imaging software (Molecular Devices, Sunnyvale, Calif.) at a temporal resolution of 66.7 ms and spatial resolution of 10 nm (nominal pixel resolution 0.156 μm/pixel) for 20 s. The tracking resolution was determined by tracking the displacements of particles immobilized with a strong adhesive, following a previously described method (Apgar et al., Biophys. J. 79:1095 (2000)). Particle trajectories were analyzed using MetaMorph software as described previously (Lai et al., Proc. Natl. Acad. Sci. U.S.A. 104:1482 (2007); Lai et al., J Virol. 83:11196 (2009); Lai et al., Proc. Natl. Acad. Sci. U.S.A. 107:598 (2010)); image contrast was adjusted to improve particle visibility, but the same contrast level was applied throughout the entire video and did not bias toward any particle population. Sub-pixel tracking resolution is obtained by determining the precise location of the particle centroid by light-intensity-weighted averaging of neighboring pixels. Trapped particles were defined as those with effective diffusivity (Deff)<0.01 μm2/s at a time scale (t) of 1 s (i.e., particles move less than their diameter within 1 s). In a subset of experiments, it was confirmed that particles defined as trapped over the course of 20 s based on this criterion remain confined to the same locations over more than 15 min. The slope, α, of the log-log mean square displacement (<MSD>) vs. time scale plot provides a further measure of particle mobility: α=1 for pure unobstructed Brownian diffusion, e.g., particles in water, a becomes smaller as obstruction to particle diffusion increases, and a is zero for permanently trapped particles. At least five independent experiments in CVM from different donors, with n≥100 particles per experiment, were performed for each condition. For a subset of donors, similar observations were made at least twice in samples obtained on separate days to ensure reproducibility, but only one sample was used for analysis.
Mouse Vaginal HSV-2 Challenge Model: All experiments conducted with mice were performed in accordance with protocols approved by the Johns Hopkins University Animal Care and Use Committee satisfying the requirements of the E.E.C. Guidelines (1986) and U.S. Federal Guidelines (1985). Female CF-1 mice (6-8 weeks old; Harlan, Frederick, Md.) were treated with Depo-Provera′ (medroxyprogesterone acetate, 2.5 mg/mouse) by subcutaneous injection into the right flank 6-8 days prior to use, Depo-Provera® synchronizes mice in a prolonged diestrus-like state, in which the vaginal epithelium thins and susceptibility of the tissue to infection increases (Cone et al., BMC Infect. Dis. 6:90 (2006)). Depo-Provera®-treated mice were randomly divided into groups of ten. The mouse vagina is pH neutral (Meysick et al., J. Parasitol. 78:157 (1992)); therefore, no attempt was made to modify vaginal pH prior to inoculation. Inocula were prepared by mixing HSV-2 (final dose 2 ID50; strain G, ATCC, Manassas, Va.) with medium or different concentrations of control (anti-biotin) or anti-gG IgG (8.F.141; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), and incubating for 1 hr at 37° C. Mice were inoculated with 20 μL, of HSV-2 solution, delivered to the vagina using a 50 μL Wiretrol (Drummond, Broomall, Pa.), fire-polished to avoid damage to the vaginal epithelium. In some studies, the mouse vagina was gently washed with ˜10 mL of normal saline delivered at 1 mL/min through a smooth ball-tipped gavage needle connected to a syringe pump, prior to HSV-2 challenge. Removal of mucus by this process was measured using a fluorimetrie mucin assay, as previously described (Crowther et al., Anal. Biochem. 163:170 (1987)). Importantly, the gentle wash did not damage the vaginal epithelium, as confirmed by microscopy with a fluorescence-based dead cell stain (YOYO-1) that assesses membrane integrity (Cone et al., 2006), compared to conventional lavage and/or vaginal swabbing with a cotton tip, which induced significant epithelial damage. YOYO-1 has been previously used to reveal toxicity caused by detergent-based microbicides that led to increased susceptibility to HSV infection (Cone et al., BMC Infect. Dis. 6:90 (2006)). Tissue sectioning and H&E staining was performed by the Animal Histopathology lab at the University of North Carolina—Chapel Hill. Infection was assayed three days post-inoculation by detection of virus in vaginal lavages. Briefly, 50 μL of medium was pipetted in and out of the vagina 20 times, diluted to 0.2 mL and placed on target cells (ELVIS® HSV Test System; Diagnostic Hybrids, Athens, Ohio); infected cells (foci) were identified one day later, following manufacturer protocol. Scores for virus shedding were assigned on a scale of 0-4 based on the approximate density of foci observed (“0”: 0; “0.5”: <100; “1”: 100-500; “2”: 500-1000; “3”: 1000+; “4”; saturated). At least three independent experiments were performed for each condition, with n=10 animals per experimental group (n≥30 total).
Statistical Analysis: correlation between endogenous anti-HSV-1 IgG levels and average particle or virus Deff in individual CVM samples was measured using Pearson's correlation coefficient (r). Statistical comparisons were limited to two groups (test group compared with the appropriate control group performed during the same experiment). Fisher's exact test was used to determine the statistical significance of reductions in % mice infected. A two-tailed Student's t-test (paired for comparisons of Ab-treated vs, native CVM for the same CVM samples) was used for all other comparisons. Differences were deemed significant at an alpha level of 0.05. All values are reported as mean±SEM unless otherwise indicated.
Trapping of HSV in Cervicovaginal Mucus
The hypothesis of trapping-in-mucus was explored using HSV-1 (d˜180 nm), a highly prevalent sexually transmitted virus. Fresh, undiluted CVM was obtained predominantly from donors with normal lactobacillus-dominated vaginal microbiota, as confirmed by Nugent scoring (Table 1). HSV-1 virions expressing a VP22-GFP tegument protein construct, packaged at high copy numbers while maintaining native viral envelope integrity, were mixed into CVM pH-neutralized to mimic neutralization by alkaline seminal fluid. Time-lapse microscopy of virion motions in real-time was then performed with high spatiotemporal resolution, and virion mobility was quantified using multiple particle tracking over a long time scale. Substantial differences in HSV-1 mobility were observed in CVM samples from different donors (
1Cycle day calculated as the number of days from the first day of the last menstrual period normalized by the cycle length to a 28 day cycle.
2N/A= hormonal contraceptive.
3Cycle phase estimated based on normalized cycle day; no samples were ovulatory based on absence of spinnbarkeitby visual inspection.
4A Nugent score of 0-3 corresponds to “normal” (lactobacilli-dominated) microflora, 4-6 to “intermediate”, and 7-10 to “bacterial vaginosis” (BV)-a condition associated with greater risk of STI acquisition. Assessment of Nugent scores was independently confirmed by the Clinical Microbiology and Immunology Lab at UNC.
5Values are expressed as a mean ± SEM.
Since IgG is the predominant immunoglobulin in human CVM (Usala et al., J. Reprod. Med. 34:292 (1989)), it was examined whether virion mobility correlated with endogenous virus-specific IgG in all 12 CVM samples using a whole-virus ELISA assay (Table 2). In good agreement with the hypothesis, HSV-1 diffused rapidly through all CVM samples that had little or no detectable endogenous anti-HSV-1 IgG (<0.2 μg/mL; detection limit 0.017 μg/mL) at rates only several-fold lower than their expected rates in water (
A well-recognized mechanism of mucosal immune defense is ‘immune exclusion’ in which microorganisms in the gut are agglutinated by secreted polyvalent IgA and IgM into clusters too large to diffuse through mucus (Hamburger et al., Curr. Top. Microbiol. Immunol. 308:173 (2006); Mantis et al., Mucosal Immunol. 4:603 (2011)). However, little to no agglutinated HSV-1 was in these experiments, consistent with previous findings that IgG is a relatively poor agglutinator (Berzofsky et al., In Fundamental Immunology. W. E. Paul, editor The Raven Press, New York, N.Y. 421 (1993)). Together, these observations suggest that individual HSV-1 virions in samples with elevated endogenous levels of anti-HSV-1 IgG are slowed or trapped by multiple low-affinity bonds with CVM rather than by physical (steric) obstruction.
To confirm that trapping of HSV-1 in CVM was mediated specifically by IgG bound to virions and not by any other component in mucus that might be associated with elevated endogenous anti-HSV-1 IgG, HSV-1 specific IgG was affinity-purified from human intravenous immunoglobulin (starting with a pure clinical IgG preparation), and the purified IgG was mixed into CVM samples that had low endogenous anti-HSV-1 IgG. It was found that addition of 1 μg/mL anti-HSV-1 IgG trapped HSV-1 with a potency comparable to that of endogenous anti-HSV-1 IgG (
It was next sought to determine the biochemical basis of the low-affinity bonds between IgG and CVM, The Fc domain of all IgGs harbors a conserved N-glycosylation site at Asn297, and many IgG effector functions are Fe- and Asn297 glycan-dependent (Ha et al., Glycobiology 21:1087 (2011)). Thus, F(ab′)2 fragments (
To determine whether trapping viruses in mucus can protect against infection in vivo, the ability of a non-neutralizing monoclonal IgG1 to reduce HSV-2 transmission in the pH neutral mouse vagina was evaluated. This monoclonal IgG1binds to the relatively sparse gG surface glycoprotein, and exhibited no neutralization activity across all concentrations tested in vitro (
The first evidence of antibody-mucin affinity can be traced back more than 30 years, when Kremer and Jager noted that infertility in humans is often caused by anti-sperm antibodies (Jager et al., Fertil. Steril. 36:792 (1981); Kremer et al., Fertil. Steril. 27:335 (1976)). In cervical mucus samples with high levels of anti-sperm Ab, they found that both individual and agglutinated sperm make no forward progress and shake in place for hours until they die, despite vigorous flagellar motility. More recently, Phalipon et al. suggested secretory IgA can aggregate pathogenic Shigella flexneri in mouse nasal mucus secretions via the secretory component, anchoring the bacteria to the mucus gel and thereby ‘excluding’ them from infectious entry (Phalipon et al., Immunity 17:107 (2002)).
In both of the above instances, the authors assumed that the antibodies were attached firmly to the mucins. Similarly, the mucin-like Fey binding protein (FcγBP) has been proposed to serve an immunological role in mucus through its ability to bind strongly to IgG Fc (Kobayashi et at, Gut 51:169 (2002)). Nevertheless, more recent evidence indicates the primary function of FcγBP is instead to stabilize gastrointestinal mucus gel by covalently cross-linking Muc2 mucins (Johansson et at, Proc. Natl. Acad. Set. U.S.A. 108 Suppl 1:4659 (2011); Johansson et at, J. Proteome Res. 8:3549 (2009)). An IgG Fc-FcγBP-mucin crosslinking mechanism also directly contradicts numerous prior efforts that have failed to detect any significant binding of individual Ab to mucins (Clamp, Biochem. Soc. Trans. 5:1579 (1977); Cone, In Handbook of Mucosal Immunology. P. L. Ogra et al., editors. Academic Press, San Diego, Calif. 43-64 (1999); Crowther et al., Fed. Proc. 44:691 (1985); Olmsted et al., Biophys. J. 81:1930 (2001); Saltzman et al., Biophys. J. 66:508 (1994)). Indeed, previous FRAP experiments (Olmsted et al., Biophys. J. 81:1930 (2001)) and those here demonstrate that IgG and other Ab diffuse rapidly in human genital mucus, slowed only slightly compared to their diffusion in water. This can only be explained by weak and short-lived adhesive interactions between IgG and the mucin mesh, and not by the strong binding of IgG by FcγBP (Kobayashi et al., J. Immunol. 143:2567 (1989)). FcγBP also binds broadly to all IgG, and the interaction is thus subject to competitive inhibition (Kobayashi et at, J. Immunol. 143:2567 (1989)). In the present experiments where exogenous HSV-1 specific IgG was added to CVM, total levels of IgG already present in the samples were hundreds- to thousands-fold higher than the HSV-1 specific IgG doses added. Thus, for FcγBP to be responsible for the observed trapping phenomena, FcγBP must have been present in greater molar quantities than native IgG, which is unlikely given that the protein has not been routinely identified in proteomic screens of human genital tract fluid (see (Andersch-Bjorkman et al., Mol. Cell. Proteomics 6:708(2007)) vs (Dasari et al., J. Proteome Res. 6:1258 (2007); Shaw et al., J. Proteome Res. 6:2859 (2007); Tang et at, J. Proteome Res. 6:2874 (2007)). In contrast to previous studies, by examining the effect of IgG on virions in mucus gel rather than probing directly for interactions between individual IgG molecules and mucins, the present results document not only the potent trapping of individual virions by multiple surface-bound IgG, but also that the IgG-mucin interactions are Fc- and glycan-dependent.
Trapping virions in genital tract mucus should markedly reduce heterosexual transmission of viral infections. Women acquire many of the major sexually transmitted viral infections (e.g., HIV, HPV, and HSV) at rates on the order of 1 per 100 to 1,000 sex acts on average. This suggests few if any of these virions are able to infect target cells per intercourse, and therefore any reduction in the flux of virions that reach target cells should proportionally reduce transmission rates. Blocking initial infection altogether, rather than attempting to clear initial infections, may be especially critical for infections that are difficult, if not impossible, to cure once established (e.g., HSV, HIV). In the recent gD2-AS04 HSV vaccine trial (Belshe et al., New Engl J. Med. 366:34 (2012)), protective efficacy was initially observed in seronegative women but not in men or seropositive women, and a larger study of seronegative women revealed only moderate efficacy against HSV-1 (˜35% efficacy against HSV-1 infection and 58% efficacy against HSV-1 genital disease) but interestingly no protection against HSV-2. In both studies, the vaccine elicited neutralizing serum Ab against HSV as well as HSV-specific cellular immune responses in all women and men. However, because mucosal levels of Ab were not monitored, it remains unclear whether what little protection was observed could have correlated with mucosal Ab response. It is likely that generating sufficient mucosal Ab levels remains a major bottleneck to an effective HSV vaccine. Inducing secreted Ab that bind to ‘non-neutralizing’ surface epitopes should trap pathogens as effectively as those that bind to neutralizing epitopes, a prospect that may broaden potential antigen targets for vaccine development, especially against virions with rapidly evolving neutralizing epitopes.
Trapping of Salmonella typhimurium in Gastrointestinal Mucus
Fe-mediated trapping of pathogens in mucus, which directly blocks infections at the portals of entry, may represent an exceptionally potent mechanism by which the immune system can rapidly adapt and reinforce multiple mucosal surfaces against diverse and rapidly evolving pathogens. A microscopy setup was developed that enables measuring bacterial mobility in real time directly in mucus overlaying intestinal epithelial tissue excised from mice (
Role of N-Glycans on IgG-Mucin Interactions
To further evaluate the role of N-glycans on IgG-mucin interactions, a number of anti-gD antibodies (“HSV8”) with distinct glycosylation patterns were tested in the CVM assay of Example 1. Antibodies with distinct glycosylation profiles were produced in Nicotiana benthamiana plants using a viral-based transient expression system (magnICON). Transgenic Nicotiana benthamiana, in which plant-specific N-glycans (with core α1,3 fucose and β1,2 xylose) are reduced by RNAi inhibition of plant-specific glycosyltransferases and specific glycosyltransferases were over-expressed (e.g., β1,4 galactosyltransferase, see jbc.org/content/284/31/20479.full), were used as the host plant. Extract was clarified and IgG purified by protein A chromatography. Antibodies that were tested include HSV8-GnGn, in which contains about 95% of the antibodies contain the core structure with GlcNAc, mannose and terminal GlcNAc; HSV8-Gal, in which about 63% of the antibodies have either one or two terminal galactose sugar residues and about 5% have the GnGn core structure; HSV8-Agly, which is free of any glycosylation; and HSV8 produced in 293 cell cultures, which have the general diversity in glycosylation profiles found in humans. Note that HSV8-Gal represents the major serum IgG glycoform. We found that surprisingly, HSV8-Gal were substantially less potent at trapping viruses as HSV8-GnGn, which suggests antibodies that are engineered to have greater amount/extent of GnGn glycosylation vs. the naturally found Gal-terminated glycosylation is expected to be more potent at trapping virions. This observation not only further confirms the dependence on glycosylation, but underscores the importance of the precise nature of the glycosylation pattern.
As discussed above, the present invention is also based, in part, on the discovery and characterization of weak binding interactions between antibodies and mucins and the ability of such antibodies to stop the penetration of virions through mucus layers of the respiratory tract, as generally illustrated in
As mentioned above, the presently-disclosed subject matter includes antibodies, compositions, and methods for inhibiting and/or treating viral infection, and/or eliminating virions from a mucosal surface of the respiratory tract. In particular, the presently-disclosed subject matter relates to antibodies and compositions capable of trapping virions in mucus, thereby inhibiting transport of virions across or through mucus secretions.
One aspect of the invention relates to an antibody, e.g., a recombinant monoclonal antibody molecule, comprising a human Fc portion and a set of CDRs with specific affinity for a virion present in the lung of a subject.
In some embodiments, the antibody binds specifically to a single epitope on a single virion. In some embodiments, the antibody is a multimeric construct, e.g., wherein 2 or more Fabs (e.g., 2, 3, 4, 5, or more) are associated with a single Fc domain. The Fabs may be the same or different, and may recognize the same epitope on a virion, different epitopes on the same virion, or epitopes on different virions. The multimeric constructs are expected to facilitate more effective agglutination of both virus and/or virus-infected cells, resulting in more effective elimination from mucosal surfaces. Such multimeric antibody constructs can be prepared by linking multiple Fab domains via a flexible peptide linker. The heavy- and light-chain gene sequences for IgG control antibodies and each of the Fab-based multimeric antibodies may first be optionally codon-optimized, then synthesized and cloned into mammalian expression vectors (such as those offered by, e.g., Integrated DNA Technologies). For each format, Fab-components may be separated by a flexible peptide linker (e.g., amino acids comprising about 6 repeated units of GSSSS (SEQ ID NO:13), e.g., 2, 3, 4, 5, 6, 7, or 8 repeated units).
In some embodiments, the antibody may be a bispecific or multispecific construct against more than one virion. In one example, the construct may bind both RSV and metapneumovirus (MPV). In some embodiments, a bispecific antibody (e.g., Fab-IgG1, where an extra Fab is introduced in the N-terminus immediately adjacent to the native Fab of IgG) can be produced, e.g., by introducing separate orthogonal mutation sets (Lewis et al., Nature Biotechnol. 2014) into Fab A versus Fab B to ensure proper pairing of heavy and light chains. Fab A and Fab B may be combined with CH1/CL and Fc regions of human IgG1 Ab.
The CDRs may be any set of CDRs known in the art or later identified that specifically bind a virion.
In some embodiments, the virus is Ebola virus and the CDRs are selected from any combination of the CDRs found in known anti-Ebola virus antibodies.
In some embodiments, the virus is RSV and the CDRs are selected from any combination of the CDRs found in known anti-RSV antibodies. In some embodiments, the CDRs are from any of the antibody sequences disclosed in U.S. Pat. No. 8,562,996 (referred to as palivizumab, or SYNAGIS), incorporated by referenced herein in its entirety, or derivatives thereof (e.g., motavizumab, Wu H et al, 2007, J. Mol. Biol. 368, 652-665). In some embodiments, the CDRs may be one or more of the CDRs found in any of the heavy and light chain sequences below.
DIWWDDKKDYNPSLKS RLTISKDTSKNQVVLKVTNMDPADTATYYCAR
SMITNWYFDV WGAGTTVTVSS (SEQ ID NO: 3)
DTSKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC FQGSGYPFT
Any of the antibodies described herein may be muco-trapping antibodies, configured to transiently bind to mucus, as described above. For example, in some embodiments, the antibody comprises an oligosaccharide at a glycosylation site, the oligosaccharide comprising, consisting essentially of, or consisting of a pattern correlating with (providing) enhanced trapping potency of the antibody in mucus, and wherein the antibody specifically binds an epitope of a target virion. The unique glycosylation pattern/unique oligosaccharide component of the antibody is designed to maximize trapping potency of the antibody once a plurality of antibodies are bound to the target virion, without unduly hindering the ability of the unbound antibody to diffuse readily through mucus to rapidly bind a target virion. In certain embodiments, the antibody is one that exhibits a mobility in mucus that is reduced no more than about 50%, e.g., no more than about 40%, 30, 20%, 10%, or 5%, relative to its mobility in solution (e.g., saline or water) and effectively traps a target virion in mucus based on a plurality of bound antibodies (e.g., at least 50% of virions slowed by at least 90%). In some embodiments, the antibody reduces the mobility of at least 50% of the virions, e.g., at least 60%, 70%, 80%, or 90% or more of the virions by at least 90%, e.g., at least 95%, 96%, 97%, 98%, or 99% or more. In other embodiments, the antibody reduces the percentage of virions that can penetrate mucus by at least 10%, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Based on the disclosure herein, one of skill in the art can readily identify/design oligosaccharide patterns that provide the desired trapping potency. In other embodiments, the antibody has a sufficient binding rate to an epitope of the target virion to accumulate on the surface of the target virion at sufficient levels to trap the target virion in mucus within one hour (e.g., within 30 minutes or 15 minutes) at an antibody concentration in the mucus of less than 5 μg/mL (e.g., less than 1 μg/mL or 0.1 μg/mL).
In some embodiments, the oligosaccharide component is bound to an N-linked glycosylation site in an Fc region of the antibody. The N-linked glycosylation site can be an asparagine residue on the Fc region of the antibody, for example, the Asn 297 asparagine residue. The amino acid numbering is with respect to the standard amino acid structure of a human IgG molecule.
As described in greater detail above, the N-glycan structure on human IgG-Fc is typically dominated by a biantennary core structure that shares a common core sugar sequence, Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser/Thr, with “antennae” initiated by N-acetylglucosaminyltransferases (GlcNAcTs) that help attach additional sugars to the core. In IgG found in human serum, the most common structures are those that contain both N-acetylglucosamine on each branch with one terminal galactose (39%), two terminal galactose (20%), or one terminal galactose and one terminal sialic acid (15%). Together, antibodies that comprise at least one terminal galactose represents about 74% of the IgG-Fc glycoforms. A pure GnGn form (with terminal N-acetylglucosamine on each branch without terminal galactose or sialic acid) may represent about 26% of the IgG-Fc glycoforms.
In some embodiments, the glycan does not contain any galactose residues. Without being limited by theory, it is believed that the presence of galactose compromises trapping potency. Antibodies with glycoforms that do not contain galactose represent just a small fraction of the entire repertoire of glycoforms found in nature. The use of a population of antibodies enriched with desirable glycoforms (whether naturally occurring or modified glycans) is advantageously used for trapping pathogens in mucus.
In some embodiments, the antibody of the invention is a mixture of antibodies having different oligosaccharide components. In some embodiments, the mixture of antibodies comprises at least about 20% (e.g., about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, etc.) antibodies having the core glycan structure described above (e.g., GnGn) with or without a fucose residue, e.g., at least about 40%, 50%, 60%, 70%, 80%, 90% or more.
In some embodiments, the mixture of antibodies is the mixture generated in a human cell line, e.g., a 293 cell line, e.g., a 293T cell line. In some embodiments, the mixture of antibodies is the mixture generated in a plant cell or in a plant.
The antibody may be useful for binding target virions to trap the pathogen in mucus of the respiratory tract to inhibit infection by the virions. Target virions of the antibody can include any virus that can infect a subject through a mucus membrane. Target virions further include synthetic systems comprising an antigen having an epitope, for example particles or particulates (e.g., polystyrene beads) comprising attached proteins, e.g., as might be used for bioterrorism.
Viruses include those that cause respiratory diseases, including, without limitation, influenza (including influenza A, B, and C); severe acute respiratory syndrome (SARS); respiratory syncytial virus (RSV); metapneumovirus; parainfluenza; adenovirus; human rhinovirus; coronavirus; and norovirus. Viruses also include those that do not necessarily cause respiratory disease but can infect a subject by entry through the respiratory tract, e.g., Ebola virus.
Viruses include those that affect non-human animals, such as livestock, e.g., swine (e.g., porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza, porcine circovirus), ruminants (e.g., RSV, adenovirus, reovirus), ungulates (e.g., bocaparvovirus), horses (e.g., equine herpesvirus-1, equine herpesvirus-4, equine influenza virus), poultry (e.g., avian influenza virus, avian infectious bronchitis virus (IBV)), and the like.
The terms virus and viral pathogen are used interchangeably herein, and further refer to various strains of virus, e.g., influenza is inclusive of new strains of influenza, which would be readily identifiable to one of ordinary skill in the art.
In some embodiments, it is contemplated that an antibody according to the presently-disclosed subject matter is capable of broadly binding to viruses containing lipid envelopes, which are not necessarily specific to one virus.
It was surprisingly discovered that sub-neutralization doses of an antibody can be used to effectively trap a target virion in mucus. As such, in some embodiments, wherein the antibody specifically binds a neutralizing epitope of the target virion, a sub-neutralization dose can be used. A sub-neutralization doses is a dose below that which would be needed to achieve effective neutralization.
As will be recognized by one of skill in the art, appropriate doses may differ between virions, between mucosal surfaces, and also between individuals. It will also be recognized that different subjects and different mucosal surfaces may have different optimal glycan patterns and optimal antibody-mucin affinities, contributing to different optimal doses.
It is further proposed herein that antibodies that selectively bind non-neutralizing epitopes of a target virion can be used to effectively trap the target virion in mucus. As such, in some embodiments, the antibody specifically binds a non-neutralizing epitope, e.g., one or more non-neutralizing epitopes.
The presently-disclosed subject matter further includes an antibody that selectively binds a conserved epitope of a target virion. A benefit of targeting a conserved epitope would be to preserve efficacy of the antibody as against new strains of the virus. Targeting such epitopes has been avoided at times in the past because they were viewed as being ineffective targets; however, in view of the disclosure herein that non-neutralizing epitopes can serve as effective targets and/or that sub-neutralization doses can be effective for inhibiting infection, previously dismissed conserved epitopes of target virions can be seen as effective targets.
As noted above, it was determined that the low-affinity binding interactions that an antibody forms with mucins are influenced by antibody glycosylation, and are also Fc-dependent. As such, the presently-disclosed subject matter includes antibodies having a preserved and/or engineered Fc region. Such antibodies can be, for example, one or more of IgG, IgA, IgM, IgD, or IgE. In certain embodiments, the antibodies are IgG. In some embodiments, the antibodies are one or more subclasses of IgG, e.g., IgG1, IgG2, IgG3, IgG4, or any combination thereof.
In some embodiments, the antibody has a sufficient binding rate and/or binding affinity to an epitope of the target virion to accumulate on the surface of the virion at sufficient levels to trap the virion within one hour after administration of the antibody at an antibody concentration of less than about 5 μg/mL, although excess antibody may be used (e.g., about 5 μg/mL or more, 10 μg/mL or more, 15 μg/mL or more, 20 μg/mL or more, 25 μg/mL or more, 30 μg/mL or more, 40 μg/mL or more, 50 μg/mL or more, 60 μg/mL or more, 70 μg/mL or more, 80 μg/mL or more, 90 μg/mL or more, 100 μg/mL or more, etc.). The term “trap” in this instance refers to reduction of further movement through the mucus. In some embodiments, the target virion may be trapped within about 30 minutes, e.g., about 25, 20, 15, or 10 minutes after administration of the antibody. In some embodiments, the antibody traps the target virion at an antibody concentration of less than about 4, 3, 2, or 1 μg/mL.
As mentioned, formulations for administration, including intranasal administration, etc., are contemplated for use in connection with the presently-disclosed subject matter. All formulations, devices, and methods known to one of skill in the art which are appropriate for delivering the antibody or composition containing the antibody to one or more mucus membranes of the respiratory tract of a subject can be used in connection with the presently-disclosed subject matter. The antibody can be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the antibody, which the subject inhales. The respirable particles can be liquid or solid. The term “aerosol” includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth. 27:143 (1992). Aerosols of liquid particles comprising the antibody can be produced by any suitable means, such as with a vibrating mesh nebulizer, a pressure-driven aerosol nebulizer, or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles comprising the antibody can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art. In some embodiments, the nebulizer and nebulization conditions are selected to produce aerosol particles of a desired size. In one embodiment, the nebulized composition has a Mass Median Aerodynamic Diameter (MMAD) in the 2-5 mm range, as measured using a Next Generation Impactor.
In some embodiments of the presently-disclosed subject matter, a composition includes a first antibody and a second antibody, as disclosed herein, wherein the first antibody specifically binds a first epitope of the target virion and the second antibody specifically binds a second epitope of the target virion, wherein said first epitope is distinct from the second epitope. In certain embodiments, the composition includes three or more different antibodies, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different antibodies, wherein each antibody specifically binds a different epitope of the target virion.
It may also be desirable to provide a composition that can provide treatment or prevention of infection due to more than one target virus. In some embodiments of the presently-disclosed subject matter, a composition includes a first antibody and a second antibody, as disclosed herein, wherein the first antibody specifically binds an epitope of a first target virion and the second antibody specifically binds an epitope of second target virion. In certain embodiments, the composition includes three or more different antibodies, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different antibodies, wherein each antibody specifically binds an epitope of a different target virion.
In some embodiments, the pharmaceutical composition can further include an additional active agent, e.g., a prophylactic or therapeutic agent. For example, the additional active agent can be an antimicrobial agent, as would be known to one of skill in the art. The antimicrobial agent may be active against algae, bacteria, fungi, parasites (helminths, protozoa), viruses, and subviral agents. Accordingly, the antimicrobial agent may be an antibacterial, antifungal, antiviral, antiparasitic, or antiprotozoal agent. The antimicrobial agent is preferably active against infectious diseases. Examples of suitable antimicrobial agents (e.g., one or more antibacterial, antifungal, antiviral, antiparasitic, or antiprotozoal agents) include those described above.
The presently-disclosed subject matter further includes methods of treating, inhibiting, or preventing a viral infection in a subject in need thereof, the viral infection characterized by a virion in the lung of the subject, the method comprising administering, via an inhaled route, a recombinant monoclonal antibody molecule comprising a human Fc portion and a set of CDRs with specific affinity for the virion, thereby treating, inhibiting, or preventing the infection. The recombinant monoclonal antibody molecule can be an antibody and/or composition as disclosed herein. In certain embodiments, the methods comprise additional steps such as one or more of isolating the antibodies, preparing a composition of the isolated antibodies, determining the level of antibodies in the mucus of the subject before administering the antibodies, and determining the level of antibodies in the mucus of the subject after administering the antibodies.
The virion may be any virus as discussed herein. In some embodiments, the virion is Ebola virus. In some embodiments, the virion is RSV. In some embodiments, the antibody comprises any of the CDR sequences disclosed herein.
The antibodies and compositions of the present invention according to the methods described herein are administered or otherwise applied by delivering the composition, e.g., to the lungs, e.g., by inhalation. The subject may be one where an infection is already present in the lungs (an actual site of infection) or where an infection is likely to occur in the lungs (a potential site of site of infection in an uninfected individual). In some embodiments, the antibodies and compositions may be delivered to the respiratory tract, e.g., the nasal cavity and the lungs, by any method known in the art to be effective. In some embodiments, the antibodies and compositions may be delivered directly to the respiratory tract. In other embodiments, the antibodies and compositions may be systemically delivered such that the antibodies are secreted into the mucus of the subject. Accordingly, the compositions as described above may be delivered to a mucosal surface of the respiratory tract.
In some embodiments, the antibody is formulated for delivery to the respiratory tract. In some embodiments, the molecule is formulated as an aerosol composition. In some embodiments, the aerosol composition is suitable for nebulization. Any type of suitable nebulizer may be used. In one embodiment, the aerosol composition is nebulized by a vibrating mesh nebulizer. In some embodiments, the nebulizer and nebulization conditions are selected to produce aerosol particles of a desired size. In one embodiment, the nebulized composition has a Mass Median Aerodynamic Diameter (MMAD) in the 2-5 mm range, as measured using a Next Generation Impactor.
An effective amount of the antibody can be administered. As used herein, an “effective amount” of the antibody for inhibition or prevention of infection refers to a dosage sufficient to inhibit or prevent infection by the target virion. As used herein, an “effective amount” of the antibody for treatment of infection refers to a dosage sufficient to inhibit spread of the target virion from infected cells to non-infected cells in the subject and/or to inhibit spread of the target virion from the infected subject to another subject, e.g., a non-infected subject. The effective amount can be an amount sufficient to trap an amount of the target virion in mucus. As will be recognized by one of skill in the art, the amount can vary depending on the patient and the target virion. The exact amount that is required will vary from subject to subject, depending on the species, age, and general condition of the subject, the particular carrier or adjuvant being used, mode of administration, and the like. As such, the effective amount will vary based on the particular circumstances, and an appropriate effective amount can be determined in a particular case by one of skill in the art using only routine experimentation. In some instances, an effective amount of the antibody that specifically binds the target virion can be an amount that achieves a concentration of the antibody in the mucus of about 50 ng/mL to about 1000 μg/mL, e.g., about 0.1 μg/mL to about 100 μg/mL, about 0.5 μg/mL to about 100 μg/mL, about 1 μg/mL to about 50 μg/mL, about 5 μg/mL to about 500 μg/mL, about 5 μg/mL to about 300 μg/mL, about 20 μg/mL to about 200 μg/mL, or any range therein (e.g., between 0.5 μg/mL to about 20 μg/mL).
In some embodiments, the antibody may be administered in two or more stages with different doses in each stage. For example, higher doses can be administered initially in order to clear target virions that are present in the mucus of exposed or infected subjects and ensure that sufficient amounts of antibody remain in the mucus to provide protection, e.g., for about 24 hours. In later stages, lower doses can be administered to maintain protective levels of the antibody. In other embodiments, protective doses can be administered to subjects that are likely to be exposed to a virus and higher doses can be administered if infection occurs.
In some embodiments, the antibody or composition is administered at regular intervals until an effect is achieved. The interval may be e.g., multiple times a day (e.g., every 1, 2, 3, 4, 5, 6, 8, 12, 18 or 24 hours, e.g., every 3-24 hours)), once every 24, 48, or 72 hours, or once a week.
As will be recognized by one of skill in the art, the term “inhibiting” or “inhibition” or “preventing” or “prevention” does not refer to the ability to completely eliminate the possibility of infection in all cases. Rather, the skilled artisan will understand that the term “inhibiting” or “preventing” refers to reducing the chances of virions moving through mucus beyond the mucus membrane such that infection of a subject can occur, such as reducing chances of infection by a virion when such virion is bound to trapping antibodies in mucus. Such decrease in infection potential can be determined relative to a control that has not been administered the antibodies of the invention. In some embodiments, the decrease of inhibition potential relative to a control can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease.
In some embodiments inhibiting or treating an infection in a subject can comprise trapping a virion in mucus. As such, in some embodiments a method of trapping a target virion in mucus is provided, which method includes administering to a mucosa of the respiratory tract of the subject an antibody or composition as described herein.
In some embodiments, a method of inhibiting or treating an infection in a subject, and/or trapping a virion in the mucus of a subject, involves administering to a mucosa of the respiratory tract of the subject a composition comprising an isolated antibody that specifically binds a non-neutralizing epitope of a target virion. The antibody can be a non-neutralizing antibody. In some embodiments, the non-neutralizing antibody is provided at a concentration above a predetermined amount.
In some embodiments, a method of inhibiting or treating an infection in a subject, and/or trapping a virion in the mucus of a subject, involves administering to a mucosa of the respiratory tract of the subject a composition comprising an isolated antibody that specifically binds a neutralizing epitope of a target virion, wherein the antibody is provided at a sub-neutralization dose.
Having described the present invention, the same will be explained in greater detail in the following, examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.
Blocking Penetration of Ebola Viruses in Human Airway Mucus
Ebola virus, a member of the filovirus family of viruses that cause severe and often fatal hemorrhagic fevers, readily infects many cell types, including immune cells, fibroblasts, endothelial cells and epithelial cells. Given the broad tissue tropism of Ebola, effective treatments of systemic Ebola infection generally require high doses of therapeutic molecules administered systemically. For instance, ZMapp™, a cocktail of three chimeric monoclonal antibodies, was evaluated at 50 mg/kg in a randomized, controlled clinical trial in Guinea, Sierra Leone and Liberia during the 2014-2016 Ebola outbreak.
Rather than treating Ebola infections systemically, a potential alternative strategy is to block or treat infections at the portals of entry before virions proliferate and spread throughout the body. In addition to transmission by direct contact with the blood, bodily fluids, or skin of Ebola-positive individuals, it is possible that Ebola may be transmitted via virus-laden droplets generated from a heavily infected individual by coughing, sneezing, vomiting or medical procedures that are directly propelled onto the mucus membranes of a nearby person. The term droplet-based aerosol transmission may be used to differentiate this potential mechanism from strict airborne transmission of individual viruses, which is generally considered an unlikely mechanism of Ebola transmission. While aerosol transmission of Ebola has not been ascertained in humans, it has been demonstrated in multiple animal studies, including with non-human primates. Given the possibility of aerosol transmission of Ebola to humans, as well as the potential threat of aerosolized filovirus-based biowarfare agents, we sought to investigate the fate of Ebola deposited at mucosal surfaces.
Mucus membranes are characterized by a layer of mucus secretions that can trap diverse foreign particles and pathogens, facilitate their elimination via natural mucus clearance mechanisms, and consequently reduce the flux of pathogens reaching target cells. Human airway mucus (AM) is likely responsible in part for the relatively modest transmission rates of many respiratory viruses, but it is also likely that AM can be reinforced to further limit the flux of pathogens reaching the underlying epithelium. As described above, IgG antibodies (Ab) in cervicovaginal mucus can trap viruses in mucus via multiple low-affinity Fc-mucin bonds between IgG accumulated on the virus surface and mucins, akin to a Velcro® patch. Further, the immobilization of H1N1 and H3N2 influenza viruses in human AM may be correlated to the presence of influenza-binding IgG and IgA. Here, we investigate whether topically dosed IgG against Ebola may similarly trap Ebola in AM and facilitate their elimination from the airways.
Ebola virus-like particles (VLP) were prepared by transfecting 293T cells with plasmids encoding Gag-mCherry and Ebola GP. 293T cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (FBS) and 2 mM L-glutamine (DMEM-10). All cell cultures were maintained at 37° C. in a humidified 5% CO2 atmosphere. 293T cells (2.0×106) were seeded in a 25 cm2 flask (Thermo Scientific, Rochester, NY) and transfected with the expression plasmids using X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics, Indianapolis, IN). Gag-mCherry and Ebola GP plasmids were mixed in a 1:1 ratio (1.5 μg of each), and added to 500 μL of serum free DMEM with 9 μL of X-tremeGENE HP DNA Transfection Reagent. The mixture was incubated at room temperature for 30 min before being added to the culture of 293T cells. After 3 to 5 hr incubation at 37° C. in 5% CO2, transfected cells were washed extensively with DMEM and incubated for additional 24-48 hr with 2 mL of DMEM-10 at 37° C. in 5% CO2. Supernatants from virus particle-producing cultures were then collected and clarified by centrifugation for 10 min at 300×g, filtered through a low protein binding 0.45 μM syringe filter (Millipore, Bedford, MA) and partially purified through 25% w/v sucrose in Hepes-NaCl buffer by centrifugation at 221,630×g at 4° C. for 2.5 hr. The pellet was resuspended overnight at 4° C. in 10% sucrose in Hepes-NaCl buffer, aliquoted, and stored at −80° C.
To measure incorporation of Ebola GP protein into the VLP, sucrose gradient-purified VLP were lysed in buffer containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2% Triton X-100 and protease inhibitors at 4° C. for 30 min. Recombinant Ebola glycoprotein rGPd™ (#0501-001; IBT Bioservices, Rockville, MD) was run as a positive control. Samples were boiled in SDS-PAGE sample buffer with 2-mercaptoethanol and separated on 4%-12% Tris-Glycine gradient gels (Invitrogen, Grand Island, NY), transferred to nitrocellulose membranes, and probed with Mouse Ebola GPII Monoclonal Antibody (MyBiosource, San Diego, CA) at a concentration of 1:2,000, as shown in
Both the ZMapp™ and any of the individual Ebola-binding mAbs used herein were modified to enhance musical binding, by enriching the population of Ebola-binding mAbs to include a majority (e.g., 50% or more, 55% or more, 60% or more, 65% or more, 70% or more 75% or more; 80% or more 85% or more, etc. such as 85%-95% etc.) having a G0 glycosylation pattern on the Fc region (e.g., a glycosylation pattern comprising the biantennary core glycan structure Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminal N-acetylglucosamine on each branch). Native anti-Ebola mAb (e.g., human anti-Ebola mAb) has a much lower G0 content.
Fluorescent, carboxyl-modified polystyrene beads (PS-COOH) and PEGylated nanoparticles (PS-PEG) sized approximately 100 nm were prepared and characterized. Fluorescent, carboxyl-modified polystyrene beads (PS-COOH) sized 100 nm were purchased from Molecular Probes (Eugene, OR). PEGylated nanoparticles (PS-PEG) were prepared by conjugating 2 kDa amine-modified PEG (Rapp Polymere, Tuebingen, Germany) to PS-COOH particles via a carboxyl-amine reaction. Particle size and -potential were determined by dynamic light scattering and laser Doppler anemometry, respectively, using a Zetasizer Nano ZS (Malvern Instruments, Southborough, MA). Size measurements were performed at 25° C. at a scattering angle of 90°. Samples were diluted in 10 mM NaCl solution, and measurements were performed according to instrument instructions. PEG conjugation was confirmed by a near-neutral ζ-potential (Table 3).
Fresh human airway mucus (AM) was obtained from healthy adult patients (Table 4) intubated for general anesthesia during elective surgery, following a protocol that was deemed non-human subjects research by the University of North Carolina at Chapel Hill Institutional Review Board. After surgery, the endotracheal tube was removed from the patient, and mucus coating the tube was collected by gentle centrifugation. AM that was non-uniform in color or consistency or had visible blood contamination was discarded. Samples were treated with protease inhibitor immediately after collection to minimize potential enzymatic degradation, and stored at 4° C. until microscopy, typically within 24 hr.
Total immunoglobulin levels in AM were quantified by conventional ELISA and confirmed using the Human Isotyping Kit on Luminex (HGAMMAG-301K; Millipore, Billerica, MA) according to manufacturer protocol. Briefly, 20× stock isotyping beads were vortexed, sonicated, diluted to 1×, and incubated with 50 μL of serially diluted AM supernatant at 1:2 beads:AM volume ratio. After 1 hr, the beads were separated from AM supernatant using a magnetic plate, and washed twice with wash buffer. The beads were then incubated with 25 μL of 1× anti-Human Kappa and Lambda-PE for 1 hr, washed twice, and resuspended in Luminex Drive Fluid. Fluorescence intensities indicative of immunoglobulin levels present in AM were measured using the Luminex MAGPIX system, and data analysis was performed using Milliplex Analyst (v3.5.5.0; Vigene Tech Inc., Carlisle, MA). All incubations were carried out at room temperature in the dark with vigorous agitation. Total Ig and IgG isotypes levels are shown in (Table 5).
Dilute particle solutions (˜108-109 particles/mL, 1 μL) and different Ab (2 μL, to a final concentration of 22 μg/mL) were added to 20 μL of fresh, undiluted AM in custom-made chambers, and samples were incubated 1 hr at 37° C. before microscopy for Multiple particle tracking analysis. All conditions were tested in aliquots of the same AM samples, allowing direct comparison between conditions. The trajectories of the fluorescent particles in AM were recorded using an EMCCD camera (Evolve 512; Photometrics, Tucson, AZ) mounted on an inverted epifluorescence microscope (AxioObserver D1; Zeiss, Thornwood, NY), equipped with an Alpha Plan-Apo 100×/1.46 NA objective, environmental (temperature and CO2) control chamber and an LED light source (Lumencor Light Engine DAPI/GFP/543/623/690). Videos (512×512, 16-bit image depth) were captured with MetaMorph imaging software (Molecular Devices, Sunnyvale, CA) at a temporal resolution of 66.7 ms and spatial resolution of 10 nm (nominal pixel resolution 0.156 μm/pixel). The tracking resolution was determined by tracking the displacements of particles immobilized with a strong adhesive. Particle trajectories were analyzed using Video SpotTracker (University of North Carolina, Chapel Hill, NC). Sub-pixel tracking resolution was achieved by determining the precise location of the particle centroid by light-intensity-weighted averaging of neighboring pixels. Trajectories of n≥130 particles per frame on average (typically corresponding to n≥300 total traces) were analyzed for each experiment, and 8-9 independent experiments for each condition were performed in AM collected from unique subjects. The coordinates of particle centroids were transformed into time-averaged mean squared displacements (MSD), calculated as <Δr2(τ)>=[x(t+τ)−x(t)]2+[y(t+τ)−y(t)]2 (where τ=time scale or time lag), from which distributions of MSDs and Deff were calculated. Mobile particles were defined as those moving more than approximately 200 nm (i.e. roughly twice the particle diameter) within 0.2667 s.
MSD may also be expressed as MSD=4D0τα, where α, the slope of the curve on a log-log scale, is a measure of the extent of impediment to particle diffusion (α=1 for pure unobstructed Brownian diffusion; α<1 indicates increasing impediment to particle movement as a decreases). A limitation with 15D particle tracking is the inability to track rapidly diffusing species due to diffusion out of the focal plane, particularly with sub 200 nm species. Consequently, MSD calculations at longer time scales will underestimate the true mobility of these species. Therefore, a was calculated only over the linear portion of the log-log MSD curve (here, up to τ=0.6 s). Calculating a to span τ=1 s results in a value of 0.71 vs. 0.80 for Ebola VLP in AM with no Ab, but had negligible impact on a values for Ebola VLP in AM treated with ZMapp™ or individual mAb.
In order to predict particle population behavior, we performed a first passage time analysis, calculating the expected time for 10% and 50% of a particle population to pass through a 50 μm thick layer of mucus. Given the diffusivity D of a particle, the probability that the particle has not passed through a layer of thickness L as of a given time t may be described by an explicit “survival function”. Using T to denote the time it takes for the particle to pass through the layer, the formula for this “survival function” is
Suppose that a heterogeneous population of particles have individual diffusivities {Di} with respective weights {wi} where i ranges from 1 to the number of particles N. The expected fraction of particles that remain in the fluid layer as of time t is then equivalent to the following weighted survival function:
We set wi to be the number of frames in which the ith particle is present. For each weighting, we calculated the times t10 and t50 for which P(T>t10)=0.9 and P(T>t50)=0.5.
A slightly hypotonic buffer, prepared by diluting Gibco® 1×PBS with an equal volume of Milli-Q® water to ˜150 mOsm/kg, was used to deliver Ebola VLP to the mouse lung (hypotonic solutions were found to promote advective flow to the epithelium, allowing us to more rigorously evaluate whether ZMapp™ Ab can prevent Ebola from crossing the AM layer). ZMapp™ cocktail (c2G4, c116C6FR1 and c4G7 mixed at a 1:1:1 ratio, total 25 μL per mouse) or hypotonic PBS was first loaded into a Penn Century Microsprayer (FMJ-250; Penn Century, Inc., Wyndmoor, PA), aerosolized and delivered to the airways of Balb/c mice (female, 8-10 weeks) anesthetized in an isoflurane chamber. After 15 min, Ebola VLP prepared in hypotonic PBS were instilled using the microsprayer at 25 μL per mouse, for both ZMapp™-treated and control mice. A third set of mice were treated with two instillations of hypotonic PBS to measure the autofluorescence of mouse lung tissue. Experimental and control groups had n=3 mice each. All experimental protocols were approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee, and conform to the Declaration of Helsinki conventions for the use and care of animals.
To investigate the distribution of Ebola VLP, mice were euthanized 30 min after the final microsprayer instillation and the entire lung, including trachea, was dissected. The dissected lung was kept intact, washed with 1×PBS, and embedded in 100% optimal cutting temperature (OCT) compound before freezing at −80° C. After overnight freezing, 10 μM thick transverse cross sections of the upper airways were obtained via cryosectioning at −20° C. and stained with DAPI. Cryosections were imaged using an Olympus FV1000 MPE laser scanning confocal microscope (Olympus Life Science Solutions, Center Valley, PA) at 20× magnification and the following excitation/emission spectrum: TRITC (559/603 nm) and DAPI (405/422 nm). Fluorescence intensity was quantified using ImageJ (National Institutes of Health, Bethesda, MD) for on average 10 cross sections per mouse, and background fluorescence was subtracted.
Data averages are presented as means with standard error of the mean (SEM) indicated. Statistical comparisons were limited to two groups. A one-tailed, paired Student's t-test was used for all comparisons, since different conditions were tested in aliquots of the same mucus samples. Differences were deemed significant at an alpha level of 0.05.
The Ebola virus-like particles were found to quickly penetrate human airway mucus. Wild-type Ebola virus requires Biosafety Level 4 containment that few laboratories have access to. Therefore, to investigate the fate of Ebola in mucus, we prepared fluorescent, non-infectious Ebola VLP comprised of HIV-1 Gag-mCherry capsid proteins in the core and the Zaire glycoprotein (GP), from the same species as in the West Africa epidemic in 2014-2016, on the surface. The same strategy was previously used to prepare both HIV and influenza VLP. GP incorporation into Ebola VLP was confirmed via Western blot (
To avoid the effects of dilution with using hypertonic saline to induce sputum expectoration, we obtained undiluted human airway mucus directly from freshly extubated endotracheal tubes. Ebola VLP were readily diffusive in all AM secretions tested, as shown in
Along the major conducting airways, the approximate thickness of mucus lining the bronchial airways is ˜50 μm, and the entire mucus blanket can be renewed in as little as 15-30 minutes. Therefore, we performed a first passage time estimate, using the measured diffusivities of individual VLP in AM to determine the time needed for viruses to diffuse across a 50 μm thick AM layer. We found that upon airway deposition, nearly 10% of Ebola virions can penetrate the luminal AM layer within approximately 5 minutes, and nearly 50% can penetrate the AM layer in as little as 30 minutes (
The treatment of human airway mucus with ZMapp™ may immobilize Ebola virus-like particles. While total Ig and IgG isotype levels varied substantially across AM samples (Table 5), the rapid diffusion of Ebola VLP in AM was remarkably consistent, suggesting endogenous antibody did not impact Ebola VLP mobility. We next evaluated whether Ebola-binding IgG, in the form of the three chimeric monoclonal antibody (mAb) cocktail ZMapp™, could enhance the diffusional barrier properties of AM against Ebola. The addition of modest levels of Ebola-binding IgG into AM effectively reduced the mobility of Ebola VLP, with the majority of VLP moving much less than their diameters over at least 20 s (
To determine whether any particular mAb (c2G4, c116C6FR1, c4G7) within the ZMapp™ cocktail may confer superior “muco-trapping” potency, we measured the mobility of Ebola VLP in different aliquots of the same AM specimens treated with the individual mAb. Interestingly, all three mAb, including one with Door neutralizing activity against Ebola, were similarly effective in reducing the mobility of Ebola VLP in AM, as shown in
To better illustrate how the aforementioned changes in VLP mobility might alter the flux of virions reaching target cells, we performed first passage time analysis as described above. The predicted time for 10% of Ebola VLP to diffuse across a 50 μm thick mucus layer increased from ˜0.1 hours (i.e., approximately 5 minutes) for native AM to 2.2, 2.1, 1.6, and 1.7 hours for ZMapp™-, c2G4-, c116C6FR1- and c4G7-treated AM, respectively. Similarly, the estimated time for 50% of VLP to cross the mucus layer increased from 0.5 hours for native AM to 23, 22, 20, and 22 hours, respectively. Since mucociliary clearance occurs on the order of 15-30 minutes, these results suggest that, with the exception of native AM that is devoid of Ebola-binding IgG, the vast majority of Ebola virions would be quickly trapped and eliminated from ZMapp™-treated airways before they could penetrate AM.
The topical delivery of ZMapp™ may facilitate the rapid elimination of Ebola from the lung airways: Lastly, we sought to evaluate whether ZMapp™-induced trapping of viruses in mucus secretions ex vivo would translate to an improved barrier against Ebola penetration of AM and consequently altered distribution in the lung airways in vivo. Using an aerosol delivery device (e.g., PennCentury microsprayer), we administered ZMapp™ or PBS to the mouse lung, followed by addition of fluorescent Ebola VLP 30 minutes later. In control mice treated, we observed substantial red fluorescence associated with Ebola VLP throughout the lung airways, including fluorescence indicative of mucosal penetration and accumulation in the underlying airway epithelium, as shown in
Ebola VLP readily penetrate fresh human AM secretions, but can be effectively immobilized in AM by antigen-specific IgG (e.g., ZMapp™). Trapping in turn facilitated rapid elimination of Ebola VLP from the mouse airways. Although Ebola is generally not considered an airborne pathogen, aerosol transmission of Ebola virus is biologically plausible. Ebola virus is present in saliva, feces, blood, and other body fluids that can be aerosolized through Ebola symptoms (e.g., coughing, vomiting, diarrhea) and through health care delivery (e.g., intubation, suctioning, delivery of nebulized medications). Large droplets from a human sneeze can travel up to 1-2 m, while smaller droplets can travel up to 6-8 m away within seconds to a few minutes. Studies have also found Ebola to survive in aerosol form for tens of minutes if not hours. Ebola can initiate infection in cells present in the respiratory tract, and fatal respiratory infection has been observed in guinea pigs and non-human primates following intranasal and aerosol exposure. Furthermore, Ebola transmission has been shown between infected and healthy macaques and between infected pigs and macaques without direct physical contact, likely via aerosol or droplet transmission, although potential cross-contamination during animal husbandry practices could not be discounted entirely. A study frequently cited in arguments against airborne transmission found no detectable Ebola transmission when monkeys inoculated intramuscularly were housed in neighboring open-barred cages separated by a Plexiglas® divider that prevented direct contact; nevertheless, high viral titers were only detected in the blood, suggesting virus titers in the lungs of these animals may not have achieved the critical titers necessary for respiratory transmission before the animals succumbed to the systemic effects of infection. In contrast, recent studies in humans have reported substantial quantities of Ebola in respiratory secretions, and pathology studies have also found viral antigen in lung tissue. Finally, aerosol dissemination of weaponized forms of these viruses, a version of which has reportedly already been developed for Marburg virus, presents a significant threat to both the military and potentially the general public. Altogether, these reports substantiate continued concern over aerosol transmission of Ebola virus, and the need to explore strategies to prevent and treat Ebola transmission at mucosal membranes, particularly if Ebola or other filoviruses become weaponized.
While the secretion of mucus can increase in response to infection, mucus is generally viewed as a passive rather than adaptive barrier against pathogens, and consequently overlooked in most studies of mucosal infection. The notion that Ab can work in tandem with mucus to reinforce the diffusional barrier properties of mucus has remained largely unexplored, despite the fact that large quantities of Ab, including both IgG and IgA, are secreted into AM. Here, in good agreement with our recent discovery that IgG can trap HSV-1 in human cervicovaginal mucus, we showed that Ebola-binding mAb, in the form of ZMapp™, were able to facilitate effective trapping of the majority of Ebola VLP in AM. Ab-mediated trapping of Ebola VLP markedly reduced the fraction of virions predicted to penetrate the mucus layer over the first few minutes of exposure. Since trapped viruses are quickly eliminated by natural mucociliary clearance in the airways, ZMapp™-mediated trapping of Ebola would likely reduce the total flux of viruses arriving at the airway epithelium and thus the likelihood and/or severity of infection, rather than simply delaying the onset of infection. Consistent with this hypothesis, we observed topical delivery (e.g., via aerosol) of ZMapp™ into the mouse lung greatly reduced the amount of fluorescent Ebola VLP retained in the conducting airways within 30 minutes. IgG-mediated trapping of viruses in mucus appears to be a universal protective immune function across different mucosal surfaces that enables protection directly at the portals of entry for viral transmission.
The concept of mucosal Ab prophylaxis and/or therapy based on Ab designed to work together with mucus to trap pathogens represents a unique and complementary approach in the arsenal of protective methods against infectious disease. First, the concept radically shifts the first line of defense against respiratory viruses to extracellular mucus gels instead of cellular targets, which is especially important against viruses that are either exceptionally virulent (e.g., Ebola) and/or without a cure (e.g., HIV, Ebola). Second, Ab that trap viruses in mucus need not bind to neutralizing epitopes; this greatly broadens the potential antigen targets that can be exploited to achieve protection. Indeed, one of the mAb in the ZMapp™ cocktail is actually a poor neutralizer. Third, since the viral load during the transmission episode at mucus membranes is likely low, the overall dose of mAb needed at mucosal surfaces, either before or immediately following a high-risk exposure event, may be substantially less than the mAb dose needed to treat a proliferating systemic infection. Thus, ZMapp™ delivered topically may be a particularly useful preventative measure or emergency intervention for populations at the highest risks of acquiring Ebola infections, such as healthcare workers, for reducing both the odds of becoming sick as well as the viral load entering the circulation following an exposure event.
Blocking Penetration of Respiratory Syncytial Viruses in Human Airway Mucus
In the U.S., RSV is the leading cause of bronchiolitis, pneumonia and viral death, with >3.5 million RSV infections and >125,000 hospitalizations in children under 2 each year. RSV infection in nursing homes ranges between 5-10%, leading to 177,000 hospitalizations and significant rates of pneumonia (10-20%) and death (14,000 deaths annually) among the infected elderly. Palivizumab (also referred to as SYNAGIS) is a mAb developed for treatment of RSV. Despite its modest efficacy in reducing risk of RSV-associated hospitalization, SYNAGIS is only given to a very small subset of the pediatric population. Per the CDC, there is currently no treatment for RSV.
SYNAGIS is typically applied by injection. Although it may decrease the risk of developing RSV, SYNAGIS is not effective in treating RSV infection. Surprisingly, based on the inventions described herein, this is likely because the amount of mAb extravasating into the airways is inadequate. In other words, there is likely just enough mAb reaching the lung lumen with IM injection to modestly reduce the rate of acquiring RSV infection, especially since the viral titers of typical RSV exposure are low. However, once infection is established, the amount of mAb is inadequate to contain the spread of higher titers of RSV. Given the unique pathophysiology of RSV that sheds infectious virions into the lumen, we believe concentrating mAb in the lung would be a more effective therapeutic approach than systemic or IM delivery. In cotton rat studies, pulmonary delivery of polyclonal IgG was 160-fold more effective in treating RSV than intramuscular delivery.
To visualize individual RSV virions in AM, we fluorescently tagged RSV by conjugating AF488 dye to the virus surface, which reduced the total mAb that could bind RSV by only ˜10%-20%. When mixed into fresh, physiological human AM collected from intubated endotracheal tubes, a substantial fraction of RSV (˜28%±7%) exhibited rapid diffusion in all 12 independent AM specimens tested (
The “muco-trapping” palivizumab described herein includes the heavy and light chain variable regions of palivizumab and includes a glycosylation pattern on the Fc region that enhances mucous trapping. For example, the muco-trapping palivizumab may be a population of mAb in which at least some percentage (e.g., at least 50%, at least 60%, etc.) of the mAb having the heavy and/or light chain variable regions of palivizumab (or a derivative of palivizumab) contains a glycosolyation pattern of G0/G0F (i.e. GnGn) such that they facilitate trapping of viruses in mucus. For example, in some variations the population of muco-trapping antibody (e.g., muco-trapping palivizumab) has at least 60% of the recombinant antibodies in the population having a glycosylation pattern comprising the biantennary core glycan structure Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminal N-acetylglucosamine on each branch.
Further, muco-trapping mAb against RSV rapidly eliminates RSV from mouse airways. To verify that trapping in mucus facilitates rapid elimination from the airways, we performed an in vivo study where fluorescent RSV was dosed to the airways of Balb/c mice (8-10 weeks), followed by either PBS or muco-trapping Ab against RSV, and finally euthanasia and cryo-sectioning of the trachea and airways 30 mins later. As shown by cryosectioned images, topical delivery of the RSV-trapping IgG effectively reduces the amount of RSV remaining in the mouse airways as little as 30 mins post-delivery compared to PBS control, reaching levels comparable to mouse treated with the same RSV-trapping IgG 15 mins prior to RSV inoculation (n=6;
Intranasally dosed “muco-trapping” mAb against RSV effectively reduces infectious RSV titers in cotton rats. Based on our observations above that muco-trapping mAb against RSV was able to trap RSV in human AM and facilitate rapid elimination of RSV in the mouse lung, we next proceeded to evaluate whether topical delivery of the same mAb can be used to treat RSV infections. Cotton rats remains the gold standard as a first in vivo model for RSV infection. In this study, we inoculated RSV Strain A2 (100 μL; 1×106 PFU per rat) on Day 0. After a 48 hr period to allow the infection to proliferate, we treated 3 groups of mice with either 2 daily doses (i.e., once each on Day 2 and Day 3) of the muco-trapping mAb against RSV (possessing same Fab as SYNAGIS, modified to have enhanced mucosal trapping as described herein; n=9), control IgG (e.g., HGN-194, a mAb against HIV; n=8), or vehicle control (e.g., saline; n=8). All treatments were administered intranasally, and lungs were harvested from all rats on Day 4, snap frozen, and finally homogenized for viremia analysis via immunoplaque assay. An anti-RSV antibody having enhanced mucosal trapping (Muco-trapping mAb) mediated on average >100-fold reduction in infectious viral titers in the cotton rat lung, including 3 rats that showed no detectable viremia, underscoring the potential effectiveness of this treatment. This is illustrated in,
To demonstrate that we can deliver mAb to the lung airways via nebulization, we performed initial characterization of nebulization using PARI's off-the-shelf eRapid system. Using the Copley Next Generation Impactor (NGI), shown in
Nebulized mAb
Inertial Impaction and Aerodynamic Particle Size Distribution (APSD) was determined by inertial impaction (NGI, MSP Corp., USA) operated at 15 L/min for 3 minutes of actuation. No pre-separator was used and the NGI was cooled for at least 90 minutes in a refrigerator to reduce evaporation of nebulized product during dosing. Privigen (CSL Behring) was added to the nebulizer (PART eFlow, as will described for
Although purified human or humanized monoclonal antibody (mAb) was nebulized using a vibrating mesh nebulizer, any appropriate nebulizer may be used, including (but not limited to): jet nebulizer, vibrating net nebulizer, etc. Further, although the methods and examples described herein illustrate ‘wet’ nebulizer formulations, in some variations dry/powder inhaler formulations may be used.
As shown in
Lower concentrations of mAb (e.g., mAb directed against respiratory virons, such as IN-001 and IN-002), were not as effectively delivered. For example,
The Applicants have found that there appears to be an optimal concentration range for treating or preventing Respiratory syncytial virus (RSV) in a subject inhaling the nebulized solution. In particular, when the nebulized solution includes a recombinant antibody (e.g., such as palivizumab, or a variant of palivizumab like motavizumab) having a human or humanized Fc region and an oligosaccharide having a glycosylation pattern that enhances the trapping potency of the recombinant antibody in mucus, wherein the nebulized solution comprises particles having a mass median aerodynamic diameter (MMAD). The optimal concentration of antibody within particles in this MMAD size range is between about 10 mg/mL and about 100 mg/mL, specifically greater than 10 mg/mL and less than 100 mg/mL (e.g., between 11 mg/mL and about 100 mg/ml, between 12 mg/mL and 100 mg/mL, between 13 mg/mL and 100 mg/mL, between 14 mg/mL and 100 mg/mL, between 15 mg/mL and 100 mg/mL). Below this range, the treatment less effective or even ineffective. In some variations this treatment is delivered and is optimized for delivery only after 2-3 days post-infection.
PARI LC Sprint™ nebulizers were used to administer RSV virus (e.g., 6 mL of RSV M37 strain at 1.1×107 FFU/mL in media containing 20% sucrose) or control media (media from HEp-2 cells lacking RSV) to individual lambs. Three 2-mL aliquots of virus-containing media or control media were administered to each animal over the course of 23 minutes resulting in a total inhalation of about 3 mL by each lamb. The RSV-inoculated lambs each received a calculated effective dose of 0.1.29×107 total FFU/animal. Aerogen Solo nebulizers were used to deliver the nebulized solutions described above.
On either day 2 or day 3 post-infection, neonatal lambs were treated with either saline or two different muco-trapping mAb, IN-001 and IN-002. Nebulized IN-001 (beginning on day 3, and dosed also on day 4 and 5) were delivered at a target inhaled dose of ˜0.9 mg/kg. Nebulized IN-002 (beginning on day 3, and dosed also on day 4 and 5) were delivered at a target inhaled dose of ˜0.8 mg/kg. Nebulized IN-002 (low doses) were given beginning on day 2, and also on day 3, 4 and 5, at a target inhaled dose of ˜0.3 mg/kg. Target inhaled doses were calculated (DOI: 10.1080/19420862.2018.1470727).
The thorax was opened, lungs removed and gross lesions were scored, as shown in
As shown in
Bronchoalveolar Lavage Fluid (BALF) samples from each animal were collected immediately after euthanasia at day 6. Right caudal lobe BALF was collected in 5 mL of cold double-modified Iscove's media (DMIM) containing 42.5% Iscove's modified Dulbecco's medium, 7.5% glycerol, 1% heat-inactivated FBS, 49% DMEM, and 5 μg/mL kanamycin sulfate. For BALF, immediately after lung dissection, the right caudal lung lobe from lambs 1 to 20 were flushed through its major bronchus five times (with a single 5 mL aliquot of DMIM) using a 10 mL serological pipet fitted to an electronic pipettor and about 2.6-3.0 mL were collected. 100 μL Right caudal lobe BALF was used for qRT-PCR assessment of RSV load by ISU and 1.2 mL (from which 850 μL was used) for FFU analysis by ISU; as described below. In addition, BALF was also collected from the right middle lung lobe of each animal by flushing it five times (using a 1 mL pipet) through its major bronchus with five separate 1 mL aliquots of sterile 0.9% saline, about 2-3.5 mL of which were recovered in most cases.
Infectious focus-forming unit (FFU) assays on BALF and Lung Tissue were performed, the results of which are illustrated in
In addition, prior to use in the infectious focus assay, 0.5 g of collected lung samples (pooled from 0.1 g from each of 5 lobes for each animal; right cranial, accessory, left cranial, left middle, and left caudal) were immediately homogenized in 5 mL of DMIM on ice for 80 seconds using an OMNI TH homogenizer. 1 mL of each homogenate was transferred to a sterile 1.5 mL vial and microfuged at 1780×g for 5 minutes to pellet large debris. 800-850 μL of each clarified supernatant was removed by pipet and applied to a new SPIN-X column which was spun for 5 minutes at 15,600×g. This was repeated two or three more times (using a new SPIN-X column each time) for each lung sample. At this point, the lung sample homogenates were considered ready for infectious focus assay.
Briefly, HEp-2 cells were grown to 70% confluence in 12-well culture plates in DMEM media supplemented to 10% with heat-inactivated fetal bovine serum (FBS) and 50 μg/mL kanamycin sulfate. Collected BALF samples were microfuged for 5 minutes at 3000×g to pellet large debris. 850 μL of each supernatant was removed and spun through 850 μL-capacity 0.45 μm Costar SPIN-X filters at 15,600×g for 5 minutes. Each filter-clarified BALF sample was analyzed at full-strength and at four additional serial-dilutions of 1:10, 1:100, 1:1,000 and 1:10,000. 200 μL of each sample dilution were added in duplicate to wells of a 12-well plate (e.g.; each sample used an entire 12-well culture plate); two control wells on each plate received virus-free cell culture medium. Plates will be incubated for 80 minutes in a CO2 incubator at 37° C. and 5% CO2 with manual rocking every 20 minutes. 1 mL of culture medium was added to each well and cells were allowed to incubate for 48 hours after which medium was removed and cells fixed with 60% acetone/40% methanol solution for 1 minute. The fixing solution was removed and plates were allowed to air-dry for 2 minutes after which each well was rehydrated with 1 mL TBS-0.05% Tween 20, pH 7.4-7.6 (TBST) for 1 minute with mild rotation. To block non-specific binding, 1 mL of 3% BSA (Fisher Scientific, Hanover Park, IL, USA) in TBST was added to all wells at room temperature with gentle rocking for 30 minutes. Primary polyclonal goat anti-RSV (all antigens) antibody (EMD Millipore, Billerica, MA) were diluted 1:800 in TBST containing 3% BSA; 325 μL of this was added to each well and plates were allowed to incubate overnight at 4° C. with gentle rocking. The next day, plates were behed gently three times for 5 minutes each with TBST, then 325 μL secondary antibody [Alexa Fluor® 488 F (ab′) 2 fragment of rabbit anti-goat IgG (H+L), Molecular Probes/Life Technologies] diluted 1:800 in TBST containing 3% BSA was added to each well and allowed to incubate at room temperature for 30 minutes with gentle orbital rotation. Plates were rinsed two times for 5 minutes each with TBST and 1 mL of TBST was added back to each well prior to microscopic inspection. Plates were examined for the presence of fluorescing infectious foci using the FITC/GFP filter on an inverted stage fluorescence microscope (Olympus CKX41, Center Valley, PA, USA). Clusters of 5 or more fluorescing cells were counted as single infectious focal events. Titers will be calculated using the following formula: an average of 20 counts in a 1:100-diluted (duplicate) sample indicated that the original BALF sample had a “titer” of 10,000 since [20 counts×dilution factor of 100×1,000 μL/mL]/200 μL assessed=10,000 infectious focus-forming units/mL (FFU/mL). For lung homogenate calculations: an average of 20 counts in a 1:100-diluted sample indicates that the original lung sample had a titer of 110,000 FFU/g lung tissue since 11×[20 counts×dilution of 100×1000 μL/mL]/200 μL assessed=110,000 infectious foci “FFU”/g. These results will be multiplied by fraction of sample loss
As shown in
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.
This patent application is a 35 U.S.C. § 371 national phase application of PCT Application PCT/US2019/023174 filed Mar. 20, 2019, which claims priority to U.S. provisional patent application No. 62/646,220, filed Mar. 21, 2018, titled “Compositions and Methods for Inhibiting Pathogen Infection in the Lung,” the entire contents of each of which incorporated by reference herein in its entirety.
This invention was made with Government support under Grant Nos. UL1TR001111 and R42AI138728, awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
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| PCT/US2019/023174 | 3/20/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/183228 | 9/26/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 10100102 | Lai | Oct 2018 | B2 |
| 10829543 | Lai | Nov 2020 | B2 |
| 20130164338 | Lipp | Jun 2013 | A1 |
| 20150284451 | Lai | Oct 2015 | A1 |
| 20170267780 | Tsui | Sep 2017 | A1 |
| 20210002355 | Lai | Jan 2021 | A1 |
| Number | Date | Country |
|---|---|---|
| 0539522 | May 1993 | EP |
| 2011056697 | May 2011 | WO |
| 2011056899 | May 2011 | WO |
| 2012006596 | Jan 2012 | WO |
| 2014070786 | May 2014 | WO |
| Entry |
|---|
| “International Preliminary Report on Patentability corresponding to International Application No. PCT/US2019/023174 mailed Oct. 1, 2020”. |
| “International Search Report and Written Opinion corresponding to International Application No. PCT/US2019/023174 mailed Jul. 4, 2019”. |
| Robbie, Gabriel J., et al., “A Novel Investigational Fc-Modified Humanized Monoclonal Antibody, Motavizumab-YTE, Has an Extended Half-Life in Healthy Adults”, Antimicrob. Agents Chemother. 57(12):6147-6153 (2013). |
| “Extended European Search Report corresponding to European Application No. 19771963.6 dated Dec. 16, 2021”. |
| Jacobino, Shamir R, et al., “Reformatting palivizumab and motavizumab from IgG human IgA impairs their efficacy against RSV infection in vitro and in vivo”, MABS 10(3):453-462 (Mar. 19, 2018). |
| Mayor, Alexie , et al., “Inhaled antibodies: formulations require specific development to overcome instability due to nebulization”, Drug Delivery and Translational Research 11:1625-1633 (Mar. 25, 2021). |
| Sécher, Thomas , et al., “Barriers for orally inhaled therapeutic antibodies”, Expert Opinion on Drug Delivery 20(8):1071-1084 (Aug. 25, 2023). |
| Fahy, John V, et al., “Effect of Aerosolized Anti-IgE (E25) on Airway Response to Inhaled Allergen in Asthmatic Subjects”, Am. J. Respir. Crit. Care Med. 160:1023-1027 (1999). |
| Number | Date | Country | |
|---|---|---|---|
| 20210002355 A1 | Jan 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62646220 | Mar 2018 | US |
