Monoclonal Antibodies (IgG) for Intranasal Prophylaxis of ......Monoclonal Antibodies are a class of...
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Confidential No duplication or distribution without the expressed written approval of Diomics Corporation. Monoclonal Antibodies (IgG) for Intranasal Prophylaxis of COVID-19 Introduction It has become increasingly clear that the nasal cavity is a major site of infection by SARS- CoV-2. Viral shedding is highest from the nose with significant transmission and spread of the disease. The objective of this project is to develop a prophylactic nasal antibody spray which provides soldiers with passive immunity to SARS-CoV-2 during exposure in high risk areas and confined spaces. SARS-CoV-2 (red) infected ciliated cells. In a study by the UNC School of Medicine and the UNC Gillings School of Global Public Health the specific ways in which SARS-CoV-2 infects the nasal cavity has been characterized. The findings suggest the virus tends to become firmly established first in the nasal cavity and scientists also found that ACE2 - the cell surface receptor that the virus uses to get into cells - was more abundant on nasal-lining cells and less abundant on the surface of lower airway cells. This difference could explain, at least in part, why upper airway nasal-lining cells were more susceptible to infection. They found a striking pattern of continuous variation, or gradient, from a relatively high infectivity of SARS-CoV-2 in cells lining the nasal passages, to less infectivity in cells lining the throat and bronchia, to relatively low infectivity in lung cells. We believe that a prophylactic response to the virus could positively impact both the infection patterns and severity of infections by way of an administration of a longer- duration IgG nasal product. One Hundred Seventy-Eight human monoclonal antibodies have been isolated from 11 recently recovered COVID-19 patients. From this library of clones, 17 antibodies were isolated that inhibit viral infection in Vero-E6 cell lines. From the 17 neutralizing clones, #414-1 was selected as the candidate to inhibit viral infection as a prophylactic nasal spray. This clone shows robust authentic viral neutralization in Vero-E6 cells with an IC50 below 1.75 nM, and with an affinity greater than that of the virus to the ACE2 receptor.
Transcript of Monoclonal Antibodies (IgG) for Intranasal Prophylaxis of ......Monoclonal Antibodies are a class of...
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MonoclonalAntibodies(IgG)forIntranasalProphylaxisofCOVID-19
Introduction
IthasbecomeincreasinglyclearthatthenasalcavityisamajorsiteofinfectionbySARS-CoV-2.Viralsheddingishighestfromthenosewithsignificanttransmissionandspreadofthedisease.TheobjectiveofthisprojectistodevelopaprophylacticnasalantibodyspraywhichprovidessoldierswithpassiveimmunitytoSARS-CoV-2duringexposureinhighriskareasandconfinedspaces.
SARS-CoV-2 (red) infected ciliated cells.
InastudybytheUNCSchoolofMedicineandtheUNCGillingsSchoolofGlobalPublic
HealththespecificwaysinwhichSARS-CoV-2infectsthenasalcavityhasbeencharacterized.ThefindingssuggestthevirustendstobecomefirmlyestablishedfirstinthenasalcavityandscientistsalsofoundthatACE2-thecellsurfacereceptorthatthevirususestogetintocells-wasmoreabundantonnasal-liningcellsandlessabundantonthesurfaceoflowerairwaycells.Thisdifferencecouldexplain,atleastinpart,whyupperairwaynasal-liningcellsweremoresusceptibletoinfection.
Theyfoundastrikingpatternofcontinuousvariation,orgradient,fromarelativelyhighinfectivityofSARS-CoV-2incellsliningthenasalpassages,tolessinfectivityincellsliningthethroatandbronchia,torelativelylowinfectivityinlungcells.
Webelievethataprophylacticresponsetotheviruscouldpositivelyimpactboththeinfectionpatternsandseverityofinfectionsbywayofanadministrationofalonger-durationIgGnasalproduct.OneHundredSeventy-Eighthumanmonoclonalantibodieshavebeenisolatedfrom11
recentlyrecoveredCOVID-19patients.Fromthislibraryofclones,17antibodieswereisolatedthatinhibitviralinfectioninVero-E6celllines.Fromthe17neutralizingclones,#414-1wasselectedasthecandidatetoinhibitviral
infectionasaprophylacticnasalspray.ThiscloneshowsrobustauthenticviralneutralizationinVero-E6cellswithanIC50below1.75nM,andwithanaffinitygreaterthanthatofthevirustotheACE2receptor.
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AntibodybindinghasalsobeenconfirmedbyX-raycrystallography.Thecrystal
structureinconjunctionwithsitedirectedmutagenesisstudiesshowedthebindingofthemAbtoallthreeRBDintheS1tetramerandtooneofthefourknownneutralizationmotifsoftheS1regionofSARS-COV-2,locatedatthetopofRBDwheredirectACE2interactionoccurs.
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StructureandneutralizationstudiesclearlyshowsthattheAntibodychosenclearlyavoidstheN-andO-linkedglycosylationsitesofthespikeglycoproteinthatdistinguishitfromtheSARSvirusandunderlinesshieldingandcamouflageofCOVID-19fromthehostdefensesystem.Wearecouplingthesehumanmonoclonalantibodiestoourproprietary,hydrophilic
microbeadsinordertoincreasetheefficacyofviralneutralizationinthenasalmucosaandtopreventrapidantibodyclearancevianasalcilia.Studiessuggestabsorptionoftheantibody-microbeadsintothenasalmucosawillincreasetheantibodyresidencetimeinthenasalcavityascomparedwithsalineformulations.,ThisprovidesalocalizedconcentrationofpotentSARS-CoV-2neutralizingantibodiesatthesiteofprimaryinfection.
MonoclonalAntibodySafety.
MonoclonalAntibodiesareaclassoftherapeuticproteinswidelyinvestigatedastreatmentsfornumerousindications.Overonehundredhavebeenapprovedforhumantherapeuticusetodate,andoverfivehundredhavebeenusedasinvestigationaldrugs.1Themajorityofthemarehumanorhumanizedantibodies.Humanizedantibodieshaveaproteinsequencemodifiedtomakethemmoresimilartonormalhumanvariantsandmakethemlessimmunogenicforhumantherapeuticuse.BecauseofthelargenumberoftherapeuticusesforhumanmAbsitisgenerallybelieved
thattheiruseissafe,andthatsideeffectsandtoxicitiesaregenerallyattributedtothespecifictargetormechanismofaction.ManyhumanorhumanizedmAbsareapprovedforuseagainstawiderangeofcancersorinseveralauto-immunediseases(e.g.arthritis,asthma,Crohn’sdisease,psoriasis).ThemAbsunderinvestigationinDioguard™wereisolatedfromrecoveredCOVID-19
patientsthroughstate-of-the-artmolecularbiologymethodsandarefullyhuman.2BecausetheirproteinsequenceisfullyhumanandtheirmodeofactionisbindingwithhighspecificitytotheSARS-CoV2ReceptorBindingDomain(RBD)itisbelievedthesewillbegenerallysafe.Studiestoconfirmtheirsafetyfortheinitiationofhumanclinicalstudiesareunderway.
MonoclonalAntibodyEfficacyagainstSARS-CoV2.
Group Tested Beads Used(mg)
Approx. Protein in Bead Sample (ug)
1st Abs./2nd Measurement
Average Abs.
Untreated 30-micron Beads
0.02 0 0.145/0.141 0.143
Untreated 30-micron Beads
+ Coupled RBD
0.02 5.14 0.259/0.261 0.26
Diobead (60-minutes Hydroxylation)+ Coupled RBD
0.02 5.89 0.371/0.384 0.3775
Figure 1. Sample Standard with respective ABS value for [RBD] provided by Active Motif staff.
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TherearestrongprecedentsfortheuseofmAbsastreatmentsorpreventionofrespiratoryviraldiseases.3PalivizumabisahumanizedmAbapprovedforthepreventionofrespiratorysyncytialvirus(RSV)inpediatricpatients.OthermAbsforthisindicationhavebeenproveneffectiveinanimalmodels.4In2012,theMERS-CoVepidemiccausedmorethan1300humaninfectionsand500deathsworldwide.MERScausesinfectionofthelowerrespiratorytract,similartotheSevereAcuteRespiratorySyndromeCoV(SARS-CoV)thatappearedinChinain2002.Followingthatoutbreak,neutralizinghumanmAbsagainstMERS-CoVwereisolatedandclonedfromsinglehumanB-cellsandshowntobeeffectiveinmousemodelsthroughintraperitonealinjectionandintranasaladministration.5AsimilarexperimentalapproachwasusedintheisolationoftheSAS-CoV-2neutralizing
mAbsusedintheDioguard™product.2Theseantibodieswereshowntobehighlyactiveinpseudoviralinfectionassays.TheywerealsoshowntoneutralizeauthenticviralentryinVero-E6cells.ThebestoftheseblockedviralentrywithanIC50of1.75nMandshowedabindingaffinitytotheviralRBD(KD)of0.4nM.TheDioguard™polymerisanintegralpartoftheproductdesign,andbyanalogytoother
reportsisintendedtoallowforextendedresidencetimeinthenasalcavityandincreasetheefficacyoftheproduct.6AnimalefficacymodelshavebeendevelopedforSARS-CoV-2therapeuticand
vaccinationstudies,andthesecanbeadaptedforpre-clinicalefficacytestingoftheDioguard™antibodies.
Conclusions
InviewofthetolerabilityofhumanmAbsinmultiplesystemicapplicationsandthein-vitroefficacydemonstratedwiththeDioguard™antibodieswebelievethereissignificantpotentialforaneffectiveandwell-toleratedintranasalproductforuseinthepreventionofCOVID-19.OtherthanbeingapotentneutralizeroflifeSARS-COVID-2virustherecombinantclone
isaverygoodexpressormakingtheprojectfeasiblefinanciallyandproductsexceptionallymanufacturableandscalable.
References
1) https://en.wikipedia.org/wiki/List_of_therapeutic_monoclonal_antibodies 2) Wan et al., 2020, Cell Reports 32, 107918. Human-IgG-Neutralizing Monoclonal Antibodies Block the SARS-CoV-2 Infection. 3 ) Weltzin et al., CLINICAL MICROBIOLOGY REVIEWS, July 1999, p. 383–393. Intranasal Antibody Prophylaxis for Protection against Viral Disease. 4 ) Zeitlin Et Al. https://doi.org/10.4161/mabs.23281. Prophylactic and therapeutic testing of Nicotiana-derived RSV-neutralizing human monoclonal antibodies in the cotton rat model. 5 ) Corti et al., www.pnas.org/cgi/doi/10.1073/pnas.1510199112 , Prophylactic and postexposure efficacy of a potent human monoclonal antibody against MERS coronavirus. 6 ) Walsh et al., Pharmaceutical Research, Vol. 21, No. 10, October 2004. Extended Nasal Residence Time of Lysostaphin and an Anti-Staphylococcal Monoclonal Antibody by Delivery in Semisolid or Polymeric Carriers.
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7 ) https://www.medpagetoday.com/infectiousdisease/covid19/88343 Monoclonal antibodies could hold promise in COVID-19 treatment and prevention if the results bear out in clinical trials for efficacy, the nation's leading infectious diseases expert. 8) Major study of coronavirus infection in human airways adds to evidence that wearing a mask is an important protective step toward limiting transmission of COVID-19 UNIVERSITY OF NORTH CAROLINA HEALTH CARE KeyResourcesTable
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