WO2022084817A1 - Protein drugs for prevention and treatment of covid-19 - Google Patents

Protein drugs for prevention and treatment of covid-19 Download PDF

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Publication number
WO2022084817A1
WO2022084817A1 PCT/IB2021/059541 IB2021059541W WO2022084817A1 WO 2022084817 A1 WO2022084817 A1 WO 2022084817A1 IB 2021059541 W IB2021059541 W IB 2021059541W WO 2022084817 A1 WO2022084817 A1 WO 2022084817A1
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antibody
igy
rbd
formulation
covid
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PCT/IB2021/059541
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French (fr)
Inventor
Uday Saxena
Satishchandran CHANDRASEKHAR
Subrahmanyam VANGALA
Subramanian Iyer
Gopi KADIYALA
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Lay Sciences Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is related to a protein drug consisting of chicken IgY antibodies against Sars CoV-2 spike protein receptor binding domain (RBD) which is used prophylactically or therapeutically against COVID- 19 infections.
  • RBD Sars CoV-2 spike protein receptor binding domain
  • COVID-19 has created havoc globally both with its mortality and its debilitating effects seen in patients. Previously it was thought that the viral infection mostly impairs lung cells, but now it has been shown in patients that other cell/tissues/organs such as endothelium, kidney and heart are also majorly impacted. Importantly, abnormal blood clotting and inflammation are being now thought as important causes of the damage caused by the viral infections.
  • Passive immunity provides immediate and transient protection against the pathogen to interfere with the onset of infection.
  • SARS-CoV-2 initial entry into the human body is through the oral/nasal/eye mucosa. The initial amplification of the virus appears to be in the oropharyngeal cavity.
  • preformed antibodies that can neutralize the binding of the virus to its human cell receptors in oral and pharyngeal cavity will serve as prophylactic protein drugs to prevent onset of infection and therapeutically to prevent progression.
  • Passive immunotherapy also serves to reduce transmission of virus, by reducing viral load.
  • the anti-RBD chicken IgY antibodies currently developed by the Applicant neutralizes the binding of RBD to ACE2 in both ELISA and dot blot assays.
  • the chicken IGY antibodies are polyclonal in nature and therefore offer high likelihood of neutralizing the binding of virus to human cells.
  • Chicken IgY also offer other advantages over the traditional animal derived IgG such as 1) they can be raised in gram quantities at low cost for continuous supply 2) Being polyclonal in nature they offer wider neutralizing capability 3) They do not activate the complement pathway and 4) usually generate higher titers of antibodies to an antigen given that they are further removed from the humans in evolution than other mammals. These attributes make chicken IgY very attractive as therapeutics.
  • Figure 1 Titre evaluation of anti-RBD IgY antibodies developed in hyper immunized chicken eggs.
  • Figure 4 Activity of the anti-RBD containing liquid drink as shown by dot blot assay.
  • the present invention provides a formulation of IgY polyclonal antibodies against Sars CoV-2 proteins (S, Spike especially Receptor Binding Domain, RBD) singularly or in combination, which are used prophylactically and/or therapeutically against COVID-19 infections.
  • the IgY polyclonal antibody of the invention is combined with antivirals such as remdesivir or lopinavir/flapinavir, anti-inflammatory, steroid and anti-thrombotic agents.
  • the formulation is designed to neutralize the virus, as antibodies bind to surface proteins of the virus and interfere with the onset of infection.
  • the formulation is developed as a whole hyperimmune egg, hyperimmune egg yolk or purified antibodies (IgY).
  • the formulation of the invention is used as a prophylactic and/or therapeutic against COVID-19 viral infection.
  • the formulation of the invention is beneficial as a prophylactic and/or therapeutic against COVID-19 viral infection caused by strains selected from the group consisting of (B.1.1.7 (UK), B.1.351 (SA), P.l (Brazil), B.1.617.3 (India), B.1.429 and B.1.427 (USA), also referred to as YP_009724397.2, Alpha_B.1.1.7_Q.l-Q.8, Beta_B.1.351.1- 351.5, Delta_B.1.617.2_AY.l-AY.38, Gamma_P.l-P.1.9 and any other strain hitherto unknown.
  • the antibodies are raised in chickens (polyclonal IgY antibodies) and harvested from chicken eggs immunized with the viral proteins, together or separately (and the yolks pooled).
  • chicken is immunized with DNA (Innovio, Cadilla vaccines) or RNA (Pfizer BionTech and Moderna COVID vaccines) encoding the proteins as in DNA and RNA-based vaccines, other genetic constructs using viral vectors (Adeno or Adeno associated virus or Lentivirus or other as in Astra Zeneca Covishield), peptides of the proteins, or constructs with all proteins strung together (chimeric) or whole inactivated SARS COV-2 virus (COVAXIN, SINOVAC). All of the above antigenic designs may be mixed with adjuvants (Freunds - complete or incomplete, MPL etc) or with TLR agonists or with cytokines to improve immune responses in chickens.
  • adjuvants Feunds - complete or incomplete, MPL etc
  • TLR agonists or with cytokines to improve immune responses in chickens.
  • the harvested antibodies from hyperimmunized chickens are used as prophylactic formulation directly as whole IgY containing egg yolk, whole egg powder or may be further purified as pure IgY antibodies for use for oral administration or as purified IgY polyclonal protein therapeutically when administered parenterally.
  • the Sars Cov2 protein Spike proteins was used for the immunization of chicken IgY antibodies.
  • the full protein sequences of the Spike proteins are: domain shown in MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWI FGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SAS
  • GS linker sequence included to prevent protein folding problems.
  • Poly histidine sequence included to facilitate affinity purification.
  • a plasmid construct as designed above was used to express the proteins as designed.
  • Purified RBD protein were used to vaccinate chickens and IgY antibodies isolated from vaccinated chicken eggs. Chicken vaccination and isolation of IgY are as described in (https://doi.org/10.1016Zi.eips.2017.05.069).
  • the antibodies of the invention are useful for the treatment of disease conditions in subjects, mammals in particular, including humans.
  • the protein drug is used for the treatment of viral infections and CO VID 19.
  • the drug is also effective as both a prophylactic and therapeutic for viral infections
  • the protein drug of the present invention is delivered to the subjects by forms suitable for each administration route.
  • the drug is administered as tablets, capsules, injection, drops, inhaler, ointment, foams suppository.
  • the route of administration is oral, parenteral or intranasal.
  • Nasal formulation includes powders, sprays, patches, inhalants and nebulizers.
  • composition of the present invention is presented in unit dosage form generally in an amount that produces a therapeutic effect in the subject.
  • the compounds of the present invention are administered at a daily dose that is the lowest dose effective to produce a therapeutic effect.
  • the dosage will affect from about 0.0001 to about lOOmg per kg body weight per day.
  • the dosage will range from about 0.001 to 75mg per kg body weight per day and more preferably, the dosage will range from about 0.1 to about 50mg per kg body weight per day.
  • Each unit dose may be, for example, 5, 10, 25, 50, 100, 125, 150, 200 or 250 mg of the compound of the invention.
  • the effective daily dose of the compound is administered as two, three, four or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms.
  • the protein drug of the present invention may be administered by any method known in the art.
  • suitable modes of administration include oral, intravenous, intramuscular, intranasal or any other parenteral mode of administration.
  • the present invention is directed to a method of formulating compounds of the present invention in a pharmaceutically acceptable carrier or excipient and may be administered in a wide variety of different dosage forms e.g. tablets, capsules, sprays, creams, lotions, ointments, aqueous suspensions syrups, and the like.
  • a pharmaceutically acceptable carrier or excipient may be administered in a wide variety of different dosage forms e.g. tablets, capsules, sprays, creams, lotions, ointments, aqueous suspensions syrups, and the like.
  • Such carriers may include one or more of solid diluents or fillers, sterile aqueous media, and various nontoxic organic solvents, etc.
  • tablets may contain various excipients such as one or more of microcrystalline cellulose, sodium citrate, calcium carbonate and the like, along with various disintegrants such as starch and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose and the like.
  • solid compositions of a similar type may also be employed as fillers in gelatin capsules, nano emulsions, microfilms that dissolves on the tongue or as chewing gum or edible candy.
  • Oral administration also includes liquid formulations such as a oral rinse, digestible drinks or flavored powders in a sachet that can be formulated in a liquid form.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluents or solvent.
  • the acceptable vehicles and solvents that may be employed are water, Ringer’s solution and isotonic sodium chloride solution.
  • sterile fixed oils are conventionally employed including synthetic mono or diglycerides.
  • fatty acids such as oleic acid find in the preparation of injectables.
  • These aqueous solutions may be suitable for intravenous injection purposes.
  • the oily solutions may be suitable for intra articular, intramuscular, and/or subcutaneous injection purposes.
  • the compounds of the present invention may be administered topically that include transdermal, buccal, or sublingual application.
  • therapeutic compounds may be suitably admixed in a pharmacologically inert topical carrier such as a gel, an ointment, a lotion, and/or a cream.
  • topical carriers may include water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, and/or mineral oils.
  • the timing of the administration of the pharmaceutical composition may also be regulated.
  • the compounds may be administered intermittently or by controlled release.
  • the protein drug described here can be combined with other monoclonal antibodies targeting other SarsCov2 proteins, drugs, peptides that can interfere with viral entry and replication or antiinflammatory, steroid and anti-thrombotic agents that are used for COVID prevention and treatment.
  • RBD domain peptide of SARS CoV-2 S protein (seq ID 2), were used to vaccinate chickens and boosted several times to achieve high titer. Titer was determined using partially (>85%) purified total IgYs from yolk. The ELISA data is shown below.
  • ELISA was carried out as per the standard protocol.
  • RBD (lug) proteins was coated on the plate (in wells Al through B9 for Ih at 37C.
  • Anti-RBD was diluted as shown and applied to the plate to bind RBD protein.
  • PC positive control where a known antigen X was coated on the plate and anti-X-IgY was applied to bind to antigen X.
  • CPC Only anti-RBD or anti-X antibodies were applied. No antigen.
  • NC Antigen X was coated on the plate and anti-RBD IgY antibodies are applied to check if there is any cross reactivity. The plate was blocked and washed following each addition as per standard ELISA protocol.
  • the color was developed by complexing HRP conjugated anti-IgY antibody and HRP standard as per standard ELISA protocols.
  • the Salmonella and yeast contaminants in the solution were sterilized by heat treatment.
  • the preferred sterilization temperature at which the IgY titer is unaffected is 61 °C and the sterilization time required was 3 to 5 minutes.
  • the whole egg powder or isolated IgY was formulated into a readily water-soluble powder form.
  • the formulation by which the IgY activity was unaffected was a blend of maltodextrin (10-60%), Whole egg powder (1-10%), Dextrose (10-30%) and Aerosil (1-10%), Orange or any other flavoring agent (1-10%) and stevia or any other sweetener (0.1-10%) (Table 2).
  • the formulation includes whole egg or yolk powder or IgY antibodies isolated (0.001-10%), Ashwagandha extract (1-10%), Immuno stimmulants for example Inulin (10- 40%), citric acid (0.1-10%), sodium benzoate (0.1-10%), flavors including but not limited to lichee, orange, strawberry or chocolate (0.1-10%) and sweetener including but not limited to Sucralose or stevia (Table 3, 4 and 5).
  • Table 3 Oral Liquid formulation of IgY liquid.
  • Table 4 Powder formulation of Sweetened IgY powder (Sachet format) solubilized in water prior to oral administration.
  • Table 5 Powder formulation of Unsweetened IgY powder (Sachet format) solubilized in water prior to oral administration.
  • sample A binding of biotinylated RBD to ACE2, as indicated by the stain density
  • sample B binding of biotinylated RBD
  • sample C A known compound NAC which blocks the binding of RBD binding to ACE2 was also tested (sample C), which inhibited the binding as expected.
  • Sample D is the anti- RBD IgY in a drink which is used as a oral rinse and it showed strong inhibition of RBD binding to ACE2, thus confirming that it may be used as an oral rinse.
  • sample E and F are unknown peptides used as negative controls and did not show any activity. The oral rinse is thus used in studies described below.
  • Anti-RBD IgY neutralizes the binding of delta strain RBD to ACE2
  • the delta strain of SarsCov2 virus has emerged as the dominant strain in the world currently and is found in 80% of the people infected globally.
  • the delta strain RBD is mutated with three amino acid changes from the native strain. These mutations make this strain more transmissible and infective.
  • Applicants tested whether the IgY antibody (of the invention) raised as above will a) bind to delta RBD and b) neutralize the binding of delta RBD to ACE2 using ELISA and dot blot assays respectively.
  • Figure 5 shows the head to head comparison of direct binding of our IgY to either native or delta RBD coated on an ELISA plate. Bound IgY was detected using and biotinylated anti-IgY antibody. As seen in the Figure, the IgY bound directly to both native and delta RBD effectively with binding to delta RBD being almost 4-fold higher suggesting that the IgY does recognize the mutated delta strain RBD.
  • saliva neutralizing activity we designed a simple protocol. Healthy volunteers were provided with 10 ml of drink containing 16.6 ug/ml of purified anti-RBD IgY as an oral rinse. The volunteers rinsed their oral cavity for 3 minutes and then spit out the rinse. Saliva samples were collected at zero time (basal sample before oral rinse) and at 30, 1 hour, 2 hours and 3 hours after the rinse and stored at 4°C till the dot blot assay was performed. Just prior to use, the saliva was gently centrifuged at 5000 RPM for 2 minutes to settle any debris and the supernatant (10 p l) was used in the dot blot. Data are reported as increase in neutralizing activity over the basal levels.
  • Figure 7 shows the activity in saliva samples from a volunteer who was not vaccinated and was RTPCR negative for COVID 19. The data shows that there was a time-dependent increase in neutralizing activity up to 60% increase over baseline, with persistence of some activity till 2 hours after oral rinse.
  • Example 10 The effect of IgY was also examined in SarsCov2 in vero cells in a plaque reduction neutralization test (PRNT) and PRNT50 was determined ( dilution of IgY at which there was 50% reduction in plaque formation) . Since the IgY neutralize the binding of RBD to ACE2, it is expected that there will be reduction in viral entry into the cells. Table 7 below shows that at two concentrations tested, IgY was able to reduce plaque formation effectively. Table 7. Plaque formation reduced by IgY antibody

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Abstract

The present invention provides a protein drug consisting of chicken IgY antibodies against Sars CoV-2 spike protein receptor binding domain (RBD) which is used prophylactically and/or therapeutically against COVID-19 infections.

Description

Protein drags for prevention and treatment of COVID-19
RELATED APPLICATION
This application is related to and takes priority from the Indian Application No.202041045470 filed 19th October 2020 (19/10/2020) and is incorporated herein in its entirety.
FIELD OF INVENTION
The present invention is related to a protein drug consisting of chicken IgY antibodies against Sars CoV-2 spike protein receptor binding domain (RBD) which is used prophylactically or therapeutically against COVID- 19 infections.
BACKGROUND OF THE INVENTION
COVID-19 has created havoc globally both with its mortality and its debilitating effects seen in patients. Previously it was thought that the viral infection mostly impairs lung cells, but now it has been shown in patients that other cell/tissues/organs such as endothelium, kidney and heart are also majorly impacted. Importantly, abnormal blood clotting and inflammation are being now thought as important causes of the damage caused by the viral infections.
Administration of pre-made active immunoglobulins to provide neutralizing activity against pathogens to prevent and/or treat diseases caused by pathogens is passive immunotherapy.
• Passive immunity provides immediate and transient protection against the pathogen to interfere with the onset of infection.
• SARS-CoV-2’s initial entry into the human body is through the oral/nasal/eye mucosa. The initial amplification of the virus appears to be in the oropharyngeal cavity.
• To this end, preformed antibodies that can neutralize the binding of the virus to its human cell receptors in oral and pharyngeal cavity will serve as prophylactic protein drugs to prevent onset of infection and therapeutically to prevent progression.
• Passive immunotherapy also serves to reduce transmission of virus, by reducing viral load.
• In acute therapeutic use, it may be delivered parenterally multiple times prior to any immune responses against the drug product; immune responses against the product might be expected in 1-3 weeks following administration of the 1st dose when delivered parenterally (SC, IM, IV, ID).
• At present there are no marketed oral virus neutralizing prophylactic treatments for COVID- 19 or other directly targeted therapies for this disease. Currently the available treatment options being used as ‘off-label’ for COVID-19 include
• Remdesivir
• Prednisone/Dexamethasone
• Hydroxy chloroquine
• Convalescent plasma with inconsistent results as these drugs were not originally designed for COVID treatment. The only treatment that appears to be effective in acute setting utilizes parenteral administration of a mixture of 2 or more SARS COV-2 RBD binding monoclonal antibodies, and this has been shown to be effective when administered in the early stages of infection with the onset of early stage symptoms (cough, sore throat, fever, breathlessness etc.).
The rapid and continued spread of COVID 19 disease has prompted many approaches to prevention and treatment of the disease. Currently, vaccinations are the most advanced preventive measures (https://www.medicalnewstoday.com/articles/new-sars-cov-2-variants-how-can-vaccines-be- adapted#Do-we-really-need-second-generation-vaccines?). There are several vaccines available and they have successfully prevented severe disease, hospitalizations and death. Yet the emergence of newer variants such as delta variant has created a need for supplemental protection after vaccinations or especially those people who are not vaccinated or have no access to the vaccines yet. More importantly alternate strategies may be useful in children and elderly who may not be eligible for the vaccines for various reasons.
That said, there are a variety of products available today that could be used as preventive measures besides the vaccine (https://www.covidl9treatmentguidelines.nih.gov/overview/prevention-of-sars- cov-2/). There are mild detergents, enzymes and aseptic nasal or oral sprays and washes that sold as preventive measures. Typically, these are could be used in situations where the individual may anticipate an exposure to the virus such as going to crowded places, hospitals or when taking care of COVID recovering persons at home. Children may use these when going to school etc.
While such approaches are useful, they are generic in nature and do not necessarily target SarsCov2, the virus responsible for COVID 19. As such their efficacy may be less than desirable and there may be some side effects as well. In view of this we have designed a SARSCov2 specific preventive oral wash that could play a significant role in the fight against prevention of this infection. Specifically, applicants have developed chicken IgY antibodies to the receptor binding domain (RBD) of SarsCov2 spike protein which mediates the binding to human cell ACE2 receptors (Hoffmann et al., 2020, Cell 181, 1-10 April 16, 2020).
The anti-RBD chicken IgY antibodies currently developed by the Applicant neutralizes the binding of RBD to ACE2 in both ELISA and dot blot assays. The chicken IGY antibodies are polyclonal in nature and therefore offer high likelihood of neutralizing the binding of virus to human cells. Chicken IgY also offer other advantages over the traditional animal derived IgG such as 1) they can be raised in gram quantities at low cost for continuous supply 2) Being polyclonal in nature they offer wider neutralizing capability 3) They do not activate the complement pathway and 4) usually generate higher titers of antibodies to an antigen given that they are further removed from the humans in evolution than other mammals. These attributes make chicken IgY very attractive as therapeutics.
DESCRIPTION OF THE DRAWINGS
Scheme 1. Workflow to produce anti-COVID19 formulation of the invention
Figure 1. Titre evaluation of anti-RBD IgY antibodies developed in hyper immunized chicken eggs.
Figure 2. Inhibition by Anti-RBD IgY antibody at different concentrations using Genscript ePass ELISA kit (A) and DOT BLOT assay (B).
Figure 3. Concentration response curve of Homoharringtonine (inhibitor of RBD binding to ACE2).
Figure 4. Activity of the anti-RBD containing liquid drink as shown by dot blot assay.
Figure 5. IgY directly binds to native and delta strain RBD.
Figure 6. Inhibition of ACE2 interaction with both native and delta RBD by IgY antibody
Figure 7. Neutralizing activity of oral rinse of IgY antibody containing drink in unvaccinated volunteer.
Figure 8. Neutralizing activity of oral rinse of IgY antibody containing drink in vaccinated (single shot) volunteer.
Figure 9. Neutralizing activity of oral rinse of IgY antibody containing drink in vaccinated (both shots) volunteer.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a formulation of IgY polyclonal antibodies against Sars CoV-2 proteins (S, Spike especially Receptor Binding Domain, RBD) singularly or in combination, which are used prophylactically and/or therapeutically against COVID-19 infections. In one embodiment, the IgY polyclonal antibody of the invention is combined with antivirals such as remdesivir or lopinavir/flapinavir, anti-inflammatory, steroid and anti-thrombotic agents.
The formulation is designed to neutralize the virus, as antibodies bind to surface proteins of the virus and interfere with the onset of infection.
In one embodiment, the formulation is developed as a whole hyperimmune egg, hyperimmune egg yolk or purified antibodies (IgY).
In a preferred embodiment the formulation of the invention is used as a prophylactic and/or therapeutic against COVID-19 viral infection. The formulation of the invention is beneficial as a prophylactic and/or therapeutic against COVID-19 viral infection caused by strains selected from the group consisting of (B.1.1.7 (UK), B.1.351 (SA), P.l (Brazil), B.1.617.3 (India), B.1.429 and B.1.427 (USA), also referred to as YP_009724397.2, Alpha_B.1.1.7_Q.l-Q.8, Beta_B.1.351.1- 351.5, Delta_B.1.617.2_AY.l-AY.38, Gamma_P.l-P.1.9 and any other strain hitherto unknown.
In another embodiment, the antibodies are raised in chickens (polyclonal IgY antibodies) and harvested from chicken eggs immunized with the viral proteins, together or separately (and the yolks pooled).
In yet another embodiment, chicken is immunized with DNA (Innovio, Cadilla vaccines) or RNA (Pfizer BionTech and Moderna COVID vaccines) encoding the proteins as in DNA and RNA-based vaccines, other genetic constructs using viral vectors (Adeno or Adeno associated virus or Lentivirus or other as in Astra Zeneca Covishield), peptides of the proteins, or constructs with all proteins strung together (chimeric) or whole inactivated SARS COV-2 virus (COVAXIN, SINOVAC). All of the above antigenic designs may be mixed with adjuvants (Freunds - complete or incomplete, MPL etc) or with TLR agonists or with cytokines to improve immune responses in chickens. The harvested antibodies from hyperimmunized chickens are used as prophylactic formulation directly as whole IgY containing egg yolk, whole egg powder or may be further purified as pure IgY antibodies for use for oral administration or as purified IgY polyclonal protein therapeutically when administered parenterally.
Typical work flow that is used for generation of the anti-COVID-19 protein drug formulation is shown below in Scheme 1.
The following sequences and methods are used in the invention:
The Sars Cov2 protein: Spike proteins was used for the immunization of chicken IgY antibodies.
The full protein sequences of the Spike proteins are: domain shown in
Figure imgf000005_0001
MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWI FGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV WLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP GTNTSNQVAV LYQDVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY ECDIPIGAGI CASYQTQTNS PRRARSVASQ SIIAYTMSLG AENSVAYSNN SIAIPTNFTI SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE VFAQVKQIYK TPPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GAALQIPFAM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TASALGKLQD VVNQNAQALN TLVKQLSSNF GAISSVLNDI LSRLDKVEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA QEKNFTTAPA ICHDGKAHFP REGVFVSNGT HWFVTQRNFY EPQIITTDNT FVSGNCDVVI GIVNNTVYDP LQPELDSFKE ELDKYFKNHT SPDVDLGDIS GINASVVNIQ KEIDRLNEVA KNLNESLIDL QELGKYEQYI KWPWYIWLGF IAGLIAIVMV TIMLCCMTSC CSCLKGCCSC GSCCKFDEDD SEPVLKGVKL HYT 1273
Designed RBD Domain: SEQ ID NO. 2 used for raising IgY
ASV YAWNR KRISN CVADY SVLYN SASFS TFKCY GVSPT KLNDL CFTNV YADSF VIRGD
EVRQI APGQT GKIAD YNYKL PDDFT GCVIA WNSNN LDSKV GGNYN YLYRL FRKSN LKPFE
RDIST EIYQA GSTPC NGVEG FNCYF PLQSY GFQPT NGVGY QPYRV VVLSF ELLHA PATVC
GPKKS TNLVK NKCVN FNFNG LTGTG VLTES NKKFL PFQQF GRDIA DTTDA VRDPQ TLEIL
DITPC SFGGV SVITP GTNTS NQVAV LYQDV NCTEV PVAIH ADQLT PTWRV YSTGS NVFQT
GGSGGGSGGGS HHHHHH
Note: Provided in Bold is Poly glycine-serine (GS) linker sequence included to prevent protein folding problems. Provided in italics are Poly histidine sequence included to facilitate affinity purification.
A plasmid construct as designed above was used to express the proteins as designed. Purified RBD protein were used to vaccinate chickens and IgY antibodies isolated from vaccinated chicken eggs. Chicken vaccination and isolation of IgY are as described in (https://doi.org/10.1016Zi.eips.2017.05.069).
Uses
The antibodies of the invention are useful for the treatment of disease conditions in subjects, mammals in particular, including humans. In one embodiment, the protein drug is used for the treatment of viral infections and CO VID 19. In another embodiment the drug is also effective as both a prophylactic and therapeutic for viral infections
Route of Administration
The protein drug of the present invention is delivered to the subjects by forms suitable for each administration route. For example, the drug is administered as tablets, capsules, injection, drops, inhaler, ointment, foams suppository. In a preferred embodiment, the route of administration is oral, parenteral or intranasal. Nasal formulation includes powders, sprays, patches, inhalants and nebulizers.
Dosage Forms
The composition of the present invention is presented in unit dosage form generally in an amount that produces a therapeutic effect in the subject.
The compounds of the present invention are administered at a daily dose that is the lowest dose effective to produce a therapeutic effect. Generally, the dosage will affect from about 0.0001 to about lOOmg per kg body weight per day. Preferably, the dosage will range from about 0.001 to 75mg per kg body weight per day and more preferably, the dosage will range from about 0.1 to about 50mg per kg body weight per day. Each unit dose may be, for example, 5, 10, 25, 50, 100, 125, 150, 200 or 250 mg of the compound of the invention. As per the requirement of the subject, the effective daily dose of the compound is administered as two, three, four or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms.
Formulation
The protein drug of the present invention may be administered by any method known in the art. Some examples of suitable modes of administration include oral, intravenous, intramuscular, intranasal or any other parenteral mode of administration.
In certain embodiments, the present invention is directed to a method of formulating compounds of the present invention in a pharmaceutically acceptable carrier or excipient and may be administered in a wide variety of different dosage forms e.g. tablets, capsules, sprays, creams, lotions, ointments, aqueous suspensions syrups, and the like. Such carriers may include one or more of solid diluents or fillers, sterile aqueous media, and various nontoxic organic solvents, etc.
For oral administration, tablets may contain various excipients such as one or more of microcrystalline cellulose, sodium citrate, calcium carbonate and the like, along with various disintegrants such as starch and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose and the like. Solid compositions of a similar type may also be employed as fillers in gelatin capsules, nano emulsions, microfilms that dissolves on the tongue or as chewing gum or edible candy.
Oral administration also includes liquid formulations such as a oral rinse, digestible drinks or flavored powders in a sachet that can be formulated in a liquid form.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluents or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid find in the preparation of injectables. These aqueous solutions may be suitable for intravenous injection purposes. The oily solutions may be suitable for intra articular, intramuscular, and/or subcutaneous injection purposes.
In another embodiment, the compounds of the present invention may be administered topically that include transdermal, buccal, or sublingual application. For topical applications, therapeutic compounds may be suitably admixed in a pharmacologically inert topical carrier such as a gel, an ointment, a lotion, and/or a cream. Such topical carriers may include water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, and/or mineral oils.
The timing of the administration of the pharmaceutical composition may also be regulated. For example the compounds may be administered intermittently or by controlled release.
Combinations
The protein drug described here can be combined with other monoclonal antibodies targeting other SarsCov2 proteins, drugs, peptides that can interfere with viral entry and replication or antiinflammatory, steroid and anti-thrombotic agents that are used for COVID prevention and treatment.
The invention is further elucidated via Examples. The Examples though are for illustrative purposes and for better understanding and in no way to be construed as limiting the scope of the invention.
EXAMPLES
Example 1: Development of anti-RBD IgY
RBD domain peptide of SARS CoV-2 S protein (seq ID 2), were used to vaccinate chickens and boosted several times to achieve high titer. Titer was determined using partially (>85%) purified total IgYs from yolk. The ELISA data is shown below.
ELISA was carried out as per the standard protocol. RBD (lug) proteins was coated on the plate (in wells Al through B9 for Ih at 37C. Anti-RBD was diluted as shown and applied to the plate to bind RBD protein. PC = positive control where a known antigen X was coated on the plate and anti-X-IgY was applied to bind to antigen X.
CPC= Only anti-RBD or anti-X antibodies were applied. No antigen.
NC = Antigen X was coated on the plate and anti-RBD IgY antibodies are applied to check if there is any cross reactivity. The plate was blocked and washed following each addition as per standard ELISA protocol.
The color was developed by complexing HRP conjugated anti-IgY antibody and HRP standard as per standard ELISA protocols.
The results demonstrate that high titer IgY antibodies (>160,000) are obtained against RBD (Table 1, Figure 1). Ability to get high titer antibodies is prerequisite to the development of an effective therapeutics.
Table 1: Titer values obtained in I-ELISA assay
Figure imgf000009_0001
Example 2 Pretreatment of whole egg liquid containing IgY antibody
Before processing the egg yolk or whole egg solution, the Salmonella and yeast contaminants in the solution were sterilized by heat treatment. The preferred sterilization temperature at which the IgY titer is unaffected is 61 °C and the sterilization time required was 3 to 5 minutes. Example 3
Profiling the anti-RBD IGY antibody
Applicants used two separate methods to characterize the neutralizing activity of the anti-RBD IgY - a commercially available competition ELISA assay and a proprietary dot blot assay.
As shown in Figure 2 below, a comparison of the two methods shows similar neutralizing activity by the two assays. Using a concentration curve, we were able to determine that the IgY inhibited the binding of RBD to ACE2 similarly with IC50 of inhibition being 0.2 mg/ml in the ELISA assay and 0.1 mg/ml in the dot blot assay.
In another experiment the inhibition of a drug Homoharringtonine of RBD binding to ACE2 was examined. This is a drug which applicants have previously reported (BioRxIV https://doi.org/10.1101/2021.05.02.442384) to inhibit RBD binding to ACE2. As shown in Figure 3, at 50-500 pM concentrations a linear concentration response of inhibition was found suggesting that the dot blot assay is responsive over a range of inhibitory activities from 20-80% inhibition.
Example 4
To examine the utility of the IgY antibodies in preventing SarsCov2 infection, we developed liquid and dry powder formulations of the antibodies to be used as a drink or a sachet containing a powder as described below. a) Dry powder preparation of whole egg liquid containing antigen specific IgY antibodies
The whole egg liquid which has been pasteurized at 61 °C for 5 minutes was spray dried to prepare a powder. Spray drying was achieved by spraying the sterilized whole egg liquid or egg yolk liquid for 1 to 5 seconds at an inlet temperature 160°C to 190°C and drying the chamber at a temperature of 60°C to 90°C. b) Powder Formulation of IgY product
In order to make the anti-RBD IgY containing egg powder into a consumable and safe product, the whole egg powder or isolated IgY was formulated into a readily water-soluble powder form. The formulation by which the IgY activity was unaffected was a blend of maltodextrin (10-60%), Whole egg powder (1-10%), Dextrose (10-30%) and Aerosil (1-10%), Orange or any other flavoring agent (1-10%) and stevia or any other sweetener (0.1-10%) (Table 2).
Table 2: Original formulation of IgY powder in sachet
Figure imgf000010_0001
Figure imgf000011_0001
c) Liquid formulation of IgY Product
In order to make the anti-RBD IgY containing egg powder into a consumable and safe product, we have formulated the whole egg or yolk powder or isolated IgY into a readily drinkable beverages with different flavors. The formulation includes whole egg or yolk powder or IgY antibodies isolated (0.001-10%), Ashwagandha extract (1-10%), Immuno stimmulants for example Inulin (10- 40%), citric acid (0.1-10%), sodium benzoate (0.1-10%), flavors including but not limited to lichee, orange, strawberry or chocolate (0.1-10%) and sweetener including but not limited to Sucralose or stevia (Table 3, 4 and 5). Table 3: Oral Liquid formulation of IgY liquid.
Figure imgf000011_0002
Table 4: Powder formulation of Sweetened IgY powder (Sachet format) solubilized in water prior to oral administration.
Figure imgf000011_0003
Figure imgf000012_0001
Table 5: Powder formulation of Unsweetened IgY powder (Sachet format) solubilized in water prior to oral administration.
Figure imgf000012_0002
Example 5
RBD-ACE2 binding neutralizing activity of IgY prepared in a drink
Using the anti-RBD IgY of the invention, applicants used dot blot to measure the binding of RBD to ACE2 and the effect of saliva on the binding. Dot blot assay developed in-house was used for examining the binding of biotinylated RBD to ACE2. The dot blot is a competitive assay wherein higher the neutralizing activity results in lower blot color formation. The dot blot stain color intensity was captured and quantitated using by ImageJ software analysis.
The activity of the anti-RBD containing liquid drink is shown in Figure 4. As can be observed in the dot blot, control binding in sample A (binding of biotinylated RBD to ACE2, as indicated by the stain density) was maximum and addition of native anti-RBD IgY markedly reduced the binding of biotinylated RBD (sample B). A known compound NAC which blocks the binding of RBD binding to ACE2 was also tested (sample C), which inhibited the binding as expected. Sample D is the anti- RBD IgY in a drink which is used as a oral rinse and it showed strong inhibition of RBD binding to ACE2, thus confirming that it may be used as an oral rinse. Finally, sample E and F are unknown peptides used as negative controls and did not show any activity. The oral rinse is thus used in studies described below.
Example 6
Anti-RBD IgY neutralizes the binding of delta strain RBD to ACE2
The delta strain of SarsCov2 virus has emerged as the dominant strain in the world currently and is found in 80% of the people infected globally. The delta strain RBD is mutated with three amino acid changes from the native strain. These mutations make this strain more transmissible and infective. Applicants tested whether the IgY antibody (of the invention) raised as above will a) bind to delta RBD and b) neutralize the binding of delta RBD to ACE2 using ELISA and dot blot assays respectively.
Figure 5 shows the head to head comparison of direct binding of our IgY to either native or delta RBD coated on an ELISA plate. Bound IgY was detected using and biotinylated anti-IgY antibody. As seen in the Figure, the IgY bound directly to both native and delta RBD effectively with binding to delta RBD being almost 4-fold higher suggesting that the IgY does recognize the mutated delta strain RBD.
Example 7
Using the dot blot assay we also determined if the IgY antibody of the invention can inhibit the binding of ACE2 to delta RBD. As shown in Figure 6, at the concentrations of IgY used (1 pg/ml and 0.4 pg/ml) we found that IgY antibody inhibited ACE2 interaction with both native and delta RBD suggesting its utility in prevention of treatment of infections caused by delta strain.
Example 8
Oral rinse with IgY containing drink and detection of neutralizing activity in saliva
Applicants designed a study to test the ability of IgY formulated as a drink to boost RBD-ACE2 neutralizing activity in saliva after a single oral rinse with a leechi flavored drink containing IgY. To determine the increase in saliva neutralizing activity we designed a simple protocol. Healthy volunteers were provided with 10 ml of drink containing 16.6 ug/ml of purified anti-RBD IgY as an oral rinse. The volunteers rinsed their oral cavity for 3 minutes and then spit out the rinse. Saliva samples were collected at zero time (basal sample before oral rinse) and at 30, 1 hour, 2 hours and 3 hours after the rinse and stored at 4°C till the dot blot assay was performed. Just prior to use, the saliva was gently centrifuged at 5000 RPM for 2 minutes to settle any debris and the supernatant (10 p l) was used in the dot blot. Data are reported as increase in neutralizing activity over the basal levels.
The data of RBD-ACE2 binding neutralizing activity is shown below. Figure 7 shows the activity in saliva samples from a volunteer who was not vaccinated and was RTPCR negative for COVID 19. The data shows that there was a time-dependent increase in neutralizing activity up to 60% increase over baseline, with persistence of some activity till 2 hours after oral rinse.
The Applicants then looked at another volunteer who was vaccinated one month ago and did not have COVID 19. As shown in Figure 8, there was up to a 30% increase in neutralizing activity above baseline and there was demonstrable activity even at 3 hours after the oral rinse. These data suggest that even in an individual who had one shot of vaccine there was still an increase in neutralizing over base line.
The effect of oral rise on saliva neutralizing activity in another volunteer who had received both shots of vaccine a month before was also examined. Interestingly in this case the increase in saliva neutralizing activity was not detectable at 30 minutes but was clearly present at 1, 2 and 3 hours with peak activity of 30% increase at 2 hours (Figure 9). This suggest that even in fully vaccinated individuals, there is still an increase in saliva neutralizing activity by oral rinse with the IgY beverage.
Finally the impact of oral rinse on an individual who had recently recovered from COVID 19 and was RTPCR negative at the time of this study was examined. There was no increase in the neutralizing activity at any time point after the oral rinse. It can be deduced that lack of increase in neutralizing activity was due to high levels of neutralizing activity in this individual with recent COVID 19 infection, but that remains to be tested.
Example 9
The applicants also examined the impact of various sachet formulations for their neutralizing activity. Four formulations with Orange 1, Orange 2, coconut and blueberry dissolved in water were compared for their neutralizing activity in the dot blot assay. While all of the flavored sachet retained neutralizing activity, orange flavor 2 retained (98%) activity similar to anti-RBD IgY, suggesting its superiority over other flavors (Table 6). Table 6. Sachet Formulations: Neutralizing Activity
Figure imgf000015_0001
Example 10 The effect of IgY was also examined in SarsCov2 in vero cells in a plaque reduction neutralization test (PRNT) and PRNT50 was determined ( dilution of IgY at which there was 50% reduction in plaque formation) . Since the IgY neutralize the binding of RBD to ACE2, it is expected that there will be reduction in viral entry into the cells. Table 7 below shows that at two concentrations tested, IgY was able to reduce plaque formation effectively. Table 7. Plaque formation reduced by IgY antibody
Figure imgf000015_0002

Claims

CLAIMS We Claim:
1. An antibody characterized in that it binds to the receptor binding domain (RBD) of SarsCov2 spike protein comprising a sequence set forth in SEQ ID NO. 1.
2. The antibody as claimed in claim 1 wherein the said antibody is selected from IgY antibody, IgG antibody, nanobody, polyclonal, monoclonal or truncated antibody derived from phage display.
3. The antibody as claimed in claim 1 wherein the said antibody is a IgY antibody
4. The antibody as claimed in claim 1 wherein the said antibody is a raised in chicken or other avian species.
5. A formulation comprising the antibody as claimed in claim 1 wherein the said formulation is formulated as a powder or liquid.
6. The formulation as claimed in claim 5 wherein the said formulation is selected from the group consisting of nanoparticles, nano-emulsions, tablets, enteric coated capsules, nebulizer, oral rinse, oral drink and microfilm.
7. The formulation as claimed in claim 5 wherein the said formulation is prepared using a whole hyperimmune egg, hyperimmune egg yolk, whole egg powder or purified antibodies.
8. Use of antibody characterized in that it binds to the receptor binding domain (RBD) of SarsCov2 spike protein comprising a sequence set forth in SEQ ID NO. 1 as a prophylactic or therapeutic for the treatment of COVID- 19 viral infections.
9. Use of antibody as claimed in claim 8 wherein the said COVID- 19 viral infection is caused by variants selected from the group of strains consisting of B.1.1.7, B.1.351, P.l, B.1.617.3, B.1.429, B.1.427, YP_009724397.2, Alpha_B.1.1.7_Q.l-Q.8, Beta_B.1.351.1-351.5, Delta_B.1.617.2_AY.l-AY.38 and Gamma_P.l-P.1.9.
10. Use of antibody as claimed in claim 8 in combination with monoclonal or antibodies targeting SarsCov2 proteins, drugs, peptides or compounds that interfere with viral entry and replication.
11. Use of antibody as claimed in claim 8 in combination with antivirals, anti-inflammatory, steroid and anti-thrombotic agents.
12. Use of antibody as claimed in claim 8 for the treatment of human subjects wherein the said subject is vaccinated against COVID-19 virus or unvaccinated or infected with the any of the strains of COVID-19 virus as claimed in claim 8.
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Publication number Priority date Publication date Assignee Title
US10787501B1 (en) * 2020-04-02 2020-09-29 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments

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Publication number Priority date Publication date Assignee Title
US10787501B1 (en) * 2020-04-02 2020-09-29 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments

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Title
LU ET AL.: "Generation of Chicken IgY against SARS-COV-2 Spike Protein and Epitope Mapping", J IMMUNOL RES., vol. 2020, no. 9465398, 17 October 2020 (2020-10-17), pages 1 - 8, XP055872677, DOI: 10.1155/2020/9465398 *

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