WO2021216954A1 - Compositions et méthodes de traitement ou de prévention d'une infection virale - Google Patents

Compositions et méthodes de traitement ou de prévention d'une infection virale Download PDF

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WO2021216954A1
WO2021216954A1 PCT/US2021/028772 US2021028772W WO2021216954A1 WO 2021216954 A1 WO2021216954 A1 WO 2021216954A1 US 2021028772 W US2021028772 W US 2021028772W WO 2021216954 A1 WO2021216954 A1 WO 2021216954A1
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antibody
integrin
integrin antagonist
fragment
derivative
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PCT/US2021/028772
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David Cheresh
Sara WEIS
Stephen Mccormack
Thomas Rogers
Tami VON SCHALCHA
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Alpha Beta Holdings, Llc
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Publication of WO2021216954A1 publication Critical patent/WO2021216954A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2848—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • 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
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention further relates to methods of treating, reducing the severity of, or preventing RGD and/or RLD-dependent virus infections using integrin antagonists, such as antibodies or fragments or derivatives thereof, peptides, or peptidomimetics targeted to alpha V-containing integrins that recognize RGD binding motifs, integrin ⁇ M ⁇ 2 that recognizes RLD binding motifs, or integrin ⁇ v ⁇ 3 that recognizes both RGD and RLD binding motifs.
  • integrin antagonists such as antibodies or fragments or derivatives thereof, peptides, or peptidomimetics targeted to alpha V-containing integrins that recognize RGD binding motifs, integrin ⁇ M ⁇ 2 that recognizes RLD binding motifs, or integrin ⁇ v ⁇ 3 that recognizes both RGD and RLD binding motifs.
  • the invention additionally includes compositions useful for carrying out the methods of the invention.
  • SARS-CoV-2 belongs to a family of viruses that includes SARS-CoV that causes severe acute respiratory syndrome and MERS-CoV that causes Middle East respiratory syndrome, both of which are the causes of major epidemics.
  • Full-length genomic sequencing of the new SARS-CoV-2 virus reveals 79.6% sequence identity with SARS-CoV, and this study also confirmed that SARS-CoV-2 utilizes the same cell entry receptor (angiotensin converting enzyme II, ACE2) as SARS-CoV (Zhou, Yang et al.2020).
  • ACE2 angiotensin converting enzyme II
  • S proteins form a spike that the virus uses to infect host cells by binding to receptors on the surface of host cells.
  • the biological events that govern the ability of this spike protein to interact with host cells represents a potential opportunity to block virus attachment, fusion, and entry.
  • the S protein contains a receptor binding domain (RBD) in the S1 subunit that contains binding sites recognized by different receptors.
  • RBD receptor binding domain
  • SARS-CoV-2 virus that is lacking in the other SARS-like coronaviruses is the presence of a site on the S protein that can be cleaved by the enzyme furin (Coutard, Valle et al.2020). Furin is highly expressed in lungs, and its ability to cleave a site on the S protein of SARS-CoV-2 may contribute to the more aggressive pathogenicity of the new virus, since cleavage can release the S1 subunit that allows SARS-CoV-2 to bind to angiotensin converting enzyme (ACE2) more tightly than the SARS virus.
  • ACE2 angiotensin converting enzyme
  • RGD sequence Adjacent to the ACE2 binding sequence on the spike protein is an “RGD sequence” that is the minimal peptide sequence recognized by the family of alpha V integrins, cell surface receptors with diverse biological functions. Since this RGD sequence is present in SARS-CoV-2 but absent from all other coronaviruses examined, it has been suggested that the new virus may have gained the ability to utilize integrins as cell receptors to mediate virus entry (Sigrist, Bridge et al.2020). A high-throughput virtual screen was performed to search chemical libraries for agents capable of preventing the interaction of the S protein with both ACE2 and integrins, producing a list of potential drug candidates (Yan, Sun et al.2020).
  • compositions and methods of using such compositions, to treat or prevent virus infections, including in particular SARS-CoV-2 and other viruses that depend on RGD binding to mediate entry into cells.
  • virus infections including in particular SARS-CoV-2 and other viruses that depend on RGD binding to mediate entry into cells.
  • adenoviruses via an RGD adhesion sequence in their penton base coat protein
  • alpha V integrins on mammalian cells to facilitate viral uptake (Wickham, Mathias et al. 1993, Nemerow, Cheresh et al.1994, Wickham, Filardo et al.
  • the RLD motif mediates binding to only integrins ⁇ v ⁇ 3 and ⁇ M ⁇ 2 (also known as CD11b/CD18 or Mac-1). A relationship between the RLD motif and virus entry has not been recognized previously. An analysis of potential integrin binding sites in coronavirus S proteins did not identify the RLD motif (Tresoldi, Sangiuolo et al. (2020)). The RLD integrin binding motif is present in the heptad repeat 1 (HR1) domain of the S protein of all coronaviruses. The HR1 domain mediates membrane fusion. An analysis of the three-dimensional structure of the S protein of SARS- CoV-2 shows that the RGD and RLD sequences, while in different parts of the linear amino acid sequence (S1 vs.
  • integrin ⁇ v ⁇ 3 mediates entry of rotaviruses, but their entry cannot be blocked by RGD peptides (Guerrero, Méndez et al. 2000).
  • the present invention reveals that many viruses also have an RLD motif on a surface protein, including viruses that have been linked to ⁇ v ⁇ 3 for entry and internalization.
  • the RLD motif may represent an important mechanism for viral entry and internalization that functions independently or in addition to an RGD motif.
  • the RLD motif is a new target for disruption of viral entry for coronaviruses and other viruses.
  • the RLD motif is recognized by only two integrins (integrin ⁇ v ⁇ 3 and ⁇ M ⁇ 2), it represents a more selective target than the RGD motif that is recognized by many integrins, including those containing ⁇ 1 or ⁇ v (Ruoslahti 1996).
  • the present inventors have studied integrin ⁇ 3 expression, particularly ⁇ v ⁇ 3, and elucidated expression changes that may play a critical role in the sensitivity of subjects to infection by RGD and/or RLD-dependent viruses such as SARS-CoV-2. These findings also provide guidance for effective treatment of infected subjects as well as prevention of infection.
  • Subjects that are more likely to be infected and/or more likely to incur a severe infection can be identified based on integrin expression levels and conditions that are known to elevate integrin expression levels, such as tissue injury and inflammation. These identified subjects also are the ones most likely to benefit from a targeted treatment aimed at inhibiting the specific integrin(s) responsible for enhancing viral infection and its consequences.
  • One example is the relationship between the severity of COVID-19 in people with preexisting health issues and the ability of SARS-CoV-2 to enter cells. In particular, patients with cancer are more likely to be infected with COVID-19 (Leslie 2020).
  • integrin ⁇ v ⁇ 3 a driver of metastasis, stemness, and drug resistance in multiple types of solid tumors.
  • integrin ⁇ v ⁇ 3 a driver of metastasis, stemness, and drug resistance in multiple types of solid tumors.
  • An enrichment of integrin ⁇ v ⁇ 3 expression on lung cancers that had gained resistance to EGFR blockade was previously reported (Seguin, Kato et al.2014), and new data presented here reveals the induction of ⁇ 3 expression when normal or malignant epithelial cells are exposed to cellular stresses or inflammatory cytokines.
  • one aspect of the invention relates to a method of inhibiting uptake of an RGD and/or RLD-dependent virus into a cell, comprising contacting the cell with an effective amount of an integrin antagonist.
  • Another aspect of the invention relates to a method of treating, inhibiting the severity of, or preventing an RGD and/or RLD-dependent virus infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an integrin antagonist, thereby treating, inhibiting the severity of, or preventing RGD and/or RLD-dependent virus infection.
  • a further aspect of the invention relates to a method of treating, inhibiting the severity of, or preventing an RGD and/or RLD-dependent virus infection in a subject in need thereof, comprising the steps of: a) identifying a subject that has a condition that increases integrin expression; and b) if the subject has a condition that increases integrin expression, administering to the subject a therapeutically effective amount of an integrin antagonist, thereby treating, inhibiting the severity of, or preventing RGD and/or RLD-dependent virus infection.
  • An additional aspect of the invention relates to an integrin antagonist, e.g., one that inhibits entry of an RGD and/or RLD-dependent virus into a cell.
  • a further aspect of the invention relates to a polynucleotide encoding the integrin antagonist of the invention and a vector or host cell comprising the polynucleotide.
  • Another aspect of the invention relates to a composition, e.g., a pharmaceutical composition, comprising the integrin antagonist of the invention and a carrier.
  • An additional aspect of the invention relates to a kit comprising the integrin antagonist of the invention.
  • FIGS.1A-1B show anti- ⁇ v ⁇ 3 (mIgG1, hIgG1, or hLM609-hIgG4-S228P) blocks SARS-CoV-2 infection in human HeLa-ACE2 cells.
  • Live SARS-CoV-2 virus was added to HeLa-ACE2 cells in the presence of integrin function-blocking antibodies that recognize the ⁇ 1 integrin subunit, the ⁇ v ⁇ 3 integrin heterodimer, or the ⁇ v ⁇ 5 integrin heterodimer. After 24 h, cells were fixed, permeabilized, and stained using serum from a COVID-19 patient to identify the extent of virus infection.
  • Fig.1A Representative images and full dose range for hLM609- hIgG4-S228P are shown in Fig.1A. Immunoblot for HeLa-ACE2 and VERO cell lysates confirms integrin ⁇ 3 expression.
  • Fig.1B shows control plus three doses each for three different anti- ⁇ v ⁇ 3 antibodies (hLM609-hIgG4-S228P, hLM609-hIgG1, and LM609- mIgG1), two antibodies targeting other RGD-dependent integrins ( ⁇ v ⁇ 5 and ⁇ 1), and two peptide antagonists (cilengitide and cRGDFV).
  • FIGS.2A-2C show that integrin ⁇ 3 expression is absent on normal lung bronchial epithelium but upregulated by injury, stress, or inflammation.
  • HCC827 human lung cancer cells were grown for 24 h in the presence of vehicle control or 4 ng/ml TGF ⁇ 1. qPCR analysis for ITGB3 mRNA expression is shown as fold change relative to control (PBS). Right, H358 and HCC827 human lung cancer cells were grown in the presence of 10 ng/ml TGF ⁇ 1 for 48 h, then protein expression was analyzed by immunoblot. Graphs show mean ⁇ SD. [0025] FIG.3 shows integrin ⁇ 3 protein expression is absent on normal pancreas but upregulated in pancreatitis and cancer.
  • FIGS.4A-4D show the effect of inflammation on integrin ⁇ 3 expression in pancreatic cells.
  • A) Mouse pancreatic acinar cells were treated with vehicle control (PBS), a combination of cytokines (50 ng/ml TNF ⁇ and 50 ng/ml CCL5 for 5 days), or the pancreatitis-inducing agent caerulein (10-100nM for 48 hours). mRNA expression was analyzed by qPCR.
  • FIG.5 is a schematic showing the location of RGD and RLD integrin binding motifs in the SARS-CoV-2 S protein amino acid sequence (SEQ ID NO:16).
  • FIG.6 is a sequence alignment showing unique expression of the RGD motif on SARS-CoV-2.
  • the RGD integrin binding motif that is recognized by ⁇ v-containing integrins is present at amino acids 403-405 in the SARS-CoV-2 virus, but not found in any of the other betacoronaviruses examined.
  • FIG.7 is a sequence alignment showing the highly conserved RLD motif for betacoronaviruses.
  • the RLD integrin binding motif that is recognized by integrin ⁇ v ⁇ 3 and ⁇ M ⁇ 2 is highly conserved across betacoronaviruses except for the bat coronavirus HKU9.
  • FIG.8 shows the locations of the RGD and RLD binding motifs on a 3D structural visualization of the SARS-CoV-2 virus spike protein trimer. Top, Three RGD motifs are shown at the center of the spike.
  • RGD motif is predicted to be exposed in the up promoter, while somewhat concealed on the two down promoters, to participate in receptor binding.
  • An RLD motif located at the apex of the HR1 region is shown to act as a pedestal upon which the RBD from an adjacent down promoter sits. This region may impact the metastability of the spike before cleavage, after which the exposed RLD binding motif may interact with integrin ⁇ v ⁇ 3 to facilitate membrane fusion and/or virus internalization. Integrins are known to be robustly activated by binding to multivalent ligands, such as the conformation of RGD/RLD motifs predicted by the 3D model.
  • FIGS.9A-9B are schematics showing the implications for ⁇ v ⁇ 3 expression on susceptibility and progression of COVID-19, and the proposed effect of anti-integrin therapy on SARS-CoV-2 virus internalization.
  • integrin ⁇ v ⁇ 3 expression may already be elevated and contribute to an elevated susceptibility to infection and/or a more rapid disease progression that could be further exacerbated by the release of cytokines.
  • This scenario provides the rationale for targeting integrin ⁇ v ⁇ 3 as a novel therapeutic opportunity to protect host cells from viral infection. Such a strategy could be used in combination with other approaches to directly target the virus and/or suppress the release of cytokines.
  • the SARS-CoV-2 virus enters host cells by first engaging host cell surface receptors, including ACE2.
  • alpha V integrins bind to the virus spike proteins that each contain three RGD sequences, and this multivalent interaction promotes integrin clustering and activation.
  • SARS-CoV-2 utilizes alpha V integrins to mediate internalization into host cells.
  • An antibody, peptide, organic molecule, or other naturally agent that disrupts the ligand binding capacity of an alpha V-containing integrin, such as ⁇ v ⁇ 3, can prevent SARS-CoV-2 uptake and internalization.
  • DETAILED DESCRIPTION [0032] 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.
  • 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 ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • 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.
  • 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 term “consists essentially of” (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5’ and/or 3’ or N-terminal and/or C-terminal ends of the recited sequence or between the two ends (e.g., between domains) such that the function of the polynucleotide or polypeptide is not materially altered.
  • SEQ ID NO a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • a “peptide” refers to a polypeptide containing less than 20 amino acid residues, e.g., less than 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acid residues, and incudes linear and cyclic peptides.
  • chimeric refers to a molecule having two or more portions that are not naturally found together in the same molecule.
  • 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.
  • isolated means a molecule, e.g., a protein, polynucleotide, or cell, 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 molecule. The term also encompasses molecules that have been prepared synthetically.
  • treat 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.
  • the terms “prevent,” “prevents,” or “prevention” and “inhibit,” “inhibits,” or “inhibition” 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.
  • 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.
  • a “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.
  • the effects need not be complete or curative, as long as some benefit is provided to the subject.
  • the term “bind specifically” or “specifically binds” in reference to an antibody or a fragment or derivative thereof of the invention means that the agent will bind with an epitope (including one or more epitopes) of a target, but does not substantially bind to other unrelated epitopes or molecules.
  • the term refers to an agent 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.
  • Integrin antagonists e.g., antibodies and fragments and derivatives thereof, peptides, peptidomimetics
  • a first aspect of the invention relates to integrin antagonists (e.g., antibodies and fragments and derivatives thereof, peptides, peptidomimetics) that bind integrin and can be used in methods of inhibiting uptake of an RGD and/or RLD-dependent virus into a cell and methods of treating, inhibiting the severity of, or preventing an RGD and/or RLD-dependent virus infection in a subject.
  • an RGD and/or RLD-dependent virus is any virus that depends at least in part on the presence of an RGD sequence, an RLD sequence, or both sequences on the surface of the virus (e.g., on a structural protein) for attachment and/or entry into cells.
  • the integrin antagonist can inhibit the uptake of an RGD-dependent virus.
  • the integrin antagonist can inhibit the uptake of an RLD-dependent virus.
  • the integrin antagonist can inhibit the uptake of an RGD and RLD-dependent virus.
  • RGD and/or RLD-dependent viruses attach to a cell by binding a cell surface receptor (e.g., ACE2 for SARS-CoV-2), followed by the virus binding to integrins, causing the integrins to cluster and facilitate virus internalization (See FIG.9B).
  • a cell surface receptor e.g., ACE2 for SARS-CoV-2
  • integrin antagonists blocks virus- integrin binding to prevents internalization, even if the virus has initially attached to the cell surface receptor.
  • the integrin antagonist can be one that binds to a specific integrin or binds to a class of integrins.
  • the integrin may be any integrin that is known or later identified to mediate entry of an RGD and/or RLD-dependent virus into a cell.
  • the integrin antagonist specifically binds integrin ⁇ v.
  • the integrin antagonist specifically binds integrin ⁇ 3.
  • the integrin antagonist specifically binds a single integrin heterodimer such as ⁇ v ⁇ 3, ⁇ M ⁇ 2, or ⁇ v ⁇ 5.
  • the integrin antagonist may be any structure that is capable of binding to an integrin on the surface of a cell and inhibiting virus attachment and/or entry into the cell.
  • the integrin antagonist may be an RGD peptide or an analog or derivative thereof.
  • the integrin antagonist may be an RLD peptide or an analog or derivative thereof.
  • the integrin antagonist is a cyclic peptide, e.g., the cyclic RGD peptide cilengitide or analogs thereof. See, e.g., Meena, Singh et al. (2020), incorporated by reference herein in its entirety.
  • the cyclic peptide is an RLD version of cilengitide or analogs thereof.
  • the integrin antagonist is a peptidomimetic, e.g., of an RGD or RLD peptide.
  • the peptidomimetic may be one that has increased stability relative to a peptide, e.g., by replacing one or more peptidic bond or using one or more non-naturally occurring amino acids.
  • analog is used to refer to a peptide which differs from a disclosed peptide by modifications to the peptide, but which significantly retains a biological activity of the disclosed peptide.
  • Minor modifications include, without limitation, changes in one or a few amino acid side chains, changes to one or a few amino acids (including deletions, insertions, and substitutions), changes in stereochemistry of one or a few atoms, and minor derivatizations, including, without limitation, methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation, and addition of glycosylphosphatidyl inositol.
  • substantially retains refers to a fragment, analog, or other variant of a peptide that retains at least about 20% of the activity of the naturally occurring peptide (e.g., binding to an integrin), e.g., about 30%, 40%, 50% or more.
  • Peptides and analogs or fragments of the invention can be modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant peptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular interaction or uptake.
  • blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by any suitable methods. For example, one or more non- naturally occurring amino acids, such as D-alanine, can be added to the termini. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety.
  • the peptide terminus can be modified, e.g., by acetylation of the N- terminus and/or amidation of the C-terminus.
  • the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable “carrier” proteins prior to administration.
  • the integrin antagonist may be a small molecule, e.g., a compound having a molecular mass less than 1000 Da.
  • the integrin antagonist may be an antibody or a fragment or derivative thereof.
  • the antibody or a fragment or derivative thereof is an antibody or an antigen-binding fragment thereof.
  • the antibody or a fragment or derivative thereof comprises one or more first domains corresponding to a Fab domain. In some embodiments, the antibody or a fragment or derivative thereof further comprises one or more second domains corresponding to an Fc domain. In some embodiments, one or both domains of the antibody or a fragment or derivative thereof is a non-immunoglobulin scaffold, an aptamer, a small molecule (e.g., a receptor ligand), or other binding moiety. [0061] In certain embodiments, the first domain of the antibody or a fragment or derivative thereof is an antibody domain. In certain embodiments, the second domain of the antibody or a fragment or derivative thereof is an antibody domain. In some embodiments, both domains are antibody domains.
  • the first domain is a humanized or human antibody domain.
  • the second domain is a humanized or human antibody domain.
  • the first domain and the second domain are humanized or human antibody domains.
  • the antibody or a fragment or derivative thereof may be a bispecific antibody or a fragment or derivative thereof.
  • the bispecific antibody or a fragment or derivative thereof may bind a second antigen present on a cell comprising an integrin, e.g., another cell surface component utilized by an RGD and/or RLD-dependent virus to attach to and/or enter a cell.
  • the second antigen is ACE2.
  • the second antigen could be derived from a neutralizing or non-neutralizing antibody isolated from a patient who has recovered from COVID-19.
  • the first domain comprises, consists essentially of, or consists of a Fab domain of an antibody.
  • the Fab domain may be from any antibody isotype.
  • the first domain comprises a Fab domain of an IgG antibody, e.g., an IgG1 or IgG4 antibody.
  • the first domain comprises the amino acid sequence of the light chain of hLM609-hIgG4-S228P (SEQ ID NO:2) and the Fab portion (also known as the Fd fragment) of the heavy chain of hLM609-hIgG4-S228P (SEQ ID NO:3) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the first domain comprises the amino acid sequence of a superhumanized variant of shLM609-hIgG1-WT, e.g., the LM609_7 Fab domain of heavy chain (SEQ ID NO:5) and light chain (SEQ ID NO:6) or the JC7U Fab domain of heavy chain (SEQ ID NO:7) and light chain (SEQ ID NO:8) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • a superhumanized variant of shLM609-hIgG1-WT e.g., the LM609_7 Fab domain of heavy chain (SEQ ID NO:5) and light chain (SEQ ID NO:6) or the JC7U Fab domain of heavy chain (SEQ ID NO:7) and light chain (SEQ ID NO:8) or a sequence at least 90% identical thereto, e.g., at
  • the first domain comprises the amino acid sequence of the light chain of hLM609-hIgG1-WT (SEQ ID NO:9) and the Fab portion of the heavy chain of hLM609-hIgG1-WT (SEQ ID NO:10) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the second domain does not significantly engage natural killer (NK) cells.
  • the second domain does not significantly engage one or more types of lymphocytes, e.g., NK cells, B cells, or T cells. “Does not significantly engage,” as used herein, refers to less than 30% of the total engaged cells being the indicated cell type, e.g., less than 25%, 20%, 15%, 10%, or 5%.
  • the second domain specifically binds a protein on the surface of a myeloid-derived cell to mediate antibody-dependent cytotoxicity of cells expressing the target antigen.
  • the protein is not present or only present at low levels on other cell types, e.g., natural killer cells.
  • the second domain specifically binds to an Fc-gamma receptor.
  • the second domain specifically binds Fc-gamma receptor 1 (Fc ⁇ R1, CD64).
  • the second domain specifically binds Fc-gamma receptor IIA (Fc ⁇ RIIA, CD32) or Fc-gamma receptor IIIA (Fc ⁇ RIIIA, CD16a).
  • the second domain does not bind Fc-gamma receptor IIB (Fc ⁇ RIIB).
  • the second domain comprises, consists essentially of, or consists of a Fc domain of an antibody.
  • the Fc domain may be from any antibody isotype.
  • the first domain comprises a Fc domain of an IgG antibody, e.g., an IgG1 antibody or an IgG4 antibody.
  • the second domain comprises a Fc domain of an IgA or IgE antibody.
  • the second domain further comprises a hinge domain of an antibody.
  • the second domain comprises the amino acid sequence of the heavy chain Fc domain and hinge domain of hLM609-hIgG4-S228P (SEQ ID NO:4) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the second domain comprises the amino acid sequence of the heavy chain Fc domain and hinge domain of hLM609-hIgG1-WT (SEQ ID NO:9) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the antibody or a fragment or derivative thereof comprises the amino acid sequence of the hLM609-hIgG4-S228P heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:2) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the antibody or a fragment or derivative thereof comprises the amino acid sequence of the hLM609-hIgG1-WT heavy chain (SEQ ID NO:9) and light chain (SEQ ID NO:10) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the antibody or a fragment or derivative thereof comprises the amino acid sequence of the shLM609- hIgG1-LALA PG YTE heavy chain (SEQ ID NO:17).
  • the antibody or a fragment or derivative thereof is any antibody known to bind to one or more integrins.
  • the antibody or a fragment or derivative thereof is etaracizumab/MEDI-522 (ABEGRINTM), MEDI-523 (VITAXINTM), intetumumab/CNTO 95, or an antibody or a fragment or derivative thereof comprising a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the antibody or a fragment or derivative thereof may include sequence modifications that are known to enhance the characteristics of an antibody, e.g., stability, or alter the binding of the antibody to Fc-gamma receptors.
  • the amino acid sequence of the antibody or a fragment or derivative thereof comprises a S228P (Eu numbering system) mutation in the hinge region.
  • the amino acid sequence comprises a mutation selected from: a) S239D/A330L/I332E; b) I332E; c) G236A/S239D/I332E; d) G236A; e) N297A/E382V/M428I; f) M252Y/S254T/T256E; g) Q295R/L328W/A330V/P331A/I332Y/E382V/M428I; h) L234A/L235A/P329G; i) M428L/N434S; j) L234A/L235A/P331S; k) L234A/L235A/P329G/M252Y/S254T/T256E;
  • antibody refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE.
  • the antibody can be monoclonal, oligoclonal, or polyclonal and can be of any species of origin, including (for example) mouse, rat, hamster, rabbit, horse, cow, goat, sheep, pig, camel, monkey, 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.
  • 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.
  • 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)).
  • antibody fragment may also include any protein construct that is capable of binding a target antigen.
  • Antibodies of the invention may be altered or mutated for compatibility with species other than the species in which the antibody was produced.
  • 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.
  • CDR complementarity determining region
  • donor antibody non-human species
  • 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.
  • 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 can be a superhumanized antibody where only two CDRs are non-human (US Patent No.7,087,409).
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), 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)).
  • Fc immunoglobulin constant region
  • 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.
  • 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.
  • 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.
  • Recombinant or synthetic polypeptides and peptides are the preferred immunogens for the production of monoclonal or polyclonal antibodies.
  • 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®.
  • an immunogen e.g., a purified or synthetic peptide, a peptide coupled to an appropriate carrier (e.g., glutathione-S-transferase, keyhole limpet hemocyanin, 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.
  • an immunogen e.g., a purified or synthetic peptide, a peptide coupled to an appropriate carrier (e.g., glutathione-S-transferase, keyhole limpet hemocyanin, 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
  • 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.
  • 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.
  • 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.
  • a carrier protein e.g., as a fusion protein
  • 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.
  • monoclonal antibodies from various mammalian hosts, such as rodents, lagomorphs, 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 are fused with myeloma cells. The result is a hybrid cell or “hybridoma” that is capable of reproducing in vitro.
  • an immunogen e.g., an immunogenic peptide either alone or optionally linked to a carrier protein.
  • myeloma cells myeloma cells
  • the population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen.
  • 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.
  • 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.
  • the present invention additionally provides polynucleotides encoding the integrin antagonist (e.g., the antibody or a fragment or derivative thereof) of this invention.
  • the polynucleotides comprises a heavy chain encoding nucleotide sequence of SEQ ID NO:13 and a light chain encoding sequence of SEQ ID NO:14 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical.
  • the polynucleotides comprises a heavy chain encoding nucleotide sequence of SEQ ID NO:15 and a light chain encoding sequence of SEQ ID NO:14 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical.
  • a vector comprising the polynucleotide of the invention.
  • Vectors include, but are not limited to, plasmid vectors, phage vectors, virus vectors, or cosmid vectors.
  • the present invention provides a host cell comprising the polynucleotide and/or vector of this invention.
  • the host cell can be a eukaryotic or prokaryotic cell and may be used for expressing the antibody or a fragment or derivative thereof or other purposes.
  • a further aspect of the invention relates to a composition comprising the integrin antagonist (e.g., the antibody or a fragment or derivative thereof) of the invention and a carrier.
  • the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further comprise an additional therapeutic agent, e.g., an antiviral agent.
  • Antiviral agents include, without limitation, remdesivir, gimsilumab, REGN3048, REGEN3051, Kevzara, AdCOVID, EIDD- 2801, favipiravir (Avigan), umifenovir (Arbidol), lopinavir, ritonavir, kaletra (a combination of lopinavir and ritonavir), danoprevir+ritonavir, falidesivir, oseltamivir, emtricitabine/tenofovir, nelfinavir, or darunavir.
  • the additional therapeutic agent is one that inhibits the interaction of the RGD and/or RLD-dependent virus with a cell surface receptor used for attachment.
  • the binding of SARS-CoV-2 to ACE2 or the function of ACE2 may be inhibited, e.g., using hesperidin, curcumin, brazilin, galangin, nafamostat, desmethylcurcumin, bisdesmethylcurcumin, tangeretin, hesperetin, nobiletin, naringenin, brailein, aceto cavicol acetate, rutin, diosmin, apiin, diacetyl curcumin, rescinnamine, iloprost, prazosin, posaconazole, itraconazole, sulfasalazine, azlocillin, penicillin, cefsulodin, dabigatran etexilate, licoflavonol, cos
  • kits comprising the integrin antagonist (e.g., the antibody or a fragment or derivative thereof) of the invention or cells for producing the integrin antagonist (e.g., the antibody or a fragment or derivative thereof, peptide, or peptidomimetic) of the invention.
  • the kit can include multiple integrin antagonists and/or compositions containing such agents.
  • each of the multiple integrin antagonists provided in such a kit can specifically bind to a different antigen and/or inhibit a different RGD and/or RLD-dependent virus.
  • the kit can further include an additional active agent, e.g., an antiviral agent as would be known to one of skill in the art.
  • the kit can further include additional reagents, buffers, containers, instructions, etc.
  • Another aspect of the invention relates to a method of inhibiting uptake of an RGD and/or RLD-dependent virus into a cell, comprising contacting the cell with an effective amount of an integrin antagonist (e.g., an antibody or a fragment or derivative thereof, peptide, or peptidomimetic), e.g., an integrin antagonist of the invention.
  • an integrin antagonist e.g., an antibody or a fragment or derivative thereof, peptide, or peptidomimetic
  • An additional aspect of the invention relates to a method of treating, inhibiting the severity of, or preventing an RGD and/or RLD-dependent virus infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an integrin antagonist (e.g., an antibody or a fragment or derivative thereof that binds integrin, peptide, or peptidomimetic), e.g., the integrin antagonist or the pharmaceutical composition of the invention, thereby treating, inhibiting the severity of, or preventing RGD and/or RLD- dependent virus infection.
  • an integrin antagonist e.g., an antibody or a fragment or derivative thereof that binds integrin, peptide, or peptidomimetic
  • the onset of infection is delayed relative to the timing in the absence of the method.
  • the severity of the infection e.g., the number of symptoms or the severity of the symptoms, is reduced relative to the level in the absence of the method.
  • the percentage of subjects exposed to the virus that actually get infected is reduced relative to the percentage in the absence of the method.
  • the recovery from the infection is faster relative to the recovery in the absence of the method.
  • the subject is one that has tested positive for an RGD and/or RLD-dependent virus infection. In some embodiments, the subject is one that has or may have been exposed to an RGD and/or RLD-dependent virus.
  • the subject is one that will potentially be exposed to an RGD and/or RLD-dependent virus, e.g., health care workers, emergency medical technicians, law enforcement officers, medical research personnel, etc.
  • the subject may be one that is at increased risk of infection with an RGD and/or RLD-dependent virus due to underlying or preexisting conditions, e.g., conditions that increase the level of integrins on cells.
  • the subject has tissue injury or tissue inflammation, e.g., epithelial tissue injury or inflammation.
  • the subject has systemic or local inflammation, which is known to increase integrin levels.
  • the subject has lung inflammation, rendering the subject more susceptible to a respiratory virus such as SARS-CoV-2.
  • the subject has a cancer, which is known to increase integrin levels.
  • Another aspect of the invention relates to a method of treating, inhibiting the severity of, or preventing an RGD and/or RLD-dependent virus infection in a subject in need thereof, comprising the steps of: a) identifying a subject that has a condition that increases integrin expression; and b) if the subject has a condition that increases integrin expression, administering to the subject a therapeutically effective amount of an integrin antagonist, thereby treating, inhibiting the severity of, or preventing RGD and/or RLD-dependent virus infection.
  • the methods of the invention may be used for whole populations of subjects, or the majority of the population, to treat and/or prevent infection. In this scenario, the methods are likely to be effective in some but not all subjects.
  • the step of identifying a subject that has a condition that increases integrin expression may be used to provide a subpopulation of subjects in which the methods of the present invention may be most effective.
  • the method advantageously may also help identify the appropriate integrin to target, e.g., if a subject has a condition known to increase ⁇ v ⁇ 3 expression, an antagonist of ⁇ v ⁇ 3 would be the most appropriate treatment.
  • the method may, e.g., prevent a subject from getting infected upon exposure to the virus, limit the infection to one that is asymptomatic or mildly symptomatic, limit the symptoms from progressing to severe levels, and/or allow a quicker recovery from the infection.
  • the method may prevent subjects from undergoing a downward spiral in which a subject has elevated integrin levels, gets infected with a virus in part because of the elevated integrin levels, the infection causes further tissue injury and/or inflammation causing integrin levels to further increase, allowing even more virus particles to enter cells, making the infection even more severe.
  • Identifying a subject that has a condition that increases integrin expression may be carried out by identifying subjects that have certain conditions that are known to increase integrin expression, such as ⁇ v ⁇ 3. These include subjects have systemic or local tissue inflammation or tissue injury, e.g., epithelial tissue inflammation or injury, e.g., in the lungs or other organs.
  • the subject may have a disease such as cancer.
  • the subject may have an acute or chronic inflammatory disease, e.g., pancreatitis.
  • the subject may have hypercytokinemia or “cytokine storm”, indicative of tissue injury and/or inflammation.
  • the subject may be exposed to noxious chemicals, such as cigarette smoke or pollutants, leading to lung injury.
  • the subject may have a lung disease that causes cellular injury and/or inflammation, such as asthma, emphysema, chronic obstructive pulmonary disorder, cystic fibrosis, etc.
  • a lung disease that causes cellular injury and/or inflammation such as asthma, emphysema, chronic obstructive pulmonary disorder, cystic fibrosis, etc.
  • Identifying a subject that has a condition that increases integrin expression may be carried out by identifying subjects that have functional limitations indicative of tissue injury or inflammation. For example, subjects may be tested for oxygen saturation, e.g., using a finger pulse oximeter or measuring arterial blood gases, as an indication of lung injury and/or inflammation. An oxygen saturation of less than 95%, e.g., less than 90% or 85%, is indicative of a subject likely to have increased integrin expression levels in the lungs.
  • the virus is one that contains at least one RGD motif. In some embodiments, the virus is one that contains at least one RLD motif. In some embodiments, the virus is one that contains both at least one RGD motif and at least one RLD motif.
  • An appropriate integrin antagonist may be selected for use in the methods of the invention based on the binding motifs present on the virus. An integrin antagonist targeted to integrins that bind the RGD motif may be used for RGD-containing viruses. An integrin antagonist targeted to integrins that bind the RLD motif may be used for RGD-containing viruses.
  • the methods of the invention may comprise using two or more integrin antagonists, including any combination of at least one targeted to RGD binding integrins and one targeted to RLD binding-integrins.
  • an integrin antagonist that targets integrins that bind both binding motifs may be used, e.g., an ⁇ v ⁇ 3 antagonist.
  • the RGD and/or RLD-dependent virus may be, without limitation, any of the virus families or viruses listed in Table 1. Table 1
  • the RGD and/or RLD-dependent virus is a coronavirus (e.g., SARS-CoV-2), adenovirus (e.g., type 2/5), human cytomegalovirus, Kaposi’s sarcoma- associated herpesvirus, Epstein-Barr virus, human immunodeficiency virus-1, HPS- associated hantavirus NY-1, Sin Nombre virus, rotavirus, echovirus type 1, echovirus type 9, foot-and-mouth disease virus, coxsackievirus A9, murine polyomavirus, vaccinia virus, West Nile virus, simian virus 40, Ross River virus, human papillomavirus, Zika virus, or Ebola virus.
  • coronavirus e.g., SARS-CoV-2
  • adenovirus e.g., type 2/5
  • human cytomegalovirus e.g., Kaposi’s sarcoma- associated herpesvirus
  • Epstein-Barr virus
  • the RGD-dependent virus is SARS-CoV-2.
  • Examples of specific integrins associated with RGD and/or RLD-dependent viruses and the role of the integrins are shown in Table 2. This information allows one of skill in the art to select the appropriate integrin to target and the appropriate integrin antagonist (e.g., antibody or fragment or derivative thereof, peptide, or peptidomimetic) to use to protect against a given virus.
  • Table 2 [0101]
  • viruses that contain RGD and/or RLD integrin binding motifs are shown in Table 3. Each of the sequences associated with the listed accession number is incorporated by reference herein in its entirety.
  • the methods of the invention may further comprise administering to the subject an additional therapeutic agent or treatment.
  • the additional therapeutic agent or treatment is an antiviral agent, e.g., remdesivir, gimsilumab, REGN3048, REGEN3051, Kevzara, AdCOVID, EIDD-2801, favipiravir (Avigan), umifenovir (Arbidol), lopinavir, ritonavir, kaletra (a combination of lopinavir and ritonavir), danoprevir+ritonavir, falidesivir, oseltamivir, emtricitabine/tenofovir, or darunavir.
  • an antiviral agent e.g., remdesivir, gimsilumab, REGN3048, REGEN3051, Kevzara, AdCOVID, EIDD-2801, favipiravir (Avigan), umifenovir (Arbidol), lopinavir, ritonavir, kal
  • the additional therapeutic agent is one that inhibits the interaction of the RGD and/or RLD-dependent virus with a cell surface receptor used for attachment.
  • the binding of SARS-CoV-2 to ACE2 or the function of ACE2 may be inhibited, e.g., using hesperidin, curcumin, brazilin, galangin, nafamostat, desmethylcurcumin, bisdesmethylcurcumin, tangeretin, hesperetin, nobiletin, naringenin, brailein, aceto cavicol acetate, rutin, diosmin, apiin, diacetyl curcumin, rescinnamine, iloprost, prazosin, posaconazole, itraconazole, sulfasalazine, azlocillin, penicillin, cefsulodin, dabigatran etexilate, licoflavonol, cos
  • integrin antagonist e.g., the antibody or a fragment or derivative thereof used in the methods of the present invention is administered directly to a subject.
  • the integrin antagonist will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or administered subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
  • a pharmaceutically-acceptable carrier e.g., physiological saline
  • the intratracheal or intrapulmonary delivery can be accomplished using a standard nebulizer, jet nebulizer, wire mesh nebulizer, dry powder inhaler, or metered dose inhaler to deliver an aerosol.
  • the agents can be delivered locally, e.g., directly to the site of the disease or disorder, such as lungs, kidney, or intestines, e.g., injected in situ into or near a tumor.
  • the agents can be delivered to the mucosa.
  • the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient’s illness; the subject’s size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician.
  • Suitable dosages for each agent are in the range of 0.01-100 ⁇ g/kg. Wide variations in the needed dosage are to be expected in view of the variety of agents available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-; 20-, 50-, 100-, 150-, or more fold). Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or nanoparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
  • a suitable delivery vehicle e.g., polymeric microparticles or nanoparticles or implantable devices
  • compositions of the invention can optionally comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.
  • the integrin antagonist of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (21 * Ed.2006). In the manufacture of a pharmaceutical formulation according to the invention, the agent is typically admixed with, inter alia, an acceptable carrier.
  • the carrier can be a solid or a liquid, or both, and may be formulated with the agent as a unit-dose formulation, for example, a capsule or vial, which can contain from 0.01 or 0.5% to 95% or 99% by weight of the agent.
  • One or more agents can be incorporated in the formulations of the invention, which can be prepared by any of the well-known techniques of pharmacy.
  • the formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical (i.e., both skin and mucosal surfaces, including airway surfaces), intranasal, transdermal, intraarticular, intrathecal, and inhalation administration, administration to the liver by intraportal delivery, as well as direct organ injection (e.g., into the liver, into the brain for delivery to the central nervous system, or into the pancreas) or injection into a body cavity.
  • buccal e.g., sub-lingual
  • vaginal e.g., parenteral
  • parenteral e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal
  • the carrier will typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.).
  • the carrier can be either solid or liquid.
  • the agent can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • Agents can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.
  • inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.
  • 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.
  • Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the agent in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the agent in an inert base such as gelatin and glycerin or sucrose and acacia.
  • Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the agent, which preparations are preferably isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents.
  • the formulations can be presented in unit/dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.
  • sterile liquid carrier for example, saline or water-for-injection immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.
  • an injectable, stable, sterile composition comprising an agent of the invention, in a unit dosage form in a sealed container.
  • the agent is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject.
  • the unit dosage form typically comprises from about 1 mg to about 10 grams of the agent.
  • a sufficient amount of emulsifying agent which is pharmaceutically acceptable can be employed in sufficient quantity to emulsify the agent in an aqueous carrier.
  • One such useful emulsifying agent is phosphatidyl choline.
  • Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by admixing the agent with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
  • Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • Formulations suitable for transdermal administration can be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Tyle, Pharm. Res.3:318 (1986)) and typically take the form of an optionally buffered aqueous solution of the compounds. Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M of the compound.
  • the agent can alternatively 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 agent, which the subject inhales.
  • the respirable particles can be liquid or solid.
  • aerosol includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages.
  • 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 another 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 agent can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No.4,501,729. Aerosols of solid particles comprising the agent can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art. [0117] Alternatively, one can administer the compound in a local rather than systemic manner, for example, in a depot or sustained-release formulation. [0118] Further, the present invention provides liposomal formulations of the agents disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art.
  • the compound or salt thereof is an aqueous-soluble salt
  • the same can be incorporated into lipid vesicles.
  • the agent due to the water solubility of the agent, the agent will be substantially entrained within the hydrophilic center or core of the liposomes.
  • the lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free.
  • the salt can be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced can be reduced in size, as through the use of standard sonication and homogenization techniques.
  • the liposomal formulations containing the agent can be lyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
  • a pharmaceutical composition can be prepared containing the water-insoluble agent, such as for example, in an aqueous base emulsion.
  • the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the agent.
  • Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.
  • the integrin antagonist is administered to the subject in a therapeutically effective amount, as that term is defined above.
  • Dosages of pharmaceutically active agents can be determined by methods known in the art, see, e.g., Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa).
  • the therapeutically effective dosage of any specific agent will vary somewhat from agent to agent, and patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the agent.
  • Toxicity concerns at the higher level can restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the agent.
  • a dosage from about 10 mg/kg to about 50 mg/kg can be employed for oral administration.
  • a dosage from about 0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection.
  • Particular dosages are about 1 ⁇ mol/kg to 50 ⁇ mol/kg, and more particularly to about 22 ⁇ mol/kg and to 33 ⁇ mol/kg of the agent for intravenous or oral administration, respectively.
  • more than one administration can be employed over a variety of time intervals (e.g., hourly, daily, weekly, monthly, etc.) to achieve therapeutic effects.
  • time intervals e.g., hourly, daily, weekly, monthly, etc.
  • the present invention finds use in veterinary and medical applications. Suitable subjects include both avians and mammals, with mammals being preferred.
  • mammals include both avians and mammals, with mammals being preferred.
  • the term “mammal” as used herein includes, but is not limited to, humans, primates, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc. Human subjects include neonates, infants, juveniles, and adults.
  • the subject may be one in need of the methods of the invention, e.g., a subject that has or is suspected of having an infection or likely to be exposed to a virus.
  • the subject may be a laboratory animal, e.g., an animal model of a disease.
  • the present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.
  • Example 1 [0125] The presence of an RGD sequence in the S protein of the SARS-CoV-2 virus that was not present in the closely related SARS virus raised the question of whether an alpha V integrin may participate in the attachment or entry of the SARS-CoV-2 virus (Sigrist, Bridge et al. 2020).
  • HeLa-ACE2 human cervical carcinoma cells with ectopic expression of ACE2
  • HLa-ACE2 human cervical carcinoma cells with ectopic expression of ACE2
  • viral infection was evaluated by immunostaining fixed and permeabilized cells with sera from a convalescent COVID-19 patient.
  • SARS-CoV-2 infection for HeLA-ACE2 cells with endogenous ⁇ 3 expression was dose-dependently blocked by an antibody recognizing the alpha V-containing integrin ⁇ v ⁇ 3 (FIG.1A), but not antibodies that recognize the ⁇ 1 integrin subunit or the ⁇ v ⁇ 5 integrin heterodimer (FIG.1B).
  • Integrin ⁇ v ⁇ 3 is largely absent on all cell types in the healthy adult, but its expression and function emerges during tissue remodeling events including wound healing, angiogenesis, or cancer (Weis and Cheresh 2011). There is new evidence that the severity of progression of COVID-19 may be related to a “cytokine storm” that emerges during infection (Ye, 2020). This state has been shown to arise in patients who did not show severe clinical manifestations during early stages of disease, but who rapidly and suddenly deteriorate.
  • integrin ⁇ v ⁇ 3 may be upregulated in lung epithelial cells exposed to various forms of injury or inflammatory stimuli.
  • FIG.2A For a mouse model of lung injury, there is the expected absence of integrin ⁇ 3 expression on epithelial cells in the adult mouse lung at baseline (Day 0) (FIG.2A). Analysis of lung tissue from mice two days after treatment with naphthalene showed that the injury induced a rapid gain of integrin ⁇ 3 expression (Day 2) that was then downregulated as the lung epithelium is repaired (Day 14). Similarly, primary human lung epithelial cells showed a dose-dependent increase in ITGB3 mRNA in response to serum deprivation, a form of nutrient stress (FIG.
  • integrin ⁇ 3 protein expression was examined by immunohistochemical staining for a pancreas disease spectrum microarray slide, a tissue for which the chronically inflamed state of pancreatitis represents a major risk factor for pancreatic cancer (Pierro, Minici et al.2003, DiMagno and DiMagno 2016). While undetectable in normal adult pancreatic tissues, integrin ⁇ 3 expression not only increased from mild to chronic pancreatitis, but also from low to high grade pancreatic ductal adenocarcinoma (FIG.3).
  • pancreatic epithelial cells also upregulated the integrin ⁇ 3 subunit, but not other integrin ⁇ subunits, in response to TNF ⁇ as well as dose-dependently increase ITGB3 mRNA in response to nutrient stress (FIG.4C). These results highlight how normal pancreatic cells that are negative for integrin ⁇ 3 in adult tissues gain expression of this integrin when exposed to pro-inflammatory stimuli known to enhance the emergence of pancreatic cancer.
  • pancreatic cancer cell lines with low endogenous expression of integrin ⁇ 3 also upregulated its expression after 72 hours of culture in serum-free media (FIG.4D), suggesting that multiple cell types within the pancreas, including cancer cells, respond to inflammatory cytokines or cellular stress by upregulating integrin ⁇ 3.
  • FOG.4D serum-free media
  • these examples reveal that cells from epithelial tissues such as the lung and pancreas are generally lacking in expression of integrin ⁇ v ⁇ 3 in the healthy, resting state, but that they rapidly respond to injury, inflammation, or cellular stress by upregulating mRNA expression of the ⁇ 3 subunit leading to expression of the intact avb3 heterodimer.
  • integrin alpha V While integrin alpha V is generally expressed by most cell types in the body, the modulation of ⁇ 3 expression in response to various stimuli suggests a role for ⁇ 3 integrin as a “stress- inducible” gene that when heterodimerized with alpha V can be broadly induced as a mechanism to mitigate stress by a variety of cell types.
  • cytokine storm that emerges during infection (Ye, 2020)
  • a cocktail of cytokines that includes those highly enriched in COVID-19 patients (including INF ⁇ , TNF ⁇ , and IL-6)
  • cytokines that includes those highly enriched in COVID-19 patients (including INF ⁇ , TNF ⁇ , and IL-6)
  • INF ⁇ , TNF ⁇ , and IL-6 those highly enriched in COVID-19 patients
  • integrin ⁇ 3 expression that serves as an internalization receptor for viral infection.
  • Patients with underlying health conditions especially those that involve elevated cytokines or lung inflammation, may be especially susceptible to SARS-CoV-2 infection and/or at high risk for severe disease progression.
  • Integrins are cell surface receptors that interact with a variety of ligands via binding to specific sequences or motifs found on extracellular matrix proteins, such as the Arg-Gly- Asp (RGD) motif recognized by alpha V-containing integrins (Ruoslahti and Pierschbacher 1987). Aside from their role as cell-matrix receptors, integrins can also bind to non-matrix ligands to mediate cell-cell interactions or to serve as receptors for soluble factors including growth factors or hormones (LaFoya, Munroe et al.2018).
  • integrins Some functions of integrins have also been exploited by cancer cells to mitigate environmental stresses, immune surveillance, and to escape the effects of cancer therapy (Seguin, Desgrosellier et al.2015), and by bacteria and viruses to support various aspects of infection (LaFoya, Munroe et al.2018).
  • the alpha V containing integrins ⁇ v ⁇ 3 and ⁇ v ⁇ 5 were discovered to promote adenovirus internalization, but not virus attachment (Wickham, Mathias et al.1993, Nemerow, Cheresh et al.1994, Wickham, Filardo et al.1994).
  • viruses utilize a variety of integrins for cell attachment, entry, or both (Hussein, Walker et al. 2015). While many viruses contain one or more RGD motifs that serve as integrin- binding sites, disrupting RGD binding can prevent infection of certain viruses but not others. Examples of viruses that utilize integrin ⁇ v ⁇ 3 for internalization but do not rely on RGD binding include the hantaviruses NY-1 and Sin Nombre Virus (Grajovskaya, Shepley et al. 1998) and rotaviruses (Guerrero, Méndez et al.2000). [0133] Integrins recognize certain binding motifs to achieve receptor-ligand specificity.
  • integrin ⁇ v ⁇ 3 the receptor that this invention links to both cytokine storm and SARS-CoV-2 infection.
  • integrin ⁇ v ⁇ 3 was shown to recognize two motifs, Arg-Gly-Asp (RGD) and Arg- Leu-Asp (RLD) (Ruoslahti 1996). While, the RGD motif is recognized by a variety of ⁇ v and ⁇ 1 containing integrins, the RLD and KRLDGS motifs are recognized by only two integrins, ⁇ v ⁇ 3 and ⁇ M ⁇ 2 (Ruoslahti 1996).
  • ⁇ v ⁇ 3 and ⁇ M ⁇ 2 are the only integrins that bind to fibrinogen, and this occurs through recognition of an RLD motif (Altieri, Plescia et al. 1993).
  • An RGD motif has been identified in the SARS-CoV-2 S protein that was not present in other coronaviruses including SARS, but there have been opposing views on whether this motif enhances or prevents viral infection (Luan, Lu et al.2020, Sigrist, Bridge et al. 2020).
  • the Leu-Asp-Ile (LDI) motif is present in the S proteins of both SARS-CoV-2 and SARS (Tresoldi, Sangiuolo et al.).
  • FIG.5 shows the existence of an RLD motif in the SARS-CoV-2 S protein at amino acids 983-985.
  • the RGD sequence is located on the S1 subunit within the receptor binding domain (RBD)
  • the RLD sequence is located within the heptad repeat 1 (HR1) domain of the S2 subunit that participates in membrane fusion.
  • the RGD motif is a unique sequence found in SARS-CoV-2 that is not present in other betacoronavirus family members (FIG.6).
  • the RLD motif is highly conserved across betacoronavirus S proteins, except for one exception (the bat coronavirus HKU9) (FIG.7).
  • This particular region of the HR1 that contains the RLD motif is highly conserved among coronaviruses from diverse species, as well as sequences from many individuals with COVID-19 (Xia, Liu et al.2020). If viruses generally utilize an RLD motif for fusion or internalization, agents targeting this mechanism may have broad applications across many viral families.
  • the combination of both RGD and RLD motifs may provide a mechanistic explanation for the aggressive nature of SARS-CoV-2. [0135] 3D structural analysis of the SARS-CoV-2 virus (FIG.8) reveals that one RGD motif is be exposed in the up promoter, while somewhat concealed on the two down promoters, to participate in receptor binding.
  • RLD motif located at the apex of the HR1 region may act as a pedestal upon which the RBD from an adjacent down promoter sits. This region could impact the metastability of the spike protein.
  • the exposed RLD binding motif may interact with integrin ⁇ v ⁇ 3 to facilitate membrane fusion and/or virus internalization.
  • integrins are known to be robustly activated by binding to multivalent ligands, such as the conformation of RGD/RLD motifs predicted by the 3D model. Integrin ⁇ v ⁇ 3 may be particularly important for SARS-CoV-2 viral infection by virtue of its ability to recognize both the RGD and RLD binding motifs.
  • an alpha V integrin such as integrin ⁇ v ⁇ 3 can become activated and clustered when the SARS-CoV-2 or another RGD and/or RLD-dependent virus engages receptors such as ACE2 that mediates initial virus attachment.
  • An activated alpha V integrin would then facilitate virus internalization to enable viral replication.
  • the right side of FIG.9B provides a potential therapeutic intervention using an integrin antagonizing peptide, organic molecule, or function-blocking antibody to block the interaction between the virus and integrin, thereby preventing viral entry.
  • integrin ⁇ v ⁇ 3 blockade using a humanized monoclonal antibody is able to block viral infection of cultured human cells.
  • the structural motif glycine 190-valine 202 of the fibrinogen gamma chain interacts with CD11b/CD18 integrin (alpha M beta 2, Mac-1) and promotes leukocyte adhesion.” Journal of Biological Chemistry 268(3): 1847-1853. Chen, C. A., J. M. Chang, E. E. Chang, H. C. Chen and Y. L. Yang (2016). "TGF- ⁇ 1 modulates podocyte migration by regulating the expression of integrin- ⁇ 1 and - ⁇ 3 through different signaling pathways.” Biomed Pharmacother 105: 974-980. Coutard, B., C. Valle, X. de Lamballerie, B. Canard, N. G. Seidah and E.
  • hLM609-hIgG4-S228P (humanized LM609) Heavy chain (SEQ ID NO:1) Light chain (SEQ ID NO:2) Fab domain of heavy chain (SEQ ID NO:3) Fc and hinge domain of heavy chain (SEQ ID NO:4) shLM609-hIgG1-WT (super-humanized LM609_7) Fab domain of heavy chain (SEQ ID NO:5) Light chain (SEQ ID NO:6) shLM609-hIgG1-WT (super-humanized JC7U) Fab domain of heavy chain (SEQ ID NO:7) Light chain (SEQ ID NO:8) hLM609-hIgG1-WT (humanized LM609) Heavy chain (SEQ ID NO:9) Light chain (SEQ ID NO:10) mAb LM609-mIgG1-kappa Heavy chain (SEQ ID NO:11) Light chain (SEQ ID NO:12) hLM609-hIgG4-S228

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Abstract

La présente invention concerne des procédés d'inhibition virale qui dépendent de motifs de liaison à l'intégrine RGD et/ou RLD sur les protéines structurales virales pour entrer dans des cellules. L'invention concerne en outre des méthodes de traitement, de réduction de la gravité, ou de prévention d'infections virales dépendant de RGD et/ou de RLD à l'aide d'antagonistes d'intégrine tels que des anticorps ou des fragments ou des dérivés de ceux-ci, des peptides ou des peptidomimétiques ciblés sur des intégrines alpha V qui reconnaissent les motifs de liaison RGD, l'intégrine αMβ2 qui reconnaît les motifs de liaison RLD, ou l'intégrine αvβ3 qui reconnaît à la fois les motifs de liaison RGD et RLD. L'invention concerne en outre des compositions utiles pour la mise en œuvre des méthodes selon l'invention.
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WO2007048127A2 (fr) * 2005-10-20 2007-04-26 The Scripps Research Institute MARQUAGE DE PARTIES Fc POUR L'IMMUNOCOLORATION ET L'IMMUNOCIBLAGE

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WO2007048127A2 (fr) * 2005-10-20 2007-04-26 The Scripps Research Institute MARQUAGE DE PARTIES Fc POUR L'IMMUNOCOLORATION ET L'IMMUNOCIBLAGE

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HUSSEIN HOSNI A. M.; WALKER LIA R.; ABDEL-RAOUF USAMA M.; DESOUKY SAYED A.; MONTASSER ABDEL KHALEK M.; AKULA SHAW M.: "Beyond RGD: virus interactions with integrins", ARCHIVES OF VIROLOGY, SPRINGER WIEN, AT, vol. 160, no. 11, 1 September 2015 (2015-09-01), AT , pages 2669 - 2681, XP037066974, ISSN: 0304-8608, DOI: 10.1007/s00705-015-2579-8 *
SIGRIST CHRISTIAN JA; BRIDGE ALAN; LE MERCIER PHILIPPE: "A potential role for integrins in host cell entry by SARS-CoV-2", ANTIVIRAL RESEARCH, ELSEVIER BV, NL, vol. 177, 1 March 2020 (2020-03-01), NL , XP086133689, ISSN: 0166-3542, DOI: 10.1016/j.antiviral.2020.104759 *
WILLIAMS ÇIĞDEM H., KAJANDER TOMMI, HYYPIÄ TIMO, JACKSON TERRY, SHEPPARD DEAN, STANWAY GLYN: "Integrin α v β 6 Is an RGD-Dependent Receptor for Coxsackievirus A9", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 78, no. 13, 1 July 2004 (2004-07-01), US , pages 6967 - 6973, XP055860781, ISSN: 0022-538X, DOI: 10.1128/JVI.78.13.6967-6973.2004 *

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