WO2025085675A1 - Conjugués, compositions et méthodes de traitement d'une infection par le vrs - Google Patents

Conjugués, compositions et méthodes de traitement d'une infection par le vrs Download PDF

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WO2025085675A1
WO2025085675A1 PCT/US2024/051835 US2024051835W WO2025085675A1 WO 2025085675 A1 WO2025085675 A1 WO 2025085675A1 US 2024051835 W US2024051835 W US 2024051835W WO 2025085675 A1 WO2025085675 A1 WO 2025085675A1
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compound
alkyl
rsv
ligand
mmol
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Mary Lynn Niedrauer
Jeffery Jay Howard NIELSEN
Imrul SHAHRIAR
Ananda Kumar KANDULURU
Philip Stewart Low
Madduri SRINIVASARAO
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug

Definitions

  • the F glycoprotein (“F protein”) is a class I fusion protein that mediates viral entry of RSV into a cell by fusing the viral envelope with a host cell membrane.
  • the F protein consists of two disulfide-linked subunits – F1 and F2.
  • the F1 subunit contains a hydrophobic ⁇ fusion peptide at its N terminus followed by two heptad repeats – HR1 and HR2, which are separated by an intervening region of about 300 amino acids.
  • the G glycoprotein (“G protein”) exists in both membrane-bound and secreted forms, and is primarily responsible for viral attachment to host cells.
  • T is a radical of a ligand for a target protein of a RSV or a RSV-infected cell
  • L 1 , L 2 , and L 3 are each, an independently selected linker
  • a 1 and A 2 are each, a radical of an independently selected hapten.
  • compositions for use in treating RSV infection comprising one or more compounds of Formula I: ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ Formula I and pharmaceutically acceptable salts and solvates thereof, and optionally including one or more diluents, carriers, or excipients, or combinations thereof; wherein: ⁇ T is a radical of a ligand for a target protein of a RSV or a RSV-infected cell; L 1 , L 2 , and L 3 are each, an independently selected linker; and A 1 and A 2 are each, a radical of an independently selected hapten.
  • unit doses for use in treating RSV infection comprising a therapeutically effective amount of one or more compounds of Formula I: ⁇ Formula I and pharmaceutically acceptable salts and solvates thereof, and optionally including one or more diluents, carriers, or excipients, or combinations thereof; wherein: T is a radical of a ligand for a target protein of a RSV or a RSV-infected cell; ⁇ L 1 , L 2 , and L 3 are each, an independently selected linker; and A 1 and A 2 are each, a radical of an independently selected hapten.
  • Described herein are methods for treating RSV infection the methods comprising administering a therapeutically effective amount of one or more compounds of Formula I: ⁇ Formula I and pharmaceutically acceptable salts and solvates thereof, compositions thereof optionally including one or more diluents, carriers, or excipients, or combinations thereof, or unit doses thereof to a host to a host animal having a RSV infection; wherein: T is a radical of a ligand for a target protein of a RSV or a RSV-infected cell; ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ L 1 , L 2 , and L 3 are each, an independently selected linker; and A 1 and A 2 are each, a radical of an independently selected hapten.
  • Described herein are uses of one or more compounds of Formula I: ⁇ Formula I and pharmaceutically acceptable salts and solvates thereof, and optionally including one or more diluents, carriers, or excipients, or combinations thereof in the manufacture of a medicament for use in treating a RSV infection in a host animal; wherein: T is a radical of a ligand for a target protein of a RSV or a RSV-infected cell; ⁇ L 1 , L 2 , and L 3 are each, an independently selected linker; and A 1 and A 2 are each, a radical of an independently selected hapten.
  • T is a radical of a RSV fusion (F) glycoprotein.
  • T is a radical of a RSV attachment glycoprotein (G).
  • the haptens recruit endogenous antibodies ⁇ present in the host animal.
  • compounds, compositions, and unit doses described herein are used in methods for treating RSV infection in a host animal, where the compounds, compositions, and unit doses are administered to the host animal, and after administration A 1 and A 2 , or any epitope of the foregoing are each bound by one or more ⁇ antibodies.
  • each of L 1 , L 2 , and L 3 is independently selected, and each may comprise a single divalent atom, or a chain of atoms.
  • L 2 may be a bond attaching L 1 to A 1 .
  • ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ each of L 3 may be a bond attaching L 1 to A 2 .
  • the compounds, compositions, unit doses, methods, and uses described herein are useful in treating RSV infection. It is to be understood that such treatment includes prophylactic and preventative treatment of host animals, including host animals at risk of ⁇ RSV infection, or host animals exposed to RSV infection, including when symptoms of RSV infection have not been observed.
  • the compounds, compositions, unit doses, methods, and uses described herein are useful in preventing the onset of RSV infection, and/or decreasing the severity of later arising symptoms associated with RSV infection.
  • FIG.1 shows example compounds.
  • FIG.2 shows a reaction scheme for preparing Compound 47.
  • FIG.3 shows an immunofluorescence assay of Compound 47 in Hep2 cells.
  • FIG. 4 shows the reduction of the virus cytopathic effect by Compound 47 in Hep2 ⁇ cells. Higher absorbance (Y axis) denotes greater cell viability and a reduction in a virus induced cytopathic effect.
  • FIG.5 shows the induction of ADCC by Compound 47 in human cells infected with RSV.
  • FIG.25 shows the induction of ADCC by Compound 47 in human cells infected with RSV.
  • FIG. 6 shows the in vivo efficacy of Compound 47 against RSV in rats following a ⁇ single dose compared to ALS-008176 and vehicle.
  • FIG. 7 shows the in vivo efficacy of Compound 26 against RSV in rats following a single dose compared to ALS-008176 and vehicle.
  • FIG.8 shows the ADCP Assay of Compound 47 against RSV-infected cells.
  • FIG.9 shows the CDC Assay of Compound 47 against RSV-infected cells.
  • FIG.10 shows the in vivo efficacy of Compound 27 against RSV in rats following a single dose compared to vehicle.
  • T is a radical of a ligand for a target protein of a RSV or a RSV-infected cell
  • L 1 , L 2 , and L 3 are each, an independently selected linker
  • ⁇ A 1 and A 2 are each, a radical of an independently selected hapten.
  • the compound of clause 1 wherein the ligand is an inhibitor of RSV attachment glycoprotein (G). 6.
  • the compound of clause 1 wherein the ligand is of the formula where X is CH2, O, S, or SO2; n is 1 or 2 with the proviso that if X is not CH2, n is 2; and Y is alkyl, halo, CN, alkoxy, alkyl-OH, or H.
  • the compound of clause 1 wherein the ligand is TMC353121 ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ or an analog or derivative thereof.
  • the compound of clause 1 wherein the ligand is ⁇ or an analog or derivative thereof.
  • the compound of clause 1 wherein the ligand is or an analog or derivative thereof. 10.
  • the compound of clause 1 wherein the ligand is ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ or an analog or derivative thereof. 14. The compound of clause 1 wherein the ligand is susinatovir ⁇ or an analog or derivative thereof. 15. The compound of clause 1 wherein the ligand is or an analog or derivative thereof. ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ 16. The compound of clause 1 wherein the ligand is VP-14637 or an analog or derivative thereof. 17. The compound of clause 1 wherein the ligand is of RFI-641 or an analog or derivative thereof. 18.
  • the compound of clause 1 wherein the ligand is SEQ ID NO: 34 (T205).
  • the compound of any preceding clause wherein A 1 and A 2 are the same hapten.
  • hapten is selected from a rhamnose, a nitrophenyl, a nitrophenol, a nitroaniline, a dinitrophenyl (DNP), a dinitrophenol, a dinitroaniline, a trinitrophenyl (TNP), a trinitrophenol, a trinitroaniline, chloronitrophenyl, a chloronitrophenol, a chloronitroaniline, an iodonitrophenyl, an iodonitrophenol, an iodonitroaniline, a nitrotyrosine, an hydroxynitrotyrosine, an ⁇ aminonitrotyrosine, 4-hydroxy-3-nitrophenyl acetic acid, an ⁇ -galactosyl moiety, a sulfated Gal, a phosphorylcholine, a bacterial antigen, a viral antigen, A 1 and A 2 are each an independently selected radical of a rhamnose, ⁇ -galacto
  • a 1 and/or A 2 is a radical of a ⁇ dinitrophenyl (DNP), a dinitrophenol, or a dinitroaniline.
  • DNP dinitrodinitrophenyl
  • a 1 and/or A 2 is a radical of a rhamnose, including L-rhamnose.
  • one of A 1 and A 2 is a radical of L- rhamnose; and the other of A 1 and A 2 is a radical of a dinitroaniline.
  • ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ 31 The compound of any preceding clause wherein the hapten has an epitope that has an endogenous Ab, B or T cell. 32.
  • L 1 , L 2 , and L 3 is a ⁇ single divalent atom selected from N, O, P, and S, where N and P are optionally substituted. 39.
  • one or more of L 1 , L 2 , and L 3 is a chain of atoms, where the length of each chain is independently selected and in the range of about 2 to about 60. ⁇ 40.
  • L 1 , L 2 , and L 3 includes or also includes ethoxy, ethylamino, ethylene glycol, aza-ethylene glycol, (PEG)n, or aza-(PEG) n , or a combination thereof, where n is in the range from 2 to about 36. 41.
  • each of L 1 , L 2 , and L 3 includes or also includes ethoxy, ethylamino, ethylene glycol, aza-ethylene glycol, (PEG)n, or aza- (PEG)n, or a combination thereof, where n is 2-36. ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ 43.
  • L 1 , L 2 , and L 3 ⁇ includes or also includes O-alkyl-C(O), N-alkyl-C(O), O-alkyl-N-alkyl-C(O), N-alkyl-O- alkyl-C(O), or C(O)alkyl-C(O), or a combination thereof.
  • L 1 includes or also includes O- alkyl-C(O)N-diyl, O-alkyl-O-alkyl-C(O)N-diyl, or N-alkyl-O-alkyl-O-alkyl-C(O)N-diyl. 46.
  • L 1 , L 2 , and L 3 ⁇ includes or also includes one or more amino acids. 47.
  • hydrophilic amino acids selected from Arg, Asn, Asp, Cys, Glu, Gln, His, Lys, Met, Orn, Ser, or Thr, including the naturally occurring L- enantiomers of each of the foregoing.
  • L 1 , L 2 , and L 3 includes or also includes one or more hydrophilic amino acids selected from ⁇ -NH 2 -Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Lys, Met, Orn, Ser, or Thr, including the naturally occurring L-enantiomers of each of the foregoing.
  • one or more of L 1 , L 2 , and ⁇ includes or also includes one or more amino acids selected from glycine, serine, proline, ornithine, and lysine. 50.
  • L 1 , L 2 , and L 3 includes or also includes 1,2,3-triazol-1,4-diyl, 1,2,3-triazol-1,4-diyl, or a combination ⁇ thereof.
  • one or more of L 1 , L 2 , and L 3 includes or also includes a maleimid-diyl or thiomaleimid-N,S-diyl.
  • L 2 and/or L 3 are hydrophilic. ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ 68.
  • L 1 includes a region that is capable of forming an ⁇ -helical conformation.
  • the extended conformation of L 2 and/or L 3 is at least 8 ⁇ , 9 ⁇ , 10 ⁇ , 11 ⁇ , 12 ⁇ , 13 ⁇ , 14 ⁇ , 15 ⁇ , 20 ⁇ , 25 ⁇ , 30 ⁇ , 35 ⁇ , ⁇ or 40 ⁇ , in length. 70.
  • L 2 and/or L 3 includes or also includes (PEG) n or aza-(PEG) n , where n is in the range from about 3 to about 36, from about 4 to about 36, from about 5 to about 36, from about 6 to about 36, or from about 7 to about 36.
  • L 2 and/or L 3 includes or also includes one or more divalent cycloalkyl, including adamantyl, heterocyclyl, including maleimidyl, aryl, heteroaryl including triazolyl, , stilbene, oligoproline, or oligopiperidine groups.. 72.
  • the compound of any preceding clause wherein the extended conformation of T-L 1 ⁇ is at least 10 ⁇ , 12 ⁇ , 15 ⁇ , 20 ⁇ , 25 ⁇ , 30 ⁇ , 35 ⁇ , 40 ⁇ , 45 ⁇ , or 50 ⁇ in length.
  • L 1 includes or also includes a linear, unbranched chain of atoms having an extended conformation of at least 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , 10 ⁇ , 11 ⁇ , 12 ⁇ , 13 ⁇ , 14 ⁇ , or 15 ⁇ in length. 74.
  • L 1 includes or also includes ⁇ (PEG)n or aza-(PEG)n, where n is in the range from about 8 to about 36, from about 9 to about 36, from about 10 to about 36, from about 12 to about 36, from about 12 to about 36.
  • L 1 includes or also includes an hydrophobic region attached to T, and having an extended conformation of at least 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , 10 ⁇ , 11 ⁇ , 12 ⁇ , or 13 ⁇ in length.
  • ⁇ 76 A compound of any formula shown in FIG 1 or a pharmaceutically acceptable salt or solvate thereof.
  • the foregoing illustrative compounds, as defined by clauses 1-81 may be included in any of the compositions, unit doses, uses, or methods described herein.
  • the conjugate compounds described herein may be formed from a ligand that is capable of targeting a protein of a RSV-infected cell.
  • the protein is ⁇ on the surface of a RSV-infected cell, or is otherwise accessible from the surface of a RSV-infected cell.
  • the conjugates described herein may also be formed from two or more haptens, each comprising an immune system responsive epitope or antigen.
  • the conjugates described herein may also be formed from a polyvalent linker that covalently attaches the ligand to the two or more haptens. Described herein are numerous selections ⁇ for each of the ligand, the haptens, and the linker.
  • each of the ligands and haptens described herein may be proteinaceous or small molecules.
  • Each of the linkers described herein may be proteinaceous or small molecules, or combinations thereof. It is to be understood that any ligand, any haptens, and any linker described herein may be combined to form the conjugates.
  • the linkers themselves are formed from various ⁇ building blocks, including single atoms, functional groups, and chemical fragments. It is to be understood that every combination of building blocks is described herein for forming the linkers included in the conjugates.
  • the conjugates have a dual mechanism of action, where the conjugate inhibits the virus, and also labels or decorates virus-infected cells for ⁇ intervention by the immune system of the host animal, including humans.
  • the conjugates ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ elicit host animal immune response against the virus or virus-infected cell by recruiting antibodies in the host animal.
  • the antibodies are endogenous.
  • the anti-hapten antibodies bind to the hapten and activate the innate immune system against the target ⁇ virus and virus-infected cells.
  • the haptens may be the same or different. In the conjugates where the haptens are the same, the immune system of the host animal may be multiplied. In the conjugates where the haptens are different, the immune system of the host animal may respond in multiple ways.
  • the conjugates described herein provide a therapeutic delivery system for selectively or specifically delivering haptens to target virus and virus- ⁇ infected cells.
  • the haptens are selected to activate the innate immune system of the subject to recruit immune cells and/or otherwise leverage the subject’s own immune system against the virus.
  • the targeting ligand can selectively or specifically recognize a target protein or receptor, such as an envelope protein of a virus, which can be highly or exclusively expressed on the virus, the surface of an infected cell, ⁇ or accessible from the surface of an infected cell.
  • the conjugate compound of Formula I can have fragments L 2 -A 1 and L 3 -A 2 bound to the same atom on L 1 .
  • the same atom is not a carbon atom. In some embodiments, the same atom is a nitrogen atom.
  • the conjugate of Formula I can have fragments L 2 -A 1 and L 3 -A 2 bound to a different atom ⁇ on L 1 .
  • the method described herein can ⁇ elicit an immune response leading to clearance of an antibody (Ab)-coated virus or an Ab- coated-virally infected cell via Ab-dependent cellular phagocytosis (ADCP), Ab-dependent cellular cytotoxicity (ADCC), and/or complement-dependent cytotoxicity (CDC) which works in conjunction with the inhibition of viral budding by neuraminidase inhibition leading to viral eradication.
  • the method for activating an immune response can further ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ comprise administering to the subject autologous antibodies, allogeneic IgG antibodies, or human IVIG. In these and other embodiments, the subject may be further treated with anti- hapten antibodies.
  • the present disclosure includes conjugates comprising small-molecule inhibitors of ⁇ F glycoprotein (F protein) of respiratory syncytial virus (RSV) and their use in the treatment of RSV.
  • the small-molecule inhibitors of F protein are used as ligands to target a conjugated agent to F protein present on the surface of RSV and RSV-infected cells.
  • the conjugate binds to F protein on the virus, it prevents the virus from binding to nucleolin on the surface of a cell and entering the cell.
  • the RSV targeting ligand is a RSV fusion (F) glycoprotein ligand, or a compound capable of binding to a fusion (F) glycoprotein.
  • the RSV targeting ligand is a RSV attachment glycoprotein (G) ligand, or a compound capable of binding to a RSV attachment glycoprotein (G).
  • illustrative ligands include compounds that target RSV fusion ⁇ (F) glycoprotein.
  • Illustrative ligands also include compounds that target RSV attachment glycoprotein (G).
  • illustrative ligands include compounds that are inhibitors of RSV fusion (F) glycoprotein, including RSV fusion inhibitors and compounds that inhibit the six-helix bundle formation.
  • Illustrative ligands include compounds that are inhibitors of ⁇ RSV attachment glycoprotein (G).
  • the ligand is of the formula where X is CH 2 , O, S, or SO 2 ; n is 1 or 2 with the proviso that if X is not CH 2 , n is 2; and Y is alkyl, halo, CN, alkoxy, alkyl-OH, or H.
  • the ligand is TMC353121 ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ or an analog or derivative thereof, such as compounds disclosed in WO 2005/058869, WO 2005/058871, WO 20025/058874, and the like.
  • the ligand is of the formula ⁇ or analogs or derivatives thereof, such as compounds disclosed in Roymans et al. PNAS 107(1):208-13 (2010), such as compounds disclosed in WO 2005/058869, WO 2005/058871, WO 20025/058874, and the like and the like.
  • T is a radical of the formula ⁇ or an analog or derivative thereof.
  • the ligand is of the formula ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ or an analog or derivative thereof.
  • the ligand is of the formula ⁇ where R1 is NH2, NHRn, where Rn is alky, amino, hydroxyalkyl, OR, or OH; R2 is alkyl (C-1 to C-4), halo, CN, N 3 , OR; R 3 is alkyl (C-1 to C-4); and R 4 is alkyl (C-1 to C-4), halo, CN, N 3 , OR.
  • the ligand is presatovir ⁇ or an analog or derivative thereof, such as compounds disclosed in Mackman et al. J Med Chem 58:1630-43 (2015), WO 2011/163518, and the like.
  • T is a radical of the formula ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇
  • the ligand is of the formula wherein Ra and Rb are independently selected from the group alkyl (C1- to C4), alkoxyl ⁇ (C1- to C4), halo, cyano, and azido; and n is 1, 2, or 3.
  • the ligand is of the formula or an analog or derivative thereof, such as compounds disclosed in Bonfanti et al. J Med Chem 51:875-96 (2008), Andries at al. J Antiviral Res 60:209-19 (2003), WO ⁇ 2001/000611, and the like.
  • T is a radical of the formula ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇
  • the ligand is susinatovir or an analog or derivative thereof, such as compounds disclosed in Cockerill et al. J Med ⁇ Chem 64:3658-76 (2021), and the like.
  • T is a radical of the formula [0056]
  • the ligand is JNJ-49153390 ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ or an analog or derivative thereof.
  • T is a radical of the formula ⁇ [0058]
  • the ligand is of the formula or an analog or derivative thereof.
  • T is a radical of the formula .
  • the ligand is BMS-433771 ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ or an analog or derivative thereof.
  • T is a radical of the formula ⁇
  • the ligand is of the formula or an analog or derivative thereof, such as compounds disclosed in Yamaguchi-Sasaki et al. ACS Med Chem Lett 11:1145-51 (2020), and the like.
  • the ligand is Ziresovir ⁇ or an analog or derivative thereof.
  • T is a radical of the formula ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ .
  • the ligand is VP-14637 or an analog or derivative thereof.
  • T is a radical of the formula [0067]
  • the ligand is RFI-641 ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ or an analog or derivative thereof.
  • the ligand is risetovir ⁇ or an analog or derivative thereof.
  • the ligand is selected from risetovir, BMS-433771, rilematovir, ziresovir, or an analog or derivative of the foregoing, such as compounds disclosed in Zheng et al. J Med Chem 62:6003-14 (2019), and the like, RSV-IN-1, RSV-IN- 5, or an analog or derivative of the foregoing, such as compounds disclosed in Lundin et al. ⁇ Antiviral Res 88:317-24 (2010), and the like, YM 53403, or BTA-9981, and analogs and derivatives thereof.
  • a radical may be formed on any atom of any of the foregoing ligands to form the radical T.
  • the ligand is not a folate.
  • the ligand is a peptide, such as a peptide having SEQ ID NO: 1, or a fragment thereof, including compounds described in Lambert et al. PNAS 93:2186- 91 (1996); or a peptide having SEQ ID NO: 18, or a fragment thereof, including compounds ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ described in Outlaw et al. JACS 142:2140-44 (2020).
  • the ligand is SEQ ID NO: 16 (T118); SEQ ID NO: 34 (T205), SEQ ID NO: 40 (VIQKI), or a substitution isomer selected from VIQKI-I456F (SEQ ID NO: 41) and VIQKI-I454F/I456F SEQ ID NO: 42).
  • each hapten can be bound by an antibody. After administration to a subject, each hapten (A 1 and A 2 ) can be bound by an antibody. In ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ certain embodiments, the two haptens, A 1 and A 2 can each be bound by a different antibody after administration to a subject. It is to be understood that any hapten, including peptide sequences, that elicit an immunological response in the host animal may be used to form the conjugates described herein.
  • a 1 and A 2 are each an independently selected radical of an antigen where for each antigen there is an endogenous antibody present in the host animal.
  • Illustrative haptens include, but are not limited to, a rhamnose, including L-rhamnose, a ⁇ nitrophenyl, a nitrophenol, a nitroaniline, a dinitrophenyl (DNP), a dinitrophenol, a dinitroaniline, a trinitrophenyl (TNP), a trinitrophenol, a trinitroaniline, chloronitrophenyl, a chloronitrophenol, a chloronitroaniline, an iodonitrophenyl, an iodonitrophenol, an iodonitroaniline, a nitrotyrosine, an hydroxynitrotyrosine, an aminonitrotyrosine, 4- hydroxy-3-nitrophenyl acetic acid, an ⁇ -galactosyl moiety, a sulfated Gal, compounds of the , a phosphorylcholine, a bacterial antigen, a viral antigen, and the like, and any combination of two or more
  • the hapten is selected from a dinitrophenyl (DNP), a ⁇ dinitrophenol, and a dinitroaniline.
  • a 1 and/or A 2 is a radical of a ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ dinitrophenyl (DNP), a dinitrophenol, or a dinitroaniline, including radicals of the formulae where L 2 or L 3 is attached at (*).
  • the hapten is selected from a rhamnose, including L- ⁇ rhamnose.
  • a 1 and/or A 2 is a radical of a rhamnose, including L- rhamnose, and including a radicals of the formulae where L 2 or L 3 is attached at (*).
  • a 1 is a DNP radical and A 2 is a rhamnose radical, or vice ⁇ versa.
  • a 1 is a DNP radical and A 2 is an L-rhamnose radical, or vice versa.
  • neither A 1 nor A 2 comprises a fluorescein, such as FITC, and the like.
  • linker generally refers to a chain of atoms that connects ⁇ two or more functional parts of a molecule to form a conjugate.
  • the chain of atoms is selected from C, N, O, S, Si, and P, or C, N, O, S, and P, or C, N, O, and S.
  • the chain of atoms covalently connects different functional capabilities of the conjugate.
  • the linker may have a wide variety of lengths, such as in the range from about 2 to about 100 atoms in the contiguous backbone.
  • the atoms used in forming the linker may be combined ⁇ in all chemically relevant ways, such as chains of carbon atoms forming alkylene, alkenylene, and alkynylene groups, and the like; chains of carbon and oxygen atoms forming ethers, polyoxyalkylene groups, or when combined with carbonyl groups forming esters and carbonates, and the like; chains of carbon and nitrogen atoms forming amines, imines, polyamines, hydrazines, hydrazones, or when combined with carbonyl groups ⁇ forming amides, ureas, semicarbazides, carbazides, and the like; chains of carbon, ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ nitrogen, and oxygen atoms forming alkoxyamines, alkoxylamines, or when combined with carbonyl groups forming urethanes, amino acids, acyloxylamines, hydroxamic acids, and the like; and many others.
  • the atoms forming the chain in each of the foregoing illustrative embodiments may be either saturated or ⁇ unsaturated, thus forming single, double, or triple bonds, such that for example, alkanes, alkenes, alkynes, imines, and the like may be radicals that are included in the linker.
  • the atoms forming the linker may also be cyclized upon each other or be part of cyclic structure to form divalent cyclic structures that form the linker, including cycloalkanes, cyclic ethers, cyclic amines, and other heterocycles, ⁇ arylenes, heteroarylenes, and the like in the linker.
  • the linker length may be defined by any pathway through the one or more cyclic structures.
  • the linker length is defined by the shortest pathway through the each one of the cyclic structures.
  • the linkers may be optionally substituted at any one or more of the open valences along the chain of atoms, ⁇ such as optional substituents on any of the carbon, nitrogen, silicon, or phosphorus atoms.
  • the linker may connect the two or more functional parts of a molecule to form a conjugate at any open valence, and it is not necessary that any of the two or more functional parts of a molecule forming the conjugate are attached at any apparent end of the linker.
  • L 1 , L 2 , and L 3 each, independently comprises a chain of atoms from 3 atoms to about 60 atoms in length. In many embodiments, L 1 , L 2 , and L 3 each, independently comprises a chain of atoms from about 4 ⁇ to about 72 ⁇ in length.
  • the chain of atoms are part of the backbone of the conjugate of Formula I.
  • the term “backbone” of the linker L refers to the shortest chain of ⁇ contiguous atoms forming a covalently bonded connection between T and L 2 , between T and L 3 , between A 1 and L 1 , or between A 2 and L 1 .
  • L 1 , L 2 , or L 3 each, independently, comprise a chain of atoms at least 3 atoms in length, at least 7 atoms in length, at least 10 atoms in length, at least 14 atoms in length, or at least 20 atoms in length.
  • L 1 , ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ L 2 , or L 3 each, independently, have a chain of between 3 and 7 atoms in length, between 7 and 10 atoms in length, between 10 and 14 atoms in length, between 14 and 20 atoms in length, between 20 and 30 atoms in length, between 30 and 40 atoms in length, between 40 and 50 atoms in length, or between 50 and 60 atoms in length.
  • L 1 , L 2 , or L 3 each, independently, comprise a chain of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 atoms in length.
  • the term “between” is inclusive of the endpoints meaning that between 3 and 5 atoms in a chain length ⁇ includes 3 atoms, 4 atoms, and 5 atoms.
  • L 2 and L 3 are covalently attached to L 1
  • both L 2 and L 3 may be attached to the same atom of L 1
  • L 2 and L 3 may each be attached to different atoms of L 1
  • L 2 and L 3 are attached to the same atom on L 1 where that atom is not a carbon atom.
  • L 1 , L 2 , or L 3 can each, independently, comprise one or more amino acid or peptide residues.
  • amino acid refers generally to beta, gamma, and longer amino acids, and including cyclic groups, that have both an amino group and an acid group from each of which a radical can be formed.
  • Illustrative acyclic amino acids include the ⁇ formula -N(R)-(CR ⁇ R ⁇ )q-C(O)- where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting group, R ⁇ and R ⁇ are hydrogen or a substituent, each of which is independently selected in each occurrence, and q is an integer such as 1, 2, 3, 4, or 5.
  • R ⁇ and/or R ⁇ independently ⁇ correspond to, but are not limited to, hydrogen or the side chains present on naturally occurring amino acids, such as methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, and derivatives and protected derivatives thereof.
  • the above described formula includes all stereoisomeric variations.
  • the amino acid may be selected from asparagine, aspartic acid, cysteine, glutamic acid, ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ lysine, glutamine, arginine, serine, ornithine, threonine, and the like.
  • L 1 , L 2 , or L 3 can each, independently, comprise one or more lysine residues, each of which is independently optionally substituted.
  • L 1 can comprise a lysine residue of the formula wherein A 1 or A 2 is attached to the lysine residue via a linker.
  • the lysine residue is L-Lys.
  • L 1 comprises at least one lysine residue, such as L-Lys.
  • a linker comprises one or more PEG moieties where all carbon and oxygen atoms of the one or more PEG moieties are part of the backbone of the linker.
  • L 1 , L 2 , or L 3 can each, independently, comprise one or more alkyl groups.
  • L 1 , L 2 , or L 3 can each, independently, comprise one or more or sugar moieties, glycan residues, or peptidoglycan residues.
  • bonds connecting atoms in the chain can be either saturated or unsaturated, such that for example, alkanes, alkenes, alkynes, cycloalkanes, arylenes, imides, and the like can be divalent radicals that are included in ⁇ L.
  • the atoms of L in the chain can be substituted or unsubstituted.
  • the atoms forming the linker may also be cyclized to form saturated or unsaturated divalent cyclic radicals in the linker, such as radicals of the formulae: ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ wherein each X 1 is independently CH2, NR’, or O wherein R’ is alkyl or hydrogen and each X 2 is independently S, O, N, NH, CR” wherein R” is alkyl or hydrogen.
  • radicals include: ⁇
  • L 1 , L 2 , or L 3 comprises suitable substituents that change the hydrophobicity or hydrophilicity of the linker.
  • Illustrative hydrophobic groups include alkyl, cycloalkyl, aryl, and arylalkyl, each of which is optionally substituted.
  • L 1 , L 2 , or L 3 can each, independently, comprise alkylene-amino- alkylenecarbonyl, alkylene-thio-(carbonylalkylsuccinimid-3-yl) moieties, including the following formulae: wherein x and y are each independently 1, 2, 3, 4, or 5, where the asterisk identifies ⁇ points of attachment either to other linker fragments of to T or A 1 or A 2 .
  • linkers include 1- alkylsuccinimid-3-yl, carbonyl, thionocarbonyl, alkyl, cycloalkyl, alkylcycloalkyl, alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-alkylsuccinimid-3-yl, 1- (carbonylalkyl)succinimid-3-yl, alkylsulfoxyl, sulfonylalkyl, alkylsulfoxylalkyl, ⁇ alkylsulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1- (carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and 1- (carbonyltetrahydrofuranyl)succinimid
  • one or more of the aforementioned groups can be used in combination (or more than once) (e.g., -alkyl-C(O)-alkyl) and may further ⁇ comprise an additional nitrogen (e.g., alkyl-C(O)-NH-, -NH-alkyl-C(O)- or -NH- alkyl-), oxygen (e.g., -alkyl-O-alkyl-) or sulfur (e.g., -alkyl-S-alkyl-).
  • an additional nitrogen e.g., alkyl-C(O)-NH-, -NH-alkyl-C(O)- or -NH- alkyl-
  • oxygen e.g., -alkyl-O-alkyl-
  • sulfur e.g., -alkyl-S-alkyl-
  • L 1 , L 2 , or L 3 can be formed via click chemistry or be click chemistry-derived.
  • L 1 , L 2 , or L 3 can be derived from copper- catalyzed azide-alkyne cycloaddition (CuAAC), strain promoted azide-alkyne cycloaddition (SPAAC), inverse electron demand Diels-Alder reaction (IEDDA), and Staudinger ligation (SL).
  • CuAAC copper- catalyzed azide-alkyne cycloaddition
  • SPAAC strain promoted azide-alkyne cycloaddition
  • IEDDA inverse electron demand Diels-Alder reaction
  • SL Staudinger ligation
  • T can be a moiety of the formula T-N3.
  • T-N3 ⁇ can then be reacted with an alkyne as shown in the following Scheme: where the wavy line connected to T and to A 1 /A 2 represents a linker between T and A 1 /A 2 and the groups to which they are attached.
  • L 1 , L 2 , or L 3 can include ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ where x is an integer from 0 to 50 and y is an integer from 0 to 50 [0101] In other embodiments, L 1 , L 2 , or L 3 can include ⁇ wherein each of R 2 and R 3 is independently H or C1-6alkyl; and z is an integer from 1 to 8.
  • L 1 , L 2 , or L 3 can include an amide, ester, urea, carbonate, carbamate, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl cycloalkyl, aryl, ⁇ heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof.
  • L 1 , L 2 , or L 3 can include a glycosylated amino acid.
  • L 1 , L 2 , or L 3 can include one or more monosaccharide, disaccharide, polysaccharide, glycan, or peptidoglycan. In some embodiments, L 1 , L 2 , and L 3 do not comprise a glycan. In some embodiments, L 1 , L 2 , ⁇ and L 3 do not comprise a sugar. [0103] In some embodiments, L 1 , L 2 , or L 3 can include a rigid functionality such as an oligoproline or oligopiperidine.
  • an oligoproline or oligopiperidine has about two up to and including about fifty, about two to about forty, about two to about thirty, about two to ⁇ about twenty, about two to about fifteen, about two to about ten, or about two to about six repeating units (e.g., prolines or piperidines).
  • L 1 , L 2 , or L 3 can comprise (–CH 2 CH 2 -O-) n , where n is an integer between and including 1 and 36 (e.g., 1 to 2, 2 to 6, 3 to 8, 6 to 12, and 4 to 10) a peptide, an alkylamido group (e.g., C(O)N(H)C2-C18 alkyl- or C2-C18 alkyl-C(O)N(H)-), an alkylamidoalkyl group (e.g., a C2-C18 alkyl-C(O)N(C2-C18 alkyl)2 or a C2-C18 alkyl- ⁇ C(O)N(H)-C2-C18 alkyl group, such as a -CH2CH2C(O)N(CH2CH2)2 or a - CH 2 CH 2 C(O)N(H)(CH 2 CH 2 )
  • n is an integer between and including 1 and 36 (e.g., 1 to
  • L 1 , L 2 , or L 3 can comprise wherein m is an integer from 0 to 20, such as from 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8 or ⁇ 3 to 9.
  • m can be 0 or 1.
  • m can be 5 or 6.
  • m can be 7 or 8.
  • L 1 , L 2 , or L 3 can comprise wherein p and q are each, independently, an integer from 0 to 20, such as from 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8 or 3 to 9.
  • p can be 2 or 3.
  • q can be 2 ⁇ or 3.
  • L 1 , L 2 , or L 3 can comprise wherein d is an integer from 0 to 20, such as from 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8 or 3 to 9.
  • d can be 1, 2 or 3.
  • L 1 , L 2 , and L 3 taken together can comprise ⁇ ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ such as wherein m, p, d, and q are each, independently, an integer from 0 to 20, such as from 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8 or 3 to 9.
  • m can be 0 or 1.
  • m ⁇ can be 5 or 6.
  • m can be 7 or 8.
  • p can be 2 or 3.
  • L 1 , L 2 , and L 3 taken together can comprise ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ [0109]
  • L 1 , L 2 , and/or L 3 can increase the water-solubility of the conjugate.
  • L 1 , L 2 , and L 3 can each, independently, comprise one or more of an amino acid residue, a polyethylene glycol (PEG) monomer group, a PEG oligomer group, a PEG ⁇ polymer group, or a combination of any of the foregoing.
  • PEG polyethylene glycol
  • L 1 , L 2 , and L 3 can each, independently, comprise an oligomer of peptidoglycan groups, glycan groups, or a combination of any of the foregoing. In many embodiments, L 1 , L 2 , and L 3 can each, independently, comprise at least one 2,3-diaminopropionic acid group, at least one glutamic acid group, at least one cysteine group, or a combination of two or more of the foregoing. ⁇ [0110] In many embodiments, one or more of L 1 , L 2 , and L 3 independently comprise one or more rigid or conformationally restricting linkers.
  • rigid and conformationally restricting linkers include oligoproline, oligoalkyne, oligophenyl and oligopiperidine groups.
  • one or more of L 1 , L 2 , and L 3 independently comprise one or more of an oligoethylene group, a PEG group, an alkyl, oligopeptide group, polypeptide ⁇ group, a peptidoglycan group, an oligoproline group, an oligopiperidine group or any combination thereof.
  • one or more of L 1 , L 2 , and L 3 independently comprise an oligoethylene glycol group or a PEG group.
  • one or more of L 1 , L 2 , and L 3 independently comprise an oligoethylene glycol group. In some embodiments, one or more of L 1 , L 2 , and L 3 independently comprise a PEG ⁇ group. [0111] In some embodiments, when any of L 1 , L 2 , and L 3 is an oligoproline or oligopiperidine group, then such oligoproline or oligopiperidine group has between (and including the end points of the ranges) 2 and 50 , 2 to 40, or 2 to 30, or 2 to 20, or 2 to 15, to 2 to 10, or 2 to 6 repeating units.
  • such repeating units are prolines ⁇ (for oligoprolines) and piperedines (for oligopiperidine). ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ [0112]
  • such oligoproline or oligopiperidine group has between (and including the end points of the ranges) 2 and 50 , 2 to 40, or 2 to 30, or 2 to 20, or 2 to 15, to 2 to 10, or 2 to 6 repeating units.
  • such repeating units are prolines ⁇ (for oligoprolines) and piperedines (for oligopiperidine).
  • one or more of L 1 , L 2 , and L 3 may independently comprise a moiety comprising an ester, a phosphate, an oxime, an acetal, a pyrophosphate, a polyphosphate, a sulfate, a hydrazide, an imine, a carbonate, a carbamate which may, in turn, be used to bind A 1 or A 2 to L 3 and L 2 , respectively.
  • one or more of L 1 , L 2 , and L 3 may independently comprise a moiety comprising an ester, a phosphate, an oxime, an acetal, a pyrophosphate, a polyphosphate, a sulfate, a hydrazide, an imine, a carbonate, a carbamate which may, in turn, be used to bind A 1 or A 2 to L 3 and L 2 , respectively.
  • a 1 can be bound to L 3 by a moiety comprising an ester, a phosphate, an oxime, an acetal, a pyrophosphate, a ⁇ polyphosphate, a sulfate, a hydrazide, an imine, a carbonate, a carbamate; and
  • a 2 can be bound to L 2 by a moiety comprising an ester, a phosphate, an oxime, an acetal, a pyrophosphate, a polyphosphate, a sulfate, a hydrazide, an imine, a carbonate, a carbamate.
  • L 1 , L 2 , and/or L 3 may be substituted so as to alter the affect the hydrophobicity or hydrophilicity of L 1 , L 2 , and/or L 3 .
  • L 1 , L 2 , and/or ⁇ L 3 may have a hydrophobic side chain group, such as an alkyl, cycloalkyl, aryl, arylalkyl, or like group, each of which is optionally substituted.
  • L 1 , L 2 , and/or L 3 comprise or further comprise one or more amino acids with side chains, such as phenylalanine (Phe) and tyrosine (Tyr), including substituted variants thereof, and analogs and derivatives of such side chains.
  • side chains such as phenylalanine (Phe) and tyrosine (Tyr), including substituted variants thereof, and analogs and derivatives of such side chains.
  • amino acid side chains include: .
  • L 1 , L 2 , and/or L 3 comprise portions that are neutral under physiological conditions.
  • L 1 , L 2 , and/or L 3 comprise portions that can ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ be protonated or deprotonated to carry one or more positive or one or more negative charges, respectively.
  • L 1 , L 2 , and/or L 3 comprises neutral portions and portions that may be protonated to carry one or more positive charges.
  • neutral portions include polyhydroxyl groups, such as sugars, carbohydrates, saccharides, inositols, ⁇ and the like, and/or polyether groups, such as polyoxyalkylene groups, including polyoxyethylene, polyoxypropylene, and the like.
  • portions that can be protonated to carry one or more positive charges include amino groups, such as polyaminoalkylenes, including ethylene diamines, propylene diamines, butylene diamines and the like, and/or heterocycles, including pyrrolidines, piperidines, piperazines, and other ⁇ amino groups, each of which can be optionally substituted.
  • portions that can be deprotonated to carry one or more negative charges include carboxylic acids, such as aspartic acid, glutamic acid, and longer chain carboxylic acid groups, and sulfuric acid esters, such as alkyl esters of sulfuric acid.
  • the conjugate can be water-soluble.
  • L 1 , L 2 , and/or L 3 can increase the water- ⁇ solubility of the conjugate.
  • the total combined length of L 1 + L 2 + L 3 is between about 17 and 100 ⁇ or between about 17 to about 75 ⁇ .
  • L 1 , L 2 , and L 3 can each, independently, comprise one or more of an amino acid residue, a polyethylene glycol (PEG) monomer group, a PEG oligomer group, a PEG polymer group, or a combination of any of the foregoing.
  • L 1 , L 2 , and L 3 can each, independently, comprise at least one 2,3-diaminopropionic acid group, at least one glutamic acid group, at least one cysteine group, or a combination of two or more of the foregoing.
  • one or more of L 1 , L 2 , and L 3 independently comprise ⁇ carbonyl, thionocarbonyl, alkylene, cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl, 1- (carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl, alkylenesulfoxylalkyl, alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1- ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ (carbonyltetrahydro-2H-pyranyl)succinimid-3-yl or 1- (carbonyltetrahydrofuranyl)succinimid-3-yl,
  • one or more of L 1 , L 2 , and L 3 independently comprise or further comprise a nitrogen such that one or more of L, L 1 , and L 2 comprise ⁇ alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl or 1- (carbonylalkyl)succinimid-3-yl groups, each of which is optionally substituted, bonded to said nitrogen to form an amide.
  • one or more of L 1 , L 2 , and L 3 independently comprise or further comprise a sulfur atom and alkylene or cycloalkylene groups, each of which is optionally substituted with carboxy, and is bonded to the sulfur to ⁇ form a thiol.
  • L 1 , L 2 , and L 3 independently comprise or further comprise a sulfur atom and 1-alkylenesuccinimid-3-yl and 1-(carbonylalkyl)succinimid-3- yl groups bonded to the sulfur to form a succinimid-3-ylthiol.
  • one or more of L 1 , L 2 , and/or L 3 can each, independently, comprise an amino acid such as an amino acid selected from Lys, Asn, Thr, Ser, Ile, Met, ⁇ Pro, His, Gln, Arg, Gly, Asp, Glu, Ala, Val, Phe, Leu, Tyr, Cys, and Trp.
  • the compounds described herein may contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers. It is to be understood that in one embodiment, the invention described herein is not limited to any particular stereochemical requirement, and that the compounds, and compositions, methods, uses, ⁇ and medicaments that include them may be optically pure, or may be any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. It is also to be understood that such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration at one or more other ⁇ chiral centers.
  • the compounds described herein may include geometric centers, such as cis, trans, E, and Z double bonds, or spatial arrangements, such as cis, trans, syn, and anti, relative configurations on a ring. It is to be understood that in another embodiment, the invention described herein is not limited to any particular geometric isomer requirement, ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ and that the compounds, and compositions, methods, uses, and medicaments that include them may be pure, or may be any of a variety of geometric isomer mixtures. It is also to be understood that such mixtures of geometric isomers may include a single configuration at one or more double bonds, while including mixtures of geometry at one or more other ⁇ double bonds.
  • the conjugates hereof can be “deuterated,” meaning one or more hydrogen atoms can be replaced with deuterium. As deuterium and hydrogen have nearly the same physical properties, deuterium substitution is the smallest structural change that can be made. Deuteration is well known to those of ordinary skill in the art.
  • solvates refers to compounds described herein complexed with a solvent molecule. It is appreciated that compounds described herein may form such complexes with solvents by simply mixing the compounds with a solvent, or dissolving the compounds in a solvent. It is appreciated that where the compounds are to be used as pharmaceuticals, such solvents are pharmaceutically acceptable solvents.
  • the relative amount of solvent that forms the solvate should be less than established guidelines for such pharmaceutical uses, such as less than International Conference on Harmonization (ICH) Guidelines. It is to be understood that the solvates may be isolated from excess solvent by evaporation, precipitation, and/or crystallization. In some embodiments, the ⁇ solvates are amorphous, and in other embodiments, the solvates are crystalline. When the solvent is water, the solvate is termed a hydrate. [0125] As used herein, the term “composition” generally refers to any product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified ⁇ amounts.
  • compositions described herein may be prepared from isolated compounds described herein or from salts, solutions, hydrates, solvates, and other forms of the compounds described ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ herein. It is also to be understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and/or other solid forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various hydrates and/or solvates of the compounds described herein.
  • compositions may be prepared from various co- crystals of the compounds described herein. Accordingly, such pharmaceutical compositions that recite compounds described herein are to be understood to include each of, or any combination of, the various solid forms and/or solvate or hydrate forms of the compounds described herein.
  • Pharmaceutical compositions can be prepared by combining one or more conjugates with one or more pharmaceutically acceptable excipients, carriers, or diluents, or any combination thereof, and, optionally, one or more additional pharmaceutically active agents DEFINITIONS ⁇ [0127]
  • the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise.
  • n ⁇ is an integer from 0 to 8 also describes each and every subrange, each of which may for the basis of a further embodiment, such as n is an integer from 1 to 8, from 1 to 7, from 1 to 6, from 2 to 8, from 2 to 7, from 1 to 3, from 2 to 4, etc.
  • n is an integer from 1 to 8, from 1 to 7, from 1 to 6, from 2 to 8, from 2 to 7, from 1 to 3, from 2 to 4, etc.
  • the recitation of the number 1 with a single significant figure is understood to properly refer to a range of values from 0.5 to 1.4.
  • the recitation ⁇ of the number 1.0 with two significant figures is understood to properly refer to a range of values from 0.95 to 1.04.
  • the relative precision of the numerical value can be further indicated by modifying with the term “about” to indicate that the modified number has lower precision.
  • the term “about” when used with numerical values or limits ⁇ generally means that the number is approximate and that, as recited, it is understood to include a range of values.
  • a real number that is recited with a single significant figure would by definition include a so-called rounding range; the number about 5 would at the very least include the range 4.5-5.4, as each of those values rounds to 5.
  • the same is to be understood for real numbers expressed with additional significant ⁇ figures, where the corresponding rounding range applies to the last significant figure.
  • Integers are to be understood to at least include the values ⁇ 1 for single-digit numbers, ⁇ 10 for two-digit numbers, etc.
  • the term “about” is also interpreted to contemplate a range based on a percentage of the recited number, such as about 5 construed to include 5 ⁇ 10% or 5 ⁇ 20%.
  • radical refers to a ligand for a target protein or hapten, respectively, as described herein, where one or more atoms or groups, such as a hydrogen atom, or an alkyl group on a heteroatom, and the like, is removed to provide a radical for covalent linking or ⁇ conjugation to the polyvalent linkers L 1 , L 2 , and L 3 .
  • one or more groups such as a hydrogen atom, or an alkyl group on a heteroatom, and the like
  • Illustrative analogs include, but are not limited to, those compounds that share functional and in some cases structural similarity to those compounds described hereinIt is to be understood that such radicals can also be formed on acid, ester, or amide groups, such ⁇ as carboxy, phosphoryl, and sulfuryl acids, by removing the OH, ester, or amide group. It is also to be understood that such radicals can be formed by removing other fragments, such as halo, alkoxy, amino, heterocyclyl, or heteroaryl groups.
  • alkyl group is a saturated, partially saturated, or unsaturated straight chain or branched non-cyclic hydrocarbon having from 1 to 10 carbon atoms (C1-C10 alkyl), from 1 ⁇ to 8 carbons (C1-C8 alkyl), from 1 to 6 (C1-C6 alkyl), 1 to 4 (C1-C4 alkyl), 1 to 3 (C1-C3 alkyl), or 2 to 6 (C2-C6 alkyl) carbon atoms.
  • the alkyl group has monovalency. Examples of alkyl groups with monovalency include -CH 3 , -CH 2 CH 3 , and the like.
  • Monovalent alkyls may be found on substitutions in the chain of linker, L, for example.
  • the alkyl group has bivalency, such as when found in the ⁇ chain of the linker, L. Examples of alkyl groups with bivalency include, but are not limited to, -CH2-, -CH2CH2-, and the like.
  • the alkyl group is a saturated alkyl group.
  • an alkyl group is an unsaturated alkyl group, also termed an alkenyl group or an alkynyl group.
  • heteroalkyl by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain, or combination(s) thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be ⁇ oxidized, and the nitrogen heteroatom may optionally be quarternized.
  • the heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
  • aryl includes monocyclic and polycyclic aromatic carbocyclic groups, each of which may be optionally substituted.
  • Illustrative aromatic ⁇ carbocyclic groups described herein include, but are not limited to, phenyl, naphthyl, and the like.
  • heteroaryl includes aromatic heterocyclic groups, each of which may be optionally substituted.
  • Illustrative aromatic heterocyclic groups include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, ⁇ isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl, and the like.
  • optionally substituted includes the replacement of hydrogen atoms with other functional groups on the radical that is optionally substituted.
  • Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, ⁇ halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof, and the like.
  • any of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is optionally substituted.
  • Illustrative substituents include, but are not limited to, a radical -(CH 2 ) x Z X , where x is an integer from 0-6 and Z X is selected from halogen, hydroxy, alkanoyloxy, including C1-C6 alkanoyloxy, optionally substituted aroyloxy, alkyl, including C1-C6 alkyl, alkoxy, including C1-C6 alkoxy, cycloalkyl, including C3-C8 cycloalkyl, cycloalkoxy, including C3- ⁇ C8 cycloalkoxy, alkenyl, including C2-C6 alkenyl, alkynyl, including C2-C6 alkynyl, haloalkyl, including C 1 -C 6 haloalkyl, haloalkoxy, including C 1 -C 6 haloalkoxy, halocycloalkyl,
  • the compounds described herein can be used for both human clinical medicine and ⁇ veterinary applications.
  • the host animal treated with the compounds described herein can be human or, in the case of veterinary applications, can be a laboratory, agricultural, domestic, or wild animal.
  • the present invention can be applied to host animals including, but not limited to, humans, laboratory animals such rodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats, ⁇ and rabbits, agricultural animals such as cows, horses, pigs, sheep, goats, and wild animals in captivity such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.
  • rodents e.g., mice, rats, hamsters, etc.
  • rabbits, monkeys, chimpanzees domestic animals
  • domestic animals such as dogs, cats, ⁇ and rabbits
  • agricultural animals such
  • the term “therapeutically effective amount” as used herein, refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response ⁇ in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
  • the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit/risk ratio applicable to any medical treatment.
  • ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically-effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the ⁇ specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient: the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well known to the researcher, veterinarian, medical doctor or ⁇ other clinician of ordinary skill.
  • the conjugates and compositions may be delivered by suitable methods of delivery including, for example but not limited to, intranasally, orally, and intravenously.
  • the method can further comprise administering autologous antibodies or allogeneic Immunoglobulin G (IgG) antibodies.
  • IgG Immunoglobulin G
  • the monotherapy may include co-administration of one or more carriers, vehicles, diluents, adjuvants, excipients, and the like, and combinations thereof, and/or include co- administration of one or more additional active pharmaceutical ingredients
  • additional active pharmaceutical ingredients are to be understood to be for treating diseases and/or symptoms distinct from treating the underlying conditions described herein, such as the treatment of the viral infection itself.
  • additional active pharmaceutical ingredients may include, for example, active ingredients for treating pain, inflammation, cough, congestion, and the like.
  • ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ EXAMPLES [0143] serve to further illustrate the invention described herein.
  • Example 2 Compound 2 ⁇ ⁇ ⁇ [0148] To a solution of commercially available 1-chloro-2,4-dinitrobenzene (0.5 g, 2.47 mmol) and 3-(2-aminoethoxy)propanoic acid, 0.33 g, 2.47 mmol) dissolved in EtOH (25 mL) was added TEA (1.38 mL, 9.87 mmol). The reaction mixture was heated to 55oC for ⁇ 16h.
  • the resulting oil (0.50 g, 1.50 mmol) was dissolved in DCM (DCM) (7.5 mL) prior to addition of commercially available amino- PEG4-OH , (0.35 g, 1.81 mmol, 1.2 eq.) under N2 atmosphere.
  • DCM DCM
  • the reaction flask was placed in an ice bath, and boron trifluoride diethyletherate (0.56 mL, 4.51 mmol, 3.0 eq.) was added ⁇ dropwise over 30 min.
  • the reaction mixture was stirred for 2h, then allowed to reach ambient temperature. After 16h, reaction was complete, and the reaction mixture was poured into ice water and extracted with DCM.
  • Example 3 Compound 4 ⁇
  • Tosyl-PEG12-Azide (27.0mg, 0.0376 mmol) in DMSO (0.2 mL).
  • the reaction mixture was heated to 60 o C and stirred for 2h.
  • the reaction mixture was cooled to ambient temperature and charged with water (2mL) and purified by RP-HPLC (30%-95% ACN in 10mM aq.
  • Example 4 Compound 6 [0158] To a solution of commercially available Sisunatovir (10.0 mg, 0.0224 mmol) and K 2 CO 3 (17.9 mg, 0.448 mmol) in DMSO (0.2mL) was added Tosyl-PEG12-Azide ( 17.9mg, ⁇ 0.0246 mmol) in DMSO (0.2mL). The reaction mixture was heated to 60 o C and stirred for 2h. The reaction mixture was cooled to ambient temperature and charged with water (2mL) forming a solid precipitate.
  • Example 5 Compound 8A ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ [0160] To a solution of commercially available JNJ2408068 (Compound 14, 10.0 mg, 0.0253 mmol) and Boc Anhydride (11.0 mg, 0.0507 mmol) in DMF (1 mL) was added ⁇ Potassium carbonate (13.9 mg, 0.101 mmol). The reaction mixture was stirred for 2h.
  • Compound 21 Compound 21.
  • Compound 8a is prepared according to the process described in Bonfanti et al. J Med Chem 51:875-96 (2008).
  • a solution of 8a (0.910 g) in MeOH is treated with 10% Pd/C under H2 atmosphere. ⁇ Compound 8b is isolated (600 mg, 62% yield).
  • a solution of compound 8b (600 mg) in DMF is treated with Br-(CH2)2-NH-Boc (9) and TEA at 40°C for 24 to give a mixture of compound 8c and an O,N-dialkylated product.
  • Compound 8c is isolated (0.310 g, 27% yield).
  • a solution of compound 8c (0.310 g) in DCM is treated with MnO 2 for 12h.
  • ⁇ Compound 8d is isolated (230 mg, 63% yield).
  • a solution of compound 8d (230 mg) in ACN is treated with 3-(2-amino-4- methylphen-1-yl)propanol (10) and sodium cyanoborohydride and acetic acid for 6h.
  • Compound 11 is isolated (140 mg, 41% yield).
  • a solution of compound 11 (140 mg) in DCM is treated with TFA.
  • Compound 18 is isolated as a TFA salt (70 mg, 84% yield). The free amine of compound 18 is isolated by HPLC.
  • Example 7 N-((S)-25-((2,4-dinitrophenyl)amino)-13,20-dioxo-1- ⁇ (((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,23- tetraoxa-12,19-diazapentacosan-14-yl)-1-((2-((6-(((2-(3-hydroxypropyl)-5- methylphenyl)amino)methyl)-2-((3-morpholinopropyl)amino)-1H-benzo[d]imidazol-1- yl)methyl)-6-methylpyridin-3-yl)oxy)-3,6,9,12,15,18,21,24,27,30,33- undecaoxahexatriacontan-36-amide (Compound 26).
  • Compound 2 was prepared using the same procedure used to make compound 2 in Example 7.
  • Compound 2 was prepared from compound 1 (BLD Pharma, 0.5 ⁇ g, 0.793 mmol) in DCM (5 mL), and TEA (0.252 mL, 1.74 mmol) with p- (chlorosulfonyl)toluene (0.166 g, 0.872 mmol), and was purified by column chromatography using 2-5% MeOH/DCM as eluent to afford compound 2 as a viscous colourless liquid. Yield: 0.480 g, 77 %.
  • reaction mixture 60 mg, 47.8 ⁇ mol was combined with compound 6 (57.0 mg, 0.049 mmol, see Example 6, compound 21) in additional DMF (1 mL), HATU (21.8 mg, 1.2 eq., 57.4 ⁇ mol), 1H-1,2,3-benzotriazol-4-ol (0.008 g, 0.0574 mmol) and DIPEA (0.013 mL, 0.0717 mmol) were added.
  • the reaction mixture was stirred for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was evaporated under ⁇ high vacuum, and the remainder was purified by reverse phase column chromatography using C18 column with 10-100% H2O:ACN gradient.
  • Compound 1 The 41-mer protected peptide (1) was synthesized on 2-CTC support resin using a conventional automatic peptide synthesizer and Fmoc/OtBu protocol. ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ [0190] Compound 2. 2CTC resin loaded with protected peptide 1 (350 mg) was suspended in 20% 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in DCM (20 mL) in a 50 mL peptide reaction vessel. The reaction vessel was shaken for 30 min. The resin was filtered and washed three times with 10 mL portions of 20% HFIP in DCM.
  • HFIP 1,1,1,3,3,3-hexafluoroisopropanol
  • Compound 10 was prepared from compound 8 (0.15 g, 0.13 mmol) and compound 9 (0.12 g, 0.13 mmol, see Example 6, compound 21) in DMF (5.0 ⁇ mL) with HATU (0.057 g, 0.15 mmol) and DIPEA (0.12 mL, 0.68 mmol), and was purified by silica gel chromatography using 8-10% MeOH/DCM as eluent to afford compound 10 as a yellowish liquid. Yield: 0.09 g, 33.66 %; LCMS (ESI) m/z 1954.45 [M+1] + . [0215] Compound 53.
  • METHOD EXAMPLE 1 Immunofluorescence Assay (IFA) – FIG.3 ⁇
  • IFA Immunofluorescence Assay
  • FIG.3 Hep2 cells were seeded into 12 well plates at a density of 0.1 x 10 6 cells per well 48 hours prior to the assay and were incubated at 37qC overnight. The next day selected wells were infected with RSV Long Strain (ATCC – VR-26) at a multiplicity of infection (MOI) of 3 in 250 Pl of complete media, consisting of 2% Low IgG FBS Serum (Corning – MT35073CV), 1x EMEM (ATCC – 30-2003) and 1x PenStrep (Fisher – 15-140-148) for 2 ⁇ hours at 37qC with rocking.
  • RSV Long Strain ATCC – VR-26
  • MOI multiplicity of infection
  • Secondary fluorescent antibodies (Alexa Fluor 488 Goat-anti-Human IgG – Invitrogen – A11013) were then added at a dilution of 1:500 to target the anti-RSV F Primary antibodies at RT for 1 hour in the dark. After washing the cells 3 times, 500 ⁇ l of 4 ⁇ ,6-diamidino-2- phenylindole (DAPI), a fluorescent dye that binds to DNA, solution in blocking buffer (1 ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ Pg/ml working concentration [Invitrogen – 62248]) was added to each well.
  • DAPI 4 ⁇ ,6-diamidino-2- phenylindole
  • the row labelled RSV F Protein in each image shows the signal ⁇ generated from the anti F protein antibody showing where RSV F protein is located in green.
  • the row labelled Compound 47 shows the signal generated from anti DNP antibody binding to Compound 47 showing where compound 47 is bound in red.
  • the row labelled DAPI Signal shows where DNA is present staining the nucleus of healthy cells in blue to show where cells are present.
  • the row labelled Overlay shows all the of the signals combined to ⁇ show colocalization and where the drug is specifically binding.
  • FIG. 3 shows that upon addition of 100-fold TMC353121 (the free targeting ligand) to compete with compound 47 at the same binding site as compound 47, the interaction of ⁇ compound 47 with the binding site is specific and completable with excess free targeting ligand, suggesting that the signal seen in the absence of ligand competition is due to a specific interaction not due to nonspecific binding.
  • TMC353121 the free targeting ligand
  • Compounds 47 was also evaluated in absence of the anti-DNP antibody as indicated by the column labeled Infection Only compound 47, which shows that compound 47 did not activate the immune system in the ⁇ absence of the anti-hapten antibodies, and that the signal seen in the other columns is due to anti-DNP binding to compound 47 which is bound to the infected cells and not due to extraneous binding of the secondary antibody to compound 47.
  • the final column labelled No Infection shows a situation in which compound 47 and anti-DNP antibody are present but virus has not been added. The data indicate that compound 47 does not bind or ⁇ accumulate to healthy non-infected cells. Compound 47 is shown to very specifically bind to infected cells and trigger immunological mediated clearance of those cells.
  • ADCC Antibody Dependent Cellular Cytotoxicity
  • 25 Pl of anti-DNP IgG antibodies (Acro Biosystems – DNP-M2-25mg) were added to selected wells at a concentration of 33.3 Pg/ml ⁇ in RPMI media and plates were incubated again at 37qC for 30 minutes. Finally, 25 Pl of ADCC effector cells were then added to the infected cells, and the plates were incubated at 37qC for 6 hours.
  • 25 Pl of RPMI media containing 2x10 5 TCID50/ml of clarified RSV Long strain virus was added to each well in ⁇ place of infected cells. The rest of the assay was performed as described for infected cells.
  • CDC Compliment Dependent Cytotoxicity Assay ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇
  • Hep2 cells were seeded into 96 well white-walled plates at a density of 0.2 x 10 5 cells per well 48 hours prior to the assay and were infected as described in the Immunofluorescence Assay.
  • Hep2 cells were serially diluted 2-fold from 20,000 cells per well down to 312.5 cells per well in triplicate to produce a standard curve from ⁇ which to determine % cell death upon the completion of the assay. After 24 hours of infection. Media was removed and cells were washed twice with 1x PBS. After washing, 25 Pl of serum-free RPMI 1640 media (Promega – G708A) was added to the cells.
  • % cell death of compound treated cells was determined using the amount of luminescence from the cell dilution standard curve and the ethanol treated cells were used as the baseline of 100% cell death.
  • the EC 50 curves were calculated using GraphPad Prism (version 10.3.1).
  • ⁇ [0236] As shown in FIG. 9, compound 47 was found to enable complement dependent cell death upon binding to RSV infected cells in the presence of anti-hapten antibodies. This interaction was found to be specific. No activation was identified in infection alone without Compound 47, both in the presence and absence of antibody, suggesting Compound 47 functions to bridge the complement proteins to RSV infected cells, facilitating the death of ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ infected cells.
  • ALS-008176 was dissolved in phosphate buffered saline and 2% Sucrose and administered orally with a rodent feeding tube. 48 hours post infection the animals were euthanized and the lungs were weighed and removed in a sterile ⁇ environment. 1 lung was placed in a triazol solution for RNA extraction. The other was placed in to PBS and flash frozen with liquid nitrogen. [0241] The lung tissue was automatically homogenized using a Fisherbrand Bead Mill 4 Mini Homogenizer. Viral RNA was extracted following the product protocol forymo ran extraction kit .
  • RNA was then quantified with qPCR with primers specific to RSV f protein ⁇ and GAPDH using Bio-Rad's iTaq Universal SYBR Green SuperMix and a Biorad CFX thermocyler. Relative amounts of Viral RNA were standardized to the amount of the housekeeping gene GAPDH and compared. Compound 47 was found to significantly decrease the viral RNA.
  • the other lobe was subjected to a quantification of live virus in via TCID50 quantification. Lung tissue designated for TCID50 was placed into 1 ml of complete ⁇ media and flash frozen in liquid nitrogen.
  • Frozen samples were thawed and homogenized using a FisherbrandTM Bead Mill 4 Mini Homogenizer (Fisher – 15-340-164) for 2 minutes at level 5 speed.
  • the homogenate was clarified using a microcentrifuge at 10,000 x g for 10 minutes.
  • the clarified homogenate was collected in a fresh microcentrifuge tube for TCID50 analysis.
  • Hep2 cells were seeded into 96 well plates at a density of 0.2 x ⁇ 105 cells per well and were roughly 85% confluent at the start of the assay. In separate 96 well plates, serial 10-fold dilutions were made of the clarified lung homogenate samples into complete media.
  • the media was removed from the Hep2 cells, and the cells were washed twice with 1x PBS. Next, 25 ⁇ l of the homogenate dilutions or control groups were added onto the cells and the infection was allowed to progress for 2 hours at 37oC with rocking. ⁇ After the incubation period, 75 ⁇ l of complete media was added to each well, and the assay was allowed to progress for 5 days. The media and inoculum was removed after 120 hours post infection, and 100 ⁇ l of fresh complete media was added to all wells. Then, 20 ml of MTS reagent (Promega – G5430) was added to the cells, and the plates were incubated at 37°C for 2 hours.
  • MTS reagent Promega – G5430
  • cotton rats were treated with Vehicle (20mg/ml Avicel ⁇ 491(Sigma - 11363), 6mg/ml n-dodecyl ⁇ -D-maltoside (Sigma - 69227-93-6), 1x Phosphate buffered saline (Corning – MT21040CV)), or Compound 26 1.5 ⁇ mol/kg, or ALS- 008176(MecChem Express) 100umol/kg. Vehicle and Compound 26 were administered intranasally to cotton rats which had been anesthetized via isoflurane inhalation with a dose volume of 0.2ml/kg.
  • Vehicle 20mg/ml Avicel ⁇ 491(Sigma - 11363), 6mg/ml n-dodecyl ⁇ -D-maltoside (Sigma - 69227-93-6), 1x Phosphate buffered saline (Corning – MT21040CV)
  • Compound 26 1.5 ⁇
  • ALS-008176 was dissolved in phosphate buffered saline and 2% ⁇ Sucrose and administered orally with a rodent feeding tube.
  • Cotton Rats were euthanized 24 hours post treatment with compounds, and their lungs were harvested under sterile conditions. After removal, the lungs were rinsed in 1x PBS containing 1x PenStrep to remove any surface exposed bacteria and were weighed to the nearest mg.
  • Lung tissue designated for TCID 50 was placed into 1 ml of complete media and flash frozen in liquid ⁇ nitrogen. The lung tissue was subjected to a quantification of live virus in via TCID50 quantification. Lung tissue designated for TCID50 was placed into 1 ml of complete media and flash frozen in liquid nitrogen.
  • Frozen samples were thawed and homogenized using a FisherbrandTM Bead Mill 4 Mini Homogenizer for 2 minutes at level 5 speed.
  • the homogenate was clarified using a microcentrifuge at 10,000 x g for 10 minutes.
  • the clarified ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ homogenate was collected in a fresh microcentrifuge tube for TCID50 analysis.
  • TCID50 curves were calculated using GraphPad Prism (version 10.3.1), and the final titer values were produced by normalizing the TCID50 output ⁇ to the weight of total lung tissue homogenized (TCID50/mg) and the dilution factors used during infection. [TCID50 value produced in Prism / Total weight of lung tissue (mg)] x [dilution factor of homogenate volume (40)].
  • Vehicle and Compound 26 were administered intranasally to ⁇ cotton rats which had been anesthetized via isoflurane inhalation with a dose volume of 0.2ml/kg.
  • Cotton Rats were euthanized 4 days post infection and their lungs were harvested under sterile conditions. Lungs were flash frozen in liquid nitrogen immediately upon collection. The lung tissue for RNA analysis was automatically homogenized using a Fisherbrand Bead Mill 4 Mini Homogenizer (Fisher – 15-340-164). Viral RNA was ⁇ ⁇ ⁇ 70403-03 ⁇ ⁇ extracted following the product protocol Zymo RNA extraction kit.
  • RNA was then quantified with qPCR with primers specific to RSV F protein and GAPDH using Bio- Rad's ⁇ iTaq ⁇ Universal SYBR Green SuperMix and a Biorad CFX Thermocyler. Relative amounts of Viral RNA were standardized to the amount of the housekeeping gene GAPDH ⁇ and compared. Compound 26 was found to significantly decrease the viral RNA. [0246] Compound 27 was also found to significantly decrease the viral RNA. [0247] METHOD EXAMPLE 9. Cytokine release assay. [0248] A cytokine release assay was performed on compound 47 to assess the risk of causing a cytokine storm in humans.
  • human PBMCs pooled from at least 4 ⁇ donors were plated at 2.5x10 5 cells/well.1 ⁇ M of compound 47, 1 ⁇ M TLR7 agonist, or RPMI was added to the wells. All groups were supplemented with 10mg/mL Immune Globulin Injection (IVIG) to provide anti-DNP and anti-rhamnose antibodies.
  • IVIG Immune Globulin Injection
  • the PBMCs were then incubated at 37 C, 5% CO2 for 24 hours. Cells were centrifuged at 450xg for 10 min and supernatant was collected. Cytokines in the supernatant were measured using a ⁇ LEGENDplexTM Human Inflammation Panel 1 (biolegend, cat. #740809) following manufacturers protocol.
  • the 9 different proteins measured were IL-1 ⁇ , IFN- ⁇ 2, IFN- ⁇ , TNF- ⁇ , MCP-1 (CCL2), IL-10, IL-12p70, IL-18, IL-23, and IL-33. Fluorescence was measured using an Attune NxT flow cytometer (Fisher), and the concentration of each protein was quantified using LEGENDplexTM software (biolegend). Data was graphed ⁇ using GraphPad prism.. An unpaired t test was performed to determine the significance of the difference in cytokines levels between media and compound 47. There was no significant difference for any of the cytokines measured.

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Abstract

L'invention concerne un composé conjugué comprenant (i) un ligand, qui cible une protéine de VRS ou une cellule infectée par le VRS, (ii) un lieur ou des lieurs, et (iii) au moins deux haptènes ; une composition pharmaceutique comprenant le composé conjugué ; et une méthode d'administration de l'agent au VRS ou à une cellule infectée par le VRS pour une thérapie.
PCT/US2024/051835 2023-10-17 2024-10-17 Conjugués, compositions et méthodes de traitement d'une infection par le vrs Pending WO2025085675A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021050612A1 (fr) * 2019-09-09 2021-03-18 Cidara Therapeutics, Inc. Compositions et méthodes pour le traitement du virus respiratoire syncytial
WO2023141500A2 (fr) * 2022-01-19 2023-07-27 The Children's Medical Center Corporation Produits d'addition de nanocorps-médicament et leurs utilisations

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021050612A1 (fr) * 2019-09-09 2021-03-18 Cidara Therapeutics, Inc. Compositions et méthodes pour le traitement du virus respiratoire syncytial
WO2023141500A2 (fr) * 2022-01-19 2023-07-27 The Children's Medical Center Corporation Produits d'addition de nanocorps-médicament et leurs utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIET ET AL.: "Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells", CHEM. EUR. J., vol. 25, 2019, pages 15508 - 15515, XP071850342, DOI: 10.1002/chem.201903327 *

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