WO2022133281A1 - Compositions et méthodes pour le traitement du virus de l'immunodéficience humaine - Google Patents

Compositions et méthodes pour le traitement du virus de l'immunodéficience humaine Download PDF

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WO2022133281A1
WO2022133281A1 PCT/US2021/064151 US2021064151W WO2022133281A1 WO 2022133281 A1 WO2022133281 A1 WO 2022133281A1 US 2021064151 W US2021064151 W US 2021064151W WO 2022133281 A1 WO2022133281 A1 WO 2022133281A1
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optionally substituted
conjugate
amino acid
acid sequence
formula
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PCT/US2021/064151
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English (en)
Inventor
Allen Borchardt
Thomas P. Brady
Hongyuan Chen
Zhi-yong CHEN
Quyen-Quyen Thuy Do
Travis James HAUSSENER
Robert Michael Hughes
Alain Noncovich
Leslie W. TARI
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Cidara Therapeutics, Inc.
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Publication of WO2022133281A1 publication Critical patent/WO2022133281A1/fr

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    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • HIV antiviral inhibitors come in many several classes targeting distinct steps of the HIV cycle.
  • One class of antivirals nucleoside reverse transcriptase inhibitors (NRTIs) inhibit viral replication by chain termination after being incorporated into growing DNA strands by HIV reverse transcriptase.
  • NRTIs nucleoside reverse transcriptase inhibitors
  • NRTIs non-nucleoside reverse transcription inhibitors
  • a different class of antivirals, integrase inhibitors inhibit viral DNA insertion into the host cellular genome.
  • Protease inhibitors are agents that inhibit the protease enzyme, a key enzyme in the assembly of new virus particles.
  • One class of antivirals, known as viral entry inhibitors contains agents that interfere in viral entry into the cell by binding to HIV envelope (Env) glycoprotein. In particular, viral entry inhibitors target the surface subunit gp120 receptor of the HIV virus.
  • the disclosure relates to conjugates, compositions, and methods for inhibiting viral growth, methods for the treatment of viral infections, and methods of synthesizing conjugates.
  • such conjugates contain monomers or dimers of a moiety that inhibits human immunodeficiency virus, for example by binding to the gp120 glycoprotein (e.g., a gp120 binder such as temsavir, BMS-818251, DMJ- II-121, BNM-IV-147, or analogs thereof), conjugated to Fc monomers, Fc domains, Fc-binding peptides, albumin proteins, or albumin protein-binding peptides.
  • gp120 glycoprotein e.g., a gp120 binder such as temsavir, BMS-818251, DMJ- II-121, BNM-IV-147, or analogs thereof
  • the HIV targeting moiety e.g., temsavir, BMS-818251, DMJ-II-121, BNM-IV-147, or analogs thereof
  • the HIV targeting moiety targets a protein encoded by the HIV Env gene, in particular gp120 glycoprotein on the surface of the viral particle, thereby preventing viral attachment to the host CD4+ T cell and entry into the host immune cell.
  • the Fc monomers or Fc domains in the conjugates bind to Fc ⁇ Rs (e.g., FcRn, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb) on immune cells, e.g., neutrophils, to activate phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), thus leading to the engulfment and destruction of viral particles by immune cells and further enhancing the antiviral activity of the conjugates.
  • Fc ⁇ Rs e.g., FcRn, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb
  • immune cells e.g., neutrophils
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the albumin or albumin-binding peptide may extend the half-life of the conjugate, for example, by binding of albumin to the recycling neon
  • compositions are useful in methods for the inhibition of viral growth and in methods for the treatment of viral infections, such as those caused by an HIV-1 and HIV-2.
  • the disclosure features a conjugate described by any one of formulas (D-I), (M-I), (1), or (2): wherein each A 1 and each A 2 is independently described by formula (A-I) or (A-II): wherein Q is selected from the group consisting of: S is selected from the group consisting of: ; R 1 , R 2 , R 3 , are each independently selected from H, OH, halogen, nitrile, nitro, optionally substituted amine, optionally substituted sulfhydryl, optionally substituted carboxyl, optionally substituted C 1 -C 20 alkyl, optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 alkenyl, optionally substituted C 3 -C 20 cycloalkenyl, optionally substituted C 2 -C
  • each R 8 is independently selected from H, optionally substituted C 1 -C 20 alkyl, optionally substituted C 1 -C 20 alkylene, optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 heterocycloalkyl, optionally substituted C 5 -C 15 aryl, and optionally substituted C 2 -C 15 heteroaryl; each R 9 is independently selected from optionally substituted C 1 -C 20 alkylene, optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 heterocycloalkyl, optionally substituted C 5 -C 15 aryl, and optionally substituted C 2 -C 15 heteroaryl; x is 1 or 2; k is 0, 1, 2, 3, 4, or 5; Ar is selected from the group consisting of optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 heterocycloalkyl, optionally substituted C 5 -C
  • each A 1 -L or each A 1 -L-A 2 may be independently selected (e.g., independently selected from any of the A 1 -L or A 1 -L-A 2 structures described herein).
  • n is 1 and each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153), an albumin protein (e.g., an albumin protein having the sequence of any one of SEQ ID NOs: 96-98), an albumin protein-binding peptide, or an Fc-binding peptide.
  • n is 2 and each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153), wherein the Fc domain monomers dimerize to form and Fc domain.
  • Fc domain monomer e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153
  • the Fc domain monomers dimerize to form and Fc domain.
  • x is 2.
  • x is 2.
  • each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153).
  • each A 1 and each A 2 is independently described by any one of formulas (A-Ia)-(A-Ih): wherein each X is independently C or N; or a pharmaceutically acceptable salt thereof.
  • each A 1 and each A 2 is independently described by any one of formulas (A-Ia-i)-(A-Ih-i): or a pharmaceutically acceptable salt thereof.
  • each A 1 and each A 2 is independently described by any one of formulas (A-Ia-ii)-(A-Ih-ii):
  • each A 1 and each A 2 is independently described by any one of formulas (A-Ic-i) and (A-Ic-ii): or a pharmaceutically acceptable salt thereof.
  • each A 1 and each A 2 is independently described by any one of formulas (A-Ii)-(A-Ip): wherein each X is independently C or N; or a pharmaceutically acceptable salt thereof.
  • each A 1 and each A 2 is independently described by any one of formulas (A-Iq)-(A-Ix): or a pharmaceutically acceptable salt thereof.
  • each A 1 and each A 2 is independently described by any one of formulas (A-Iq-i)-(A-Ix-i): or a pharmaceutically acceptable salt thereof.
  • each A 1 and each A 2 is independently described by any one of formulas (A-Iaa)-(A-Ihh): or a pharmaceutically acceptable salt thereof.
  • each A 1 and each A 2 is independently described by any one of formulas (A-Iii)-(A-Ipp): wherein each X is independently C or N; or a pharmaceutically acceptable salt thereof.
  • each A 1 and each A 2 is independently described by any one of formulas (A-Iii-i)-(A-Ipp-i): or a pharmaceutically acceptable salt thereof.
  • R 1 is H.
  • R 2 is H.
  • R 2 is -OCH 3 .
  • R 3 is H.
  • R 4 is H.
  • R 5 is H.
  • R 7 is a carbonyl.
  • X is N.
  • X is C.
  • each A 1 and each A 2 is independently described by any one of formulas (A-IIa)-(A-IId):
  • each A 1 and each A 2 is independently described by any one of formulas (A-IIa-i)-(A-IId-i): wherein U5 is C 1 -C 10 alkyl; or a pharmaceutically acceptable salt thereof.
  • each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153);
  • L in each A 1 -L-A 2 is a linker covalently attached to a sulfur atom of a cysteine or a nitrogen atom of a lysine in E and to each of A 1 and A 2 ;
  • n is 1 or 2 (e.g., when n is 2, the two Fc domain monomers dimerize to form and Fc domain);
  • T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the squiggly line connected to the E indicates that each A 1 -L-A 2 is covalently attached (e.g., by way of a covalent bond or linker) to a sulfur atom of a hinge cysteine in E, or
  • each A 1 -L-A 2 may be independently selected (e.g., independently selected from any of the A 1 -L-A 2 structures described herein).
  • each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153);
  • L in each L-A 1 is a linker covalently attached to a sulfur atom of a cysteine or a nitrogen atom of a lysine in E and to A 1 ;
  • n is 1 or 2 (e.g., when n is 2, the two Fc domain monomers dimerize to form and Fc domain);
  • T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and the squiggly line connected to E indicates that each L-A 1 is covalently attached (e.g., by way of a covalent bond or linker) to the sulfur atom of the hinge cysteine in E, or a pharmaceutically acceptable salt thereof.
  • each A 1 may be independently selected from any structure described by formula (A-I).
  • each E includes an Fc domain monomer.
  • each E includes an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153.
  • the conjugate forms a homodimer including an Fc domain.
  • E homodimerizes with another E to form an Fc domain.
  • each E includes an albumin protein having the sequence of any one of SEQ ID NOs: 96-98.
  • T is 1 and L-A 1 is covalently attached to the sulfur atom corresponding to Cys34 of SEQ ID NO: 96.
  • the disclosure features an intermediate (Int) of Table 1. These intermediates comprise one or more gp120 binders and a linker (e.g., a PEG 2 -PEG 20 linker) and may be used in the synthesis of a conjugate described herein.
  • Intermediates of Table 1 may be conjugated to, for example, an Fc domain or Fc domain monomer, albumin protein, albumin protein-binding peptide, or Fc-binding peptide (e.g., by way of a linker) by any suitable methods known to those of skill in the art, including any of the methods described or exemplified herein.
  • the conjugate (e.g., a conjugate described by any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)) includes E, wherein E is an Fc domain monomer or an Fc domain (e.g., an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, the sequence of any one of SEQ ID NOs: 1-95 and 125-153).
  • E is an Fc domain monomer or an Fc domain (e.g., an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, the sequence of any one of SEQ ID NOs: 1-95 and 125-153).
  • one or more nitrogen atoms of one or more surface exposed lysine residues of E or one or more sulfur atoms of one or more surface exposed cysteines in E is covalently conjugated to a linker (e.g., a PEG 2 -PEG 20 linker).
  • the linker conjugated to E may be functionalized such that it may react to form a covalent bond with any of the Ints described herein (e.g., an Int of Table 1).
  • E is conjugated to a linker functionalized with an azido group and the Int (e.g., an Int of Table 1) is functionalized with an alkyne group.
  • Conjugation (e.g., by click chemistry) of the linker-azido of E and linker-alkyne of the Int forms a conjugate of the invention, for example a conjugate described by any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII).
  • E is conjugated to a linker functionalized with an alkyne group and the Int (e.g., an Int of Table 1) is functionalized with an azido group.
  • Conjugation e.g., by click chemistry
  • the linker-alkyne of E and the linker-azido of the Int forms a conjugate of the invention, for example a conjugate described by any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII).
  • the Int e.g., an Int of Table 1
  • a phenyl ester group e.g., a trifluorophenyl ester group or a tetrafluorophenyl ester group.
  • Conjugation e.g., by acylation
  • linker-phenyl ester e.g., trifluorophenyl ester or tetrafluorophenyl ester
  • the linker-phenyl ester e.g., trifluorophenyl ester or tetrafluorophenyl ester
  • M-I M-XVII
  • the disclosure further features a composition (e.g., a pre-conjugation intermediate) having the structure of any one of Int-1 to Int-190 (e.g., Int-1 to Int-93, Int-1 to Int-27, Int-28 to Int-93, Int-94 to Int- 141, Int-142 to Int-190 or Int-28 to Int-190).
  • a composition e.g., a pre-conjugation intermediate
  • the disclosure features a composition (e.g., a pre-conjugation intermediate) having the structure of any one of Int-28 to Int-190 (e.g., Int-28 to Int-93, Int-94 to Int-141, Int-142 to Int-190 or Int-28 to Int-190).
  • the disclosure provides a method of making an antiviral-Fc conjugate by conjugating (e.g., via a linker) any one of Int-1 to Int-190 (e.g., Int-1 to Int-93, Int-1 to Int-27, Int-28 to Int-93, Int-94 to Int-141, Int- 142 to Int-190, or Int-28 to Int-190) to an Fc domain monomer or an Fc domain.
  • Int-1 to Int-190 e.g., Int-1 to Int-93, Int-1 to Int-27, Int-28 to Int-93, Int-94 to Int-141, Int- 142 to Int-190, or Int-28 to Int-190
  • the disclosure features a conjugate, wherein the conjugate comprises a small molecule targeting agent, wherein the targeting agent is described by any one of Int-1 to Int-190 (e.g., Int-1 to Int-93, Int-1 to Int-27, Int-28 to Int-93, Int-94 to Int-141, Int-142 to Int-190, or Int-28 to Int-190), which is conjugated to an Fc (e.g., via a linker).
  • Table 1 Intermediates
  • the disclosure features a conjugate of Table 2.
  • Each conjugate of Table 2 corresponds to a conjugate of either formula (M-I) or formula (D-I), as indicated.
  • Conjugates of Table 2 include conjugates formed by the covalent reaction of an Int of Table 1 with a linker which is in turn conjugated to E (e.g., an Fc domain monomer, an albumin protein, an albumin protein-binding peptide, or an Fc-binding peptide).
  • E e.g., an Fc domain monomer, an albumin protein, an albumin protein-binding peptide, or an Fc-binding peptide.
  • the reactive moiety of the Int (e.g., the alkyne or azido group) reacts with a corresponding reactive group (e.g., an alkyne or azido) of a linker (represented by L’) covalently attached to E, such that an Int of Table 1 is covalently attached to E.
  • a corresponding reactive group e.g., an alkyne or azido
  • L linker
  • the reactive moiety of the Int (e.g., the phenyl ester group, e.g., tetrafluorophenyl ester or trifluorophenyl ester group) reacts with a corresponding reactive group (e.g., a nitrogen or sulfur atom) of an amino acid side chain of E, such that an Int of Table 1 is covalently attached to E.
  • a corresponding reactive group e.g., a nitrogen or sulfur atom
  • L’ corresponds to the remainder of L as defined in (M-I) or (D-I) (e.g., L’ is a linker that covalently joins the Int and E).
  • L’ may include a triazole (formed by the click chemistry reaction between the Int and a linker conjugated to E) and a linker (e.g., a PEG 2 -PEG 20 linker) which in turn is conjugated to an amino acid side chain of E.
  • n is 1 or 2.
  • each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153), an albumin protein (e.g., an albumin protein having the sequence of any one of SEQ ID NOs: 96-98), an albumin protein-binding peptide, or an Fc-binding peptide.
  • each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153), and the Fc domain monomers dimerize to form and Fc domain.
  • T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • the disclosure also provides a population of any of the conjugates of Table 2 wherein the average value of T is 1 to 20 (e.g., the average value of T is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, or 15 to 20).
  • the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the average T is 1 to 10 (e.g., 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10).
  • the average T is 1 to 5 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5).
  • the average T is 5 to 10 (e.g., 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10).
  • the average T is 2.5 to 7.5 (e.g., 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, or 7.5).
  • the squiggly line in the conjugates of Table 2 indicates that each L’-Int is covalently attached to an amino acid side chain in E (e.g., the nitrogen atom of a surface exposed lysine or the sulfur atom of a surface exposed cysteine in E), or a pharmaceutically acceptable salt thereof.
  • E amino acid side chain
  • the disclosure further features a conjugate of Table 2, wherein the conjugate is produced by conjugation (e.g., via a linker) of any one of Int-1 to Int-190 to an Fc domain or an Fc domain monomer.
  • Table 2 Conjugates Corresponding to Intermediates of Table 1
  • each E includes an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153.
  • the disclosure features a conjugate including (i) a first moiety, A 1 ; (ii) a second moiety, A 2 ; (iii) an Fc domain monomer or an Fc domain; and (iv) a linker covalently attached to A 1 and A 2 , and to the Fc domain monomer or the Fc domain; wherein each A 1 and each A 2 is independently selected from any structure described by formula (A-I) or (A-II).
  • x is 2.
  • the disclosure features a conjugate including (i) a first moiety, Int; (ii) an Fc domain monomer or an Fc domain; and (iv) a linker covalently attached to Int, and to the Fc domain monomer or the Fc domain; wherein each Int is independently selected from any one of the intermediates of Table 1.
  • the disclosure features a conjugate including (i) a first moiety, A 1 ; (ii) a second moiety, A 2 ; (iii) an albumin protein, an albumin protein-binding peptide, or an Fc-binding peptide; and (iv) a linker covalently attached to A 1 and A 2 , and to the Fc domain monomer or the Fc domain; wherein each A 1 and each A 2 is independently selected from any structure described by formula (A-I) or (A-II). In a preferred embodiment of the above, x is 2.
  • each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153), an albumin protein (e.g., an albumin protein having the sequence of any one of SEQ ID NOs: 96-98), an albumin protein-binding peptide, or an Fc-binding peptide;
  • n is 1 or 2;
  • T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (e.g., T is the total number of A 1 -L-A 2 moieties conjugated to (E)n); and L is a linker covalently attached to each of E, A 1 , and A 2 , or a pharmaceutically acceptable salt thereof.
  • each A 1 -L-A 2 may be independently selected (e.g., independently selected from any of the A 1 -L-A 2 structures described herein).
  • x is 2.
  • the conjugate is described by formulas (e.g., (D-I)-(D-XVII)) found in WO 2020/252393.
  • the conjugate is described by formula (D-IV-7): or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (D-IV-8): or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (D-IV-9): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (D-V-7): or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (D-V-8): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (D-V-9): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (D-VI-8): wherein L’ is the remainder of L, or a pharmaceutically acceptable salt thereof.
  • L or L’ includes one or more optionally substituted C 1 -C 20 alkylene, optionally substituted C 1 -C 20 heteroalkylene, optionally substituted C 2 -C 20 alkenylene, optionally substituted C 2 -C 20 heteroalkenylene, optionally substituted C 2 -C 20 alkynylene, optionally substituted C 2 -C 20 heteroalkynylene, optionally substituted C 3 -C 20 cycloalkylene, optionally substituted C 2 -C 20 heterocycloalkylene, optionally substituted C 4 -C 20 cycloalkenylene, optionally substituted C 4 -C 20 heterocycloalkenylene, optionally substituted C 8 -C 20 cycloalkynylene, optionally substituted C 8 -C 20 heterocycloalkynylene, optionally substituted C 5 -C 15 arylene, optionally substituted C 3 -C 15 heteroarylene, O, S,
  • the backbone of L or L’ consists of one or more optionally substituted C 1 -C 20 alkylene, optionally substituted C 1 -C 20 heteroalkylene, optionally substituted C 2 -C 20 alkenylene, optionally substituted C 2 -C 20 heteroalkenylene, optionally substituted C 2 - C 20 alkynylene, optionally substituted C 2 -C 20 heteroalkynylene, optionally substituted C 3 -C 20 cycloalkylene, optionally substituted C 2 -C 20 heterocycloalkylene, optionally substituted C 4 -C 20 cycloalkenylene, optionally substituted C 4 -C 20 heterocycloalkenylene, optionally substituted C 8 -C 20 cycloalkynylene, optionally substituted C 8 -C 20 heterocycloalkynylene, optionally substituted C 5 -C 15 arylene, optionally substituted C 3 -C 15 heteroarylene
  • L or L’ is oxo substituted.
  • the backbone of L or L’ includes no more than 250 atoms.
  • the backbone of L or L’ includes between 1 and 250 atoms (e.g., between 5 and 250 atoms, between 10 and 250 atoms, between 50 and 250 atoms, between 10 and 200 atoms, between 10 and 100 atoms, between 10 and 50 atoms, between 5 and 100 atoms, between 5 and 50 atoms, between 5 and 30 atoms).
  • L or L’ is capable of forming an amide, a carbamate, a sulfonyl, or a urea linkage.
  • L or L’ is a bond. In some embodiments, L or L’ is an atom. In some embodiments of any of the aspects described herein, each L is described by formula (D- L-I): wherein L A is described by formula G A1 -(Z A1 )g1-(Y A1 )h1-(Z A2 )i1-(Y A2 )j1-(Z A3 )k1-(Y A3 )l1-(Z A4 )m1-(Y A4 )n1-(Z A5 )o1- G A2 ; L B is described by formula G B1 -(Z B1 )g2-(Y B1 )h2-(Z B2 )i2-(Y B2 )j2-(Z B3 )k2-(Y B3 )l2-(Z B4 )m2-(Y B4 )n2-(Z B5 )o2-G B2 ; L C is described by formula G C1 -(Z C
  • optionally substituted includes substitution with a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • a PEG has a repeating unit structure (-CH 2 CH 2 O-)n, wherein n is an integer from 2 to 100.
  • a polyethylene glycol may be selected any one of PEG 2 to PEG 100 (e.g., PEG 2 , PEG 3 , PEG 4 , PEG 5 , PEG 5 -PEG 10 , PEG 10 -PEG 20 , PEG 20 - PEG 30 , PEG 30 -PEG 40 , PEG 50 -PEG 60 , PEG 60 -PEG 70 , PEG 70 -PEG 80 , PEG 80 - PEG 90 , PEG 90 -PEG 100 ).
  • L C may have two points of attachment to the Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide (e.g., two G C2 ).
  • L includes a polyethylene glycol (PEG) linker.
  • a PEG linker includes a linker having the repeating unit structure (-CH 2 CH 2 O-)n, wherein n is an integer from 2 to 100.
  • a polyethylene glycol linker may covalently join a gp120 binder and E (e.g., in a conjugate of any one of formulas (M-I)-(M-XVII)).
  • a polyethylene glycol linker may covalently join a first gp120 binder and a second gp120 binder (e.g., in a conjugate of any one of formulas (D-I)-(D-XVII)).
  • a polyethylene glycol linker may covalently join a gp120 binder dimer and E (e.g., in a conjugate of any one of formulas (D-I)-(D-XVII)).
  • a polyethylene glycol linker may be selected from any one of PEG 2 to PEG 100 (e.g., PEG 2 , PEG 3 , PEG 4 , PEG 5 , PEG 5 -PEG 10 , PEG 10 -PEG 20 , PEG 20 -PEG 30 , PEG 30 -PEG 40 , PEG 50 -PEG 60 , PEG 60 -PEG 70 , PEG 70 -PEG 80 , PEG 80 -PEG 90 , PEG 90 -PEG 100 ).
  • L c includes a PEG linker, where L C is covalently attached to each of Q i and E.
  • each A 1 is independently described by formula (A-I); each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153), an albumin protein (e.g., an albumin protein having the sequence of any one of SEQ ID NOs: 96-98), an albumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2; T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (e.g., T is the total number of A 1 -L moieties conjugated to (E)n); and L is a linker covalently attached to each of E and A 1 , or a pharmaceutically acceptable salt thereof.
  • Fc domain monomer e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153
  • each A 1 may be independently selected from any structure described by formula (A-I).
  • x is 2.
  • the conjugate is described by formula (M-II): wherein X is C, O, or N, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-III): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-III-1): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-III-2): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-III-3): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-III-4): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-III-5): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-III-6): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IV): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IV-1): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IV-2): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IV-3): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IV-4): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IV-5): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IV-6): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IV-7): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IV-8): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IV-9): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-V): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-V-1): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-V-2): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-V-3): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-V-4): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-V-5): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-V-6): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-V-7): or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (M-V-8): or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (M-V-9): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (M-VI): or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (M-VI-1): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-VI-2): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-VI-3): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-VI-4): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-VI-5): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-VI-6): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-VI-7): wherein L’ is the remainder of L, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-VI-8): wherein L’ is the remainder of L, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-VII): wherein X is C, O, or N, or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (M-VIII): or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (M-VIII-1): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (M-IX): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-IX-1): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-X): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-X-1): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XI): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XI-1): wherein L’ is the remainder of L, and y 1 is an integer from 1-20 (e.g., y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XII): wherein X is C, O, or N, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XII-1): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XII-2): or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (M-XIII): or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (M-XIII-1): or a pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is described by formula (M-XIII-2): or a pharmaceutically acceptable salt thereof.
  • the disclosure features a conjugate described by formula (M-I): wherein each A 1 is independently described by formula (A-II); each E comprises an Fc domain monomer; the squiggly line connected to the E indicates that each A 1 -L-A 2 is covalently attached to E, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XIV): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XIV-1): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XIV-2): wherein L’ is the remainder of L and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XIV-3): wherein L’ is the remainder of L; e 1 is an integer from 1-10; and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XIV-4): wherein L’ is the remainder of L; e 1 and e 3 are each independently an integer from 1-10; and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XIV-5): wherein L’ is the remainder of L; e 1 and e 3 are each independently an integer from 1-10; and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XV): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XV-1): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XV-2): wherein L’ is the remainder of L and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XV-3): wherein L’ is the remainder of L; e 1 is an integer from 1-10; and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XV-4): wherein L’ is the remainder of L; e 1 and e 3 are each independently an integer from 1-10; and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XV-5): wherein L’ is the remainder of L; e 1 and e 3 are each independently an integer from 1-10; and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVI): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVI-1): wherein U5 is C 1 -C 10 alkyl, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVI-2): wherein L’ is the remainder of L and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVI-3): wherein L’ is the remainder of L; e 1 is an integer from 1-10; and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVI-4): wherein L’ is the remainder of L; e 1 and e 3 are each independently an integer from 1-10; y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVI-5): wherein L’ is the remainder of L; e 1 and e 3 are each independently an integer from 1-10; y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVII): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVII-1): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVII-2): wherein L’ is the remainder of L and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVII-3): wherein L’ is the remainder of L; e 1 is an integer from 1-10; and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVII-4): wherein L’ is the remainder of L; e 1 and e 3 are each independently an integer from 1-10; and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-XVII-5): wherein L’ is the remainder of L; e 1 and e 3 are each independently an integer from 1-10; and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • L or L’ includes one or more optionally substituted C 1 -C 20 alkylene, optionally substituted C 1 -C 20 heteroalkylene, optionally substituted C 2 -C 20 alkenylene, optionally substituted C 2 -C 20 heteroalkenylene, optionally substituted C 2 -C 20 alkynylene, optionally substituted C 2 -C 20 heteroalkynylene, optionally substituted C 3 -C 20 cycloalkylene, optionally substituted C 2 -C 20 heterocycloalkylene, optionally substituted C 4 -C 20 cycloalkenylene, optionally substituted C 4 -C 20 heterocycloalkenylene, optionally substituted C 8 -C 20 cycloalkynylene, optionally substituted C 8 -C 20 heterocycloalkynylene, optionally substituted C 5 -C 15 arylene, optionally substituted C 3 -C 15 heteroarylene, O, S,
  • the backbone of L or L’ consists of one or more optionally substituted C 1 -C 20 alkylene, optionally substituted C 1 -C 20 heteroalkylene, optionally substituted C 2 -C 20 alkenylene, optionally substituted C 2 -C 20 heteroalkenylene, optionally substituted C 2 - C 20 alkynylene, optionally substituted C 2 -C 20 heteroalkynylene, optionally substituted C 3 -C 20 cycloalkylene, optionally substituted C 2 -C 20 heterocycloalkylene, optionally substituted C 4 -C 20 cycloalkenylene, optionally substituted C 4 -C 20 heterocycloalkenylene, optionally substituted C 8 -C 20 cycloalkynylene, optionally substituted C 8 -C 20 heterocycloalkynylene, optionally substituted C 5 -C 15 arylene, optionally substituted C 3 -C 15 heteroarylene
  • L or L’ is oxo substituted.
  • the backbone of L or L’ includes no more than 250 atoms.
  • the backbone of L or L’ includes between 1 and 250 atoms (e.g., between 5 and 250 atoms, between 10 and 250 atoms, between 50 and 250 atoms, between 10 and 200 atoms, between 10 and 100 atoms, between 10 and 50 atoms, between 5 and 100 atoms, between 5 and 50 atoms, between 5 and 30 atoms).
  • L or L’ is capable of forming an amide, a carbamate, a sulfonyl, or a urea linkage.
  • L or L’ is a bond. In some embodiments, L or L’ is an atom. In some embodiments, L’ is a nitrogen atom. In some embodiments, each L is described by formula (M-L): J 1 -(Q 1 )g-(T 1 )h-(Q 2 )i-(T 2 )j-(Q 3 )k-(T 3 )l-(Q 4 )m-(T 4 )n-(Q 5 )o-J 2 wherein J 1 is a bond attached to A 1 ; J 2 is a bond attached to E or a functional group capable of reacting with a functional group conjugated to E (e.g., maleimide and cysteine, amine and activated carboxylic acid (e.g., carboxylic acid activated by tetrafluorophenol or trifluorophenol), thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyn
  • each of g, h, i, j, k, l, m, n, and o is, independently, 0 or 1.
  • each L is described by formula (M-L-I): J 1 -(Q 1 )g-(T 1 )h-(Q 2 )i-(T 2 )j-(Q 3 )k-(T 3 )l-(Q 4 )m-(T 4 )n-(Q 5 )o-J 2 wherein: J 1 is a bond attached to A 1 ; J 2 is a bond attached to E or a functional group capable of reacting with a functional group conjugated to E (e.g., maleimide and cysteine, amine and activated carboxylic acid (e.g., carboxylic acid activated by tetrafluorophenol or trifluorophenol), thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and
  • optionally substituted includes substitution with a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • a PEG has a repeating unit structure (-CH 2 CH 2 O-)n, wherein n is an integer from 2 to 100.
  • a polyethylene glycol may be selected from any one of PEG 2 to PEG 100 (e.g., PEG 2 , PEG 3 , PEG 4 , PEG 5 , PEG 5 -PEG 10 , PEG 10 -PEG 20 , PEG 20 -PEG 30 , PEG 30 -PEG 40 , PEG 50 -PEG 60 , PEG 60 -PEG 70 , PEG 70 -PEG 80 , PEG 80 -PEG 90 , PEG 90 -PEG 100 ).
  • J 2 may have two points of attachment to the Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide (e.g., two J 2 ).
  • L is , wherein d is an integer from 1 to 20 (e.g., d is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, L is , , , ,
  • g is 0 or 1; h is 0; i is 0 or 1; j is 0 or 1; k is 0 or 1; l is 0; m is 0 or 1; n is 0; and o is 0 or 1.
  • g is 1; h is 0; i is 1; j is 0; k is 1; l is 0; m is 1; n is 0; and o is 1.
  • g is 0; h is 0; i is 1; j is 0; k is 1; l is 0; m is 1; n is 0; and o is 1.
  • g is 1; h is 0; i is 0; j is 0; k is 1; l is 0; m is 1; n is 0; and o is 1.
  • g is 0; h is 0; i is 1; j is 1; k is 1; l is 0; m is 1; n is 0; and o is 1.
  • L is a linker described by formula (M-L-Ia): J 1 -Q 1 -Q 2 -Q 3 -Q 4 -Q 5 -J 2 .
  • L is a linker described by formula (M-L-Ib): J 1 -Q 2 -Q 3 -Q 4 -Q 5 -J 2 .
  • L is a linker described by formula (M-L-Ic): J 1 -Q 1 -Q 3 -Q 4 -Q 5 -J 2 .
  • L is a linker described by formula (M-L-Id): J 1 -Q 2 -T 2 -Q 3 -Q 4 -Q 5 -J 2 .
  • Q 1 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 1 -C 20 heteroalkylene, optionally substituted C 2 -C 20 alkenylene, optionally substituted C 2 -C 20 heteroalkenylene, optionally substituted C 2 -C 20 alkynylene, or optionally substituted C 2 -C 20 heteroalkynylene.
  • Q 1 is optionally substituted C 1 -C 20 alkylene or optionally substituted C 1 -C 20 heteroalkylene.
  • Q 1 is optionally substituted C 1 -C 20 alkylene.
  • Q 1 is optionally substituted C 1 -C 20 heteroalkylene.
  • Q 2 is optionally substituted C 3 -C 20 cycloalkylene, optionally substituted C 2 - C 20 heterocycloalkylene, optionally substituted C 4 -C 20 cycloalkenylene, optionally substituted C 4 -C 20 heterocycloalkenylene, optionally substituted C 8 -C 20 cycloalkynylene, optionally substituted C 8 -C 20 heterocycloalkynylene, optionally substituted C 5 -C 15 arylene, or optionally substituted C 2 -C 15 heteroarylene.
  • Q 2 is optionally substituted C 3 -C 20 cycloalkylene, optionally substituted C 2 -C 20 heterocycloalkylene, optionally substituted C 4 -C 20 cycloalkenylene, optionally substituted C 4 -C 20 heterocycloalkenylene, optionally substituted C 8 -C 20 cycloalkynylene, or optionally substituted C 8 -C 20 heterocycloalkynylene.
  • Q 2 is optionally substituted C 3 -C 20 cycloalkylene or optionally substituted C 2 -C 20 heterocycloalkylene.
  • Q 2 is optionally substituted C 3 -C 20 cycloalkylene.
  • Q 2 is optionally substituted C 2 -C 20 heterocycloalkylene.
  • Q 2 is a five-membered C 2 -C 4 heterocycloalkylene or six- membered C 2 -C 5 heterocycloalkylene.
  • Q 3 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 1 -C 20 heteroalkylene, optionally substituted C 2 -C 20 alkenylene, optionally substituted C 2 -C 20 heteroalkenylene, optionally substituted C 2 -C 20 alkynylene, or optionally substituted C 2 -C 20 heteroalkynylene.
  • Q 3 is optionally substituted C 1 -C 20 alkylene or optionally substituted C 1 -C 20 heteroalkylene. In some embodiments, Q 3 is optionally substituted C 1 -C 20 alkylene. In some embodiments, Q 3 is optionally substituted C 1 -C 20 heteroalkylene. In some embodiments, Q 3 is , where e4 is an integer from 1 to 10. In some embodiments, Q 4 is optionally substituted C 5 -C 15 arylene or optionally substituted C 2 -C 15 heteroarylene. In some embodiments, Q 4 is optionally substituted C 5 -C 15 arylene. In some embodiments, Q 4 is optionally substituted C 2 -C 15 heteroarylene.
  • Q 4 is a five-membered optionally substituted C 2 -C 4 heteroarylene. In some embodiments, Q 4 is a five-membered C 2 -C 4 heteroarylene. In some embodiments, Q 4 is a five- membered C 2 heteroarylene. In some embodiments, Q 5 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 1 -C 20 heteroalkylene, optionally substituted C 2 -C 20 alkenylene, optionally substituted C 2 -C 20 heteroalkenylene, optionally substituted C 2 -C 20 alkynylene, or optionally substituted C 2 -C 20 heteroalkynylene.
  • Q 5 is optionally substituted C 1 -C 20 alkylene or optionally substituted C 1 -C 20 heteroalkylene. In some embodiments, Q 5 is optionally substituted C 1 -C 20 alkylene. In some embodiments, Q 5 is optionally substituted C 1 -C 20 heteroalkylene. In some embodiments, Q 5 is , where e5 is an integer from 1 to 8; and e6 is an integer from 1 to 16. In some embodiments of any of the aspects described herein, L includes (e.g., a portion of L that joins A 1 and E includes): , , ,
  • L includes (e.g., a portion of L that joins A 1 and E includes):
  • L is: , ,
  • L includes a polyethylene glycol (PEG) linker.
  • PEG linker includes a linker having the repeating unit structure (-CH 2 CH 2 O-) n , wherein n is an integer from 2 to 100.
  • a polyethylene glycol linker may covalently join a gp120 binder and E (e.g., in a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)).
  • a polyethylene glycol linker may covalently join a first gp120 binder and a second gp120 binder (e.g., in a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)).
  • a polyethylene glycol linker may covalently join a gp120 binder dimer and E (e.g., in a conjugate of any one of formulas).
  • a polyethylene glycol linker may be selected from any one of PEG 2 to PEG 100 (e.g., PEG 2 , PEG 3 , PEG 4 , PEG 5 , PEG 5 -PEG 10 , PEG 10 - PEG 20 , PEG 20 -PEG 30 , PEG 30 -PEG 40 , PEG 50 -PEG 60 , PEG 60 -PEG 70 , PEG 70 -PEG 80 , PEG 80 -PEG 90 , PEG 90 - PEG 100 ).
  • L c includes a PEG linker, where L C is covalently attached to each of Q i and E.
  • L is covalently attached to the nitrogen atom of a surface exposed lysine of E or L is covalently attached to the sulfur atom of a surface exposed cysteine of E.
  • E is an Fc domain monomer.
  • n is 2 and each E dimerizes to form an Fc domain.
  • n 2
  • each E is an Fc domain monomer, each E dimerizes to form an Fc domain, and the conjugate is described by formula (D-I-1): wherein J is an Fc domain; and T is an integer from 1 to 20 (e.g., T is 1 ,2 ,3 ,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • each E is an Fc domain monomer, each E dimerizes to form an Fc domain, and the conjugate is described by formula (M-I-1): wherein J is an Fc domain; and T is an integer from 1 to 20 (e.g., T is 1 ,2 ,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.
  • each E independently includes an amino acid sequence having at least 90% (e.g., at least 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of any one of SEQ ID Nos: 1-95 and 125-153.
  • each E includes the sequence of any one of SEQ ID NOs: 1-95 and 125-153.
  • E is an albumin protein, an albumin protein-binding peptide, or an Fc-binding peptide.
  • n is 1.
  • n is 1, E is an albumin protein, an albumin protein-binding peptide, or an Fc-binding peptide and the conjugate is described by formula (D-I-2): wherein E is an albumin protein, an albumin protein-binding peptide, or Fc-binding peptide; and T is an integer from 1 to 20, or a pharmaceutically acceptable salt thereof.
  • n 1, E is an albumin protein, an albumin protein-binding peptide, or an Fc-binding peptide, and the conjugate is described by formula (M-I-2): wherein E is an albumin protein, an albumin protein-binding peptide, or an Fc-binding peptide; and T is an integer from 1 to 20, or a pharmaceutically acceptable salt thereof.
  • E is an albumin protein having the sequence of any one of SEQ ID NOs: 96-98.
  • T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • the disclosure provides a population of conjugates having the structure of any of the conjugates described herein (e.g., a population of conjugates having the formula of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)), wherein the average value of T is 1 to 20 (e.g., the average value of T is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, or 15 to 20). In some embodiments, the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising any of the conjugates described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M- I)-(M-XVII)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • the disclosure provides a method for the treatment of a subject having a viral infection or presumed to have a viral infection, the method comprising administering to the subject an effective amount of any of the conjugates or compositions described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)).
  • an effective amount of any of the conjugates or compositions described herein e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)).
  • the disclosure provides a method for the prophylactic treatment of a viral infection in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the conjugates or compositions described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)).
  • the viral infection is caused by HIV.
  • the viral infection is HIV-1 or HIV-2.
  • the subject is immunocompromised.
  • the subject has been diagnosed with humoral immune deficiency, T cell deficiency, neutropenia, asplenia, or complement deficiency.
  • the subject is being treated or is about to be treated with an immunosuppressive therapy.
  • the subject has been diagnosed with a disease which causes immunosuppression.
  • the disease is cancer or acquired immunodeficiency syndrome.
  • the cancer is leukemia, lymphoma, or multiple myeloma.
  • the subject has undergone or is about to undergo hematopoietic stem cell transplantation. In some embodiments, wherein the subject has undergone or is about to undergo an organ transplant.
  • the conjugate of composition is administered intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, by inhalation, by injection, or by infusion.
  • the subject is treated with a second therapeutic agent.
  • the second therapeutic agent is an antiviral agent.
  • the second therapeutic agent is a viral vaccine.
  • the viral vaccine elicits an immune response in the subject against HIV (e.g., HIV-1 or HIV-2).
  • HIV e.g., HIV-1 or HIV-2.
  • an Fc-domain-containing composition may be substituted for an Fc domain and an Fc-domain-monomer-containing composition may be substituted for an Fc domain monomer in any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII), when n is 1, E is an Fc- domain-monomer-containing composition.
  • E is an Fc-domain-containing composition.
  • the Fc-domain-containing composition is an antibody or an antibody fragment.
  • An antibody may include any form of immunoglobulin, heavy chain antibody, light chain antibody, LRR-based antibody, or other protein scaffold with antibody-like properties, as well as any other immunological binding moiety known in the art, including antibody fragments (e.g., a Fab, Fab', Fab’2, F(ab')2, Fd, Fv, Feb, scFv, or SMIP).
  • An antibody fragment may include a binding moiety that includes a portion derived from or having significant homology to an antibody, such as the antigen- determining region of an antibody.
  • Exemplary antibody fragments include Fab, Fab', Fab’2, F(ab')2, Fd, Fv, Feb, scFv, and SMIP.
  • the antibody or antibody fragment is a human, mouse, camelid (e.g., llama, alpaca, or camel), goat, sheep, rabbit, chicken, guinea pig, hamster, horse, or rat antibody or antibody fragment.
  • the antibody is an IgG, IgA, IgD, IgE, IgM, or intrabody.
  • the antibody fragment includes an scFv, sdAb, dAb, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, or SMIP.
  • the Fc-domain-containing composition e.g., an antibody or antibody fragment
  • confers binding specificity to a one or more targets e.g., an antigen, such as an antigen associated with HIV.
  • HIV-targeting antibodies are known in the art, for example, as described in Wibmer et al. Curr. Opin.
  • the one or more targets (e.g., an antigen) bound by the Fc-domain- containing composition is a viral (e.g., HIV) protein such as gp41 or gp120 receptor.
  • the antibody or antibody fragment recognizes a viral surface antigen.
  • E e.g., each E includes the amino acid sequence of SEQ ID NO: 1.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.
  • E includes the amino acid sequence of SEQ ID NO: 2.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2.
  • E includes the amino acid sequence of SEQ ID NO: 3.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3.
  • E includes the amino acid sequence of SEQ ID NO: 4.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4.
  • E includes the amino acid sequence of SEQ ID NO: 5.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 6.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6.
  • E includes the amino acid sequence of SEQ ID NO: 7.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7.
  • E includes the amino acid sequence of SEQ ID NO: 8.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.
  • E includes the amino acid sequence of SEQ ID NO: 9.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9.
  • E includes the amino acid sequence of SEQ ID NO: 10.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10.
  • E includes the amino acid sequence of SEQ ID NO: 11.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.
  • E includes the amino acid sequence of SEQ ID NO: 12.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12.
  • E includes the amino acid sequence of SEQ ID NO: 13.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13.
  • E includes the amino acid sequence of SEQ ID NO: 14.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14.
  • E includes the amino acid sequence of SEQ ID NO: 15.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15.
  • E includes the amino acid sequence of SEQ ID NO: 16.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.
  • E includes the amino acid sequence of SEQ ID NO: 17.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17.
  • E includes the amino acid sequence of SEQ ID NO: 18.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18.
  • E includes the amino acid sequence of SEQ ID NO: 19.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19.
  • E includes the amino acid sequence of SEQ ID NO: 20.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20.
  • E includes the amino acid sequence of SEQ ID NO: 21.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21.
  • E includes the amino acid sequence of SEQ ID NO: 22.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.
  • E includes the amino acid sequence of SEQ ID NO: 23.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.
  • E includes the amino acid sequence of SEQ ID NO: 24.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24.
  • E includes the amino acid sequence of SEQ ID NO: 25.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.
  • E includes the amino acid sequence of SEQ ID NO: 26.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26.
  • E includes the amino acid sequence of SEQ ID NO: 27.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27.
  • E includes the amino acid sequence of SEQ ID NO: 28.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28.
  • E includes the amino acid sequence of SEQ ID NO: 29.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.
  • E includes the amino acid sequence of SEQ ID NO: 30.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30.
  • E includes the amino acid sequence of SEQ ID NO: 31.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31.
  • E includes the amino acid sequence of SEQ ID NO: 32.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32.
  • E includes the amino acid sequence of SEQ ID NO: 33.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33.
  • E includes the amino acid sequence of SEQ ID NO: 34.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34.
  • E includes the amino acid sequence of SEQ ID NO: 35.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35.
  • E includes the amino acid sequence of SEQ ID NO: 36.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 36.
  • E includes the amino acid sequence of SEQ ID NO: 37.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37.
  • E includes the amino acid sequence of SEQ ID NO: 38.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 38.
  • E includes the amino acid sequence of SEQ ID NO: 39.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39.
  • E includes the amino acid sequence of SEQ ID NO: 40.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40.
  • E includes the amino acid sequence of SEQ ID NO: 41.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41.
  • E includes the amino acid sequence of SEQ ID NO: 42.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42.
  • E includes the amino acid sequence of SEQ ID NO: 43.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43.
  • E includes the amino acid sequence of SEQ ID NO: 44.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45.
  • E includes the amino acid sequence of SEQ ID NO: 46.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46.
  • E includes the amino acid sequence of SEQ ID NO: 47.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47.
  • E includes the amino acid sequence of SEQ ID NO: 48.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48.
  • E includes the amino acid sequence of SEQ ID NO: 49.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49.
  • E includes the amino acid sequence of SEQ ID NO: 50.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50.
  • E includes the amino acid sequence of SEQ ID NO: 51.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51.
  • E includes the amino acid sequence of SEQ ID NO: 52.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52.
  • E includes the amino acid sequence of SEQ ID NO: 53.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53.
  • E includes the amino acid sequence of SEQ ID NO: 54.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54.
  • E includes the amino acid sequence of SEQ ID NO: 55.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55.
  • E includes the amino acid sequence of SEQ ID NO: 56.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 57.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 57.
  • E includes the amino acid sequence of SEQ ID NO: 58.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 58.
  • E includes the amino acid sequence of SEQ ID NO: 59.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 59.
  • E includes the amino acid sequence of SEQ ID NO: 60.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60.
  • E includes the amino acid sequence of SEQ ID NO: 61.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 61.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 62.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62.
  • E includes the amino acid sequence of SEQ ID NO: 63. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments of any of the aspects described herein, E (e.g., each E) includes the amino acid sequence of SEQ ID NO: 64. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64.
  • E includes the amino acid sequence of SEQ ID NO: 65. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65. In some embodiments of any of the aspects described herein, E (e.g., each E) includes the amino acid sequence of SEQ ID NO: 66. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 66.
  • E includes the amino acid sequence of SEQ ID NO: 67.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67.
  • E includes the amino acid sequence of SEQ ID NO: 68.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 68.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 69.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 69.
  • E includes the amino acid sequence of SEQ ID NO: 70. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 70. In some embodiments of any of the aspects described herein, E (e.g., each E) includes the amino acid sequence of SEQ ID NO: 71. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 71.
  • E includes the amino acid sequence of SEQ ID NO: 72. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments of any of the aspects described herein, E (e.g., each E) includes the amino acid sequence of SEQ ID NO: 73. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73.
  • E includes the amino acid sequence of SEQ ID NO: 74. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 74. In some embodiments of any of the aspects described herein, E (e.g., each E) includes the amino acid sequence of SEQ ID NO: 75. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 75.
  • E includes the amino acid sequence of SEQ ID NO: 76.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 76.
  • E includes the amino acid sequence of SEQ ID NO: 77.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 77.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 78.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 78.
  • E includes the amino acid sequence of SEQ ID NO: 79. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 79. In some embodiments of any of the aspects described herein, E (e.g., each E) includes the amino acid sequence of SEQ ID NO: 80. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 80.
  • E includes the amino acid sequence of SEQ ID NO: 81.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 81.
  • E includes the amino acid sequence of SEQ ID NO: 82.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 82.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 83.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 83.
  • E includes the amino acid sequence of SEQ ID NO: 84. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 84. In some embodiments of any of the aspects described herein, E (e.g., each E) includes the amino acid sequence of SEQ ID NO: 85. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 85.
  • E includes the amino acid sequence of SEQ ID NO: 86.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 86.
  • E includes the amino acid sequence of SEQ ID NO: 87.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 87.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 88.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 88.
  • E includes the amino acid sequence of SEQ ID NO: 89. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments of any of the aspects described herein, E (e.g., each E) includes the amino acid sequence of SEQ ID NO: 90. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 90.
  • E includes the amino acid sequence of SEQ ID NO: 91.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 91.
  • E includes the amino acid sequence of SEQ ID NO: 92.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 92.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 93.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 93.
  • E includes the amino acid sequence of SEQ ID NO: 94. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 94. In some embodiments of any of the aspects described herein, E (e.g., each E) includes the amino acid sequence of SEQ ID NO: 95. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 95.
  • E includes the amino acid sequence of SEQ ID NO: 96.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96.
  • E includes the amino acid sequence of SEQ ID NO: 97.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 97.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 98.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 98.
  • E includes the amino acid sequence of SEQ ID NO: 125.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125.
  • E includes the amino acid sequence of SEQ ID NO: 126.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 127.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 127.
  • E includes the amino acid sequence of SEQ ID NO: 128.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 128.
  • E includes the amino acid sequence of SEQ ID NO: 129.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 129.
  • E includes the amino acid sequence of SEQ ID NO: 130.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 130.
  • E includes the amino acid sequence of SEQ ID NO: 131.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 131.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 132.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 132.
  • E includes the amino acid sequence of SEQ ID NO: 133.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 133.
  • E includes the amino acid sequence of SEQ ID NO: 134.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 134.
  • E includes the amino acid sequence of SEQ ID NO: 135.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 135.
  • E includes the amino acid sequence of SEQ ID NO: 136.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 136.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 137.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137.
  • E includes the amino acid sequence of SEQ ID NO: 138.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 138.
  • E includes the amino acid sequence of SEQ ID NO: 139.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 139.
  • E includes the amino acid sequence of SEQ ID NO: 140.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 140.
  • E includes the amino acid sequence of SEQ ID NO: 141.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 141.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 142.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 142.
  • E includes the amino acid sequence of SEQ ID NO: 143.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 143.
  • E includes the amino acid sequence of SEQ ID NO: 144.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 144.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 145.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 145.
  • E includes the amino acid sequence of SEQ ID NO: 146.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 146.
  • E includes the amino acid sequence of SEQ ID NO: 147.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 147.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 148.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 148.
  • E includes the amino acid sequence of SEQ ID NO: 149. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 149. In some embodiments of any of the aspects described herein, E (e.g., each E) includes the amino acid sequence of SEQ ID NO: 150. In some embodiments, E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 150.
  • E includes the amino acid sequence of SEQ ID NO: 151.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 151.
  • E includes the amino acid sequence of SEQ ID NO: 152.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 152.
  • E e.g., each E
  • E includes the amino acid sequence of SEQ ID NO: 153.
  • E includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 153.
  • the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153) includes a triple mutation corresponding to M252Y/S254T/T256E (YTE).
  • YTE M252Y/S254T/T256E
  • an amino acid “corresponding to” a particular amino acid residue should be understood to include any amino acid residue that one of skill in the art would understand to align to the particular residue (e.g., of the particular sequence).
  • any one of SEQ ID NOs: 1-95 and 125-153 may be mutated to include a YTE mutation.
  • the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153) includes a double mutant corresponding to M428L/N434S (LS).
  • an amino acid “corresponding to” a particular amino acid residue e.g., or a particular SEQ ID NO.
  • any one of SEQ ID NOs: 1-95 and 125-153 may be mutated to include a LS mutation.
  • the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153) includes a mutant corresponding to N434H.
  • an amino acid “corresponding to” a particular amino acid residue e.g., of a particular SEQ ID NO.
  • any one of SEQ ID NOs: 1-95 and 125- 153 may be mutated to include an N434H mutation.
  • the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153) includes a mutant corresponding to C220S.
  • an amino acid “corresponding to” a particular amino acid residue e.g., or a particular SEQ ID NO.
  • any one of SEQ ID NOs: 1-95 and 125- 153 may be mutated to include a C220S mutation.
  • the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-138 or 168-197) includes a mutant corresponding to K246X, where X is not Lys (e.g., K246X is K246S, K246G, K246A, K246T, K246N, K246Q, K246R, K246H, K246E, or K246D).
  • X is not Lys
  • an amino acid “corresponding to” a particular amino acid residue should be understood to include any amino acid residue that one of skill in the art would understand to align to the particular residue (e.g., of the particular sequence).
  • any one of SEQ ID NOs: 1-95 or 125-153 may be mutated to at position 246 such that position 246 is not a Lys.
  • any one of SEQ ID NOs: 1-95 or 125-153 may be mutated to include a K246S mutation.
  • the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 and 125-153) is a fragment of the Fc domain monomer (e.g., a fragment of at least 25 (e.g., 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 or more), at least 50 (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or more), at least 75 (e.g., 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
  • the Fc domain monomer is a fragment of the Fc domain monomer (e.g
  • one or more nitrogen atoms of one or more surface exposed lysine residues of E or one or more sulfur atoms of one or more surface exposed cysteines in E is covalently conjugated to a linker (e.g., a PEG 2 -PEG 20 linker).
  • the linker conjugated to E may be functionalized such that it may react to form a covalent bond with the L of any A 1 -L or any A 2 -L-A 1 described herein.
  • E is conjugated to a linker functionalized with an azido group and the L of A 1 -L or any A 2 -L-A 1 is functionalized with an alkyne group.
  • Conjugation e.g., by click chemistry
  • the linker-azido of E and the linker-alkyne of A 1 -L or A 2 -L-A 1 forms a conjugate of the invention, for example a conjugate described by any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)- (M-XVII).
  • E is conjugated to a linker functionalized with an alkyne group and L of an A 1 -L or of any A 2 -L-A 1 is functionalized with an azido group.
  • Conjugation e.g., by click chemistry
  • linker-alkyne of E and linker-azido of A 1 -L or of any A 2 -L-A 1 forms a conjugate of the invention, for example a conjugate described by any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII).
  • the squiggly line of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII) represents a covalent bond between the L of A 1 -L or A 2 -L-A 1 or L’ of A 1 -L’ or A 1 -L’-A 2 .
  • the squiggly line of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII) represents that one or more amino acid side chains of E (e.g., one or more nitrogen atoms of one or more surface exposed lysine residues of E or one or more sulfur atoms of one or more surface exposed cysteines in E) have been conjugated to a linker (e.g., a PEG 2 -PEG 20 linker) wherein the linker has been functionalized with a reactive moiety, such that the reactive moiety forms a covalent bond with the L of any A 1 -L or any A 2 -L-A 1 described herein (e.g., by click chemistry between an azido functionalized linker and an alkyne functionalized linker, as described above).
  • a linker e.g., a PEG 2 -PEG 20 linker
  • a 1 and/or A 2 have the structure described by (A-I): In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: .
  • a 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: .
  • a 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: .
  • a 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: . In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by: .
  • a 1 and/or A 2 have the structure described by (A-II): In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by (A-IIaa): In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by (A-IIbb): In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by (A-IIcc): In some embodiments of any of the aspects described herein, A 1 and/or A 2 have the structure described by (A-IIdd): In another aspect, the disclosure features a conjugate selected from any one of conjugates 1-76 (e.g., Conjugates 1-4, 5a, 5b, 6-8, 9a, 9b, 10, 11, 12a, 12b, 13a, 13b, 14a, 14b, 15-28, 29a, 29b, 30a, 30b, 31-36
  • the disclosure features a conjugate selected from any one of conjugates 1-48 (e.g., Conjugates 1-4, 5a, 5b, 6-8, 9a, 9b, 10, 11, 12a, 12b, 13a, 13b, 14a, 14b, 15-28, 29a, 29b, 30a, 30b, 31-36, 37a, 37b, and 38-48).
  • the conjugate is selected from any one of conjugates 5b, 9b, 12b, 13b, 14b, and 15-48 (e.g., Conjugates 15-28, 29a, 29b, and 30-48).
  • the conjugate is selected from any one of conjugates 5b, 29a, 29b, 30a, and 37a.
  • the conjugate is conjugate 1, or any regioisomer thereof, and the drug-to- antibody ratio (DAR) (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 2, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 3, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 4, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 5a, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 6, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 7, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 8, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 9a, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 10, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 11, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 12a, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 13a, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 14a, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 5b, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 9b, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 12b, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 13b, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 14b, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 29b, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 15, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 16, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 17, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 18, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 19, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 20, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 21, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 22, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 23, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 24, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 25, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 26, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 27, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 28, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 29a, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 29b, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 30a, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 31, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 32, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 33, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 34, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 35, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 36, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 37a, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 38, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 39, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 40, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 41, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 42, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 43, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 44, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 45, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 46, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 47, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 48, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 30b, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 37b, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 49, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 50, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 51, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 52, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 53, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 54, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 55, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 56, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 57, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 58, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 59, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 60, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 61, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 62, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 63, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 64, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 65, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 66, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 67, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 68, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 69, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 70, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 71, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 72, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 73, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 74, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 75, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 76, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 77, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 78, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 79, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 80, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 81, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 82, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 83, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 84, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 85, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 86, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 87, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 88, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 89, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 90, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 91, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 92, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 93, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 94, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 95, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 96, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 97, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 98, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 99, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 100, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 101, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 102, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 103, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 104, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 105, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 106, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 107, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 108, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 109, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 110, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 111, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 112, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 113, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 114, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 115, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 116, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 117, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 118, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 119, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 120, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • the conjugate is conjugate 121, or any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.
  • a population of conjugates described herein has a DAR (e.g., T) of between 1 and 2, 2 and 4, 4 and 6, 6 and 8, 8 and 10, 1 and 10, 1 and 20, 1 and 5, 3 and 7, 5 and 10, or 10 and 20.
  • DAR e.g., T
  • the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.
  • the Fc domain monomer includes less than about 300 amino acid residues (e.g., less than about 300, less than about 295, less than about 290, less than about 285, less than about 280, less than about 275, less than about 270, less than about 265, less than about 260, less than about 255, less than about 250, less than about 245, less than about 240, less than about 235, less than about 230, less than about 225, or less than about 220 amino acid residues).
  • the Fc domain monomer is less than about 40 kDa (e.g., less than about 35kDa, less than about 30kDa, less than about 25kDa).
  • the Fc domain monomer includes at least 200 amino acid residues (e.g., at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 amino residues). In some embodiments, the Fc domain monomer is at least 20 kDa (e.g., at least 25 kDa, at least 30 kDa, or at least 35 kDa). In some embodiments, the Fc domain monomer includes 200 to 400 amino acid residues (e.g., 200 to 250, 250 to 300, 300 to 350, 350 to 400, 200 to 300, 250 to 350, or 300 to 400 amino acid residues).
  • the Fc domain monomer is 20 to 40 kDa (e.g., 20 to 25 kDa, 25 to 30 kDa, 35 to 40 kDa, 20 to 30 kDa, 25 to 35 kDa, or 30 to 40 KDa).
  • the Fc domain monomer includes an amino acid sequence at least 90% identical (e.g., at least 95%, at least 98%) to the sequence of any one of SEQ ID NOs: 1-95 and 125-153, or a region thereof.
  • the Fc domain monomer includes the amino acid sequence of any one of SEQ ID NOs: 1-95 and 125-153, or a region thereof.
  • the Fc domain monomer includes a region of any one of SEQ ID NOs: 1- 95 and 125-153, wherein the region includes positions 220, 252, 254, and 256.
  • the region includes at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acids residues, at least 80 amino acids residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 110 amino acid residues, at least 120 amino residues, at least 130 amino acid residues, at least 140 amino acid residues, at least 150 amino acid residues, at least 160 amino acid residues, at least 170 amino acid residues, at least 180 amino acid residues, at least 190 amino acid residues, or at least 200 amino acid residues.
  • the disclosure features a method of synthesizing a conjugate of formula (D-I): wherein each A 1 and each A 2 is independently selected from any one of formulas (A-I)-(A-VI) as described herein; n is 1 or 2; each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 or 125-153), an albumin protein (e.g., an albumin protein having the sequence of any one of SEQ ID NOs: 96-98), an albumin protein-binding peptide, or an Fc-binding peptide; L is a linker covalently attached to E and to Y of each of A 1 and A 2 ; T is an integer from 1 to 20; and each squiggly line in formula (D-I) indicates that L is covalently attached (e.g., by way of a covalent bond or linker) to each E, the method including the steps of: (a) providing an Fc
  • the disclosure features a method of synthesizing a conjugate of formula (M-I): wherein each A 1 is selected from any one of formulas (A-I)-(A-VI) as described herein; n is 1 or 2; each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 or 125-153), an albumin protein (e.g., an albumin protein having the sequence of any one of SEQ ID NOs: 96-98), an albumin protein-binding peptide, or an Fc-binding peptide; L is a linker covalently attached to E and to Y of A 1 ; T is an integer from 1 to 20; and each squiggly line in formula (M-I) indicates that L is covalently attached (e.g., by way of a covalent bond or linker) to each E, the method including the steps of: (a) providing a first composition including E; (b)
  • L’ includes G, wherein G is optionally substituted C 1 -C 6 alkylene, optionally substituted C 1 -C 6 heteroalkylene, optionally substituted C 2 -C 6 alkenylene, optionally substituted C 2 -C 6 heteroalkenylene, optionally substituted C 2 -C 6 alkynylene, optionally substituted C 2 -C 6 heteroalkynylene, optionally substituted C 3 -C 10 cycloalkylene, optionally substituted C 2 -C 10 heterocycloalkylene, optionally substituted C 6 -C 10 arylene, or optionally substituted C 2 -C 10 heteroarylene.
  • a compound of formula (MF-I) or salt thereof has the structure of any one of Int-1 to Int-140 (e.g., Int-56, Int-57, and Int-74). In some embodiments, a compound of formula (MF-I) or salt thereof includes the structure of any one of Int-1 to Int-140 (e.g., Int-56, Int-57, and Int-74).
  • a compound of formula (MF-I) or salt thereof is synthesized from the structure of any one of Int-1 to Int-140 (e.g., Int-56, Int-57, and Int-74).
  • a compound of formula (DF-I) or (MF-I), where each R is halo (e.g., F) provides technical advantages (e.g., increased stability) in methods of synthesizing protein-drug conjugates (e.g., the methods described herein).
  • the increased stability allows for purification by reverse phase chromatography.
  • the increased stability allows for lyophilization with minimal hydrolysis of the activated ester.
  • a compound of formula (DF-I) or (MF-I), where m is 3, provides technical advantages (e.g., increased stability) in methods of synthesizing protein-drug conjugates (e.g., the methods described herein).
  • the increased stability allows for purification by reverse phase chromatography.
  • the increased stability allows for lyophilization with minimal hydrolysis of the activated ester.
  • a compound of formula (DF-I) or (MF-I), where m is 3 and each R is halo (e.g., F) provides technical advantages (e.g., increased stability) in methods of synthesizing protein-drug conjugates (e.g., the methods described herein).
  • the increased stability allows for purification by reverse phase chromatography. In some embodiments, the increased stability allows for lyophilization with minimal hydrolysis of the activated ester.
  • the disclosure features a method of synthesizing a conjugate of formula (D-I): wherein each A 1 and each A 2 is independently selected from any one of formulas (A-I)-(A-VI) as described herein; n is 1 or 2; each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 or 125-153), an albumin protein (e.g., an albumin protein having the sequence of any one of SEQ ID NOs: 96-98), an albumin protein-binding peptide, or an Fc-binding peptide; L is a linker covalently attached to E and to Y of each of A 1 and A 2 ; T is an integer from 1 to 20; and each squiggly line in formula (D-I)
  • G is optionally substituted C 1 -C 6 heteroalkylene or optionally substituted C 2 -C 10 heteroarylene. In some embodiments, G is optionally substituted C 1 -C 6 heteroalkylene. In some embodiments, G is , where R a is H, optionally substituted C 1 - C 20 alkylene (e.g., optionally substituted C 1 -C 6 alkylene), or optionally substituted C 1 -C 20 heteroalkylene (e.g., optionally substituted C 1 -C 6 heteroalkylene). In some embodiments, G is optionally substituted C 2 -C 10 heteroarylene. In some embodiments, G is optionally substituted C 2 -C 5 heteroarylene.
  • G is a 5-membered or 6- membered optionally substituted C 2 -C 5 heteroarylene. In some embodiments, G is a triazolylene.
  • the conjugate of formula (D-I) has the structure of: , and the method includes the steps of: (a) providing a first composition including E; (b) providing a second composition including a compound of formula (DF-II-A) or salt thereof: and (c) combining the first composition, the second composition, and a buffer to form a mixture.
  • the synthesis of compound of formula (DF-II-A) includes: (d) providing a third composition including formula (D-G1-A) or salt thereof: (e) providing a fourth composition including formula (D-G1-B) or salt thereof: and (f) combining the third composition and the fourth composition to form a mixture.
  • the conjugate of formula (D-I) has the structure of: , and the method includes the steps of: (a) providing a first composition including E; (b) providing a second composition including a compound of formula (DF-II-B) or salt thereof: and (c) combining the first composition, the second composition, and a buffer to form a mixture.
  • the synthesis of compound of formula (DF-II-B) includes: (d) providing a third composition including formula (D-G2-A) or salt thereof: (e) providing a fourth composition including formula (D-G2-B) or salt thereof: and (f) combining the third composition and the fourth composition to form a mixture.
  • step (f) includes the use of a Cu(I) source.
  • the disclosure features a method of synthesizing a conjugate of formula (M-I): wherein each A 1 is selected from any one of formulas (A-I)-(A-VI) as described herein; n is 1 or 2; each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 or 125-153), an albumin protein (e.g., an albumin protein having the sequence of any one of SEQ ID NOs: 96-98), an albumin protein-binding peptide, or an Fc-binding peptide; L is a linker covalently attached to E and to Y of A 1 ; T is an integer from 1 to 20; and each squiggly line in formula (M-I) indicates that L is covalently attached (e.g., by way of a covalent bond or linker) to each E, the method including the steps of: (a) providing a first composition including E; (b)
  • G is optionally substituted C 1 -C 6 heteroalkylene or optionally substituted C 2 -C 10 heteroarylene. In some embodiments, G is optionally substituted C 1 -C 6 heteroalkylene. In some embodiments, G is , where R a is H, optionally substituted C 1 - C 20 alkylene (e.g., optionally substituted C 1 -C 6 alkylene), or optionally substituted C 1 -C 20 heteroalkylene (e.g., optionally substituted C 1 -C 6 heteroalkylene). In some embodiments, G is optionally substituted C 2 -C 10 heteroarylene. In some embodiments, G is optionally substituted C 2 -C 5 heteroarylene.
  • G is a 5-membered or 6- membered optionally substituted C 2 -C 5 heteroarylene.
  • G is a triazolylene.
  • the conjugate of formula (M-I) has the structure of: , and the method including the steps of: (a) providing a first composition including E; (b) providing a second composition including a compound of formula (MF-II-A) or salt thereof: and (c) combining the first composition, the second composition, and a buffer to form a mixture.
  • the synthesis of compound of formula (MF-II-A) includes: (d) providing a third composition including formula (M-G1-A) or salt thereof: ( ) (e) providing a fourth composition including formula (M-G1-B) or salt thereof: and (f) combining the third composition and the fourth composition to form a mixture.
  • the conjugate of formula (M-I) has the structure of: , and the method includes the steps of: (a) providing a first composition including E; (b) providing a second composition including a compound of formula (MF-II-B) or salt thereof: and (c) combining the first composition, the second composition, and a buffer to form a mixture.
  • the synthesis of compound of formula (MF-II-B) includes: (d) providing a third composition including formula (M-G2-A) or salt thereof: (e) providing a fourth composition including formula (M-G2-B) or salt thereof: and (f) combining the third composition and the fourth composition to form a mixture.
  • step (f) includes the use of a Cu(I) source.
  • the disclosure features a method of synthesizing a conjugate of formula (D-I): , wherein each A 1 and each A 2 is independently selected from any one of formulas (A-I)-(A-VI) as described herein; n is 1 or 2; each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 or 125-153), an albumin protein (e.g., an albumin protein having the sequence of any one of SEQ ID NOs: 96-98), an albumin protein-binding peptide, or an Fc-binding peptidee; L is a linker covalently attached to E and to Y of each of A 1 and A 2 ; T is an integer from 1 to 20; and each squiggly line in formula (D-I) indicates that L is covalently attached (e.g., by way of a covalent bond or linker) to each E, the method including the steps of: (
  • step (c) includes the use of a Cu(I) source.
  • the method further includes: (d) providing a third composition including E; and (e) combining the third composition, the first mixture, and a buffer to form a second mixture.
  • the disclosure features a method of synthesizing a conjugate of formula (M-I): wherein each A 1 is selected from any one of formulas (A-I)-(A-VI) as described herein; n is 1 or 2; each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95 or 125-153), an albumin protein (e.g., an albumin protein having the sequence of any one of SEQ ID NOs: 96-98), an albumin protein-binding peptide, or an Fc-binding peptide; L is a linker covalently attached to E and to Y of A 1 ; T is an integer from 1 to 20; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), where when T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • step (c) includes the use of a Cu(I) source.
  • the method further includes: (d) providing a third composition including E; and (e) combining the third composition, the first mixture, and a buffer to form a second mixture.
  • G a includes optionally substituted amino.
  • G b includes a carbonyl.
  • G a includes a carbonyl.
  • G b includes optionally substituted amino.
  • G a includes an azido group.
  • G b includes an alknyl group.
  • G a includes an alkynyl group.
  • G b includes an azido group.
  • a compound of formula (MF-II) or salt thereof has the structure of any one of Int-1 to Int-140 (e.g., Int-56, Int-57, and Int-74)
  • a compound of formula (MF-II) or salt thereof includes the structure of any one of Int-1 to Int-140 (e.g., Int-56, Int-57, and Int-74).
  • a compound of formula (MF-II) or salt thereof is synthesized from the structure of any one of Int-1 to Int-140 (e.g., Int 110, Int-133, or Int- 148).
  • a compound of formula (DF-II) e.g., a compound of formula (DF-II-A) or (DF-II-B) and/or a compound of formula (D-G1-A) or (D-G2-A), where each R is halo (e.g., F)
  • a compound of formula (DF-II) e.g., a compound of formula (DF-II-A) or (DF-II-B) and/or a compound of formula (D-G1-A) or (D-G2-A), where each R is halo (e.g., F)
  • the increased stability allows for purification by reverse phase chromatography. In some embodiments, the increased stability allows for lyophilization with minimal hydrolysis of the activated ester.
  • a compound of formula (DF-II) e.g., a compound of formula (DF-II-A) or (DF-II-B) and/or a compound of formula (D-G1-A) or (D-G2-A), where m is 3, provides technical advantages (e.g., increased stability) in methods of synthesizing protein-drug conjugates (e.g., the methods described herein).
  • the increased stability allows for purification by reverse phase chromatography.
  • the increased stability allows for lyophilization with minimal hydrolysis of the activated ester.
  • a compound of formula (DF-II) e.g., a compound of formula (DF-II-A) or (DF-II-B) and/or a compound of formula (D-G1-A) or (D-G2-A), where m is 3 and each R is halo (e.g., F)
  • DF-II a compound of formula (DF-II)
  • D-G1-A) or (DF-G2-A) where m is 3 and each R is halo (e.g., F)
  • the increased stability allows for purification by reverse phase chromatography.
  • the increased stability allows for lyophilization with minimal hydrolysis of the activated ester.
  • a compound of formula (MF-II) e.g., a compound of formula (MF-II-A) or (MF-II-B) and/or a compound of formula (M-G1-A) or (M-G2-A), where each R is halo (e.g., F)
  • MF-II a compound of formula
  • M-G1-A e.g., F
  • the increased stability allows for purification by reverse phase chromatography.
  • the increased stability allows for lyophilization with minimal hydrolysis of the activated ester.
  • a compound of formula (MF-II) (e.g., a compound of formula (MF-II-A) or (MF-II-B) and/or a compound of formula (M-G1-A) or (M-G2-A), where m is 3, provides technical advantages (e.g., increased stability) in methods of synthesizing protein-drug conjugates (e.g., the methods described herein).
  • the increased stability allows for purification by reverse phase chromatography.
  • the increased stability allows for lyophilization with minimal hydrolysis of the activated ester.
  • a compound of formula (MF-II) (e.g., a compound of formula (MF-II-A) or (MF-II-B) and/or a compound of formula (M-G1-A) or (M-G2-A), where m is 3 and each R is halo (e.g., F), provides technical advantages (e.g., increased stability) in methods of synthesizing protein-drug conjugates (e.g., the methods described herein).
  • the increased stability allows for purification by reverse phase chromatography.
  • the increased stability allows for lyophilization with minimal hydrolysis of the activated ester.
  • the first composition including E is an Fc domain (e.g., n is 2, each E is an Fc domain monomer, and the Fc domain monomers dimerize to form an Fc domain).
  • E includes at least one lysine residue.
  • the squiggly line in formula (D-I) or (M-I) is covalently bound to a lysine residue of each E.
  • E includes at least one cysteine residue.
  • the squiggly line in formula (D-I) or (M-I) is covalently bound to a cysteine residue of each E.
  • each R is, independently, halo, cyano, nitro, haloalkyl, or , where R z is optionally substituted C 1 -C 5 alkyl group or optionally substituted C 1 -C 5 heteroalkyl group.
  • each R is, independently, halo, cyano, nitro, or haloalkyl.
  • each R is, independently, F, Cl, Br, or I.
  • each R is F.
  • m is 3 or 4.
  • m is 3. In some embodiments, m is 4.
  • the buffer includes borate or carbonate. In some embodiments, the buffer includes borate. In some embodiments, the buffer includes carbonate. In some embodiments, the buffer has a pH of about 7.0 to 10.0 (e.g., about 7.0 to 7.5, 7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, 9.5 to 10.0, 7.0 to 8.0, 7.5 to 8.5, 8.0 to 9.0, 8.5 to 9.5, 9.0 to 10.0, 7.0 to 9.0, 7.5 to 9.5, or 8.0 to 10.0).
  • 7.0 to 10.0 e.g., about 7.0 to 7.5, 7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, 9.5 to 10.0, 7.0 to 8.0, 7.5 to 8.5, 9.5, 9.0 to 10.0, 7.0 to 9.0, 7.5 to 9.5, or 8.0 to 10.0.
  • the buffer has a pH of about 7.0. In some embodiments, the buffer has a pH of about 7.1. In some embodiments, the buffer has a pH of about 7.2. In some embodiments, the buffer has a pH of about 7.3. In some embodiments, the buffer has a pH of about 7.4. In some embodiments, the buffer has a pH of about 7.5. In some embodiments, the buffer has a pH of about 7.6. In some embodiments, the buffer has a pH of about 7.7. In some embodiments, the buffer has a pH of about 7.8. In some embodiments, the buffer has a pH of about 7.9. In some embodiments, the buffer has a pH of about 8.0. In some embodiments, the buffer has a pH of about 8.1.
  • the buffer has a pH of about 8.2. In some embodiments, the buffer has a pH of about 8.3. In some embodiments, the buffer has a pH of about 8.4. In some embodiments, the buffer has a pH of about 8.5. In some embodiments, the buffer has a pH of about 8.6. In some embodiments, the buffer has a pH of about 8.7. In some embodiments, the buffer has a pH of about 8.8. In some embodiments, the buffer has a pH of about 8.9. In some embodiments, the buffer has a pH of about 9.0. In some embodiments, the buffer has a pH of about 9.5. In some embodiments, the buffer has a pH of about 9.6. In some embodiments, the buffer has a pH of about 9.7.
  • the buffer has a pH of about 9.8. In some embodiments, the buffer has a pH of about 9.9. In some embodiments, the buffer has a pH of about 10.0. In some embodiments of any of the aspects described herein, step (c) or step (e) is conducted at a temperature of 5 to 50 °C, such as 20 to 30 °C (e.g., 20 to 25, 21 to 26, 22 to 27, 23 to 28, 24 to 29, or 25 to 30 °C). In some embodiments, step (c) or step (e) is conducted at a temperature of about 25 °C.
  • step (c) or step (e) is conducted for about 1 to 24 hours, such as 1 to 12 hours (e.g., 1 to 2, 1 to 5, 2 to 3, 2 to 5, 2 to 10, 2 to 12, 3 to 4, 4 to 5, 1 to 3, 2 to 4, or 3 to 5 hours).
  • step (c) or step (e) is conducted for about 2 hours.
  • step (c) or step (e) is conducted for about 3 hours.
  • step (c) or step (e) is conducted for about 4 hours.
  • step (c) or step (e) is conducted for about 5 hours.
  • step (c) or step (e) is conducted for about 6 hours.
  • step (c) or step (e) is conducted for about 7 hours. In some embodiments, step (c) or step (e) is conducted for about 8 hours. In some embodiments, step (c) or step (e) is conducted for about 9 hours. In some embodiments, step (c) or step (e) is conducted for about 10 hours. In some embodiments, step (c) or step (e) is conducted for about 11 hours. In some embodiments, step (c) or step (e) is conducted for about 12 hours.
  • the first composition or third composition includes phosphate-buffered saline buffer.
  • the buffer has a pH of about 7.0 to 8.0 (e.g., about 7.0 to 7.5, 7.5 to 8.0, 7.0 to 7.2, 7.2 to 7.4, 7.4 to 7.6, 7.6 to 7.8, or 7.8 to 8.0). In some embodiments, the buffer has a pH of about 7.5. In some embodiments, the second composition or the first mixture includes DMF. In some embodiments, the method further includes a purification step. In some embodiments, the purification step includes dialysis in arginine buffer. In some embodiments, the purification step includes a buffer exchange. In some embodiments, T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • the average value of T is 1 to 20 (e.g., the average value of T is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, or 15 to 20). In some embodiments, the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the average T is 1 to 10 (e.g., 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10).
  • the average T is 1 to 5 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5).
  • the average T is 5 to 10 (e.g., 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10).
  • the average T is 2.5 to 7.5 (e.g., 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, or 7.5). Definitions To facilitate the understanding of this invention, a number of terms are defined below.
  • viral infection is meant the pathogenic growth of a virus (e.g., the human immunodeficiency virus) in a host organism (e.g., a human subject).
  • a viral infection can be any situation in which the presence of a viral population(s) is damaging to a host body.
  • Fc domain monomer refers to a polypeptide chain that includes at least a hinge domain and second and third antibody constant domains (CH 2 and CH3) or functional fragments thereof (e.g., fragments that that capable of (i) dimerizing with another Fc domain monomer to form an Fc domain, and (ii) binding to an Fc receptor.
  • the Fc domain monomer can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD (e.g., IgG). Additionally, the Fc domain monomer can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4) (e.g., IgG1). An Fc domain monomer does not include any portion of an immunoglobulin that is capable of acting as an antigen-recognition region, e.g., a variable domain or a complementarity determining region (CDR).
  • CDR complementarity determining region
  • Fc domain monomers in the conjugates as described herein can contain one or more changes from a wild- type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions, or deletions) that alter the interaction between an Fc domain and an Fc receptor. Examples of suitable changes are known in the art.
  • a human Fc domain monomer e.g., an IgG heavy chain, such as IgG1
  • IgG heavy chain such as IgG1
  • a human Fc domain monomer includes a region that extends from any of Asn208, Glu216, Asp221, Lys222, or Cys226 to the carboxyl-terminus of the heavy chain at Lys447.
  • C-terminal Lys447 of the Fc region may or may not be present, without affecting the structure or stability of the Fc region.
  • numbering of amino acid residues in the IgG or Fc domain monomer is according to the EU numbering system for antibodies, also called the Kabat EU index, as described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • the term “Fc domain” refers to a dimer of two Fc domain monomers that is capable of binding an Fc receptor.
  • the two Fc domain monomers dimerize by the interaction between the two CH3 antibody constant domains, in some embodiments, one or more disulfide bonds form between the hinge domains of the two dimerizing Fc domain monomers.
  • covalently attached refers to two parts of a conjugate that are linked to each other by a covalent bond formed between two atoms in the two parts of the conjugate.
  • Fc-binding peptide refers to a polypeptide having an amino acid sequence of 5 to 50 (e.g., 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or 10 to 20) amino acid residues that has affinity for and functions to bind an Fc domain, such as any of the Fc domain described herein.
  • An Fc-binding peptide can be of different origins, e.g., synthetic, human, mouse, or rat.
  • Fc-binding peptides of the invention include Fc-binding peptides which have been engineered to include one or more (e.g., two, three, four, or five) solvent-exposed cysteine or lysine residues, which may provide a site for conjugation to a compound of the invention (e.g., conjugation to a gp120 binder monomer or dimer, including by way of a linker). Most preferably, the Fc-binding peptide will contain a single solvent-exposed cysteine or lysine, thus enabling site-specific conjugation of a compound of the invention. Fc-binding peptides may include only naturally occurring amino acid residues, or may include one or more non-naturally occurring amino acid residues.
  • a non-naturally occurring amino acid residue e.g., the side chain of a non-naturally occurring amino acid residue
  • a compound of the invention e.g., a gp120 binder monomer or dimer, including by way of a linker
  • Fc-binding peptides of the invention may be linear or cyclic.
  • Fc-binding peptides of the invention include any Fc-binding peptides known to one of skill in the art.
  • albumin protein refers to a polypeptide comprising an amino acid sequence corresponding to a naturally-occurring albumin protein (e.g., human serum albumin) or a variant thereof, such as an engineered variant of a naturally-occurring albumin protein.
  • Variants of albumin proteins include polymorphisms, fragments such as domains and sub-domains, and fusion proteins (e.g., an albumin protein having a C-terminal or N-terminal fusion, such as a polypeptide linker).
  • the albumin protein has the amino acid sequence of human serum albumin (HSA) or a variant or fragment thereof, most preferably a functional variant or fragment thereof.
  • Albumin proteins of the invention include proteins having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 96-98.
  • Albumin proteins of the invention include albumin proteins which have been engineered to include one or more (e.g., two, three, four, or five) solvent-exposed cysteine or lysine residues, which may provide a site for conjugation to a compound of the invention (e.g., conjugation to a gp120 binder monomer or dimer, including by way of a linker).
  • albumin protein will contain a single solvent-exposed cysteine or lysine, thus enabling site- specific conjugation of a compound of the invention.
  • Albumin proteins may include only naturally occurring amino acid residues, or may include one or more non-naturally occurring amino acid residues. Where included, a non-naturally occurring amino acid residue (e.g., the side chain of a non-naturally occurring amino acid residue) may be used as the point of attachment for a compound of the invention (e.g., a gp120 binder monomer or dimer, including by way of a linker).
  • albumin protein-binding peptide refers to a polypeptide having an amino acid sequence of 5 to 50 (e.g., 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or 10 to 20) amino acid residues that has affinity for and functions to bind an albumin protein, such as any of the albumin proteins described herein.
  • the albumin protein-binding peptide binds to a naturally- occurring serum albumin, most preferably human serum albumin.
  • An albumin protein-binding peptide can be of different origins, e.g., synthetic, human, mouse, or rat.
  • Albumin protein-binding peptides of the invention include albumin protein-binding peptides which have been engineered to include one or more (e.g., two, three, four, or five) solvent-exposed cysteine or lysine residues, which may provide a site for conjugation to a compound of the invention (e.g., conjugation to a gp120 binder monomer or dimer, including by way of a linker). Most preferably, the albumin protein-binding peptide will contain a single solvent-exposed cysteine or lysine, thus enabling site-specific conjugation of a compound of the invention.
  • Albumin protein-binding peptides may include only naturally occurring amino acid residues, or may include one or more non-naturally occurring amino acid residues. Where included, a non-naturally occurring amino acid residue (e.g., the side chain of a non-naturally occurring amino acid residue) may be used as the point of attachment for a compound of the invention (e.g., a gp120 binder monomer or dimer, including by way of a linker). Albumin protein-binding peptides of the invention may be linear or cyclic. Albumin protein-binding peptide of the invention include any albumin protein-binding peptides known to one of skill in the art, examples of which, are provided herein.
  • albumin protein-binding peptides are provided in U.S. Patent Application No.2005/0287153, which is incorporated herein by reference in its entirety.
  • a “surface exposed amino acid” or “solvent-exposed amino acid,” such as a surface exposed cysteine or a surface exposed lysine refers to an amino acid that is accessible to the solvent surrounding the protein.
  • a surface exposed amino acid may be a naturally-occurring or an engineered variant (e.g., a substitution or insertion) of the protein.
  • a surface exposed amino acid is an amino acid that when substituted does not substantially change the three- dimensional structure of the protein.
  • linker refers to a covalent linkage or connection between two or more components in a conjugate (e.g., between two gp120 binders in a conjugate described herein, between a gp120 binder and an Fc domain or albumin protein in a conjugate described herein, and between a dimer of two gp120 binders and an Fc domain or an albumin protein in a conjugate described herein).
  • a conjugate described herein may contain a linker that has a trivalent structure (e.g., a trivalent linker).
  • a trivalent linker has three arms, in which each arm is covalently linked to a component of the conjugate (e.g., a first arm conjugated to a first gp120 binder, a second arm conjugated to a second gp120 binder, and a third arm conjugated to an Fc domain or an albumin protein).
  • a component of the conjugate e.g., a first arm conjugated to a first gp120 binder, a second arm conjugated to a second gp120 binder, and a third arm conjugated to an Fc domain or an albumin protein.
  • Molecules that may be used as linkers include at least two functional groups, which may be the same or different, e.g., two carboxylic acid groups, two amine groups, two sulfonic acid groups, a carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid group, an amine group and a maleimide group, an amine group and an alkyne group, or an amine group and a sulfonic acid group.
  • two functional groups which may be the same or different, e.g., two carboxylic acid groups, two amine groups, two sulfonic acid groups, a carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid
  • the first functional group may form a covalent linkage with a first component in the conjugate and the second functional group may form a covalent linkage with the second component in the conjugate.
  • two arms of a linker may contain two dicarboxylic acids, in which the first carboxylic acid may form a covalent linkage with the first gp120 binder in the conjugate and the second carboxylic acid may form a covalent linkage with the second gp120 binder in the conjugate, and the third arm of the linker may for a covalent linkage with an Fc domain or albumin protein in the conjugate. Examples of dicarboxylic acids are described further herein.
  • a molecule containing one or more maleimide groups may be used as a linker, in which the maleimide group may form a carbon-sulfur linkage with a cysteine in a component (e.g., an Fc domain monomer, an Fc domain, or an albumin protein) in the conjugate.
  • a molecule containing one or more alkyne groups may be used as a linker, in which the alkyne group may form a 1,2,3-triazole linkage with an azide in a component (e.g., an Fc domain monomer, an Fc domain, or an albumin protein) in the conjugate.
  • a molecule containing one or more azide groups may be used as a linker, in which the azide group may form a 1,2,3-triazole linkage with an alkyne in a component (e.g., an Fc domain monomer, an Fc domain, or an albumin protein) in the conjugate.
  • a molecule containing one or more bis-sulfone groups may be used as a linker, in which the bis-sulfone group may form a linkage with an amine group a component (e.g., an Fc domain monomer, an Fc domain, or an albumin protein) in the conjugate.
  • a molecule containing one or more sulfonic acid groups may be used as a linker, in which the sulfonic acid group may form a sulfonamide linkage with a component in the conjugate.
  • a molecule containing one or more isocyanate groups may be used as a linker, in which the isocyanate group may form a urea linkage with a component in the conjugate.
  • a molecule containing one or more haloalkyl groups may be used as a linker, in which the haloalkyl group may form a covalent linkage, e.g., C-N and C-O linkages, with a component in the conjugate.
  • a molecule containing one or more phenyl ester groups may be used as a linker, in which the phenyl ester group (e.g., triflurophenyl ester group or tetrafluorophenyl ester group) may form an amide with an amine in a component (e.g., a fusion protein) in the conjugate.
  • a linker provides space, rigidity, and/or flexibility between the two or more components.
  • a linker may be a bond, e.g., a covalent bond.
  • a linker refers to a chemical bond, e.g., an amide bond, a disulfide bond, a C-O bond, a C-N bond, a N-N bond, a C-S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation.
  • a linker includes no more than 250 atoms. In some embodiments, a linker includes no more than 250 non-hydrogen atoms. In some embodiments, the backbone of a linker includes no more than 250 atoms.
  • the “backbone” of a linker refers to the atoms in the linker that together form the shortest path from one part of a conjugate to another part of the conjugate (e.g., the shortest path linking a first gp120 binder and a second gp120 binder).
  • the atoms in the backbone of the linker are directly involved in linking one part of a conjugate to another part of the conjugate (e.g., linking a first gp120 binder and a second gp120 binder).
  • hydrogen atoms attached to carbons in the backbone of the linker are not considered as directly involved in linking one part of the conjugate to another part of the conjugate.
  • a linker may comprise a synthetic group derived from, e.g., a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer).
  • a linker may comprise one or more amino acid residues, such as D- or L-amino acid residues.
  • a linker may be a residue of an amino acid sequence (e.g., a 1-25 amino acid, 1-10 amino acid, 1-9 amino acid, 1-8 amino acid, 1-7 amino acid, 1-6 amino acid, 1-5 amino acid, 1-4 amino acid, 1-3 amino acid, 1-2 amino acid, or 1 amino acid sequence).
  • a linker may comprise one or more, e.g., 1-100, 1-50, 1-25, 1-10, 1-5, or 1-3, optionally substituted alkylene, optionally substituted heteroalkylene (e.g., a PEG unit), optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted alkynylene, optionally substituted heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted cycloalkenylene, optionally substituted heterocycloalkenylene, optionally substituted cycloalkynylene, optionally substituted heterocycloalkynylene, optionally substituted arylene, optionally substituted heteroarylene (e.g., pyridine), O, S, NR i , , (each R i is, independently, H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl,
  • a linker may comprise one or more optionally substituted C 1 -C 20 alkylene, optionally substituted C 1 -C 20 heteroalkylene (e.g., a PEG unit), optionally substituted C 2 -C 20 alkenylene (e.g., C 2 alkenylene), optionally substituted C 2 -C 20 heteroalkenylene, optionally substituted C 2 -C 20 alkynylene, optionally substituted C 2 -C 20 heteroalkynylene, optionally substituted C 3 -C 20 cycloalkylene (e.g., cyclopropylene, cyclobutylene), optionally substituted C 2 -C 20 heterocycloalkylene, optionally substituted C 4 -C 20 cycloalkenylene, optionally substituted C 4 -C 20 heterocycloalkenylene, optionally substituted C 8 -C 20 cycloalkynylene, optionally substituted C 8 -C 20 heterocycloalkynylene,
  • alkyl straight-chain and branched- chain monovalent substituents, as well as combinations of these, containing only C and H when unsubstituted.
  • alkyl group includes at least one carbon-carbon double bond or carbon-carbon triple bond, the alkyl group can be referred to as an “alkenyl” or “alkynyl” group respectively.
  • alkenyl or alkynyl group respectively.
  • the monovalency of an alkyl, alkenyl, or alkynyl group does not include the optional substituents on the alkyl, alkenyl, or alkynyl group.
  • alkyl, alkenyl, or alkynyl group is attached to a compound
  • monovalency of the alkyl, alkenyl, or alkynyl group refers to its attachment to the compound and does not include any additional substituents that may be present on the alkyl, alkenyl, or alkynyl group.
  • the alkyl or heteroalkyl group may contain, e.g., 1-20.1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1- 6, 1-4, or 1-2 carbon atoms (e.g., C 1 -C 20 , C 1 -C 18 , C 1 -C 16 , C 1 -C 14 , C 1 -C 12 , C 1 -C 10 , C 1 -C 8 , C 1 -C 6 , C 1 -C 4 , or C 1 -C 2 ).
  • 1-20.1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1- 6, 1-4, or 1-2 carbon atoms e.g., C 1 -C 20 , C 1 -C 18 , C 1 -C 16 , C 1 -C 14 , C 1 -C 12 , C 1 -C 10 , C 1 -C 8 , C 1 -C 6 , C 1 -C 4 , or C 1
  • the alkenyl, heteroalkenyl, alkynyl, or heteroalkynyl group may contain, e.g., 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, or 2-4 carbon atoms (e.g., C 2 -C 20 , C 2 -C 18 , C 2 -C 16 , C 2 -C 14 , C 2 -C 12 , C 2 -C 10 , C 2 -C 8 , C 2 -C 6 , or C 2 -C 4 ).
  • carbon atoms e.g., C 2 -C 20 , C 2 -C 18 , C 2 -C 16 , C 2 -C 14 , C 2 -C 12 , C 2 -C 10 , C 2 -C 8 , C 2 -C 6 , or C 2 -C 4 ).
  • cycloalkyl represents a monovalent saturated or unsaturated non- aromatic cyclic alkyl group.
  • a cycloalkyl may have, e.g., three to twenty carbons (e.g., a C 3 -C 7 , C 3 -C 8 , C 3 - C 9 , C 3 -C 10 , C 3 -C 11 , C 3 -C 12 , C 3 -C 14 , C 3 -C 16 , C 3 -C 18 , or C 3 -C 20 cycloalkyl).
  • Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • the cycloalkyl group When the cycloalkyl group includes at least one carbon-carbon double bond, the cycloalkyl group can be referred to as a “cycloalkenyl” group.
  • a cycloalkenyl may have, e.g., four to twenty carbons (e.g., a C 4 -C 7 , C 4 -C 8 , C 4 - C 9 , C 4 -C 10 , C 4 -C 11 , C 4 -C 12 , C 4 -C 14 , C 4 -C 16 , C 4 -C 18 , or C 4 -C 20 cycloalkenyl).
  • Exemplary cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexenyl, and cycloheptenyl.
  • the cycloalkyl group includes at least one carbon-carbon triple bond, the cycloalkyl group can be referred to as a “cycloalkynyl” group.
  • a cycloalkynyl may have, e.g., eight to twenty carbons (e.g., a C 8 -C 9 , C 8 -C 10 , C 8 -C 11 , C 8 -C 12 , C 8 - C 14 , C 8 -C 16 , C 8 -C 18 , or C 8 -C 20 cycloalkynyl).
  • the term “cycloalkyl” also includes a cyclic compound having a bridged multicyclic structure in which one or more carbons bridges two non-adjacent members of a monocyclic ring, e.g., bicyclo[2.2.1.]heptyl and adamantane.
  • cycloalkyl also includes bicyclic, tricyclic, and tetracyclic fused ring structures, e.g., decalin and spiro cyclic compounds.
  • a “heterocycloalkyl,” “heterocycloalkenyl,” or “heterocycloalkynyl” group refers to a cycloalkyl, cycloalkenyl, or cycloalkynyl group having one or more rings (e.g., 1, 2, 3, 4 or more rings) that has one or more heteroatoms independently selected from, e.g., N, O, and S.
  • heterocycloalkyl groups include pyrrolidine, thiophene, thiolane, tetrahydrofuran, piperidine, tetrahydropyran, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, indole, benzothiophene, benzofuran, isoindole, benzo[c]thiophene, isobenzofuran, benzimidazole, benzoxazole, benzothiazole, 1H-indazole, 1,2,benzisoxazole, 1,2-benzisothiazole, 2,1-benzisothiazole, 2,1-benzisoxazole, purine, pyrrolizidine, indene, fluorene, carbazole, dibenzofuran, acridine, phenazine, and phenoxazine.
  • aryl refers to any monocyclic or fused ring bicyclic or tricyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system, e.g., phenyl, naphthyl, or phenanthrene.
  • a ring system contains 5-15 ring member atoms or 5-10 ring member atoms.
  • An aryl group may have, e.g., five to fifteen carbons (e.g., a C 5 -C 6 , C 5 -C 7 , C 5 -C 8 , C 5 -C 9 , C 5 -C 10 , C 5 -C 11 , C 5 -C 12 , C 5 -C 13 , C 5 - C 14 , or C 5 -C 15 aryl).
  • the term “heteroaryl” also refers to such monocyclic or fused bicyclic ring systems containing one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms selected from O, S and N.
  • a heteroaryl group may have, e.g., two to fifteen ring member atoms (e.g., a C 2 -C 3 , C 2 -C 4 , C 2 -C 5 , C 2 -C 6 , C 2 -C 7 , C 2 -C 8 , C 2 -C 9 , C 2 -C 10 , C 2 -C 11 , C 2 -C 12 , C 2 -C 13 , C 2 -C 14 , or C 3 -C 15 heteroaryl).
  • the inclusion of a heteroatom permits inclusion of 5-membered rings to be considered aromatic as well as 6-membered rings.
  • heteroaryl systems include, e.g., pyridyl, pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, triazolyl (e.g., 1,2,3- or 1,2,4-triazolyl) oxazolyl, isoxazolyl, benzoxazolyl, benzoisoxazolyl, and imidazolyl. Because tautomers are possible, a group such as phthalimido is also considered heteroaryl.
  • the aryl or heteroaryl group is a 5- or 6-membered aromatic rings system optionally containing 1-2 nitrogen atoms.
  • the aryl or heteroaryl group is an optionally substituted phenyl, pyridyl, indolyl, pyrimidyl, pyridazinyl, benzothiazolyl, benzimidazolyl, pyrazolyl, imidazolyl, isoxazolyl, thiazolyl, or imidazopyridinyl.
  • the aryl group is phenyl.
  • an aryl group may be optionally substituted with a substituent such an aryl substituent, e.g., biphenyl.
  • aryl substituent e.g., biphenyl.
  • alkaryl refers to an aryl group that is connected to an alkylene, alkenylene, or alkynylene group. In general, if a compound is attached to an alkaryl group, the alkylene, alkenylene, or alkynylene portion of the alkaryl is attached to the compound.
  • an alkaryl is C 6 -C 3 5 alkaryl (e.g., C 6 -C 16 , C 6 -C 14 , C 6 -C 12 , C 6 -C 10 , C 6 -C 9 , C 6 -C 8 , C 7 , or C 6 alkaryl), in which the number of carbons indicates the total number of carbons in both the aryl portion and the alkylene, alkenylene, or alkynylene portion of the alkaryl.
  • C 6 -C 3 5 alkaryl e.g., C 6 -C 16 , C 6 -C 14 , C 6 -C 12 , C 6 -C 10 , C 6 -C 9 , C 6 -C 8 , C 7 , or C 6 alkaryl
  • alkaryls include, but are not limited to, (C 1 -C 8 )alkylene(C 6 - C 12 )aryl, (C 2 -C 8 )alkenylene(C 6 -C 12 )aryl, or (C 2 -C 8 )alkynylene(C 6 -C 12 )aryl.
  • an alkaryl is benzyl or phenethyl.
  • one or more heteroatoms selected from N, O, and S may be present in the alkylene, alkenylene, or alkynylene portion of the alkaryl group and/or may be present in the aryl portion of the alkaryl group.
  • the substituent may be present on the alkylene, alkenylene, or alkynylene portion of the alkaryl group and/or may be present on the aryl portion of the alkaryl group.
  • the term “amino,” as used herein, represents –N(R x ) 2 or –N + (R x ) 3 , where each R x is, independently, H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R x combine to form a heterocycloalkyl.
  • the amino group is -NH 2 .
  • alkamino refers to an amino group, described herein, that is attached to an alkylene (e.g., C 1 -C 5 alkylene), alkenylene (e.g., C 2 -C 5 alkenylene), or alkynylene group (e.g., C 2 -C 5 alkenylene).
  • alkylene e.g., C 1 -C 5 alkylene
  • alkenylene e.g., C 2 -C 5 alkenylene
  • alkynylene group e.g., C 2 -C 5 alkenylene
  • the amino portion of an alkamino refers to –N(R x ) 2 or –N + (R x ) 3 , where each R x is, independently, H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R x combine to form a heterocycloalkyl.
  • the amino portion of an alkamino is -NH 2 .
  • An example of an alkamino group is C 1 -C 5 alkamino, e.g., C 2 alkamino (e.g., CH 2 CH 2 NH 2 or CH 2 CH 2 N(CH 3 ) 2 ).
  • heteroalkamino group one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms selected from N, O, and S may be present in the alkylene, alkenylene, or alkynylene portion of the heteroalkamino group.
  • an alkamino group may be optionally substituted.
  • the substituent may be present on the alkylene, alkenylene, or alkynylene portion of the alkamino group and/or may be present on the amino portion of the alkamino group.
  • alkamide refers to an amide group that is attached to an alkylene (e.g., C 1 -C 5 alkylene), alkenylene (e.g., C 2 -C 5 alkenylene), or alkynylene (e.g., C 2 -C 5 alkenylene) group.
  • alkylene e.g., C 1 -C 5 alkylene
  • alkenylene e.g., C 2 -C 5 alkenylene
  • alkynylene e.g., C 2 -C 5 alkenylene
  • the amide portion of an alkamide refers to – C(O)-N(R x ) 2 , where each R x is, independently, H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R x combine to form a heterocycloalkyl.
  • the amide portion of an alkamide is -C(O)NH 2 .
  • An alkamide group may be -(CH 2 ) 2 -C(O)NH 2 or -CH 2 -C(O)NH 2 .
  • heteroalkamide group one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms selected from N, O, and S may be present in the alkylene, alkenylene, or alkynylene portion of the heteroalkamide group.
  • an alkamide group may be optionally substituted.
  • the substituent may be present on the alkylene, alkenylene, or alkynylene portion of the alkamide group and/or may be present on the amide portion of the alkamide group.
  • alkylene alkenylene
  • alkynylene refer to divalent groups having a specified size.
  • an alkylene may contain, e.g., 1-20, 1-18, 1-16, 1-14, 1- 12, 1-10, 1-8, 1-6, 1-4, or 1-2 carbon atoms (e.g., C 1 -C 20 , C 1 -C 18 , C 1 -C 16 , C 1 -C 14 , C 1 -C 12 , C 1 -C 10 , C 1 -C 8 , C 1 -C 6 , C 1 -C 4 , or C 1 -C 2 ).
  • carbon atoms e.g., C 1 -C 20 , C 1 -C 18 , C 1 -C 16 , C 1 -C 14 , C 1 -C 12 , C 1 -C 10 , C 1 -C 8 , C 1 -C 6 , C 1 -C 4 , or C 1 -C 2 ).
  • an alkenylene or alkynylene may contain, e.g., 2-20, 2- 18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, or 2-4 carbon atoms (e.g., C 2 -C 20 , C 2 -C 18 , C 2 -C 16 , C 2 -C 14 , C 2 -C 12 , C 2 - C 10 , C 2 -C 8 , C 2 -C 6 , or C 2 -C 4 ).
  • Alkylene, alkenylene, and/or alkynylene includes straight-chain and branched-chain forms, as well as combinations of these.
  • the divalency of an alkylene, alkenylene, or alkynylene group does not include the optional substituents on the alkylene, alkenylene, or alkynylene group.
  • two gp120 binders may be attached to each other by way of a linker that includes alkylene, alkenylene, and/or alkynylene, or combinations thereof.
  • Each of the alkylene, alkenylene, and/or alkynylene groups in the linker is considered divalent with respect to the two attachments on either end of alkylene, alkenylene, and/or alkynylene group.
  • a linker includes -(optionally substituted alkylene)-(optionally substituted alkenylene)-(optionally substituted alkylene)-
  • the alkenylene is considered divalent with respect to its attachments to the two alkylenes at the ends of the linker.
  • the optional substituents on the alkenylene are not included in the divalency of the alkenylene.
  • the divalent nature of an alkylene, alkenylene, or alkynylene group refers to both of the ends of the group and does not include optional substituents that may be present in an alkylene, alkenylene, or alkynylene group.
  • Alkylene, alkenylene, and/or alkynylene groups can be substituted by the groups typically suitable as substituents for alkyl, alkenyl and alkynyl groups as set forth herein.
  • -HCR-C ⁇ C- may be considered as an optionally substituted alkynylene and is considered a divalent group even though it has an optional substituent, R.
  • Heteroalkylene, heteroalkenylene, and/or heteroalkynylene groups refer to alkylene, alkenylene, and/or alkynylene groups including one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms, e.g., N, O, and S.
  • a polyethylene glycol (PEG) polymer or a PEG unit -(CH 2 ) 2 -O- in a PEG polymer is considered a heteroalkylene containing one or more oxygen atoms.
  • PEG polyethylene glycol
  • cycloalkylene refers to a divalent cyclic group linking together two parts of a compound.
  • one carbon within the cycloalkylene group may be linked to one part of the compound, while another carbon within the cycloalkylene group may be linked to another part of the compound.
  • a cycloalkylene group may include saturated or unsaturated non-aromatic cyclic groups.
  • a cycloalkylene may have, e.g., three to twenty carbons in the cyclic portion of the cycloalkylene (e.g., a C 3 -C 7 , C 3 -C 8 , C 3 -C 9 , C 3 -C 10 , C 3 -C 11 , C 3 -C 12 , C 3 -C 14 , C 3 -C 16 , C 3 -C 18 , or C 3 -C 20 cycloalkylene).
  • the cycloalkylene group includes at least one carbon-carbon double bond
  • the cycloalkylene group can be referred to as a “cycloalkenylene” group.
  • a cycloalkenylene may have, e.g., four to twenty carbons in the cyclic portion of the cycloalkenylene (e.g., a C 4 -C 7 , C 4 -C 8 , C 4 -C 9 . C 4 -C 10 , C 4 -C 11 , C 4 -C 12 , C 4 -C 14 , C 4 -C 16 , C 4 -C 18 , or C 4 -C 20 cycloalkenylene).
  • the cycloalkylene group includes at least one carbon-carbon triple bond
  • the cycloalkylene group can be referred to as a “cycloalkynylene” group.
  • a cycloalkynylene may have, e.g., four to twenty carbons in the cyclic portion of the cycloalkynylene (e.g., a C 4 -C 7 , C 4 -C 8 , C 4 -C 9 , C 4 -C 10 , C 4 -C 11 , C 4 -C 12 , C 4 -C 14 , C 4 -C 16 , C 4 -C 18 , or C 8 -C 20 cycloalkynylene).
  • a cycloalkylene group can be substituted by the groups typically suitable as substituents for alkyl, alkenyl and alkynyl groups as set forth herein.
  • Heterocycloalkylene refers to a cycloalkylene group including one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms, e.g., N, O, and S.
  • Examples of cycloalkylenes include, but are not limited to, cyclopropylene and cyclobutylene.
  • a tetrahydrofuran may be considered as a heterocycloalkylene.
  • arylene refers to a multivalent (e.g., divalent or trivalent) aryl group linking together multiple (e.g., two or three) parts of a compound.
  • one carbon within the arylene group may be linked to one part of the compound, while another carbon within the arylene group may be linked to another part of the compound.
  • An arylene may have, e.g., five to fifteen carbons in the aryl portion of the arylene (e.g., a C 5 -C 6 , C 5 -C 7 , C 5 -C 8 , C 5 -C 9 , C 5 -C 10 , C 5 -C 11 , C 5 -C 12 , C 5 -C 13 , C 5 -C 14 , or C 5 - C 15 arylene).
  • An arylene group can be substituted by the groups typically suitable as substituents for alkyl, alkenyl and alkynyl groups as set forth herein.
  • Heteroarylene refers to an aromatic group including one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms, e.g., N, O, and S.
  • a heteroarylene group may have, e.g., two to fifteen carbons (e.g., a C 2 -C 3 , C 2 -C 4 , C 2 -C 5 , C 2 -C 6 , C 2 -C 7 , C 2 -C 8 , C 2 -C 9 .
  • Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, alkaryl, acyl, heteroaryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroalkaryl, halogen, oxo, cyano, nitro, amino, alkamino, hydroxy, alkoxy, alkanoyl, carbonyl, carbamoyl, guanidinyl, ureido, amidinyl, any of the groups or moieties described above, and hetero versions of any of the groups or moieties described above.
  • Substituents include, but are not limited to, F, Cl, methyl, phenyl, benzyl, OR, NR 2 , SR, SOR, SO 2 R, OCOR, NRCOR, NRCONR 2 , NRCOOR, OCONR 2 , RCO, COOR, alkyl-OOCR, SO 3 R, CONR 2 , SO 2 NR 2 , NRSO 2 NR 2 , CN, CF 3 , O CF 3 , SiR 3 , and NO 2 , wherein each R is, independently, H, alkyl, alkenyl, aryl, heteroalkyl, heteroalkenyl, or heteroaryl, and wherein two of the optional substituents on the same or adjacent atoms can be joined to form a fused, optionally substituted aromatic or nonaromatic, saturated or unsaturated ring which contains 3–8 members, or two of the optional substituents on the same atom can be joined to form an optionally substituted aromatic or non
  • an optionally substituted group or moiety refers to a group or moiety (e.g., any one of the groups or moieties described above) in which one of the atoms (e.g., a hydrogen atom) is optionally replaced with another substituent.
  • an optionally substituted alkyl may be an optionally substituted methyl, in which a hydrogen atom of the methyl group is replaced by, e.g., OH.
  • a substituent on a heteroalkyl or its divalent counterpart, heteroalkylene may replace a hydrogen on a carbon or a hydrogen on a heteroatom such as N.
  • the hydrogen atom in the group -R-NH-R- may be substituted with an alkamide substituent, e.g., -R-N[(CH 2 C(O)N(CH 3 ) 2 ]-R.
  • an optional substituent is a noninterfering substituent.
  • a “noninterfering substituent” refers to a substituent that leaves the ability of the conjugates described herein (e.g., conjugates of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)) to either bind to viral gp41 or gp120 receptor or to inhibit the proliferation of HIV.
  • the substituent may alter the degree of such activity.
  • the conjugate retains the ability to bind to viral gp41 or gp120 receptor or to inhibit HIV proliferation, the substituent will be classified as “noninterfering.”
  • the noninterfering substituent would leave the ability of the compound to provide antiviral efficacy based on an IC 5 0 value of 10 ⁇ M or less in a viral plaque reduction assay.
  • the substituent may alter the degree of inhibition based on plaque reduction or gp120 receptor inhibition.
  • hetero when used to describe a chemical group or moiety, refers to having at least one heteroatom that is not a carbon or a hydrogen, e.g., N, O, and S. Any one of the groups or moieties described above may be referred to as hetero if it contains at least one heteroatom.
  • a heterocycloalkyl, heterocycloalkenyl, or heterocycloalkynyl group refers to a cycloalkyl, cycloalkenyl, or cycloalkynyl group that has one or more heteroatoms independently selected from, e.g., N, O, and S.
  • An example of a heterocycloalkenyl group is a maleimido.
  • a heteroaryl group refers to an aromatic group that has one or more heteroatoms independently selected from, e.g., N, O, and S.
  • One or more heteroatoms may also be included in a substituent that replaced a hydrogen atom in a group or moiety as described herein.
  • the substituent may also contain one or more heteroatoms (e.g., methanol).
  • acyl refers to a group having the structure: , wherein R z is an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, alkamino, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaryl, heteroalkaryl, or heteroalkamino.
  • halo or “halogen,” as used herein, refers to any halogen atom, e.g., F, Cl, Br, or I.
  • halo moiety if it contains at least one halogen atom, such as haloalkyl.
  • haloalkyl refers to an alkyl group substituted with one or more (e.g., one, two, three, four, five, six, or more) halo groups.
  • Haloalkyl groups include, but are not limited to, fluoroalkyl (e.g., trifluoromethyl and pentafluoroethyl) and chloroalkyl.
  • hydroxyl represents an -OH group.
  • carbonyl refers to a group having the structure:
  • thiocarbonyl refers to a group having the structure: .
  • phosphate represents the group having the structure:
  • phosphoryl represents the group having the structure:
  • sulfonyl represents the group having the structure:
  • amino represents the group having the structure: , wherein R is an optional substituent.
  • N-protecting group represents those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 5th Edition (John Wiley & Sons, New York, 2014), which is incorporated herein by reference.
  • N-protecting groups include, e.g., acyl, aryloyl, and carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthaloyl, o-nitrophenoxyacetyl, ⁇ -chlorobutyryl, benzoyl, carboxybenzyl (CBz), 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acid residues such as alanine, leucine, phenylalanine; sulfonyl-containing groups such as benzenesulfonyl and p-toluenesulfonyl; carba
  • amino acid means naturally occurring amino acids and non-naturally occurring amino acids.
  • naturally occurring amino acids means amino acids including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
  • non-naturally occurring amino acid means an alpha amino acid that is not naturally produced or found in a mammal.
  • non-naturally occurring amino acids include D-amino acids; an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine; a pegylated amino acid; the omega amino acids of the formula NH 2 (CH 2 )nCOOH where n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine; oxymethionine; phenylglycine; citrulline; methionine sulfoxide; cysteic acid; ornithine; diaminobutyric acid; 3-aminoalanine; 3-hydroxy-D-proline; 2,4-diaminobutyric acid; 2-aminopentanoic acid; 2-aminooctanoic acid, 2-carboxy piperazine; piperazine-2-carboxylic acid, 2-amino
  • amino acids are ⁇ -aminobutyric acid, ⁇ -amino- ⁇ - methylbutyrate, aminocyclopropane-carboxylate, aminoisobutyric acid, aminonorbornyl-carboxylate, L- cyclohexylalanine, cyclopentylalanine, L-N-methylleucine, L-N-methylmethionine, L-N-methylnorvaline, L- N-methylphenylalanine, L-N-methylproline, L-N-methylserine, L-N-methyltryptophan, D-ornithine, L-N- methylethylglycine, L-norleucine, ⁇ -methyl-aminoisobutyrate, ⁇ -methylcyclohexylalanine, D- ⁇ - methylalanine, D- ⁇ -methylarginine, D- ⁇ -methylasparagine, D- ⁇ -methylaspartate, D- ⁇ -methylcysteine
  • amino acid residues may be charged or polar.
  • Charged amino acids include alanine, lysine, aspartic acid, or glutamic acid, or non-naturally occurring analogs thereof.
  • Polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, or tryptophan, or non-naturally occurring analogs thereof. It is specifically contemplated that in some embodiments, a terminal amino group in the amino acid may be an amido group or a carbamate group.
  • percent (%) identity refers to the percentage of amino acid residues of a candidate sequence, e.g., an Fc-IgG, or fragment thereof, that are identical to the amino acid residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software.
  • the percent amino acid sequence identity of a given candidate sequence to, with, or against a given reference sequence is calculated as follows: 100 x (fraction of A/B) where A is the number of amino acid residues scored as identical in the alignment of the candidate sequence and the reference sequence, and where B is the total number of amino acid residues in the reference sequence.
  • the percent amino acid sequence identity of the candidate sequence to the reference sequence would not equal to the percent amino acid sequence identity of the reference sequence to the candidate sequence.
  • Two polynucleotide or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described above. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
  • a “comparison window” as used herein refers to a segment of at least about 15 contiguous positions, about 20 contiguous positions, about 25 contiguous positions, or more (e.g., about 30 to about 75 contiguous positions, or about 40 to about 50 contiguous positions), in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the term “treating” or “to treat,” as used herein, refers to a therapeutic treatment of a viral infection (e.g., a viral infection such as an HIV infection) in a subject. In some embodiments, a therapeutic treatment may slow the progression of the viral infection, improve the subject’s outcome, and/or eliminate the infection.
  • a therapeutic treatment of a viral infection in a subject may alleviate or ameliorate of one or more symptoms or conditions associated with the viral infection, diminish the extent of the viral, stabilize (i.e., not worsening) the state of the viral infection, prevent the spread of the viral infection, and/or delay or slow the progress of the viral infection, as compare the state and/or the condition of the viral infection in the absence of the therapeutic treatment.
  • the average number of monomers of gp120 binder or dimers of gp120 binders conjugated to an Fc domain monomer may be from 1 to 20 (e.g., the average value of T is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, or 15 to 20). In some embodiments, the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • the term “subject,” as used herein, can be a human or non-human primate, or other mammal, such as but not limited to dog, cat, horse, cow, pig, turkey, goat, fish, monkey, chicken, rat, mouse, or sheep.
  • terapéuticaally effective amount refers to an amount, e.g., pharmaceutical dose, effective in inducing a desired effect in a subject or in treating a subject having a condition or disorder described herein (e.g., a viral infection, such as an HIV infection). It is also to be understood herein that a “therapeutically effective amount” may be interpreted as an amount giving a desired therapeutic and/or preventative effect, taken in one or more doses or in any dosage or route, and/or taken alone or in combination with other therapeutic agents (e.g., an antiviral agent described herein).
  • an effective amount of a conjugate is, for example, an amount sufficient to prevent, slow down, or reverse the progression of the viral infection as compared to the response obtained without administration of the conjugate.
  • the term “pharmaceutical composition” refers to a medicinal or pharmaceutical formulation that contains at least one active ingredient (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)) as well as one or more excipients and diluents to enable the active ingredient suitable for the method of administration.
  • the pharmaceutical composition of the present disclosure includes pharmaceutically acceptable components that are compatible with a conjugate described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)).
  • a pharmaceutically acceptable carrier refers to an excipient or diluent in a pharmaceutical composition.
  • a pharmaceutically acceptable carrier may be a vehicle capable of suspending or dissolving the active conjugate (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)).
  • the pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient.
  • the pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to a conjugate described herein. The nature of the carrier differs with the mode of administration.
  • a solid carrier for oral administration, a solid carrier is preferred; for intravenous administration, an aqueous solution carrier (e.g., WFI, and/or a buffered solution) is generally used.
  • a pharmaceutically acceptable salt represents salts of the conjugates described herein (e.g., conjugates of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)) that are, within the scope of sound medical judgment, suitable for use in methods described herein without undue toxicity, irritation, and/or allergic response.
  • Pharmaceutically acceptable salts are well known in the art.
  • gp120 binder refers to a moiety, such as a small molecule (e.g., temsavir, BMS-818251, DMJ-II-121, BNM-IV-147 or analogs thereof) that binds to the HIV gp120 glycoprotein.
  • Gp120 binders of the invention include compounds described by formula (A-I), preferably temsavir, BMS-818251, DMJ-II-121, BNM-IV-147, or an analog thereof.
  • the term “about,” as used herein, indicates a deviation of ⁇ 5%. For example, about 10% refers to from 9.5% to 10.5%. Any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.
  • the term “(1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)”, as used herein, represents the formulas of any one of (D-IV-7), (D-IV-8), (D-V-6), (D-V-7), (D-V-8), (D-V-9), (D-VI-8), (M-I), (M-II), (M-III), (M-III-1), (M-III-2), (M-III-3), (M-III-4), (M-III-5), (M-III-6), (M-IV), (M-IV-1), (M-IV-2), (M-IV-3), (M-IV-4), (M-IV-5), (M- IV-6), (M-IV-7), (M-IV-8), (M-IV-9), (M-V), (M-V-1), (M-V-2), (M-V-3), (M-V-4), (M-V-5), (M-V-6), (M-V-7), (M-
  • FIG.1 is an image depicting exemplary methods of conjugating a gp120 receptor inhibitor monomer or dimer, e.g., by way of a linker, to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide.
  • FIG.2 is an image depicting a method of conjugating a gp120 binder monomer or dimer, e.g., by way of a linker, to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide by oxime conjugation to an amino acid residue, e.g., a nitrogen atom of a surface exposed lysine.
  • an amino acid residue e.g., a nitrogen atom of a surface exposed lysine.
  • FIG.3 is an image depicting a method of conjugating a gp120 binder monomer or dimer, e.g., by way of a linker, to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide by thioether conjugation to an amino acid residue, e.g., a nitrogen atom of a surface exposed lysine.
  • FIG.4 is an image depicting a method of conjugating a gp120 binder monomer or dimer, e.g., by way of a linker, to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide by rebridged cysteine conjugation, e.g., rebridged cysteine conjugation to a pair of sulfur atoms of two hinge cysteines in an Fc domain monomer or Fc domain.
  • FIG.5 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence of SEQ ID NO: 1.
  • FIG.6 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence of SEQ ID NO: 3.
  • FIG.7 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence of SEQ ID NO: 5.
  • FIG.8 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence of SEQ ID NO: 7.
  • FIG.9 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence of SEQ ID NO: 9.
  • FIG.10 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence of SEQ ID NO: 12.
  • FIG.11 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence of SEQ ID NO: 14.
  • FIG.12 is a graph showing the binding of conjugates containing gp120 binders to the gp120 protein compared to a polyclonal goat anti-gp120 HRP (PA 1 -73097, Invitrogen) positive control and an unconjugated Fc molecule negative control.
  • PA 1 -73097 polyclonal goat anti-gp120 HRP
  • FIG.13 is a graph showing plasma levels of a conjugate including an Fc domain having a C220S mutation (SEQ ID NO: 64) (2 mpk IV) compared to a conjugate including an Fc domain having a C220S mutation and a YTE triple mutation (SEQ ID NO: 67) (2 mpk IV) in non-human primate PK studies determined by Fc capture. This study was performed as described in Example 40.
  • FIG.14 is an image depicting exemplary conjugates including a gp120 binder monomer or dimer and an Fc domain monomer or an Fc domain.
  • T is representative of the drug-to-antibody ratio (DAR) and depicts that multiple monomers or dimers can be conjugated to an Fc domain monomer or an Fc domain.
  • FIG.15 is a graph showing the 7-day mouse PK profiles of Conjugate 5b and Fc control (SEQ ID NO:73).
  • FIG.16 is a graph showing the percent reduction in viral cytopathic effect (CPE) of HIV-1IIIB and cell viability of CEM-SS cells by AZT at a concentration of 2,000 pM, 5,000 pM, 20,000 pM, 50,000 pM, 160,000 pM, and 500,000 pM.
  • CPE viral cytopathic effect
  • FIG.17 is a graph showing the percent reduction in viral CPE of HIV-1IIIB and cell viability of CEM- SS cells by the temsivir buffer at a dilution of 0, 00000002, and 0.000001.
  • FIG.18 is a graph showing the percent reduction in viral CPE of HIV-1IIIB and cell viability of CEM- SS cells by temsavir at a concentration of 0.64 pM, 3.2 pM, 16 pM, 80 pM, 400 pM, and 2,000 pM.
  • FIG.19 is a graph showing the percent reduction in viral CPE of HIV-1IIIB and cell viability of CEM- SS cells by the conjugate buffer at a dilution of 0, 0.0000001, 0.0000004, 0.000002, 0.00001, and 0.00005.
  • FIG.20 is a graph showing the percent reduction in viral CPE of HIV-1IIIB and cell viability of CEM- SS cells by Conjugate 5b at a concentration of 0.64 pM, 3.2 pM, 16 pM, 80 pM, 400 pM, and 2,000 pM.
  • FIG.21 is a graph showing the percent reduction in viral CPE of HIV-1IIIB and cell viability of CEM- SS cells by Conjugate 29a at a concentration of 0.64 pM, 3.2 pM, 16 pM, 80 pM, 400 pM, and 2,000 pM.
  • FIG.22 is a graph showing the percent reduction in viral CPE of HIV-1IIIB and cell viability of CEM- SS cells by Conjugate 29b at a concentration of 0.64 pM, 3.2 pM, 16 pM, 80 pM, 400 pM, and 2,000 pM.
  • FIG.23 is a graph showing the percent reduction in viral CPE of HIV-1IIIB and cell viability of CEM- SS cells by Conjugate 30a at a concentration of 0.64 pM, 3.2 pM, 16 pM, 80 pM, 400 pM, and 2,000 pM.
  • FIG.24 is a graph showing the percent reduction in viral CPE of HIV-1IIIB and cell viability of CEM- SS cells by Conjugate 37a at a concentration of 0.64 pM, 3.2 pM, 16 pM, 80 pM, 400 pM, and 2,000 pM.
  • FIG.25 is a graph showing the 7-day mouse PK profiles of Conjugate 5b, Conjugate 29a, and Fc control (SEQ ID NO: 72).
  • the disclosure features conjugates, compositions, methods for the treatment of viral infections (e.g., human immunodeficiency viral infections), and methods of synthesizing conjugates.
  • the conjugates disclosed herein include monomers or dimers of viral gp120 binders (e.g., temsavir, BMS- 818251, DMJ-II-121, BNM-IV-147, or analogs thereof) conjugated to Fc monomers, Fc domains, Fc- binding peptides, albumin proteins, or albumin protein-binding peptides.
  • the gp120 binder e.g., temsavir, BMS-818251, DMJ-II-121, BNM-IV-147, or analogs thereof
  • the gp120 binder targets the gp120 receptor on the surface of the viral particle.
  • the Fc monomers or Fc domains in the conjugates bind to Fc ⁇ Rs (e.g., FcRn, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb) on immune cells, e.g., neutrophils, to activate phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), thus leading to the engulfment and destruction of viral particles by immune cells and further enhancing the antiviral activity of the conjugates.
  • Fc ⁇ Rs e.g., FcRn, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb
  • immune cells e.g., neutrophils
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the albumin or albumin-binding peptide may extend the half-life of the conjugate, for example, by binding of albumin to the recycling neon
  • compositions are useful in methods for the inhibition of viral growth and in methods for the treatment of viral infections, such as those caused by an HIV-1 or HIV-2.
  • Viral Infections The compounds and pharmaceutical compositions described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)) can be used to treat a viral infection (e.g., an HIV-1 or HIV-2 viral infection).
  • a viral infection e.g., an HIV-1 or HIV-2 viral infection.
  • Viral infection refers to the pathogenic growth of a virus (e.g., the human immunodeficiency virus) in a host organism (e.g., a human subject).
  • a viral infection can be any situation in which the presence of a viral population(s) is damaging to a host body.
  • a subject is suffering from a viral infection when an excessive amount of a viral population is present in or on the subject’s body, or when the presence of a viral population(s) is damaging the cells or other tissue of the subject.
  • the human immunodeficiency viruses are two species of Lentivirus (a subgroup of retrovirus) that causes HIV infection and over time acquired immunodeficiency syndrome (AIDS).
  • AIDS is a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. Without treatment, average survival time after infection with HIV is estimated to be 9 to 11 years, depending on the HIV subtype.
  • HIV is a sexually transmitted infection and occurs by contact with or transfer of blood, pre-ejaculate, semen, and vaginal fluids.
  • Two types of HIV have been characterized: HIV-1 and HIV-2. HIV infects vital cells in the human immune system, such as helper T cells (specifically CD4+ T cells), macrophages, and dendritic cells. HIV infection leads to low levels of CD4+ T cells through a number of mechanisms, including pyroptosis of abortively infected T cells, apoptosis of uninfected bystander cells, direct viral killing of infected cells, and killing of infected CD4+ T cells by CD8+ cytotoxic lymphocytes that recognize infected cells.
  • helper T cells specifically CD4+ T cells
  • macrophages specifically dendritic cells
  • dendritic cells dendritic cells. HIV infection leads to low levels of CD4+ T cells through a number of mechanisms, including pyroptosis of abortively infected T cells
  • conjugates of the Disclosure Provided herein are synthetic conjugates useful in the treatment of viral infections (e.g., HIV infections).
  • the conjugates disclosed herein include an Fc domain monomer, an Fc domain, or an albumin protein conjugated to one or more monomers gp120 binders or one or more dimers of two gp120 binders (e.g., gp120 binders selected from temsavir, BMS-818251, DMJ-II-121, BNM-IV-147, or analogs thereof).
  • the dimers of two gp120 binders include a gp120 binder (e.g., a first gp120 binder of formula (A-I) or (A-II)) and a second gp120 binder (e.g., a second gp120 binder of formula(A-I) or (A-II)).
  • the first and second gp120 binders are linked to each other by way of a linker.
  • conjugates described herein bind to the surface of a viral particle (e.g., bind to viral gp120 receptor on the surface on an human immunodeficiency virus particle) through the interactions between the gp120 binder moieties in the conjugates and proteins on the surface of the viral particle.
  • the gp120 binder disrupts gp120, an envelope glycoprotein that binds with the CD4 receptor, particularly on helper T cells. Binding to CD4 initiates a cascade of conformational changes in gp120 and gp41 that lead to the fusion of the viral membrane with the host cell membrane, allowing the spread of the virus.
  • Conjugates of the invention include gp120 binder monomers and dimers conjugated to an Fc domain, Fc monomer, or Fc-binding peptide.
  • the Fc domain in the conjugates described herein binds to the Fc ⁇ Rs (e.g., FcRn, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb) on immune cells.
  • Conjugates of the invention include gp120 binder monomers and dimers conjugated to an albumin protein or an albumin protein-binding peptide.
  • the albumin protein or albumin protein-binding peptide may extend the half-life of the conjugate, for example, by binding of albumin to the recycling neonatal Fc receptor.
  • Conjugates provided herein are described by any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII).
  • the conjugates described herein include one or more monomers of gp120 binders conjugated to an Fc domain or an albumin protein.
  • the conjugates described herein include one or more dimers of gp120 binders conjugated to an Fc domain monomer, an Fc domain, or an albumin protein.
  • n is 2
  • E an Fc domain monomer
  • Conjugates described herein may be synthesized using available chemical synthesis techniques in the art.
  • a molecule may be derivatized using conventional chemical synthesis techniques that are well known in the art.
  • the conjugates described herein contain one or more chiral centers.
  • the conjugates include each of the isolated stereoisomeric forms as well as mixtures of stereoisomers in varying degrees of chiral purity, including racemic mixtures. It also encompasses the various diastereomers, enantiomers, and tautomers that can be formed.
  • Gp120 binders A component of the conjugates described herein is an HIV gp120 binder moiety. The gp120 binder disrupts gp120, an envelope glycoprotein that binds with the CD4 receptor, particularly on helper T-Cells.
  • Binding to CD4 initiates a cascade of conformational changes in gp120 and gp41 that lead to the fusion of the viral membrane with the host cell membrane, allowing the spread of the virus.
  • gp120 binders include temsavir, BMS-818251, DMJ-II-121, and BNM-IV-147.
  • derivatives of temsavir, BMS-818251, DMJ-II-121, and BNM-IV-147 such as those found in the literature, have gp120 binder activity and are useful as gp120 inhibitor moieties of the compounds herein (see, for example, Lu et al. Curr. Top. Med. Chem.16(10): 1074-1090).
  • Conjugates described herein are separated into two types: (1) one or more dimers of gp120 binders conjugated to an Fc domain monomer, an Fc domain, or an albumin protein and (2) one or more monomers of gp120 binders conjugated to an Fc domain monomer, an Fc domain, or an albumin protein.
  • the dimers of gp120 binders are linked to each other by way of a linker, such as the linkers described herein.
  • Viral gp120 binders of the invention include temsavir, BMS-818251, DMJ-II-121, BNM-IV-147, and analogs thereof, such as the viral gp120 binders of formula (A-I) and (A-II): wherein Q is selected from the group consisting of: , S is selected from the group consisting of: R 1 , R 2 , R 3 , are each independently selected from H, OH, halogen, nitrile, nitro, optionally substituted amine, optionally substituted sulfhydryl, optionally substituted carboxyl, optionally substituted C 1 -C 20 alkyl, optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 alkenyl, optionally substituted C 3 -C 20 cycloalkenyl, optionally substituted C 2 -C 20 alkynyl, optionally substituted C 5 -C 20 aryl, optionally substitute
  • each R 8 is independently selected from H, optionally substituted C 1 -C 20 alkyl, optionally substituted C 1 -C 20 alkylene, optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 heterocycloalkyl, optionally substituted C 5 -C 15 aryl, and optionally substituted C 2 -C 15 heteroaryl; each R 9 is independently selected from optionally substituted C 1 -C 20 alkylene, optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 heterocycloalkyl, optionally substituted C 5 -C 15 aryl, and optionally substituted C 2 -C 15 heteroaryl; x is 1 or 2; k is 0, 1, 2, 3, 4, or 5; Ar is selected from the group consisting of optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 heterocycloalkyl, optionally substituted C 5 -C
  • x is 2.
  • the gp120 inhibitor is selected from temsavir, BMS-818251, DMJ-II-121, or BNM-IV- 147: Conjugates of dimers of gp120 binders linked to an Fc domain or an albumin protein
  • the conjugates described herein include an Fc domain monomer, an Fc domain, an Fc-binding peptide, and albumin protein, or an albumin protein-binding peptide covalently linked to one or more dimers of gp120 binders.
  • the dimers of two gp120 binders include a first gp120 binder (e.g., a first viral gp120 binder of formula (A-I) or (A-II)) and a second gp120 binder (e.g., a second viral gp120 binder of formula (A-I) or (A-II)).
  • the first and second gp120 binders are linked to each other by way of a linker, such as a linker described herein.
  • the first and second gp120 binders are the same.
  • the first and second gp120 binders are different.
  • each A 1 -L-A 2 may be independently selected (e.g., independently selected from any of the A 1 -L-A 2 structures described herein).
  • E may be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different A 1 -L-A 2 moieties.
  • E is conjugated to a first A 1 -L-A 2 moiety, and a second A 1 -L-A 2 , moiety.
  • each of A 1 and A 2 of the first A 1 -L-A 2 moiety and of the second A 1 -L-A 2 moiety are independently selected from any structure described by formula (A-I) and (A-II):
  • x is 2.
  • the first A 1 -L-A 2 moiety is conjugated specifically to lysine residues of E (e.g., the nitrogen atoms of surface exposed lysine residues of E), and the second A 1 -L-A 2 moiety is conjugated specifically to cysteine residues of E (e.g., the sulfur atoms of surface exposed cysteine residues of E).
  • the first A 1 -L-A 2 moiety is conjugated specifically to cysteine residues of E (e.g., the sulfur atoms of surface exposed cysteine residues of E), and the second A 1 -L-A 2 moiety is conjugated specifically to lysine residues of E (e.g., the nitrogen atoms of surface exposed lysine residues of E).
  • the disclosure provides a conjugate, or a pharmaceutically acceptable salt thereof, described by any one any one of formulas (D-I), (D-II), (D-III), (D-III-1), (D-III-2), (D-III-3), (D-III- 4), (D-III-5), (D-III-6), (D-IV), (D-IV-1), (D-IV-2), (D-IV-3), (D-IV-4), (D-IV-5), (D-IV-6), (D-IV-7), (D-IV-8), (D-IV-9), (D-V), (D-V-1), (D-V-2), (D-V-3), (D-V-4), (D-V-5), (D-V-6), (D-V-7), (D-V-8), (D-V-9), (D-VI), (D- VI-1), (D-VI-2), (D-VI-3), (D-VI-4), (D-VI-5), (D-VI-6), (D-VI-7), (D-VI-8), (D-V
  • the squiggly line connected to E indicates that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) dimers of gp120 binders may be attached to an Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein- binding peptide.
  • one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) dimers of gp120 binders may be attached to an Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide.
  • one or more dimers of gp120 binders may be attached to an Fc domain.
  • the squiggly line in the conjugates described herein is not to be construed as a single bond between one or more dimers of gp120 binders and an atom in the Fc domain monomer, Fc domain, or albumin protein.
  • T when T is 1, one dimer of gp120 binders may be attached to an atom in the Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide.
  • two dimers of gp120 binders may be attached to an atom in the Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide.
  • a linker in a conjugate described herein e.g., L or L’
  • a linker in a conjugate described herein e.g., L or L’
  • the linker when the linker has three arms, two of the arms may be attached to the first and second gp120 binders and the third arm may be attached to the Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide.
  • conjugates having an Fc domain covalently linked to one or more dimers of gp120 binders as represented by the formulae above, when n is 2, two Fc domain monomers (each Fc domain monomer is represented by E) dimerize to form an Fc domain.
  • Conjugates of monomers of gp120 binders linked to an Fc domain monomer, an Fc domain, or an albumin protein include an Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide covalently linked to one or more monomers of gp120 binders.
  • Conjugates of an Fc domain monomer or albumin protein and one or more monomers of gp120 binders may be formed by linking the Fc domain monomer, Fc domain, or albumin protein to each of the monomers of gp120 binders through a linker, such as any of the linkers described herein.
  • the squiggly line connected to E indicates that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) monomers of gp120 binders may be attached to an Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide.
  • n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) monomers of gp120 binders may be attached to an Fc domain monomer, Fc domain, or an albumin protein.
  • one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) monomers of gp120 binders may be attached to an Fc domain.
  • the squiggly line in the conjugates described herein is not to be construed as a single bond between one or more monomers of gp120 binders and an atom in the Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide.
  • T when T is 1, one monomer of gp120 binder may be attached to an atom in the Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide. In some embodiments, when T is 2, two monomers of gp120 binders may be attached to an atom in the Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide.
  • each A 1 -L may be independently selected (e.g., independently selected from any of the A 1 -L structures described herein).
  • E may be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different A 1 -L moieties.
  • E is conjugated to a first A 1 -L moiety, and a second A 1 -L, moiety.
  • a 1 of each of the first A 1 -L moiety and of the second A 1 -L moiety is independently selected from any structure described by formula (A-I) or (A-II):
  • x is 2.
  • the first A 1 -L moiety is conjugated specifically to lysine residues of E (e.g., the nitrogen atoms of surface exposed lysine residues of E), and the second A 1 -L moiety is conjugated specifically to cysteine residues of E (e.g., the sulfur atoms of surface exposed cysteine residues of E).
  • the first A 1 -L moiety is conjugated specifically to cysteine residues of E (e.g., the sulfur atoms of surface exposed cysteine residues of E), and the second A 1 -L moiety is conjugated specifically to lysine residues of E (e.g., the nitrogen atoms of surface exposed lysine residues of E).
  • a linker in a conjugate having an Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide covalently linked to one or more monomers of the gp120 binders described herein (e.g., L or L’) may be a divalent structure having two arms.
  • One arm in a divalent linker may be attached to the monomer of the gp120 binder and the other arm may be attached to the Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide.
  • a conjugate containing an Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide covalently linked to one or more monomers of gp120 binders provided herein is described by any one of formulas (M-I), (M-II), (M-III), (M-III-1), (M-III- 2), (M-III-3), (M-III-4), (M-III-5), (M-III-6), (M-IV), (M-IV-1), (M-IV-2), (M-IV-3), (M-IV-4), (M-IV-5), (M-IV-6), (M-IV-7), (M-IV-8), (M-IV-9), (M-V), (M-V-1), (M-V-2), (M-V-3), (M-V-4), (M-V-5), (M-V-6), (M-V-7), (M-V- 8), (M-V-9), (M-VI), (M-VI), (M-
  • Fc domain monomers and Fc domains An Fc domain monomer includes a hinge domain, a C H 2 antibody constant domain, and a C H 3 antibody constant domain.
  • the Fc domain monomer can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD.
  • the Fc domain monomer can also be of any immunoglobulin antibody isotype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4).
  • the Fc domain monomer can be of any immunoglobulin antibody allotype (e.g., IGHG1*01 (i.e., G1m(za)), IGHG1*07 (i.e., G1m(zax)), IGHG1*04 (i.e., G1m(zav)), IGHG1*03 (G1m(f)), IGHG1*08 (i.e., G1m(fa)), IGHG2*01, IGHG2*06, IGHG2*02, IGHG3*01, IGHG3*05, IGHG3*10, IGHG3*04, IGHG3*09, IGHG3*11, IGHG3*12, IGHG3*06, IGHG3*07,
  • the Fc domain monomer can also be of any species, e.g., human, murine, or mouse.
  • a dimer of Fc domain monomers is an Fc domain that can bind to an Fc receptor, which is a receptor located on the surface of leukocytes.
  • an Fc domain monomer in the conjugates described herein may contain one or more amino acid substitutions, additions, and/or deletion relative to an Fc domain monomer having a sequence of any one of SEQ ID NOs: 1-95 and 125-153.
  • an Asn in an Fc domain monomer in the conjugates as described herein may be replaced by Ala in order to prevent N- linked glycosylation (see, e.g., SEQ ID NOs: 12-15, where Asn to Ala substitution is labeled with *).
  • an Fc domain monomer in the conjugates described herein may also containing additional Cys additions (see, e.g., SEQ ID NOs: 9, 10, and 11, where Cys additions are labeled with *).
  • an Fc domain monomer in the conjugates as described herein includes an additional moiety, e.g., an albumin-binding peptide, a purification peptide (e.g., a hexa-histidine peptide (HHHHHH (SEQ ID NO: 99)), or a signal sequence (e.g., IL2 signal sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 100)) attached to the N- or C-terminus of the Fc domain monomer.
  • an additional moiety e.g., an albumin-binding peptide, a purification peptide (e.g., a hexa-histidine peptide (HHHHHH (SEQ ID NO: 99)), or a signal sequence (e.g., IL2 signal sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 100)) attached to the N- or C-terminus of the Fc domain monomer.
  • an Fc domain monomer in the conjugate does not contain any type of antibody variable region, e.g., VH, VL, a complementarity determining region (CDR), or a hypervariable region (HVR).
  • an Fc domain monomer in the conjugates as described herein may have a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to the sequence of any one of SEQ ID NOs: 1-95 and 125-153 shown below.
  • an Fc domain monomer in the conjugates as described herein may have a sequence of any one of SEQ ID NOs: 1-95 and 125-153 shown below.
  • SEQ ID NO: 1 murine Fc-IgG2a with IL2 signal sequence at the N-terminus (bold) MYRMQLLSCIALSLALVTNSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 2: mature murine Fc-IgG2a PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTLRVVSA
  • the variant Fc domain monomer includes at least the following mutations K246X, M252Y, S254T, and T256E, where X is not Lys. In some embodiments, the variant Fc domain monomer includes at least the following mutations K246X, V309D, Q311H, and N434S, where X is not Lys. In some embodiments, the variant Fc domain monomer includes at least the following mutations K246X, M428L, and N434S, where X is not Lys. In some embodiments, the variant Fc domain further includes a mutation of position 220, e.g., a C220S mutation.
  • a variant Fc domain monomer includes a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to the sequence of any one of SEQ ID NOs: 125-153 shown below.
  • a variant Fc domain monomer includes the sequence of any one of SEQ ID NOs: 125-153 shown below.
  • a variant Fc domain monomer includes at least the following mutations K246X, M252Y, S254T, and T256E, where X is not Lys.
  • a variant Fc domain monomer includes at least the following mutations K246X, V309D, Q311H, and N434S, where X is not Lys. In some embodiments, a variant Fc domain monomer includes at least the following mutations K246X, M428L, and N434S, where X is not Lys. In some embodiments, the substitution at K246X is selected from Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp. In some embodiments, the substitution at K246X is Ser.
  • SEQ ID NO: 125 mature human IgG1 Fc; X 1 (position 201) is Asn or absent; X 2 (position 220) is Cys or Ser; X 3 (position 246) is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X 4 (position 252) is Met or Tyr; X 5 (position 254) is Ser or Thr; X 6 (position 256) is Thr or Glu; X 7 (position 297) is Asn or Ala; X 8 (position 309) is Leu or Asp; X 9 (position 311) is Gln or His; X 10 (position 356) is Asp or Glu; and X 11 (position 358) is Leu or Met; X 12 (position 428) is Met or Leu; X 13 (position 434) is Asn or Ser; X 14 (position 447) is Lys or absent; N-terminal Fab residues are underlined; hinge residues are italicized X 1
  • An Fc domain forms the minimum structure that binds to an Fc receptor, e.g., Fc-gamma receptors (i.e., Fc ⁇ receptors (Fc ⁇ R)), Fc-alpha receptors (i.e., Fc ⁇ receptors (Fc ⁇ R)), Fc-epsilon receptors (i.e., Fc ⁇ receptors (Fc ⁇ R)), and/or the neonatal Fc receptor (FcRn).
  • Fc-gamma receptors i.e., Fc ⁇ receptors (Fc ⁇ R)
  • Fc-alpha receptors i.e., Fc ⁇ receptors (Fc ⁇ R)
  • Fc-epsilon receptors i.e., Fc ⁇ receptors (Fc ⁇ R)
  • FcRn neonatal Fc receptor
  • an Fc domain of the present invention binds to an Fc ⁇ receptor (e.g., FcRn, Fc ⁇ RI (CD64), Fc ⁇ RIIa (CD32), Fc ⁇ RIIb (CD32), Fc ⁇ RIIIa (CD16a), Fc ⁇ RIIIb (CD16b)), and/or Fc ⁇ RIV and/or the neonatal Fc receptor (FcRn).
  • FcRn Fc ⁇ receptor
  • FcRn Fc ⁇ RI
  • Fc ⁇ RI CD64
  • Fc ⁇ RIIa CD32
  • Fc ⁇ RIIb CD32
  • Fc ⁇ RIIIa CD16a
  • Fc ⁇ RIIIb CD16b
  • FcRn neonatal Fc receptor
  • the Fc domain monomer or Fc domain of the invention is an aglycosylated Fc domain monomer or Fc domain (e.g., an Fc domain monomer or an Fc domain that maintains engagement to an Fc receptor (e.g., FcRn
  • the Fc domain is an aglycosylated IgG1 variants that maintains engagement to an Fc receptor (e.g., an IgG1 having an amino acid substitution at N297 and/or T299 of the glycosylation motif).
  • an Fc receptor e.g., an IgG1 having an amino acid substitution at N297 and/or T299 of the glycosylation motif.
  • Exemplary aglycosylated Fc domains and methods for making aglycosylated Fc domains are known in the art, for example, as described in Sazinsky S.L. et al., Aglycosylated immunoglobulin G1 variants productively engage activating Fc receptors, PNAS, 2008, 105(51):20167-20172, which is incorporated herein in its entirety.
  • the Fc domain or Fc domain monomer of the invention is engineered to enhance binding to the neonatal Fc receptor (FcRn).
  • the Fc domain may include the triple mutation corresponding to M252Y/S254T/T256E (YTE) (e.g., an IgG1, such as a human or humanized IgG1 having a YTE mutation, for example SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57).
  • YTE M252Y/S254T/T256E
  • the Fc domain may include the double mutant corresponding to M428L/N434S (LS) (e.g., an IgG1, such as a human or humanized IgG1 having an LS mutation, such as SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 59).
  • the Fc domain may include the single mutant corresponding to N434H (e.g., an IgG1, such as a human or humanized IgG1 having an N434H mutation).
  • the Fc domain may include the single mutant corresponding to C220S (e.g., and IgG1, such as a human or humanized IgG1 having a C220S mutation, such as SEQ ID NO: 34, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68).
  • C220S e.g., and IgG1, such as a human or humanized IgG1 having a C220S mutation, such as SEQ ID NO: 34, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, S
  • the Fc domain may include a combination of one or more of the above-described mutations that enhance binding to the FcRn.
  • Enhanced binding to the FcRn may increase the half-life Fc domain-containing conjugate.
  • incorporation of one or more amino acid mutations that increase binding to the FcRn e.g., a YTE mutation, an LS mutation, or an N434H mutation
  • Exemplary Fc domains with enhanced binding to the FcRN and methods for making Fc domains having enhanced binding to the FcRN are known in the art, for example, as described in Maeda, A. et al., Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody, MABS, 2017, 9(5):844-853, which is incorporated herein in its entirety.
  • an amino acid “corresponding to” a particular amino acid residue e.g., of a particular SEQ ID NO.
  • any one of SEQ ID NOs: 1-95 and 125- 153 may be mutated to include a YTE mutation, an LS mutation, and/or an N434H mutation by mutating the “corresponding residues” of the amino acid sequence.
  • a sulfur atom “corresponding to” a particular cysteine residue of a particular SEQ ID NO. should be understood to include the sulfur atom of any cysteine residue that one of skill in the art would understand to align to the particular cysteine of the particular sequence.
  • the protein sequence alignment of human IgG1 (UniProtKB: P01857; SEQ ID NO: 121), human IgG2 (UniProtKB: P01859; SEQ ID NO: 122), human IgG3 (UniProtKB: P01860; SEQ ID NO: 123), and human IgG4 (UniProtKB: P01861; SEQ ID NO: 124) is provided below (aligned with Clustal Omega Multiple Pairwise Alignment).
  • the alignment indicates cysteine residues (e.g., sulfur atoms of cysteine residues) that “correspond to” one another (in boxes and indicated by the • symbol).
  • Cys10 of SEQ ID NO: 10 corresponds to, for example, Cys109 of IgG1, Cys106 of IgG2, Cys156 of IgG3, Cys29 of SEQ ID NO: 1, Cys9 of SEQ ID NO: 2, Cys30 of SEQ ID NO: 3, or Cys10 of SEQ ID NO: 10.
  • the Fc domain or Fc domain monomer of the invention has the sequence of any one of SEQ ID NOs: 39-95 may further include additional amino acids at the N-terminus (Xaa)x and/or additional amino acids at the C-terminus (Xaa)z, wherein Xaa is any amino acid and x and z are a whole number greater than or equal to zero, generally less than 100, preferably less than 10 and more preferably 0, 1, 2, 3, 4, or 5.
  • the additional amino acids are least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to one or more consecutive amino acids of SEQ ID NO: 103.
  • the additional amino acids may be a single amino acid on the C- terminus corresponding to Lys330 of IgG1 (SEQ ID NO: 121).
  • a nitrogen atom “corresponding to” a particular lysine residue of a particular SEQ ID NO. should be understood to include the nitrogen atom of any lysine residue that one of skill in the art would understand to align to the particular lysine of the particular sequence.
  • human IgG1 UniProtKB: P01857; SEQ ID NO: 121
  • human IgG2 UniProtKB: P01859; SEQ ID NO: 122
  • human IgG3 UniProtKB: P01860; SEQ ID NO: 123
  • human IgG4 UniProtKB: P01861; SEQ ID NO: 1244
  • the alignment indicates lysine residues (e.g., nitrogen atoms of lysine residues) that “correspond to” one another (in boxes and indicated by the * symbol).
  • Lys35 of SEQ ID NO: 10 corresponds to, for example, Lys129 of IgG1, Lys126 of IgG2, Lys176 of IgG3, Lys51 of SEQ ID NO: 1, Lys31 of SEQ ID NO: 2, Lys50 of SEQ ID NO: 3, or Lys30 of SEQ ID NO: 10.
  • the Fc domain monomer includes less than about 300 amino acid residues (e.g., less than about 300, less than about 295, less than about 290, less than about 285, less than about 280, less than about 275, less than about 270, less than about 265, less than about 260, less than about 255, less than about 250, less than about 245, less than about 240, less than about 235, less than about 230, less than about 225, or less than about 220 amino acid residues).
  • amino acid residues e.g., less than about 300, less than about 295, less than about 290, less than about 285, less than about 280, less than about 275, less than about 270, less than about 265, less than about 260, less than about 255, less than about 250, less than about 245, less than about 240, less than about 235, less than about 230, less than about 225, or less than about 220 amino acid residues.
  • the Fc domain monomer is less than about 40 kDa (e.g., less than about 35kDa, less than about 30kDa, less than about 25kDa).
  • the Fc domain monomer includes at least 200 amino acid residues (e.g., at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 amino residues).
  • the Fc domain monomer is at least 20 kDa (e.g., at least 25 kDa, at least 30 kDa, or at least 35 kDa).
  • the Fc domain monomer includes 200 to 400 amino acid residues (e.g., 200 to 250, 250 to 300, 300 to 350, 350 to 400, 200 to 300, 250 to 350, or 300 to 400 amino acid residues).
  • the Fc domain monomer is 20 to 40 kDa (e.g., 20 to 25 kDa, 25 to 30 kDa, 35 to 40 kDa, 20 to 30 kDa, 25 to 35 kDa, or 30 to 40 KDa).
  • the Fc domain monomer includes an amino acid sequence at least 90% identical (e.g., at least 95%, at least 98%) to the sequence of any one of SEQ ID NOs: 1-95 and 125-153, or a region thereof. In some embodiments, the Fc domain monomer includes the amino acid sequence of any one of SEQ ID NOs: 1-95 and 125-153, or a region thereof. In some embodiments, the Fc domain monomer includes a region of any one of SEQ ID NOs: 1- 95 and 125-153, wherein the region includes positions 220, 252, 254, and 256.
  • the region includes at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acids residues, at least 80 amino acids residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 110 amino acid residues, at least 120 amino residues, at least 130 amino acid residues, at least 140 amino acid residues, at least 150 amino acid residues, at least 160 amino acid residues, at least 170 amino acid residues, at least 180 amino acid residues, at least 190 amino acid residues, or at least 200 amino acid residues.
  • Fc-gamma receptors bind the Fc portion of immunoglobulin G (IgG) and play important roles in immune activation and regulation.
  • IgG immunoglobulin G
  • ICs immune complexes
  • the human Fc ⁇ R family contains several activating receptors (Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb) and one inhibitory receptor (Fc ⁇ RIIb).
  • Fc ⁇ R signaling is mediated by intracellular domains that contain immune tyrosine activating motifs (ITAMs) for activating Fc ⁇ Rs and immune tyrosine inhibitory motifs (ITIM) for inhibitory receptor Fc ⁇ RIIb.
  • ITAMs immune tyrosine activating motifs
  • ITIM immune tyrosine inhibitory motifs
  • Fc ⁇ R binding by Fc domains results in ITAM phosphorylation by Src family kinases; this activates Syk family kinases and induces downstream signaling networks, which include PI3K and Ras pathways.
  • the portion of the conjugates including monomers or dimers of gp120 binders bind to and inhibits viral gp120 receptor leading to inhibition of viral replication, while the Fc domain portion of the conjugates bind to Fc ⁇ Rs (e.g., FcRn, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb) on immune cells and activate phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), thus leading to the engulfment and destruction of viral particles by immune cells and further enhancing the antiviral activity of the conjugates.
  • Fc ⁇ Rs e.g., FcRn, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • immune cells that may be activated by the conjugates described herein include, but are not limited to, macrophages, neutrophils, eosinophils, basophils, lymphocytes, follicular dendritic cells, natural killer cells, and mast cells.
  • Tissue distribution After a therapeutic enters the systemic circulation, it is distributed to the body’s tissues. Distribution is generally uneven because of different in blood perfusion, tissue binding, regional pH, and permeability of cell membranes. The entry rate of a drug into a tissue depends on the rate of blood flow to the tissue, tissue mass, and partition characteristics between blood and tissue.
  • the conjugates described herein may be optimized to distribute to lung tissue.
  • the conjugates have a concentration ratio of distribution in epithelial lining fluid of at least 30% the concentration of the conjugate in plasma within 2 hours after administration.
  • ratio of the concentration is at least 45% within 2 hours after administration.
  • the ratio of concentration is at least 55% within 2 hours after administration.
  • the ratio of concentration is at least 60% within 2 hours after administration.
  • a conjugate having an Fc domain (SEQ ID NO: 64) decorated with one or more small molecule antiviral inhibitors ELF levels are surprisingly ⁇ 60% of plasma exposure levels as measured by AUC across the rest of the time course indicating nearly immediate partitioning of the conjugate from plasma to the ELF in the lung.
  • An albumin protein of the invention may be a naturally-occurring albumin or a variant thereof, such as an engineered variant of a naturally-occurring albumin protein.
  • Variants include polymorphisms, fragments such as domains and sub-domains, and fusion proteins.
  • An albumin protein may include the sequence of an albumin protein obtained from any source. Preferably the source is mammalian, such as human or bovine. Most preferably, the albumin protein is human serum albumin (HSA), or a variant thereof. Human serum albumins include any albumin protein having an amino acid sequence naturally occurring in humans, and variants thereof.
  • An albumin protein coding sequence is obtainable by methods know to those of skill in the art for isolating and sequencing cDNA corresponding to human genes.
  • An albumin protein of the invention may include the amino acid sequence of human serum albumin (HSA), provided in SEQ ID NO: 96 or SEQ ID NO: 97, or the amino acid sequence of mouse serum albumin (MSA), provided in SEQ ID NO: 98, or a variant or fragment thereof, preferably a functional variant or fragment thereof.
  • a fragment or variant may or may not be functional, or may retain the function of albumin to some degree.
  • a fragment or variant may retain the ability to bind to an albumin receptor, such as HSA or MSA, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 105% of the ability of the parent albumin (e.g., the parent albumin from which the fragment or variant is derived).
  • the albumin protein may be a naturally-occurring polymorphic variant of an albumin protein, such as human serum albumin. Generally, variants or fragments of human serum albumin will have at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, or 70%, and preferably 80%, 90%, 95%, 100%, or 105% or more of human serum albumin or mouse serum albumin’s ligand binding activity.
  • the albumin protein may include the amino acid sequence of bovine serum albumin.
  • Bovine serum albumin proteins include any albumin having an amino acid sequence naturally occurring in cows, for example, as described by Swissprot accession number P02769, and variants thereof as defined herein.
  • Bovine serum albumin proteins also includes fragments of full-length bovine serum albumin or variants thereof, as defined herein.
  • the albumin protein may comprise the sequence of an albumin derived from one of serum albumin from dog (e.g., Swissprot accession number P49822-1), pig (e.g., Swissprot accession number P08835-1), goat (e.g., Sigma product no.
  • cat e.g., Swissprot accession number P49064-1
  • chicken e.g., Swissprot accession number P19121-1
  • ovalbumin e.g., chicken ovalbumin
  • turkey ovalbumin e.g., Swissprot accession number O73860-1
  • donkey e.g., Swissprot accession number Q5XLE4-1
  • guinea pig e.g., Swissprot accession number Q6WDN9-1
  • hamster e.g., as described in DeMarco et al.
  • horse e.g., Swissprot accession number P35747-1
  • rhesus monkey e.g., Swissprot accession number Q28522-1
  • mouse e.g., Swissprot accession number P07724-1
  • pigeon e.g., as defined by Khan et al. Int. J. Biol. Macromol.30(3-4),171-8 (2002)
  • rabbit e.g., Swissprot accession number P49065-1
  • rat e.g., Swissprot accession number P02770-1
  • sheep e.g., Swissprot accession number P14639-1
  • albumin proteins of the invention include variants of naturally-occurring albumin proteins.
  • a variant albumin refers to an albumin protein having at least one amino acid mutation, such as an amino acid mutation generated by an insertion, deletion, or substitution, either conservative or non-conservative, provided that such changes result in an albumin protein for which at least one basic property has not been significantly altered (e.g., has not been altered by more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%).
  • Exemplary properties which may define the activity of an albumin protein include binding activity (e.g., including binding specificity or affinity to bilirubin, or a fatty acid such as a long-chain fatty acid), osmolarity, or behavior in a certain pH-range.
  • an albumin protein variant will have at least 40%, at least 50%, at least 60%, and preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with a naturally-occurring albumin protein, such as the albumin protein of any one of SEQ ID NOs: 96-98.
  • Methods for the production and purification of recombinant human albumins are well-established (Sleep et al. Biotechnology, 8(1):42-6 (1990)), and include the production of recombinant human albumin for pharmaceutical applications (Bosse et al. J Clin Pharmacol 45(1):57-67 (2005)).
  • HSA three-dimensional structure of HSA has been elucidated by X-ray crystallography (Carter et al. Science. 244(4909): 1195-8(1998)); Sugio et al. Protein Eng.12(6):439-46 (1999)).
  • the HSA polypeptide chain has 35 cysteine residues, which form 17 disulfide bonds, and one unpaired (e.g., free) cysteine at position 34 of the mature protein. Cys-34 of HSA has been used for conjugation of molecules to albumin (Leger et al. Bioorg Med Chem Lett 14(17):4395-8 (2004); Thibaudeau et al. Bioconjug Chem 16(4):1000-8 (2005)), and provides a site for site-specific conjugation.
  • SEQ ID NO: 96 Human serum albumin (HSA), variant 1) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLF GDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYL YEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGER AFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECC EKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRL AKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRY
  • the albumin protein may be conjugated to any compound of the invention by any method well-known to those of skill in the art for producing small-molecule-protein conjugates. This may include covalent conjugation to a solvent-exposed amino acid, such as a solvent exposed cysteine or lysine.
  • human serum albumin may be conjugated to a compound of the invention by covalent linkage to the sulfur atom corresponding to Cys34 of SEQ ID NO: 96 or Cys40 of SEQ ID NO: 97.
  • An albumin protein of the invention may be conjugated to any compound of the invention by way of an amino acid located within 10 amino acid residues of the C-terminal or N-terminal end of the albumin protein.
  • An albumin protein may include a C-terminal or N-terminal polypeptide fusion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more amino acid.
  • the C-terminal or N-terminal polypeptide fusion may include one or more solvent-exposed cysteine or lysine residues, which may be used for covalent conjugation of a compound of the invention (e.g., conjugation to a gp120 binder monomer or dimer, including by way of a linker).
  • Albumin proteins of the invention include any albumin protein which has been engineered to include one or more solvent-exposed cysteine or lysine residues, which may provide a site for conjugation to a compound of the invention (e.g., conjugation to a gp120 binder monomer or dimer, including by way of a linker). Most preferably, the albumin protein will contain a single solvent-exposed cysteine or lysine, thus enabling site-specific conjugation of a compound of the invention. Exemplary methods for the production of engineered variants of albumin proteins that include one or more conjugation-competent cysteine residues are provided in U.S. Patent Application No. 2017/0081389, which is incorporated herein by reference in its entirety.
  • albumin protein variants are those comprising a single, solvent-exposed, unpaired (e.g., free) cysteine residue, thus enabling site-specific conjugation of a linker to the cysteine residue.
  • Albumin proteins which have been engineered to enable chemical conjugation to a solvent- exposed, unpaired cysteine residue include the following albumin protein variants: (a) an albumin protein having a substitution of a non-cysteine amino acid residue with a cysteine at an amino acid residue corresponding to any of L585, D1, A2, D562, A364, A504, E505, T79, E86, D129, D549, A581, D121, E82, S270, Q397, and A578 of SEQ ID NO: 96; (b) an albumin protein having an insertion of a cysteine at a position adjacent the N- or C-terminal side of an amino acid residue corresponding to any of L585, D1, A2, D562, A364, A504, E505, T79,
  • the net result of the substitution, deletion, addition, or insertion events of (a), (b), (c) and/or (d) is that the number of conjugation competent cysteine residues of the polypeptide sequence is increased relative to the parent albumin sequence.
  • the net result of the substitution, deletion, addition, or insertion events of (a), (b), (c) and/or (d) is that the number of conjugation competent-cysteine residues of the polypeptide sequence is one, thus enabling site-specific conjugation.
  • Preferred albumin protein variants also include albumin proteins having a single solvent-exposed lysine residue, thus enabling site-specific conjugation of a linker to the lysine residue.
  • Such variants may be generated by engineering an albumin protein, including any of the methods previously described (e.g., insertion, deletion, substitution, or C-terminal or N-terminal fusion).
  • Albumin protein-binding peptides Conjugation of a biologically-active compound to an albumin protein-binding peptide can alter the pharmacodynamics of the biologically-active compound, including the alteration of tissue uptake, penetration, and diffusion.
  • conjugation of an albumin protein-binding peptide to a compound of the invention e.g., a gp120 binder monomer or dimer, by way of a linker
  • Albumin protein-binding peptides of the invention include any polypeptide having an amino acid sequence of 5 to 50 (e.g., 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or 10 to 20) amino acid residues that has affinity for and functions to bind an albumin protein, such as any of the albumin proteins described herein.
  • the albumin protein-binding peptide binds to a naturally occurring serum albumin, most preferably human serum albumin.
  • An albumin protein-binding peptide can be of different origins, e.g., synthetic, human, mouse, or rat.
  • Albumin protein-binding peptides of the invention include albumin protein-binding peptides which have been engineered to include one or more (e.g., two, three, four, or five) solvent-exposed cysteine or lysine residues, which may provide a site for conjugation to a compound of the invention (e.g., conjugation to a gp120 binder monomer or dimer, including by way of a linker). Most preferably, the albumin protein-binding peptide will contain a single solvent-exposed cysteine or lysine, thus enabling site-specific conjugation of a compound of the invention.
  • Albumin protein-binding peptides may include only naturally occurring amino acid residues, or may include one or more non-naturally occurring amino acid residues. Where included, a non-naturally occurring amino acid residue (e.g., the side chain of a non-naturally occurring amino acid residue) may be used as the point of attachment for a compound of the invention (e.g., a gp120 binder monomer or dimer, including by way of a linker). Albumin protein-binding peptides of the invention may be linear or cyclic. Albumin protein- binding peptides of the invention include any albumin protein-binding peptides known to one of skill in the art, examples of which, are provided herein.
  • Albumin protein-binding peptide and conjugates including an albumin protein-binding peptide, preferably bind an albumin protein (e.g., human serum albumin) with an affinity characterized by a dissociation constant, Kd, that is less than about 100 ⁇ M, preferably less than about 100 nM, and most preferably do not substantially bind other plasma proteins.
  • an albumin protein e.g., human serum albumin
  • Kd dissociation constant
  • Specific examples of such compounds are linear or cyclic peptides, preferably between about 10 and 20 amino acid residues in length, optionally modified at the N-terminus or C-terminus or both.
  • Albumin protein-binding peptides include linear and cyclic peptides comprising the following general formulae, wherein Xaa is any amino acid: SEQ ID NO: 101 Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys SEQ ID NO: 102 Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe SEQ ID NO: 103 Cys-Tyr-Xaa-Pro-Gly-Xaa-Cys SEQ ID NO: 104 Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp SEQ ID NO: 105 Trp-Cys-
  • albumin protein-binding peptides are provided in U.S. Patent Application No. 2005/0287153, which is incorporated herein by reference in its entirety.
  • Conjugation of albumin protein-binding peptides An albumin protein-binding peptide of the invention may be conjugated to (e.g., by way of a covalent bond) to any compound of the invention (e.g., by way of the linker portion of a gp120 binder monomer or dimer).
  • the albumin protein-binding peptide may be conjugated to any compound of the invention by any method known to those of skill in the art for producing peptide-small molecule conjugates.
  • This may include covalent conjugation to the side chain group of an amino acid residue, such as a cysteine, a lysine, or a non-natural amino acid.
  • covalent conjugation may occur at the C-terminus (e.g., to the C-terminal carboxylic acid, or to the side chain group of the C-terminal residue) or at the N-terminus (e.g., to the N-terminal amino group, or to the side chain group of the N- terminal amino acid).
  • Linkers A linker refers to a linkage or connection between two or more components in a conjugate described herein (e.g., between two gp120 binders in a conjugate described herein, between a gp120 binder and an Fc domain monomer, an Fc domain, or an albumin protein in a conjugate described herein, and between a dimer of two gp120 binders and an Fc domain monomer, an Fc domain or an albumin protein in a conjugate described herein).
  • Linkers in conjugates having an Fc domain monomer, an Fc domain, or an albumin protein covalently linked to dimers of gp120 binders In a conjugate containing an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide covalently linked to one or more dimers of gp120 binders as described herein, a linker in the conjugate (e.g., L or L’) may be a branched structure.
  • a linker in a conjugate described herein may be a multivalent structure, e.g., a divalent or trivalent structure having two or three arms, respectively.
  • the linker when the linker has three arms, two of the arms may be attached to the first and second gp120 binders and the third arm may be attached to an Fc domain monomer, an Fc domain, an Fc- binding peptide, an albumin protein, or an albumin protein-binding peptide.
  • one arm may be attached to an Fc domain monomer, an Fc domain, or an albumin protein and the other arm may be attached to one of the two gp120 binders.
  • a linker with three arms may be used to attach the two gp120 binders on a conjugate containing an Fc domain monomer, an Fc domain, or albumin protein covalently linked to one or more dimers of gp120 binders.
  • a linker in a conjugate having an Fc domain monomer, an Fc domain, or an albumin protein covalently linked to one or more dimers of gp120 binders is described by formula (D-L- I): wherein L A is described by formula G A1 -(Z A1 )g1-(Y A1 )h1-(Z A2 )i1-(Y A2 )j1-(Z A3 )k1-(Y A3 )l1-(Z A4 )m1-(Y A4 )n1-(Z A5 )o1- G A2 ; L B is described by formula G B1 -(Z B1 ) g2 -(Y B1 ) h2 -(Z B2 ) i2 -(Y B2 ) j2 -(Z B3 ) k2 -(Y B3 ) l2 -(Z B4 ) m2 -(Y B4 )
  • optionally substituted includes substitution with a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • a PEG has a repeating unit structure (-CH 2 CH 2 O-)n, wherein n is an integer from 2 to 100.
  • a polyethylene glycol may be selected from any one of PEG 2 to PEG 100 (e.g., PEG 2 , PEG 3 , PEG 4 , PEG 5 , PEG 5 -PEG 10 , PEG 10 -PEG 20 , PEG 20 -PEG 30 , PEG 30 -PEG 40 , PEG 50 -PEG 60 , PEG 60 -PEG 70 , PEG 70 -PEG 80 , PEG 80 -PEG 90 , PEG 90 -PEG 100 ).
  • L C may have two points of attachment to the Fc domain (e.g., two G C2 ).
  • L includes a polyethylene glycol (PEG) linker.
  • a PEG linker includes a linker having the repeating unit structure (-CH 2 CH 2 O-)n, where n is an integer from 2 to 100.
  • a polyethylene glycol linker may covalently join a gp120 binder and E (e.g., in a conjugate of any one of formulas (M-I)-(M-X)).
  • a polyethylene glycol linker may covalently join a first gp120 binder and a second gp120 binder (e.g., in a conjugate of any one of formulas (D-I)-(D-X)).
  • a polyethylene glycol linker may covalently join a gp120 binder dimer and E (e.g., in a conjugate of any one of formulas (D-I)-(D-X)).
  • a polyethylene glycol linker may be selected from any one of PEG 2 to PEG 100 (e.g., PEG 2 , PEG 3 , PEG 4 , PEG 5 , PEG 5 -PEG 10 , PEG 10 -PEG 20 , PEG 20 -PEG 30 , PEG 30 -PEG 40 , PEG 50 -PEG 60 , PEG 60 -PEG 70 , PEG 70 - PEG 80 , PEG 80 -PEG 90 , PEG 90 -PEG 100 ).
  • L c includes a PEG linker, where L C is covalently attached to each of Q i and E.
  • Linkers in conjugates having an Fc domain monomer, an Fc domain, or an albumin protein covalently linked to monomers of gp120 binders In a conjugate containing an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide covalently linked to one or more monomers of gp120 binders as described herein, a linker in the conjugate (e.g., L, or L’) may be a divalent structure having two arms.
  • One arm in a divalent linker may be attached to the monomer of gp120 binder and the other arm may be attached to the Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide.
  • the one or more monomers of gp120 binders in the conjugates described herein may each be, independently, connected to an atom in the Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein- binding peptide.
  • each L is described by formula (M-L): J 1 -(Q 1 )g-(T 1 )h-(Q 2 )i-(T 2 )j-(Q 3 )k-(T 3 )l-(Q 4 )m-(T 4 )n-(Q 5 )o-J 2 wherein J 1 is a bond attached to A 1 ; J 2 is a bond attached to E or a functional group capable of reacting with a functional group conjugated to E (e.g., maleimide and cysteine, amine and activated carboxylic acid (e.g., carboxylic acid activated by tetrafluorophenol or trifluorophenol), thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrazine); each of Q 1 , Q 2 , Q 3 , Q 4 , and Q 5 is, independently, optionally substitute
  • each of g, h, i, j, k, l, m, n, and o is, independently, 0 or 1.
  • a linker is described by formula (M-L-I): J 1 -(Q 1 )g-(T 1 )h-(Q 2 )i-(T 2 )j-(Q 3 )k-(T 3 )l-(Q 4 )m-(T 4 )n-(Q 5 )o-J 2 wherein J 1 is a bond attached to a gp120 binder; J 2 is a bond attached to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide, or a functional group capable of reacting with a functional group conjugated to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide (e
  • optionally substituted includes substitution with a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • a PEG has a repeating unit structure (-CH 2 CH 2 O-) n , wherein n is an integer from 2 to 100.
  • a polyethylene glycol may be selected from any one of PEG 2 to PEG 100 (e.g., PEG 2 , PEG 3 , PEG 4 , PEG 5 , PEG 5 -PEG 10 , PEG 10 -PEG 20 , PEG 20 -PEG 30 , PEG 30 -PEG 40 , PEG 50 -PEG 60 , PEG 60 -PEG 70 , PEG 70 -PEG 80 , PEG 80 -PEG 90 , PEG 90 -PEG 100 ).
  • J 2 may have two points of attachment to the Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide (e.g., two J 2 ).
  • Linking groups In some embodiments, a linker provides space, rigidity, and/or flexibility between the gp120 binders and the Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide in the conjugates described here or between two gp120 binders in the conjugates described herein.
  • a linker may be a bond, e.g., a covalent bond, e.g., an amide bond, a disulfide bond, a C-O bond, a C-N bond, a N-N bond, a C-S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation.
  • a covalent bond e.g., an amide bond, a disulfide bond, a C-O bond, a C-N bond, a N-N bond, a C-S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation.
  • a linker (L or L’ as shown in any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII) includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1- 180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60
  • a linker includes no more than 250 non-hydrogen atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1- 20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 non- hydrogen atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-hydrogen
  • the backbone of a linker includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1- 50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)).
  • the “backbone” of a linker refers to the atoms in the linker that together form the shortest path from one part of the conjugate to another part of the conjugate.
  • the atoms in the backbone of the linker are directly involved in linking one part of the conjugate to another part of the conjugate.
  • hydrogen atoms attached to carbons in the backbone of the linker are not considered as directly involved in linking one part of the conjugate to another part of the conjugate.
  • Molecules that may be used to make linkers (L or L’) include at least two functional groups, e.g., two carboxylic acid groups.
  • two arms of a linker may contain two dicarboxylic acids, in which the first carboxylic acid may form a covalent linkage with the first gp120 binder in the conjugate and the second carboxylic acid may form a covalent linkage with the second gp120 binder in the conjugate, and the third arm of the linker may for a covalent linkage (e.g., a C-O bond) with an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide in the conjugate.
  • a covalent linkage e.g., a C-O bond
  • the divalent linker may contain two carboxylic acids, in which the first carboxylic acid may form a covalent linkage with one component (e.g., a gp120 binder) in the conjugate and the second carboxylic acid may form a covalent linkage (e.g., a C-S bond or a C-N bond) with another component (e.g., an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide) in the conjugate.
  • dicarboxylic acid molecules may be used as linkers (e.g., a dicarboxylic acid linker).
  • the first carboxylic acid in a dicarboxylic acid molecule may form a covalent linkage with a hydroxyl or amine group of the first gp120 binder and the second carboxylic acid may form a covalent linkage with a hydroxyl or amine group of the second gp120 binder.
  • dicarboxylic acids molecules that may be used to linkers are found in WO 2020/252393, hereby incorporated by reference.
  • dicarboxylic acid molecules such as the ones described herein, may be further functionalized to contain one or more additional functional groups.
  • Dicarboxylic acids may be further functionalized, for example, to provide an attachment point to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide (e.g., by way of a linker, such as a PEG linker).
  • the linking group when the gp120 binder is attached to Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide, the linking group may comprise a moiety comprising a carboxylic acid moiety and an amino moiety that are spaced by from 1 to 25 atoms. Examples of such linking groups re found in WO 2020/252393, hereby incorporated by reference.
  • a linking group may include a moiety including a carboxylic acid moiety and an amino moiety, such as the ones described herein, may be further functionalized to contain one or more additional functional groups.
  • linking groups may be further functionalized, for example, to provide an attachment point to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide (e.g., by way of a linker, such as a PEG linker).
  • a linker such as a PEG linker
  • the linking group may comprise a moiety comprising two or amino moieties (e.g., a diamino moiety) that are spaced by from 1 to 25 atoms.
  • a linking group may include a diamino moiety, such as the ones described herein, may be further functionalized to contain one or more additional functional groups.
  • Such diamino linking groups may be further functionalized, for example, to provide an attachment point to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide (e.g., by way of a linker, such as a PEG linker).
  • a molecule containing an azide group may be used to form a linker, in which the azide group may undergo cycloaddition with an alkyne to form a 1,2,3-triazole linkage.
  • a molecule containing an alkyne group may be used to form a linker, in which the alkyne group may undergo cycloaddition with an azide to form a 1,2,3-triazole linkage.
  • a molecule containing a maleimide group may be used to form a linker, in which the maleimide group may react with a cysteine to form a C-S linkage.
  • a molecule containing one or more haloalkyl groups may be used to form a linker, in which the haloalkyl group may form a covalent linkage, e.g., C-N and C-O linkages, with a gp120 binder.
  • a linker (L or L’) may comprise a synthetic group derived from, e.g., a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer).
  • a linker may comprise one or more amino acid residues.
  • a linker may be an amino acid sequence (e.g., a 1-25 amino acid, 1-10 amino acid, 1-9 amino acid, 1-8 amino acid, 1-7 amino acid, 1-6 amino acid, 1-5 amino acid, 1-4 amino acid, 1-3 amino acid, 1-2 amino acid, or 1 amino acid sequence).
  • a linker may include one or more optionally substituted C 1 -C 20 alkylene, optionally substituted C 1 -C 20 heteroalkylene (e.g., a PEG unit), optionally substituted C 2 -C 20 alkenylene (e.g., C 2 alkenylene), optionally substituted C 2 -C 20 heteroalkenylene, optionally substituted C 2 -C 20 alkynylene, optionally substituted C 2 -C 20 heteroalkynylene, optionally substituted C 3 -C 20 cycloalkylene (e.g., cyclopropylene, cyclobutylene), optionally substituted C 2 -C 20 heterocycloalkylene, optionally substituted C 4 -C 20 cycloalkenylene, optionally substituted C 4 -C 20 heterocycloalkenylene, optionally substituted C 8 -C 20 cycloalkynylene, optionally substituted C 8 -C 20 heteroalkynylene, optionally substituted
  • Conjugation chemistries Gp120 binder monomers or dimers (e.g., in a conjugate of any one of formulas (1), (2), (D-I), (D- IV)-(D-VI), or (M-I)-(M-XVII)) may be conjugated to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide, e.g., by way of a linker, by any standard conjugation chemistries known to those of skill in the art.
  • conjugation chemistries are specifically contemplated, e.g., for conjugation of a PEG linker (e.g., a functionalized PEG linker) to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein- binding peptide.
  • a PEG linker e.g., a functionalized PEG linker
  • Covalent conjugation of two or more components in a conjugate using a linker may be accomplished using well-known organic chemical synthesis techniques and methods. Complementary functional groups on two components may react with each other to form a covalent bond.
  • complementary reactive functional groups include, but are not limited to, e.g., maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrazine.
  • Site-specific conjugation to a polypeptide e.g., an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein- binding peptide
  • Exemplary techniques for site- specific conjugation of a small molecule to an Fc domain are provided in Agarwall. P., et al.
  • amino-reactive acylating groups include, e.g., (i) an isocyanate and an isothiocyanate; (ii) a sulfonyl chloride; (iii) an acid halide; (iv) an active ester, e.g., a nitrophenylester or N- hydroxysuccinimidyl ester; (v) an acid anhydride, e.g., a mixed, symmetrical, or N-carboxyanhydride; (vi) an acylazide; and (vii) an imidoester. Aldehydes and ketones may be reacted with amines to form Schiff’s bases, which may be stabilized through reductive amination.
  • a linker of the invention e.g., L or L’, such as L C of D-L-I
  • E e.g., an Fc domain monomer, an Fc domain, or albumin protein
  • a linker e.g., an active ester, e.g., a nitrophenylester or N- hydroxysuccinimidyl ester, or derivatives thereof (e.g., a functionalized PEG linker (e.g., azido-PEG 2 - PEG 40 -NHS ester)
  • a T of e.g., DAR
  • 0.5 and 10.0 e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,
  • the E-(PEG 2 -PEG 40 )-azide can react with an Int having a terminal alkyne linker (e.g., L, or L’, such as L C of D-L-I) through click conjugation.
  • an Int having a terminal alkyne linker e.g., L, or L’, such as L C of D-L-I
  • the linker conjugated to E is a terminal alkyne and is conjugated to an Int having a terminal azide.
  • Exemplary preparations of preparations of E-(PEG 2 -PEG 40 )-azide are described in Examples 2, 3, and 12.
  • One of skill in the art would readily understand the final product from a click chemistry conjugation.
  • Exemplary linking strategies e.g., methods for linking a monomer or a dimer of a neuraminidase inhibitor to E, such as, by way of a linker are further depicted in FIGS.1-4 and 14. VI.
  • one or more antiviral agents may be administered in combination (e.g., administered substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions) or administered separately at different times) with a conjugate described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)).
  • a conjugate described herein e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)
  • the antiviral agent is an antiviral agent for the treatment of HIV.
  • the antiviral agent may be a nucleoside/nucleotide reverse transcriptase inhibitor, a gp120 inhibitor, a polymerase inhibitor, or a fusion protein inhibitor.
  • the antiviral agent may target either the virus or the host subject.
  • the antiviral agent for the treatment of HIV used in combination with a conjugate described herein may be selected from an integrase inhibitor (e.g., dolutegravir, elvitegravir, or raltegravir), a nucleoside reverse transcriptase inhibitor (NRTI) (e.g., abacavir, lamivudine, zidovudine, emtricitabine, tenofovir, emtricitabine, didanosine, or stavudine), a non-nucleoside reverse transcriptase inhibitor (NNRTI) (e.g., efavirenz, etravirine, nevirapine, rilpivirine, or delavirdine), a protease inhibitor (e.g., a nucleoside reverse transcriptase inhibitor (NRTI) (e.g., abacavir, lamivudine, zidovudine, emtricita
  • any one of conjugates described herein e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)
  • an antiviral vaccine e.g., a composition that elicits an immune response in a subject directed against a virus.
  • the antiviral vaccine may be administered substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions) as the conjugates, or may be administered prior to or following the conjugates (e.g., within a period of 1 day, 2, days, 5, days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 6 months, or 12 months, or more).
  • the viral vaccine includes an immunogen that elicits an immune response in the subject against HIV-1 or HIV-2.
  • the vaccine is administered as a nasal spray. VII. Methods Methods described herein include, e.g., methods of protecting against or treating a viral infection (e.g., an HIV infection) in a subject and methods of preventing, stabilizing, or inhibiting the growth of viral particles.
  • a method of treating a viral infection (e.g., an HIV infection) in a subject includes administering to the subject a conjugate described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)- (D-VI), or (M-I)-(M-XVII)) or a pharmaceutical composition thereof.
  • the viral infection is cause by the human immunodeficiency virus (e.g., HIV-1 or HIV-2).
  • the viral infection is caused by a resistant strain of virus.
  • a method of preventing, stabilizing, or inhibiting the growth of viral particles or preventing the replication and spread of the virus includes contacting the virus or a site susceptible to viral growth with a conjugate described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)) or a pharmaceutical composition thereof.
  • methods described herein also include methods of protecting against or treating viral infection in a subject by administering to the subject a conjugate described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)).
  • the method further includes administering to the subject an antiviral agent or an antiviral vaccine.
  • Methods described herein also include methods of protecting against or treating a viral infection in a subject by administering to said subject (1) a conjugate described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)) and (2) an antiviral agent or an antiviral vaccine.
  • Methods described herein also include methods of preventing, stabilizing, or inhibiting the growth of viral particles or preventing the replication or spread of a virus, by contacting the virus or a site susceptible to viral growth with (1) a conjugate described herein (e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)) and (2) an antiviral agent or an antiviral vaccine.
  • a conjugate described herein e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)
  • the conjugate described herein e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)
  • the conjugate described herein is administered first, followed by administering of the antiviral agent or antiviral vaccine alone.
  • the antiviral agent or antiviral vaccine is administered first, followed by administering of the conjugate described herein alone.
  • the conjugate described herein and the antiviral agent or antiviral vaccine are administered substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions).
  • the conjugate described herein or the antiviral agent or antiviral vaccine is administered first, followed by administering of the conjugate described herein and the antiviral agent or antiviral vaccine substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, the conjugate described herein and the antiviral agent or antiviral vaccine are administered first substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions), followed by administering of the conjugate described herein or the antiviral agent or antiviral vaccine alone.
  • a conjugate described herein e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)
  • an antiviral agent or antiviral vaccine when administered together (e.g., substantially simultaneously in the same or separate pharmaceutical compositions, or separately in the same treatment regimen), inhibition of viral replication of each of the conjugate and the antiviral agent or antiviral vaccine may be greater (e.g., occur at a lower concentration) than inhibition of viral replication of each of the conjugate and the antiviral agent or antiviral vaccine when each is used alone in a treatment regimen.
  • a conjugate described herein may be formulated in a pharmaceutical composition for use in the methods described herein.
  • a conjugate described herein may be formulated in a pharmaceutical composition alone.
  • a conjugate described herein may be formulated in combination with an antiviral agent or antiviral vaccine in a pharmaceutical composition.
  • the pharmaceutical composition includes a conjugate described herein (e.g., a conjugate described by any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)) and pharmaceutically acceptable carriers and excipients.
  • Acceptable carriers and excipients in the pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed.
  • Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acid residues such as glycine, glutamine, histidine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol.
  • buffers such as phosphate, citrate, HEPES, and TAE
  • antioxidants such as ascorbic acid and methionine
  • preservatives such as hex
  • excipients examples include, but are not limited to, antiadherents, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, sorbents, suspensing or dispersing agents, or sweeteners.
  • excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylit
  • the conjugates herein may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts.
  • These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the conjugates herein be prepared from inorganic or organic bases.
  • the conjugates are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases.
  • Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like for forming basic salts.
  • Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, n
  • alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.
  • a conjugate herein or a pharmaceutical composition thereof used in the methods described herein will be formulated into suitable pharmaceutical compositions to permit facile delivery.
  • a conjugate e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)
  • a pharmaceutical composition thereof may be formulated to be administered intramuscularly, intravenously (e.g., as a sterile solution and in a solvent system suitable for intravenous use), intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally (e.g., a tablet, capsule, caplet, gelcap, or syrup), topically (e.g., as a cream, gel,
  • a conjugate herein or a pharmaceutical composition thereof may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols.
  • the compositions may be formulated according to conventional pharmaceutical practice.
  • a conjugate described herein may be formulated in a variety of ways that are known in the art.
  • a conjugate described herein can be formulated as pharmaceutical or veterinary compositions.
  • a conjugate described herein is formulated in ways consonant with these parameters.
  • a summary of such techniques is found in Remington: The Science and Practice of Pharmacy, 22nd Edition, Lippincott Williams & Wilkins (2012); and Encyclopedia of Pharmaceutical Technology, 4th Edition, J. Swarbrick and J. C. Boylan, Marcel Dekker, New York (2013), each of which is incorporated herein by reference.
  • Formulations may be prepared in a manner suitable for systemic administration or topical or local administration.
  • Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration.
  • the formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, and preservatives.
  • the conjugates can be administered also in liposomal compositions or as microemulsions.
  • Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration.
  • Oral administration is also suitable for conjugates herein. Suitable forms include syrups, capsules, and tablets, as is understood in the art.
  • compositions can be administered parenterally in the form of an injectable formulation.
  • Pharmaceutical compositions for injection can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle.
  • Formulations may be prepared as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions.
  • Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco’s Modified Eagle Medium (DMEM), ⁇ -Modified Eagles Medium ( ⁇ -MEM), F-12 medium).
  • Such injectable compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, such as sodium acetate and sorbitan monolaurate.
  • Formulation methods are known in the art, see e.g., Pharmaceutical Preformulation and Formulation, 2nd Edition, M. Gibson, Taylor & Francis Group, CRC Press (2009).
  • the pharmaceutical compositions can be prepared in the form of an oral formulation.
  • Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients.
  • excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiad
  • Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example, peanut oil, liquid paraffin, or olive oil.
  • Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus, or a spray drying equipment.
  • Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin
  • an oil medium for example, peanut oil, liquid paraffin, or olive oil.
  • Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.
  • Dissolution or diffusion controlled release of a conjugate described herein e.g., a conjugate of any one of (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)
  • a pharmaceutical composition thereof can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of the conjugate, or by incorporating the conjugate into an appropriate matrix.
  • a controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols.
  • shellac beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glyce
  • the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.
  • the pharmaceutical composition may be formed in a unit dose form as needed.
  • the amount of active component, e.g., a conjugate described herein e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)
  • a suitable dose within the designated range is provided (e.g., a dose within the range of 0.01-100 mg/kg of body weight).
  • conjugates herein may be administered by any appropriate route for treating or protecting against a viral infection (e.g., an HIV infection), or for preventing, stabilizing, or inhibiting the proliferation or spread of a virus (e.g., an HIV virus).
  • Conjugates described herein may be administered to humans, domestic pets, livestock, or other animals with a pharmaceutically acceptable diluent, carrier, or excipient.
  • administering includes administration of any of the conjugates described herein (e.g., conjugates of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)) or compositions intramuscularly, intravenously (e.g., as a sterile solution and in a solvent system suitable for intravenous use), intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intra
  • an antiviral agent if an antiviral agent is also administered in addition to a conjugate described herein, the antiviral agent or a pharmaceutical composition thereof may also be administered in any of the routes of administration described herein.
  • the dosage of a conjugate described herein e.g., a conjugate of any one of formulas (1), (2), (D- I), (D-IV)-(D-VI), or (M-I)-(M-XVII)
  • pharmaceutical compositions thereof depends on factors including the route of administration, the disease to be treated (e.g., the extent and/or condition of the viral infection), and physical characteristics, e.g., age, weight, general health, of the subject.
  • the amount of the conjugate or the pharmaceutical composition thereof contained within a single dose may be an amount that effectively prevents, delays, or treats the viral infection without inducing significant toxicity.
  • a pharmaceutical composition may include a dosage of a conjugate described herein ranging from 0.01 to 500 mg/kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg) and, in a more specific embodiment, about 0.1 to about 30 mg/kg and, in a more specific embodiment, about 1 to about 30 mg/kg.
  • a conjugate described herein e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D-VI), or (M-I)-(M-XVII)
  • an antiviral agent or antiviral vaccine are administered in combination (e.g., substantially simultaneously in the same or separate pharmaceutical compositions, or separately in the same treatment regimen)
  • the dosage needed of the conjugate described herein may be lower than the dosage needed of the conjugate if the conjugate was used alone in a treatment regimen.
  • a conjugate described herein e.g., a conjugate of any one of formulas (1), (2), (D-I), (D-IV)-(D- VI), or (M-I)-(M-XVII)
  • a pharmaceutical composition thereof may be administered to a subject in need thereof, for example, one or more times (e.g., 1-10 times or more; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times) daily, weekly, monthly, biannually, annually, or as medically necessary. Dosages may be provided in either a single or multiple dosage regimens. The timing between administrations may decrease as the medical condition improves or increase as the health of the patient declines.
  • Example 1 Preparation of Fc constructs Reverse translations of the amino acids comprising the protein constructs (SEQ ID NOs: 1, 3, 5, 7, 9, 12, and 14) were synthesized by solid-phase synthesis.
  • the oligonucleotide templates were cloned into pcDNA3.1 (Life Technologies, Carlsbad, CA, USA) at the cloning sites BamHI and XhoI (New England Biolabs, Ipswich, MA, USA) and included signal sequences derived from the human Interleukin-2 or human albumin.
  • the pcDNA3.1 plasmids were transformed into Top10 E. coli cells (LifeTech). DNA was amplified, extracted, and purified using the PURELINK® HiPure Plasmid Filter Maxiprep Kit (LifeTech). The plasmid DNA is delivered, using the EXPIFECTAMINETM 293 Transfection Kit (LifeTech), into HEK-293 cells per the manufacturer’s protocol.
  • FIGs.5-11 show non-reducing and reducing SDS-PAGE of an Fc domain formed from Fc domain monomers having the sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 12, and 14, respectively.
  • Example 2
  • PEG4-azido Fc 0.05M PEG4-azidoNHS ester PBS buffer solution (9.88 mL, 494.0 ⁇ mol, 9.5 equivalents) was added to a solution of h-IgG1 Fc (SEQ ID NO: 4) (3027 mg in 213.0 mL of pH 7.4 PBS, MW ⁇ 58,200 Da, 16.5 ⁇ mol) and the mixture was shaken gently for 2 hours at ambient temperature. The solution was concentrated by using 10 centrifugal concentrators (30,000 MWCO, 15 mL) to a volume of ⁇ 1.5 mL. The crude mixture was diluted 1:10 in PBS pH 7.4, and concentrated again. This wash procedure was repeated for total of three times.
  • the small molecule reagent was removed with this wash procedure.
  • the concentrated Fc-PEG4-azide (SEQ ID NO: 4) was diluted to 213.0 mL with pH 7.4 PBS 1x buffer and ready for Click conjugation.
  • the purified material was quantified using a NANODROPTM UV visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1). Yield is quantitative after purification.
  • the Fc-PEG4-azide (SEQ ID NO: 35) was prepared analogously.
  • Conjugates and properties *The terminal Lys residue of the Fc domain may be cleaved upon expression and purification, e.g., SEQ ID NO: 64 coverts to SEQ ID NO: 73
  • Example 9 General procedure for purification of conjugates. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA), followed by size exclusion chromatography. (HiLoad 26/600 Superdex200 pg, GE Healthcare, Chicago, IL, USA). Fractions containing purified conjugate were pooled and concentrated to approximately 20 mg/mL using a centrifugal concentrator (30,000 MWCO).
  • Purified material was quantified using a NANODROPTM UV visible spectrophotometer using a calculated extinction coefficient based on the amino acid sequence of hIgG1 Fc(myc).
  • Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1 ⁇ g of each molecule into the gel, and staining using Instant Blue (Expedeon, San Diego, CA, USA). Each gel included a molecular weight ladder with the indicated molecular weight standards. Yields were calculated and purity determined by Agilent Analytical HPLC. Product peak and MW were found by MALDI MS and a final DAR calculated.
  • Example 10 Example 10
  • gp120 glycoprotein binding assay Nunc MaxiSorp flat-bottom 96-well plates (12-565-136, Fisher Scientific) were coated with recombinant HIV-1 GP120 (SAE0071, Sigma) at 2 ⁇ g/mL in PBS (pH 7.4) (10-010-049, Fisher Scientific) overnight at 4°C (100 ⁇ L, 0.2 ⁇ g/well). Plates were washed (5 x 300 ⁇ L) with wash buffer (PBS 0.05% Tween 20) and blocked with 1% BSA (A5611-10G, Sigma; 200 ⁇ L/well) in wash buffer for 1 h at room temp on an orbital microplate shaker at 500 rpm (BT908, BT LabSystems).
  • the blocking agent was removed and wells incubated with 3-fold serial dilutions of conjugate in sample diluent (0.5% BSA in PBS 0.025% Tween 20) starting at 1 ⁇ M for 1 h with shaking at room temp. After 5 x 300 ⁇ L washes, the plates were incubated with HRP conjugated donkey anti-human IgG Fc F(ab’)2 (709-036-098, Jackson ImmunoResearch) secondary antibody diluted 1:1,000 in sample diluent for 1 h with shaking at room temp. Plates were then washed (8 x 300 ⁇ L) and developed with TMB substrate (BD555214, Fisher Scientific) for 3-5 minutes at room temp.
  • sample diluent (0.5% BSA in PBS 0.025% Tween 20
  • HRP conjugated donkey anti-human IgG Fc F(ab’)2 709-036-098, Jackson ImmunoResearch
  • Activity of pre-conjugation intermediate (Int) compounds in an in vitro cell fusion assay Activity of HIV compounds was determined in an assay designed to measure the inhibition of cell- cell fusion which is an important step in the HIV infection process. Briefly, this assay measures the fusion of two cell lines, HeLa-CD4-LTR- ⁇ -Gal (catalog #1294) and HL2/3 cells (catalog #1294), obtained from the AIDS Research Reagent and Reference Program (Rockville, MD).
  • HeLa-CD4-LTR- ⁇ -Gal cells were plated at a density of 5 x 10 3 cells per well in a volume of 50 ⁇ L with 50 ⁇ L of nine serial logarithmic dilutions of compound in triplicate for one hour at 37oC/5% CO 2 . Following the incubation, 100 ⁇ L of HL2/3 cells were added to the plates. The cultures were incubated for an additional 48 hours at 37oC/5% CO 2 . Following the incubation, efficacy plates were evaluated for ⁇ -galactosidase production using a chemiluminescent substrate and toxicity plates were stained with XTT to evaluate cell viability. In these studies, cytotoxicity was also evaluated (TC 50 ).
  • Test materials were derived by measuring the reduction of the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide).
  • XTT in metabolically active cells is metabolized by the mitochondrial enzyme NADPH oxidase to a soluble formazan product.
  • XTT solution was prepared daily as a stock of 1 mg/mL in RPMI-1640 without additives.
  • Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in DPBS and stored in the dark at -20°C.
  • XTT/PMS stock was prepared immediately before use by adding 40 ⁇ L of PMS per mL of XTT solution.50 ⁇ L of XTT/PMS was added to each well of the plate and the plate incubated for 4 hours at 37°C. The 4 hour incubation has been empirically determined to be within the linear response range for XTT dye reduction with the indicated numbers of cells for each assay.
  • the plates were sealed and inverted several times to mix the soluble formazan product and the plate was read at 450 nm (650 nm reference wavelength) with a Molecular Devices SpectraMax Plus 38496 well plate format spectrophotometer. This assay identified several compounds with EC 50 values within 10-fold of the benchmark compound (Temsavir) (Table 5).
  • This solution was used for preparing other PEG4-azido Fc with a variety of DAR values by adjusting the equivalents of this PEG4-azido NHS ester PBS solution.
  • the concentrated Fc-PEG4-azide was diluted to 8.80 mL with pH 7.4 PBS buffer and ready for Click conjugation.
  • the purified material was quantified using a NANODROPTM UV visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1). Yield was quantitative after purification.
  • Example 13 Synthesis of Int-12 Int-12 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • Example 14 Synthesis of Int-13 Int-13 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • HeLa-CD4-LTR- ⁇ -Gal cells were plated at a density of 5 x 10 3 cells per well in a volume of 50 ⁇ L, with 50 ⁇ L of nine serial half-logarithmic dilutions of compound in triplicate for one hour at 37oC/5% CO 2 . Following the incubation, 100 ⁇ L of HL2/3 cells were added to the plates. The cultures were incubated for an additional 48 hours at 37oC/5% CO 2 . Following the incubation, efficacy plates were evaluated for ⁇ -galactosidase production using a chemiluminescent substrate and toxicity plates were stained with XTT to evaluate cell viability. In these studies, cytotoxicity was also evaluated (TC 50 ).
  • Test materials were derived by measuring the reduction of the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide).
  • XTT in metabolically active cells is metabolized by the mitochondrial enzyme NADPH oxidase to a soluble formazan product.
  • XTT solution was prepared daily as a stock of 1 mg/mL in RPMI-1640 without additives.
  • Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in DPBS and stored in the dark at -20°C.
  • XTT/PMS stock was prepared immediately before use by adding 40 ⁇ L of PMS per mL of XTT solution. Fifty ⁇ L (50 ⁇ L) of XTT/PMS was added to each well of the plate and the plate incubated for 4 hours at 37°C. The 4 hour incubation has been empirically determined to be within the linear response range for XTT dye reduction with the indicated numbers of cells for each assay. The plates were sealed and inverted several times to mix the soluble formazan product and the plate was read at 450 nm (650 nm reference wavelength) with a Molecular Devices SpectraMax Plus 38496 well plate format spectrophotometer.
  • This assay identified four compounds with EC 50 values approximately equal to the benchmark compound (Temsavir) (Table 6). These compounds were highly potent at inhibiting cell fusion with EC 50 values of less than 0.9 nM. One of these compounds, Int-17, also demonstrated no apparent loss of activity upon conjugation to an hIgG1 Fc (conjugate 5); this was an important finding. Lastly, no compounds showed cytotoxicity at the concentrations tested in this study. Therefore, for the most active compounds the difference between EC 50 and cytotoxicity is greater than 10,000-fold. A prior fusion inhibition study also identified several highly active compounds (Int-2 and Int-4). However, both compounds lost significant potency upon conjugation (conjugates 2 and 3, respectively), further emphasizing the significance of conjugate 4. Table 6.
  • Example 20 Synthesis of DMJ-II-121 DMJ-II-12 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • Example 21 Synthesis of Conjugate 8 A solution of azido functionalized Fc (50 mg, 28.43 mL, 0.862 ⁇ mol, 1.76 mg/mL; SEQ ID NO: 64, Example 2) was added to a 50 mL centrifuge tube following by addition of alkyne derivatized small molecule (15.83 mg, 0.012 mmol, Int-15, Example 16) in EPPES at pH 8.5, and a solution of copper (II) sulfate (1.1 mg, 0.0043 mmol) in water mixed with THTPA (0.43 mL, 0.0216 mmol, 50nM in water), aminoguanidine HCl (2.16 mL, 100 mM in water), and sodium ascorbate (2.16 mL, 100 mM in water).
  • THTPA 0.
  • Example 22 Synthesis of Int-18 Int-18 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • Example 23 Synthesis of Int-19 Int-19 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • Example 24 Synthesis of Int-20 Int-20 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • Example 25 Synthesis of Int-20 Int-20 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • Example 28 Synthesis of Int-23 Int-23 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • Example 29 Synthesis of Int-24 Int-24 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • Example 30 Synthesis of Int-25 Int-25 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • Example 31 Synthesis of Int-25 Int-25 was synthesized according to the procedure found in WO 2020/252393, hereby incorporated by reference.
  • Example 33 Synthesis of Int-53 To a -15 °C stirring solution of N ⁇ -Boc-N ⁇ -Cbz-L-ornithine (1.00 g, 2.729 mmol) and N- methylmorpholine (300 uL, 2.729 mmol) in THF (10.0 mL), it was added isobutylchlorofromate (355 uL, 2.729 mmol). After stirring for 5 minutes, a freshly prepared solution of sodium borohydride (310 mg, 8.188 mmol) in water (4.0 mL) was added. Upon reaction completion, water (10 mL) was added and the temperature raised to ambient, while stirring continued for 1 h.
  • sodium borohydride 310 mg, 8.188 mmol
  • Step b Under hydrogen atmosphere, a suspension of the product from step a (2.729 mmol, theoretical) and 20% palladium hydroxide on carbon (500 mg) in MeOH (20 mL), was stirred until full consumption of the starting material. The mixture was filtered and the filtrate concentrated per rotatory evaporation. Residual volatiles were evaporated under high vacuum. This material was used in the next step without further purification.
  • Step c To a 0 °C stirring solution of step b product (150 mg, 0.687 mmol), propargyl-PEG4-acid (179 mg, 0.687 mmol), and DIPEA (0.359 mL, 2.061 mmol) in DMF (4.0 mL) and DCM (0.5 mL), was added HATU (266 mg, 0.701 mmol). The temperature was raised to ambient and stirring was continued until complete as determined by LCMS. All the volatiles were removed per rotatory evaporation.
  • Step e To a 0 °C stirring solution of step d product (31 mg, 0.078 mmol), example XYZ step XYYY (40 mg, 0.078 mmol), HOBt hydrate (36 mg, 0.235 mmol, ⁇ 80%) and DIPEA (0.082 mL, 0.469 mmol) in DMF (3.0 mL) and DCM (0.5 mL), was added HATU (89 mg, 0.235 mmol). The temperature was raised to ambient and stirring was continued until complete as determined by LCMS. All the volatiles were removed per rotatory evaporation.
  • step b To a 0 °C stirring solution of step b product (150 mg, 0.687 mmol), propargyl-PEG4-acid (179 mg, 0.687 mmol), and DIPEA (0.359 mL, 2.061 mmol) in DMF (4.0 mL) and DCM (0.5 mL), was added HATU (266 mg, 0.701 mmol). The temperature was raised to ambient and stirring was continued until complete as determined by LCMS. All the volatiles were removed per rotatory evaporation. The residue was purified by RP-C18 column using an Isco CombiFlash liquid chromatography eluted with 0% to 100% water and methanol, no modifier. Yield 0.186 g, 59%.
  • step d product 31 mg, 0.078 mmol
  • the triazole acid described in Example 5 40 mg, 0.078 mmol
  • HOBt hydrate 36 mg, 0.235 mmol, ⁇ 80%
  • DIPEA 0.082 mL, 0.469 mmol
  • HATU 89 mg, 0.235 mmol
  • Step b To a solution of the step-a product (1.56 g, 4.57 mmol) in anhydrous DMF (5 ml) was added K 2 CO 3 (691 mg, 5 mmol) and 2,2-dimethyl-1,3-dioxan-5-amine (779.5 mg, 5.94 mmol). The mixture was heated at 70°C overnight, then cooled to room temperature.
  • Step c To a solution of the step-b product (760.9 mg, 1.94 mmol) in anhydrous DMF (3 ml) was added DIPEA (501.5 mg, 3.88 mmol), 3(-(Boc)-aminopropyl bromide (695.3 mg, 2.92 mmol) and NaI (75 mg, 0.5 mmol). The mixture was stirred at 50°C for 3 days.
  • Step d The step-c product (679.6 mg, 1.24 mmol) was dissolved in MeOH (15 ml) and added with AcOH (74.3 mg, 1.24 mmol)) and Pd(OH)2 (20% on carbon, 350 mg). The resulting mixture was stirred under hydrogen atmosphere for 3 hours. Pd(OH) 2 /C was filtered off, and the filtrate was concentrated by rotary evaporation and further dried under high vacuum. Yield 588.5 mg, quantitative yield.
  • Step e To a solution of the step-d product (588.5 mg, 1.24 mmol) in anhydrous DMF (2 ml) and THF (2 ml) was added K 2 CO 3 (514 mg 3.72 mmol) and propargyl-PEG4-mesyl ester (465.5 mg, 1.5 mmol). The resulting mixture was heated at 70°C for 1 day. The salt was filtered off, and the filtrate was concentrated and purified by HPLC (0% to 70% acetonitrile and water). Yield 253.2 mg, 32.5%.
  • Step f The step-e product (253.2 mg, 0.403 mmol) was dissolved in acetonitrile/water (1:1, 4 ml) and added with 6N HCl aqueous solution (0.5 ml, 3 mmol). The reaction was stirred at 60°C for 5 hours, then cooled to room temperature. After the pH was adjusted to about 8 with 1M KOH (3.5 ml), the solution was lyophilized. The residue was then re-dissolved in MeOH and KCl was filtered off. The filtrate was concentrated by rotary evaporation and further dried under high vacuum.
  • TFA 15 ⁇ l was added, followed by the addition of Azido-PEG4-trifluorophenyl ester ( 12.2 mg, 0.029 mmol), a pre-mixed solution of THPTA (3.5 mg, 0.008 mmol) and sodium ascorbate (40 mg, 0.202 mmol) in water (0.5 ml) and Cu 2 SO 4 (1.6 mg, 0.01 mmol).
  • the ice-water bath was removed, and the reaction mixture was stirred for 30 minutes. It was then directly purified by RPLC (50 g, 5% to 80% acetonitrile and water, using 0.1% TFA as modifier). Yield 30.7 mg, 68.8%.
  • HIV lead compounds in an in vitro cell fusion assay
  • Activity of HIV compounds was determined in an assay designed to measure the inhibition of cell- cell fusion mediated by gp120 and CD4 interaction which is an important step in the HIV infection process. Briefly, this assay measures the fusion of two cell lines, HeLa-CD4-LTR- ⁇ -Gal (catalog #1294) and HL2/3 cells (catalog #1299), obtained from the AIDS Research Reagent and Reference Program (Rockville, MD).
  • HeLa-CD4-LTR- ⁇ -Gal cells were plated at a density of 5 x 10 3 cells per well in a volume of 50 ⁇ L, with 50 ⁇ L of nine serial half-logarithmic dilutions of compound in triplicate for one hour at 37 oC/5% CO 2 . Following the incubation, 100 ⁇ L of HL2/3 cells were added to the plates. The cultures were incubated for an additional 48 hours at 37 oC/5% CO 2 . Following the incubation, efficacy plates were evaluated for ⁇ -galactosidase production using a chemiluminescent substrate and toxicity plates were stained with XTT to evaluate cell viability. In these studies cytotoxicity was also evaluated (TC 50 ).
  • Test materials were derived by measuring the reduction of the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide).
  • XTT in metabolically active cells is metabolized by the mitochondrial enzyme NADPH oxidase to a soluble formazan product.
  • XTT solution was prepared daily as a stock of 1 mg/mL in RPMI-1640 without additives.
  • Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in DPBS and stored in the dark at -20°C.
  • XTT/PMS stock was prepared immediately before use by adding 40 ⁇ L of PMS per mL of XTT solution. Fifty ⁇ L (50 ⁇ L) of XTT/PMS was added to each well of the plate and the plate incubated for 4 hours at 37°C. The 4 hour incubation has been empirically determined to be within the linear response range for XTT dye reduction with the indicated numbers of cells for each assay. The plates were sealed and inverted several times to mix the soluble formazan product and the plate was read at 450 nm (650 nm reference wavelength) with a Molecular Devices SpectraMax Plus 38496 well plate format spectrophotometer.
  • Example 40 30-day comparative non-human primate PK study following IV administration of a conjugate including an Fc domain having a C220S/YTE quadruple mutant
  • a conjugate including an Fc domain having a C220S mutation and a YTE mutation was synthesized as described in Example 39.
  • a non-human primate PK study was performed to compare IV administration of the C220S/YTE Fc conjugate (SEQ ID NO: 67) to a conjugate including an Fc domain having a C220S mutation alone (SEQ ID NO: 64).
  • Non-human primate (NHP) PK studies were performed by BTS Research (San Diego, CA) using male and female cynomolgus monkeys 5-9 years old with body weights ranging from 3.5-8.5 kg. NHPs were injected IV with 2 mg/kg of test article (0.4 mL/kg dose volume). Animals were housed under standard IACUC approved housing conditions. At appropriate times animals were non-terminally bled (via femoral or cephalic veins) with blood collected in K 2 EDTA tubes to prevent coagulation. Collected blood was centrifuged (2,000 x g, for 10 minutes) and plasma withdrawn for analysis of test article concentrations over time.
  • the plasma concentrations for the C220S/YTE Fc conjugate and the C220S conjugate at each time point were measured by sandwich ELISA. Briefly, test articles were captured on Fc-coated plates and then detected using a HRP-conjugated anti-human IgG-Fc antibody. Protein concentrations were calculated in GraphPad Prism using 4PL non-linear regression of the C220S/YTE Fc conjugate or C220S conjugate standard curves. A more detailed method description is provided above. The corresponding curves are shown in FIG.13.
  • the C220S/YTE Fc conjugate demonstrates a significantly improved terminal half-life of ⁇ 45 days compared with ⁇ 10 days for the C220S Fc conjugate. AUCs for the C220S/YTE Fc conjugate are 2X greater than the AUCs for The C220S conjugate (Table 8).
  • HeLa-CD4-LTR- ⁇ -Gal cells were plated at a density of 5 x 10 3 cells per well in a volume of 50 ⁇ L, with 50 ⁇ L of nine serial half-logarithmic dilutions of compound in triplicate for one hour at 37 oC/5% CO 2 . Following the incubation, 100 ⁇ L of HL2/3 cells were added to the plates. The cultures were incubated for an additional 48 hours at 37 °C/5% CO 2 . Following the incubation, efficacy plates were evaluated for ⁇ -galactosidase production using a chemiluminescent substrate and toxicity plates were stained with XTT to evaluate cell viability. In these studies, cytotoxicity was also evaluated (TC50).
  • Test materials were derived by measuring the reduction of the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide).
  • XTT in metabolically active cells is metabolized by the mitochondrial enzyme NADPH oxidase to a soluble formazan product.
  • XTT solution was prepared daily as a stock of 1 mg/mL in RPMI-1640 without additives.
  • Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in DPBS and stored in the dark at -20°C.
  • XTT/PMS stock was prepared immediately before use by adding 40 ⁇ L of PMS per mL of XTT solution. Fifty ⁇ L (50 ⁇ L) of XTT/PMS was added to each well of the plate and the plate incubated for 4 hours at 37 °C. The 4 hour incubation has been empirically determined to be within the linear response range for XTT dye reduction with the indicated numbers of cells for each assay. The plates were sealed and inverted several times to mix the soluble formazan product and the plate was read at 450 nm (650 nm reference wavelength) with a Molecular Devices SpectraMax Plus 38496 well plate format spectrophotometer.
  • Example 42 Activity of lead compounds in a cell fusion assay (EC50) and cytotoxicity (TC50)
  • DAR is determined by Maldi TOF of the purified conjugates and is typically 2 to 5.
  • the yields and properties for conjugates synthesized using this general procedure are listed in Table 12 below. Table 12.
  • this assay measures the fusion of two cell lines, HeLa-CD4-LTR- ⁇ -Gal (catalog #1470) and HL2/3 cells (catalog #1294), obtained from the NIH AIDS Research Reagent and Reference Program (Rockville, MD).
  • HeLa-CD4-LTR- ⁇ -Gal cells were plated at a density of 5 x 10 3 cells per well in a volume of 50 ⁇ L, with 50 ⁇ L of nine serial half-logarithmic dilutions of compound in triplicate for 1 h at 37oC/5% CO 2 . Following the incubation, 100 ⁇ L of HL2/3 cells were added to the plates. The cultures were incubated for an additional 48 h at 37oC/5% CO 2 .
  • the concentration required to induce 50% toxicity was derived by measuring the reduction of the tetrazolium dye XTT (2,3-bis(2-methoxy-4- nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide).
  • XTT in metabolically active cells is metabolized by the mitochondrial enzyme NADPH oxidase to a soluble formazan product.
  • XTT solution was prepared daily as a stock of 1 mg/mL in RPMI-1640 without additives.
  • Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in DPBS and stored in the dark at -20°C.
  • XTT/PMS stock was prepared immediately before use by adding 40 ⁇ L of PMS per mL of XTT solution. Fifty ⁇ L (50 ⁇ L) of XTT/PMS was added to each well of the plate and the plate incubated for 4 h at 37°C. The 4 h incubation has been empirically determined to be within the linear response range for XTT dye reduction with the indicated numbers of cells for each assay. The plates were sealed and inverted several times to mix the soluble formazan product and the plate was read at 450 nm (650 nm reference wavelength) with a Molecular Devices SpectraMax Plus 38496 well plate format spectrophotometer.
  • Compounds Conjugate 29a, Conjugate 30a, and Conjugate 34 were the most active with EC50 values of 7.06 nM, 10.5 nM and 14.9 nM, respectively.
  • Compounds Conjugate 5b, Conjugate 32, and Conjugate 36 were slightly less active than the most active compounds with EC50 values ranging from 22.8 nM to 43.6 nM. The remaining compounds had EC50 values ranging from 144 nM to 915 nM. All of the compounds were non-toxic up to the highest concentration evaluated (2000 nM with the exception of 1800 nM for compound Conjugate 15) (Table 13). Table 13. Evaluation of inhibition of cell to cell fusion in HeLa-CD4-LTR- ⁇ -Gal and HL2/3 cells.
  • this assay measures the fusion of two cell lines, HeLa-CD4-LTR- ⁇ -Gal (catalog #1470) and HL2/3 cells (catalog #1294), obtained from the NIH AIDS Research Reagent and Reference Program (Rockville, MD).
  • HL2/3 cells were plated at a density of 2 x 10 4 cells per well in a volume of 50 ⁇ L, with 50 ⁇ L of nine serial half-logarithmic dilutions of compound in triplicate for 1 h at 37oC/5% CO 2 . Following the incubation, 100 ⁇ L of HeLa-CD4-LTR- ⁇ -Gal cells were added to the plates. The cultures were incubated for an additional 48 h at 37oC/5% CO 2 .
  • CSB Chicago Sky Blue
  • Temsavir Temsavir
  • conjugates were evaluated for the ability to inhibit HIV-1-induced cell to cell fusion.
  • the average EC50 values defined in the assays ranged from 1.9E-01 to 6.0E+03 nM (Table 15).
  • Conjugate 14b was the most potent conjugate with an EC 50 value of 1.99E-01 nM (Table 15).
  • Several conjugates had single-digit nM potency, including Conjugate 27, Conjugate 29a, Conjugate 12b, Conjugate 37a, Conjugate 5b, and Conjugate 30a.
  • the remaining compounds had EC 50 values ranging from 1.14E+01 nM to 6.02E+03 nM (Table 15). Table 15.
  • mice were non-terminally bled (retro-orbital, cheek, or by tail vein) with blood collected in K2EDTA tubes to prevent coagulation. Collected blood was centrifuged (2,000 x g, for 10 min) and plasma withdrawn for analysis of test article concentrations over time. The Fc plasma concentrations at each time point were measured by Fc-capture sandwich ELISA as follows.
  • Nunc Maxisorp 96-well plates (cat no.12-565-136, Fisher Scientific) were coated overnight at 4 ⁇ C with 0.1 ⁇ g/100 ⁇ L/well of goat anti-human IgG (Fc ⁇ fragment specific; cat no.109-005-098, Jackson Immunoresearch) in carbonate buffer (cat no. C3041, MilliporeSigma). Plates were washed 5x with 300 ⁇ L/well PBST and blocked with 200 ⁇ L/well 5% non-fat dry milk (cat no.9999S, Cell Signaling) in PBST for 1 h at room temperature with shaking.
  • sample diluent 2.5% non-fat dry milk in PBS 0.025% Tween 20 + na ⁇ ve mouse plasma final concentration of 1:900.
  • Compound standard curves ranging from 0.03 to 55 ng/mL in duplicate, were run on each plate. Following the 2 h incubation, plates were washed 5x with 300 ⁇ L/well PBST.
  • Test article in plasma samples was interpolated using GraphPad Prism Version 8 following nonlinear regression analysis (Sigmoidal, 4PL analysis) of the standard curves. The resulting mean plasma concentrations were then used to calculate the total AUC for each plasma concentration-time profile.
  • the 7-day mouse PK profiles of Conjugate 5b and Fc control (SEQ ID NO:73) are shown in FIG. 15. Following IV administration of compound at 5 mg/kg, the average plasma exposure levels of Conjugate 5b were noninferior to SEQ ID NO:73, with AUCs of 2922 and 1766, respectively (FIG.15).
  • Step e A solution of the step-c product (35.8 mg, 0.0409 mmol) in DMF (1 ml) was mixed with TFA (20 ⁇ l) and cooled in an ice-water bath. It was then mixed with the step-d product (29.3 mg, 0.0491 mmol) and a premixed solution of THPTA (3.5 mg, 0.008 mmol) and sodium ascorbate (29 mg, 0.146 mmol) in water (0.5 ml), followed by Cu 2 SO 4 (1.6 mg, 0.01 mmol). The ice-water bath was removed, and the resulting mixture was stirred for 40 minutes.
  • Example 52 Synthesis of Int-115 To a cold solution of dimethyl 2-(3-((tert-butoxycarbonyl)amino)propyl)malonate (500 mg, 1.72 mmol, prepared as described in Angewandte Chemie, International Edition (2016), 57(22), 6527-6531) in DMF (6 ml) was added sodium hydride (72.5 mg, 1.81 mmol), followed N-(bromomethyl)phthalimide (0.45 mg, 1.9 mmol). The resulting suspension was stirred for 2.5 h at 0°C, and partitioned between water (6 ml) and dichloromethane (40 ml). The organic layer was separated, washed with water and sat. aq.
  • Step b To a solution of step-a product (300 mg, 0.66 mmol) in methanol (3 ml) was added hydrazine hydrate (0.18 ml, 3.34 mmol). The solution was stirred under reflux for 4 hrs. It was then cooled, filtered, concentrated and purified by RPLC (5% to 95% acetonitrile and water, using 0.1% TFA as modifier) to afford product. Yield 160 mg, 75 %.
  • step-c product 142 mg, 0.25 mmol
  • THF 2.5 ml
  • LiBH4 23.2 mg, 1.0 mmol
  • Step e The step-d product (91 mg, 0.17 mmol) wascooled and dissolved in HCl (2 ml, 4N in dioxane) and reaction was stirred for 2 hrs.
  • Step f To a solution of triazole-carboxylic acid (77.4 mg, 0.151 mmol) in DMF (1 mL) was added HSPyU (124 mg, 0.30 mmol) and DIPEA (0.15 ml, 0.90 mmol) and the mixture was stirred for 30 min. Step-e product (72 mg, 0.181 mmol) was then added to above solution and LCMS after 30min shows complete consumption of the triazole-carboxylic acid.
  • Step g A solution of step-f product (39 mg, 0.043 mmol) and azido-Peg4-trifluorophenol ester (23.8 mg, 0.056 mmol) in DMF (0.6 mL), was treated with a solution of copper(II) sulfate (1.73 mg, 0.010 mmol), sodium ascorbate (8.6 mg, 0.043 mmol), and THPTA (9.43 mg, 0.021 mmol) in water (0.3 mL).
  • Step f A solution of product from the previous step (0.030 g, 0.033 mmol), and azido-PEG4- trifluorophenol ester (0.018 g, 0.043 mmol) dissolved in DMF (1.0 mL), was treated with a solution of copper(II) sulfate (0.001 g, 0.008 mmol), and sodium ascorbate (0.006 g, 0.033 mmol), dissolved in water (1.0 mL), and stirred at room temperature for 30 min.
  • Example 54 Synthesis of Int-119 Step a. A mixture of t-butyl(5-formyl-2,2-dimethyl-1,3-dioxan-5-yl) carbamate (1.9 g, 7.3 mmol) and ammonium acetate (5.6 g, 73.2 mmoL)in methanol (30 mL) were stirred at ambient temperature for 1 hour. Sodium cyanoborohydride (1.2 g, 18.3 mmol) was added and the reaction was stirred for 12 hours then concentrated to a volume of approx.5 mL.
  • the amine was taken up in acetonitrile (25 mL) and DIPEA (2.5 mL, 14.7 mmol) and Fmoc-OSu (2.7 g, 8.1 mmoL was added and the mixture was stirred for 1 hour, diluted with water (50 mL) and extracted into ethyl acetate (3x, 30 mL0. The combined organic extracts were dried over sodium sulfate and concentrated.
  • the amine-HCl salt was dissolved in methanol (30 mL) and CBZ-amino- propanal (227 mg, 1.1 mmol) was added and the mixture was stirred for 30 minutes at which point sodium cyanoborohydride (140 mg, 2.2 mmol) was added and the mixture was stirred for 12 hours at ambient temperature.
  • the intermediate was stirred in methanol in the presence of 5% Pd/C (75 mg) for 2 hours.
  • Step c HATU (67 mg, 0.18 mmol) was added to a stirring mixture of the intermediate from step b. of this example (76 mg, 0.19 mmol), and the triazole carboxylic acid (75 mg, 0.15 mmol described in Example 5 of Int- 2), and DIPEA (0.10 uL, 0.58 mmol) in DMF (2 mL). The reaction was stirred for 45 minutes and then purified by reversed phase HPLC (0-95% ACN in DI water, 0.1% TFA modifier, 30 minute gradient).
  • Step d DBU (60 uL, 0.41 mmol) was added to a stirring mixture of the intermediate described in step c. of this example (75 mg, 0.084 mmol) in DMF (2 mL) and the mixture was stirred for 20 minutes. Propargyl-PEG4-carboxylic acid (33 mg, 0.13 mmol) was added followed by HATU (42 mg 0.11 mmol) and the reaction was stirred for 45 minutes.
  • Copper sulfate (0.5 mg, 0.003 mmol), sodium ascorbate (17 mg, 0.089 mmol), and BTTA (2.5 mg, 0.006 mmol), were pre-mixed in DI water (0.5 mL) and then added to a mixture of the alkyne intermediate (27 mg, 0.029mmol), and azido-peg4-trifluorophenol-ester (12 mg, 0.29 mmol), in DMF/H 2 O (3:1, 1 mL) , cooled to 0°C via and ice water bath.
  • Step b To a solution of the step-a crude product in DCM (20 ml) was added 3- [benzyloxycarbonyl)amino]-1-propanal (2.49 g, 12 mmol) and acetic acid (900 mg, 15 mmol). The resulting mixture was stirred at room temperature for 30 minutes, then sodium triacetoxyborohydride (3.15 g, 15 mmol) was added in portions.
  • Step c To a solution of the step-b product (1.05 g, 1.93 mmol) in MeOH (25 ml) was added Pd(OH) 2 on carbon (850 mg).
  • Step d To a solution of the step-c product (1.25 g, 3.33 mmol) in anhydrous DMF (4 ml) was added benzyl bis(2-chloroethyl)carbamate (1.38 g, 5 mmol), triethylamine (12 ml) and NaI (150 mg, 1 mmol).
  • Step f To a solution of the step-e product (245 mg, 0.55 mmol) in anhydrous DMF (1 ml) was added propargyl-PEG4-mesyl ester (223.5 mg, 0.72 mmol) and DIEPA (129.2 mg, 1 mmol). The resulting mixture was heated at 600C for 1 day, then directly purified by RPLC (50 g, 5 to 70% acetonitrile and water). Yield 129 mg, 35.6%.
  • step-c product 101.4 mg, 0.28 mmol
  • anhydrous DMF 0.5 ml
  • propargyl-PEG4-NHS ester 121.3 mg, 0.34 mmol
  • DIPEA 65 mg, 0.5 mmol
  • Step e The step-d product (192 mg, 0.268 mmol) was dissolved in acetonitrile (1 ml) and water (0.5 ml).
  • Step f A mixture of the triazole acid described herein (51.2 mg, 0.1 mmol) and the step-e product (53.8 mg, 0.1 mmol) was dissolved in anhydrous DMF (1 ml) and Et 3 N (606 mg, 6 mmol) by gently heated with a heat gun.
  • Step b To a solution of the step-a product (2.55 g, 4.95 mmol) in acetonitrile (5 ml) was added water (5 ml) and 6N HCl aqueous solution (4 ml). The reaction mixture was heated at 600C for 2 hours, then concentrated by rotary evaporation. Excess HCl was further removed by azeotrope/evaporation with acetonitrile. The residue was further dried under high vacuum.
  • step-c product 505 mg, 1.1 mmol
  • water 2 ml
  • 6N HCl aqueous solution 1 ml
  • Step h A solution of the step-g product (35.4 mg, 0.025 mmol) in DMF (1 ml) was treated with 40 ⁇ l of TFA, and cooled in an ice-water bath. It was then mixed with azido-PEG4-trifluorophenyl ester (12.7 mg, 0.3 mmol) and a premixed solution of THPTA (3.5 mg, 0.008 mmol) and sodium ascorbate (29 mg, 0.146 mmol) in water (0.5 ml), followed by Cu 2 SO 4 (1.6 mg, 0.01 mmol).
  • Step c HATU (72 mg, 0.19 mmol) was added to a stirring mixture of the intermediate from step b. of this example (75 mg, 0.16 mmol), and the triazole carboxylic acid, (described in example 5 of Int- 2, 81 mg, 0.16 mmol), and DIPEA (0.11 uL, 0.64 mmol) in DMF (2 mL). The reaction was stirred for 45 minutes and purified directly by reversed phase HPLC (0-95% ACN in DI water, 0.1% TFA modifier, 30 minute gradient).
  • Step d DBU (73 uL, 0.52 mmol) was added to a stirring mixture of the intermediate described in step c. of this example (100 mg, 0.10 mmol) in DMF (2 mL) and the mixture was stirred for 20 minutes. Propargyl-peg4-carboxylic acid (41 mg, 0.16 mmol) was added followed by HATU (47 mg 0.12 mmol) and the reaction was stirred for 45 minutes. The mixture was purified by reversed phase HPLC (0-95% ACN in DI water, 0.1% TFA modifier, 30 minute gradient).
  • Step h A solution of product from the previous step (0.095g, 0.147 mmol), and triazole-carboxylic acid (0.050g, 0.098 mmol, described herein) dissolved in DMF (2 mL), was treated with DIEA (0.170 mL, 0.98 mmol), HOBT (0.026 g, 0.196), and HATU (0.074 g, 0.196 mmol).
  • LCMS after 30min shows complete consumption of the triazole-carboxylic acid.
  • the reaction was concentrated and purified by semi- preparative HPLC (5% to 100% ACN/water with 0.1% TFA). Yield 0.060g, 46%.
  • Step b To a solution of the step-a product (944.7 mg, 2.77 mmol) in anhydrous DMF (3 ml) was added K 2 CO 3 (459 mg, 3.32 mmol) and ethanolamine (338.5 mg, 5.54 mmol). The mixture was heated at 70°C for 3 hours, then cooled to room temperature.
  • Step c To a solution of the step-b product (708 mg, 2.19 mmol) in anhydrous DMF (2.5 ml) was added DIPEA (568.7 mg, 4.4 mmol), 3-(Boc)-aminopropyl bromide (785.7 mg, 3.3 mmol) and NaI (105 mg, 0.7 mmol).
  • Step d To a solution of the step-c product (793 mg, 1.66 mmol) in MeOH (15 ml) was added acetic acid (99.4 mg, 1.66 mmol)) and Pd(OH) 2 (20% on carbon, 350 mg). The resulting mixture was stirred under hydrogen atmosphere for 3 hours. Pd(OH) 2 /C was filtered off, and the filtrate was concentrated by rotary evaporation.
  • Step e To a solution of the step-d (618 mg, 1.53 mmol) in anhydrous THF (3 ml) and DMF (1 ml) was added K 2 CO 3 (624.3 mg 4.59 mmol), propargyl-PEG4-mesyl ester (552.5 mg, 1.78 mmol) and NaI (45 mg, 0.3 mmol). The resulting mixture was heated at 70°C overnight.
  • Step f The step-e product (262.4 mg, 0.47 mmol) was dissolved in acetonitrile/water (1:1, 4 ml) and mixed with 6N HCl aqueous solution (0.4 ml, 2.4 mmol). The reaction was heated at 50°C for 6 hours, then cooled to room temperature. After the pH was adjusted to about 8 with 1M KOH (2.4 ml), the solution was concentrated by rotary evaporation.
  • a solution of the step-g product (20 mg, 0.0142 mmol) in DMF:MeOH (1:1, 1 ml) was mixed with TFA (20 ⁇ l) and cooled in an ice-water bath. It was then mixed with azido-PEG4-trifluorophenyl ester (7.2 mg, 0.017 mmol) and a premixed solution of THPTA (3.5 mg, 0.008 mmol) and sodium ascorbate (29 mg, 0.146 mmol) in water (0.5 ml), followed by Cu 2 SO 4 (1.6 mg, 0.01 mmol). The ice-water bath was removed, and the resulting mixture was stirred for 40 minutes.
  • step-c product 100 mg, 0.15 mmol
  • THF 1.5 ml
  • LiBH4 14.5 mg, 0.63 mmol
  • Step e The step-d product (42 mg, 0.052 mmol) was dissolved in HCl (4N in dioxane).
  • Step f To a solution of step-e product (35 mg, 0.052 mmol) in DMF (0.5 ml) was added triazole- carboxylic acid (32.1 mg, 0.062 mmol), DIPEA (0.089 mL, 0.51 mmol), HOBT (14.1 mg, 0.10 mmol), and HATU (39.8 mg, 0.10 mmol). LCMS after 30min shows complete consumption of the triazole-carboxylic acid. The reaction was concentrated and purified by semi-preparative HPLC (5% to 100% ACN/water).
  • Step g A solution of step-f product (12 mg, 0.011 mmol), and azido-Peg4-trifluorophenol ester (6 mg, 0.014 mmol) in DMF (0.6 mL), was treated with a solution of copper (II) sulfate (0.4 mg, 0.00277 mmol), sodium ascorbate (2.19 mg, 0.011 mmol), and THPTA (2.41 mg, 0.0055 mmol) in water (0.3 mL), and stirred at room temperature for 30 min. Reaction progress was monitored by LCMS. The product was purified by semi-preparative HPLC (5% to 100% ACN/water).
  • step-c product 169 mg, 0.34 mmol
  • DMF dimethyl methoxyethyl-N-(2-aminoethyl)
  • step-c product 169 mg, 0.34 mmol
  • Et3N 0.05 ml, 0.34 mmol
  • Step f Step-e product (31 mg, 0.06 mmol) was dissolved in a cool solution of HCl (1.5 ml, 4N in dioxane) and reaction was stirred for 2 hr.
  • Example 74 Activity against HIV-1 IIIB Strain (HIV-1IIIB) in a cytopathic effects assay using CEM-SS Cells Materials. This study determined the activity of conjugates, comparators, and buffers against HIV-1IIIB in a CPE assay with a T-cell lymphoblastic cell line obtained from the AIDS Research and Reference Program (Rockville, MD).
  • Controls consisted of AZT (Sigma-Aldrich), Temsavir (Astatech, PA), conjugate buffer (200 mM Arginine, 120 mM NaCl, 1% Sucrose, pH 6.0), and Temsavir buffer (DMSO/PEG300/Tween-80/PBS).
  • Methods – Evaluation of Inhibition of HIV-1 IIIB and Cellular Toxicity in CEM-SS Cells Fifty microliters (50 ⁇ L) of CEM-SS cells at a density of 2.5 x10 3 cells/well in 10% complete RPMI-1640 (10% FBS with 1% L-glutamine and 1% Penicillin/Streptomycin) media were plated in a 96-well round bottom plate.
  • One-hundred microliters (100 ⁇ L) of each compound at 6 concentrations were added in triplicate followed by 50 ⁇ L of HIV-1IIIB at a pre-determined titer. The cultures were incubated for 6 days at 37°C/5% CO 2 . Following the incubation, the cells were stained with XTT for evaluation of compound efficacy and cellular toxicity, as described below. AZT was evaluated in parallel as an assay positive control compound. Data Analysis and Evaluation. Microsoft Excel 2010 in combination with XLfit4 was used to analyze and graph data.
  • EC 25 , EC 50 and EC 95 (25%, 50% and 95% inhibition of virus replication), TC 25 , TC 50 and TC 95 (25%, 50%, and 95% reduction in cell viability) and a therapeutic index (TI, TC 25 /EC 25 , TC 50 /EC 50 , TC 95 / EC 95 ) were calculated.
  • TI TC 25 /EC 25 , TC 50 /EC 50 , TC 95 / EC 95
  • AZT was evaluated in parallel as a positive control compound and was active at the expected concentration.
  • Example 75 Activity of Conjugate 29a and Conjugate 29b against 5 HIV-1 strains in a cell-based assay with human peripheral blood mononuclear cells (PMBCs) Materials. This study determined the activity of Conjugate 29a and Conjugate 29b against wild type HIV-1 isolates covering 3 different clades and co-receptor specificities obtained from the AIDS Research and Reference Reagent Program (Rockville, MD) (Table 18).
  • Controls consisted of AZT (Sigma-Aldrich), Temsavir (Astatech, PA), and compound buffer (200 mM Arginine, 120 mM NaCl, 1% Sucrose, pH 6.0)
  • Human PBMCs were isolated from whole blood from three donors. The leukophoresed blood was diluted 1:1 with DPBS and was layered over 15 mL of Ficoll-Hypaque density gradient. These tubes were centrifuged for 30 min at 1800 rpm. Banded PBMCs were gently aspirated from the resulting interface and subsequently washed three times with DPBS by low-speed centrifugation.
  • tissue culture medium RPMI 1640 with 15% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin and 3.6 ng/mL recombinant human IL-2).
  • the cultures were then maintained until use by half culture volume change with fresh IL-2 containing tissue culture medium every 3 to 4 days. Assay conditions. Assays were initiated with PBMCs that had been induced to proliferate with PHA-P for 72 hours.
  • PHA-P stimulated PBMCs from three donors were pooled together to minimize the variability that occurs when cells from individual donors are used, resuspended in fresh tissue culture medium at 1 x 10 6 cells/mL and plated in the interior wells of a 96-well round bottom microtiter plate at 50 ⁇ L/well.
  • One-hundred microliters (100 ⁇ L) of each two times the concentration of compound in assay media was added to designated wells of the round-bottom 96-well plate containing the cells, in triplicate.
  • 50 ⁇ L of a pre- determined dilution of virus was added.
  • HIV-1 replication was quantified by the measurement of cell-free HIV-1 RT activity in the tissue culture supernatant. Cytotoxicity was evaluated using the tetrazolium dye XTT following well known standard methods. Quantification of reverse transcriptase (RT) activity. Virus production by untreated and treated cells was quantified by measuring reverse transcriptase in cell-free supernatants using a standard radioactive incorporation polymerization assay. Tritiated thymidine triphosphate (TTP) was purchased at 1 Ci/mL and 1 ⁇ L was used per enzyme reaction.
  • TTP Tritiated thymidine triphosphate
  • Poly rA and oligo dT were prepared at concentrations of 0.5 mg/mL and 1.7 Units/mL, respectively, from a stock solution which was kept at -20°C.
  • the RT reaction buffer was prepared fresh and consists of 125 ⁇ L of 1 M EGTA, 125 ⁇ L of dH 2 O, 125 ⁇ L of 20% Triton X-100, 50 ⁇ L of 1 M Tris (pH 7.4), 50 ⁇ L of 1 M DTT, and 40 ⁇ L of 1 M MgCl 2 .
  • 1 ⁇ L of TTP, 4 ⁇ L of dH 2 O, 2.5 ⁇ L of rAdT and 2.5 ⁇ L of reaction buffer were mixed.
  • Conjugate 29a and Conjugate 29b were not cytotoxicity with Conjugate 29a and Conjugate 29b against this, or any strain, in the panel (TC 50 > 250 nM).
  • Conjugate demonstrated single digit potency. Potency against HIV-1HT/92/594 was 0.49 nM for Conjugate 1.
  • Conjugate 1 also displayed potent activity against the Subtype C strain (HIV-1ZA/97/009). Likewise, Conjugate 1 demonstrated significant activity against the A Subtype (HIV-1UG/92/037). Significantly, Conjugate 1 was more potent than the Temsavir comparator against the A Subtype.
  • Conjugate 29a and Conjugate 29b were highly active against 5 clinically relevant HIV-1 strains with EC 50 values in the single digit (or better) nM range.
  • the HIV-1 panel tested in this study also included 3 different Subtypes with 3 different co-receptor tropisms indicating broad coverage by Conjugate 29a and Conjugate 29b.
  • this assay was conducted with actual human PBMCs isolated from 3 different donors, making the assay, and results, all the more meaningful.
  • Example 76 Screening of HIV antiviral small molecules and conjugates in an in vitro cell fusion inhibition assay Activity of compounds was determined by using an assay designed to measure the inhibition of cell-cell fusion mediated by gp120 and CD4, a key step in the HIV infection process. Briefly, this assay measures the fusion of two cell lines, HeLa-CD4-LTR- ⁇ -Gal (catalog #1470) and HL2/3 cells (catalog #1294), obtained from the NIH AIDS Research Reagent and Reference Program (Rockville, MD).
  • HL2/3 cells were plated at a density of 2 x 10 4 cells per well in a volume of 50 ⁇ L, with 50 ⁇ L of nine serial half- logarithmic dilutions of compound in triplicate for 1 h at 37oC/5% CO 2 .
  • 100 ⁇ L of HeLa-CD4-LTR- ⁇ -Gal cells were added to the plates.
  • the cultures were incubated for an additional 48 h at 37oC/5% CO 2 .
  • plates were evaluated for ⁇ -galactosidase production using a chemiluminescent substrate and buffer (ThermoFisher). Briefly, all media was removed from the efficacy plates and replaced with 100 ⁇ L of DPBS.
  • Table 20 Evaluation of inhibition of cell to cell fusion in HeLa-CD4-LTR- ⁇ -Gal and HL2/3 cells. All compounds inhibited HIV-induced cell to cell fusion with EC50 values ranging from 1.1E-05 to 3.1E+03 nM.
  • the conjugates were also evaluated for the ability to inhibit HIV-1-induced cell to cell fusion.
  • the EC 50 values defined in the assays are shown below in Table 21.
  • DAR drug-to-antibody ratio
  • Example 77.7-day mouse PK study comparing IV administration of Conjugate 5b, Conjugate 29a and Fc control (SEQ ID NO: 72) at 5 mg/kg
  • mice were injected intravenously (IV) via the tail vein with 5 mg/kg of test article (5 mL/kg dose volume). Animals were housed under standard IACUC approved housing conditions. At indicated times (0.25, 1, 2, 4, 24, 48, 120, 144, and 168 h), animals were non-terminally bled (retro-orbital, cheek, or by tail vein) with blood collected in K2EDTA tubes to prevent coagulation. Collected blood was centrifuged (2,000 x g, for 10 min) and plasma withdrawn for analysis of test article concentrations over time. The Fc plasma concentrations at each time point were measured by Fc-capture sandwich ELISA as described above.
  • PBMCs Banded PBMCs were gently aspirated from the resulting interface and subsequently washed three times with DPBS by low-speed centrifugation. After the final wash, cells were enumerated by Trypan Blue dye exclusion and re-suspended at 1 x 10 6 cells/mL in RPMI 1640 with 15% Fetal Bovine Serum (FBS), 2 mmol/L L-glutamine, 2 ⁇ g/mL PHA-P, 100 Units/mL penicillin and 100 ⁇ g/mL streptomycin were incubated for 72 hours at 37 o C/5% CO 2 .
  • FBS Fetal Bovine Serum
  • tissue culture medium RPMI 1640 with 15% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin and 3.6 ng/mL recombinant human IL-2).
  • the cultures were then maintained until use by half culture volume change with fresh IL-2 containing tissue culture medium every 3 to 4 days. Assay conditions. Assays were initiated with PBMCs that had been induced to proliferate with PHA-P for 72 hours.
  • PHA-P stimulated PBMCs from three donors were pooled together to minimize the variability that occurs when cells from individual donors are used, resuspended in fresh tissue culture medium at 1 x 10 6 cells/mL and plated in the interior wells of a 96-well round bottom microtiter plate at 50 ⁇ L/well.
  • One-hundred microliters (100 ⁇ L) of each two times the concentration of compound in assay media was added to designated wells of the round-bottom 96-well plate containing the cells, in triplicate.
  • 50 ⁇ L of a pre- determined dilution of virus was added.
  • HIV-1 replication was quantified by the measurement of cell-free HIV-1 RT activity in the tissue culture supernatant. Cytotoxicity was evaluated using the tetrazolium dye XTT following well known standard methods. Quantification of reverse transcriptase (RT) activity. Virus production by untreated and treated cells was quantified by measuring reverse transcriptase in cell-free supernatants using a standard radioactive incorporation polymerization assay. Tritiated thymidine triphosphate (TTP) was purchased at 1 Ci/mL and 1 ⁇ L was used per enzyme reaction.
  • TTP Tritiated thymidine triphosphate
  • Poly rA and oligo dT were prepared at concentrations of 0.5 mg/mL and 1.7 Units/mL, respectively, from a stock solution which was kept at -20°C.
  • the RT reaction buffer was prepared fresh and consists of 125 ⁇ L of 1 M EGTA, 125 ⁇ L of dH 2 O, 125 ⁇ L of 20% Triton X-100, 50 ⁇ L of 1 M Tris (pH 7.4), 50 ⁇ L of 1 M DTT, and 40 ⁇ L of 1 M MgCl 2 .
  • 1 ⁇ L of TTP, 4 ⁇ L of dH 2 O, 2.5 ⁇ L of rAdT and 2.5 ⁇ L of reaction buffer were mixed.
  • Conjugate 71 is a conjugate with single small molecule (SM) targeting moieties (TM), which generated EC 50 values ranging from 0.00828 to >100 (Table 26 and Table 27).
  • SM single small molecule
  • step-a product 500 mg, 0.75 mmol
  • DMF dimethyl methoxysulfoxide
  • 4-imidazolecarboxylic acid 85mg, 0.75mmol
  • HATU hydroxybenzyl
  • step-b product 500 mg, 0.94 mmol
  • 20% Pd(OH) 2 100 mg
  • MeOH 10 ml
  • step d A mixture of step-c product (298 mg, 0.75 mmol), propargyl-Peg4-mesylate (257 mg, 0.83 mmol), and potassium carbonate (310 mg, 2.26 mmol) in acetonitrile (2 mL) were heated at 65 o C for 12 h.
  • Step A mixture of product-f (40 mg, 0.032 mmol), and azido-Peg4-trifluorophenol ester (18 mg, 0.042 mmol) in DMF (0.6 mL) was treated with a solution of copper(II) sulfate (1.2 mg, 0.008 mmol), sodium ascorbate (6.4 mg, 0.032 mmol), and THPTA (7 mg, 16.2 mmol) in water (0.6 mL), and stirred room temperature for 30 min. Reaction progress was monitored by LCMS. The product was purified by semi- preparative HPLC (5% to 100% ACN/water). Yield mg, %. LCMS: [(M + 2H)/2] + 712.7. Example 81.
  • step-a product 160 mg, 0.71 mmol
  • 20% Pd(OH) 2 32 mg
  • MeOH 5 ml
  • step c A mixture of step-b product (115 mg, 0.25 mmol), propargyl-Peg4-mesylate (95 mg, 0.3 mmol), and potassium carbonate (213 mg, 1.5 mmol) were dissolved in acetonitrile (2 mL) and heated at 65 o C for 12h.
  • NUMBERED EMBODIMENTS 1.
  • R 8 are each independently selected from H, optionally substituted C 1 -C 20 alkyl, optionally substituted C 1 -C 20 alkylene, optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 heterocycloalkyl, optionally substituted C 5 -C 15 aryl, and optionally substituted C 2 -C 15 heteroaryl;
  • R 9 are each independently selected from optionally substituted C 1 -C 20 alkylene, optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 heterocycloalkyl, optionally substituted C 5 -C 15 aryl, and optionally substituted C 2 -C 15 heteroaryl;
  • x is 1 or 2;
  • k is 0, 1, 2, 3, 4, or 5;
  • Ar is selected from the group consisting of optionally substituted C 3 -C 20 cycloalkyl, optionally substituted C 2 -C 20 heterocycloalkyl, optional
  • each X is independently C or N; or a pharmaceutically acceptable salt thereof.
  • each A 1 and each A 2 is independently described by any one of formulas (A-Iq)-(A-Ix): or a pharmaceutically acceptable salt thereof.
  • the conjugate is described by formula (M-I): wherein each A 1 is independently described by formula (A-I); each E comprises an Fc domain monomer, and the squiggly line connected to the E indicates that each A 1 -L is covalently attached to E; or a pharmaceutically acceptable salt thereof.
  • the conjugate of embodiment 18, wherein L’ is a nitrogen atom.
  • 20. The conjugate of embodiment 9, wherein the conjugate is described by formula (M-IV): or a pharmaceutically acceptable salt thereof.
  • 21. The conjugate of embodiment 20, wherein the conjugate is described by formula (M-IV-1): or a pharmaceutically acceptable salt thereof.
  • 22. The conjugate of embodiment 21, wherein the conjugate is described by formula (M-IV-2): wherein L’ is the remainder of L, and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • 23. The conjugate of embodiment 22, wherein L’ is a nitrogen atom.
  • 24. The conjugate of embodiment 20, wherein the conjugate is described by formula (M-IV-3): or a pharmaceutically acceptable salt thereof. 25.
  • the conjugate of embodiment 48, wherein L’ is a nitrogen atom.
  • the conjugate of embodiment 46, wherein the conjugate is described by formula (M-VI-3): or a pharmaceutically acceptable salt thereof.
  • the conjugate of embodiment 46, wherein the conjugate is described by formula (M-VI-4): wherein L’ is the remainder of L, and y 1 is an integer from 1-20, or a pharmaceutically acceptable salt thereof.
  • 52. The conjugate of embodiment 51, wherein L’ is a nitrogen atom.
  • the conjugate of embodiment 46, wherein the conjugate is described by formula (M-VI-5): or a pharmaceutically acceptable salt thereof. 54.
  • L or L’ comprises one or more optionally substituted C 1 -C 20 alkylene, optionally substituted C 1 -C 20 heteroalkylene, optionally substituted C 2 -C 20 alkenylene, optionally substituted C 2 -C 20 heteroalkenylene, optionally substituted C 2 -C 20 alkynylene, optionally substituted C 2 -C 20 heteroalkynylene, optionally substituted C 3 -C 20 cycloalkylene, optionally substituted C 2 -C 20 heterocycloalkylene, optionally substituted C 4 -C 20 cycloalkenylene, optionally substituted C 4 -C 20 heterocycloalkenylene, optionally substituted C 8 -C 20 cycloalkynylene, optionally substituted C 8 -C 20 heterocycloalkynylene, optionally substituted C 5 -C 15 arylene, optionally substituted C 3 -C 15 heteroarylene, O, S,
  • each L is described by formula (M- L): J 1 -(Q 1 )g-(T 1 )h-(Q 2 )i-(T 2 )j-(Q 3 )k-(T 3 )l-(Q 4 )m-(T 4 )n-(Q 5 )o-J 2 wherein J 1 is a bond attached to A 1 ; J 2 is a bond attached to E or a functional group capable of reacting with a functional group conjugated to E (e.g., maleimide and cysteine, amine and activated carboxylic acid (e.g., carboxylic acid activated by tetrafluorophenol or trifluorophenol), thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrazine); each of Q 1 , Q 2 , Q 3 , Q 4 , and
  • each E dimerizes to form an Fc domain.
  • 92 The conjugate of any one of embodiments 1-91, wherein each E has the sequence of any one of SEQ ID NOs: 1-95 and 125-153.
  • 93 The conjugate of embodiment 92, wherein each E comprises the sequence of SEQ ID NO: 72 or SEQ ID NO: 73.
  • 94 A conjugate selected from any one of conjugates 1-121.
  • 95 The conjugate of embodiment 94, wherein the conjugate is selected from any one of conjugates 5b, 9b, 12b, 13b, 14b, 15-28, 29a, 29b, 30a, 31-36, 37a, and 38-48.
  • 96 The conjugate of embodiment 90, wherein n is 2, and each E dimerizes to form an Fc domain.
  • the conjugate of embodiment 94, wherein the conjugate is selected from any one of conjugates 30b, 37b, and 49-76. 97.
  • the conjugate of embodiment 94, wherein the conjugate is selected from any one of conjugates 77-121. 98.
  • the conjugate of any one of embodiments 1-98, wherein T is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • 100. A population of conjugates of any one of embodiments 1-98, wherein the average value of T is 1 to 10.
  • 101. A population of conjugates of embodiment 100, wherein the average value of T is 1 to 5. 102.
  • a pharmaceutical composition comprising a conjugate of any one of embodiments 1-99, or a population of conjugates of embodiments 100 or 101, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • a method for the treatment of a subject having a viral infection or presumed to have a viral infection comprising administering to the subject an effective amount of a conjugate of any one of embodiments 1-99, a population of conjugates of embodiments 100 or 101, or a composition of embodiment 102. 104.
  • a method for the prophylactic treatment of a viral infection in a subject in need thereof comprising administering to the subject an effective amount of a conjugate of any one of embodiments 1-99, a population of conjugates of embodiments 100 or 101, or a composition of embodiment 102.
  • 105. The method of embodiment 103 or 104, wherein the viral infection is caused by human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • 106 The method of embodiment 105, wherein the HIV is HIV-1 or HIV-2.
  • 107. The method of any one of embodiments 103-106, wherein the subject is immunocompromised. 108.
  • the method of embodiment 110, wherein the disease is cancer.
  • the method of embodiment 111, wherein the cancer is leukemia, lymphoma, or multiple myeloma. 113.
  • any one of embodiments 103-114 wherein the conjugate of composition is administered intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, by inhalation, by injection, or by infusion. 116.
  • the antiviral agent is selected from an integrase inhibitor, a nucleoside reverse transcriptase inhibitor (NRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor, an inhibitor of viral entry, a CCR5 antagonist, or a CYP3A inhibitor.
  • NRTI nucleoside reverse transcriptase inhibitor
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • protease inhibitor an inhibitor of viral entry, a CCR5 antagonist, or a CYP3A inhibitor.
  • the integrase inhibitor is selected from dolutegravir, elvitegravir, or raltegravir. 120.
  • nucleoside reverse transcriptase inhibitor is selected from abacavir, lamivudine, zidovudine, emtricitabine, tenofovir, emtricitabine, didanosine, or stavudine.
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • the method of embodiment 118, wherein the protease inhibitor is selected from atazanavir, cobicistat, darunavir, cobicistat, lopinavir, ritonavir, fosamprenavir, tipranavir, nelfinavir, indinavir, or saquinavir. 123.
  • the method of embodiment 118, wherein the inhibitor of viral entry is enfuvirtide.
  • the CCR5 antagonist is maraviroc.
  • the method of embodiment 118, wherein the CYP3A inhibitor is cobicistat or ritonavir. 126.
  • a method of synthesizing a conjugate of any one of embodiments 1-99, a population of conjugates of any one of embodiments 100 or 101, or pharmaceutical composition of embodiment 102 comprising: (a) providing a first composition including E; (b) providing a second composition including a compound of formula (DF-I) or salt thereof: wherein L’ is the remainder of L; m is 0, 1, 2, 3, or 4; and each R is, independently, halo, cyano, nitro, optionally substituted C 1 -C 6 alkyl group, or optionally substituted C 1 -C 6 heteroalkyl group; and (c) combining the first composition, the second composition, and a buffer to form a mixture.
  • DF-I formula
  • a method of synthesizing a conjugate of any one of embodiments 1-99, a population of conjugates of any one of embodiments 100 or 101, or pharmaceutical composition of embodiment 102 comprising: (a) providing a first composition including E; (b) providing a second composition including a compound of formula (MF-I) or salt thereof: wherein L’ is the remainder of L; m is 0, 1, 2, 3, or 4; and each R is, independently, halo, cyano, nitro, optionally substituted C 1 -C 6 alkyl group, or optionally substituted C 1 -C 6 heteroalkyl group; and (c) combining the first composition, the second composition, and a buffer to form a mixture. 128.
  • a method of synthesizing a conjugate of any one of embodiments 1-99, a population of conjugates of any one of embodiments 100 or 101, or pharmaceutical composition of embodiment 102 comprising: (a) providing a first composition including E; (b) providing a second composition including a compound of formula (DF-II) or salt thereof: wherein G is optionally substituted C 1 -C 6 alkylene, optionally substituted C 1 -C 6 heteroalkylene, optionally substituted C 2 -C 6 alkenylene, optionally substituted C 2 -C 6 heteroalkenylene, optionally substituted C 2 -C 6 alkynylene, optionally substituted C 2 -C 6 heteroalkynylene, optionally substituted C 3 -C 10 cycloalkylene, optionally substituted C 2 -C 10 heterocycloalkylene, optionally substituted C 6 -C 10 arylene, or optionally substituted C 2 -C 10 heteroarylene; L’-G-L’’ is the remainder
  • a method of synthesizing a conjugate of any one of embodiments 1-99, a population of conjugates of any one of embodiments 100 or 101, or pharmaceutical composition of embodiment 102 comprising: (a) providing a first composition including formula (D-G3-A) or a salt thereof: where G a is a functional group that reacts with G b to form G; (b) providing a second composition including formula (D-G3-B) or a salt thereof: where G b is a functional group that reacts with G a to form G; and (c) combining the first composition and the second composition to form a first mixture, where m is 0, 1, 2, 3, or 4; and each R is, independently, halo, cyano, nitro, optionally substituted C 1 -C 6 alkyl group, or optionally substituted C 1 -C 6 heteroalkyl group.
  • a method of synthesizing a conjugate of any one of embodiments 1-99, a population of conjugates of any one of embodiments 100 or 101, or pharmaceutical composition of embodiment 102 comprising: (a) providing a first composition including formula (M-G3-A) or a salt thereof: where G a is a functional group that reacts with G b to form G; (b) providing a second composition including formula (M-G3-B) or a salt thereof: where G b is a functional group that reacts with G a to form G; and (c) combining the first composition and the second composition to form a first mixture.

Abstract

Des compositions et des méthodes pour le traitement d'infections virales font intervenir des conjugués contenant des inhibiteurs du récepteur gp120 viral (par ex., temsavir, BMS-818251, DMJ-ll-121, BNM-IV-147, ou analogues de ceux-ci) liés à un monomère Fc, un domaine Fc et un peptide de liaison à Fc, une protéine d'albumine, ou un peptide de liaison à l'albumine. En particulier, les conjugués peuvent être utilisés dans le traitement d'infections virales (par exemple, des infections par le VIH).
PCT/US2021/064151 2020-12-17 2021-12-17 Compositions et méthodes pour le traitement du virus de l'immunodéficience humaine WO2022133281A1 (fr)

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