RU2816717C2 - Compositions and methods for treating viral infections - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a group of inventions comprising a conjugate for treating a viral infection caused by an influenza or parainfluenza virus, or a pharmaceutically acceptable salt thereof, the use of said conjugate for treating a viral infection caused by an influenza virus or a parainfluenza virus, a pharmaceutical composition for treating a viral infection caused by an influenza virus or a parainfluenza virus, containing a conjugate or a pharmaceutically acceptable salt thereof, a method of treating a subject having a viral infection caused by an influenza virus or a parainfluenza virus, and a method for preventive treatment of a viral infection caused by an influenza virus or a parainfluenza virus in a subject in need thereof.

EFFECT: invention extends the range of products for treating viral infection caused by influenza virus or parainfluenza virus.

30 cl, 68 dwg, 65 tbl, 147 ex

Description

Prerequisites for creating the invention

The need for new antiviral drugs to treat influenza is significant and particularly critical in the medical field. Influenza virus, the causative agent of influenza or acute respiratory viral infection, is responsible for three to five million cases of severe illness each year and approximately 500,000 deaths worldwide. Although most people recover completely from the flu in about one to two weeks, others develop life-threatening complications such as pneumonia. Thus, the flu can be deadly, especially for the young, old, or chronically ill. People with weak or compromised immune systems, such as people with advanced HIV infection or transplant patients whose immune systems are drug-suppressed to prevent organ transplant rejection, are at higher risk for flu-related complications. Pregnant women and young children are also at high risk of complications.

The development of antiviral drugs to treat influenza remains challenging. Several antiviral agents against influenza have been approved for clinical use, and these agents play an important role in modulating disease severity and controlling pandemics while vaccines are being developed. However, drug-resistant strains have emerged among the most commonly used inhibitors.

Influenza antiviral agents primarily target proteins present on the surface of the influenza virus particle. The influenza virus envelope contains two immunodominant glycoproteins, hemagglutinin and neuraminidase, which play key roles in viral infection and spread. Hemagglutinin influences viral attachment to the host cell by interacting with surface sialic acids, thereby initiating entry. Neuraminidase is an exoglycosidase enzyme that cleaves sialic acids (terminal neuramic acid residues) from glycan structures on the surface of infected host cells, releasing progeny viruses and allowing the virus to spread from the host cell to uninfected surrounding cells. Thus, inhibition of neuraminidase serves as a pharmacological target for antiviral drugs. Viral neuraminidase inhibitors have been identified and are used to reduce viral spread, including oseltamivir (Tamiflu™), zanamivir (Relenza™), and peramivir (Rapivab™).

However, influenza in transplant recipients is still characterized by prolonged viral shedding, increasing the likelihood of the development of drug-resistant strains. New, more effective therapies are needed to treat influenza.

Brief description of the invention

The disclosure relates to conjugates, compositions and methods for inhibiting the growth of viruses, as well as methods for treating viral infections. In particular, such conjugates contain monomers or dimers of a moiety that inhibits influenza virus neuraminidase (eg, zanamivir, peramivir, or analogs thereof) conjugated to Fc monomers, Fc domains, Fc-binding peptides, albumin proteins, or protein albumin-binding peptides. A neuraminidase inhibitor (for example, zanamivir, peramivir or their analogs) in conjugates specifically targets neuraminidase on the surface of the viral particle. Fc monomers or Fc domains in conjugates bind to Fcγ receptors (FcγRs) (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells, such as neutrophils, to activate phagocytosis and effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), which leads to the uptake and destruction of viral particles by immune cells and further enhances the antiviral activity of the conjugates. Albumin or albumin-binding peptide can increase the half-life of the conjugate, for example, by binding albumin to the recycling neonatal Fc receptor. Such compositions are useful in methods of inhibiting the growth of viruses and in methods of treating viral infections such as infections caused by influenza A virus, influenza B virus and influenza C virus.

In one aspect, the disclosure provides a conjugate described by formula (1):

where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (eg, an Fc domain monomer having the sequence shown in any of SEQ ID NO: 1-68); L in each A ₁ -LA ₂ is a linker covalently attached to the sulfur atom of the hinge cysteine in each E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the two wavy lines connected to the two E's indicate that each A ₁ -LA ₂ is covalently attached (eg, via a covalent bond or linker) to a pair of sulfur atoms of the two hinge cysteines in the two E's, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the disclosure provides a conjugate described by formula (1):

where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (eg, an Fc domain monomer having the sequence shown in any of SEQ ID NO: 1-68); L in each A ₁ -LA ₂ is a linker covalently attached to the sulfur atom of the hinge cysteine in each E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the two wavy lines connected to the two E's indicate that each A ₁ -LA ₂ is covalently attached (e.g., by a covalent bond or linker) to a pair of sulfur atoms of the two hinge cysteines in the two E's, or its pharmaceutically acceptable salt. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the disclosure provides a conjugate described by formula (1):

where each A ₁ and each A ₂ are independently selected from any of formulas (A-VI) - (A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (e.g., an Fc domain monomer having the sequence represented by any of SEQ ID NO: 1-68); L in each A ₁ -LA ₂ is a linker covalently attached to the sulfur atom of the hinge cysteine in each E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, or 20), and two wavy lines connected to two E's indicate that each A ₁ -LA ₂ is covalently attached (e.g., via a covalent bond or linker) to a pair of sulfur atoms two hinge cysteines in two E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (2):

where each A ₁ is independently selected from any of the formulas (AI)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (eg, an Fc domain monomer having the sequence shown in any of SEQ ID NO: 1-68); L in each LA ₁ is a linker covalently attached to the sulfur atom of the hinge cysteine in each E and to A ₁ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and two wavy lines connected to two sulfur atoms indicate that each LA ₁ is covalently attached (eg, by a covalent bond or linker) to a pair of sulfur atoms of two hinge cysteines in two E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (AI)-(A-XII).

In another aspect, the invention relates to a conjugate represented by formula (2):

where each A ₁ is independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (eg, an Fc domain monomer having the sequence shown in any of SEQ ID NO: 1-68); L in each LA ₁ is a linker covalently attached to the sulfur atom of the hinge cysteine in each E and to A ₁ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and two wavy lines connected to two sulfur atoms indicate that each LA ₁ is covalently attached (eg, by a covalent bond or linker) to a pair of sulfur atoms of two hinge cysteines in two E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (AI)-(AV).

In another aspect, the invention relates to a conjugate represented by formula (2):

where each A ₁ is independently selected from any of formulas (A-VI)-(A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₁ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (eg, an Fc domain monomer having the sequence shown in any of SEQ ID NO: 1-68); L in each LA ₁ is a linker covalently attached to the sulfur atom of the hinge cysteine in each E and to A ₁ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and two wavy lines connected to two sulfur atoms indicate that each LA ₁ is covalently attached (eg, by a covalent bond or linker) to a pair of sulfur atoms of two hinge cysteines in two E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (A-VI)-(A-IX).

In some embodiments of any of the above embodiments, each E includes an Fc domain monomer having the sequence presented in any of SEQ ID NO: 1-68.

In some embodiments, each E contains the sequence MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (SEQ ID NO: 10).

In some embodiments, each E contains the sequence MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

In some embodiments, at least one of the pair of sulfur atoms is a sulfur atom corresponding to (eg, a sulfur atom) a hinge cysteine of SEQ ID NO: 10 or SEQ ID NO: 11, i.e. Cys10, Cys13, Cys16, or Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the pair of sulfur atoms is the sulfur atoms corresponding to (e.g., the sulfur atoms) Cys10 and Cys13 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys10 and Cys16 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys30 and Cys18 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys13 and Cys36 in SEQ ID NO: 10 or SEQ ID NO : 11, Cys13 and Cys38 in SEQ ID NO: 10 or SEQ ID NO: 11, and/or Cys36 and Cys38 in SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, when T is 2, the pair of sulfur atoms is (e.g., sulfur atoms corresponding to) Cys10 and Cys13 in SEQ ID NO: 10 or SEQ ID NO: 11, or Cys36 and Cys38 in SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, the pair of sulfur atoms includes one cysteine sulfur atom from each E, i.e. L-A, together with the sulfur atoms to which it is attached, forms a bridge between two Fc domains (eg, two Fc domains containing the sequence SEQ ID NO: 10 or SEQ ID NO: 11). In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, the sulfur atom pair is a sulfur atom corresponding to (e.g., the sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (e.g., the sulfur atom) Cys16 of the sequence SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, the pair of sulfur atoms represents is a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E.

In some embodiments, when T is 2, the sulfur atom pairs are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (e.g., a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, the sulfur atom pairs are a sulfur atom corresponding to (e.g., a sulfur atom ) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom, corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10, or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of another E. In some embodiments, when T is 2, the pairs of sulfur atoms are a sulfur atom corresponding to (eg, a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E , and a sulfur atom corresponding to (for example, the sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, when T is 2, the sulfur atom pairs are a sulfur atom corresponding to (e.g., the sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (e.g., a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, the sulfur atom pairs are a sulfur atom corresponding to (e.g., a sulfur atom ) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom, corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, when T is 2, the sulfur atom pairs are a sulfur atom corresponding to (e.g., the sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (e.g., the sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, when T is 3, the sulfur atom pairs are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., sulfur atom) Cys10 of the sequence SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E. In some embodiments, when T is 3, the pairs of sulfur atoms are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (eg, a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E. In some embodiments, when T is 3, the pairs of sulfur atoms are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (eg, the sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E. In some embodiments, when T is 3, the pairs of sulfur atoms are a sulfur atom corresponding to (e.g., a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (eg, the sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E.

In some embodiments, when T is 3, the sulfur atom pairs are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., sulfur atom) Cys10 of the sequence SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E.

In some embodiments, the conjugate has the structure:

where each of a, b, c and d is independently equal to 0 or 1, and wherein when a, b, c or d is equal to O, the two sulfur atoms form a disulfide bond.

In some embodiments, a is 1, and b, c, and d are 0. In some embodiments, a and b are 1, and c, and d are 0. In some embodiments, a and c are 1, and b and d are 0 In some embodiments, a and d are equal to 1, and b and c are equal to 0. In some embodiments, a, b, and c are equal to 1, and d is equal to 0. In some embodiments, a, b, and d are equal to 1, and c is equal to 0. In some embodiments, a, c, and d are equal to 1, and b is equal to 0. In some embodiments, b and c are equal to 1, and a and d are equal to 0. In some embodiments, b and d are equal to 1, and a and c are equal to 0. In some embodiments, b, c, and d are equal to 1, and a is equal to 0. In some embodiments, c and d are equal to 1, and a and b are equal to 0. In some embodiments, a, b, c, and d are equal to 1 .

In some embodiments, each E comprises the sequence MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4).

In some embodiments, each E comprises the sequence MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33).

In some embodiments, at least one of the pair of sulfur atoms is a sulfur atom corresponding to (eg, a sulfur atom) a hinge cysteine of SEQ ID NO: 4 or SEQ ID NO: 33, i.e. Cys10 and/or Cys13. In some embodiments, the sulfur atom pair is the sulfur atoms corresponding to (e.g., the sulfur atoms) Cys10 and Cys13 in SEQ ID NO: 4 or SEQ ID NO: 33.

In some embodiments, the pair of sulfur atoms includes one cysteine sulfur atom from each E, i.e. L-A, together with the sulfur atoms to which it is attached, forms a bridge between two Fc domains (eg, two Fc domains containing the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 33). In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 of one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E. In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., the sulfur atom) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from one E, and a sulfur atom corresponding to (e.g., the sulfur atom) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E. In some embodiments, when T is 2, the sulfur atom pairs represent a sulfur atom corresponding to (e.g., sulfur atom) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E, and an atom a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E.

In some embodiments, the conjugate has the structure:

where each of a and b is independently equal to 0 or 1, and wherein when a or b is equal to 0, the two sulfur atoms form a disulfide bond. In some embodiments, a is 1 and b is 0. In some embodiments, a is 0 and b is 1. In some embodiments, a and b are 1.

In some embodiments, each E comprises the sequence MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSVGKHHHHHH (SEQ ID NO: 8).

In some embodiments, at least one of the pair of sulfur atoms is a sulfur atom corresponding to (eg, a sulfur atom) the hinge cysteine of SEQ ID NO: 8, i.e. Cys10 and/or Cys13. In some embodiments, the sulfur atom pair is sulfur atoms corresponding to (e.g., the sulfur atoms) Cys10 and Cys13 in SEQ ID NO: 8.

In some embodiments, the pair of sulfur atoms includes one cysteine sulfur atom from each E, i.e. L-A, together with the sulfur atoms to which it is attached, forms a bridge between two Fc domains (eg, two Fc domains containing the sequence shown in SEQ ID NO: 8). In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 8 from one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 8 from another E. In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., the sulfur atom) Cys13 of SEQ ID NO: 8 of one E, and a sulfur atom corresponding to (e.g., the sulfur atom) Cys143 of SEQ ID NO: 8 from another E. In some embodiments, when T is 2, the pairs of sulfur atoms are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of the sequence SEQ ID NO: 8 from one E, and a sulfur atom corresponding to (e.g., a sulfur atom ) Cys10 of SEQ ID NO: 8 from another E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 8 from one E, and a sulfur atom corresponding to (eg, sulfur atom) Cys13 of SEQ ID NO : 8 from another E.

In some embodiments, the conjugate has the structure:

where each of a and b is independently equal to 0 or 1, and wherein when a or b is equal to 0, the two sulfur atoms form a disulfide bond. In some embodiments, a is 1 and b is 0. In some embodiments, a is 0 and b is 1. In some embodiments, a and b are 1.

In another aspect, the invention also provides a group of conjugates described in any of the previous aspects, in which the average T value is 1-20 (for example, the average T value is 1-2, 1-3, 1-4, 1-5, 5 -10, 10-15 or 15-20). In some embodiments, the average T value is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In another aspect, the invention relates to a conjugate represented by formula (3):

where each A ₁ and each A ₂ are independently selected from any of formulas (AI)-(A-VII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each A ₁ -LA ₂ is a linker covalently attached to the sulfur atom of the hinge cysteine in E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and two wavy lines connected to E indicate that each A ₁ -LA ₂ is covalently attached (eg, through a covalent bond or linker) to the sulfur atom of the hinge cysteine in E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (3):

where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each A ₁ -LA ₂ is a linker covalently attached to the sulfur atom of the hinge cysteine in E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and two wavy lines connected to E indicate that each A ₁ -LA ₂ is covalently attached (eg, through a covalent bond or linker) to the sulfur atom of the hinge cysteine in E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (3):

where each A ₁ and each A ₂ are independently selected from any of formulas (A-VI)-(A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each A ₁ -LA ₂ is a linker covalently attached to the sulfur atom of the hinge cysteine in E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and two wavy lines connected to E indicate that each A ₁ -LA ₂ is covalently attached (eg, through a covalent bond or linker) to the sulfur atom of the hinge cysteine in E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In some embodiments, each E includes an Fc domain monomer having the sequence presented in any of SEQ ID NO: 1-68.

In some embodiments, each E contains the sequence MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (SEQ ID NO: 10).

In some embodiments, each E contains the sequence MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

In some embodiments, at least one of the pair of sulfur atoms is a sulfur atom corresponding to (eg, a sulfur atom) a hinge cysteine of SEQ ID NO: 10 or SEQ ID NO: 11, i.e. Cys10, Cys13, Cys16, or Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the pair of sulfur atoms is the sulfur atoms corresponding to (e.g., the sulfur atoms) Cys10 and Cys13 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys10 and Cys16 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys30 and Cys18 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys13 and Cys36 in SEQ ID NO: 10 or SEQ ID NO : 11, Cys13 and Cys38 in SEQ ID NO: 10 or SEQ ID NO: 11, and/or Cys36 and Cys38 in SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, when T is 2, the sulfur atom pair is sulfur atoms corresponding to (e.g., the sulfur atoms) Cys10 and Cys13 in SEQ ID NO: 10 or SEQ ID NO: 11, and Cys36 and Cys38 in SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, the pair of sulfur atoms includes one cysteine sulfur atom from each E, i.e. L-A, together with the sulfur atoms to which it is attached, forms a bridge between two Fc domains (eg, two Fc domains containing the sequence SEQ ID NO: 10 or SEQ ID NO: 11). In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, the sulfur atom pair is a sulfur atom corresponding to (e.g., the sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (e.g., the sulfur atom) Cys16 of the sequence SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, the pair of sulfur atoms represents is a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E.

In some embodiments, when T is 2, the sulfur atom pairs are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (e.g., a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, the sulfur atom pairs are a sulfur atom corresponding to (e.g., a sulfur atom ) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom, corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, the pairs of sulfur atoms are a sulfur atom corresponding to (eg, a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to ( e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (eg, the sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, when T is 2, the sulfur atom pairs are a sulfur atom corresponding to (e.g., the sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (e.g., a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, the sulfur atom pairs are a sulfur atom corresponding to (e.g., a sulfur atom ) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom, corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, when T is 2, the sulfur atom pairs are a sulfur atom corresponding to (e.g., the sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (e.g., the sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, when T is 3, the sulfur atom pairs are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., sulfur atom) Cys10 of the sequence SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E. In some embodiments, when T is 3, the pairs of sulfur atoms are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (eg, the sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E. In some embodiments, when T is 3, the pairs of sulfur atoms are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (eg, the sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E. In some embodiments, when T is 3, the pairs of sulfur atoms are a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E, and an atom sulfur corresponding to (eg, the sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E.

In some embodiments, when T is 3, the sulfur atom pairs are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (e.g., sulfur atom) Cys10 of the sequence SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys18 of SEQ ID NO: 10 or SEQ ID NO : 11 from another E.

In some embodiments, the conjugate has the structure:

wherein each of a, b, c and d is independently equal to 0 or 1, and wherein when a, b, c or d is equal to 0, the two sulfur atoms form a disulfide bond.

In some embodiments, a is 1, and b, c, and d are 0. In some embodiments, a and b are 1, and c, and d are 0. In some embodiments, a and c are 1, and b and d are 0 In some embodiments, a and d are equal to 1, and b and c are equal to 0. In some embodiments, a, b, and c are equal to 1, and d is equal to 0. In some embodiments, a, b, and d are equal to 1, and c is equal to 0. In some embodiments, a, c, and d are equal to 1, and b is equal to 0. In some embodiments, b and c are equal to 1, and a and d are equal to 0. In some embodiments, b and d are equal to 1, and a and c are equal to 0. In some embodiments, b, c, and d are equal to 1, and a is equal to 0. In some embodiments, c and d are equal to 1, and a and b are equal to 0. In some embodiments, a, b, c, and d are equal to 1 .

In some embodiments, each E comprises the sequence MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4).

In some embodiments, each E comprises the sequence MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33).

In some embodiments, at least one of the pair of sulfur atoms is a sulfur atom corresponding to (eg, a sulfur atom) a hinge cysteine of SEQ ID NO: 4 or SEQ ID NO: 33, i.e. Cys10 and/or Cys13. In some embodiments, the sulfur atom pair is the sulfur atoms corresponding to (e.g., the sulfur atoms) Cys10 and Cys13 in SEQ ID NO: 4 or SEQ ID NO: 33.

In some embodiments, the pair of sulfur atoms includes one cysteine sulfur atom from each E, i.e. L-A, together with the sulfur atoms to which it is attached, forms a bridge between two Fc domains (eg, two Fc domains containing the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 33). In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 of one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E. In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., the sulfur atom) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from one E, and a sulfur atom corresponding to (e.g., the sulfur atom) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E. In some embodiments, when T is 2, the sulfur atom pairs represent a sulfur atom corresponding to (e.g., sulfur atom) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E, and an atom a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 of one E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E.

In some embodiments, the conjugate has the structure:

where each of a and b is independently equal to 0 or 1, and wherein when a or b is equal to 0, the two sulfur atoms form a disulfide bond. In some embodiments, a is 1 and b is 0. In some embodiments, a is 0 and b is 1. In some embodiments, a and b are 1.

In some embodiments, each E comprises the sequence MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSVGKHHHHHH (SEQ ID NO: 8)

In some embodiments, at least one of the pair of sulfur atoms is a sulfur atom corresponding to (eg, a sulfur atom) the hinge cysteine of SEQ ID NO: 8, i.e. Cys10 and/or Cys13. In some embodiments, the sulfur atom pair is sulfur atoms corresponding to (e.g., the sulfur atoms) Cys10 and Cys13 in SEQ ID NO: 8.

In some embodiments, the pair of sulfur atoms includes one cysteine sulfur atom from each E, i.e. L-A, together with the sulfur atoms to which it is attached, forms a bridge between two Fc domains (eg, two Fc domains containing the sequence shown in SEQ ID NO: 8). In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 8 from one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of SEQ ID NO: 8 from another E. In some embodiments, the pair of sulfur atoms is a sulfur atom corresponding to (e.g., a sulfur atom) Cys13 of SEQ ID NO: 8 of one E, and a sulfur atom corresponding to (e.g., a sulfur atom) Cys13 of SEQ ID NO: 8 from another E. In some embodiments, when T is 2, the pairs of sulfur atoms are a sulfur atom corresponding to (e.g., a sulfur atom) Cys10 of the sequence SEQ ID NO: 8 from one E, and a sulfur atom corresponding to (e.g., a sulfur atom ) Cys10 of SEQ ID NO: 8 from another E, and a sulfur atom corresponding to (eg, a sulfur atom) Cys13 of SEQ ID NO: 8 from one E, and a sulfur atom corresponding to (eg, sulfur atom) Cys13 of SEQ ID NO : 8 from another E.

In some embodiments, the conjugate has the structure:

where each of a and b is independently equal to 0 or 1, and wherein when a or b is equal to 0, the two sulfur atoms form a disulfide bond. In some embodiments, a is 1 and b is 0. In some embodiments, a is 0 and b is 1. In some embodiments, a and b are 1.

In another aspect, the invention relates to a conjugate represented by formula (4):

where each A ₁ is independently selected from any of the formulas (AI)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each LA ₁ is a linker covalently attached to the sulfur atom of the hinge cysteine in E and to A ₁ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and two wavy lines connected to E indicate that each LA ₁ is covalently attached (eg, through a covalent bond or linker) to the sulfur atom of the hinge cysteine in E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (AI)-(A-XII).

In another aspect, the invention relates to a conjugate represented by formula (4):

where each A ₁ is independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each LA ₁ is a linker covalently attached to the sulfur atom of the hinge cysteine in E and to A ₁ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and a wavy line connected to E indicates that each LA ₁ is covalently attached (eg, through a covalent bond or linker) to the sulfur atom of the hinge cysteine in E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (AI)-(AV). In another aspect, the invention relates to a conjugate represented by formula (4):

where each A ₁ is independently selected from any of formulas (A-VI)-(A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each LA ₁ is a linker covalently attached to the sulfur atom of the hinge cysteine in E and to A ₁ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and a wavy line connected to E indicates that each LA ₁ is covalently attached (eg, through a covalent bond or linker) to the sulfur atom of the hinge cysteine in E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (A-VI)-(A-IX).

In some embodiments, each E includes an Fc domain monomer having the sequence presented in any of SEQ ID NO: 1-68.

In some embodiments, each E comprises the sequence MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4).

In some embodiments, each E comprises the sequence MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33).

In some embodiments, at least one of the sulfur atoms is a sulfur atom corresponding to (eg, a sulfur atom) a hinge cysteine of SEQ ID NO: 4 or SEQ ID NO: 33, i.e. Cys10 and/or Cys13. In some embodiments, the sulfur atoms are a sulfur atom corresponding to (e.g., the sulfur atom) Cys10 in SEQ ID NO: 4 or SEQ ID NO: 33. In some embodiments, the sulfur atom corresponds to (e.g., the sulfur atom) Cys10 in SEQ ID NO: : 4 or SEQ ID NO: 33.

In some embodiments, the conjugate has the structure:

where each of a and b is independently equal to 0 or 1, and wherein when a or b is equal to 0, the sulfur atoms represent a thiol. In some embodiments, a is 1 and b is 0. In some embodiments, a is 0 and b is 1. In some embodiments, a and b are 1.

In another aspect, the invention relates to a group of conjugates in which the average T value is 1-20 (for example, the average T value is 1-2, 1-3, 1-4, 1-5, 5-10, 10-15 or 15 -20). In some embodiments, the average T value is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In another aspect, the invention relates to a conjugate represented by formula (3):

where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each A ₁ -LA ₂ is a linker covalently attached to the surface-exposed lysine nitrogen atom in E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the wavy line connected to E indicates that each A ₁ -LA ₂ is covalently attached (eg, through a covalent bond or linker) to the surface-exposed lysine nitrogen atom of E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (3):

where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each A ₁ -LA ₂ is a linker covalently attached to the surface-exposed lysine nitrogen atom in E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the wavy line connected to E indicates that each A ₁ -LA ₂ is covalently attached (eg, through a covalent bond or linker) to the surface-exposed lysine nitrogen atom of E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (3):

where each A ₁ and each A ₂ are independently selected from any of formulas (A-VI)-(A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each A ₁ -LA ₂ is a linker covalently attached to the surface-exposed lysine nitrogen atom in E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the wavy line connected to E indicates that each A ₁ -LA ₂ is covalently attached (eg, through a covalent bond or linker) to the surface-exposed lysine nitrogen atom of E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (4):

where each A ₁ is independently selected from any of the formulas (AI)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C2-C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each LA ₁ is a linker covalently attached to the surface-exposed lysine nitrogen atom in E and to A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and a wavy line connected to E indicates that each LA ₁ is covalently attached (eg, through a covalent bond or linker) to the surface-exposed lysine nitrogen atom of E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A may be independently selected from any of formulas (AI)-(A-XII).

In another aspect, the invention relates to a conjugate represented by formula (4):

where each A ₁ is independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each LA ₁ is a linker covalently attached to the surface-exposed nitrogen atom of the lysine in E and to A ₁ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and a wavy line connected to E indicates that each LA ₁ is covalently attached (eg, through a covalent bond or linker) to the surface-exposed lysine nitrogen atom of E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (AI)-(AV).

In another aspect, the invention relates to a conjugate represented by formula (4):

where each A ₁ is independently selected from any of formulas (A-VI)-(A-IX):

where R ₁ is selected from -OH, -Nth, -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); L in each LA ₁ is a linker covalently attached to the surface exposed nitrogen atom of lysine in E and to A; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and a wavy line connected to E indicates that each LA ₁ is covalently attached (eg, through a covalent bond or linker) to the surface-exposed lysine nitrogen atom of E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (A-VI)-(A-IX).

In another aspect, the invention relates to a conjugate represented by formula (5):

where each Int is independently selected from any of the intermediates presented in Table 1; E contains an Fc domain monomer or an Fc domain (eg, an Fc domain monomer or an Fc domain, each Fc domain monomer having, independently, a sequence set forth in any of SEQ ID NOs: 1-68); The L in each L-Int is a linker covalently attached to the surface-exposed nitrogen atom of the lysine in E and to the Int; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), a wavy line connected to E indicates that each L-Int is covalently attached (e.g., through a covalent bond or linker) to the surface-exposed lysine nitrogen atom or the surface-exposed cysteine sulfur atom in E, or its pharmaceutical acceptable salt. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each Int may be independently selected from any of the intermediates presented in Table 1.

The intermediates in Table 1 can be conjugated to an Fc domain or Fc domain monomer (eg, via a linker) using any suitable methods known to those skilled in the art, including any of the methods described or illustrated herein. In some embodiments, the conjugate (e.g., the conjugate described by formula (5)) comprises E, wherein E is an Fc domain monomer or an Fc domain (e.g., an Fc domain monomer or an Fc domain, wherein each Fc domain monomer has, independently, the sequence presented in any of SEQ ID NO: 1-68). In preferred embodiments, one or more nitrogen atoms of one or more surface exposed lysine residues in E, or one or more sulfur atoms of one or more surface exposed cysteines in E, are covalently conjugated to a linker (eg, a PEG ₂ -PEG ₂₀ linker). The E-conjugated linker may be functionalized such that it can react to form a covalent bond with any of the Ints described herein (eg, the Int of Table 1). In preferred embodiments, E is conjugated to a linker functionalized with an azido group, and Int (eg, Int from Table 1) is functionalized with an alkyne group. When conjugated (eg by click chemistry) the azido linker E and the alkyne linker Int, a conjugate of the invention is formed, for example the conjugate described by formula (5). In yet other embodiments, E is conjugated to a linker functionalized with an alkyne group, and Int (eg, Int from Table 1) is functionalized with an azido group. When conjugated (for example, by click chemistry) linker-alkyne E and linker-azido Int, a conjugate according to the invention is formed, for example, the conjugate described by formula (5).

In some embodiments, each E includes an Fc domain monomer having the sequence presented in any of SEQ ID NO: 1-68. In some embodiments, E includes the sequence

In some embodiments, each E includes the sequence

In some embodiments, E includes the sequence

In some embodiments, each E contains the sequence

In some embodiments of the previous three aspects, the nitrogen atom is a surface exposed nitrogen of lysine, for example, a nitrogen atom corresponding to (e.g., a nitrogen atom) Lys35, Lys63, Lys77, Lys79, Lys106, Lysl23, Lysl29, Lysl81, Lys203, Lys228, or Lys236 sequences of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the nitrogen atom is a nitrogen atom corresponding to (e.g., the nitrogen atom) Lys65, Lys79, Lys108, Lys230, and/or Lys238 of the sequence SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, the conjugate has the structure:

wherein each of a, b, c, d and e is independently equal to 0 or 1, and wherein when a, b, c, d or e is equal to 0, the two nitrogen atoms represent NH ₂ . In some embodiments, a is 1, and b, c, d, and e are 0. In some embodiments, b is 1, and a, c, d, and e are 0. In some embodiments, c is 1, and a, b , d, and e are 0. In some embodiments, d is 1, and a, b, c, and e are 0. In some embodiments, e is 1, and a, b, c, and d are 0. In some embodiments, a and b are 1, and c, d, and e are 0. In some embodiments, a and c are 1, and b, d, and e are 0. In some embodiments, a and d are 1, and b, these are 0. In some embodiments, a and e are 1, and b, c, and d are 0. In some embodiments, b and c are 1, and a, d, and e are 0. In some embodiments, b and d are 1, and a , c, and e are equal to 0. In some embodiments, b and e are equal to 1, and a, c, and d are equal to 0. In some embodiments, e and d are equal to 1, and a, b, and e are equal to 0. In some embodiments, c and e are 1, and a, b, and d are 0. In some embodiments, d and e are 1, and a, b, and c are 0. In some embodiments, a, b, and c are 1, and d and e are equal 0. In some embodiments, a, b, and d are equal to 1, and c, and e are equal to 0. In some embodiments, a, b, and e are equal to 1, and c, and d are equal to 0. In some embodiments, a, c, and d are equal 1, and b and e are equal to 0. In some embodiments, a, c, and e are equal to 1, and b and d are equal to 0. In some embodiments, a, d, and e are equal to 1, and b and c are equal to 0. In some embodiments embodiments, b, c, and d are 1, and a, and e are 0. In some embodiments, b, d, and e are 1, and a, and c are 0. In some embodiments, c, d, and e are 1, and a and b are equal to 0.

In another aspect, the invention relates to a group of conjugates described in any of the previous aspects, where the average T value is 1-20 (for example, the average T value is 1-2, 1-3, 1-4, 1-5, 5-10 , 10-15 or 15-20). In some embodiments, the average T value 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 of the conjugates described herein, the conjugate forms a homodimer including an Fc domain.

In some embodiments of the conjugates described herein, E homodimerizes with another E to form an Fc domain.

In another aspect, the invention relates to a conjugate represented by formula (3):

where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an albumin protein (eg, an albumin protein having the sequence shown in any of SEQ ID NOs: 69-71), an albumin-binding peptide protein or an Fc-binding peptide; L in each A ₁ -LA ₂ is a linker covalently attached to the surface-exposed cysteine sulfur atom or the surface-exposed lysine nitrogen atom in E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the wavy line connected to E indicates that each A ₁ -LA ₂ is independently covalently attached (eg, via a bond or linker) to a solvent-exposed cysteine sulfur atom or solvent-exposed lysine nitrogen atom in E, or its pharmaceutically acceptable salt. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (5):

where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an albumin protein (eg, an albumin protein having the sequence shown in any of SEQ ID NOs: 69-71), an albumin-binding peptide protein or an Fc-binding peptide; L in each A ₁ -LA ₂ is a linker independently covalently attached to the surface-exposed cysteine sulfur atom or the surface-exposed lysine nitrogen atom in E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the wavy line connected to E indicates that each A ₁ -LA ₂ is independently covalently attached (eg, via a bond or linker) to a solvent-exposed cysteine sulfur atom or solvent-exposed lysine nitrogen atom in E, or its pharmaceutically acceptable salt. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (3):

where each A ₁ and each A ₂ are independently selected from any of formulas (A-VI)-(A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an albumin protein (eg, an albumin protein having the sequence shown in any of SEQ ID NOs: 69-71), an albumin-binding peptide protein or an Fc-binding peptide; L in each A ₁ -LA ₂ is a linker independently covalently attached to the surface-exposed cysteine sulfur atom or the surface-exposed lysine nitrogen atom in E and to each of A ₁ and A ₂ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the wavy line connected to E indicates that each A ₁ -LA ₂ is independently covalently attached (eg, via a bond or linker) to a solvent-exposed cysteine sulfur atom or solvent-exposed lysine nitrogen atom in E, or its pharmaceutically acceptable salt. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (4):

where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an albumin protein (eg, an albumin protein having the sequence shown in any of SEQ ID NOs: 69-71), an albumin-binding peptide protein or an Fc-binding peptide; L in each LA ₁ is a linker independently covalently attached to the surface-exposed cysteine sulfur atom or the surface-exposed lysine nitrogen atom in E and to A ₁ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the wavy line connected to E indicates that each LA ₁ is independently covalently attached (eg, via a bond or linker) to a solvent-exposed cysteine sulfur atom or solvent-exposed lysine nitrogen atom in E, or a pharmaceutically acceptable salt thereof . When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A may be independently selected from any of formulas (AI)-(A-XII).

In another aspect, the invention relates to a conjugate represented by formula (4):

where each A ₁ is independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an albumin protein (eg, an albumin protein having the sequence shown in any of SEQ ID NOs: 69-71), an albumin-binding peptide protein or an Fc-binding peptide; L in each LA ₁ is a linker independently covalently attached to the surface-exposed cysteine sulfur atom or the surface-exposed lysine nitrogen atom in E and to A ₁ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the wavy line connected to E indicates that each LA ₁ is independently covalently attached (eg, via a bond or linker) to a solvent-exposed cysteine sulfur atom or solvent-exposed lysine nitrogen atom in E, or a pharmaceutically acceptable salt thereof . When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (AI)-(AV).

In another aspect, the invention relates to a conjugate represented by formula (4):

where each A ₁ is independently selected from any of formulas (A-VI)-(A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; E contains an albumin protein (eg, an albumin protein having the sequence shown in any of SEQ ID NOs: 69-71), an albumin-binding peptide protein, or an Fc-binding peptide; L in each LA ₁ is a linker independently covalently attached to the surface-exposed cysteine sulfur atom or the surface-exposed lysine nitrogen atom in E and to A ₁ ; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the wavy line connected to E indicates that each LA ₁ is independently covalently attached (eg, via a bond or linker) to a solvent-exposed cysteine sulfur atom or solvent-exposed lysine nitrogen atom in E, or a pharmaceutically acceptable salt thereof . When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (A-VI)-(A-IX).

In another aspect, the invention relates to a conjugate represented by formula (5):

where each Int is independently selected from any of the intermediates from Table 1; E contains an albumin protein (eg, an albumin protein having the sequence shown in any of SEQ ID NOs: 69-71), an albumin-binding peptide protein or an Fc-binding peptide; The L in each L-Int is a linker independently covalently attached to the surface-exposed cysteine sulfur atom or the surface-exposed lysine nitrogen atom in E and to the Int; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and a wavy line connected to E indicates that each L-Int is independently covalently attached to a solvent-exposed cysteine sulfur atom or solvent-exposed lysine nitrogen atom in E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each Int may be independently selected from any of the intermediates in Table 1.

The intermediates of Table 1 can be conjugated to protein albumin, protein albumin-binding peptide, or Fc-binding peptide (eg, via a linker), using any suitable methods known to those skilled in the art, including any of the methods described or cited as an example in this document. In some embodiments, the conjugate (e.g., the conjugate described by formula (5)) contains E, where E is an albumin protein (e.g., an albumin protein having the sequence set forth in any of SEQ ID NOs: 69-71), an albumin binding protein peptide or Fc-binding peptide. In preferred embodiments, one or more nitrogen atoms of one or more surface exposed lysine residues in E, or one or more sulfur atoms of one or more surface exposed cysteines in E, are covalently conjugated to a linker (eg, a PEG ₂ -PEG ₂₀ linker). The E-conjugated linker may be functionalized such that it can react to form a covalent bond with any of the Ints described herein (eg, the Int of Table 1). In preferred embodiments, E is conjugated to a linker functionalized with an azido group, and Int (eg, Int from Table 1) is functionalized with an alkyne group. Conjugation (eg by click chemistry) of the azido linker E and the alkyne linker Int results in the formation of the conjugate of the invention, for example the conjugate described by formula (5). In still other embodiments, E is conjugated to a linker functionalized with an alkyne group, and Int (eg, Int from Table 1) is functionalized with an azido group. Conjugation (eg, by click chemistry) of the alkyne linker E and the azido linker Int results in the formation of the conjugate of the invention, for example the conjugate described by formula (5).

In some embodiments, each E includes an albumin protein having the sequence set forth in any of SEQ ID NOs: 69-71.

In some embodiments, T is 1 and L-A is covalently attached to the sulfur atom corresponding to Cys34 of SEQ ID NO: 69.

In another aspect, the invention relates to a group of conjugates described in any of the previous aspects, where the average T value is 1-20 (for example, the average T value is 1-2, 1-3, 1-4, 1-5, 5-10 , 10-15 or 15-20). In some embodiments, the average T value is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In another aspect, the invention provides a conjugate comprising (i) a first moiety, A ₁ ; (ii) second fragment, A ₂ ; (iii) an Fc domain monomer or Fc domain; and (iv) a linker covalently attached to A ₁ and A ₂ and to the Fc domain monomer or Fc domain; where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO2H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a conjugate comprising (i) a first moiety, A ₁ ; (ii) second fragment, A ₂ ; (iii) an Fc domain monomer or Fc domain; and (iv) a linker covalently attached to A ₁ and A ₂ and to the Fc domain monomer or Fc domain; where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a conjugate comprising (i) a first moiety, A ₁ ; (ii) second fragment, A ₂ ; (iii) an Fc domain monomer or Fc domain; and (iv) a linker covalently attached to A ₁ i and A ₂ and to the Fc domain monomer or Fc domain; where each A ₁ and each A ₂ are independently selected from any of formulas (A-VI)-(A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ o)cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to a conjugate comprising (i) a first moiety, Int; (ii) an Fc domain monomer or Fc domain; and (iv) a linker covalently attached to Int and to the Fc domain monomer or Fc domain; where each Int is independently selected from any of the intermediates from Table 1.

In another aspect, the invention provides a conjugate comprising (i) a first moiety, A ₁ ; (ii) second fragment, A ₂ ; (iii) albumin protein, albumin-binding peptide or Fc-binding peptide; and (iv) a linker covalently attached to A ₁ and A ₂ and to albumin protein, albumin binding peptide or Fc binding peptide; where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a conjugate comprising (i) a first moiety, A ₁ ; (ii) second fragment, A ₂ ; (iii) albumin protein, albumin-binding peptide or Fc-binding peptide; and (iv) a linker covalently attached to A ₁ and A ₂ and to albumin protein, albumin binding peptide or Fc binding peptide; where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a conjugate comprising (i) a first moiety, A ₁ ; (ii) second fragment, A ₂ ; (iii) albumin protein, albumin-binding peptide or Fc-binding peptide; and (iv) a linker covalently attached to A ₁ and A ₂ and to albumin protein, albumin binding peptide or Fc binding peptide; where each A ₁ and each A ₂ are independently selected from any of formulas (A-VI)-(A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to a conjugate represented by formula (D-I):

where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68), an albumin protein (e.g., an albumin protein having the sequence set forth in any of SEQ ID NOs: 69-71), albumin-binding peptide or Fc-binding peptide protein; n is equal to 1 or 2; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and L is a linker covalently attached to each of E, A ₁ and A ₂ , or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (D-I):

where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68), an albumin protein (e.g., an albumin protein having the sequence set forth in any of SEQ ID NOs: 69-71), albumin-binding peptide or Fc-binding peptide protein; n is equal to 1 or 2; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and L is a linker covalently attached to each of E, A ₁ and A ₂ , or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In another aspect, the invention relates to a conjugate represented by formula (D-I):

where each A ₁ and each A ₂ are independently selected from any of formulas (A-VI) - (A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -C1 and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH3, -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68), an albumin protein (e.g., an albumin protein having the sequence set forth in any of SEQ ID NOs: 69-71), albumin-binding peptide or Fc-binding peptide protein; n is 1 or 2; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and L is a linker covalently attached to each of E, A and A ₂ , or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein).

In some embodiments, the conjugate is described by formula (D-II): (E)n

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-2);

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-3):

where L' is the remainder of L, ay ₁ and y ₂ are each independently an integer equal to 1-20 (for example, y ₁ and y ₂ are each independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate has a structure selected from

In some embodiments, the conjugate is described by formula (D-II-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-5):

where L' is the remainder of L, ay ₁ and y ₂ are each independently an integer equal to 1-20 (for example, y ₁ and y ₂ are each independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate has a structure selected from:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has a structure selected from:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-6):

where R7 is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-7):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-8):

where L' is the remainder of L, ay ₁ and y ₂ are each independently an integer equal to 1-20 (for example, y ₁ and y ₂ are each independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate has the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-9):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-10):

where L' is the remainder of L, ay ₁ and y ₂ are each independently an integer equal to 1-20 (for example, y ₁ and y ₂ are each independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate has the structure:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-3):

where L' is the remainder of L, ay ₁ and y ₂ are each independently an integer equal to 1-20 (for example, y ₁ and y ₂ are each independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate is described by formula (D-III-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-5):

where L' is the remainder of L, ay ₁ and y ₂ are each independently an integer equal to 1-20 (for example, y ₁ and y ₂ are each independently equal to 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, L' is a nitrogen atom

In some embodiments, the conjugate is described by formula (D-III-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-7):

where L' is the remainder of L, ay ₁ and y ₂ are each independently an integer equal to 1-20 (for example, y ₁ and y ₂ are each independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate is described by formula (D-III-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-9):

where L' is the remainder of L, ay ₁ and y ₂ are each independently an integer equal to 1-20 (for example, y ₁ and y ₂ are each independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate is described by formula (D-IV):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IV-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IV-2):

where L' is the remainder of L, ay ₁ and y ₂ are each independently an integer equal to 1-20 (for example, y ₁ and y ₂ are each independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate is described by formula (D-V):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-3):

where L' is the remainder of L, ay ₁ and y ₂ are each independently an integer equal to 1-20 (for example, y ₁ and y ₂ are each independently equal to 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, L' is a nitrogen atom. In some embodiments, each y ₁ and y ₂ is 1, each y ₁ and y ₂ is 2, or each y ₁ and y ₂ is 3.

In some embodiments, the conjugate is described by formula (D-V-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-5):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom. In some embodiments, each y ₁ and y ₂ is 1, each y ₁ and y ₂ is 2, or each y ₁ and y ₂ is 3.

In some embodiments, the conjugate is described by formula (D-V-6):

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):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom. In some embodiments, _y1 and _y2 are each equal to 1, _y1 and _y2 are each equal to 2, or y1 and y2 are each equal to 3.

In some embodiments, the conjugate is described by formula (D-V-9):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-10):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom. In some embodiments, y ₁ and y ₂ are each 1; each y ₁ and y ₂ is equal to 2, or each y ₁ and y ₂ is equal to 3.

In some embodiments, the conjugate is described by formula (D-VI):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-3):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom. In some embodiments, each y ₁ and y ₂ is 1, each y ₁ and y ₂ is 2, or each y ₁ and y ₂ is 3.

In some embodiments, the conjugate is described by formula (D-VI-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-5):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom. In some embodiments, each y ₁ and y ₂ is 1, each y ₁ and y ₂ is 2, or each y ₁ and y ₂ is 3.

In some embodiments, the conjugate is described by formula (D-VI-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-7):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom. In some embodiments, each y ₁ and y ₂ is 1, each y ₁ and y ₂ is 2, or each y ₁ and y ₂ is 3.

In some embodiments, the conjugate is described by formula (D-VI-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-9):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom. In some embodiments, each y ₁ and y ₂ is 1, each y ₁ and y ₂ is 2, or each y ₁ and y ₂ is 3.

In some embodiments, the conjugate is described by formula (D-VII):

or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, r ₁ represents OH. In some embodiments of any of the aspects described herein, R ₁ is NH ₂ . In some embodiments of any of the aspects described herein, R ₁ is -NHC(=NH)NH ₂ .

In some embodiments, the conjugate is described by formula (D-VIII):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-3):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate has a structure selected from

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-5):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate has a structure; selected from:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-7):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate is described by formula (D-VIII-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-9):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1-20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate is described by formula (D-VIII-10):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-II):

where L' represents the remainder of L, and each y ₁ and y ₂ is independently an integer equal to 1 through 20 (for example, each y ₁ and y ₂ is independently equal to 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, L' is a nitrogen atom.

In some embodiments, the conjugate is described by formula (D-IX):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (D-IX-I):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (D-IX-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IX-3):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IX-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IX-5):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (D-IX-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-X):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (D-X-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (D-X-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-X-3):

or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, L or L' includes one or more of an optionally substituted (C ₁ -C ₂₀ )alkylene, an optionally substituted (C ₁ -C ₂₀ )heteroalkylene, an 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 )nicloalkylene, optional substituted (C ₃ -C ₂₀ )heterocycloalkylene, optionally substituted (C ₄ -C ₂₀ )nicloalkenylene, 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 ₂ -C ₁₅ )heteroarylene, O, S, NR ⁱ , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino, where R ⁱ represents H, optionally substituted (C ₁ -C ₂₀ )alkyl, optionally substituted (C ₁ -C ₂₀ )heteroalkyl, optionally substituted (C ₂ -C ₂₀ )alkenyl, optionally substituted (C ₂ -C ₂₀ )heteroalkenyl, optionally substituted (C ₂ -C ₂₀ )alkynyl, optionally substituted (C ₂ -C ₂₀ )heteroalkynyl, optionally substituted (C ₃ -C ₂₀ )cycloalkyl, optionally substituted (C ₃ -C ₂₀ )heterocycloalkyl, optionally substituted (C ₄ -C ₂₀ )cycloalkenyl, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenyl, optionally substituted (C ₈ -C ₂₀ )cycloalkynyl, optionally substituted (C ₈ -C ₂₀ )heterocycloalkynyl, optionally substituted (C ₅ -C ₁₅ )aryl or optionally substituted ( C ₂ -C ₁₅ )heteroaryl.

In some embodiments of any of the aspects described herein, the backbone of L or L' consists of one or more of an optionally substituted (C ₁ -C ₂₀ )alkylene, an optionally substituted (C ₁ -C ₂₀ ) heteroalkylene, an 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 ) picloalkylene, optionally substituted (C ₃ -C ₂₀ )heterocycloalkylene, optionally substituted (C ₄ -C ₂₀ )picloalkenylene, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenylene, optionally substituted (C ₈ -C ₂₀ )picloalkynylene, optionally substituted (C ₈ -C ₂₀ )heterocycloalkynylene, optionally substituted (C ₅ -C ₁₅ )arylene, optionally substituted (C ₂ -C ₁₅ )heteroarylene, O, S, NR ⁱ , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino, where R ⁱ represents H, optionally substituted (C ₁ -C ₂₀ )alkyl, optionally substituted (C ₁ -C ₂₀ )heteroalkyl, optionally substituted (C ₂ -C ₂₀ )alkenyl, optionally substituted (C ₂ -C ₂₀ )heteroalkenyl , optionally substituted (C ₂ -C ₂₀ )alkynyl, optionally substituted (C ₂ -C ₂₀ )heteroalkynyl; optionally substituted (C ₃ -C ₂₀ )cycloalkyl, optionally substituted (C ₃ -C ₂₀ )heterocycloalkyl, optionally substituted (C ₄ -C ₂₀ )cycloalkenyl, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenyl, optionally substituted (C ₈ - C ₂₀ )cycloalkynyl, optionally substituted (C ₈ -C ₂₀ )heterocycloalkynyl, optionally substituted (C ₅ -C ₁₅ )aryl or optionally substituted (C ₂ -C ₁₅ )heteroaryl.

In some embodiments of any of the aspects described herein, L or L' is oxo-substituted. In some embodiments, the backbone of L or L' contains no more than 250 atoms. In some embodiments, L or L' is capable of forming an amide, carbamate, sulfonyl, or urea bond. In some embodiments, 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 the formula (D-L-I):

where L ^A is described by the 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 the 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 the formula G ^C1 -(Z ^C1 ) _g3 -(Y ^C1 ) _h3 -(Z ^C2 ) _i3 -(Y ^C2 ) _j3 -(Z ^C3 ) _k3 -(Y ^C3 ) _l3 -(Y ^C4 ) _m3 -( Y ^C4 ) _n3 -(Z ^C5 ) _o3 -G ^C2 ; G ^A1 represents the bond attached to Q; G ^A2 represents the bond attached to A1; G ^B1 represents the bond attached to Q); G ^B2 represents the bond attached to A2; G ^C1 represents the bond attached to Q; G ^C2 represents a bond attached to E, or a functional group capable of reacting with a functional group conjugated to E (for example, maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, as well as alkene and tetrazine); each of Z ^A1 , Z ^A2 , Z ^A3 , Z ^A4 , Z ^A5 , Z ^B1 , Z ^B2 , Z ^B3 , Z ^B4 , Z ^B5 , Z ^Cl , Z ^C2 , Z ^C3 , Z ^C4 and Z ^C5 represents, independently , 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 )cycloalkynylene, optionally substituted (C ₃ -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; each of Y ^A1 , Y ^A2 , Y ^A3 , Y ^A4 , Y ^B1 , Y ^B2 , Y ^B3 , Y ^B4 , Y ^C1 , Y ^C2 , Y ^C3 and Y ^C4 represents, independently, O, S, NR ⁱ , P , carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino; R ⁱ represents H, optionally substituted (C ₁ -C ₂₀ )alkyl, optionally substituted (C ₁ -C ₂₀ )heteroalkyl, optionally substituted (C ₂ -C ₂₀ )alkenyl, optionally substituted (C ₂ -C ₂₀ )heteroalkenyl, optionally substituted (C ₂ -C ₂₀ )alkynyl, optionally substituted (C ₂ -C ₂₀ )heteroalkynyl, optionally substituted (C ₃ -C ₂₀ )cycloalkynyl, optionally substituted (C ₃ -C ₂₀ )heterocycloalkyl, optionally substituted (C ₄ - C ₂₀ )cycloalkenyl, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenyl, optionally substituted (C ₈ -C ₂₀ )cycloalkynyl, optionally substituted (C ₈ -C ₂₀ )heterocycloalkynyl, optionally substituted (C ₅ -C ₁₅ )aryl or optionally substituted (C ₂ -C ₁₅ )heteroaryl; each of g1, h1, i1, j1, k1, l1, m1, n1, o1, g2, h2, i2, j2, k2, l2, m2, n2, o2, g3, h3, i3, j3, k3, l3, m3, n3 and o3 are equal, independently, to 0 or 1; Q represents a nitrogen atom, 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 ₃ -C ₂₀ )heterocycloalkylene, optionally substituted (C ₄ - C ₂₀ )picloalkenylene, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenylene, optionally substituted (C ₈ -C ₂₀ )picloalkynylene, optionally substituted (C ₈ -C ₂₀ )heterocycloalkynylene, optionally substituted (C ₅ -C ₁₅ )arylene or optionally substituted (C ₂ -C ₁₅ )heteroarylene.

In some embodiments, the L ^C may have two attachment points to the Fc domain, Fc binding peptide, albumin protein, or albumin binding peptide (eg, two G ^C2 ). In some embodiments of any of the aspects described herein, L includes a polyethylene glycol (PEG) linker. The PEG linker is a linker having the structure of repeating units (-CH ₂ CH ₂ O-)n, where n is an integer from 2 to 100. The polyethylene glycol linker can be covalently attached to the neuraminidase inhibitor and E (for example, in a conjugate of any of the formulas (MI)-(MX)). The polyethylene glycol linker may be covalently attached to the first neuraminidase inhibitor and the second neuraminidase inhibitor (eg, in a conjugate of any of formulas (DI)-(DX)). The polyethylene glycol linker can be covalently attached to the neuraminidase inhibitor dimer and E (eg, in a conjugate of any of formulas (DI)-(DX)). The polyethylene glycol linker may be selected from any of PEG ₂ -PEG ₁₀₀ (for example, PEG ₂ , RES ₃ , PEG ₄ , PEG ₅ , PEG ₅ -PEG ₁₀ , PEG ₁₀ -PEG ₂₀ , PEG ₂₀ -PEG ₁₀ , RES ₃₀ -PEG ₄₀ , PEG ₅₀ -PEG ₆₀ , PEG ₆₀ -PEG ₇₀ , PEG ₇₀ -PEG ₈₀ , PEG ₈₀ -PEG ₉₀ , PEG ₉₀ -PEG ₁₀₀ ). In some embodiments, L ^C includes a PEG linker, wherein L ^C is covalently attached to each of Q and E.

In some embodiments, L is:

where each z ₁ and z ₂ are, independently, an integer from 1 to 20; and R ₉ is selected from H; (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl; (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl.

In some embodiments, L is

where R* represents a bond or includes one or more of 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 ₃ -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 )heterocycloalkylene, optionally substituted (C ₅ - C ₁₅ )arylene, optionally substituted (C ₂ -C ₁₅ )heteroarylene, O, S, NR ⁱ , P, carbonyl, thiocarbonyl, sulfonyl, phosphate and imino, and where R ⁱ represents H, optionally substituted (C ₁ -C ₂₀ )alkyl, optionally substituted (C ₁ -C ₂₀ )heteroalkyl, optionally substituted (C ₂ -C ₂₀ )alkenyl, optionally substituted (C ₂ -C ₂₀ )heteroalkenyl, optionally substituted (C ₂ -C ₂₀ )alkynyl, optionally substituted (C ₂ -C ₂₀ )heteroalkynyl, optionally substituted (C ₃ -C ₂₀ )cycloalkyl, optionally substituted (C ₃ -C ₂₀ )heterocycloalkyl, optionally substituted (C ₄ -C ₂₀ )cycloalkenyl, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenyl, optionally substituted (C ₈ -C ₂₀ )cycloalkynyl, optionally substituted (C ₈ -C ₂₀ )heterocycloalkynyl, optionally substituted (C ₅ -C ₁ 5)aryl or optionally substituted (C ₂ -C ₁ 5)heteroaryl.

In some embodiments, Y is: (-NH(C=O)O-) and L represents:

In some embodiments, Y is: (-NH(C=O)O-) and L represents:

In some embodiments, Y is: (-NH(C=O)O) and L represents:

In some embodiments, Y is: (-O-) and L represents:

In another aspect, the invention relates to a conjugate represented by formula (M-I):

where each A1 is independently selected from any of formulas (A-I)-(A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68), an albumin protein (e.g., an albumin protein having the sequence set forth in any of SEQ ID NOs: 69-71), albumin-binding peptide or Fc-binding peptide protein; n is 1 or 2; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and L is a linker covalently attached to each of E and A ₁ , or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (AI)-(A-XII).

In another aspect, the invention relates to a conjugate represented by formula (M-I):

where each A ₁ is independently selected from any of the formulas (AI)-(AV):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68), an albumin protein (e.g., an albumin protein having the sequence set forth in any of SEQ ID NOs: 69-71), albumin-binding peptide or Fc-binding peptide protein; n is 1 or 2; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and L is a linker covalently attached to each of E and A ₁ , or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (AI)-(AV). In another aspect, the invention relates to a conjugate represented by formula (MI):

where each A ₁ is independently selected from any of formulas (A-VI)-(A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68), an albumin protein (e.g., an albumin protein having the sequence set forth in any of SEQ ID NOs: 69-71), albumin-binding peptide or Fc-binding peptide protein; n is 1 or 2; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and L is a linker covalently attached to each of E and A ₁ , or a pharmaceutically acceptable salt thereof. When T is greater than 1 (for example, T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A ₁ may be independently selected from any of formulas (A-VI)-(A-IX).

In some embodiments, the conjugate is described by formula (M-II):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-II-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-II-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-3):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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 the formula (M-II-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-5):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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 has the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-6):

where R7 is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-7):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-8):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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 has the structure

In some embodiments, the conjugate is described by formula (M-II-9):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-II-10):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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 has the structure:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-3):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-III-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-5):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-III-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-7):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-III-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-9):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-IV):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IV-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IV-2):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-V):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-3):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-V-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-5):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-V-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, 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):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-V-9):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-10):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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.

In some embodiments, the conjugate is described by formula (M-VI-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-3):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-5):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-7):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-9):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-VII):

or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, R ₁ represents OH. In some embodiments of any of the aspects described herein, R ₁ is NH ₂ . In some embodiments of any of the aspects described herein, R ₁ is -NHC(=NH)NH ₂ .

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):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-3):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-VIII-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-5):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20, or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-7):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-VIII-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-9):

where L represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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-VIII-10):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-11):

where L' represents the remainder of L, and y ₁ represents an integer equal to 1-20 (for example, 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.

In some embodiments, the conjugate is described by the formula (M-IX-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-IX-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-IX-3):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-IX-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IX-5):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-IX-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-X):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-X-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-X-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the formula (M-X-3):

or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, L or L' contains one or more of an optionally substituted (C ₁ -C ₂₀ )alkylene, an optionally substituted (C ₁ -C ₂₀ ) heteroalkylene, an 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, optional substituted (C ₃ -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 ₂ -C ₁₅ heteroarylene, O, S, NR ⁱ , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino, where R ⁱ represents is H, optionally substituted (C ₁ -C ₂₀ )alkyl, optionally substituted (C ₁ -C ₂₀ )heteroalkyl, optionally substituted (C ₂ -C ₂₀ )alkenyl, optionally substituted (C ₂ -C ₂₀ )heteroalkenyl, optionally substituted ( C ₂ -C ₂₀ )alkynyl, optionally substituted (C ₂ -C ₂₀ )heteroalkynyl, optionally substituted (C ₃ -C ₂₀ )cycloalkyl, optionally substituted (C ₃ -C ₂₀ )heterocycloalkyl, optionally substituted (C ₄ -C ₂₀ ) cycloalkenyl, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenyl, optionally substituted (C ₈ -C ₂₀ )cycloalkynyl, optionally substituted (C ₈ -C ₂₀ )heterocycloalkynyl, optionally substituted (C ₅ -C ₁₅ )aryl or optionally substituted (C ₂ -C ₁₅ )heteroaryl.

In some embodiments of any of the aspects described herein, the backbone of L or L' consists of one or more of an optionally substituted (C ₁ -C ₂₀ )alkylene, an optionally substituted (C ₁ -C ₂₀ ) heteroalkylene, an 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 ₃ -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 ₂ -C ₁₅ )heteroarylene, O, S, NR ⁱ , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino, where R ⁱ represents H, optionally substituted (C ₁ -C ₂₀ )alkyl, optionally substituted (C ₁ -C ₂₀ )heteroalkyl, optionally substituted (C ₂ -C ₂₀ )alkenyl, optionally substituted (C ₂ -C ₂₀ )heteroalkenyl , optionally substituted (C ₂ -C ₂₀ )alkynyl, optionally substituted (C ₂ -C ₂₀ )heteroalkynyl, optionally substituted (C ₃ -C ₂₀ )cycloalkyl, optionally substituted (C ₃ -C ₂₀ )heterocycloalkyl, optionally substituted (C ₄ -C ₂₀ )cycloalkenyl, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenyl, optionally substituted (C ₈ -C ₂₀ )cycloalkynyl, optionally substituted (C ₈ -C ₂₀ )heterocycloalkynyl, optionally substituted (C ₅ -C ₁₅ )aryl or optionally substituted (C ₂ -C ₁₅ )heteroaryl.

In some embodiments of any of the aspects described herein, L or L' is oxo-substituted. In some embodiments, the backbone of L or L' contains no more than 250 atoms. In some embodiments, L or L' is capable of forming an amide, carbamate, sulfonyl, or urea bond. In some embodiments, L or L' is a bond. In some embodiments, L or L' is an atom.

In some embodiments, each L is described by the formula (M-L-1):

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

where: J ₁ represents the bond attached to A1; J ² represents a bond attached to E, or a functional group capable of reacting with a functional group conjugated to E (for example, maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, as well as alkene and tetrazine); each of Q ¹ , Q ² , Q ³ , Q ⁴ and Q ⁵ independently represents an optionally substituted (C ₁ -C ₂₀ )alkylene, an optionally substituted (C ₁ -C ₂₀ ) heteroalkylene, an 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 )cycloalkynylene, optionally substituted (C ₃ -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; each of T ¹ , T ² , T ³ , T ⁴ independently represents O, S, NR ⁱ , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino; R ⁱ represents H, optionally substituted (C ₁ -C ₂₀ )alkyl, optionally substituted (C ₁ -C ₂₀ )heteroalkyl, optionally substituted (C ₂ -C ₂₀ )alkenyl, optionally substituted (C ₂ -C ₂₀ )heteroalkenyl, optionally substituted (C ₂ -C ₂₀ )alkynyl, optionally substituted (C ₂ -C ₂₀ )heteroalkynyl, optionally substituted (C ₃ -C ₂₀ )cycloalkyl, optionally substituted (C ₃ -C ₂₀ )heterocycloalkyl, optionally substituted (C ₄ - C ₂₀ )cycloalkenyl, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenyl, optionally substituted (C ₈ -C ₂₀ )cycloalkynyl, optionally substituted (C ₈ -C ₂₀ )heterocycloalkynyl, optionally substituted (C ₅ -C ₁₅ )aryl or optionally substituted (C ₂ -C ₁₅ )heteroaryl; and each of g, h, i, j, k, l, m, n and o is independently equal to 0 or 1; or a pharmaceutically acceptable salt thereof.

In some embodiments, J ² may have two attachment points to an Fc domain, Fc binding peptide, albumin protein, or albumin binding peptide protein (eg, two J ² ).

In some embodiments, L is:

where d is an integer from 1 to 20 (for example, 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:

where each of d and e is independently an integer from 1 to 26; or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, L includes a polyethylene glycol (PEG) linker. The PEG linker is a linker having a repeating unit structure (-CH ₂ CH ₂ O-) _n , where n is an integer from 2 to 100. The polyethylene glycol linker can be covalently attached to the neuraminidase inhibitor and E (for example, in a conjugate of any of the formulas (MI)-(MX)). The polyethylene glycol linker may be covalently attached to the first neuraminidase inhibitor and the second neuraminidase inhibitor (eg, in a conjugate of any of formulas (DI)-(DX)). The polyethylene glycol linker can be covalently attached to the neuraminidase inhibitor dimer and E (eg, in a conjugate of any of formulas (DI)-(DX)). The polyethylene glycol linker may be selected from any of PEG ₂ -PEG ₁₀₀ (eg 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 ). In some embodiments, L ^c includes a PEG linker, where L ^C is covalently attached to each of Q and E.

In some embodiments of any of the aspects described herein, R ₁ is -NHC(=NH)NH ₂ . In some embodiments of any of the aspects described herein, R ₂ is -F. In some embodiments of any of the aspects described herein, R ₃ is -F. In some embodiments of any of the aspects described herein, R ₄ is -CO ₂ H. In some embodiments of any of the aspects described herein, R ₅ is -COCH ₃ .

In some embodiments of any of the aspects described herein, L is covalently attached to the surface-exposed lysine nitrogen atom of E or L is covalently attached to the surface-exposed cysteine sulfur atom of E.

In some embodiments of any of the aspects described herein, E is an Fc domain monomer. In some embodiments, n is 2 and each E dimerizes to form an Fc domain.

In some embodiments, n is 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):

where J represents an Fc domain; and T is an integer from 1 to 20 (for example, 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.

In some embodiments, n is 2, 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):

where J represents an Fc domain; and T is an integer from 1 to 20 (for example, 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

In some embodiments of any of the aspects described herein, E has the sequence presented in any of SEQ ID NO: 1-68.

In some embodiments of any of the aspects described herein, E is albumin protein, albumin binding peptide, or Fc binding peptide. In some embodiments, where E is albumin protein, albumin binding peptide, or Fc binding peptide, n is 1.

In some embodiments, n is 1, E is albumin protein, albumin-binding peptide, or Fc-binding peptide, and the conjugate is described by formula (D-I-2):

where E is albumin protein, albumin-binding peptide or Fc-binding peptide; and T is an integer from 1 to 20, or a pharmaceutically acceptable salt thereof.

In some embodiments, n is 1, E is albumin protein, albumin-binding peptide, or Fc-binding peptide, and the conjugate is described by the formula (M-I-2):

where E is albumin protein, albumin-binding peptide or Fc-binding peptide; and T is an integer from 1 to 20, or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, E is an albumin protein having the sequence of any of SEQ ID NO: 69-71.

In some embodiments of any of the aspects described herein, T is 1, 2, 3, 4, or 5.

In another aspect, the invention provides a group of conjugates having the structure of any of the conjugates described herein (for example, a group of conjugates having any of formulas (1)-(5), (D-I)-(D-X), (D'-I) , (M-I)-(M-X) or (M'-I)), where the average value of T is 1-20 (for example, the average value of T is 1-2, 1-3, 1-4, 1-5, 5- 10, 10-15 or 15-20). In some embodiments, the average T value 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 of any of the aspects described herein, 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, 17, 18, 19 or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein). In some embodiments, E may be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different A ₁ -LA ₂ moieties. In some embodiments, E is conjugated to a first A ₁ -LA ₂ moiety and a second A ₁ -LA ₂ moiety. In some embodiments, A ₁ and A ₂ of the first fragment A ₁ -LA ₂ are independently selected from any of formulas (A-III)-(AV):

and A ₁ and A ₂ of the second fragment A ₁ -LA ₂ are independently selected from any of formulas (AI), (A-II), (A-VI), (A-VII), (A-VIII) and (A- IX):

In some embodiments, each of the first A ₁ -LA ₂ fragments is specifically conjugated to a lysine E residue (e.g., the nitrogen atom of a surface exposed lysine E residue), and each of the second A ₁ -LA ₂ fragments is specifically conjugated to a cysteine E residue (e.g. , a sulfur atom exposed on the surface of a cysteine residue E). In some embodiments, each of the first A ₁ -LA ₂ fragments is specifically conjugated to a cysteine E residue (e.g., the sulfur atom of a surface-exposed cysteine E residue), and each of the second A ₁ -LA ₂ fragments is specifically conjugated to a lysine E residue (e.g. , the nitrogen atom exposed on the surface of the lysine residue in E).

In some embodiments, the number of first A ₁ -LA ₂ moieties conjugated to E is an integer from 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the number of second A ₁ -LA ₂ moieties conjugated to E is an integer from 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In some embodiments of any of the aspects described herein, 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, 17, 18, 19 or 20), each A ₁ -L may be independently selected (eg, independently selected from any of the A ₁ -L structures described herein). In some embodiments, E may be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different A ₁ -L moieties. In some embodiments, E is conjugated to a first A ₁ -L moiety and a second A ₁ -L moiety. In some embodiments, A ₁ from the first fragment A ₁ -L is selected from any of formulas (A-III)-(AV):

and A ₁ of the second group A ₁ -L is selected from any of formulas (AI), (A-II), (A-VI), (A-VII), (A-VIII) or (A-IX):

In some embodiments, each of the first A ₁ -L fragments is specifically conjugated to a lysine E residue (e.g., the nitrogen atom of a surface exposed lysine E residue), and each of the second A ₁ -L fragments is specifically conjugated to a cysteine E residue (e.g., a sulfur exposed on the surface of the cysteine residue E). In some embodiments, each of the first A ₁ -L fragments is specifically conjugated to a cysteine E residue (e.g., the sulfur atom of a surface-exposed cysteine E residue), and each of the second A ₁ -L fragments is specifically conjugated to a lysine E residue (e.g., a nitrogen exposed on the surface of the lysine E residue).

In some embodiments, the number of first A ₁ -L fragments conjugated to E is an integer from 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the number of second A ₁ -L moieties conjugated to E is an integer from 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In another aspect, the invention relates to a conjugate represented by formula (D'-I):

where each A ₁ is independently selected from any of formulas (A-III)-(AV):

where each A ₂ is independently selected from any of formulas (AI), (A-II), (A-VI), (A-VII), (A-VIII) and (A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68), an albumin protein (e.g., an albumin protein having the sequence set forth in any of SEQ ID NOs: 69-71), albumin-binding peptide or Fc-binding peptide protein; n is 1 or 2; T represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and L ₁ is a linker covalently conjugated to E and each A ₁ , T ₁ is an integer from 1 to 10 (for example, T ₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); L ₂ is a linker covalently conjugated to E and each A ₂ ; T ₂ is an integer from 1 to 10 (for example, T ₂ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), or a pharmaceutically acceptable salt thereof.

In some embodiments, each A ₁ -LA ₁ is specifically conjugated to a lysine E residue (e.g., a surface-exposed nitrogen atom of a lysine E residue), and each A ₂ -LA ₂ is specifically conjugated to a cysteine E residue (e.g., a surface-exposed sulfur atom cysteine residue E). In some embodiments, each A ₁ -LA ₁ fragment is specifically conjugated to a cysteine E residue (e.g., the sulfur atom of a surface-exposed cysteine E residue), and each A ₂ -LA ₂ fragment is specifically conjugated to a lysine E residue (e.g., a nitrogen atom of a surface-exposed cysteine E residue) on the surface of the lysine residue E).

In another aspect, the invention relates to a conjugate represented by formula (M'-I):

where each A ₁ is independently selected from any (M-IX) formulas (A-III)-(AV):

where each A ₂ is independently selected from any of formulas (AI), (A-II), (A-VI), (A-VII), (A-VIII) and (A-IX):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68), an albumin protein (e.g., an albumin protein having the sequence set forth in any of SEQ ID NOs: 69-71), albumin-binding peptide or Fc-binding peptide protein; n is 1 or 2; T ₁ represents an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); and L ₁ is a linker covalently conjugated to E and Ai, T ₁ is an integer from 1 to 10 (for example, T ₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) ; L ₂ is a linker covalently conjugated to E and A ₂ ; T ₂ is an integer from 1 to 10 (for example, T ₂ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), or a pharmaceutically acceptable salt thereof.

In some embodiments, each A ₁ -L is specifically conjugated to a lysine E residue (e.g., the nitrogen atom of a surface exposed lysine E residue), and each A ₂ -L is specifically conjugated to a cysteine E residue (e.g., the sulfur atom of a surface exposed cysteine residue E). In some embodiments, each A ₁ -L moiety is specifically conjugated to a cysteine E residue (e.g., a sulfur atom of a surface-exposed cysteine E residue), and each A ₂ -L moiety is specifically conjugated to a lysine E residue (e.g., a surface-exposed nitrogen atom lysine E residue).

In yet another aspect, the invention provides a pharmaceutical composition containing any of the conjugates described herein (for example, a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I )-(M-X) or (M'-I)), or a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient thereof.

In yet another aspect, the invention provides a method of treating a subject having or suspected of having 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 of formulas (1)-(5 ), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I)).

In yet another aspect, the invention provides a method of prophylactically treating 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 of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I)).

In some embodiments, the viral infection is caused by an influenza virus or parainfluenza virus. In some embodiments, the viral infection is an influenza A, B, or C virus, or a parainfluenza virus.

In some embodiments, the subject is immunocompromised.

In some embodiments, the subject is diagnosed with humoral immunodeficiency, T-cell deficiency, neutropenia, asplenia, or complement deficiency.

In some embodiments, the subject is being treated or is about to be treated with immunosuppressive therapy.

In some embodiments, the subject has been diagnosed with a disease causing immunosuppression. In some embodiments, the disease is cancer or acquired immunodeficiency syndrome. In some embodiments, the cancer is leukemia, lymphoma, or multiple myeloma.

In some embodiments, the subject has undergone or is about to undergo a hematopoietic stem cell transplant.

In some embodiments, the subject has undergone or is about to undergo an organ transplant.

In some embodiments, the composition conjugate is administered intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesional, intracranial, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, locally, intratumorally, periotoneally, subcutaneously, subconjunctivally, intravesically cular, mucosal , intrapericardial, intraumbilical, intraocular, oral, topical, inhalation, injection or infusion.

In some embodiments, the subject is treated with a second therapeutic agent. In some embodiments, the second therapeutic agent is an antiviral agent. In some embodiments, the antiviral agent is selected from oseltamivir, zanamivir, peramivir, laninamivir, amantadine, or rimantadine. In some embodiments, the second therapeutic agent is a viral vaccine. In some embodiments, the viral vaccine induces an immune response in a subject against an influenza A, B, or C virus, or a parainfluenza virus.

In some embodiments, the Fc domain-containing composition may be replaced by an Fc domain, and the Fc domain monomer-containing composition may be replaced by an Fc domain monomer in any of formulas (1)-(5), (D-I)-( D-X), (D'-I), (M-I)-(M-X) or (M'-I) (for example, any of formulas (1), (2), (3), (4), (5), (D-I), (D-II), (D-II-1), (D-II-2), (D-II-3), (D-II-4), (D-II-5), (D-II-6), (D-II-7), (D-II-8), (D-II-9), (D-II-10), (D-III), (D-III -1), (D-III-2), (D-III-3), (D-III-4), (D-III-5), (D-III-6), (D-III-7 ), (D-III-8), (D-III-9), (D-IV), (D-IV-1), (D-IV-2), (D-V), (D-V-l), (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-V-10 ), (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-VI-9), (D-VII), (D-VIII), (D-VIII-1), (D-VIII-2), (D-VIII-3), (D-VIII-4), (D-VIII-5), (D-VIII-6), (D-VIII-7), (D -VIII-8), (D-VIII-9), (D-VIII-10), (D-VIII-11), (D-IX), (D-IX-1), (D-IX-2 ), (D-IX-3), (D-IX-4), (D-IX-5), (D-IX-6), (D-X), (D-X-l), (D-X-2), (D-X -3), (D'-I), (M-I), (M-II), (M-II-1), (M-II-2), (M-II-3), (M-II- 4), (M-II-5), (M-II-6), (M-II-7), (M-II-8), (M-II-9), (M-II-10) , (M-III), (M-III-1), (M-III-2), (M-III-3), (M-III-4), (M-III-5), (M- III-6), (M-III-7), (M-III-8), (M-III-9), (M-IV), (M-IV-1), (M-IV-2) , (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-V-10), (M-VI), (M-VI-1), (M-VI-2), (M-VI-3), (M-VI- 4), (M-VI-5), (M-VI-6), (M-VI-7), (M-VI-8), (M-VI-9), (M-VII), ( M-VIII), (M-VIII-1), (M-VIII-2), (M-VIII-3), (M-VIII-4), (M-VIII-5), (M-VIII- 6), (M-VIII-7), (M-VIII-8), (M-VIII-9), (M-VIII-10), (M-VIII-11), (M-IX), ( M-IX-1), (M-IX-2), (M-IX-3), (M-IX-4), (M-IX-5), (M-IX-6), (M-X) , (M-X-1), (M-X-2), (M-X-3) or (M'-I)). In any of the formulas described herein (for example, any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M '-I)), when n is 1, E is a composition containing an Fc domain monomer. In any of the formulas described herein (for example, any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I )), when n is 2, E is an Fc domain-containing composition.

In some embodiments, the Fc domain-containing composition is an antibody or antibody fragment. The antibody may include any form of immunoglobulin, heavy chain antibody, light chain antibody, LRR 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., Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, scFv or SMIP). The subunit structures and three-dimensional configurations of various classes of antibodies are known in the art. The antibody fragment may include a binding fragment that includes a portion derived from the antibody or having significant homology to the antibody, such as the antigen-determining region of the antibody. Typical antibody fragments include Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, scFv and SMIP.

In specific embodiments, the antibody or antibody fragment is an antibody or antibody fragment from a human, mouse, camel (eg, llama, alpaca, or camel), goat, sheep, rabbit, chicken, guinea pig, hamster, horse, or rat. In specific embodiments, the antibody is an IgG, IgA, IgD, IgE, IgM, or intrabody. In some embodiments, the antibody fragment includes a scFv, sdAb, dAb, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, or SMIP.

In some embodiments, an Fc domain-containing composition (eg, an antibody or antibody fragment) confers binding specificity to one or more targets (eg, an antigen).

In some embodiments, the one or more targets (eg, antigen) bound by the Fc domain-containing composition (eg, antibody or antibody fragment) is a viral (eg, influenza) protein, such as a neuraminidase or hemagglutinin. In some embodiments, the antibody or antibody fragment recognizes a surface antigen of a virus. In some embodiments, the antibody or antibody fragment targets hemagglutinin. Hemagglutinin-targeting antibodies include monoclonal antibodies such as CR6261, CR8020, MEDI8852, MNAA4549A and VIS410. In some embodiments, the antibody or antibody fragment is a broadly neutralizing antibody or antibody fragment that targets influenza hemagglutinin (e.g., the antibody or antibody fragment described in Wu et al., J. Mol. Biol. 429: 2694 2709 (2017)) . In some embodiments, the antibody or antibody fragment targets a viral matrix protein (eg, matrix protein 2). TCN032 is a monoclonal antibody targeting matrix protein 2.

In some embodiments, the Fc domain-containing composition (eg, an antibody or antibody fragment) includes one or more single domain antibodies (sdAbs). In some embodiments, the Fc domain-containing composition is an antibody or antibody fragment comprising an sdAb with influenza A reactivity, such as an sdAb that binds to influenza A virus hemagglutinin (e.g., SD36 or SD38, described in Laursen et al. Science 362:598-602 (2018)). In some embodiments, the Fc domain-containing composition is an antibody or antibody fragment comprising an sdAb with reactivity against influenza B virus, such as an sdAb that binds to influenza B virus hemagglutinin (e.g., SD83 or SD84, as described by Laursen et al Science 362:598–602 (2018)).

In some embodiments, the Fc domain-containing composition is a multi-domain antibody (MDAb) or multi-domain antibody fragment comprising 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) sdAbs . In some embodiments, the MDAb or fragment thereof includes one or more sdABs that bind to influenza A virus hemagglutinin and one or more sdAbs that bind to influenza B virus hemagglutinin. In some embodiments, the MDAb is JNJ-7445 (also known as MD3606), which is described in Laursen et al. Science. 362:598–602 (2018). Briefly, JNJ-7445 is an MDAb that includes two sdAbs that bind to influenza A virus hemagglutinin (SD36 and SD38) and two sdAbs that bind to influenza B virus hemagglutinin (SD83 and SD84) that bind to the Fc domain (IgG1). SdAb was produced by immunizing llamas with influenza vaccine and recombinant hemagglutinin H7 and H2.

In yet another aspect, the invention includes a conjugate described by any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I) ( for example, any of the formulas (1), (2), (3), (4), (5), (D-I), (D-II), (D-II-1), (D-II-2) , (D-II-3), (D-II-4), (D-II-5), (D-11-6), (D-II-7), (D-II-8), ( D-II-9), (D-II-10), (D-III), (D-III-1), (D-III-2), (D-III-3), (D-III- 4), (D-III-5), (D-III-6), (D-III-7), (D-III-8), (D-III-9), (D-IV), ( D-IV-1), (D-IV-2), (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-V-10), (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-VI-9), (D-VII), (D-VIII), (D-VIII-1), (D-VIII-2), (D-VIII-3), (D-VIII-4) , (D-VIII-5), (D-VIII-6), (D-VIII-7), (D-VIII-8), (D-VIII-9), (D-VIII-10), ( D-VIII-11), (D-IX), (D-IX-1), (D-IX-2), (D-IX-3), (D-IX-4), (D-IX- 5), (D-IX-6), (D-X), (D-X-1), (D-X-2), (D-X-3), (D'-I), (M-I), (M-II), (M-II-1), (M-II-2), (M-II-3), (M-II-4), (M-II-5), (M-II-6), (M -II-7), (M-II-8), (M-II-9), (M-II-10), (M-III), (M-III-1), (M-III-2 ), (M-III-3), (M-III-4), (M-III-5), (M-III-6), (M-III-7), (M-III-8), (M-III-9), (M-IV), (M-IV-1), (M-IV-2), (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-V-10), (M-VI), (M-VI-1), (M-VI-2), (M-VI-3), (M-VI-4), (M-VI-5), (M-VI-6), (M -VI-7), (M-VI-8), (M-VI-9), (M-VII), (M-VIII), (M-VIII-1), (M-VIII-2), (M-VIII-3), (M-VIII-4), (M-VIII-5), (M-VIII-6), (M-VIII-7), (M-VIII-8), (M -VIII-9), (M-VIII-10), (M-VIII-11), (M-IX), (M-IX-1), (M-IX-2), (M-IX-3 ), (M-IX-4), (M-IX-5), (M-IX-6), (M-X), (M-X-1), (M-X-2), (M -X-3) or (M'-I)), where E represents an antibody or antibody fragment. In preferred embodiments, where E is an antibody or antibody fragment, n is 1. In some embodiments, the antibody or antibody fragment includes any antibody or antibody fragment described herein, such as a monoclonal antibody that binds to viral hemagglutinin (eg , CR6261, CR8020, MEDI8852, MNAA4549A or VIS410); a broadly neutralizing antibody or antibody fragment targeting viral hemagglutinin (e.g., antibodies or antibody fragments described in Wu et al., J. Mol. Biol. 429:2694-2709 (2017)); sdAb targeting viral hemagglutinin (eg, SD36, SD38, SD83, or SD84); or an MDAb or fragment thereof targeting viral hemagglutinin (eg, JNJ-7445).

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 1. 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 set forth in SEQ ID NO: 1.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 2. 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 set forth in SEQ ID NO: 2.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 3. 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 set forth in SEQ ID NO: 3.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 4. 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 set forth in SEQ ID NO: 4.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 5. 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 set forth in SEQ ID NO: 5.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 6. 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 set forth in SEQ ID NO: 6.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 7. 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 set forth in SEQ ID NO: 7.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 8. 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 set forth in SEQ ID NO: 8.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 9. 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 set forth in SEQ ID NO: 9.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 10. 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 set forth in SEQ ID NO: 10.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 11. 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 set forth in SEQ ID NO: 11.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 12. 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 set forth in SEQ ID NO: 12.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 13. 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 set forth in SEQ ID NO: 13.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 14. 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 set forth in SEQ ID NO: 14.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 15. 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 set forth in SEQ ID NO: 15.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 16. 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 set forth in SEQ ID NO: 16.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 17. 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 set forth in SEQ ID NO: 17.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 18. 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 set forth in SEQ ID NO: 18.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 19. 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 set forth in SEQ ID NO: 19.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 20. 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 set forth in SEQ ID NO: 20.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 21. 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 set forth in SEQ ID NO: 21.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 22. 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 set forth in SEQ ID NO: 22.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 23. 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 set forth in SEQ ID NO: 23.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 24. 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 set forth in SEQ ID NO: 24.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 25. 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 set forth in SEQ ID NO: 25.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 26. 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 set forth in SEQ ID NO: 26.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 27. 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 set forth in SEQ ID NO: 27.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 28. 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 set forth in SEQ ID NO: 28.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 29. 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 set forth in SEQ ID NO: 29.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 30. 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 set forth in SEQ ID NO: 30.

In some embodiments of any of the aspects described herein, E (eg, each E) includes an amino acid sequence set forth in SEQ ID NO: 31. 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 set forth in SEQ ID NO: 31.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 32. 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 set forth in SEQ ID NO: 32.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 33. 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 set forth in SEQ ID NO: 33.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 34. 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 set forth in SEQ ID NO: 34.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 35. 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 set forth in SEQ ID NO: 35.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 36. 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 set forth in SEQ ID NO: 36.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 37. 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 set forth in SEQ ID NO: 37.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 38. 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 set forth in SEQ ID NO: 38.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 39. 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 set forth in SEQ ID NO: 39.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 40. 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 set forth in SEQ ID NO: 40.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 41. 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 set forth in SEQ ID NO: 41.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 42. 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 set forth in SEQ ID NO: 42.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 43. 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 set forth in SEQ ID NO: 43.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 44. 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 set forth in SEQ ID NO: 44.

In some embodiments of any of the aspects described herein, E (eg, each E) includes an amino acid sequence set forth in SEQ ID NO: 45. 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 set forth in SEQ ID NO: 45.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 46. 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 set forth in SEQ ID NO: 46.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 47. 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 set forth in SEQ ID NO: 47.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 48. 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 set forth in SEQ ID NO: 48.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 49. 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 set forth in SEQ ID NO: 49.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 50. 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 set forth in SEQ ID NO: 51.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 52. 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 set forth in SEQ ID NO: 52.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 53. 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 set forth in SEQ ID NO: 53.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 54. 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 set forth in SEQ ID NO: 54.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 55. 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 set forth in SEQ ID NO: 55.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 56. 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 set forth in SEQ ID NO: 56.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 57. 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 set forth in SEQ ID NO: 57.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 58. 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 set forth in SEQ ID NO: 58.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 59. 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 set forth in SEQ ID NO: 59.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 60. 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 set forth in SEQ ID NO: 60.

In some embodiments of any of the aspects described herein, E (eg, each E) includes an amino acid sequence set forth in SEQ ID NO: 61. 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 set forth in SEQ ID NO: 61.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 62. 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 set forth in SEQ ID NO: 62.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in 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 set forth in SEQ ID NO: 63.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in 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 set forth in SEQ ID NO: 64.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in 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 set forth in SEQ ID NO: 65.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in 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 set forth in SEQ ID NO: 66.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 67. 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 set forth in SEQ ID NO: 67.

In some embodiments of any of the aspects described herein, E (eg, each E) includes an amino acid sequence set forth in SEQ ID NO: 68. 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 set forth in SEQ ID NO: 68.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in SEQ ID NO: 69. 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 set forth in SEQ ID NO: 69.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in 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 set forth in SEQ ID NO: 70.

In some embodiments of any of the aspects described herein, E (e.g., each E) includes an amino acid sequence set forth in 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 set forth in SEQ ID NO: 71.

In some embodiments of any of the aspects described herein, wherein E includes an Fc domain monomer, said Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NO: 1-68) includes triple mutation corresponding to M252Y/S254T/T256E (YTE). As used herein, it should be understood that an amino acid “corresponding to” a particular amino acid residue (e.g., a particular SEQ ID NO.) includes any amino acid residue that would be understood by one skilled in the art to match when aligned with the particular residue (e.g. specific sequence). For example, any of SEQ ID NO: 1-68 may be mutated to include a YTE mutation.

In some embodiments of any of the aspects described herein, wherein E includes an Fc domain monomer, the Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68) includes a double mutant corresponding to M428L/N434S (LS). As used herein, it should be understood that an amino acid “corresponding to” a particular amino acid residue (e.g., a particular SEQ ID NO.) includes any amino acid residue that would be understood by one skilled in the art to match when aligned with the particular residue (e.g. specific sequence). For example, any of SEQ ID NO: 1-68 may be mutated to include the LS mutation.

In some embodiments of any of the aspects described herein, wherein E includes an Fc domain monomer, the Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68) includes a mutant , corresponding to N434H. As used herein, it should be understood that an amino acid “corresponding to” a particular amino acid residue (e.g., a particular SEQ ID NO.) includes any amino acid residue that would be understood by one skilled in the art to match when aligned with the particular residue (e.g. specific sequence). For example, any of SEQ ID NO: 1-68 may be mutated to include the N434H mutation.

In some embodiments of any of the aspects described herein, wherein E includes an Fc domain monomer, said Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NO: 1-68) includes mutant corresponding to C220S. As used herein, it should be understood that an amino acid “corresponding to” a particular amino acid residue (e.g., a particular SEQ ID NO.) includes any amino acid residue that would be understood by one skilled in the art to match when aligned with the particular residue (e.g. specific sequence). For example, any of SEQ ID NO: 1-68 may be mutated to include the C220S mutation.

In some embodiments, any of the aspects described herein, wherein E includes an Fc domain monomer, said Fc domain monomer (e.g., an Fc domain monomer having the sequence set forth in any of SEQ ID NOs: 1-68) is a fragment of an 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 (for example, 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 (for example, 75 , 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more) consecutive amino acids in length from SEQ ID NO: 1-68.

In some embodiments, any of the aspects described herein (e.g., a conjugate of any of formulas (1)-(5), (DI)-(DX), (D'-I), (MI)-(MX), or (M'-I)), one or more nitrogen atoms of one or more surface-exposed lysine residues in E, or one or more sulfur atoms of one or more surface-exposed cysteine residues in E are covalently conjugated to a linker (e.g., a PEG2- linker PEG20). The E-conjugated linker may be functionalized such that it can react to form a covalent bond with the L of any A ₁ -L or any A ₂ -LA ₁ described herein. In preferred embodiments, E is conjugated to an azido-functionalized linker, and L in A ₁ -L or any A ₂ -LA ₁ is functionalized with an alkyne group. Conjugation (eg, by click chemistry) of an azido linker at E and an alkyne linker at A ₁ -L or A ₂ -LA ₁ results in the formation of a conjugate of the invention, for example a conjugate described by any of formulas (1)- (5), (DI)-(DX), (D'-I), (MI)-(MX) or (M'-I). In yet other embodiments, E is conjugated to a linker functionalized with an alkyne group, and L of any A ₁ -L or any A ₂ -LA ₁ is functionalized with an azido group. Conjugation (eg, by click chemistry) of an alkyne linker at E and an azido linker at A ₁ -L or A ₂ -LA ₁ results in the formation of a conjugate of the invention, for example a conjugate described by formula (1)-(5 ), (DI)-(DX), (D'-I), (MI)-(MX) or (M'-I).

In some embodiments of any of the aspects described herein, the wavy line in any of formulas (1)-(5), (DI)-(DX), (D'-I), (MI)-(MX), or (M'-I) may represent a covalent bond between E and L in A ₁ -L or A ₂ -LA ₁ .

In some embodiments of any of the aspects described herein, the wavy line in any of formulas (1)-(5), (DI)-(DX), (D'-I), (MI)-(MX), or (M'-I) may indicate that one or more amino acid side chains in E (for example, one or more nitrogen atoms of one or more surface-exposed lysine residues in E, or one or more sulfur atoms of one or more surface-exposed cysteines in E) have been conjugated to a linker (eg, a PEG ₂ -PEG ₂₀ linker), wherein the linker is functionalized with a reactive moiety such that the reactive moiety forms a covalent bond with L in any A ₁ -L or any A ₂ -LA ₁ described in herein (eg, by using click chemistry between an azido-functionalized linker and an alkyne-functionalized linker, as described above). In some embodiments of any of the aspects described herein, A ₁ and/or A ₂ have the structure described by (AI):

In preferred embodiments, where A ₁ and/or A ₂ have the structure described by (AI): R ₁ is -NHC(=NH)NH ₂ , R ₄ is -CO ₂ H, R ₅ is -COCH ₃ and/or X is -O-. In preferred embodiments, A ₁ and/or A ₂ have the zanamivir structure described as follows:

In some embodiments of any of the aspects described herein, A ₁ and/or A ₂ have the structure described by (A-II):

In preferred embodiments where A ₁ and/or A ₂ have the structure described in (A-II): R ₁ is -NFC(=NF)NH ₂ , R ₂ is H or F, R ₃ is H or F, R ₄ is -CO ₂ H, R ₅ is -COCH ₃ and/or X is -O-. In preferred embodiments, A ₁ and/or A ₂ have a structure described as follows:

In some embodiments of any of the aspects described herein, A ₁ and/or A ₂ have the structure described in (A-III):

In preferred embodiments, where A ₁ and/or A ₂ have the structure described in (A-III): R ₁ is -NHC(=NH)NH ₂ , R ₄ is -CO ₂ H and/or R ₅ is itself -COSN ₃ . In preferred embodiments, A ₁ and/or A ₂ have a peramivir structure described as follows:

In some embodiments of any of the aspects described herein, A ₁ and/or A ₂ have the structure described by (A-IV):

In preferred embodiments where A ₁ and/or A ₂ have the structure described in (A-IV): R ₁ is -NHC(=NH)NH ₂ , R ₄ is -CO ₂ H and/or R ₅ is itself -COSN ₃ . In preferred embodiments, A ₁ and/or A ₂ have a structure described as follows:

In some embodiments of any of the aspects described herein, A ₁ and/or A ₂ have the structure described by (AV):

In preferred embodiments, where A ₁ and/or A ₂ have the structure described by (AV): R ₁ is -NHC(=NH)NH ₂ , R ₄ is -CO ₂ H and/or R ₅ is - SOSN ₃ . In preferred embodiments, A ₁ and/or A ₂ have a structure described as follows:

In some embodiments of any of the aspects described herein, A ₁ and/or A ₂ have the structure described by (A-VI):

In preferred embodiments, where A ₁ and/or A ₂ have the structure described in (A-VI): R ₁ is -NHC(=NH)NH ₂ , R ₄ is -CO ₂ H, R ₅ is - COCH ₃ and/or X represents -O-. In preferred embodiments, A ₁ and/or A ₂ have the zanamivir 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-VII):

In preferred embodiments where A ₁ and/or A ₂ have the structure described in (A-VII): R ₁ is -NHC(=NH)NH ₂ , R ₂ is H or F, R ₃ is H or F, R ₄ is -CO ₂ H, R ₅ is -COCH ₃ and/or X is -O-. In preferred embodiments, 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 in (A-VIII):

In preferred embodiments where A ₁ and/or A ₂ have the structure described in (A-VIII): R ₁ is -NHC(=NH)NH ₂ , R ₅ is -COCH ₃ and/or X is - ABOUT-. In preferred embodiments, 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-IX):

In preferred embodiments where A ₁ and/or A ₂ have the structure described in (A-IX): R ₁ is -NHC(=NH)NH ₂ , R ₂ is H or F, R ₃ is H or F, R ₅ is -COCH ₃ and/or X is -O-. In preferred embodiments, 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-X):

In preferred embodiments, where A ₁ and/or A ₂ have the structure described by (A-X): R ₁ is -NHC(=NH)NH ₂ , R ₃ is H, R ₅ is -COCH ₃ and /or X represents -O-. In preferred embodiments, A ₁ and/or A ₂ have the sulfosanamivir 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-XI):

In preferred embodiments where A ₁ and/or A ₂ have the structure described in (A-XI): R ₄ is -CO ₂ H and/or R ₅ is -COCH ₃ . In preferred embodiments, the alkene is (E), (Z), or a racemic mixture (E)/(Z). In preferred embodiments, A ₁ and/or A ₂ have structure A-315675 (Abbott) described by:

In some embodiments of any of the aspects described herein, A ₁ and/or A ₂ have the structure described in (A-XIII):

In preferred embodiments, where A ₁ and/or A ₂ have the structure described in (A-XIII): R ₄ is -CO ₂ H. In preferred embodiments, A ₁ and/or A ₂ have the structure A-315675 (Abbott ), described by:

In some embodiments, the conjugate is conjugate 1 or any regioisomer thereof, and the drug to antibody ratio (DAR) (e.g., T) is from 0.5 to 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, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 2 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 3 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 4 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 5 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 6 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 7 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 8 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 9 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 10 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 2 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.263, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9 .9 or 10.0. In some embodiments, the DAR is 0.5 to 264.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 12 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 13 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 14 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 15 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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, 8.265, 8.4, 8.5, 8.6 , 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9 .9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 16 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 17 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 18 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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, 8.6 , 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9 .9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 19 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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, 2.266, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.266, 3.5, 3.6 , 3.7, 3.8, 3.9, 266, 266.1, 4.2, 266.3, 266.266, 266.5, 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, 8.6, 8.7 , 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10 ,0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 20 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 21 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 22 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 23 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 24 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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, 8.6 , 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9 .9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 25 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 26 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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, 8.6 , 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.267, 9.6, 9.7, 9.8, 9.9 or 10.0. In some embodiments, the DAR is from 0.5 to 2.0, from 2.0 to 4.0, from 4.0 to 268.0, from 6.0 to 8.0, or from 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 27 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 28 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 29 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 30 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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, 6.269, 6.8, 6.9, 269.0, 7.1, 7.2, 7.3, 269 ,4, 269.5, 269.6, 269.7, 7.8.0, 269.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6 , 8.269, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 31 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 32 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 33 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 34 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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, 2.270, 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, 8.6 , 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9 .9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 35 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 35 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 36 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 37 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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, 8.6 , 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9 .9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 38 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 39 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 40 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.271, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9 .9 or 10.0. In some embodiments, the DAR is from 0.5 to 2.0, from 2.0 to 4.0, from 4.0 to 6.0, from 6.0 to 8.0, or from 8.0 to 272.0 .

In some embodiments, the conjugate is conjugate 41 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 42 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

In some embodiments, the conjugate is conjugate 43 or any regioisomer thereof, and the DAR (e.g., T) is from 0.5 to 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 ,6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 , 9.9 or 10.0. In some embodiments, the DAR is 0.5 to 2.0, 2.0 to 4.0, 4.0 to 6.0, 6.0 to 8.0, or 8.0 to 10.0 .

Definitions

To assist in understanding this invention, definitions of a number of terms are provided below. The terms defined herein have meanings commonly understood by those skilled in the art pertaining to the present invention. Singular terms are not intended to denote only a singular entity, but include a general class, a specific example of which may be used for illustration. Terminology herein is used to describe specific embodiments of the invention, but their use does not limit the scope of the invention except as set forth in the claims.

The term "neuraminidase inhibitor" or "viral neuraminidase inhibitor" as used herein refers to compounds that reduce the activity of the influenza virus neuraminidase enzyme (eg, from influenza A, B or C virus). The neuraminidase inhibitor can be identified using methods known to those skilled in the art, for example, by reducing viral replication in an influenza virus plaque neutralization assay, for example, at concentrations less than 20 μM (e.g., less than 10 μM, 5 μM, 2 μM, 1 μM , 500 nM or 100 nM). Viral neuraminidase inhibitors known to those skilled in the art include zanamivir, sulfozanamivir, peramivir, and A-315675 (Abbott) (see, e.g., Hadhazi et al. A sulfozanamivir analogue has potent anti-influenza virus activity. ChemMedChem Comm. 13:785 -789 (2018) and In vitro characterization of A-315675, a highly potent inhibitor of A and B strain of influenze virus neuraminidases and influenza virus replication. Antimicrobial Agents and Chemotherapy 46(4): 1014-1021 (2002)). The viral neuraminidase inhibitors of the invention include zanamivir, sulfosanamivir, peramavir, A-315675 and analogs thereof, such as the viral neuraminidase inhibitors of formulas (A-I) to (A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; each R ₂ and R ₃ is independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl. As used herein, the term “inhibits neuraminidase activity” refers to an IC ₅₀ value that is 1000 nM or less, for example, as measured according to the neuraminidase inhibition assay in Example 2 herein. Specifically, the IC ₅₀ value represents the concentration of influenza neuraminidase inhibitor that is required for 50% inhibition in vitro. In some aspects, an IC ₅₀ value of 100 nM or less or 10 nM or less according to a neuraminidase inhibition assay indicates a compound that inhibits neuraminidase activity.

By “viral infection” is meant the pathogenic growth of a virus (eg, influenza virus) in a host (eg, a human subject). A viral infection can be any situation in which the presence of a viral population(s) causes harm to the host. Thus, a subject "suffers" from a viral infection when an excessive amount of the viral population is present in or on the subject's body, or when the presence of the viral population(s) damages cells or other tissue of the subject.

As used herein, the term “Fc domain monomer” refers to a polypeptide chain that includes at least the hinge domain and the second and third antibody constant domains ( _CH2 and _CH3 ) or functional fragments thereof (e.g., fragments which are 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 may be a subtype of IgG (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4) (e.g., IgG1). The Fc domain monomer does not include any portion of the immunoglobulin that capable of acting as an antigen recognition region, for example, a variable domain or a complementarity determining region (CDR).Fc domain monomers in conjugates as described herein may contain one or more changes compared to the 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 the Fc domain and the Fc receptor. Examples of suitable variations are known in the art. In some embodiments, a human Fc domain monomer (e.g., an IgG heavy chain, such as IgG1) contains a region that extends from any of Asn208, Glu216, Asp221, Lys222, or Cys226 to the carboxyl terminus of the heavy chain at Lys447. The C-terminal Lys447 of the Fc region may or may not be present without affecting the structure or stability of the Fc region. Unless otherwise indicated herein, the numbering of amino acid residues in an IgG or Fc domain monomer follows the EU numbering system for antibodies, also referred to as the EU Kabat 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.

As used herein, the term “Fc domain” refers to a dimer of two Fc domain monomers that is capable of binding an Fc receptor. In a wild-type Fc domain, two Fc domain monomers dimerize through interactions between two antibody C _H 3 constant domains, in some embodiments one or more disulfide bonds are formed between the hinge domains of the two dimerizing Fc domain monomers.

The term "covalently attached" refers to two parts of the conjugate that are linked to each other by a covalent bond formed between the two atoms in the two parts of the conjugate.

As used herein, the term “Fc binding peptide” refers to a polypeptide having an amino acid sequence of 5-50 (e.g., 5-40, 5-30, 5-20, 5-15, 5-10, 10-50, 10 -30 or 10-20) amino acid residues that has the affinity and function to bind to an Fc domain, such as any Fc domain described herein. The Fc binding peptide can be of various origins, for example synthetic, human, murine or rat. The Fc binding peptides of the invention include Fc binding peptides that have been designed to include one or more (e.g., two, three, four, or five) solvent accessible cysteine or lysine residues that can provide a site for conjugation to a compound of the invention (for example, conjugation with a monomer or dimer of a neuraminidase inhibitor, including through a linker). Most preferably, the Fc binding peptide will contain a single solvent accessible cysteine or lysine, thereby providing site-specific conjugation of the 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. If included, a non-naturally occurring amino acid residue (eg, a side chain of a non-naturally occurring amino acid residue) can be used as an attachment point for a compound of the invention (eg, a neuraminidase inhibitor monomer or dimer, including via a linker). The Fc binding peptides of the invention may be linear or cyclic. The Fc binding peptides of the invention include any Fc binding peptides known to one of ordinary skill in the art.

As used herein, the term “albumin protein” refers to a polypeptide containing an amino acid sequence corresponding to a naturally occurring albumin protein (eg, 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 subdomains, and fusion proteins (eg, an albumin protein having a C-terminal or N-terminal fusion such as a polypeptide linker). Preferably, 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. The 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 with any of SEQ ID NO: 69-71. The albumin proteins of the invention include albumin proteins that have been designed to include one or more (e.g., two, three, four or five) solvent accessible cysteine or lysine residues that can provide a site for conjugation to a compound of the invention (e.g. conjugation with a monomer or dimer of a neuraminidase inhibitor, including through a linker). Most preferably, the albumin protein contains the only solvent accessible cysteine or lysine, thereby allowing site-specific conjugation of the 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. If included, a non-naturally occurring amino acid residue (eg, a side chain of a non-naturally occurring amino acid residue) can be used as an attachment point for a compound of the invention (eg, a neuraminidase inhibitor monomer or dimer, including via a linker).

As used herein, the term “albumin-binding peptide protein” refers to a polypeptide having an amino acid sequence of 5-50 (e.g., 5-40, 5-30, 5-20, 5-15, 5-10, 10-50, 10-30 or 10-20) amino acid residues that have the affinity and function to bind an albumin protein, such as any of the albumin proteins described herein. Preferably, the albumin-binding peptide protein binds to naturally occurring serum albumin, most preferably human serum albumin. The albumin-binding peptide protein can be of various origins, for example synthetic, human, murine or rat. The protein albumin-binding peptides of the invention include protein albumin-binding peptides that have been designed to include one or more (e.g., two, three, four, or five) solvent accessible cysteine or lysine residues that can provide a site for conjugation to a compound of the invention (for example, conjugation with a monomer or dimer of a neuraminidase inhibitor, including via a linker). Most preferably, the albumin-binding peptide protein will contain a single solvent-accessible cysteine or lysine, thereby allowing site-specific conjugation of the compound of the invention. Protein albumin-binding peptides may include only naturally occurring amino acid residues, or may include one or more non-naturally occurring amino acid residues. If included, a non-naturally occurring amino acid residue (eg, a side chain of a non-naturally occurring amino acid residue) can be used as an attachment point for a compound of the invention (eg, a neuraminidase inhibitor monomer or dimer, including via a linker). The protein albumin-binding peptides of the invention can be linear or cyclic. The protein albumin binding peptide of the invention includes any protein albumin binding peptides known to one of ordinary skill in the art, examples of which are presented herein. Additional exemplary protein albumin-binding peptides are presented in US Patent Application No. 2005/0287153, which is incorporated herein by reference in its entirety.

As used herein, a “surface-exposed amino acid” or “solvent-exposed amino acid,” such as surface-exposed cysteine or surface-exposed lysine, refers to an amino acid that is accessible to the solvent surrounding the protein. The surface exposed amino acid may be a naturally occurring or engineered variant (eg, substitution or insertion) of a protein. In some embodiments, the surface-exposed amino acid is an amino acid that, when replaced, does not significantly alter the three-dimensional structure of the protein.

The terms “linker,” “L,” and “L'” as used herein refer to a covalent bond or connection between two or more components in a conjugate (e.g., between two neuraminidase inhibitors in a conjugate described herein, between a neuraminidase inhibitor and an Fc- domain or protein albumin in the conjugate described herein, and between a dimer of two neuraminidase inhibitors and the Fc domain or protein albumin in the conjugate described herein). In some embodiments, a conjugate described herein may contain a linker that has a trivalent structure (eg, a trivalent linker). The trivalent linker has three arms, where each arm is covalently linked to a conjugate component (eg, the first arm is conjugated to a first neuraminidase inhibitor, the second arm is conjugated to a second neuraminidase inhibitor, and the third arm is conjugated to an Fc domain or albumin protein).

Molecules that can be used as linkers include at least two functional groups, which may be the same or different, for example, two carboxylic acid groups, two amino 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 amino group, a carboxylic acid group and a sulfonic acid group, an amino group and a maleimide group, an amino group and an alkyne group, or an amino group and a sulfonic acid group. The first functional group may form a covalent bond with the first component in the conjugate, and the second functional group may form a covalent bond with the second component in the conjugate. In some embodiments of a trivalent linker, two arms of the linker may comprise two dicarboxylic acids, wherein the first carboxylic acid may form a covalent bond with the first neuraminidase inhibitor in the conjugate, and the second carboxylic acid can form a covalent bond with the second neuraminidase inhibitor in the conjugate, and the third arm of the linker may provide a covalent bond with the Fc domain or protein albumin in the conjugate. Examples of dicarboxylic acids are described later herein. In some embodiments, a molecule containing one or more maleimide groups can be used as a linker in which the maleimide group can form a carbon-sulfur bond with a cysteine in a component (eg, Fc domain or albumin protein) in the conjugate. In some embodiments, a molecule containing one or more alkyne groups can be used as a linker, in which the alkyne group can form a 1,2,3-triazole bond with an azide in the component (e.g., Fc domain or albumin protein) in the conjugate . In some embodiments, a molecule containing one or more azide groups can be used as a linker, in which the azide group can form a 1,2,3-triazole bond with an alkyne in a component (e.g., Fc domain or albumin protein) in a conjugate . In some embodiments, a molecule containing one or more bis-sulfone groups can be used as a linker, in which the bis-sulfone group can form a bond with an amino group in a component (eg, Fc domain or albumin protein) into a conjugate. In some embodiments, a molecule containing one or more sulfonic acid groups can be used as a linker, in which the sulfonic acid group can form a sulfonamide bond with a component in the conjugate. In some embodiments, a molecule containing one or more isocyanate groups can be used as a linker, in which the isocyanate group can form a urea linkage to a component in the conjugate. In some embodiments, a molecule containing one or more haloalkyl groups can be used as a linker in which the haloalkyl group can form a covalent bond, such as C-N and C-O bonds, with a component in the conjugate.

In some embodiments, the linker provides space, rigidity and/or flexibility between two or more components. In some embodiments, the linker may be a bond, such as a covalent bond. The term "bond" refers to a chemical bond, such as an amide bond, a disulfide bond, a C-O bond, a C-N bond, an N-N bond, a C-S bond, or any bond created by a chemical reaction, such as chemical conjugation. In some embodiments, the linker includes no more than 250 atoms. In some embodiments, the linker includes no more than 250 non-hydrogen atoms. In some embodiments, the linker backbone comprises no more than 250 atoms. The “backbone” of a linker refers to the atoms in the linker that together form a shortest path from one part of the conjugate to another part of the conjugate (eg, a shortest path linking a first neuraminidase inhibitor and a second neuraminidase inhibitor). Atoms in the linker backbone are directly involved in linking one part of the conjugate to another part of the conjugate (eg, linking a first neuraminidase inhibitor and a second neuraminidase inhibitor). For example, hydrogen atoms attached to carbon atoms in the linker backbone are not considered to be directly involved in linking one part of the conjugate to another part of the conjugate.

In some embodiments, the linker may contain a synthetic group derived from, for example, a synthetic polymer (eg, a polyethylene glycol (PEG) polymer). In some embodiments, the linker may contain one or more amino acid residues, such as D- or L-amino acid residues. In some embodiments, the linker may be a residue of an amino acid sequence (e.g., a sequence of 1-25 amino acids, 1-10 amino acids, 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids , 1-4 amino acids, 1-3 amino acids, 1-2 amino acids or 1 amino acid). In some embodiments, the linker may contain one or more, for example, 1-100, 1-50, 1-25, 1-10, 1-5, or 1-3, of 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 heterocycloal kinylene, optionally substituted arylene, optionally substituted heteroarylene ( for example, pyridine), O, S, NR ⁱ (R ⁱ represents H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted cycloalkenyl, optionally substituted heteroocycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocycloalkynyl, optionally substituted aryl or optionally substituted heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino. For example, the linker may contain one or more of an optionally substituted (C ₁ -C ₂₀ )alkylene, an optionally substituted (C ₁ -C ₂₀ )heteroalkylene (for example, a PEG unit), an optionally substituted (C ₂ -C ₂₀ )alkenylene (for example, 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 ( for example, cyclopropylene, cyclobutylene), optionally substituted (C ₃ -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 (for example, C6-arylene), optionally substituted (C ₂ -C ₁₅ )heteroarylene (for example, imidazole, pyridine), O, S, NR ⁱ (R ⁱ represents H, optionally substituted (C ₁ -C ₂₀ )alkyl, optionally substituted (C ₁ -C ₂₀ )heteroalkyl, optionally substituted (C ₂ -C ₂₀ )alkenyl, optionally substituted (C ₂ - C ₂₀ )heteroalkenyl, optionally substituted (C ₂ -C ₂₀ )alkynyl, optionally substituted (C ₂ -C ₂₀ )heteroalkynyl, optionally substituted (C ₃ -C ₂₀ )cycloalkyl, optionally substituted (C ₃ -C ₂₀ )heterocycloalkyl, optional substituted (C ₄ -C ₂₀ )cycloalkenyl, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenyl, optionally substituted (C ₈ -C ₂₀ )cycloalkynyl, optionally substituted (C ₈ -C ₂₀ )heterocycloalkenyl, optionally substituted (C ₅ -C ₁₅ )aryl or optionally substituted (C ₂ -C ₁₅ )heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino.

As used herein, the terms “alkyl,” “alkenyl,” and “alkynyl” include straight and branched chain monovalent substituents, as well as combinations thereof containing only C and H in the unsubstituted form. When the alkyl group includes at least one carbon-carbon double bond or carbon-carbon triple bond, the alkyl group may be referred to as an "alkenyl" or "alkynyl" group, respectively. The monovalency of an alkyl, alkenyl or alkynyl group does not include optional substituents on the alkyl, alkenyl or alkynyl group. For example, if an alkyl, alkenyl, or alkynyl group is attached to a compound, the 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. In some embodiments, the alkyl or heteroalkyl group may contain, for example, 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. C1-C20, C1-C18, C1- Cl6, C1-C14, C1-C12, C1-C10, C1-C8, C1-C6, C1-C4 or C1-C2). In some embodiments, an alkenyl, heteroalkenyl, alkynyl, or heteroalkynyl group may contain, for example, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, or 2 -4 carbon atoms (for example, C2-C20, C2-Cl8, C2-Cl6, C2-Cl4, C2-Cl2, C2-Cl0, C2-C8, C2-C6 or C2-C4). Examples include, but are not limited to, methyl, ethyl, isobutyl, sec-butyl, tert-butyl, 2-propenyl and 3-butynyl.

As used herein, the term “cycloalkyl” represents a monovalent saturated or unsaturated non-aromatic cyclic alkyl group. Cycloalkyl may have, for example, from three to twenty carbon atoms (for example, C3-C7, C3-C8, C3-C9, C3-C10, C3-C11, C3-C12, C3-C14, C3-C16, C3-C18 or C3-C20 cycloalkyl). Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. When a cycloalkyl group includes at least one carbon-carbon double bond, the cycloalkyl group may be referred to as a "cycloalkenyl" group. Cycloalkenyl may have, for example, from four to twenty carbon atoms (for example, C4-C7, C4-C8, C4-C9, C4-C10, C4-C11, C4-C12, C4-C14, C4-C16, C4-C18 or C4-C20 cycloalkenyl). Examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexenyl and cycloheptenyl. When a cycloalkyl group includes at least one carbon-carbon triple bond, the cycloalkyl group may be referred to as a "cycloalkynyl" group. A cycloalkynyl may have, for example, eight to twenty carbon atoms (eg C8-C9, C8-C10, C8-C11, C8-C12, C8-C14, C8-C16, C8-C18 or C8-C20 cycloalkynyl). The term "cycloalkyl" also includes a cyclic compound having a bridged polycyclic structure in which one or more carbon atoms link two non-adjacent members of a monocyclic ring, for example, bicyclo[2.2.1.]heptyl and adamantane. The term "cycloalkyl" also includes bicyclic, tricyclic and tetracyclic fused ring structures, such as decalin and pyrocyclic compounds.

As used herein, the term “aryl” refers to any monocyclic or fused ring bicyclic or tricyclic system that has aromatic characteristics in terms of the distribution of electrons throughout the ring system, such as phenyl, naphthyl, or phenanthrene. In some embodiments, the ring system contains 5-15 ring atoms or 5-10 ring atoms. The aryl group may have, for example, five to fifteen carbon atoms (for example, C5-C6, C5-C7, C5-C8, C5-C9, C5-C10, C5-C11, C5-C12, C5-C13, C5- C14 or C5-C15 aryl). The term "heteroaryl" also refers to such monocyclic or fused bicyclic ring systems containing one or more, for example, 1-4, 1-3, 1, 2, 3 or 4 heteroatoms selected from O, S and N. The heteroaryl group may have, for example, from two to fifteen carbon atoms (for example, C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C2-C9. C2-C10, C2-C11, C2 -C12, C2-C13, C2-C14 or C2-C15 heteroaryl). The inclusion of a heteroatom allows the inclusion of 5-membered rings to be considered as aromatic, as well as 6-membered rings. Thus, typical heteroaryl systems include, for example, pyridyl, pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, benzoxazolyl, benzoisoxazolyl and imidazolyl. Since tautomers are possible, a group such as phthalimido is also considered heteroaryl. In some embodiments, the aryl or heteroaryl group is a 5- or 6-membered aromatic ring system, optionally containing 1-2 nitrogen atoms. In some embodiments, the aryl or heteroaryl group is an optionally substituted phenyl, pyridyl, indolyl, pyrimidyl, pyridazinyl, benzothiazolyl, benzimidazolyl, pyrazolyl, imidazolyl, isoxazolyl, thiazolyl, or imidazopyridinyl. In some embodiments, the aryl group is phenyl. In some embodiments, the aryl group may be optionally substituted with a substituent, such as an aryl substituent, such as biphenyl.

The term "alkaryl" refers to an aryl group that is connected to an alkylene, alkenylene or alkynylene group. Typically, if a compound is attached to an alkaryl group, an alkylene, alkenylene, or alkynylene moiety of the alkaryl is attached to the compound. In some embodiments, the alkaryl is a C6-C35 alkaryl (e.g., C6-C16, C6-C14, C6-C12, C6-C10, C6-C9, C6-C8, C7, or C6 alkaryl), wherein the number of carbon atoms is the total number of carbon atoms in the aryl moiety and the alkylene, alkenylene, or alkynylene moiety of the alkaryl. Examples of alkaryls include, but are not limited to, (C1-C8)alkylene(C6-C12)aryl, (C2-C8)alkenylene(C6-C12)aryl, or (C2-C8)alkynylene(C6-C12)aryl. In some embodiments, the alkaryl is benzyl or phenethyl. In a heteroalkaryl, 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. In an optionally substituted alkaryl, a 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 independently represents H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R ^x are combined to form a heterocycloalkyl. In some embodiments, the amino group is -NH ₂ .

As used herein, the term “alkamino” refers to an amino group as described herein that is attached to an alkylene (eg, C1-C5 alkylene), alkenylene (eg, C2-C5 alkenylene) or alkynylene group (eg, C2-C5 alkenylene). Typically, if a compound is attached to an alkamino group, an alkylene, alkenylene, or alkynylene moiety of the alkamino is attached to the compound. The amino moiety 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 combined with formation of heterocycloalkyl. In some embodiments, the amino portion of the alkamino is -NH ₂ . An example of an alkamino group is a C1-C5 alkamino, for example, a C2 alkamino (for example, CH ₂ CH ₂ NH ₂ or CH ₂ CH ₂ N(CH ₃ ) ₂ ). In a heteroalkamino group, one or more, for example 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 moiety of the heteroalkamino group. In some embodiments, the alkamine group may be optionally substituted. In a substituted alkamino group, 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.

As used herein, the term “alkamide” refers to an amide group that is attached to an alkylene (eg, C1-C5 alkylene), alkenylene (eg, C2-C5 alkenylene) or alkynylene (eg, C2-C5 alkenylene) group. Typically, if a compound is attached to an alkamide group, an alkylene, alkenylene, or alkynylene moiety of the alkamide is attached to the compound. The amide moiety of an alkamide refers to -C(O)-N(R ^x ) ₂ , where each Rx is independently H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two Rx are combined to form heterocycloalkyl. In some embodiments, the amide moiety of the alkamide is -C(O)NH ₂ . The alkamide group may be -(CH ₂ ) ₂ -C(O)NH ₂ or -CH ₂ -C(O)NH ₂ . In a heteroalkamide group, one or more, for example 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 heteroalkylamide group. In some embodiments, the alkamide group may be optionally substituted. In a substituted alkamide group, 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.

As used herein, the terms "alkylene", "alkenylene" and "alkynylene" refer to divalent groups having a specific size. In some embodiments, an alkylene may contain, for example, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2 atoms carbon (for example, C1-C20, C1-C18, C1-C16, C1-C14, C1-C12, C1-C10, C1-C8, C1-C6, C1-C4 or C1-C2). In some embodiments, alkenylene or alkynylene may contain, for example, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, or 2-4 carbon atoms ( for example, C2-C20, C2-C18, C2-C16, C2-C14, C2-C12, C2-C10, C2-C8, C2-C6 or C2-C4). Alkylene, alkenylene and/or alkynylene include straight and branched chain forms, as well as combinations thereof. The divalency of an alkylene, alkenylene or alkynylene group does not include optional substituents on the alkylene, alkenylene or alkynylene group. For example, two neuraminidase inhibitors can be attached to each other via a linker that includes an 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 two attachments at either end of the alkylene, alkenylene and/or alkynylene group. For example, if the 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. Optional substituents on alkenylene are not included in the divalency of alkenylene. The divalent nature of an alkylene, alkenylene or alkynylene group (eg, an alkylene, alkenylene or alkynylene group in a linker) applies to both ends of the group and does not include optional substituents that may be present on the alkylene, alkenylene or alkynylene group. Because they are divalent, they can link together multiple (eg, two) parts of the conjugate, for example, a first neuraminidase inhibitor and a second neuraminidase inhibitor. Alkylene, alkenylene and/or alkynylene groups may be substituted with groups generally suitable as substituents for alkyl, alkenyl and alkynyl groups, as defined later herein. For example, C=O represents Cl-alkylene, which is substituted with oxo (=O). For example, -HCR-C=C- can be considered an optionally substituted alkynylene and is considered a divalent group even if it has an optional substituent, R. Heteroalkylene, heteroalkenylene and/or heteroalkynylene groups refer to alkylene, alkenylene and/or alkynylene groups, comprising one or more, for example 1-4, 1-3, 1, 2, 3 or 4 heteroatoms, for example N, O and S. For example, a polyethylene glycol (PEG) polymer or a PEG -(CH ₂ ) ₂ -O unit - in a polymer, PEG is considered a heteroalkylene containing one or more oxygen atoms.

As used herein, the term “cycloalkylene” refers to a divalent cyclic group linking two halves of a compound together. For example, one carbon atom in a cycloalkylene group may be bonded to one part of the compound, while another carbon in the cycloalkylene group may be bonded to another portion of the compound. The cycloalkylene group may include saturated or unsaturated non-aromatic cyclic groups. Cycloalkylene may have, for example, from three to twenty carbon atoms in the cyclic part of the cycloalkylene (for example, C3-C7, C3-C8, C3-C9, C3-C10, C3-C11, C3-C12, C3-C14, C3-C16 , C3-C18 or C3-C20 cycloalkylene). When the cycloalkylene group includes at least one carbon-carbon double bond, the cycloalkylene group may be referred to as a "cycloalkenyl new" group. A cycloalkenylene may have, for example, four to twenty carbon atoms in the cycloalkenylene ring moiety (e.g. C4-C7, C4-C8, C4-C9. C4-C10, C4-C11, C4-C12, C4-C14, C4- C16, C4-C18 or C4-C20 cycloalkenylene). When a cycloalkylene group includes at least one carbon-carbon triple bond, the cycloalkylene group may be referred to as a “cycloalkynylene” group. The alkynylene ring may have, for example, four to twenty carbon atoms in the cycloalkynylene ring moiety (e.g. C4-C7, C4-C8, C4-C9. C4-C10, C4-C11, C4-C12, C4-C14, C4 -C16, C4-C18 or C8-C20 cycloalkynylene). The cycloalkylene group may be substituted with groups generally suitable as substituents for alkyl, alkenyl and alkynyl groups as defined herein. Heterocycloalkylene refers to a cycloalkylene group containing one or more, for example, 1-4, 1-3, 1, 2, 3 or 4 heteroatoms, for example N, O and S. Examples of cycloalkylenes include, but are not limited to, cyclopropylene and cyclobutylene. Tetrahydrofuran can be considered as a heterocycle oalkylene.

As used herein, the term “arylene” refers to a multivalent (eg, divalent or trivalent) aryl group linking together multiple (eg, two or three) moieties of a compound. For example, one carbon in the arylene group may be bonded to one part of the compound, while another carbon in the arylene group may be bonded to another part of the compound. An arylene may have, for example, five to fifteen carbon atoms in the aryl portion of the arylene (e.g. C5-C6, C5-C7, C5-C8, C5-C9. C5-C10, C5-C11, C5-C12, C5-C13 , C5-C14 or C5-C15 arylene). The arylene group may be substituted with groups generally suitable as substituents for alkyl, alkenyl and alkynyl groups as defined herein. Heteroarylene refers to an aromatic group containing one or more, for example 1-4, 1-3, 1, 2, 3 or 4 heteroatoms, for example N, O and S. A heteroarylene group may have, for example, two to fifteen carbon atoms (for example, C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C2-C9. C2-C10, C2-C11, C2-C12, C2-C13, C2-C14 or C2-C15 heteroarylene).

As used herein, the term “optionally substituted” refers to the presence of 0, 1 or more substituents, such as 0-25, 0-20, 0-10 or 0-5 substituents. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, alkaryl, acyl, heteroaryl, heteroalkyl, heterooalkenyl, heteroalkynyl, heteroalkaryl, halogen, oxo, cyano, nitro, amino, alcamino, 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 , OCF ₃ , SiR ₃ , and NO ₂ , where each R independently represents H, alkyl, alkenyl, aryl, heteroalkyl, heteroalkenyl or heteroaryl, and wherein two of the optional substituents on the same or adjacent atoms can be combined to form a fused, optionally substituted aromatic or non-aromatic, saturated or unsaturated ring that contains 3-8 members, or two of the optional substituents on one and the same atom can be combined to form an optionally substituted aromatic or non-aromatic, saturated or unsaturated ring that contains 3-8 members.

An optionally substituted group or moiety refers to a group or moiety (eg, any of the groups or moieties described above) in which one of the atoms (eg, a hydrogen atom) is optionally replaced by another substituent. For example, the optionally substituted alkyl may be an optionally substituted methyl in which the hydrogen atom of the methyl group is replaced by, for example, OH. As another example, a substituent on a heteroalkyl or its divalent analogue, heteroalkylene, can replace hydrogen with carbon or hydrogen with a heteroatom such as N. For example, the hydrogen atom in the -R-NH-R- group can be replaced with an alkamide substituent, for example - RN[(CH ₂ C(O)N(CH ₃ ) ₂ ]-R.

Typically, an optional substituent is a non-interfering substituent. "Non-interfering substituent" refers to a substituent that leaves the ability of the conjugates described herein (for example, conjugates of any of formulas (1)-(5), (DI)-(DX), (D'-I), (MI )-(MX) or (M'-I)), either bind to viral neuraminidase or inhibit influenza virus proliferation. Thus, in some embodiments, the substituent may alter the degree of such activity. However, as long as the conjugate retains the ability to bind to viral neuraminidase or inhibit viral proliferation, the substituent will be classified as “non-interfering.” For example, a non-interfering substituent will leave the compound capable of providing antiviral efficacy based on an IC ₅₀ value of 10 μM or less in a viral plaque reduction assay, such as in Example 2, based on an anti-influenza virus neuraminidase IC ₅₀ value of less than 500 nM . Thus, the substituent may vary the degree of inhibition based on plaque reduction or inhibition of influenza virus neuraminidase. However, provided that the compounds herein, such as compounds of formulas (AI), (A-II), (A-III), (A-IV), (AV), (A-VI), (A- VII), (A-VIII), (A-IX), (A-X), (A-XI) and (A-XII), retain the ability to inhibit the activity of influenza virus neuraminidase, the substituent will be classified as “non-interfering”. A number of assays to determine viral plaque reduction or the ability of any compound to inhibit influenza virus neuraminidase are available in the art, and some of these are illustrated in the examples below.

The term "hetero", when used to describe a chemical group or moiety, refers to the presence of at least one heteroatom that is not carbon or hydrogen, such as N, O and S. Any of the groups or moieties described above may be referred to as "hetero" if it contains at least one heteroatom. For example, a heterocycloalkyl, heterocycloalkenyl or heterocycloalkynyl group refers to a cycloalkyl, cycloalkenyl or cycloalkynyl group that has one or more heteroatoms independently selected from, for example, N, O and S. An example of a heterocycloalkenyl group is maleimido. For example, a heteroaryl group refers to an aromatic group that has one or more heteroatoms independently selected from, for example, N, O, and S. One or more heteroatoms may also be included in a substituent that replaces a hydrogen atom in the group or moiety, as described in this document. For example, in an optionally substituted heteroaryl group, if one of the hydrogen atoms in the heteroaryl group is replaced by a substituent (eg, methyl), the substituent may also contain one or more heteroatoms (eg, methanol).

As used herein, the term "acyl" refers to a group having the structure: where R ^z represents an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, alkamino, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaryl, heteroalkaryl or heteroalkamino.

The term "halogen" or "halogen" as used herein refers to any halogen atom, such as F, Cl, Br or I. Any of the groups or moieties described herein may be referred to as a "halogen moiety" if it contains at least one halogen atom, such as an alkyl halo.

As used herein, the term “hydroxyl” represents a -OH group.

As used herein, the term “oxo” refers to a substituent having a =O structure where there is a double bond between an atom and an oxygen atom.

The term "carbonyl" as used herein refers to a group having the structure:

The term "thiocarbonyl" as used herein refers to a group having the structure:

The term "phosphate" as used herein refers to a group having the structure:

The term "phosphoryl" as used herein represents a group having the structure:

The term "sulfonyl" as used herein represents a group having the structure:

The term "imino" as used herein represents a group having the structure: where R represents an optional substituent.

The term "N-protecting group" as used herein represents those groups that are intended to protect the amino group from unwanted 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, for example, acyl, aryloyl and carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-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; carbamate-forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-dimethoxy benzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, tert-butyloxycarbonyl (BOC) , diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl and phenylthi ocarbonyl; alkaryl groups such as benzyl, triphenylmethyl and benzyloxymethyl; and silyl groups such as trimethylsilyl.

As used herein, the term “amino acid” refers to naturally occurring amino acids and non-naturally occurring amino acids.

As used herein, the term “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.

As used herein, the term “non-naturally occurring amino acid” means an alpha amino acid that is not naturally produced or found in a mammal. Examples of non-naturally occurring amino acids include D-amino acids; an amino acid having an acetylaminomethal group attached to a sulfur atom in cysteine; pegylated amino acid; omega amino acids of the formula NH ₂ (CH ₂ ) _n COOH, where n is 2-6, neutral non-polar amino acids such as sarcosine, tert-butylalanine, tert-butyl glycine, N-methylisoleucine 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-carboxypiperazine; piperazine-2-carboxylic acid, 2-amino-4-phenylbutanoic acid; 3-(2-naphthyl)alanine and hydroxyproline. Other amino acids are α-aminobutyric acid, α-amino-α-methylbutarate, aminocyclopropane carboxylate, aminoisobutyric acid, aminonorbornyl carboxylate, L-cyclohexylalanine, cyclopentylalanine, LN-methylleucine, LN-methylmethionine, LN-methylnorvaline, LN-methylphenylalanine, LN-methylproline, LN-methylserine, LN-methyltryptophan, D-ornithine, LN-methylethylglycine, L-norleucine, α-methyl-aminoisobutyrate, α-methylcyclohexylalanine, D-α-methylalanine, D-α-methylarginine, D-α-methylasparagine, D- α-methylaspartate, D-α-methylcysteine, D-α-methylglutamine, D-α-methylhistidine, D-α-methylisoleucine, D-α-methylleucine, D-α-methyllysine, D-α-methylmethionine, D-α- methylornithine, D-α-methylphenylalanine, D-α-methylproline, D-α-methylserine, DN-methylserine, D-α-methylthreonine, D-α-methyltryptophan, D-α-methyltyrosine, D-α-metalvaline, DN- methylalanine, DN-methylarginine, DN-methylasparagine, DN-methylaspartate, DN-methylcysteine, DN-methylglutamine, DN-methylglutamate, DN-methylhistidine, DN-methylisoleucine, DN-methylleucine, DN-methyllysine, N-methylcyclohexylalanine, DN-methyl ornithine , N-methylglycine, N-methylaminoisobutyrate, N-(1-methylpropyl)glycine, N-(2-methylpropyl)glycine, DN-methyltryptophan, DN-methyltyrosine, DN-methylvaline, γ-aminobutyric acid, Lt-butylglycine, L- ethylglycine, L-homophenylalanine, L-α-methyl arginine, L-α-methylaspartate, L-α-methylcysteine, L-α-methylglutamine, L-α-methylhistidine, L-α-methylisoleucine, L-α-methylleucine, L -α-methylmethionine, L-α-methylnorvaline, L-α-methylphenylalanine, L-α-methylserine, L-α-methyltryptophan, L-α-methylvaline, N-(N-(2,2-diphenylethyl)carbamylmethylglycine, 1 -carboxy-1-(2,2-diphenylethylamino)-cyclopropane, 4-hydroxyproline, ornithine, 2-aminobenzoyl (anthraniloyl), D-cyclohexylalanine, 4-phenyl-phenylalanine, L-citrulline, α-cyclohexylglycine, L-1, 2,3,4-tetrahydroisoquinoline-3-carboxylic acid, L-thiazolidine-4-carboxylic acid, L-homotyrosine, L-2-furylalanine, L-histidine (3-methyl), N-(3-guanidinopropyl)glycine, O-methyl-L-tyrosine, O-glycan serine, metatyrosine, nor-tyrosine, LN,N',N''-trimethyllysine, homolysine, norlysine, N-glycan asparagine, 7-hydroxy-1,2,3, 4-tetrahydro-4-fluorophenylalanine, 4-methylphenylalanine, bis-(2-picolyl)amine, pentafluorophenylalanine, indoline-2-carboxylic acid, 2-aminobenzoic acid, 3-amino-2-naphthoic acid, asymmetric dimethylarginine, L-tetrahydroisoquinoline -1-carboxylic acid, D-tetrahydroisoquinoline-1-carboxylic acid, 1-aminocyclohexaneacetic acid, D/L-allylglycine, 4-aminobenzoic acid, 1-aminocyclobutanecarboxylic acid, 2- or 3- or 4-aminocyclohexanecarboxylic acid, 1-amino -1-cyclopentanecarboxylic acid, 1-aminoindan-1-carboxylic acid, 4-aminopyrrolidine-2-carboxylic acid, 2-aminotetralin-2-carboxylic acid, azetidine-3-carboxylic acid, 4-benzylpyrrolidine-2-carboxylic acid, tert -butylglycine, b-(benzothiazolyl-2-yl)alanine, b-cyclopropylalanine, 5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, (2R,4S)4-hydroxypiperidine-2-carboxylic acid, ( 2S,4S) and (2S,4R)-4-(2-naphthylmethoxy)pyrrolidine-2-carboxylic acid, (2S,4S) and (2S,4R)4-phenoxypyrrolidine-2-carboxylic acid, (2R,5S) and (2S,5R)-5-phenylpyrrolidine-2-carboxylic acid, (2S,4S)-4-amino-1-benzoylpyrrolidine-2-carboxylic acid, tert-butylalanine, (2S,5R)-5-phenylpyrrolidine-2 -carboxylic acid, 1-aminomethylcyclohexaneacetic acid, 3,5-bis-(2-amino)-ethoxybenzoic acid, 3,5-diaminobenzoic acid, 2-methylaminobenzoic acid, N-methylanthranilic acid, LN-methylalanine, LN-methylarginine, LN -methylasparagine, LN-methylaspartic acid, LN-methylcysteine, LN-methylglutamine, LN-methylglutamic acid, LN-methylhistidine, LN-methylisoleucine, LN-methyllysine, LN-methylnorleucine, LN-methylornithine, LN-methylthreonine, LN-methyltyrosine, LN -methylvaline, LN-methyl-tert-butylglycine, L-norvaline, α-methyl-γ-aminobutyrate, 4,4'-biphenylalanine, α-methylcylcopentylalanine, α-methyl-α-naphthylalanine, α-methylpenicillamine, N-(4 -aminobutyl)glycine, N-(2-aminoethyl)glycine, N-(3-aminopropyl)glycine, N-amino-α-methylbutyrate, α-naphthylalanine, N-benzylglycine, N-(2-carbamylethyl)glycine, N- (carbamylmethyl)-glycine, N-(2-carboxyethyl)glycine, N-(carboxymethyl)glycine, N-cyclobutylglycine, N-cyclodecylglycine, N-cycloheptylglycine, N-cyclohexylglycine, N-cyclodecylglycine, N-cycododecylglycine, N-cyclooctylglycine, N-cyclopropylglycine, N-cycloundecylglycine, N-(2,2-diphenylethyl)glycine, N-(3,3-diphenylpropyl)glycine, N-(3-guanidinopropyl)glycine, N-(1-hydroxyethyl)glycine, N- (hydroxyethyl))glycine, N-(imidazolylethyl))glycine, N-(3-indolylethyl)glycine, N-methyl-γ-amino butyrate, DN-methylmethionine, N-methylcyclopentylalanine, DN-methylphenylalanine, DN-methylproline, DN- methylthreonine, N-(1-methylethyl)glycine, N-methyl-naphthylalanine, N-methylpenicillamine, N-(p-hydroxyphenyl)glycine, N-(thiomethyl)glycine, penicillamine, L-α-methylalanine, L-α-methylasparagine , L-α-methyl-tert-butylglycine, L-methylethylglycine, L-α-methylglutamate, L-α-methylhomophenylalanine, N-(2-methylthioethyl)glycine, L-α-metallysine, L-α-methylnorleucine, L- α-methylornithine, L-α-methylproline, L-α-methylthreonine, L-α-methyltyrosine, LN-methyl-homophenylalanine, N-(N-(3,3-diphenylpropyl)-carbamylmethylglycine, L-pyroglutamic acid, D- pyroglutamic acid, O-methyl-L-serine, O-methyl-L-homoserine, 5-hydroxylysine, α-carboxyglutamate, phenylglycine, L-pipecolic acid (homoproline), L-homoleucine, L-lysine (dimethyl), L- 2-naphthylalanine, L-dimethyldopa or L-dimethoxyphenylalanine, L-3-pyridylalanine, L-histidine (benzoyloxymethyl), N-cycloheptylglycine, L-diphenylalanine, O-methyl-L-homotyrosine, L-β-homolysine, O-glycan -threoine, ortho-tyrosine, LN,N'-dimethyllysine, L-homoarginine, neotryptophan, 3-benzothienylalanine, isoquinoline-3-carboxylic acid, diaminopropionic acid, homocysteine, 3,4-dimethoxyphenylalanine, 4-chlorophenylalanine, L-1, 2,3,4-tetrahydronorharmonic-3-carboxylic acid, adamantylalanine, symmetrical dimethylarginine, 3-carboxythiomorpholine, D-1,2,3,4-tetrahydronorharmonic-3-carboxylic acid, 3-aminobenzoic acid, 3-amino-1- carboxymethylpyridin-2-one, 1-amino-1-cyclohexanecarboxylic acid, 2-aminocyclopentanecarboxylic acid, 1-amino-1-cyclopropanecarboxylic acid, 2-aminoindan-2-carboxylic acid, 4-aminotetrahydrothiopyran-4-carboxylic acid, azetidine-2 -carboxylic acid, b-(benzothiazol-2-yl)-alanine, neopentylglycine, 2-carboxymethylpiperidine, b-cyclobutylalanine, allylglycine, diaminopropionic acid, homocyclohexylalanine, (2S,4R)-4-hydroxypiperidine-2-carboxylic acid, octahydroindole- 2-carboxylic acid, (2S,4R) and (2S,4R)-4-hydroxypiperidine-2-carboxylic acid, octahydroindole-2-carboxylic acid, (2S,4R) and (2S,4R)-4-(2- naphthyl), pyrrolidine-2-carboxylic acid, nipecotic acid, (2S,4R) and (2S,4R)-4-(4-phenylbenzyl)pyrrolidine-2-carboxylic acid, (3S)-1-pyrrolidine-3-carboxylic acid acid, (2S,4S)-4-tritylmercaptopyrrolidine-2-carboxylic acid, (2S,4S)-4-mercaptoproline, tert-butylglycine, N,N-bis(3-aminopropyl)glycine, 1-aminocyclohexane-1-carboxylic acid acid, N-mercaptoethylglycine and selenocysteine. In some embodiments, amino acid residues may be charged or polar. Charged amino acids include alanine, lysine, aspartic acid or glutamic acid or non-natural analogues thereof. Polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine or tryptophan, or their non-naturally occurring analogues. In particular, it is contemplated that in some embodiments, the terminal amino group on an amino acid may be an amido group or a carbamate group.

As used herein, the term “percentage (%) identity” refers to the percentage of amino acid residues of a candidate sequence, e.g., Fc-IgG or fragment thereof, that are identical to the amino acid residues of a control sequence after sequence alignment and introduction of gaps, if necessary, to achieve maximum percent identity (i.e., gaps may be introduced into one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment to determine percent identity can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, the percentage of amino acid sequence identity of a given candidate sequence to a given reference sequence, with or against a given reference sequence (which may otherwise be paraphrased as a given candidate sequence that has or includes a certain percentage of amino acid sequence identity to a given reference sequence, with given reference sequence or against a given reference sequence) is calculated as follows:

100 × (A/B share)

where A is the number of amino acid residues judged to be 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. In some embodiments, where the length of the candidate sequence is not equal to the length of the reference sequence, the percentage amino acid sequence identity of the reference sequence candidate will not be equal to the percentage amino acid sequence identity of the reference sequence to the candidate sequence.

Two polynucleotide or polypeptide sequences are said to be “identical” if the nucleotide or amino acid sequence in the two sequences is the same when aligned for maximum matching as described above. Comparisons between two sequences are typically made by comparing the sequences in a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein refers to a region 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 the sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences have been optimally aligned.

The term “treating” or “treating” as used herein refers to therapeutically treating a viral infection (eg, a viral infection such as influenza infection) in a subject. In some embodiments, the therapeutic treatment can slow the progression of a viral infection, improve a patient's outcome, and/or eliminate the infection. In some embodiments, therapeutic treatment of a viral infection in a subject may promote or alleviate one or more symptoms or conditions associated with the viral infection, reduce the spread of the virus, stabilize (i.e., not worsen) the viral infection, prevent the spread of the viral infection, and/ or delay or slow the progression of a viral infection compared to the state and/or state of a viral infection in the absence of therapeutic treatment.

The term “average T value” as used herein refers to the average number of neuraminidase inhibitor monomers or neuraminidase inhibitor dimers conjugated to the Fc domain or albumin protein in a group of conjugates. In some embodiments, in a group of conjugates, the average number of neuraminidase inhibitor monomers or neuraminidase inhibitor dimers conjugated to an Fc domain monomer may be from 1 to 20 (e.g., the average T value is 1-2, 1-3, 1-4, 1 -5, 5-10, 10-15 or 15-20). In some embodiments, the average T value is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

As used herein, the term "subject" may be a human, non-human primate, or other mammal such as, but not limited to, dog, cat, horse, cow, pig, turkey, goat, fish, monkey, chicken, rat, mouse and sheep.

The term “therapeutically effective amount” as used herein refers to an amount, e.g., a pharmaceutical dose, effective to induce a desired effect in a subject or treat a subject having a condition or disorder described herein (e.g., a viral infection, e.g., influenza infection ). As used herein, it should also be understood that a "therapeutically effective amount" may be interpreted as an amount providing the desired therapeutic and/or prophylactic effect, taken in one or more doses or in any dose or by any route, and/or taken alone or in combination with other therapeutic agents (eg, an antiviral agent described herein). For example, in the context of administering a conjugate described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or ( M'-I)), which is used to treat a viral infection, an effective amount of the conjugate is, for example, an amount sufficient to prevent, slow or stop the progression of the viral infection compared to the response obtained without administration of the conjugate.

As used herein, the term “pharmaceutical composition” refers to a drug or pharmaceutical composition that contains at least one active ingredient (for example, a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D' -I), (M-I)-(M-X) or (M'-I)), as well as one or more excipients and diluents that make the active ingredient suitable for the route of administration. The pharmaceutical composition of the present invention includes pharmaceutically acceptable components that are compatible with the conjugate described herein (for example, a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), ( M-I)-(M-X) or (M'-I)).

As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. For example, a pharmaceutically acceptable carrier may be a carrier capable of suspending or dissolving the active conjugate (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X) or (M-I)-(M-VI) ). The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not harmful to the recipient. In the present disclosure, a pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to the conjugate described herein. The nature of the carrier varies depending on the route of administration. For example, for oral administration, a solid carrier is preferred; for intravenous administration, an aqueous solution is usually used as a carrier (eg, WFI and/or buffered solution).

The term "pharmaceutically acceptable salt" as used herein represents salts of the conjugates described herein (e.g., conjugates of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I)), which, to the best of medical judgment, are suitable for use in the methods described herein without undue toxicity, irritation and/or allergic reaction. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Pharmaceutical Salts: Properties, Selection, and Use (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the conjugates described herein document, or separately, by reacting the free base group with a suitable organic acid.

The term “drug/antibody ratio” or “DAR” refers to the average number of small molecule drug moieties (e.g., the average number of small molecule drug monomers or dimers) conjugated to an antibody, Fc domain, or albumin protein described herein. In some embodiments described herein, DAR is represented by the letter "T" (for example, in formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X ) or (M'-I)). In this context, each monomeric fragment (eg, each zanamivir or peramivir monomer) or each dimeric fragment (eg, each zanamivir dimer or peramivir dimer) conjugated to an Fc domain, antibody, or albumin protein corresponds to a DAR value of 1.0 ( for example, a "T" value of 1.0). For example, an Fc domain conjugated to 4 zanamivir monomers would have a DAR value of 4.0 (eg, a "T" value of 4.0). An Fc domain conjugated to 4 zanamivir dimers (eg, a total of 8 zanamivir molecules) would also have a DAR value of 4.0 (eg, a "T" value of 4.0). The DAR value can also be calculated as the average DAR value for a group of molecules, such as a group of Fc domains. antibodies or albumin proteins. DAR values can affect the efficacy, specific activity, pharmacokinetics, or toxicity of a drug.

The term "about" as used herein indicates a deviation within ±5%. For example, about 10% means 9.5% to 10.5%.

Any values represented as a range of values include the upper and lower bounds, as well as any values contained within the upper and lower bounds.

The term "(1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I)" as used herein represents formulas according to any from (1), (2), (3), (4), (5), (D-I), (D-II), (D-II-1), (D-II-2), (D- II-3), (D-II-4), (D-II-5), (D-II-6), (D-II-7), (D-II-8), (D-II- 9), (D-II-10), (D-III), (D-III-1), (D-III-2), (D-III-3), (D-III-4), ( D-III-5), (D-III-6), (D-III-7), (D-III-8), (D-III-9), (D-IV), (D-IV- 1), (D-IV-2), (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-V-10), (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-VI- 9), (D-VII), (D-VIII), (D-VIII-1), (D-VIII-2), (D-VIII-3), (D-VIII-4), (D- VIII-5), (D-VIII-6), (D-VIII-7), (D-VIII-8), (D-VIII-9), (D-VIII-10), (D-VIII- 11), (D-GC), (D-IX-1), (D-IX-2), (D-IX-3), (D-IX-4), (D-IX-5), ( D-IX-6), (D-X), (D-X-1), (D-X-2), (D-X-3), (D'-I), (M-I), (M-P), (M-II -1), (M-II-2), (M-II-3), (M-II-4), (M-II-5), (M-II-6), (M-II-7 ), (M-II-8), (M-II-9), (M-II-10), (M-III), (M-III-1), (M-III-2), (M -III-3), (M-III-4), (M-III-5), (M-III-6), (M-III-7), (M-III-8), (M-III -9), (M-IV), (M-IV-1), (M-IV-2), (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-V-10), (M-VI), (M-VI -1), (M-VI-2), (M-VI-3), (M-VI-4), (M-VI-5), (M-VI-6), (M-VI-7 ), (M-VI-8), (M-VI-9), (M-VII), (M-VIII), (M-VIII-1), (M-VIII-2), (M-VIII -3), (M-VIII-4), (M-VIII-5), (M-VIII-6), (M-VIII-7), (M-VIII-8), (M-VIII-9 ), (M-VIII-10), (M-VIII-11), (M-IX), (M-IX-1), (M-IX-2), (M-GC-3), (M -GC-4), (M-IX-5), (M-IX-6), (M-X), (M-X-1), (M-X-2), (M-X-3) or (M'-I) ).

Other features and advantages of the conjugates described herein will be apparent from the following detailed description and claims.

Description of drawings

FIG. 1 is an image depicting exemplary methods of conjugating a neuraminidase inhibitor monomer or dimer, for example, via a linker, to an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or a protein albumin binding peptide.

FIG. 2 shows SDS-PAGE under non-reducing and reducing conditions, and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence shown in SEQ ID NO: 1.

FIG. 3 shows SDS-PAGE under non-reducing and reducing conditions, and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence shown in SEQ ID NO: 3.

FIG. 4 shows SDS-PAGE under non-reducing and reducing conditions and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence shown in SEQ ID NO: 5.

FIG. 5 shows SDS-PAGE under non-reducing and reducing conditions and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence shown in SEQ ID NO: 7.

FIG. 6 shows SDS-PAGE under non-reducing and reducing conditions, and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence shown in SEQ ID NO: 9.

FIG. 7 shows SDS-PAGE under non-reducing and reducing conditions and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence shown in SEQ ID NO: 12.

FIG. 8 shows SDS-PAGE under non-reducing and reducing conditions, and a schematic illustration of an Fc domain formed from Fc domain monomers having the sequence shown in SEQ ID NO: 14.

FIG. 9 shows SDS-PAGE under non-reducing conditions of conjugate 1.

FIG. 10 shows SDS-PAGE under non-reducing conditions of conjugate 2.

FIG. 11 shows SDS-PAGE under non-reducing conditions of conjugate 3.

FIG. 12 shows SDS-PAGE under non-reducing conditions of conjugate 4.

FIG. 13 shows SDS-PAGE under non-reducing conditions of conjugate 5.

FIG. 14 shows SDS-PAGE under non-reducing conditions of conjugate 6.

FIG. 15 is a graph showing IC50 values from the H1N1 neuraminidase inhibition assay for intermediate 2 and conjugate 1.

FIG. 16 is a graph showing IC50 values from the H1N1 neuraminidase inhibition assay for conjugates 1-6.

FIG. 17 is a graph showing IC50 values from the H3N2 neuraminidase inhibition assay for conjugates 1-6.

FIG. 18A-18C are a series of graphs showing the cellular capacity of A549 cells treated with conjugate 3 (FIG. 18A), conjugate 4 (FIG. 18B), or conjugate 6 (FIG. 18C).

FIG. 19A-19E are a series of graphs showing the ability of conjugate 3 to inhibit the growth of pathogenic influenza virus strains A/WSN/33 H1N1 (FIG. 19A), A/Wyoming/3/03 H3N2 (FIG. 19B), A/California/04/ 09 H1N1 pdm (FIG. 19C), A/Vietnam/1203/04 H5N1 HALo (FIG. 19D) or B/Lee/40 Victoria (FIG. 19E) in human epithelial cells.

FIG. 20A-20E are a series of graphs showing the ability of Conjugate 4 to inhibit the growth of pathogenic influenza virus strains A/WSN/33 H1N1 (FIG. 20A), A/Wyoming/3/03 H3N2 (FIG. 20B), A/California/04/ 09 H1N1 pdm (FIG. 20C), A/Vietnam/1203/04 H5N1 HALo (FIG. 20D) or B/Lee/40 Victoria (FIG. 20E) in human epithelial cells.

FIG. 21A-21E are a series of graphs showing the ability of conjugate 6 to inhibit the growth of pathogenic influenza virus strains A/WSN/33 H1N1 (FIG. 21A), A/Wyoming/3/03 H3N2 (FIG. 21B), A/California/04/ 09 H1N1 pdm (FIG. 21C), A/Vietnam/1203/04 H5N1 HALo (FIG. 21D) or B/Lee/40 Victoria (FIG. 21E) in human epithelial cells.

FIG. 22A-22E are a series of graphs showing the ability of conjugate 6 to inhibit the growth of pathogenic influenza virus strains A/WSN/33 H1N1 (FIG. 22A), A/Wyoming/3/03 H3N2 (FIG. 22B), A/California/04/ 09 H1N1 pdm (FIG. 22C), B/Lee/40 Victoria (FIG. 22D), or A/Vietnam/1203/04 (FIG. 22E) in human epithelial cells compared to oseltamivir.

FIG. 23 is a graph showing the relative serum concentration of conjugate 6 in mice compared to Fc (hIgG1) alone.

FIG. 24 is a graph showing the effect of conjugate 6 on mouse weight in a lethal murine influenza model. The study was carried out as described in example 29.

FIG. 25 is a graph showing the effect of conjugate 6 on survival in a lethal murine influenza model. The study was carried out as described in example 29.

FIG. 26 is a graph showing the effect of conjugate 6 on mouse weight in a lethal mouse influenza model. The study was carried out as described in example 30.

FIG. 27 is a graph showing the effect of conjugate 6 on survival in a lethal mouse influenza model. The study was carried out as described in example 30.

FIG. 28 is an image showing a method of conjugating a neuraminidase inhibitor monomer or dimer, for example via a linker, to an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide by conjugating an oxime to an amino acid residue, for example , with a nitrogen atom exposed on the surface of lysine.

FIG. 29 is an image showing a method of conjugating a neuraminidase inhibitor monomer or dimer, for example via a linker, to an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide by conjugating a thioester to an amino acid residue, e.g. , with a nitrogen atom exposed on the surface of lysine.

FIG. 30 is a view showing a method of conjugating a neuraminidase inhibitor monomer or dimer, for example via a linker, to an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide by reconjugating a cysteine, for example by reconjugating conjugation of a cysteine to a pair of sulfur atoms of two hinge cysteines in an Fc domain monomer or Fc domain.

FIG. 31A-31F are a series of graphs showing the survival of mice treated with conjugate 6 in a lethal murine influenza model. Mice were treated with either oseltamivir (Tamiflu™) control, 20 mg/kg, twice daily, starting 8 hours postinfection (FIG. 31A); conjugate 6, 50 mg/kg, 1 dose 28 days before infection (FIG. 31B); conjugate 6, 10 mg/kg, 1 dose 28 days before infection (FIG. 31C); conjugate 6.5 mg/kg, 1 dose 28 days before infection (FIG. 31D); conjugate 6, 2.5 mg/kg, 1 dose 28 days before infection (FIG. 31E); or conjugate 6, 1.25 mg/kg, 1 dose 28 days before infection (FIG. 31F). This study was performed as described in Example 33.

FIG. 32A-32F are a series of graphs showing that Conjugate 6 extends the treatment window compared to oseltamivir (Tamiflu™) as measured by survival in a lethal murine influenza model. Mice were treated with either vehicle (PBS), Fc alone at 10 mg/kg body weight, or conjugate 6, 4 hours before infection (FIG. 32A); conjugate 6 or oseltamivir (Tamiflu™) 8 hours postinfection (FIG. 32B); conjugate 6 or oseltamivir (Tamiflu™) 24 hours postinfection (FIG. 32C); conjugate 6 or oseltamivir (Tamiflu™) 48 hours postinfection (FIG. 32D); conjugate 6 or oseltamivir (Tamiflu™) 72 hours postinfection (FIG. 32E); or conjugate 6 or oseltamivir (Tamiflu™) 96 hours postinfection (FIG. 32F). The study was carried out as described in example 34.

FIG. 33 is a graph showing the lack of significant effect of body weight gain following administration of Conjugate 6 in a 14-day dose-ranging toxicity study in rats. The study was carried out as described in example 35.

FIG. 34A-34D are a series of graphs showing that Conjugate 6 extends the treatment window compared to oseltamivir (Tamiflu™) as measured by survival in a lethal murine influenza model. Mice were treated with either oseltamivir (Tamiflu™) control, 20 mg/kg, twice daily, starting 8 hours postinfection (FIG. 34A); conjugate 6 at a dose of 10 mg/kg, 1 dose 4 hours before infection (FIG. 34B); conjugate 6 at a dose of 2 mg/kg, 1 dose 4 hours before infection (FIG. 34C); or conjugate 6 at a dose of 0.4 mg/kg, 1 dose 4 days before infection (FIG. 34D). The study was carried out as described in example 37.

FIG. 35 is a graph showing the effect of conjugate 6 on mouse weight in a lethal murine influenza model. The study was carried out as described in example 37.

FIG. 36 is a graph showing a 7-day pharmacokinetic study in rats following IV administration of Conjugate 6. The study was performed as described in Example 38.

FIG. 37 is a graph showing a 14-day pharmacokinetic study in rats following IV administration of Conjugate 6. The study was performed as described in Example 39.

FIG. 38 is a graph showing a 28-day pharmacokinetic study in rats comparing IV and SC (subcutaneous) administration of Conjugate 6. The study was performed as described in Example 40.

FIG. 39 is a graph showing a 28-day pharmacokinetic study in non-human primates following IV administration of Conjugate 6. The study was conducted as described in Example 41.

FIG. 40 is a graph showing a pharmacokinetic pulmonary distribution study in mice following IV administration of conjugate 6. The study was performed as described in Example 42.

FIG. 41 is a graph showing a 5-day pharmacokinetic study in mice comparing IV, SC and IM (intramuscular) administration of Conjugate 6. The study was conducted as described in Example 43.

FIG. 42 shows SDS-PAGE under non-reducing conditions of conjugate 8. FIG. 43 shows the structure of conjugate 6.

FIG. 44 is a graph showing a 24 hour in vitro stability study in mouse plasma comparing conjugate 6 incubated at 37° C. for 24 hours with control and pure compound.

FIG. 45 is a graph showing a 24 hour in vitro stability study in human plasma comparing conjugate 6 incubated at 37° C. for 24 hours with control and pure compound.

FIG. 46 is a graph showing a 24-hour mouse liver microsomal stability study comparing Conjugate 6 incubated at 37° C. for 24 hours in mouse liver microsomal cells and heat-killed mouse liver microsomal cells as a control.

FIG. 47 is a graph showing a 24-hour human liver microsomal stability study comparing Conjugate 6 incubated at 37° C. for 24 hours in human liver microsomal cells and heat-killed liver microsomal cells as a control.

FIG. 48 is a graph showing the % change in body weight in mice over 15 days after virus infection. The study was carried out as described in example 66.

FIG. 49 is a graph showing the % survival rate in mice for 15 days after infection with the virus. The study was carried out as described in example 66.

FIG. 50 is a graph showing the binding of conjugate 6 and conjugate 12 compared to hIgG1 Fc (WT) and hIgG1 Fc (N297A) to Fcγ receptor IIIA. The study was carried out as described in example 67.

FIG. 51 is a graph showing the binding of conjugate 6 and Fcγ IIIA receptor conjugate. The study was carried out as described in example 67.

FIG. 52 is a graph showing a 7-day toxicokinetic study in non-human primates following IV administration of Conjugate 6. The study was conducted as described in Example 68.

FIG. 53 is a graph showing a 28-day pharmacokinetic study in non-human primates comparing IV and SC administration of conjugate 6. The study was conducted as described in Example 68.

FIG. 54 shows SDS-PAGE under non-reducing conditions of conjugate 12.

FIG. 55 shows SDS-PAGE under non-reducing conditions of conjugate 13 having different drug-antibody ratios (DAR) (conjugate 13a, conjugate 13b, conjugate 13c, conjugate 13d, conjugate 13e, conjugate 13f and conjugate 13g).

FIG. 56 is a graph showing the % survival of immunocompromised mice 35 days after infection with the virus. The conjugate 6 0.3 mg/kg treatment group maintained 100% survival, but is slightly offset in the graph for clarity. The study was carried out as described in example 95.

FIG. 57 is a graph showing the % change in body weight in immunocompromised mice over 35 days after virus infection. The study was carried out as described in example 95.

FIG. 58 is a graph showing that administration of conjugate 6 results in dose-dependent viral clearance in the lungs in a mouse model infected with influenza A (H1N1), and that this viral clearance is greater than PBS control, Fc control alone, or oseltamivir quality of control. This study was performed as described in Example 96.

FIG. 59 is a graph showing that administration of conjugate 6 results in a dose-dependent reduction in inflammatory cytokines in the lungs in an influenza A (H1N1)-infected mouse model. This study was performed as described in Example 97.

FIG. 60 is a graph showing the pharmacokinetics of conjugate 6 in BALB/c SCID (immunocompromised) mice and CD-1 (immunocompetent) mice. This study was performed as described in Example 98.

FIG. 61 is an image showing conjugate 33.

FIG. 62A-62B are graphs showing the change in body weight (%) in BALB/c mice intranasally infected with a 3-fold dose of LD95 adapted murine influenza virus A.PR/8/1934 (H1N1). This study was performed as described in Example 133.

FIG. 63A is a graph showing viral load on day 4 postinfection. This study was performed as described in Example 133.

FIG. 63B is a graph showing the logarithmic decline in viral load on day 4 postinfection. This study was performed as described in Example 133.

FIG. 64A is a graph showing that administration of conjugate 33 results in a dose-dependent reduction in viral load in an influenza A (H1N1)-infected mouse model compared to PBS control or Fc control alone. This study was performed as described in Example 133.

FIG. 64B is a graph showing the logarithmic decline in viral load on day 4 postinfection. This study was performed as described in Example 133.

FIG. 65A-65E are a series of histograms showing that administration of conjugate 33 results in a dose-dependent fold reduction in cytokine levels for TNF-α (FIG. 65A), IL-6 (FIG. 65B), INF-γ (FIG. 65C), MCP-1 (FIG. 65D) and MIP-1α (FIG. 65E). This study was performed as described in Example 133.

FIG. 66 is a chromatograph showing the stability of intermediate 80 compared to intermediate 4 on day 7 of incubation at 37°C and 60°C. This study was performed as described in Example 142.

FIG. 67 is a graph showing serial passage of A/CA/09 pdm in the presence of conjugate 6, oseltamivir, baloxavir, or PBS as a control in A549 cells to evaluate the possibility of drug-resistant mutant viral strains developing under selectivity pressure with viral inhibitors. This study was performed as described in Example 147.

FIG. 68 is a graph showing sequential passage of A/WSN/1933 in the presence of conjugate 6, conjugate 33, oseltamivir, baloxavir, or PBS as a control in MDCK cells to evaluate the potential for the development of drug-resistant mutant viral strains under selective pressure using viral inhibitors . This study was performed as described in Example 147.

Detailed Description of the Invention

The disclosure provides conjugates, compositions, and methods for treating viral infections (eg, infections caused by influenza viruses). Conjugates disclosed herein include monomers or dimers of viral neuraminidase inhibitors (eg, zanamivir, peramivir, or analogs thereof) conjugated to Fc monomers, Fc domains, Fc binding peptides, albumin proteins, or protein albumin binding peptides. A neuraminidase inhibitor (eg, zanamivir, peramivir, or their analogues) in conjugates targets neuraminidase on the surface of the viral particle. Fc monomers or Fc domains in conjugates bind to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells, such as neutrophils, to activate phagocytosis and effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) ), which leads to the absorption and destruction of viral particles by immune cells and further enhances the antiviral activity of the conjugates. Albumin or albumin-binding peptide can increase the half-life of the conjugate, for example, by binding albumin to the recycling neonatal Fc receptor. Such compositions are useful in methods of inhibiting the growth of viruses and in methods of treating viral infections such as infections caused by influenza A virus, influenza B virus and influenza C virus.

I. Viral infections

The compounds and pharmaceutical compositions described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M '-I)) may be used to treat a viral infection (eg, an infection caused by an influenza virus such as influenza A, B, C or parainfluenza virus).

Viral infection refers to the pathogenic growth of a virus (eg, influenza virus) in a host (eg, a human subject). A viral infection can be any situation in which the presence of a viral population(s) causes harm to the host. Thus, a subject suffers from a viral infection when an excessive amount of the viral population is present in or on the subject's body, or when the presence of the viral population(s) damages cells or other tissue of the subject.

Influenza, commonly known as acute respiratory viral infection, is an infectious disease caused by the influenza virus. Symptoms can range from mild to severe. The most common symptoms include: fever, runny nose, sore throat, muscle pain, headache, cough and feeling tired. These symptoms usually begin two days after exposure to the virus and last less than a week. However, the cough may last more than two weeks. Nausea and vomiting may occur in children, but are less common in adults. Complications of the flu may include viral pneumonia, secondary bacterial pneumonia, sinus infections, and worsening of previous health problems such as asthma or heart failure. Serious complications may occur in subjects with weakened immune systems, such as the young, the old, people with diseases that weaken the immune system, and those undergoing therapeutic treatments that weaken the immune system.

Three types of influenza viruses affect people, namely type A, type B and type C. The virus is usually spread through the air by coughing or sneezing. This is believed to occur mainly over relatively short distances. It can also be spread by touching surfaces contaminated with the virus and then touching your mouth or eyes. A person can infect others both before and during symptoms. Infection can be confirmed by virus testing in the throat, sputum, or nose. A number of rapid tests are available; however, people may have an infection if the results are negative. To diagnose an influenza virus infection, a type of polymerase chain reaction that detects the RNA of the virus can be used.

II. Conjugates by opening

Provided herein are synthetic conjugates useful for treating viral infections (eg, infections caused by influenza virus). The conjugates described herein include an Fc domain or albumin protein conjugated to one or more monomers of neuraminidase inhibitors or one or more dimers of two neuraminidase inhibitors (e.g., neuraminidase inhibitors selected from zanamivir, sulfosanamivir, peramivir, A-315675, or analogues thereof) . Dimers of two neuraminidase inhibitors include a neuraminidase inhibitor (for example, the first neuraminidase inhibitor of formula (A-I), (A-II), (A-III), (A-IV), (A-V), (A-VI), (A-VII ), (A-VIII), (A-IX), (A-X), (A-XI) or (A-XII)) and a second neuraminidase inhibitor (e.g., a second neuraminidase inhibitor of formula (A-I), (A- II), (A-III), (A-IV), (A-V), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X), ( A-XI) or (A-XII)). The first and second neuraminidase inhibitors are linked to each other via a linker.

Without being limited by theory, in some aspects, the conjugates described herein bind to the surface of a viral particle (eg, bind to the viral enzyme neuraminidase on the surface of an influenza virus particle) through interactions between neuraminidase inhibitor moieties in the conjugates and proteins on the surface of the viral particle. A neuraminidase inhibitor disrupts neuraminidase, an envelope glycoprotein that cleaves sialic acids, the terminal neuraminic acid residues, from glycan structures on the surface of infected host cells, releasing progeny viruses and allowing the virus to spread from the host cell to uninfected surrounding cells.

Conjugates of the invention include neuraminidase inhibitor monomers and dimers conjugated to an Fc domain, Fc monomer or Fc binding peptide. The Fc domain in the conjugates described herein binds to FcγRs (eg, FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb) on immune cells. Binding of the Fc domain in the FcγR conjugates described herein on immune cells activates phagocytosis and effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), resulting in uptake and destruction of viral particles by immune cells and further enhancing the antiviral activity of the conjugates.

The conjugates of the invention include neuraminidase inhibitor monomers and dimers conjugated to albumin protein or albumin binding peptide. The albumin protein or albumin-binding peptide protein can increase the half-life of the conjugate, for example, by binding albumin to the recirculating neonatal Fc receptor.

The conjugates provided herein are described by any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I). In some embodiments, the conjugates described herein include one or more neuraminidase inhibitor monomers conjugated to an Fc domain or albumin protein. In some embodiments, the conjugates described herein include one or more dimers of neuraminidase inhibitors conjugated to an Fc domain or albumin protein. In some embodiments, when n is 2, E (Fc domain monomer) dimerizes to form an Fc domain.

The conjugates described herein can be synthesized using chemical synthesis methods available in the art. In cases where the functional group is not available for conjugation, the molecule can be derivatized using conventional chemical synthesis methods well known in the art. In some embodiments, the conjugates described herein contain one or more chiral centers. Conjugates include each of the isolated stereoisomeric forms, as well as mixtures of stereoisomers with varying degrees of chiral purity, including racemic mixtures. Also covered are the various diastereomers, enantiomers and tautomers that may be formed.

Neuraminidase inhibitors

A component of the conjugates described herein is a fragment of an influenza virus neuraminidase inhibitor. The influenza virus neuraminidase inhibitor destroys neuraminidase, an envelope glycoprotein that cleaves sialic acids, i.e. terminal neuraminic acid residues, from glycan structures on the surface of infected host cells, releasing progeny viruses and allowing virus spread from the host cell to uninfected surrounding cells. Examples of influenza virus neuraminidase inhibitor include zanamivir (Relenza), sulfosanamivir, A-315675 and peramivir. In addition, derivatives of zanamivir, sulfosanamivir, A-315675 and peramivir, such as those described in the literature, have neuraminidase inhibitor activity and are useful as neuraminidase inhibitory moieties of the compounds described herein (see, for example, Hadházi et al A sulfozanamivir analogue has potent anti-influenza virus activity. ChemMedChem Comm. 13: 785-789 (2018) and In vitro characterization of A-315675, a highly potent inhibitor of A and B strain of influenze virus neuraminidases and influenza virus replication. Antimicrobial Agents and Chemotherapy 46(4): 1014-1021 (2002)).

The conjugates described herein are divided into two types: (1) one or more neuraminidase inhibitor dimers conjugated to an Fc domain or albumin protein, and (2) one or more neuraminidase inhibitor monomers conjugated to an Fc domain or albumin protein. The neuraminidase inhibitor dimers are linked to each other via a linker, such as the linkers described herein.

The viral neuraminidase inhibitors of the invention include zanamivir, sulfosanamivir, A-315675, peramavir and analogs thereof, such as the viral neuraminidase inhibitors of formulas (A-I) to (A-XII):

where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from

R ₆ selected from

R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl. Most preferably, a viral neuraminidase inhibitor of the formula (AI), (A-II), (A-III), (A-IV), (AV), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X), (A-XI) or (A-XII) is covalently attached to the conjugate through Y.

Preferably, the viral neuraminidase inhibitor is selected from zanamivir, sulfosanamivir, peramivir or A-315675:

Conjugates of neuraminidase inhibitor dimers bound to the Fc domain or albumin protein

The conjugates described herein include an Fc domain and an Fc monomer, an Fc binding peptide and an albumin protein, or an albumin binding peptide protein covalently linked to one or more neuraminidase inhibitor dimers. The dimers of the two neuraminidase inhibitors include a first neuraminidase inhibitor (eg, a first neuraminidase inhibitor of viruses of formulas (AI)-(A-XII)) and a second neuraminidase inhibitor (eg, a second neuraminidase inhibitor of viruses of formulas (AI)-(A-XII)). The first and second neuraminidase inhibitors are linked to each other via a linker, such as the linker described herein. In some embodiments of neuraminidase inhibitor dimers, the first and second neuraminidase inhibitors are the same. In some embodiments, the first and second neuraminidase inhibitors are different.

Neuraminidase inhibitor dimers include homodimers of zanamivir or analogues thereof (eg, (AI), (A-II), (A-VI), (A-VII), (A-VIII) or (A-IX)). For example, neuraminidase inhibitor dimers of the invention include dimers having the structure A ₁ -LA ₂ , wherein each Ai and each A2 is selected from (AI), (A-II), (A-VI), (A-VII), (A -VIII) and (A-IX).

Neuraminidase inhibitor dimers include homodimers of peramivir or its analogues (eg, (A-III), (A-IV) or (AV)). For example, neuraminidase inhibitor dimers of the invention include dimers having the structure A ₁ -LA ₂ , where each A ₁ and each A ₂ is selected from (A-III), (A-IV) and (AV).

Neuraminidase inhibitor dimers include heterodimers including zanamivir or analogs thereof, and peramivir or analogs thereof (eg, (AI)-(A-IX)). For example, neuraminidase inhibitor dimers of the invention include dimers having the structure A ₁ -LA ₂ , wherein each A ₁ is selected from (AI), (A-II), (A-VI), (A-VII), (A-VIII ) and (A-IX), and each A ₂ is selected from (A-III), (A-IV) and (AV).

In some embodiments, 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, 17, 18, 19, or 20), each A ₁ -LA ₂ may be independently selected (eg, independently selected from any of the A ₁ -LA ₂ structures described herein). In some embodiments, E may be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different A ₁ -LA ₂ moieties. In some embodiments, E is conjugated to a first A ₁ -LA ₂ moiety and a second A ₁ -LA ₂ moiety. In some embodiments, A ₁ and A ₂ of the first fragment A ₁ -LA ₂ are independently selected from any of formulas (A-III)-(AV):

and A ₁ and A ₂ of the second fragment A ₁ -LA ₂ are independently selected from any of formulas (AI), (A-II), (A-VI), (A-VII), (A-VIII) or (A- IX):

In some embodiments, the first A ₁ -LA ₂ fragment is specifically conjugated to lysine E residues (e.g., the nitrogen atoms of surface exposed lysine E residues), and the second A ₁ -LA ₂ fragment is specifically conjugated to cysteine E residues (e.g., the sulfur atoms of surface exposed lysine E residues). on the surface of cysteine residues E). In some embodiments, the first A ₁ -LA ₂ fragment is specifically conjugated to E cysteine residues (e.g., the sulfur atoms of surface-exposed cysteine E residues), and the second A ₁ -LA ₂ fragment is specifically conjugated to E lysine residues (e.g., the nitrogen atoms of surface-exposed cysteine E residues). on the surface of lysine E residues).

In some embodiments, the disclosure provides a conjugate or a pharmaceutically acceptable salt thereof described by the formulas below.

or a pharmaceutically acceptable salt thereof.

In the conjugates described herein, a wavy 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) neuraminidase inhibitor dimers can be attached to an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or a protein albumin binding peptide. In some embodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) neuraminidase inhibitor dimers may be attached to the Fc domain monomer, Fc- domain, Fc-binding peptide, albumin protein or albumin-binding peptide. In some embodiments, when n is 2, 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) neuraminidase inhibitor dimers can be attached to the Fc domain. The wavy line in the conjugates described herein should not be construed as a single bond between one or more neuraminidase inhibitor dimers and an atom in the Fc domain or albumin protein. In some embodiments, when T is 1, one dimer of neuraminidase inhibitors can be attached to an atom in an Fc domain monomer, Fc domain, Fc binding peptide, albumin protein, or albumin binding peptide. In some embodiments, when T is 2, two neuraminidase inhibitor dimers can be attached to an atom in an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide.

As described below, the linker in a conjugate described herein (eg, L or L') may be a branched structure. As described later herein, the linker in a conjugate described herein (eg, L or L') may be a multivalent structure, eg, a divalent or trivalent structure having two or three groups, respectively. In some embodiments, when the linker has three groups, two groups can be attached to the first and second neuraminidase inhibitors, and the third group can be attached to an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin protein. binding peptide.

In conjugates having an Fc domain covalently linked to one or more neuraminidase inhibitor dimers as represented by the formulas above, when n is 2, two Fc domain monomers (each Fc domain monomer represented by an E) dimerize to form an Fc domain.

Conjugates of neuraminidase inhibitor monomers bound to the Fc domain or albumin protein

In some embodiments, the conjugates described herein include an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide protein covalently linked to one or more neuraminidase inhibitor monomers. Conjugates of an Fc domain or albumin protein monomer and one or more neuraminidase inhibitor monomers can be formed by linking the Fc domain or albumin protein to each of the neuraminidase inhibitor monomers through a linker, such as any of the linkers described herein.

In conjugates containing an Fc domain or albumin protein covalently linked to one or more neuraminidase inhibitor monomers described herein, a wavy 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) neuraminidase inhibitor monomers can be attached to an Fc domain monomer, Fc domain, Fc- binding peptide, protein albumin, or protein albumin-binding peptide. In some embodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) neuraminidase inhibitor monomers may be attached to an Fc domain monomer or protein albumin. In some embodiments, when n is 2, 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) neuraminidase inhibitor monomers can be attached to the Fc domain. The wavy line in the conjugates described herein should not be construed as a single bond between one or more neuraminidase inhibitor monomers and an atom in an Fc domain monomer, Fc domain, Fc binding peptide, albumin protein, or protein albumin binding peptide. In some embodiments, when T is 1, one neuraminidase inhibitor monomer may be attached to an atom in an Fc domain monomer, Fc domain, Fc binding peptide, albumin protein, or albumin binding peptide. In some embodiments, when T is 2, two neuraminidase inhibitor monomers can be attached to an atom in an Fc domain monomer, Fc domain, Fc binding peptide, albumin protein, or albumin binding peptide.

In some embodiments, 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, 17, 18, 19, or 20), each A ₁ -L may be independently selected (eg, independently selected from any of the A ₁ -L structures described herein). In some embodiments, E may be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different A ₁ -L moieties. In some embodiments, E is conjugated to a first A ₁ -L moiety and a second A ₁ -L moiety. In some embodiments, A ₁ of the first fragment A ₁ -L is selected from any of formulas (A-III)-(AV):

and A ₁ of the second fragment A ₁ -L is selected from any of formulas (AI), (A-II), (A-VI), (A-VII), (A-VIII) or (A-IX):

In some embodiments, the first A ₁ -L moiety is specifically conjugated to lysine residues in E (e.g., the nitrogen atoms exposed on the surface of the lysine residues in E), and the second A ₁ -L moiety is specifically conjugated to the surface exposed cysteine residues in E (e.g. , sulfur atoms exposed on the surface of cysteine residues E). In some embodiments, the first A ₁ -L moiety is specifically conjugated to cysteine residues in E (e.g., the sulfur atoms of surface-exposed cysteine residues in E), and the second A ₁ -L moiety is specifically conjugated to lysine residues in E (e.g., nitrogen atoms surface-exposed lysine residues in E).

As described later herein, a linker in a conjugate having an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide protein covalently linked to one or more monomers of the neuraminidase inhibitors described herein ( for example, L or L') may be a divalent structure having two groups. One group of the divalent linker may be attached to the neuraminidase inhibitor monomer, and the other group may be attached to the Fc domain monomer, Fc domain, Fc binding peptide, albumin protein, or protein albumin binding peptide.

In some embodiments, a conjugate comprising an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide protein covalently linked to one or more neuraminidase inhibitor monomers provided herein is described by any of the following formulas :

or a pharmaceutically acceptable salt thereof.

In conjugates having an Fc domain covalently linked to one or more neuraminidase inhibitor monomers as represented by the formulas above, when n is 2, two Fc domain monomers (each Fc domain monomer represented by an E) dimerize to form an Fc domain.

Regioisomers of conjugates, including zanamivir or its analogues

Conjugates (eg, monomer or dimer conjugates, as described in detail herein) can be obtained as a mixture or regioisomers. A particular regioisomer or mixture of regioisomers may be preferred for reasons such as ease of synthesis, thermal stability, oxidative stability, pharmacokinetics (eg, metabolic stability or bioavailability), effector binding, or therapeutic efficacy.

In some embodiments, the conjugate of the invention includes zanamivir or an analogue thereof (e.g., any of (AI), (A-II), (A-VI), (A-VII), (A-VIII), (A-IX) or (AH)). Zanamivir or an analogue thereof may be conjugated to the Fc domain or albumin protein (eg via a linker), for example via the C7 position (see, for example, (AI), (A-II) or (AX)) or via the C9 position (see, for example, (A-VI) or (A-VII)):

The present disclosure includes a group of monomeric conjugates (eg, a group of conjugates of formula (M-I)), wherein the group of conjugates includes any of the monomeric conjugates described herein and one or more corresponding regioisomers thereof. For example, a group of conjugates may include (1) zanamivir or an analogue thereof conjugated (eg, via a linker) at position C7 to the Fc domain or albumin protein, and (2) zanamivir or an analogue thereof, conjugated (eg, via a linker) at position C9 with Fc domain or albumin protein.

The population of monomeric conjugates may have a certain ratio of C7-linked conjugate to C9-linked conjugate. For example, a group of conjugates may contain essentially 100% C-7-linked conjugate and essentially 0% C-9-linked conjugate. The population of conjugates may contain about 95% C-7-linked conjugate and about 5% C-9-linked conjugate. The population of conjugates may contain about 90% C-7-linked conjugate and about 10% C-9-linked conjugate. The population of conjugates may contain about 85% C-7-linked conjugate and about 15% C-9-linked conjugate. The population of conjugates may contain about 80% C-7-linked conjugate and about 20% C-9-linked conjugate. The population of conjugates may contain about 75% C-7-linked conjugate and about 25% C-9-linked conjugate. The population of conjugates may contain about 70% C-7-linked conjugate and about 30% C-9-linked conjugate. The population of conjugates may contain about 65% C-7-linked conjugate and about 35% C-9-linked conjugate. The population of conjugates may contain about 60% C-7-linked conjugate and about 40% C-9-linked conjugate. The population of conjugates may contain about 55% C-7-linked conjugate and about 45% C-9-linked conjugate. The population of conjugates may contain about 50% C-7-linked conjugate and about 50% C-9-linked conjugate. The population of conjugates may contain about 45% C-7-linked conjugate and about 55% C-9-linked conjugate. The population of conjugates may contain about 40% C-7-linked conjugate and about 60% C-9-linked conjugate. The population of conjugates may contain about 35% C-7-linked conjugate and about 65% C-9-linked conjugate. The population of conjugates may contain about 30% C-7-linked conjugate and about 70% C-9-linked conjugate. The population of conjugates may contain about 25% C-7-linked conjugate and about 75% C-9-linked conjugate. The population of conjugates may contain about 20% C-7-linked conjugate and about 80% C-9-linked conjugate. The conjugate population may contain about 15% C-7-linked conjugate and about 85% C-9-linked conjugate. The population of conjugates may contain about 10% C-7-linked conjugate and about 90% C-9-linked conjugate. The conjugate population may contain essentially 0% C-7-linked conjugate and essentially 100% C-9-linked conjugate.

The conjugate population may contain more than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 60%, 65%, 60%, 55%, or 50% C7-linked conjugate.

The conjugate population may contain less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% C9-linked conjugate.

The present disclosure also includes a group of dimeric conjugates population (eg, a population of group of conjugates of formula (D-I)), wherein the group of conjugates population includes any of the dimeric conjugates described herein and one or more corresponding regioisomers thereof. For example, a population of group conjugates may include (1) a C7-C7 dimer (eg, zanamivir or analogue dimer fragments are conjugated (eg, via a linker) at their respective C7 positions to the Fc domain or albumin protein), (2) a C9- dimer C9 (e.g., fragments of a zanamivir dimer or an analog thereof are conjugated (e.g., via a linker) at their respective C9 positions to the Fc domain or albumin protein), and/or (3) a C7-C7 dimer (e.g., one fragment of zanamivir or an analog thereof conjugated (eg, via a linker) to the Fc domain or albumin protein through its C7 position, and another fragment of zanamivir or an analogue thereof is conjugated (eg, via a linker) to the Fc domain or albumin protein via its C9 position).

The population of dimeric conjugates may have a specific ratio of C7-C7 linked conjugate to C7-C9 linked conjugate and C9-C9 linked conjugate. For example, a group of conjugates population may contain essentially 100% C7-C7 linked conjugate and essentially 0% C7-C9 or C9-C9 linked conjugate. The conjugate population may contain essentially 100% C9-C9 coupled conjugate and essentially 0% C7-C7 or C7-C9 coupled conjugate. The conjugate population may contain substantially 100% C7-C9 coupled conjugate and substantially 0% C7-C7 or C9-C9 coupled conjugate.

The conjugate population may contain more than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 60%, 65%, 60%, 55%, or 50% C7-C7-linked conjugate.

The conjugate population may contain less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% C9-C9 conjugate.

The conjugate population may contain less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% C7-C9 conjugate.

For any of the populations described above, the A ₁ and/or A ₂ regioisomer groups (e.g., (MI) or (DI)) may be selected from zanamivir or any of the zanamivir analogues described herein (e.g., any of (AI), (A-II), (A-VI), (A-VII), (A-VIII), (A-IX) or (AX)). In particular, C7-linked zanamivir or analogs thereof are described by (AI), (A-II) and (A-X), and C9-linked zanamivir or analogs thereof are described by (A-VI) or (A-VII). Typical methods for preparing regioisomers, for example, regioisomers connected through C7, C9, C7-C7, C7-C9 and C9-C9, are described in Examples 100-103, 123 and 124. In some cases, it may be preferable to have 95% or more 96% or more, 97% or more, 98% or more, 99% or more, or substantially 100% C7-linked monomer conjugates or C7-C7 linked dimer conjugates. In these cases, it may be preferable to prepare the intermediate by a process that produces substantially C7-linked monomeric or C7-C7-linked dimeric intermediates, such as those described, for example, in Examples 103 and 123. The method in Example 103 serves as an illustration of the methods used primarily to prepare a C7- or C7-C7-linked intermediate and can be used to prepare any intermediate described herein.

In preferred embodiments, the conjugate is a conjugate of any one of the formulas (DI)-(DX), (D'-I), (MI)-(MX) or (M'-I), wherein A ₁ and/or A ₂ described by the formula (AI), (A-II) or (A-X), and Y is where R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl. In preferred embodiments, A ₁ and/or A ₂ are described by formula (AI) (eg, zanamivir). In preferred embodiments, R ₇ is (C ₁ -C ₂₀ )alkyl (eg, -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₃ ). Such conjugates have been shown to exhibit increased stability of the C7 junction, resulting in less migration from C7 to C9. Therefore, the resulting product is expected to be more homogeneous and exhibit increased efficiency.

III. Fc domain monomers and Fc domains

The Fc domain monomer includes a hinge domain, an antibody C _H 2 constant domain, and an antibody C _H 3 constant domain. The Fc domain monomer may be an immunoglobulin antibody of the IgG, IgE, IgM, IgA or IgD isotype. The Fc domain monomer can also be of any immunoglobulin antibody isotype (eg, IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain monomer can be any allotype of immunoglobulin antibody (for example, 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, IGHG3*08, IGHG3*13, IGHG3*03, IGHG3*14, IGHG3*15, IGHG3*16 , IGHG3*17, IGHG3*18, IGHG3*19, IGHG2*04, IGHG4*01, IGHG4*03 or IGHG4*02) (as described, for example, in Vidarsson et al. IgG subclasses and allotypes: from structure to effector function Frontiers in Immunology 5(520):1-17 (2014). The Fc domain monomer can also be of any kind, for example human, rat or mouse. The dimer of Fc domain monomers is an Fc domain that can bind to the Fc receptor, which is a receptor located on the surface of leukocytes.

In some embodiments, the Fc domain monomer in the conjugates described herein may contain one or more amino acid substitutions, additions and/or deletions with respect to the Fc domain monomer having the sequence set forth in any of SEQ ID NO: 1-68. In some embodiments, the Asn in the Fc domain monomer in conjugates as described herein can be replaced with Ala to prevent N-linked glycosylation (see, e.g., SEQ ID NOs: 12-15, where the replacement of Asn with Ala marked *). In some embodiments, the Fc domain monomer in the conjugates described herein may also contain additional Cys additions (see, for example, SEQ ID NOs: 9, 10 and 11, where Cys additions are marked *).

In some embodiments, the Fc domain monomer in conjugates as described herein includes an additional moiety, e.g., an albumin binding peptide, a scavenging peptide (e.g., hexahistidine peptide (SEQ ID NO: 72)) or a signal sequence (e.g. , the IL2 signal sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 73)) appended to the N- or C-terminus of the Fc domain monomer. In some embodiments, the Fc domain monomer in the conjugate does not contain any type of antibody variable region, such as V _H , V _L , complementarity determining region (CDR) or hypervariable region (HVR).

In some embodiments, the Fc domain monomer in 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 of the SEQ IDs NO: 1-68 shown below. In some embodiments, the Fc domain monomer in conjugates as described herein may have the sequence of any of SEQ ID NO: 1-68 shown below.

SEQ ID NO: 1: mouse Fc-IgG2a with IL2 signal sequence at N-terminus (bold)

SEQ ID NO: 2: Mature Mouse Fc-IgG2a

SEQ ID NO: 3: Human Fc-IgG1 with the IL2 signal sequence at the N-terminus (bold) and added N-terminal MVRS amino acid residues (underlined).

SEQ ID NO: 4: Mature human Fc-IgG1 with N-terminal MVRS amino acid residues added (underlined)

SEQ ID NO: 5: Mouse Fc-IgG2a with IL2 signal sequence (bold) at N-terminus and hexahistidine peptide (italics) at C-terminus

SEQ ID NO: 6: Mature mouse Fc-IgG2a with hexahistidine peptide (in italics) at the C-terminus

SEQ ID NO: 7: Human Fc-IgG1 with IL2 signal sequence (bold) at N-terminus, added N-terminal amino acid residues MVRS (underlined) and hexahistidine peptide (italics) at C-terminus

SEQ ID NO: 8: Mature human Fc-IgG1 with hexahistidine peptide (italicized) at the C-terminus and added N-terminal MVRS amino acid residues (underlined)

SEQ ID NO: 9: Human Fc-IgG1 with the IL2 signal sequence (bold) at the N-terminus, added N-terminal MVRS amino acid residues (underlined), two additional cysteines in the hinge region (*) and a hexahistidine peptide (italics) at the C-terminus

SEQ ID NO: 10: mature human Fc-IgG1 with added N-terminal MVRS amino acid residues (underlined), two additional cysteines in the hinge region (*) and a hexahistidine peptide (italicized) at the C-terminus

SEQ ID NO: 11: mature human Fc-IgG1 with added N-terminal MVRS amino acid residues (underlined) and two additional cysteines in the hinge region (*)

SEQ ID NO: 12: murine Fc-IgG2a with IL2 signal sequence (bold) at N-terminus, Asn to Ala substitution (*) and hexahistidine peptide (in italics) at C-terminus

SEQ ID NO: 13: Mature murine Fc-IgG2a with Asn to Ala (*) substitution and hexahistidine peptide (in italics) at the C-terminus

SEQ ID NO: 14: human Fc-IgG1 with IL2 signal sequence (bold) at the N-terminus, added N-terminal MVRS amino acid residues (underlined), substitution of Asn with Ala (*) and hexahistidine peptide (italics) with C -end

SEQ ID NO: 15: mature human Fc-IgG1 with Asn replaced by Ala (*), added N-terminal MVRS amino acid residues (underlined) and hexahistidine peptide (italicized) at the C-terminus

SEQ ID NO: 16: Human IgG1 Fc with human serum albumin signal sequence (bold) at the N-terminus and added N-terminal amino acid residues ISAMVRS (underlined)

SEQ ID NO: 17: Human IgG1 Fc with a human serum albumin signal sequence (bold) at the N-terminus, added N-terminal amino acid residues ISAMVRS (underlined), a C-terminal G4S linker (in italics), and a C-terminal c tag -Mus (underlined, italicized)

SEQ ID NO: 18: Mature human IgG1 Fc with added N-terminal amino acid residues ISAMVRS (underlined), C-terminal G4S linker (italics) and C-terminal c-Myc tag (underlined, italics)

SEQ ID NO: 19: Human IgG1 Fc with the human serum albumin signal sequence (bold), added N-terminal amino acid residues ISAMVRS (underlined) and modification of lysine to serine (*) to prevent lysine conjugation at this site

SEQ ID NO: 20: Mature human IgG1 Fc with added N-terminal amino acid residues ISAMVRS (underlined) and modification of lysine to serine (*) to prevent lysine conjugation at this site

SEQ ID NO: 21: Human IgG1 Fc with a human serum albumin signal sequence (bold) at the N-terminus, added N-terminal amino acid residues ISAMVRS (underlined), modification of lysine to serine (*) to prevent lysine conjugation at this site, C-terminal G4S linker (in italics) and C-terminal C-Myc tag (underlined, in italics)

SEQ ID NO: 22: Mature human IgG1 Fc with added N-terminal amino acid residues ISAMVRS (underlined), modification of lysine to serine (*) to prevent lysine conjugation at this site, C-terminal G4S linker (in italics) and C-terminal labeled S-Mus (underlined, italicized)

SEQ ID NO: 23: Human IgG1 Fc with human serum albumin signal sequence (bold) at N-terminus, added N-terminal amino acid residues ISAMVRS (underlined), substitution of Asn for Ala (*), C-terminal G4S linker (highlighted italic) and C-terminal tag C-myc (underlined, italicized)

SEQ ID NO: 24: Mature human IgG1 Fc with added N-terminal amino acid residues ISAMVRS (underlined), Asn to Ala substitution (*), C-terminal G4S linker (italics) and C-terminal C-myc tag (underlined). in italics)

SEQ ID NO: 25: Human IgG1 Fc with the human serum albumin signal sequence (bold) at the N-terminus, added N-terminal amino acid residues ISAMVRS (underlined), mutations H310A (*) and H435A (*) interfering with FcRn binding, C-terminal G4S (in italics) and C-terminal C-myc tag (underlined, in italics)

SEQ ID NO: 26: Mature human IgG1 Fc with the human serum albumin signal sequence (bold) at the N-terminus, added N-terminal amino acid residues ISAMVRS (underlined), with binding-interfering mutations H310A (*) and H435A (*) FcRn, C-terminal G4S (in italics) and C-terminal C-myc tag (underlined, in italics)

SEQ ID NO: 27: Human IgG1 Fc with the human serum albumin signal sequence (bold) at the N-terminus, added N-terminal amino acid residues ISAMVRS (underlined), C-terminal G4S linker (italics) and mutated at the C-terminus (lysine to phenylalanine, bold) labeled C-myc (underlined, italicized)

SEQ ID NO: 28: Mature human IgG1 Fc with added N-terminal amino acid residues ISAMVRS (underlined), C-terminal G4S linker (italics) and C-terminal mutated (lysine to phenylalanine, bold) C-myc tag (underlined , in italics)

SEQ ID NO: 29: Human IgG1 Fc with human serum albumin signal sequence (bold) at N-terminus, added N-terminal amino acid residues ISAMVRS (underlined), Asn replaced with Ala (*), C-terminal G4S linker (italics ) and C-terminal mutated (lysine to phenylalanine, bold) tag C-myc (underlined, italicized)

SEQ ID NO: 30: Mature human IgG1 Fc with added N-terminal amino acid residues MVRS (underlined), Asn to Ala substitution (*), C-terminal G4S linker (italics) and C-terminal mutated (lysine to phenylalanine, bold font) with the label C-myc (underlined, italicized)

SEQ ID NO: 31: Human IgG1 Fc with human serum albumin signal sequence (bold) at N-terminus, G1m(fa) allotype (bold italic), C-terminal G4S linker (italics) and C-terminal mutated (lysine for phenylalanine, bold) with the label C-myc (underlined)

SEQ ID NO: 32: Human IgG1 Fc with human serum albumin signal sequence (bold) at N-terminus, G1m(fa) allotype (bold italic)

SEQ ID NO: 33: Fc of mature human IgG1 triple mutation YTE (bold and underlined) with added N-terminal amino acid residues MVRS (underlined)

SEQ ID NO: 34: Human IgG1 Fc with human serum albumin signal sequence (bold) at N-terminus, contains EPKSS residues containing full hinge region at N-terminus of mature human IgG1 Fc (underlined), replacement of Cys with Ser (# ), allotype G1m(fa) (bold italics)

SEQ ID NO: 35: Human IgG1 Fc with the mouse IgG signal sequence (bold) at the N-terminus, removing the EPKSSD hinge residues from the N-terminus of the mature human IgG1 Fc, allotype G1m(fa) (bold italics)

SEQ ID NO: 36: mature human IgG1 Fc triple mutation YTE (bold and underline), removing EPKSSD hinge residues from the N-terminus of mature human IgG1 Fc, allotype G1m(fa) (bold italic)

SEQ ID NO: 37: Mature human IgG1 Fc with double mutation LS (bold and underline), removing EPKSSD hinge residues from the N-terminus of mature human IgG1 Fc, allotype G1m(fa) (bold italics)

SEQ ID NO: 38: Mature human IgG1 Fc with human serum albumin signal sequence (bold) at N-terminus, triple YTE mutation (bold and underline), G1m(fa) allotype (bold italic), C-terminal G4S linker (italics) and C-terminal tag C-myc (underlined)

SEQ ID NO: 39: Mature human IgG1 Fc, wherein X ₁ is Met or Trp, X ₂ is Ser or Thr, X ₃ is Thr or Glu, X ₄ is Asp or Glu, and X ₅ is Leu or Met, X ₆ is Met or Leu, and X ₇ is Asn or Ser

SEQ ID NO: 40: Mature human IgG1 Fc, where X ₄ is Asp or Glu, and X ₅ is Leu or Met

SEQ ID NO: 41: YTE (bold and underline) mature human IgG1 triple mutation Fc where X ₄ is Asp or Glu and X ₅ is Leu or Met

SEQ ID NO: 42: Fc of mature human IgG1 triple mutation YTE (bold and underline), allotype G1m(fa) (bold italic)

SEQ ID NO: 43: Fc of mature human IgG1 triple mutation YTE (bold and underline), G1m(f) allotype (bold italic)

SEQ ID NO: 44: Fc of mature human IgG1 double mutation LS (bold and underline), where X ₄ is Asp or Glu, and X ₅ is Leu or Met

SEQ ID NO: 45: Fc of mature human IgG1 double mutation LS (bold and underline), allotype G1m(fa) (bold italic)

SEQ ID NO: 46: Fc of mature human IgG1 double mutation LS (bold and underline), allotype G1m(f) (bold italic)

SEQ ID NO: 47: Mature human IgG1 Fc with the mouse heavy chain MIgG Vh signal sequence (bold), replacing Cys with Ser (#), and wherein X ₁ is Met or Trp, X ₂ is Ser or Thr, X ₃ is Thr or Glu, X ₄ is Asp or Glu, and X ₅ is Leu or Met, X ₆ is Met or Leu, and X ₇ is Asn or Ser

SEQ ID NO: 48: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), Cys to Ser substitution (#), G1m(fa) allotype (bold italic)

SEQ ID NO: 49: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), Cys to Ser substitution (#), G1m allotype (f) (bold italics)

SEQ ID NO: 50: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), Cys to Ser substitution (#), mutations M428L, N434S (bold/underline), G1m(fa) allotype (bold italic )

SEQ ID NO: 51: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), Cys to Ser substitution (#), mutations M428L, N434S (bold/underline), G1m(f) allotype (bold italic )

SEQ ID NO: 52: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), Cys to Ser substitution (#), YTE triple mutation (bold and underline), G1m(fa) allotype (bold italic)

SEQ ID NO: 53: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), Cys to Ser substitution (#), YTE triple mutation (bold and underline), G1m(f) allotype (bold italic)

SEQ ID NO: 54: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), added N-terminal amino acid residues ISAMVRS (italics), mutations M428L, N434S (bold/underlined), G4S linker (highlighted) italic) and C-terminal tag C-myc (underlined), allotype G1m(f) (bold italic)

SEQ ID NO: 55: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), added N-terminal amino acid residues ISAMVRS (italics), mutations M428L, N434S (bold/underlined), G4S linker (highlighted) italics), C-terminal tag C-myc (underlined), allotype G1m(fa) (bold italics)

SEQ ID NO: 56: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), added N-terminal amino acid residues ISAMVRS (italics), triple mutant YTE (bold/underline), G4S linker (italics) ) and C-terminal tag C-myc (underlined), allotype G1m(f) (bold italics)

SEQ ID NO: 57: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), added N-terminal amino acid residues ISAMVRS (italics), YTE triple mutant (bold/underline), G4S linker (italics) ), C-terminal tag C-myc (underlined), allotype G1m(fa) (bold italics)

SEQ ID NO: 58: Mature human IgG1 with the murine MIgG1 heavy chain signal sequence (bold), Cys to Ser substitution (#), C-terminal G4S (italics) and C-terminal IgA peptide (underlined), G1m allotype( fa) (bold italics)

SEQ ID NO: 59: Mature human IgG1 with murine MIgGI heavy chain signal sequence (bold), Cys to Ser (#), mutations M428L, N434S (bold/underline), C-terminal G4S (italics) and C- terminal IgA peptide (underlining), allotype G1m(fa) (bold italics)

SEQ ID NO: 60: Mature human IgG1 Fc, Z ₁ is Cys or Ser, and where X ₁ is Met or Trp, X ₂ is Ser or Thr, X ₃ is Thr or Glu, X ₄ is Asp or Glu, and X ₅ is Leu or Met, X ₆ is Met or Leu, and X ₇ is Asn or

SEQ ID NO: 61: Mature human IgG1 Fc, replacing Cys with Ser (#), and wherein X ₁ is Met or Trp, X ₂ is Ser or Thr, X ₃ is Thr or Glu, X ₄ is Asp or Glu, and X ₅ is Leu or Met, X ₆ is Met or Leu, and X ₇ is Asn or Ser

SEQ ID NO: 62: Mature human IgG1 Fc, Cys to Ser (#), X ₄ is Asp or Glu, and X ₅ is Leu or Met

SEQ ID NO: 63: Mature human IgG1 Fc, Cys to Ser substitution (#), G1m(f) allotype (bold italics)

SEQ ID NO: 64: Mature human IgG1 Fc, Cys to Ser substitution (#), G1m(fa) allotype (bold italics)

SEQ ID NO: 65: Mature human IgG1 Fc, Cys to Ser (#), mutations M428L, N434S (bold/underline), allotype G1m(fa) (bold italic)

SEQ ID NO: 66: Mature human IgG1 Fc, Cys to Ser (#), mutations M428L, N434S (bold/underline), G1m(f) allotype (bold italic)

SEQ ID NO: 67: Mature human IgG1 Fc, Cys to Ser (#), YTE triple mutation (bold and underline), G1m(fa) allotype (bold italic)

SEQ ID NO: 68: Mature human IgG1 Fc, Cys to Ser (#), YTE triple mutation (bold and underline), G1m(f) allotype (bold italic)

As defined herein, an Fc domain includes two Fc domain monomers that dimerize through interactions between the antibody C _H 3 constant domains, as well as one or more disulfide bonds that form between the hinge domains of the two dimerizing Fc domain monomers. The Fc domain forms a minimal structure that binds to an Fc receptor, e.g., Fc gamma receptors (i.e., Fcγ receptors (FcγR)), Fc alpha receptors (i.e., Fcα receptors (FcαR)), Fc -epsilon receptors (ie Fcε receptors (FcεR)) and/or neonatal Fc receptor (FcRn). In some embodiments, the Fc domain of the present invention binds to an Fcγ receptor (e.g., FcRn, FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b)) and/or FcγRIV, and/or neonatal Fc receptor (FcRn).

In some embodiments, an Fc domain monomer or Fc domain of the present invention is an aglycosylated Fc domain monomer or Fc domain (e.g., an Fc domain monomer or Fc domain that supports interaction with an Fc receptor (e.g., FcRn) For example, an Fc domain is an aglycosylated variant of IgG1 that supports interaction with 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 producing aglycosylated Fc domains are known in this area, for example, as described in Sazinsky S. L. et al., Aglycosylated immunoglobulin Gl variants productively engage activating Fc receptors, PNAS, 2008, 105(51):20167-20172, which is incorporated herein in its entirety.

In some embodiments, the Fc domain or Fc domain monomer of the invention is designed to enhance binding to the neonatal Fc receptor (FcRn). For example, the Fc domain may include a triple mutation corresponding to M252Y/S254T/T256E (YTE) (e.g., IgG1, such as human or humanized IgG1 having a YTE mutation, e.g., 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). The Fc domain may include a double mutant corresponding to M428L/N434S (LS) (e.g., IgG1, such as 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 a single mutant corresponding to N434H (eg, IgG1, such as human or humanized IgG1 having the N434H mutation). The Fc domain may include a single mutant corresponding to C220S (e.g., IgG1, such as human or humanized IgG1 having the C220S mutation, such as SEQ ID NOs: 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, SEQ ID NO: 68). The Fc domain may include a combination of one or more of the mutations described above that enhance binding to FcRn. Enhanced binding to FcRn may increase the half-life of the Fc domain-containing conjugate. For example, the inclusion of one or more amino acid mutations that increase binding to FcRn (e.g., YTE mutation, LS mutation, or N434H mutation) can increase the half-life of the conjugate by 5%, 10%, 15%, 20%, 30%, 40% , 50%, 60%, 70%, 80%, 90%. 100%, 200%, 300%, 400%, 500% or more relative to a conjugate having the corresponding Fc domain without a mutation that enhances binding to FcRn. Exemplary Fc domains with enhanced FcRN binding and methods for producing Fc domains having enhanced FcRN binding 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. As used herein, it should be understood that an amino acid “corresponding to” a particular amino acid residue (e.g., a particular SEQ ID NO.) includes any amino acid residue that would be understood by one of ordinary skill in the art to align with the particular residue (e.g. specific sequence). For example, any of SEQ ID NO: 1-68 may be mutated to include a YTE mutation, an LS mutation, and/or an N434H mutation by mutating “corresponding residues” of the amino acid sequence.

As used herein, it should be understood that the sulfur atom “corresponding to” a particular cysteine residue of a particular SEQ ID NO. includes the sulfur atom of any cysteine residue that one of ordinary skill in the art would understand to align with a particular cysteine of a particular sequence. Protein sequence alignment of human IgG1 (UniProtKB: P01857; SEQ ID NO: 94), human IgG2 (UniProtKB: P01859; SEQ ID NO: 95), human IgG3 (UniProtKB: P01860; SEQ ID NO: 96) and human IgG4 (UniProtKB: P01861; SEQ ID NO: 97) is shown below (aligned using Clustal Omega Multiple Pairwise Alignment). Alignment indicates cysteine residues (e.g., sulfur atoms of cysteine residues) that “match” each other (in boxes and indicated by •). One skilled in the art will readily be able to perform such an alignment with any IgG variant of the invention to identify a cysteine sulfur atom that corresponds to any of the specific cysteine sulfur atoms of the particular SEQ ID NO. described herein (e.g., any of SEQ ID NO: 1- 68). For example, one skilled in the art will readily be able to determine that Cys10 of SEQ ID NO: 10 (the first cysteine of the conserved CPRS motif of the Fc domain hinge region) 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.

In some embodiments, the Fc domain or Fc domain monomer of the invention has the sequence set forth in any of SEQ ID NOs: 39-68, may further include additional amino acids at the N-terminus (Xaa)x and/or additional amino acids at the C-terminus. end (Xaa)z, where Xaa is any amino acid, and x and z are integers equal to zero or greater, typically less than 100, preferably less than 10, and more preferably equal to 0, 1, 2, 3, 4 or 5. In some embodiments, the additional amino acids are at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to one or more consecutive amino acids presented in SEQ ID NO: 94. For example, the additional amino acids may be one auxiliary amino acid at the C-terminus corresponding to Lys330 of IgG1 (SEQ ID NO: 94).

As used herein, 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 ordinary skill in the art would understand to align with a particular lysine of a particular sequence. Protein sequence alignment of human IgG1 (UniProtKB: P01857; SEQ ID NO: 94), human IgG2 (UniProtKB: P01859; SEQ ID NO: 95), human IgG3 (UniProtKB: P01860; SEQ ID NO: 96) and human IgG4 (UniProtKB: P01861; SEQ ID NO: 97) is shown below (aligned using Clustal Omega Multiple Pairwise Alignment). The alignment indicates the lysine residues (e.g., the nitrogen atoms of lysine residues) that “match” each other (in boxes and indicated by *). One of ordinary skill in the art will readily be able to perform such an alignment with any IgG variant of the invention to identify a lysine nitrogen atom that corresponds to any of the particular lysine nitrogen atoms of the particular SEQ ID NO described herein (e.g., any of SEQ ID NO: 1-68 ). For example, one skilled in the art will readily be able to determine that 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 from SEQ ID NO: 3 or Lys30 from SEQ ID NO: 10.

Protein sequence alignment of IgG1 (SEQ ID NO: 94), IgG2 (SEQ ED NO: 95), IgG3 (SEQ ED NO: 96) and IgG4 (SEQ ID NO: 97)

Activation of immune cells

Fc gamma receptors (FcγRs) bind the Fc portion of immunoglobulin G (JgG) and play an important role in the activation and regulation of the immune system. For example, IgG Fc domains in immune complexes (ICs) interact with high-avidity FcγRs, thereby triggering signaling cascades that regulate immune cell activation. The human FcγR family contains several activating receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb) and one inhibitory receptor (FcγRIIb). FcγR signaling is mediated by intracellular domains that contain immune tyrosine-based activating motifs (ITAMs) for FcγR activation and immune tyrosine-based inhibitory motifs (ITIMs) for the inhibitory receptor FcγRIIb. In some embodiments, binding of FcγRs by Fc domains results in ITAM phosphorylation by Src family kinases; this activates Syk family kinases and induces downstream signaling networks that include the PI3K and Ras pathways.

In the conjugates described herein, the portion of the conjugates comprising neuraminidase inhibitor monomers or dimers binds to and inhibits viral neuraminidase, resulting in inhibition of viral replication, while the Fc domain portion of the conjugates binds to FcγR (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb) on immune cells and activate phagocytosis and effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), leading to the uptake and destruction of viral particles by immune cells and further enhancing the antiviral activity of the conjugates. Examples of immune cells that can 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.

IV. Albumin proteins and albumin binding peptides

Albumin proteins

The 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 subdomains, and fusion proteins. The albumin protein may include an albumin protein sequence obtained from any source. Preferably the source is a mammal, such as a human or cattle. Most preferably, the albumin protein is human serum albumin (HSA) or a variant thereof. Human serum albumins include any albumin protein having the amino acid sequence naturally occurring in humans, and variants thereof. The albumin protein coding sequence can be obtained using methods known to those skilled in the art to isolate and sequence cDNA corresponding to human genes. The albumin protein of the invention may include the amino acid sequence of human serum albumin (HSA) set forth in SEQ ID NO: 69 or SEQ ID NO: 70, or the amino acid sequence of mouse serum albumin (MSA) set forth in SEQ. ID NO: 71, or a variant or fragment thereof, preferably a functional variant or fragment thereof. The fragment or variant may or may not be functional, or may retain albumin function to some extent. For example, the 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 capacity of the original albumin (eg, the original albumin from which the fragment or variant is derived). Relative binding capacity can be determined by methods known in the art, such as surface plasmon resonance.

The albumin protein may be a naturally occurring polymorphic variant of the albumin protein, such as human serum albumin. Typically, human serum albumin variants or fragments will be at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, or 70%, and preferably 80%, 90%, 95 %. %, 100% or 105% or more of the ligand binding activity of human serum albumin or murine serum albumin.

The albumin protein may comprise the amino acid sequence of bovine serum albumin. Bovine serum albumin proteins include any albumin having an amino acid sequence naturally occurring in bovines, for example, as described under Swissprot accession number P02769, and variants thereof, as defined herein. Bovine serum albumin proteins also include fragments of full-length bovine serum albumin or variants thereof, as defined herein.

The albumin protein may contain an albumin sequence derived from one of canine (e.g., Swissprot accession number P49822-1), pig (e.g., Swissprot accession number P08835-1), goat (e.g., Sigma product number A2514 or A4164), cat serum albumin (e.g. Swissprot accession number P49064-1), chicken (e.g. Swissprot accession number P19121-1), ovalbumin (e.g. chicken ovalbumin) (e.g. Swissprot accession number P01012-1), turkey ovalbumin (e.g. Swissprot accession number 073 860-1), 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. International Journal for Parasitology 37(11): 1201-1208 (2007)), 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 P4 9065-1), rat (e.g. Swissprot accession number P02770 -1) or sheep (for example, Swissprot accession number P14639-1), and includes variants and fragments thereof, as defined herein.

Many naturally occurring mutant forms of albumin are known to those skilled in the art. Naturally occurring mutant forms of albumin are described, for example, in Peters, et al. All About Albumin: Biochemistry, Genetics and Medical Applications, Academic Press, Inc., San Diego, Calif., p.170-181 (1996).

The albumin proteins of the invention include variants of naturally occurring albumin proteins. An albumin variant 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, whether conservative or non-conservative, provided that such changes result in an albumin protein for which at least one basic the property was not significantly changed (for example, was not changed by more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%). Exemplary properties that may determine the activity of an albumin protein include binding activity (eg, including binding specificity or affinity for bilirubin or a fatty acid such as a long-chain fatty acid), osmolarity, or behavior over a particular pH range.

Typically, the 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%, according to 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 of SEQ ID NOs: 69-71.

Methods for the preparation and purification of recombinant human albumin are well known (Sleep et al. Biotechnology, 8(1):42-6 (1990)) and include the preparation of recombinant human albumin for pharmaceutical use (Bosse et al. J Clin Pharmacol 45(1): 57-67 (2005)). The three-dimensional structure of HSA was revealed using 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 that form 17 disulfide bonds, and one unpaired (ie, free) cysteine at position 34 of the mature protein. Cys-34 HSA has been used to conjugate 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: 69 (Human Serum Albumin (HSA) Option 1)

SEQ ID NO: 70 (Human Serum Albumin (HSA) Option 2)

KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTK

VPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLV

RRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA

VEKCCKADDKETCFAEEGKKLVAASQAALG

SEQ ID NO: 71 (Mouse Serum Albumin (MSA))

RGVFRREAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFA KTCVADESAANCDK

SLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEA MCTS FKENPTTF

MGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKAL VSSVRQRMKCSSM

QKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAE

LAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEV

CKNYAEAKDVFLGTFLYEYSRRHPDYSV

SLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGE YGFQNAILVRYTQK

APQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSE HVTKCCSGSLVER

RPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAE QLKTVMDDFAQFL

DTCCKAADKDTCFSTEGPNLVTRCKDALA

Conjugation of albumin proteins

The albumin protein of the invention may be conjugated (eg, by a covalent bond) to any compound of the invention (eg, by a linker moiety of a neuraminidase inhibitor monomer or dimer). The albumin protein can be conjugated to any compound of the invention by any method well known to those skilled in the art to produce small molecule-protein conjugates. This may involve covalent conjugation to a solvent-exposed amino acid, such as solvent-exposed cysteine or lysine. For example, human serum albumin can be conjugated to a compound of the invention by covalently bonding to a sulfur atom corresponding to Cys34 of SEQ ID NO: 69 or Cys40 of SEQ ID NO: 70.

The albumin protein of the invention can be conjugated to any compound of the invention by an amino acid located within 10 amino acid residues of the C-terminal or N-terminal end of the albumin protein. The albumin protein may comprise a C-terminal or N-terminal fusion polypeptide of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more amino acids. The C-terminal or N-terminal fusion polypeptide may include one or more solvent-exposed cysteine or lysine residues that can be used for covalent conjugation of a compound of the invention (eg, conjugation to a neuraminidase inhibitor monomer or dimer, including through a linker).

The albumin proteins of the invention include any albumin protein that has been engineered to include one or more solvent-exposed cysteine or lysine residues that may provide a site for conjugation to a compound of the invention (e.g., conjugation to a neuraminidase inhibitor monomer or dimer, in including via a linker). Most preferably, the albumin protein contains a single solvent-exposed cysteine or lysine, thereby providing site-specific conjugation of the compound of the invention.

Exemplary methods for producing engineered variant albumin proteins that include one or more conjugation-friendly cysteine residues are presented in US Patent Application No. 2017/0081389, which is incorporated herein by reference in its entirety. Briefly, preferred albumin protein variants are those that contain a single, solvent-exposed, unpaired (eg, free) cysteine residue, thereby allowing site-specific conjugation of the linker to the cysteine residue.

Albumin proteins that have been engineered to provide 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 for a cysteine in 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 from SEQ ID NO: 69;

(b) an albumin protein having a cysteine insertion at a position adjacent to the N- or C-terminal side of 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 from SEQ ID NO: 69;

(c) an albumin protein designed to contain an unpaired cysteine having a free thiol group at a residue corresponding to any of C369, C361, C91, C177, C567, C316, C75, C169, C124, or C558 of SEQ ID NO: 69, and which may or may not be formed by removing or replacing the residue corresponding to C360, C316, C75, C168, C558, C361, C91, C124, C169 or C567 in SEQ ID NO: 69; and/or

(d) adding a cysteine to the N- or C-terminus of the albumin protein.

In some embodiments, the net result of substitution, deletion, addition, or insertion events (a), (b), (c), and/or (d) is that the number of conjugable cysteine residues of the polypeptide sequence increases relative to the original sequence albumin. In some embodiments, the net result of substitution, deletion, addition, or insertion events (a), (b), (c), and/or (d) is that the number of conjugable cysteine residues of the polypeptide sequence is one, allowing site-specific conjugation.

Preferred albumin protein variants also include albumin proteins having a single solvent-exposed lysine residue, thereby allowing site-specific conjugation of the linker to the lysine residue. Such variants can be created by engineering the albumin protein, including any of the methods described previously (eg, insertion, deletion, substitution, or C-terminal or N-terminal fusion).

Protein albumin-binding peptides

Conjugation of a bioactive compound to a protein albumin-binding peptide may alter the pharmacodynamics of the bioactive compound, including changes in tissue uptake, penetration, and diffusion. In a preferred embodiment, conjugation of a protein albumin binding peptide to a compound of the invention (eg, a neuraminidase inhibitor monomer or dimer via a linker) increases the potency or reduces the toxicity of the compound compared to the compound taken alone.

Protein albumin-binding peptides of the invention include any polypeptide having an amino acid sequence of 5-50 (for example, 5-40, 5-30, 5-20, 5-15, 5-10, 10-50, 10-30 or 10 -20) amino acid residues that have affinity for albumin protein and binding functions for albumin protein, such as any of the albumin proteins described herein. Preferably, the albumin-binding peptide protein binds to naturally occurring serum albumin, most preferably human serum albumin. The albumin-binding peptide protein can be of various origins, for example synthetic, human, murine or rat. The protein albumin-binding peptides of the invention include protein albumin-binding peptides that have been designed to include one or more (e.g., two, three, four, or five) solvent-exposed cysteine or lysine residues that can provide a site for conjugation with a compound of the invention (for example, conjugation with a monomer or dimer of a neuraminidase inhibitor, including through a linker). Most preferably, the albumin-binding peptide protein will contain a single solvent-exposed cysteine or lysine, thereby providing site-specific conjugation of the compound of the invention. Protein albumin-binding peptides may include only naturally occurring amino acid residues or may include one or more non-naturally occurring amino acid residues. If included, a non-naturally occurring amino acid residue (eg, a side chain of a non-naturally occurring amino acid residue) can be used as an attachment point for a compound of the invention (eg, a neuraminidase inhibitor monomer or dimer), including through a linker). The protein albumin-binding peptides of the invention can be linear or cyclic. The protein albumin-binding peptides of the invention include any protein albumin-binding peptides known to one of ordinary skill in the art, examples of which are presented herein.

Protein albumin binding peptide and conjugates comprising protein albumin binding peptide preferably bind albumin protein (e.g., human serum albumin) with an affinity characterized by a dissociation constant, Kd, that is less than about 100 μM, preferably less than about 100 nM, and most preferably do not substantially bind other plasma proteins. Specific examples of such compounds are linear or cyclic peptides, preferably about 10 to 20 amino acid residues in length, optionally modified at the N-terminus or the C-terminus, or both.

Protein albumin-binding peptides include linear and cyclic peptides containing the following general formulas, where Xaa is any amino acid: SEQ ID NO: 74

Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys

SEQ ID NO: 75

Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe

SEQ ID NO: 76

Cys-Tyr-Xaa-Pro-Gly-Xaa-Cys

SEQ ID NO: 77

Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp

SEQ ID NO: 78

Trp-Cys-Asp-Xaa-Xaa-Leu-Xaa-Ala-Xaa-Asp-Leu-Cys

SEQ ID NO: 79

Asp-Leu-Val-Xaa-Leu-Gly-Leu-Glu-Cys-Trp

The protein albumin-binding peptides of the invention further include any of the following peptide sequences, which may be linear or cyclic:

SEQ ID NO: 80 DLCLRDWGCLW

SEQ ID NO: 81 DICLPRWGCLW

SEQ ID NO: 82 MEDICLPRWGCLWGD

SEQ ID NO: 83 QRLMEDICLPRWGCLWEDDE

SEQ ID NO: 84 QGLIGDICLPRWGCLWGRSV

SEQ ID NO: 85 QGLIGDICLPRWGCLWGRSVK

SEQ ID NO: 86 EDICLPRWGCLWEDD

SEQ ID NO: 87 RLMEDICLPRWGCLWEDD

SEQ ID NO: 88 MEDICLPRWGCLWEDD

SEQ ID NO: 89 MEDICLPRWGCLWED

SEQ ID NO: 90 RLMEDICLARWGCLWEDD

SEQ ID NO: 91 EVRSFCTRWPAEKSCKPLRG

SEQ ID NO: 92 RAPESFVCYWETICFERSEQ

SEQ ID NO: 93 EMCYFPGICWM

The protein albumin-binding peptides set forth in SEQ ID NOs: 74-93 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 an integer that is zero or greater, typically less than 100, preferably less than 10, and more preferably equal to 0, 1.2, 3.4, or 5.

Additional exemplary protein albumin-binding peptides are presented in US Patent Application No. 2005/0287153, which is incorporated herein by reference in its entirety.

Conjugation of protein albumin-binding peptides

The albumin-binding peptide protein of the invention may be conjugated (eg, by covalent bonding) to any compound of the invention (eg, by a linker moiety of a neuraminidase inhibitor monomer or dimer). Protein albumin-binding peptides can be conjugated to any compound of the invention using any method known to those skilled in the art to produce peptide-small molecule conjugates. This may involve covalent conjugation to a side chain group of an amino acid residue such as a cysteine, lysine or unnatural amino acid. Alternatively, covalent conjugation may occur at the C-terminus (e.g., to a C-terminal carboxylic acid or to a side chain group of a C-terminal residue) or at the N-terminus (e.g., to an N-terminal amino group or to a side chain group of an N-terminal residue). amino acids).

V. Linkers

A linker refers to a linkage or connection between two or more components in a conjugate as described herein (e.g., between two neuraminidase inhibitors in a conjugate as described herein, between a neuraminidase inhibitor and an Fc domain or albumin protein in a conjugate as described herein, and between the dimer of two neuraminidase inhibitors and the Fc domain or albumin protein in the conjugate described herein).

Linkers in conjugates containing an Fc domain or albumin protein covalently linked to neuraminidase inhibitor dimers.

In a conjugate containing an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide covalently linked to one or more neuraminidase inhibitor dimers as described herein, a linker in the conjugate (e.g., L or L') may have a branched structure. As described later herein, the linker in a conjugate as described herein (eg, L or L') may have a multivalent structure, eg, a divalent or trivalent structure having two or three branches, respectively. In some embodiments, when the linker has three arms, two of the arms can be attached to the first and second neuraminidase inhibitors, and the third arm can be attached to an Fc domain monomer and an Fc domain, an Fc binding peptide, an albumin protein, or an albumin protein -binding peptide. In some embodiments, when the linker has two branches, one branch can be attached to the Fc domain or albumin protein, and the other branch can be attached to one of two neuraminidase inhibitors. In other embodiments, a two-arm linker can be used to attach two neuraminidase inhibitors to a conjugate containing an Fc domain or albumin protein covalently linked to one or more neuraminidase inhibitor dimers.

In some embodiments, the linker in a conjugate having an Fc domain or albumin protein covalently linked to one or more neuraminidase inhibitor dimers is described by the formula (D-L-I):

where L ^A is described by the formula G ^A1 -(Z ^A1 ) _g1 -(Y ^A1 ) _h1 -(Z ^A2 )ii-(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 the 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 the formula G ^C1 -(Z ^C1 ) _g3 -(Y ^C1 ) _h3 -(Z ^C2 )i3-(Y ^C2 ) _j3 -(Z ^C3 ) _k3 -(Y ^C3 ) _l3 -(Z ^C4 ) _m3 -( Y ^C4 ) _n3 -(Z ^C5 ) _O3 -G ^C2 ; G ^A1 represents the bond attached to Q in the formula (DLI); G ^A2 is a bond attached to a first neuraminidase inhibitor (eg, A ₁ ); G ^B1 represents the bond attached to Q in the formula (DLI); G ^B2 is a bond attached to a second neuraminidase inhibitor (eg, A ₂ ); G ^C1 represents the bond attached to Q in the formula (DLI); G ^C2 is a bond attached to an Fc domain monomer, Fc domain, Fc binding peptide, albumin protein or protein albumin binding peptide, or a functional group capable of interacting with a functional group conjugated to an Fc domain monomer, Fc- domain, Fc-binding peptide, protein albumin, or protein albumin-binding peptide (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); each of Z ^A1 , Z ^A2 , Z ^A3 , Z ^A4 , Z ^A5 , Z ^B1 , Z ^B2 , Z ^B3 , Z ^B4 , Z ^B5 , Z ^C1 , Z ^C2 , Z ^C3 , Z ^C4 and Z ^C5 independently represents an optional substituted (C1-C20)alkylene, optionally substituted (C1-C20)heteroalkylene, optionally substituted (C2-C20)alkenylene, optionally substituted (C2-C20)heteroalkenylene, optionally substituted (C2-C20)alkynylene, optionally substituted (C2-C20) )heteroalkynylene, optionally substituted (C3-C20)cycloalkylene, optionally substituted (C3-C20)heterocycloalkylene, optionally substituted (C4-C20)cycloalkenylene, optionally substituted (C4-C20)heterocycloalkenylene, optionally substituted (C8-C20)cycloalkynylene, optionally substituted ny (C8-C20)heterocycloalkynylene, optionally substituted (C5-C15)arylene or optionally substituted (C2-C15)heteroarylene; each of Y ^A1 , Y ^A2 , Y ^A3 , Y ^A4 , Y ^B1 , Y ^B2 , Y ^B3 , Y ^B4 , Y ^C1 , Y ^C2 , Y ^C3 and Y ^C4 independently represents O, S, NR.1, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino; R ⁱ represents H, optionally substituted (C1-C20)alkyl, optionally substituted (C1-C20)heteroalkyl, optionally substituted (C2-C20)alkenyl, optionally substituted (C2-C20)heteroalkenyl, optionally substituted (C2-C20)alkynyl , optionally substituted (C2-C20)heteroalkynyl, optionally substituted (C3-C20)cycloalkyl, optionally substituted (C3-C20)heterocycloalkyl, optionally substituted (C4-C20)cycloalkenyl, optionally substituted (C4-C20)heterocycloalkenyl, optionally substituted (C8 -C20)cycloalkynyl, optionally substituted (C8-C20)heterocycloalkynyl, optionally substituted (C5-C15)aryl or optionally substituted (C2-C15)heteroaryl; each of g1, h1, i1, j1, k1, l1, m1, n1, o1, g2, h2, i2, j2, k2, 12, m2, n2, o2, g3, h3, i3, j3, k3, l3, m3, n3 and o3 are independently equal to 0 or 1; Q represents a nitrogen atom, optionally substituted (C1-C20)alkylene, optionally substituted (C1-C20)heteroalkylene, optionally substituted (C2-C20)alkenylene, optionally substituted (C2-C20)heteroalkenylene, optionally substituted (C2-C20)alkynylene , optionally substituted (C2-C20)heteroalkynylene, optionally substituted (C3-C20)cycloalkylene, optionally substituted (C3-C20)heterocycloalkylene, optionally substituted (C4-C20)cycloalkenylene, optionally substituted (C4-C20)heterocycloalkenylene, optionally substituted (C8 -C20)cycloalkynylene, optionally substituted (C8-C20)heterocycloalkynylene, optionally substituted (C5-C15)arylene or optionally substituted (C2-C15)heteroarylene.

In some embodiments, the L ^C may have two attachment points to the Fc domain (eg, two GC2). In some embodiments, L includes a polyethylene glycol (PEG) linker. The PEG linker includes a linker having a repeating unit structure (-CH ₂ CH ₂ O-) _n , where n is an integer from 2 to 100. The polyethylene glycol linker can be covalently attached to the neuraminidase inhibitor and E (for example, in a conjugate of any of the formulas (MI)-(MX)). The polyethylene glycol linker may be covalently attached to the first neuraminidase inhibitor and the second neuraminidase inhibitor (eg, in a conjugate of any of formulas (DI)-(DX)). The polyethylene glycol linker can covalently link the neuraminidase inhibitor dimer and E (eg, in a conjugate of any of formulas (DI)-(DX)).

The polyethylene glycol linker can be selected from any of PEG ₂ to PEG ₁₀₀ (eg, 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 ). In some embodiments, L ^C includes a PEG linker, wherein L ^C is covalently attached to each of Q and E.

Linkers of formula (D-L-I) that can be used in the conjugates described herein include, but are not limited to:

where z ₁ and z ₂ are each independently an integer from 1 to 20; and R ₉ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15 aryl and C2-C15 heteroaryl.

Linkers of formula (D-L-I) may also include any of

Linkers in conjugates containing an Fc domain or albumin protein covalently linked to neuraminidase inhibitor monomers

In a conjugate containing an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide protein covalently linked to one or more neuraminidase inhibitor monomers as described herein, a linker in the conjugate (e.g., L or L') may be a divalent structure having two branches. One arm of the divalent linker may be attached to a neuraminidase inhibitor monomer, and the other arm may be attached to an Fc domain monomer and an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide. In some embodiments, one or more neuraminidase inhibitor monomers in the conjugates described herein may each be independently linked to an atom in an Fc domain monomer and an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide .

In some embodiments, the linker is described by the formula (M-L-I):

where J1 is a bond attached to a neuraminidase inhibitor; J ² is a bond attached to an Fc domain monomer, Fc domain, Fc binding peptide, albumin protein or protein albumin binding peptide, or a functional group capable of interacting with a functional group conjugated to an Fc domain monomer, Fc- domain, Fc-binding peptide, protein albumin, or protein albumin-binding peptide (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); each of Q ¹ , Q ² , Q ³ , Q ⁴ and Q ⁵ independently represents an 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 ₃ -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; each of T ¹ , T ² , T ³ , T ⁴ independently represents O, S, NR1, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino; R ⁱ represents H, optionally substituted (C ₁ -C ₂₀ )alkyl, optionally substituted (C ₁ -C ₂₀ )heteroalkyl, optionally substituted (C ₂ -C ₂₀ )alkenyl, optionally substituted (C ₂ -C ₂₀ )heteroalkenyl, optionally substituted (C ₂ -C ₂₀ )alkynyl, optionally substituted (C ₂ -C ₂₀ )heteroalkynyl, optionally substituted (C ₃ -C ₂₀ )cycloalkyl, optionally substituted (C ₃ -C ₂₀ )heterocycloalkyl, optionally substituted (C ₄ - C ₂₀ )cycloalkenyl, optionally substituted (C ₄ -C ₂₀ )heterocycloalkenyl, optionally substituted (C ₈ -C ₂₀ )cycloalkynyl, optionally substituted (C ₈ -C ₂₀ )heterocycloalkynyl, optionally substituted (C ₅ -C ₁₅ )aryl or optionally substituted (C ₂ -C ₁₅ )heteroaryl; and each of g, h, i, j, k, 1, m, n and o is independently equal to 0 or 1.

In some embodiments, J ² may have two points of attachment to the Fc domain monomer and the Fc domain, Fc binding peptide, albumin protein, or albumin binding peptide protein (eg, two J ² ).

Linkers of formula (M-L-I) that can be used in the conjugates described herein include, but are not limited to,

where d is an integer from 1 to 20 (for example, d is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19 or 20).

Linkers of formula (M-L-I) that can be used in the conjugates described herein include, but are not limited to:

where d and e are each independently an integer from 1 to 26.

Linking groups

In some embodiments, the linker provides space, rigidity, and/or flexibility between the neuraminidase inhibitors and the Fc domain monomer and the Fc domain, Fc binding peptide, albumin protein, or protein albumin binding peptide in the conjugates described herein, or between two neuraminidase inhibitors in the conjugates described herein. In some embodiments, the linker may be a bond, such as a covalent bond, such as an amide bond, a disulfide bond, a C-O bond, a C-N bond, an N-N bond, a C-S bond, or any type of bond created by a chemical reaction, such as a chemical conjugation. In some embodiments, the linker (L or L', as shown in any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M '-I)) includes no more than 250 atoms (for example, 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)). In some embodiments, the linker (L or L') 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 atoms; 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 atom(s). In some embodiments, the linker backbone (L or L') 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 atoms; 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. Atoms in the linker backbone are directly involved in linking one part of the conjugate to another part of the conjugate. For example, hydrogen atoms attached to carbon atoms in the linker backbone are not considered to be directly involved in linking one part of the conjugate to another part of the conjugate.

Molecules that can be used to form linkers (L or L') include at least two functional groups, for example two carboxylic acid groups. In some embodiments of a trivalent linker, the two arms of the linker may comprise two dicarboxylic acids, wherein the first carboxylic acid may form a covalent bond with the first neuraminidase inhibitor in the conjugate, and the second carboxylic acid may form a covalent bond with the second neuraminidase inhibitor in the conjugate, and the third arm of the linker may provide a covalent bond (eg, a C-O bond) to an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or a protein albumin binding peptide in a conjugate. In some embodiments of a divalent linker, the divalent linker may comprise two carboxylic acids, where the first carboxylic acid may form a covalent bond with one component (e.g., a neuraminidase inhibitor) in the conjugate, and the second carboxylic acid may form a covalent bond (e.g., a C-S bond or C-N bond) with another component (eg, Fc domain monomer, Fc domain, Fc binding peptide, protein albumin, or protein albumin binding peptide) in the conjugate.

In some embodiments, dicarboxylic acid molecules (eg, dicarboxylic acid linker) can be used as linkers. For example, in a conjugate containing an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or an albumin binding peptide protein covalently linked to one or more neuraminidase inhibitor dimers, the first carboxylic acid in the dicarboxylic acid molecule may form a covalent bond with the hydroxyl or amino group of the first neuraminidase inhibitor, and the second carboxylic acid may form a covalent bond with the hydroxyl or amino group of the second neuraminidase inhibitor.

Examples of dicarboxylic acid molecules that can be used to form linkers include, but are not limited to,

where n is an integer from 1 to 20 (for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

Other examples of dicarboxylic acid molecules that can be used to form linkers include, but are not limited to:

In some embodiments, dicarboxylic acid molecules, such as those described herein, can be further functionalized to contain one or more additional functional groups. The dicarboxylic acids may be further functionalized, for example, to provide a point of attachment to an Fc domain monomer, Fc domain, Fc binding peptide, albumin protein, or protein albumin binding peptide (eg, via a linker such as a PEG linker).

In some embodiments, when a neuraminidase inhibitor is attached to an Fc domain monomer, an Fc domain, an Fc binding peptide, a protein albumin, or a protein albumin binding peptide, the binding group may include a moiety comprising a carboxylic acid moiety and an amino moiety that are spaced apart from 1 to 25 atoms. Examples of such linking groups include, but are not limited to:

where n is an integer from 1 to 20 (for example, n 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, a linking group including a moiety including a carboxylic acid moiety and an amino moiety, such as those described herein, may be further functionalized to contain one or more additional functional groups. Such linking groups may be further functionalized, for example, to provide a point of attachment to an Fc domain monomer, Fc domain, Fc binding peptide, albumin protein, or protein albumin binding peptide (eg, via a linker such as a PEG linker). In some embodiments, when a neuraminidase inhibitor is attached to an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or a protein albumin binding peptide, the binding moiety may comprise a moiety containing two amino moieties (e.g., a diamino moiety), which are located in a disorder from 1 to 25 atoms. Examples of such linking groups include, but are not limited to:

where n is an integer from 1 to 20 (for example, n 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, a linking group including a diamino group, such as described herein, may be further functionalized to contain one or more additional functional groups. Such diamino-binding groups may be further functionalized, for example, to provide a point of attachment to an Fc domain monomer, Fc domain, Fc binding peptide, albumin protein, or albumin protein binding peptide (e.g., via a linker such as a PEG linker) .

In some embodiments, a molecule containing an azide group can be used to form a linker, where the azide group can undergo cycloaddition with an alkyne to form a 1,2,3-triazole bond. In some embodiments, a molecule containing an alkyne group can be used to form a linker, where the alkyne group can undergo cycloaddition with an azide to form a 1,2,3-triazole linkage. In some embodiments, a molecule containing a maleimide group can be used to form a linker, where the maleimide group can react with a cysteine to form a C-S bond. In some embodiments, a molecule containing one or more sulfonic acid groups can be used to form a linker, where the sulfonic acid group can form a sulfonamide bond to the bonding nitrogen in the neuraminidase inhibitor. In some embodiments, a molecule containing one or more isocyanate groups can be used to form a linker, where the isocyanate group can form a urea bond to the nitrogen-binding agent in the neuraminidase inhibitor. In some embodiments, a molecule containing one or more haloalkyl groups can be used to form a linker, where the haloalkyl group can form a covalent bond, such as C N and C-O bonds, with the neuraminidase inhibitor.

In some embodiments, the linker (L or L') may contain a synthetic group derived from, for example, a synthetic polymer (eg, a polyethylene glycol (PEG) polymer). In some embodiments, the linker may comprise one or more amino acid residues. In some embodiments, the linker may be an amino acid sequence (e.g., a sequence of 1-25 amino acids, 1-10 amino acids, 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, 1-2 amino acids or 1 amino acid). In some embodiments, the linker (L or L') may include one or more of an optionally substituted C1-C20 alkylene, an optionally substituted (C1-C20) heteroalkylene (e.g., a PEG unit), an optionally substituted (C2-C20)alkenylene (e.g., C2 alkenylene), optionally substituted (C2-C20)heteroalkenylene, optionally substituted (C2-C20)alkynylene, optionally substituted (C2-C20)heteroalkynylene, optionally substituted (C3-C20)cycloalkylene (e.g. cyclopropylene, cyclobutylene), optional substituted (C3-C20)heterocycloalkylene, optionally substituted (C4-C20)cycloalkenylene, optionally substituted (C4-C20)heterocycloalkenylene, optionally substituted (C8-C20)cycloalkynylene, optionally substituted (C8-C20)heterocycloalkynylene, optionally substituted (C5-C1) 5 )arylene (e.g. C6 arylene), optionally substituted (C2-C15)heteroarylene (e.g. imidazole, pyridine), O S, NR ⁱ (R ⁱ is H, optionally substituted (C1-C20)alkyl, optionally substituted (C1 -C20)heteroalkyl, optionally substituted (C2-C20)alkenyl, optionally substituted (C2-C20)heteroalkenyl, optionally substituted (C2-C20)alkynyl, optionally substituted (C2-C20)heteroalkynyl, optionally substituted (C3-C20)cycloalkyl, optionally substituted (C3-C20)heterocycloalkyl, optionally substituted (C4-C20)cycloalkenyl, optionally substituted (C4-C20)heterocycloalkenyl, optionally substituted (C8-C20)cycloalkynyl, optionally substituted (C8-C20)heterocycloalkynyl, optionally substituted (C5- C15)aryl or optionally substituted (C2-C15)heteroaryl, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino.

Chemistry of conjugation

Neuraminidase inhibitor monomer or dimers (e.g., in a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I) ) can be conjugated to an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or a protein albumin binding peptide, for example, via a linker, using any standard conjugation chemistry known to those skilled in the art. The following conjugation chemistries are specifically provided for, for example, conjugating a PEG linker (eg, a functionalized PEG linker) to an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin protein, or a protein albumin binding peptide.

Covalent conjugation of two or more components in a conjugate using a linker can be accomplished using well known organic chemical synthesis techniques and methods. Additional functional groups of the two components can interact with each other to form a covalent bond. Examples of additional reactive functional groups include, but are not limited to, 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 (eg, Fc domain monomer, Fc domain, Fc binding peptide, albumin protein, or protein albumin binding peptide) can be accomplished using methods known in the art. Typical methods for site-specific conjugation of a small molecule to an Fc domain are presented in Agarwall. P., et al. Bioconjugate Chem. 26: 176-192 (2015).

Other examples of functional groups capable of reacting with amino groups include, for example, alkylating and acylating agents. Typical alkylating agents include: (i) an α-haloacetyl group, for example, XCH ₂ CO- (where X = Br, Cl or I); (ii) an N-maleimide group, which can react with amino groups either through a Michael reaction or through acylation by adding a ring to the carbonyl group; (iii) an aryl halide, for example a nitrohaloaromatic group; (iv) an alkyl halide; (v) an aldehyde or ketone capable of forming a Schiff base with amino groups; (vi) epoxide, for example epichlorohydrin and bisoxirane, which can react with amino, sulfhydryl or phenolic hydroxyl groups; (vii) chlorine containing s-triazine, which is reactive towards nucleophiles such as amino, sufhydryl and hydroxyl groups; (viii) aziridine, which is reactive towards nucleophiles such as amino groups by ring opening; (ix) squaric acid diethyl ester; and (x) an a-haloalkyl ether.

Examples of amino-reactive acylating groups include, for example, (i) isocyanate and isothiocyanate; (ii) sulfonyl chloride; (iii) acid halide; (iv) an active ester, for example, nitrophenyl ester or N-hydroxysuccinimidyl ester, or derivatives thereof (for example, azido-PEG ₂ -PEG ₄₀ -NHS ester); (v) an acid anhydride, for example mixed, symmetric or N-carboxylic anhydride; (vi) acyl azide; and (vii) imidoester. Aldehydes and ketones can react with amines to form Schiff bases, which can be stabilized through reductive amination.

It should be understood that certain functional groups may be converted to other functional groups prior to reaction, for example, to impart additional reactivity or selectivity. Examples of methods useful for this purpose include the conversion of amines to carboxyls using reagents such as dicarboxylic acid anhydrides; converting amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane or thiol-containing succinimidyl derivatives; converting thiols to carboxyls using reagents such as α-haloacetates; converting thiols to amines using reagents such as ethyleneimine or 2-bromoethylamine; converting carboxyls to amines using reagents such as carbodiimides and then diamines; and converting alcohols to thiols using reagents such as tosyl chloride, followed by transesterification with thioacetate and hydrolysis to a thiol with sodium acetate.

In some embodiments, a linker of the invention (eg, L or L', such as L ^C in DLI) is conjugated (eg, using any of the methods described herein) to an E (eg, Fc domain or albumin protein). In preferred embodiments of the invention, the linker is conjugated via: (a) a thiourea bond (ie -NH(C=S)NH-) to a lysine at E; (b) a carbamate bond (ie -NH(C=O)-O) with the lysine in E; (c) an amine bond via reductive amination (ie -NHCH ₂ ) between lysine and E; (d) an amide bond (ie -NH-(C=O)CH ₂ ) with the lysine at E; (e) a cysteine-maleimide conjugate between the linker maleimide and the cysteine in E; (f) an amine bond by reductive amination (ie -NHCH ₂ ) between the linker and the carbohydrate in E (eg the glycosyl group of an Fc domain monomer or Fc domain); (g) a re-bridged cysteine conjugate, where the linker is conjugated to two cysteines in E; (h) an oxime bond between the linker and a carbohydrate in E (eg, a glycosyl group of an Fc domain or Fc domain monomer); (i) an oxime bond between the linker and the amino acid residue in E; (j) an azido bond between the linker and E; (j) direct acylation of the linker to E; or (1) a thioether bond between the linker and E.

In some embodiments, the linker is conjugated to E, where the linkage includes the structure -NH(C=NH)X-, where X is O, HN, or a linkage. In some embodiments, the linker is conjugated to E, where the bond between the linker residue and E includes the structure -NH(C=O)NH-.

In some embodiments, a linker (e.g., an active ester, e.g., a nitrophenyl ester or an N-hydroxysuccinimidyl ester, or derivatives thereof (e.g., a functionalized PEG linker (e.g., an azido-PEG ₂ -PEG ₄₀ -NHS ester)) is conjugated to E, wherein T (for example, DAR) is from 0.5 to 10.0, for example equal to 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, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2 , 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0 In these cases, the E-(PEG ₂ -PEG ₄₀ )-azide can react with Int having a terminal alkyne linker (for example, L or L', such as L ^C to DLI) via click conjugation. During click conjugation, a copper-catalyzed reaction of an azide (e.g., Ec-(PEG ₂ -PEG ₄₀ )-azide) with an alkyne (e.g., Int, having a terminal alkyne linker (e.g., L or L', such as L ^C in DLI) occurs ), forming a 5-membered heteroatom ring. In some embodiments, the linker conjugated to E is a terminal alkyne and is conjugated to Int having a terminal azide. Exemplary preparations of E-(PEG ₂ -PEG ₄₀ )-azide drugs are described in Examples 7 , 8, 61, 84, 88 and 124. Illustrative figures of conjugates obtained by click conjugation are shown in figures 43 and 61. The final product obtained by click conjugation will be readily understood by one skilled in the art.

Exemplary coupling strategies (eg, methods of linking a neuraminidase inhibitor monomer or dimer to E, eg, via a linker) are further depicted in Figures 1, 28, 29, 30, 43 and 61. VI. Combination therapy Antiviral agents

In some embodiments, 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 (for example, a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I)).

In some embodiments, the antiviral agent is an antiviral agent for treating influenza virus. For example, the antiviral agent may be an M2 ion channel blocker, a neuraminidase inhibitor (eg, a long-acting neuraminidase inhibitor), a polymerase inhibitor, a hemagglutinin inhibitor, a fusion protein inhibitor, a COX-2 inhibitor, or a PPAR agonist. The antiviral agent can be targeted either to a virus or to a host subject. An antiviral agent for the treatment of influenza virus used in combination with a conjugate described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I) -(M-X) or (M'-I)), may be selected from oseltamivir, zanamivir, peramivir, laninamivir, CS-8958, amantadine, rimantadine, cyanovirine-N, cap-dependent endonuclease inhibitor (eg, baloxavir marboxil), inhibitor polymerase (e.g., T-705), PB2 inhibitor (e.g., JNJ-63623872), conjugated sialidase (e.g., DAS181), thiazolide (e.g., nitazoxanide), COX inhibitor, PPAR agonist, hemagglutinin-targeting antibody (e.g., monoclonal antibody, such as CR6261, CR8020, MEDI8852, MNAA4549A or VIS410) or siRNA targeting a host or viral gene, or prodrugs thereof, or pharmaceutically acceptable salts thereof.

Antiviral vaccines

In some embodiments, any of the conjugates described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X), or (M'-I)) is administered in combination with an antiviral vaccine (eg, a composition that induces an immune response in a subject against a virus). The viral 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 before or after the conjugates (e.g., over 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).

In some embodiments, the viral vaccine contains an immunogen that induces an immune response in a subject against an influenza A, B, C, or parainfluenza virus. In some embodiments, the immunogen is an inactivated virus (eg, the vaccine is a trivalent influenza vaccine that contains purified and inactivated influenza A, B, C or parainfluenza virus material, or any combination thereof). In some embodiments, the vaccine is administered as an intramuscular injection. In some embodiments, the vaccine is a live viral vaccine that contains attenuated (weakened) live viruses. In some embodiments, the vaccine is administered as a nasal spray.

VII. Methods

The methods described herein include, for example, methods of protecting against or treating a viral infection (eg, an influenza virus infection) in a subject and methods of preventing, stabilizing, or inhibiting the growth of viral particles. A method of treating a viral infection (eg, an influenza virus infection) in a subject comprises administering to the subject a conjugate described herein (eg, a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D' -I), (M-I)-(M-X) or (M'-I)), or a pharmaceutical composition thereof. In some embodiments, the viral infection is caused by an influenza virus (eg, influenza A, B, C, or parainfluenza virus). In some embodiments, the viral infection is caused by a resistant strain of the virus. A method of preventing, stabilizing or inhibiting the growth of viral particles or preventing the replication and spread of a virus involves contacting a virus or a site susceptible to viral growth with a conjugate described herein (for example, a conjugate of any of formulas (1)-(5), (D-I) -(D-X), (D'-I), (M-I)-(M-X) or (M'-I)) or a pharmaceutical composition thereof.

Moreover, the methods described herein also include methods for protecting against or treating a viral infection in a subject by administering to the subject a conjugate described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I) -(D-X), (D'-I), (M-I)-(M-X) or (M'-I)). In some embodiments, the method further includes administering an antiviral agent or an antiviral vaccine to the subject.

The 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 of formulas (1)-(5), (D-I )-(D-X), (D'-I), (M-I)-(M-X) or (M'-I)), and (2) an antiviral agent or an antiviral vaccine. The 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 a virus or a site susceptible to viral growth with (1) a conjugate described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I)), and (2) an antiviral agent or an antiviral vaccine.

In some embodiments, a conjugate described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M' -I)) is administered first, followed by a separate antiviral agent or antiviral vaccine. In some embodiments, the antiviral agent or antiviral vaccine is administered first, followed by the administration of a single conjugate as described herein. In some embodiments, a conjugate described herein and an antiviral agent or antiviral vaccine are administered substantially simultaneously (eg, in a single pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, a conjugate described herein or an antiviral agent or an antiviral vaccine is first administered, and then the conjugate described herein and an antiviral agent or an antiviral vaccine are administered substantially simultaneously (e.g., in a single pharmaceutical composition or in separate pharmaceutical formulations). compositions). In some embodiments, a conjugate described herein and an antiviral agent or an antiviral vaccine are first administered substantially simultaneously (e.g., in a single pharmaceutical composition or in separate pharmaceutical compositions), followed by the administration of a separate conjugate described herein or an antiviral agent or an antiviral vaccine. vaccines. In some embodiments, when a conjugate described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X), or (M'-I)) and the antiviral agent or antiviral vaccine are administered together (e.g., substantially simultaneously in one or separate pharmaceutical compositions, or separately in one treatment regimen), the inhibition of viral replication of each of the conjugate and the antiviral agent or antiviral vaccine may be greater (eg, occur at a lower concentration) than the inhibition of viral replication of each of the conjugate and antiviral agent or antiviral vaccine when each is used separately in a treatment regimen. VIII. Pharmaceutical compositions and preparations

The conjugate described herein can be formulated into a pharmaceutical composition for use in the methods described herein. In some embodiments, a conjugate described herein may be formulated only into a pharmaceutical composition. In some embodiments, a conjugate described herein may be formulated in combination with an antiviral agent or an antiviral vaccine into a pharmaceutical composition. In some embodiments, the pharmaceutical composition includes a conjugate described herein (e.g., a conjugate described by any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-( M-X) or (M'-I)), and pharmaceutically acceptable carriers and excipients.

Acceptable carriers and excipients in pharmaceutical compositions are non-toxic to recipients at the dosages and concentrations used. 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, octadecyl dimethyl benzyl 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.

Examples of other excipients include, but are not limited to, release agents, binders, coatings, compression aids, disintegrants, colorants, emollients, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, flavoring agents, glidants (enhancers). fluidity), lubricants, sorbents, suspending or dispersing agents or sweeteners. Illustrative excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, carmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose , magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C and xylitol.

The conjugates herein may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts comprising inorganic or organic acids, or in the case of the acid forms of the conjugates presented herein, the salts may be derived from inorganic or organic bases. Often the conjugates are prepared or used in the form of pharmaceutically acceptable salts obtained as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art, such as hydrochloric, sulfuric, hydrobromic, acetic, lactic, citric or tartaric acids for the formation of acid addition salts, as well as potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines and so on. for the formation of basic salts. Methods for preparing the corresponding salts are well known in the art.

Typical acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanate, glycerophosphate , hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate , pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate and valerate. Typical alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium , calcium and magnesium, as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine and ethylamine.

Depending on the route of administration and dosage, the conjugate herein or the pharmaceutical composition thereof used in the methods described herein will be formulated into suitable pharmaceutical compositions to ensure easy delivery. Conjugate (for example, a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I)) or pharmaceutical composition thereof may be formulated for intramuscular, intravenous (e.g., as a sterile solution and in a diluent system suitable for intravenous use), intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal , topical, intratumoral, peritoneal, subcutaneous, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, oral (eg, tablet, capsule, hard capsule, gelatin capsule, or syrup), topical (eg, cream, gel , lotion or ointment), topical, by inhalation, by injection or infusion (eg, continuous infusion, localized perfusion with direct bathing of target cells, catheter, lavage, cream or lipid compositions). Depending on the route of administration, the conjugate of the present invention or its pharmaceutical composition may be presented in the form of, for example, tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels, including hydrogels, pastes, ointments, creams, patches, liquid dosage forms for oral administration, osmotic delivery devices, suppositories, enemas, injection solutions, implants, sprays, preparations suitable for iontopheretic delivery, or aerosols. The compositions can be formulated in accordance with standard pharmaceutical practice.

The conjugate described herein can be formulated by various methods known in the art. For use in the treatment of humans and animals, the conjugate described herein can be formulated into pharmaceutical or veterinary compositions. Depending on the subject (eg, human) to be treated, the route of administration, and the type of treatment desired, eg, prophylaxis or therapy, the conjugate described herein is formulated in ways consistent with these parameters. A summary of such methods can be 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.

The compositions can be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those intended for injection (eg, intramuscular, intravenous, or subcutaneous injection) or may be formulated for transdermal, transmucosal, or oral administration. The formulation typically includes a diluent and, in some cases, adjuvants, buffers and preservatives. The conjugates can also be administered in liposomal formulations or as microemulsions. Systemic administration may also include relatively non-invasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for the conjugates herein. Suitable forms include syrups, capsules and tablets, as known in the art.

The pharmaceutical 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 carrier. The compositions may be formulated as solid forms suitable for dissolution or suspension in liquid prior to injection, or as emulsions. Pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, and cell culture media (eg, Dulbecco's modified Eagle's medium (DMEM), α-modified Eagle's medium (α-MEM), F-12 medium). Such injectable compositions may also contain amounts of non-toxic excipients such as wetting or emulsifying agents, pH buffering agents such as sodium acetate and sorbitan monolaurate. Formulation methods are known in the art, see, for example, Pharmaceutical Preformulation and Formulation, 2nd Edition, M. Gibson, Taylor & Francis Group, CRC Press (2009).

Pharmaceutical compositions can be prepared in the form of an oral preparation. Formulations for oral use include tablets containing the active ingredient(s) in admixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (for example, 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 (eg cellulose derivatives, including microcrystalline cellulose, starches, including potato starch, croscarmellose sodium, alginates or alginic acid); binding agents (eg, sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, polyvinylpyrrolidone or polyethylene glycol); and lubricants, glidants, and release agents (eg, magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc). Oral formulations may also be presented as chewable tablets or hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (eg, potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin) or as soft gelatin capsules in which the active ingredient is mixed with water or an oily medium, such as peanut oil, liquid paraffin or olive oil. Powders, granules and pellets can be prepared using the ingredients mentioned in the tablets and capsules section above in a conventional manner using, for example, a mixer, fluid bed or spray drying equipment.

Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colors, flavors, plasticizers, humectants, and buffering agents. Oral formulations may also be presented as chewable tablets or hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (eg, potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin) or as soft gelatin capsules in which the active ingredient is mixed with water or an oily medium, such as peanut oil, liquid paraffin or olive oil. Powders, granules and pellets can be prepared using the ingredients mentioned above for tablets and capsules in a conventional manner using, for example, a mixer, fluid bed or spray drying equipment.

Dissolution- or diffusion-controlled release of a conjugate as described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or ( M'-I)), or a pharmaceutical composition thereof, can be achieved by applying a suitable coating to a tablet, capsule, pill or granular conjugate formulation, or by incorporating the conjugate into a suitable matrix. The controlled release coating may include one or more of the coating agents mentioned above and/or, for example, shellac, beeswax, glycovax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glyceryl palmitostearate, ethyl cellulose, acrylic resins, dl- polylactic acid, cellulose acetobutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxy methacrylate, methacrylate hydrogels, 1,3-butylene glycol, ethylene glycol methacrylate and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate copolymer, polyvinyl chloride, polyethylene and/or halogenated fluorocarbon.

If necessary, the pharmaceutical composition can be formulated in unit dose form. The amount of the active component, for example, a conjugate described herein (for example, a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I)) included in the pharmaceutical compositions are such that a suitable dose within the specified range is provided (for example, a dose in the range of 0.01-100 mg/kg body weight).

IX. Routes of administration and dosage

In any of the methods described herein, the conjugates herein may be administered by any suitable route to treat or protect against a viral infection (eg, an influenza virus infection) or to prevent, stabilize, or inhibit the proliferation or spread of a virus (eg , influenza virus). The conjugates described herein can be administered to humans, pets, livestock or other animals with a pharmaceutically acceptable diluent, carrier or excipient. In some embodiments, administration includes administration of any of the conjugates described herein (e.g., conjugates of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-( M-X) or (M'-I)), or compositions intramuscularly, intravenously (e.g., as a sterile solution and in a solvent system suitable for intravenous use), intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic glands, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, local, intratumoral, intramuscular, subcutaneous, subconjunctival, intravesicular, transmucosal, intrapericardial, transumbilical, intraocular, oral (e.g. tablet, capsule, tablet in capsule form , soft capsule or syrup), topically (eg, as a cream, gel, lotion, or ointment), topically, by inhalation, by injection, or by infusion (eg, continuous infusion, localized perfusion with direct bathing of target cells, catheter , lavage, in the form of a cream or in the form of lipid compositions). In some embodiments, if the antiviral agent is also administered in addition to the conjugate described herein, the antiviral agent or pharmaceutical composition thereof can also be administered by any of the routes of administration described herein.

Dosage of a conjugate described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I )) or pharmaceutical compositions thereof depends on factors including the route of administration, the disease being treated (eg, the extent and/or state of the viral infection), and physical characteristics, eg, age, weight, general health of the subject. Typically, the amount of the conjugate or pharmaceutical composition thereof contained in a single dose may be an amount that effectively prevents, delays or treats viral infection without causing significant toxicity. The pharmaceutical composition may include a dosage of the 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, from about 0.1 to about 30 mg/kg, and in a more specific embodiment, from about 1 to about 30 mg/kg. In some embodiments, when a conjugate described herein (e.g., a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X), or (M'-I)), and the antiviral agent or antiviral vaccine is administered in combination (e.g., substantially simultaneously in the same or separate pharmaceutical compositions, or separately in the same treatment regimen), the required dose of the conjugate described as used herein, may be lower than the required dose of the conjugate if the conjugate is used alone in a treatment regimen.

A conjugate described herein (for example, a conjugate of any of formulas (1)-(5), (D-I)-(D-X), (D'-I), (M-I)-(M-X) or (M'-I)) or the pharmaceutical composition thereof may be administered to a subject in need thereof, for example, one or more times (for example, 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 indicated. The dosage regimen may be provided in single or multiple dosage regimens. The time between administrations may decrease as the patient's health improves or increase as the patient's condition worsens. The dosage and frequency of administration may be adjusted by the physician according to generally accepted factors such as the degree of infection and various subject parameters.

EXAMPLES

The following examples are provided to provide those of ordinary skill in the art with a description of how the compositions and methods described herein can be used, made and evaluated, and are intended to illustrate the invention only and are not intended to limit the scope of what the inventors inventions are considered as their own invention.

Example 1: Obtaining Fc constructs

Reverse translations of amino acids containing protein constructs (SEQ ID NOs: 1, 3, 5, 7, 9, 12 and 14) were synthesized using solid phase synthesis. Oligonucleotide templates were cloned into pcDNA3.1 (Life Technologies, Carlsbad, CA, USA) at the BamHI and XhoI cloning sites (New England Biolabs, Ipswich, MA, USA) and included signal sequences derived from human interleukin-2 or human albumin. pcDNA3.1 plasmids were transformed into E. coli Top 10 cells (LifeTech). DNA was amplified, extracted, and purified using the PURELINK® HiPure Plasmid Filter Maxiprep Kit (LifeTech). Plasmid DNA was delivered using the ExpiFectamine™ 293 Transfection Kit (LifeTech) into HEK-293 cells according to the manufacturer's protocol. Cells were centrifuged, filtered, and supernatants were purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule onto the gel and staining using flash blue staining. Each gel included a molecular weight size marker with the indicated molecular weight standards (Figures 2-8). Bands under reducing and non-reducing conditions are indicated by the letters "R" and "NR". Figures 2-8 show SDS-PAGE under reducing and non-reducing conditions of an Fc domain formed from Fc domain monomers having the sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 12 and 14, respectively .

Example 2 Synthesis of zanamivir intermediate

Stage a.

Methyl 5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-B-glycero-D-galacto-non-2-enonate (4.56 g, 10.0 mmol) was dissolved in anhydrous THF (15 ml), and the solution was cooled to approximately 13°C. Triphenylphosphine (2.89 g, 11 mmol) was added in portions over 20 minutes. The resulting mixture was stirred at approximately 13°C to room temperature for 2 hours, then a solution of LiOH (24 mg, 1 mmol) in water (1.5 ml) was added dropwise. After stirring for 28 hours, N,N'-bis-boc-1-guanylpyrazole (3.26 g, 10.5 mmol) and 4-dimethylaminopyridine (244.4 mg, 2 mmol) were added to the reaction mixture. The reaction mixture was stirred for 1.5 days. It was then diluted with a 1:1 mixture of ethyl acetate (100 ml) and extracted with water (30 ml). The aqueous layer was again extracted with ethyl acetate (30 ml). The combined organic layers were concentrated on a rotary evaporator. The residue was purified by C18 reverse phase column chromatography (150 g, 25-70% acetonitrile in water). The collected fractions were concentrated on a rotary evaporator at room temperature. A cloudy aqueous solution was obtained and most of the product precipitated as a gel on the flask. The solution was then extracted with ethyl acetate (150 ml). The organic layer was used to redissolve the gel material. It was then dried over Na ₂ SO ₄ , concentrated on a rotary evaporator and further dried under high vacuum to give the title compound as a white foam. Yield 6.24 g, 92.8%. Ion detected by LCMS: [M+H]+=673.2.

Stage b.

A solution of the product obtained in step-a (6.24 g, 9.28 mmol) in anhydrous MeOH (20 ml) was cooled in an ice water bath and a 0.5 M solution of sodium methoxide in MeOH (26 ml, 13 mmol) was slowly added ). The reaction mixture was stirred for 1 hour, then its pH was carefully adjusted to 7-7.5 by adding dropwise a 4 N solution of HC1 in dioxane (3 ml). The solvent was removed using a rotary evaporator at a temperature no higher than room temperature. The residue was diluted with a 2:1 mixture of ethyl acetate and hexane (150 ml), and the resulting solution was extracted with water (20 ml). The aqueous layer was again extracted with ethyl acetate (30 ml). The combined organic layers were dried over Na ₂ SO ₄ , concentrated on a rotary evaporator and then dried under high vacuum. The product was transferred to the next step without further purification. Yield 5.03 g, 99.2%. Ion detected by LCMS: [M+H]+=547.2.

Stage c.

The product obtained from step-b (713 mg, 1.3 mmol) in anhydrous DCM (6 ml) was cooled in an ice water bath and 4-dimethylaminopyridine (159.8 mg, 1.3 mmol) and DIPEA (520 mg) were added , 4 mmol). A solution of 4-nitrophenyl chloroformate (356.6 mg, 2.2 mmol) in anhydrous DCM (2 mL) was then added dropwise to the mixture. The ice water bath was then removed and the reaction mixture was stirred for 3 hours and monitored by LCMS (additional 4-nitr o phenyl chloroformate may be added if necessary). After completion of the reaction, it was quenched with water (10 ml) and the organic layer was extracted and concentrated by rotary evaporation. The residue was purified by C18 reverse phase column chromatography (100 g, 20-70% acetonitrile in water). Acetonitrile was removed from the collected fractions by rotary evaporation at room temperature. The aqueous layer was then extracted with a 1:1 mixture of ethyl acetate and hexane (120 ml). The aqueous layer was again extracted with ethyl acetate (30 ml). The combined organic layers were dried over Na ₂ SO ₄ , concentrated by rotary evaporation and further dried under high vacuum to obtain the title compound as a white solid. Yield 520 mg, 70%. Ion detected by LCMS: [M+H]+=573.2.

Example 3 Synthesis of linker-1

Stage a.

To a mixture of ZD-glutamine γ-methyl ester (2.0 g, 6.77 mmol) and glycine methyl ester HCl (1.282 g, 10.2 mmol) in anhydrous DMF (7 ml), DIPEA (2.02 g, 15 .57 mmol) followed by HATU (2.66 g, 7.0 mmol) in portions over 25 minutes. After dissolving the HATU, additional DIPEA (1.15 g, 8.8 mmol) was added. The reaction mixture was stirred for 1.5 hours, then extracted with 5% aqueous HCl (100 ml) and EtOAc (100 ml x 2). The organic layer was concentrated on a rotary evaporator. The residue was re-extracted with water (100 ml) and EtOAc/hexanes (2:1, 150 ml). The organic layer was dried over Na2SO4, concentrated on a rotary evaporator and then dried under high vacuum to a white solid. The crude product was used in the next step without further purification. Yield 2.3 g, 93%. Ion detected by LCMS: [M+H] ⁺ =367.

Stage b.

The product obtained in step-a (2.3 g, 6.29 mmol) was dissolved in a 1:1 mixture of MeOH and THF (10 ml). After cooling the solution in an ice water bath, the solution was added in portions over 1.5 hours LiOH monohydrate (630 mg, 15 mmol) in water (9 ml). After stirring for 2 additional hours, the reaction mixture was neutralized with 4N HCl in dioxane (3.7 ml). The organic solvent was partially removed by rotary evaporation at room temperature. The remaining material was purified directly by RPLC (150 g, 0-39% acetonitrile in water). Yield 2.03 g, 88.7% in two stages. Ion detected by LCMS: [M+H] ⁺ =339.2.

Stage c.

To a mixture of the product obtained in step-b (1.41 g, 4.17 mmol) and N-Boc-1,6-diaminohexane (1.99 g, 9.2 mmol) in anhydrous DMF (6 ml) and DIPEA (1.3 g, 10 mmol) was added, a solution of HATU (3.5 g, 9.2 mmol) in DMF (10 ml) was added using a syringe pump at a rate of 11 ml/h. Once the addition of HATU was complete, the reaction mixture was stirred for an additional 30 minutes and directly purified by RPLC (150 g, 10-50% acetonitrile in water, using 0.1% TFA as modifier). Yield 1.3 g, 42.4%. Ion detected by LCMS: [M+H] ⁺ =735, [M - Boc+H] ⁺ =635.4.

Stage d.

The product obtained in step-c (1.3 g, 1.77 mmol) was dissolved in MeOH (20 ml) and Pd/C was added to the solution. The mixture was stirred under a hydrogen atmosphere for 4 hours. The Pd/C was filtered off and the filtrate was concentrated by rotary evaporation and then dried under high vacuum. Yield 1.03 g, 96.9%. Ion detected by LCMS: [M+H] ⁺ =601.

Stage e.

The flame-dried reaction flask was purged with nitrogen and charged with 4-azidobutyric acid (77 mg, 0.6 mmol), N-hydroxysuccinimide (92 mg, 0.8 mmol), and anhydrous DMF (0.5 mL). The mixture was stirred to dissolve the solids and then DCC (125.5 mg, 0.608 mmol) was added. After stirring for one hour, the product obtained in step-d (300 mg, 0.5 mmol) was added to the reaction mixture. The reaction mixture was stirred for 6 hours and purified directly by RPLC (150 g, 10-70% acetonitrile in water, using 0.1% TFA as modifier). The collected fractions were lyophilized to a white solid (LCMS: [M+H] ⁺ =712, [M - Boc + H] ⁺ =612). The material was redissolved in DCM (~2 ml) and TFA (~1 ml) and stirred for 15 minutes. It was then concentrated by rotary evaporation and the residue was purified by RPLC (50 g, 0-40% acetonitrile in water). Yield 255 mg, 68.9%. Ions detected by LCMS: [M+H] ⁺ =512, [(M+2H)/2] ⁺ =256.

Example 4 Synthesis of Intermediate-1

Stage a.

To a solution of zanamivir intermediate (Example 2) (532.5 mg, 0.93 mmol) in anhydrous THF (2 ml) was added DMAP (490 mg, 4 mmol) followed by bis(pentafluorophenyl)carbonate (385 mg, 0.911 mmol ). After stirring overnight, a solution of linker-1 (Example 3) (245.6 mg, 0.332 mmol) in anhydrous DMF (1 ml) and DIPEA (91 mg, 0.3 mmol) was added to the reaction mixture. The reaction was continued for 2 hours, then purified by RPLC (100 g, 5-67% acetonitrile in water). Yield 135 mg, 23.8%. Ion detected by LCMS: [(M+2H)/2] ⁺ =845.9.

Stage b.

The product obtained in step-a (135 mg, 0.079 mmol) was dissolved in TFA (0.5 ml) and the solution was stirred at room temperature for 20 minutes. It was then directly purified by RPLC (50 g, 5-32% acetonitrile in water). Yield 88 mg, 85.1%. Ion detected by LCMS: [(M+2H)/2] ⁺ =654.8.

Stage c.

A solution of the product from step b (88 mg, 0.0673 mmol) in MeOH (1.5 mL) was cooled in an ice water bath and LiOH (5 mg, 0.2 mmol) in water (0.5 mL) was added. . After stirring the mixture for 5 hours, it was acidified with a solution of 4N HCl in dioxane (0.1 ml) and purified by HPLC (5-20% acetonitrile in water, using 0.1% TFA as modifier). Yield 76.4 mg, 78%. Ion detected by LCM: [(M + 2H)/2] ⁺ =614.8.

Example 5 Synthesis of Linker-2

Stage a.

HATU (558.9 mg, 1.47 mmol) was added to a solution of propyl-PEG4 acid (364.4 mg, 1.4 mmol) in anhydrous DMF (2 mL). After stirring to dissolve all of the coupling reagent, DIPEA (390 mg, 3 mmol) was added and stirred for 10 minutes. A solution of the product obtained from linker-1 step-d (Example 3, step d) (701.1 mg, 1.167 mmol) in anhydrous DMF (1 ml) was added. The resulting mixture was stirred for 30 minutes and directly purified using RPLC (100 g, 5-60% acetonitrile in water). Yield 830 mg, 84.4%. Ion detected by LCMS: [M+H] ⁺ =843.

Stage b.

The product obtained in step a was dissolved in THF (5 ml) and treated with a solution of 4N HCl in dioxane (2.5 ml). After stirring at room temperature overnight, the reaction mixture was concentrated by rotary evaporation. The residue was redissolved in acetonitrile/water (1:1, 16 ml) and the solution was lyophilized. The crude product was used in the next step without further purification. Yield 761.8 mg, 100%. Ion detected by LCMS: [M+H] ⁺ =643.8.

Example 6 Synthesis of Intermediate-2

Stage a.

The flame-dried reaction flask was purged with nitrogen and loaded with zanamivir intermediate (Example 2) (533.6 mg, 0.812 mmol), DMAP (99.8 mg, 0.81 mmol) and anhydrous DCM (1 ml). After stirring to dissolve the starting material, the solution was cooled in an ice water bath and 4-nitrophenyl chloroformate (242 mg, 1.2 mmol) was added. The resulting mixture was stirred for 5 hours, then added to a solution of Linker-2 (Example 5) (228.4 mg, 0.319 mmol) in anhydrous DMF (1 ml) and DIPEA (130 mg, 1 mmol). The reaction mixture was stirred overnight and purified by RPLC (150 g, 20-65% acetonitrile in water, using 0.1% TFA as modifier). The collected fractions were lyophilized. Yield 178.3 mg, 30.4%. Ion detected by LCMS: [(M+2H)/2] ⁺ =920.5, [(M+3H)/3] ⁺ =614.2.

Stage b.

The product from step a (178.3 mg, 0.0969 mmol) was dissolved in TFA (0.5 ml). The solution was stirred for 20 minutes, then purified directly by HPLC (0-25% acetonitrile in water, using 0.1% TFA as modifier). Yield 91.8 mg, 56.8%. Ions detected using LCMS: [(M+2H)/2] ⁺ =720.4, [(M+3H)/3] ⁺ =480.6.

Stage c.

The product from step b (91.8 mg, 0.055 mmol) was dissolved in MeOH (1 mL) and the solution was cooled in an ice water bath. LiOH monohydrate (21 mg, 0.5 mmol) in water (1 ml) was added in portions over 1 hour. After stirring for an additional 2 hours, the reaction mixture was acidified with a solution of 4N HCl in dioxane (0.3 ml) and purified by HPLC (0-20% acetonitrile in water, using 0.1% TFA as modifier). Yield 36.2 mg, 59.4%.%. Ions detected using LCMS: [(M+2H)/2] ⁺ =680.3, [(M+3H)/3] ⁺ =454.0.

Example 7. Synthesis of h-IgG1 Fc-PEG4-azide

PEG4-azidoNHS ester (98%, 180 µmol, 9.5 equivalents, 71.4 mg in 0.5 ml DMF and diluted to 3.60 ml with 1x PBS buffer pH 7.4) was added to the h-IgG1 solution Fc (SEQ ID NO: 4) (1103 mg in 70.0 ml PBS at pH 7.4, MW ~ 58000 Da, 19.0 μmol), and the mixture was shaken gently for 12 hours at ambient temperature. The solution was concentrated using a centrifugal concentrator (30,000 MWCO) to a volume of ~1.5 mL. The crude mixture was diluted 1:10 in PBS pH 7.4 and concentrated again. This washing procedure was repeated a total of three times. This washing procedure removed the low molecular weight reagent. Concentrated Ec-PEG4-azide was diluted to 70.0 ml with 1x PBS buffer at pH 7.4 and prepared for click conjugation. The purified material was quantified using a Nanodrop™ UV-visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1). Quantitative yield after purification. DAR=4.3 was determined using MALDI. The DAR value can be adjusted by varying the PEG4-azido NHS ester equivalents in a nearly linear relationship. For example, using 7.0 equivalents of PEG4 azide NHS ester, the DAR value would be 3.0.

Example 8. Synthesis of recombinant mouse serum albumin (MSA)-PEG4-azide.

PEG4-azidoNHS ester (98%, 81.7 µmol, 4.5 equivalents, 32.4 mg in 0.3 ml DMF and diluted to 1.63 ml with PBS buffer pH 7.4) was added to the recombinant solution mouse serum albumin (SEQ ID NO: 71) (1200 mg in 75.0 ml PBS pH 7.4, MW ~ 66000 Da, 18.2 μmol), and the mixture was shaken gently for 12 hours at ambient temperature. The solution was concentrated using a centrifugal concentrator (30,000 MWCO) to a volume of ~1.5 mL. The crude mixture was diluted 1:10 in PBS pH 7.4 and concentrated again. This washing procedure was repeated a total of three times. This washing procedure removed the low molecular weight reagent. Concentrated MSA-PEG4-azide was diluted to 75.0 ml with 1x PBS buffer at pH 7.4 and prepared for click conjugation. The purified material was quantified using a Nanodrop™ UV-visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1). Quantitative yield after purification. DAR=3.5 value determined using MALDI. The DAR value can be adjusted by changing PEG4-azidoNHS-ester equivalents, similar to h-IgG1 Fc (Example 7).

Example 9. Synthesis of conjugate 1

A PBS solution of h-IgG1 Ec-PEG4-azide (Example 7) (50 mg, 2.815 ml, 0.8591 µmol) was added to a 15 ml centrifuge tube containing intermediate-2 (Example 6) (35.2 mg, 0 .02217 mmol). After gently shaking the mixture to dissolve all of Intermediate-2, 344 µl of a solution of sodium L-ascorbic acid (59.4 mg, 0.3 mmol), copper(II) sulfate (10 mg, 0.05 mmol) and THPTA ( 23 mg 0.05 mmol) in PBS buffer at pH 7.4 (1 ml). The resulting mixture was shaken gently overnight. It was purified by affinity chromatography on a Protein A column followed by size exclusion chromatography as described in Example 8. Maldi TOF analysis of the purified final product gave an average mass of 63,797 Da (DAR=3.4). Yield 27.39 mg, 55%. Figure 9 shows SDS-PAGE under non-reducing conditions of conjugate 1.

Example 10 Purification of Conjugates

The crude mixture was diluted 1:10 in PBS at 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 the purified conjugate were pooled and concentrated to approximately 20 mg/ml using a centrifugal concentrator (30,000 MWCO). The purified material was quantified using a Nanodrop™ 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 μg of each molecule onto the gel and staining using Instant Blue (Expedeon, San Diego, CA, USA). Each gel included a molecular weight size marker with the indicated molecular weight standards. Yields were calculated and purity determined using Agilent Analytical HPLC. The product peak and molecular weight were detected using Maldi MS and the final DAR value was calculated.

Example 11 Synthesis of Intermediate-3

Stage a.

DIPEA (130 mg) was added to a solution of propargyl-PEG4 acid (260 mg, 1 mmol) and HATU (380.2 mg, 1 mmol) in anhydrous DMF (1 ml). After stirring for 5 minutes, NH-bis(PEG3-Boc) (500 mg, 0.881 mmol) was added and stirring was continued at room temperature overnight. It was then directly purified by RPLC (9100 g, 5-50% acetonitrile in water, using 0.1% TFA as modifier). Yield 683 mg, 95.8%. Ions detected using LCMS: [M+H] ⁺ =810.4, [M - Boc+H] ⁺ =710.4, [(M - 2 Boc+2H)/2] ⁺ =305.8.

Stage b.

The product obtained in step-a was dissolved in TFA (1 ml). The solution was stirred for 2 hours and then purified directly using RPLC (100 g, 0-30% acetonitrile in water). Yield 589 mg, 98.7%. Ion detected by LCMS: [(M+2H)/2] ⁺ =305.8.

Stage c.

The flame-dried reaction flask was purged with nitrogen and charged with zanamivir intermediate (Example 2) (572 mg, 1 mmol) and anhydrous DCM (1 ml). After stirring to dissolve the starting material, the solution was cooled in an ice water bath and 4-nitrophenyl chloroformate (302.4 mg, 1.5 mmol) was added, followed by DMAP (22.4 mg, 0.2 mmol). The resulting mixture was stirred for 5 hours, then cooled water (0.2 ml) was added. After stirring for 10 minutes, the product obtained in step-b (256.7 mg, 0.355 mmol) in anhydrous DMF (1 ml) and DIPEA (163.8 mg, 1.26 mmol) was added. Stirring was continued for 2 hours and then the reaction mixture was purified directly by RPLC (150 g, 20-65% acetonitrile in water, using 0.1% TFA as modifier). The collected fractions were lyophilized. The yield of the desired product, contaminated with some impurities, was 422.8 mg, a yield of <69%. The material was used in the next step without further purification. Ion detected by LCMS: [(M+2H)/2] ⁺ =903.9, [(M+3H)/3] ⁺ =603.2.

Stage d.

The product obtained in step-c (422.8 mg, <0.245 mmol) was dissolved in TFA (1 ml). The solution was stirred for 20 minutes, then purified directly by HPLC (5-25% acetonitrile in water, using 0.1% TFA as modifier). Yield 169.7 mg, 29.2% in two stages. Ions detected by LCMS: [(M+2H)/2] ⁺ =704.0, [(M+3H)/3] ⁺ =469.6.

Stage e.

The product obtained in step-d (169.7 mg, 0.0923 mmol) was dissolved in MeOH (1.5 ml) and the solution was cooled in an ice water bath. LiOH monohydrate (21 mg, 0.5 mmol) in water (1 ml) was added in portions over 1 hour. After stirring overnight, the reaction mixture was acidified with Dowex 50W x 8 hydrogen form and purified by RPLC (0-30% acetonitrile in water, using 0.1% TFA as modifier). Yield 107.9 mg, 66.9%. Ions detected using LCMS: [(M+2H)/2] ⁺ =663.8, [(M+3H)/3] ⁺ =442.9.

Example 12. Synthesis of conjugate 2

The title conjugate was prepared similarly to Conjugate 1 (Example 9) using Intermediate-3 (Example 11). Maldi TOF analysis of the purified final product gave an average mass of 63,561 Da (DAR=3.3). Yield 43.4 mg, yield 43%. Figure 10 shows SDS-PAGE analysis under non-reducing conditions of conjugate 2.

Example 13 Synthesis of Intermediate-4

Stage a.

The flame-dried reaction flask was purged with nitrogen and loaded with zanamivir intermediate (Example 2) (343.2 mg, 0.6 mmol) and anhydrous DCM (1.5 ml). The solution was cooled in an ice water bath and DIPEA (234 mg, 1.8 mmol) was added, followed by 4-nitrophenyl chloroformate (121 mg, 0.6 mmol) and DMAP (67.4 mg, 0.6 mmol). The resulting mixture was stirred for 15 minutes, then additional 4-nitrophenyl chloroformate (121 mg, 0.6 mmol) was added. After stirring for 2 hours, water (0.2 ml) was added to quench the unreacted chloroformate. After stirring for 10 minutes, a solution of propargyl-PEG4-amine (185 mg, 0.8 mmol) in anhydrous DMF (0.5 ml) was added to the reaction mixture. The reaction was continued for 1 hour and then directly purified by RPLC (100 g, 5-60% acetonitrile in water, using 0.1% TFA as modifier). The collected fractions were lyophilized. Yield 355 mg, 71.3%. Ion detected by LCMS: [M+H] ⁺ =830.2.

Stage b.

The product obtained in step-a (355 mg, 0.428 mmol) was dissolved in TFA (1 ml). The solution was stirred overnight, then purified directly by RPLC (5-25% acetonitrile in water, using 0.1% TFA as modifier). Yield 260.2 mg, 70.9% in two stages. Ion detected by LCMS: [M+H] ⁺ =630.2.

Stage c.

The product obtained in step-b (260.2 mg, 0.303 mmol) was dissolved in MeOH (1.5 ml). After cooling the solution in an ice water bath, a solution of LiOH monohydrate (42 mg, 1 mmol) in water (1 ml) was added in portions over 1 hour. The reaction mixture was stirred overnight, then acidified with Dowex 50W x 8 hydrogen form and purified by RPLC (0-50% acetonitrile in water, using 0.1% TFA as modifier). Yield 78.1 mg, 99%. Ions detected by LCMS: [M+H] ⁺ =590.2.

Example 14 Synthesis of conjugate 3

The title conjugate was prepared similarly to Conjugate 1 (Example 9) using Intermediate-4 (Example 13). Maldi TOF analysis of the purified final product gave an average mass of 61,182 Da (DAR=3.4). Yield 50.89 mg, yield 51%. Figure 11 shows SDS-PAGE analysis under non-reducing conditions of conjugate 3.

Example 15 Synthesis of Intermediate-5

Stage a.

The flame-dried reaction flask was purged with nitrogen and charged with (1S, 2S, 3R, 4R)-methyl-3-((S)-1-acetamido-2-ethylbutyl)-4-(tert-butoxycarbonylamino)-2-hydroxycyclopentanecarboxylate (320, 4 mg, 0.8 mmol) and anhydrous DCM (2 ml). The solution was cooled in an ice water bath and DIPEA (312 mg, 2.4 mmol) was added, followed by 4-nitrophenyl chloroformate (161.3 mg, 0.8 mmol) and DMAP (98 mg, 0.8 mmol). The resulting mixture was stirred for 15 minutes, then additional 4-nitrophenyl chloroformate (161.3 mg, 0.8 mmol) was added. The reaction mixture was stirred for 2 hours, then water (0.2 ml) was added to quench the unreacted chloroformate. After stirring for 10 minutes, a solution of propargyl-PEG4-amine (259 mg, 1.12 mmol) in anhydrous DMF (0.5 ml) was added. The reaction mixture was stirred for 1 hour and then purified directly by RPLC (100 g, 5-60% acetonitrile in water, using 0.1% TFA as modifier). The collected fractions were lyophilized. Yield 391.2 mg, 74.4%. Ion detected by LCMS: [M+H] ⁺ =658.3, [M - Boc+H] ⁺ =558.3.

Stage b.

The product obtained in step-a (391.2 mg, 0.595 mmol) was dissolved in TFA (0.8 ml) and the solution was stirred at room temperature for 20 minutes. It was then directly purified by RPLC (100 g, 5-60% acetonitrile in water). Yield 323.2 mg, 81%. Ion detected by LCMS: [M+H] ⁺ =558.3.

Stage c.

To a solution of the product obtained in step-b (323.2 mg, 0.482 mmol) in THF (2 ml) was added N,N'-bis-Boc-1-guanylpyrazole (224.4 mg, 0.723 mmol) and PIPEA (260 mg, 2 mmol). The reaction mixture was stirred for 1 day and then extracted with water (3 ml) and EtOAc/hexanes (1:1, 8 ml). The organic layer was dried over Na ₂ SO ₄ and concentrated by rotary evaporation. The residue was redissolved in TFA (~1 ml) and the solution was stirred overnight. It was then directly purified by RPLC (100 g, 0-30% acetonitrile in water, using 0.1% TFA as modifier). The collected fractions were lyophilized. Yield 254.2 mg, 83.2%. Ions detected using LCMS: [M+H] ⁺ =600.3, [M - Boc+H] ⁺ =300.6.

Stage d.

The product obtained in step-c (254.2 mg, 0.356 mmol) was dissolved in THF (1.5 ml) and the solution was cooled in an ice water bath. CaCl ₂ dihydrate (419 mg, 2.85 mmol) was added, then 1.8 ml KOH (112 mg, 2 mmol) in water (2 ml) was added in portions over 1 hour. After stirring for an additional 3 hours, the reaction mixture was acidified with Dowex 50W x 8 hydrogen form and purified by RPLC (0-30% acetonitrile in water, using 0.1% TFA as modifier). Yield 102 mg, 49.8%. Ions detected using LCMS: [M+H] ⁺ =586.4, [(M+2H)/2] ⁺ =293.8.

Example 16 Synthesis of conjugate 4

The title conjugate was prepared similarly to Conjugate 1 (Example 9) using Intermediate-5 (Example 15). Maldi-TOF analysis of the purified final product gave an average mass of 63,002 Da (DAR=3.4). Yield 49.315 mg, yield 49%. Figure 12 shows SDS-PAGE analysis under non-reducing conditions of conjugate 4.

Example 17 Synthesis of Intermediate-6

Stage a.

The flame-dried reaction flask was purged with nitrogen and charged with (1S, 2S, 3R, 4R)-methyl-3-((S)-1-acetamido-2-ethylbutyl)-4-(tert-butoxycarbonylamino)-2-hydroxycyclopentanecarboxylate (280, 4 mg, 0.7 mmol) and anhydrous DCM (1 ml). After stirring to dissolve the starting material, DMAP (85.7 mg, 0.7 mmol) was added to the solution, followed by bis(pentafluorophenyl) carbonate (295.6 mg, 0.75 mmol). The resulting mixture was stirred for 1 hour, then added to a solution of Linker-2 (Example 5) (157.5 mg, 0.22 mmol) in anhydrous DMF (1 ml) and DIPEA (130 mg, 1 mmol). The reaction mixture was stirred overnight and purified by RPLC (50 g, 5-90% acetonitrile in water). The collected fractions were lyophilized. Yield 134.1 mg, 40.7%. Ion detected by LCMS: [(M+2H)/2] ⁺ =748.6.

Stage b.

The product obtained in step-a (134.1 mg, 0.0896 mmol) was dissolved in TFA (0.5 ml). The solution was stirred for 20 minutes, then purified directly using RPLC (50 g, 5-60% acetonitrile in water). Yield 108.4 mg, 93.4%. Ions detected by LCMS: [(M+2H)/2] ⁺ =648.3, [(M+3H)/3] ⁺ =432.8.

Stage c.

To a solution of the product obtained in step-b (108.4 mg, 0.0837 mmol) in anhydrous THF (1 ml) was added N,N'-bis-boc-1-guanylpyrazole (81.3 mg, 0.285 mmol) and DIEPA (65 mg, 0.5 mmol). The reaction mixture was stirred at room temperature for 2.5 days, then purified directly using RPLC (100 g, 40-75% acetonitrile in water). Yield 73.6 mg, 49.4%. Ion detected by LCMS: [(M+3H)/3] ⁺ =594.2.

Stage d.

The product obtained in step-c (73.6 mg, 0.041 mmol) was dissolved in TFA (0.5 ml). The solution was stirred for 20 minutes, then purified directly by RPLC (50 g, 5% to 60% acetonitrile in water). Yield 62.1 mg, 94.1%. Ions detected using LCMS: [(M+2H)/2] ⁺ =690.4, [(M+3H)/3] ⁺ =460.8.

Stage e.

The product obtained from step-d (62.1 mg, 0.0386 mmol) in THF (3 ml) was cooled in an ice water bath and 45% KOH solution (0.2 ml) was added in portions over 1 hour. The reaction mixture was stirred for another 2 hours, then acidified with a solution of 4N HCl in dioxane (0.8 ml) and extracted with hexanes (10 ml) and water (1.5 ml). The aqueous layer was purified by HPLC (0-20% acetonitrile in water, using 0.1% TFA as modifier). Yield 36.2 mg, 59.4%. Ions detected using LCMS: [(M+2H)/2] ⁺ =676.5, [(M+3H)/3] ⁺ =451.4.

Example 18. Synthesis of conjugate 5

The title conjugate was prepared similarly to Conjugate 1 (Example 9) using Intermediate-6 (Example 17). Maldi-TOF analysis of the purified final product gave an average mass of 63,561 Da (DAR=3.3). Yield 43.4 mg, yield 43%. Figure 13 shows SDS-PAGE analysis under non-reducing conditions of conjugate 5

Example 19 Synthesis of Intermediate-7

Stage a.

To a solution of pro-pargyl-PEG4 acid (609 mg, 2.34 mmol) and NH-bis(PEG1-azide) (500 mg, 2.055 mmol) in anhydrous DMF (2 ml) was added HATU (889.7 mg, 2. 34 mmol) in portions over 5 minutes. After stirring to dissolve all of the coupling reagent, DIPEA (390 mg, 3 mmol) was added and stirring was continued for 1 hour. It was then purified directly by RPLC (100 g, 5-40% acetonitrile in water). Yield 918 mg, 92%. Ion detected by LCMS: [M+H] ⁺ =486.2.

Stage b.

The product obtained in step a (918 mg, 1.89 mmol) was dissolved in THF and the solution was cooled to ~13°C. Triphenylphosphine (1.141 g, 4.35 mmol) was added in portions over 10 minutes. The resulting mixture was stirred at -13°C to room temperature for 2 hours. A solution of LiOH monohydrate (42 mg, 1 mmol) in water (2 ml) and MeOH (1 ml) was added. After stirring for 20 hours, the reaction mixture was purified by RPLC (100 g, 0-30% acetonitrile in water, using 0.1% TFA as modifier). Yield 825 mg, 65.9%. Ion detected by LCMS: [M+H] ⁺ =434.4.

Stage c.

The flame-dried reaction flask was purged with nitrogen and loaded with zanamivir intermediate (Example 2) (865 mg, 1.51 mmol) and anhydrous DCM (5 ml). After stirring to dissolve the starting material, the solution was cooled in an ice water bath and 4-nitrophenyl chloroformate (365.3 mg, 1.81 mmol) was added, followed by DMAP (152.2 mg, 0.755 mmol). The ice water bath was removed and the mixture was stirred for 3 hours. Additional 4-nitrophenyl chloroformate (304.4 mg, 1.51 mmol) was added and stirring was continued for 1 hour. The reaction was then quenched with water (1 ml). After vigorous stirring for 1 hour, the reaction mixture was extracted with water (20 ml x 2) and DCM (20 ml). The organic layer was stirred overnight, dried over Na2SO4 and concentrated by rotary evaporation. The material was used in the next step without further purification. Ion detected by LCMS: [M+H] ⁺ =738.2.

Stage d.

A solution of a mixture of the product obtained in step-b (429.7 mg, 0.65 mmol) and DIPEA (260 mg, 2 mmol) in anhydrous THF (1 ml) was added dropwise to the product obtained in step-c. The resulting mixture was stirred for 2 hours, then purified directly using RPLC (100 g, 10-65% acetonitrile in water). Acetonitrile in the collected fractions was removed by rotary evaporation at room temperature. The heterogeneous aqueous layer was extracted with EtOAc (200 ml), then extracted again with EtOAc (50 ml). The combined organic layers were dried over Na ₂ SO ₄ and concentrated to dryness by rotary evaporation. Yield 442 mg, 41.7%. Ions detected by LCMS: [(M+2H)/2] ⁺ =815.8, [(M - Boc+2H)/2] ⁺ =765.8, [(M - 2 Boc+2H)/2] ⁺ =716 .

Stage e.

The product obtained in step-d (441 mg, 0.271 mmol) was dissolved in TFA (1 ml). The solution was stirred for 20 minutes, then purified directly by HPLC (5-20% acetonitrile in water, using 0.1% TFA as modifier). Yield 308 mg, 77.9%. Ions detected using LCMS: [(M+2H)/2] ⁺ =615.8, [(M+3H)/3] ⁺ =411.

Stage f.

The product obtained in step-e (308 mg, 0.211 mmol) was dissolved in MeOH (1 ml) and water (0.5 ml), and the solution was cooled in an ice water bath. A solution of LiOH monohydrate (42 mg, 1 mmol) in water (1 ml) was added in portions over 1 hour. The reaction mixture was stirred overnight, acidified with 4 N HCl in dioxane (0.25 ml) and purified by RPLC (0-20% acetonitrile in water, using 0.1% TFA as modifier). Yield 198 mg, 64%. Ions detected using LCMS: [(M+2H)/2] ⁺ =575.8, [(M+3H)/3] ⁺ =384.2.

Example 20. Synthesis of conjugate 6

The specified conjugate was prepared similarly to conjugate 1 (example 9) using intermediate compound-7 (example 19). Maldi-TOF analysis of the purified final product gave an average mass of 62,854 Da (DAR=3.1). Yield 175.4 mg, yield 50%. Figure 14 shows SDS-PAGE analysis under non-reducing conditions of conjugate 6.

Example 21 Synthesis of Intermediate-8

Stage a.

Methyl 5-acetoamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate (1,8 g, 4.0 mmol) was dissolved in 40 ml methanol and then treated with 400 mg 5% Pd/C and 1.1 g Boc anhydride (5.0 mmol), then the reaction mixture was stirred at room temperature under hydrogen atmosphere for 1 hours. Palladium on carbon was removed by filtration. The filtrate was concentrated and used in the next step without purification.

Stage b.

The product obtained in the previous step was dissolved in 20 ml of dry methanol and then treated with 2 ml of sodium methoxide in methanol (0.5 M) dropwise while cooling in an ice water bath. After 2 hours, the progress of the reaction was determined using LCMS. The reaction was quenched with 1N HCl to pH 5-6. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 5-100% acetonitrile and water, using 0.1% TFA as a modifier. Ions detected by LCMS: (M+H) ⁺ =405

Stage c.

Methyl 5-acetamido-2,6-anhydro-4-[(tert-butoxycarbonyl)amino]-3,4,5-trideoxy-D-erythronon-2-enonate (0.4 g, 1 mmol) was dissolved in 10 ml of acetone, 4 ml of 2,2-dimethoxypropane and 20 mg of p-toluenesulfonic acid hydrate (0.1 mmol). The resulting solution was stirred at room temperature overnight, then quenched with 1 ml of saturated NaHCO ₃ . The mixture was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 5-100% acetonitrile and unmodified water. Ions detected by LCMS: (M+H) ⁺ =445.

Stage d.

Sodium hydride (40 mg, 60% in oil, 1.0 mmol) was added to methyl 5-acetamido-2,6-anhydro-4-[(tert-butoxycarbonyl)amino]-3,4,5-trideoxy-8. 9-O-(1-methylethylidene)-D-erythronone-2-enonate (0.25 g, 0.50 mmol) in 5 ml dry THF while cooling in an ice water bath. The resulting solution was stirred for 0.5 hour, then benzyl bromoacetate (0.23 g, 1.0 mmol) was added. The resulting solution was stirred for 2 hours and quenched with 5 ml of 10% ammonium chloride in water. The solution was then diluted with 50 ml of ethyl acetate. The organic layer was separated and dried with sodium sulfate. The dried organic solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 5-100% acetonitrile and unmodified water. Ions detected by LCMS: (M+H) ⁺ =593.

Example 22 Synthesis of Intermediate 9

Stage a.

Methyl 5-acetoamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate (10 g, 22 mmol) was dissolved in 100 ml of methanol, and then heated to 60 ° C in an oil bath, SnCl ₂ (5.7 g, 20 mmol) was added to the solution in 3 portions (caution, gas is released). The reaction mixture was stirred for 10 minutes, after which the reaction was complete by HPLC. The reaction solution was slowly added to a solution of 50 ml of saturated NaHCO ₃ and 50 g of celite with vigorous stirring. The resulting suspension was filtered. The filtrate was treated with _BoC2O (6.6 g, 30 mmol, 1.5 eq.). After 2 hours at room temperature, the solution was concentrated to remove most of the methanol, dissolved in 200 ml DCM and extracted twice with 100 ml DCM. The combined extracts were dried over sodium sulfate, filtered and used in the next step without further purification. The yield of the crude product was 12 g, 100%. Ion(s) detected by LCMS: M+H=531.

Stage b.

The material obtained in the previous step was dissolved in 60 ml of dry methanol, then treated with 10 ml of sodium methoxide in methanol (0.5 M) while cooling in an ice water bath. The progress of the reaction was monitored using LCMS, which was completed after 2 hours. The reaction was quenched with 1N HCl to pH 5-6. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0% to 30% acetonitrile and water, using 0.1% TFA as a modifier. The product yield was 7.2 g, 80%. Ions detected by LCMS: M+H=405.

Stage c.

A solution of the product obtained in the previous step (3.5 g, 8.5 mmol), CDI (2.8 g, 2 eq.), trimethylamine (4.2 ml, 30 mmol) and DMAP (240 mg, 2 mmol) heated in acetonitrile (50 ml) overnight, then concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0-30% acetonitrile and unmodified water. The yield of the desired product was 2.3 g, 60%. Ions detected by LCMS: M+H=431.

Stage d.

Sodium hydride (400 mg, 60% in oil, 10 mmol) was added to the product obtained in the previous step (1.45 g, 3.3 mmol) in 50 ml of dry THF (moisture sensitive reaction) in an ice water bath. The resulting solution was stirred for 0.5 hours, then tert-butyl bromoacetate (2 g, 10 mmol) was added to the above solution, the resulting solution was heated to 60°C overnight and quenched with acetic acid. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0-50% acetonitrile and water with TFA as a modifier. Yield 1 g, 57%. Ions detected by LCMS: M+H=593.

Stage e.

The product obtained in the previous step (1.2 g, 2.2 mmol) was stirred with 10 ml TFA at room temperature overnight, and the progress of deprotection was monitored using LCMS. The resulting solution was concentrated and used in the next step without purification. The residue was redissolved in 20 ml THF, then N,N'-bis-boc-1-guanylpyrazole (1 g, 3.3 mmol), 4-dimethylaminopyridine (120 mg, 1 mmol) and triethylamine (0.7 ml, 5 mmol), and the resulting solution was heated to 60°C for 2 hours. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0-50% acetonitrile and unmodified water. Yield 700 mg, 84%. Ions detected by LCMS: M+H=631.

Stage f.

To a solution of linker-3 (prepared as described in example 19) (73 mg, 0.14 mmol) and the product obtained in the previous step (200 mg, 0.32 mmol, 2.2 eq.) in DMF (30 ml ) EDC (100 mg, 0.5 mmol), HOAt (65 mg, 3 mmol) and DIEA (0.14 ml, 1 mmol) were added at room temperature. The solution was stirred overnight. The resulting solution was concentrated, purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with gradients of 0-50% acetonitrile in water without modifier. Yield 120 mg, 52%. Ions detected by LCMS: M/2+H=830.

Stage g.

Lithium hydroxide (24 mg, 1 mmol) in 2 ml H ₂ O was added to a solution of the product obtained in the previous step (120 mg, 0.07 mmol) in 2 ml THF and 1 ml MeOH. The progress of the reaction was monitored using LCMS. Once complete, AMBERLITE® IRN-77 ion exchange resin was added to the solution to adjust the pH to 1, then the resulting solution was filtered and the filtrate was concentrated and used in the next step without purification. The resulting compound was treated with 2 ml TFA at room temperature, the solution was stirred overnight at 40°C, then concentrated and purified by HPLC, eluting with a gradient of 0-20% acetonitrile in water, using TFA as a modifier. Yield 60 mg, yield 74%. Ions detected by LCMS: [M/2] ⁺ 1=589.8.

Example 23 Neuraminidase Inhibition Assay

A neuraminidase inhibition assay using the substrate 2'-(4-methylumbelliferyl)-alpha-DN-acetylneuraminic acid (MUNANA) was performed as described below. Briefly, 50 μl of purified recombinant influenza virus neuraminidase (0.1 ng/μl, 50 mM Tris, 5 mM CaCl ₂ , 200 mM NaCl, pH 7.5) was mixed with 50 μl of inhibitor and incubated for 30 minutes at room temperature. For each replicate, at least 5 concentrations of each inhibitor were used within the appropriate range. After incubation, 50 μl of 400 μM MUNANA in 50 mM Tris, 5 mM CaCl ₂ , 200 mM NaCl (pH 7.5) was added to the solution to initiate the reaction using a 12-tip pipette (Eppendorf). Positive and negative controls were included in each 12-well lane. After starting the reaction for each lane on the plate, the reaction mixture was immediately loaded into a SpectraMax M5 (Molecular Devices), where fluorescence was quantified within 25 min at an excitation wavelength of 365 nm and an emission wavelength of 445 nm. Single time points were selected where the positive control produced a fluorescent signal of approximately 1000. All assays were performed in triplicate, and IC50 values for each inhibitor were calculated by sigmoidally fitting the logarithm of [inhibitor] versus percent inhibition using GraphPad Prism. Figure 15 shows a plot of kinetic data in terms of RFU/min in the linear range for Conjugate 1 and Intermediate-2, demonstrating the greater potency of Conjugate 1 as a neuraminidase inhibitor. Figure 16 and Figure 17 show the results of the analysis of conjugates 1-6 against neuraminidase rH1N1 and neuraminidase rH3N2, respectively (Table 2).

Example 24: Cytotoxicity Assay

Conjugates 1, 2, 3 and 6 were tested for cytotoxicity. Ten two-fold serial dilutions of each conjugate, starting at 10 μM, were prepared in triplicate for inoculation with MDCK cells in 96-well culture plates. Cell viability was determined four days after treatment using the C ell Titer-GLO kit. The 50% cytotoxic concentration ( _CC50 ) was calculated using the XLfit dose-response model (Table 3). For all compounds tested, no cytotoxicity was observed up to 10 μM, with the exception of Conjugate 3. In the case of Conjugate 3, the EC ₅₀ value in the cytopathic effect (CPE) assay (see Example 23) is 725 times lower than the CC ₅₀ value in this assay.

Example 25 Analysis of cytopathic effect

CPE Based Microneutralization Assay #1

To measure the ability of neuraminidase conjugates to protect mammalian cells from influenza virus infection and destruction, microneutralization cytopathic effect (CPE) assays were performed. Briefly, twenty-two-fold serial dilutions of each conjugate, starting at 0.25 μM, were prepared in duplicate for one-hour inoculations with MDCK cells seeded in 96-well plates. INFV CA/09 virus was added to cells at a multiplicity of infection (MOI) of 0.001 for one hour incubation. On the fourth day after incubation, cells were stained with crystal violet and absorbance was read to calculate the 50% effective concentration (EC50) of each TA using the XLfit dose-response model. Zanamivir was used as a comparator and positive control. Human Fc was included as a negative control. Conjugates 1, 2, 3 and 6 all showed superior efficacy compared to the zanamivir control, demonstrating EC50 values 42-227 times lower than zanamivir (Table 4).

CPE-Based Microneutralization Assay #2

An additional CPE-based microneutralization assay was performed to further evaluate the in vitro activity of conjugate 6 and conjugate 7. Briefly, test samples were prepared at an initial concentration of 160 nM, and a total of ten 2-fold dilutions were made. Dilutions of the test substance were then premixed with influenza A virus (INFV CA/09) at a multiplicity of infection of 0.001 relative to the monolayer. After one hour, the test substance/virus mixture was added to Madin-Darby canine kidney (MDCK) cells grown to 70-80% confluency under standard conditions in a 96-well plate. Control zanamivir was treated in the same way except that the initial concentration was 9600 nM as it was expected to be less active than the conjugates tested. After four days of incubation, the monolayer was stained with crystal violet and the absorbance (reflecting the state of the monolayer) was determined to calculate the 50% effective concentration (EC50) using the XLfit dose-response model (idbs; https://www.idbs.com).

As shown in Table 5, zanamivir at a concentration of 496 nM reduced virus-mediated cytopathic effects by half. In contrast, conjugate 6 was approximately 100-fold more active with an EC50 value of only 4.64 nM. Conjugate 7 further improved activity by approximately 15-fold, reducing the EC50 to below subnanomolar levels.

Example 26 Cell viability assay.

A549 cells were seeded in 96-well plates the day before compound treatment. Cells were treated with conjugate 3 (FIG. 18A), conjugate 4 (FIG. 18B), or conjugate 6 (FIG. 18C) at concentrations of 1 μM - 10 nM for 24 hours. Cell viability was then determined using Cell Titer Glow (Promega). Results represent the mean and standard deviation (SD) of three biological replicates. No effects on cell viability were observed for any of the conjugates at any of the concentrations used in the viral growth assay (Example 27).

Example 27 Virus growth assay.

To measure the ability of neuraminidase conjugates to inhibit the growth of pathogenic influenza virus strains of interest in human epithelial cells, viral plaque reduction assays were performed. Briefly, A549 cells were seeded in 24-well plates the day before compound treatment. Cells were treated with compounds at concentrations of 1 μM - 10 nM for 2 hours and infected with the indicated viral strains at an MOI of 0.01 for a one-hour incubation. The virus was removed, the cells were washed and the compounds were reapplied at concentrations of 1 μM - 10 nM. Supernatants were collected at the indicated time points and titrated using the plaque assay method in MDCK cells. Results represent the mean and standard deviation (SD) of three biological replicates. Oseltamivir was used as a reference drug and positive control. Conjugate 3 (FIGS. 19A-19E), conjugate 4 (FIGS. 20A-E) or conjugate 6 (FIGS. 21A-21E and FIGS. 22A-22E) all showed superior performance compared to the oseltamivir control, showing similar or similar typically) superior plaque reduction compared to oseltamivir at concentrations 100 times lower. The effects of compounds (to evaluate test samples for potential cytotoxicity) on A549 cells were assessed for each test substance using a cell viability assay (Example 26). Briefly, A549 cells were seeded in 96-well plates the day before compound treatment. Cells were treated with compounds at concentrations of 1 μM - 10 nM for 24 hours. Cell viability was then determined using Cell Titer Glow (Promega). Results represent the mean and standard deviation (SD) of three biological replicates.

Example 28 Half-life in mouse serum

Female CD-I mice (Charles River Laboratories) weighing 20-22 grams were used in pharmacokinetic (PK) studies. Mice were administered intravenously (IV) via the tail vein with 50 mg/kg of test substance (dose volume 10 ml/kg). Animals were kept under conditions approved by the IACUC (Institutional Animal Care and Use Committee). At certain time points, animals were bled without definitive arrest (retroorbital, buccal, or tail vein) and collected in tubes containing EDTA to prevent coagulation. Collected blood was centrifuged (2000 x g for 10 minutes) and plasma was collected for analysis of test substance concentrations over time.

Conjugate 6 or hIgG1 Fc plasma concentrations at each time point were measured by sandwich ELISA as follows: conjugate 6 molecules were captured on either neuraminidase-coated plates or anti-hIgG1 antibody-coated plates and then detected using HRP-conjugated anti-human IgG-Fc antibody. hIgG1 was captured using anti-hIgG1 Fc antibody. Protein concentration was calculated in GraphPad Prism using a four-parameter nonlinear regression (4PL) model of conjugate 6 (or hIgG1 Fc) standard curves. A more detailed description of the method is given below.

Qiagen Ni-NTA HisSorb plates (catalog number 35061, Qiagen) were coated with either A/California/04/2009 (H1N1) virus neuraminidase (11058-VNAHC, Sino Bio) or anti-IgG1 Fc antibody in coating buffer IX from KPL (5150-0041, SeraCare). In cases where an anti-IgG1 Fc antibody was used to capture a test substance, capture and detect anti-IgG1 Fc antibodies that bind to different epitopes were selected. The plates were incubated at room temperature for 1 hour. Serial dilutions of plasma samples were added to plates and incubated at room temperature for 2 h (sample diluent: 0.5% nonfat dry milk + 3% goat serum in a solution of 0.05% Tween20 in PBS; final concentration in plasma of unexposed mice was 1:900). Standard curves of conjugate 6 or hIgG1 Fc in the range of 500-0.230 ng/ml were reproduced in duplicate on each plate.

After a 2-hour incubation, the plates were washed with 5x 300 μl PBS with 0.05% Tween20. Neuraminidase-coupled conjugate 6 (or anti-hIgG1 Fc antibody) was then labeled with HRP-conjugated anti-human IgG Fc F(ab')2 (Jackson 709-036-098) diluted 1:2000 in sample diluent for 1 hours at room temperature. The plates were then washed with 8x 300 µl PBS with 0.05% Tween20 and developed with TMB substrate for 7 minutes. The reaction was stopped with IN H2SO4. Absorbance was read at 450 nm. A similar protocol was used for hIgG1 Fc, where only the anti-hIgG1 Fc antibody was used for capture. Amounts of conjugate 6 measured at different time points using either neuraminidase or anti-hIgG1 Fc antibody capture were similar within experimental error, suggesting that the intact conjugate is stable in vivo.

Total conjugate 6 (or hIgG1 Fc) in test samples was interpolated using Graphpad Prism Version 6 after non-linear regression analysis (sigmoidal, 4Pb analysis) of conjugate 6 (or hIgG1 Fc) standard curves. DO parameters were calculated using WinNonlin software. Comparison curves between conjugate 6 and hIgG1 Fc are shown in Figure 23, and a summary of key PK parameters is presented in Table 6. Surprisingly, plasma concentrations and terminal half-life were significantly better for conjugate 6 than for wild-type hIgG1 Fc. The area under the curve (AUC) at 8 days is 3 times higher for conjugate 6 than for hIgG1 Fc, and the terminal half-life for conjugate 6 is 214 hours compared to 52 hours for human IgG1 Fc.

Example 29. Efficacy of Conjugate 6 in a lethal mouse model of influenza: Study No. 1.

Conjugate 6 was evaluated against lethal influenza INFV A H1N1 virus infection in female BALB/c mice. The experiment involved 7 groups of 5 mice. On day 0, all mice were challenged with 1xLD90 H1N1 A/Texas/36/91. Groups 1-6 received IV treatment 4 hours before infection (Table 7). Human IgG1 (Fc only) was included as an additional negative control. Group 7 received oseltamivir phosphate orally, starting 8 hours after infection, twice daily for 5 days. All mice were monitored for weight loss (FIG. 24, Table 8) and survival (FIG. 25, Table 9) for 15 days postinfection.

Mice treated with conjugate 6 showed 100% survival at single doses at all concentrations tested, compared with survival of 20% and 0% in the vehicle control group and the hIgG1 control group, respectively. The results were statistically significant compared with the vehicle group (p=0.0135), despite the small group size (n=5). Compared to the oseltamivir phosphate group, conjugate 6 demonstrated similar efficacy at 500-fold lower cumulative dose (mg/kg). Mice at all doses of conjugate 6 maintained their weight throughout the experiment compared to the oseltamivir control group.

Example 30 Efficacy of Conjugate 6 in a Lethal Mouse Model of Influenza: Study #2

Conjugate 6 was evaluated against lethal influenza virus INFV A H3N2 infection in female BALB/c mice. The experiment involved 11 groups of 5 mice. On day 0, all mice were infected with 1xLD90 H3N2 A/Hong Kong/1/68. Groups 1-10 received IV treatment 4 hours before infection (Table 10). Human IgG1 (taken as Fc alone) was included as an additional negative control. Group 11 received oseltamivir phosphate orally starting 8 hours after infection twice daily for 5 days. All mice were monitored for weight loss (FIG. 26) and survival (FIG. 27, Table 11) for 15 days postinfection.

Mice treated with conjugate 6 showed 100% survival at single doses up to 0.4 mg/kg and 80% survival at a single dose of 0.2 mg/kg, compared to 0% survival in vehicle and control groups , receiving hIgG1, respectively. In the control group receiving oseltamivir, 80% of mice survived. The results were statistically significant compared with the vehicle group (p=0.0128), despite the small group size (n=5). Compared to the oseltamivir phosphate group, conjugate 6 demonstrated similar efficacy at a dose that was 1000 times lower cumulative (in mg/kg). Mice at all doses of conjugate 6 to 0.4 mg/kg maintained their weight within 5% better than the control group receiving oseltamivir.

Example 31 Synthesis of Intermediate 10

Stage a.

Methyl 5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate (SM-1 , 10 g, 22 mmol) was dissolved in 100 ml of methanol and then heated to 60 ° C in an oil bath, SnCl ₂ (5.7 g, 20 mmol) was added to the solution in 3 portions (caution, gas is released). The reaction mixture was stirred for 10 min, after which the reaction was complete according to HPLC. The reaction solution was slowly added to a solution of 50 ml of saturated NaHCO ₃ and 50 g of celite with vigorous stirring. The resulting suspension was filtered. The filtrate was treated with Boc ₂ O (6.6 g, 30 mmol, 1.5 eq.). After 2 hours at room temperature, the solution was concentrated to remove most of the methanol, dissolved in 200 ml DCM and extracted twice with 100 ml DCM. The combined extracts were dried over sodium sulfate, filtered and used in the next step without further purification. The yield of the crude product was 12 g, 100%. Ion(s) detected by LCMS: M+H=531.

Stage b.

The material obtained in the previous step was dissolved in 60 ml of dry methanol, then treated with 10 ml of sodium methoxide in methanol (0.5 M) while cooling in an ice water bath. The reaction progress was monitored using LCMS, which was completed after 2 hours. The reaction was quenched with 1N HCl to pH 5-6. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0% to 30% acetonitrile and water, using 0.1% TFA as a modifier. The product yield was 7.2 g, 80%. Ion(s) detected by LCMS: M+H=405.

Stage c.

A solution of the intermediate from the previous step (3.5 g, 8.5 mmol), CDI (2.8 g, 2 eq), trimethylamine (4.2 ml, 30 mmol) and DMAP (240 mg, 2 mmol ) heated at 60°C in DMF (50 ml) overnight, then concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluting from 0% to 30% acetonitrile without modifier. The yield of the desired product was 2.3 g, 60%. Ion(s) detected by LCMS: M+H=431.

Stage d.

Sodium hydride (400 mg, 60% in oil, 10 mmol) was added to intermediate 4 (1.45 g, 3.3 mmol) in 50 ml dry THF (moisture sensitive reaction) under an ice bath. The resulting solution was stirred for 0.5 h, then tert-butyl bromoacetate (2 g, 10 mmol) was added to the above solution. The resulting solution was heated to 60°C overnight and quenched with acetic acid, concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0% to 50% acetonitrile and water with TFA as modifier. The yield was 1 g, 57%. Ion(s) detected by LCMS: M+H=593 (reaction quite pure by HPLC, but low isolated yield).

Stage f.

The intermediate obtained in the previous step (1.2 g, 2.2 mmol) was stirred with 10 ml TFA at room temperature overnight. The progress of deprotection was monitored using LCMS. The resulting solution was concentrated and used in the next step without purification.

The residue obtained from the previous reaction was dissolved in 20 ml of THF, then N,N'-bis-boc-1-guanylpyrazole (1 g, 3.3 mmol), 4-dimethylaminopyridine (120 mg, 1 mmol) was added to the solution. and triethylamine (0.7 ml, 5 mmol), and the resulting solution was heated to 60°C for 2 hours. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0-50% acetonitrile and unmodified water. Yield 700 mg, 84%. Ion(s) detected by LCMS: M+H=631.

Stage g.

To a solution of linker-3 (prepared as described in Example 19) (73 mg, 0.14 mmol) and intermediate obtained in the previous step (200 mg, 0.32 mmol, 2.2 eq.) in DMF (30 ml) EDC (100 mg, 0.5 mmol), HOAt (65 mg, 3 mmol) and DIEA (0.14 ml, 1 mmol) were added at room temperature. The solution was stirred overnight. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0-50% acetonitrile and unmodified water. The yield was 120 mg, 52%. Ion(s) detected by LCMS: M/2+H=830.

Stage h.

Lithium hydroxide (24 mg, 1 mmol) in 2 ml water was added to a solution of the intermediate obtained in the previous step (120 mg, 0.07 mmol) in 2 ml THF and 1 ml MeOH. Once the reaction was complete by LCMS, the solution was quenched with AMBERLITE® IRN-77 ion exchange resin to adjust the pH to 1, then the solution was filtered and the filtrate was concentrated and used in the next step without further purification. The intermediate obtained in the previous step was treated in 2 ml TFA at room temperature overnight at 40°C. The crude reaction mixture was concentrated and purified by HPLC using 0-20% acetonitrile and water using TFA as a modifier. The yield was 60 mg, yield 74%. Ion(s) detected by LCMS: [M/2]+1=589.8.

Example 32. Synthesis of conjugate 7

A solution of Ec-PEG4 azide (each Fc domain monomer having the sequence shown in SEQ ID NO: 38) in PBSx1 buffer solution (100 mg, 1.28 µmol, 6.1 ml, MW=57260 Da, DAR=3 ,6) was added to intermediate-10 (prepared as described in Example 31) (TFA salt, 44 mg, 0.031 mmol) and freshly prepared solutions in PBS pH 7.4 CuSO ₄ (0.7 ml, 50.0 mmol , 20 equiv.), tris(3-hydroxypropyltriazolyl methyl)amine (THPTA, 0.7 ml 50.0 mmol, 20 equiv.) and sodium ascorbate (1.05 ml 50.0 mmol, 30 equiv.). The resulting homogeneous solution was stirred on a shaker for 12 hours. The crude solutions were diluted with PBS pH 7.4 to a final concentration of 1 mg/ml and ultrafiltered twice (10,000 MWCO) to a volume of 1 ml. The crude mixtures were then diluted 1:10 in PBS pH 7.4 and purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA), followed by size exclusion chromatography. The purified product was quantified using a Nanodrop™ UV-visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of the Fc used for conjugation, and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 µg of each molecule onto the gel and staining using flash blue staining.Each gel included a molecular weight bar with the molecular weight standards indicated.Yields are typically 40-60%. MALDI MS analysis showed a mass range (60000-90000) with an average mass of 63633. Average DAR=4.5.

Example 33. Efficacy of conjugate 6 in a model of lethal influenza in pneumonia: study No. 3

Conjugate 6 was evaluated against lethal influenza INFV A H1N1 virus infection in female BALB/c mice. The experiment involved 7 groups of 5 mice. On day 0, all mice were challenged with 1xLD90 H1N1 A/Texas/36/91. Groups 1-6 received IV treatment 28 days before infection (Table 12). Vehicle (PBS) was included as an additional negative control. Group 7 received oseltamivir phosphate orally starting 8 hours after infection twice daily for 5 days. All mice were observed for survival (FIGS. 31A-31F) for 15 days post-infection.

Mice treated with conjugate 6 showed 100% survival at single doses at all concentrations up to 2.5 mg/kg and 80% survival at 1.25 mg/kg, compared to 0% survival in vehicle-treated controls. All mice in the control group treated with oseltamivir phosphate survived. Compared with the oseltamivir phosphate group, Conjugate 6 demonstrated similar efficacy to oseltamivir at a 20-fold lower cumulative dose (in mg/kg), despite the fact that all groups receiving Conjugate 6 were dosed once, over 28 days before infection.

Example 34: Efficacy of Conjugate 6 in a Lethal Mouse Influenza Model: Study #4

Conjugate 6 was evaluated against lethal influenza INFV A H1N1 virus infection in female BALB/c mice. The experiment involved 13 groups of 5 mice. On day 0, all mice were challenged with 1xLD90 H1N1 A/Texas/36/91. All groups receiving conjugate 6 (8-13) received single IV doses of 10 mg/kg at various times before and after infection, as shown in Table 13. Vehicle (PBS) and Fc alone were included as negative controls. Groups 3-7 received oseltamivir phosphate (20 mg/kg) by oral delivery starting at different time points after infection twice daily for 5 days, as shown in Table 13. All mice were observed for survival (FIGS. 32A-32F ) within 15 days after infection.

Mice treated with conjugate 6 showed 100% survival with single doses of 10 mg/kg when treated 24 hours postinfection, and 60 and 80% survival at 48 and 72 hours postinfection, respectively. In the group receiving oseltamivir phosphate, survival dropped sharply to 0% and 20%, respectively, in the groups in which treatment was started 48 and 72 hours after infection. These results suggest that conjugate 6 potentially has a better therapeutic window compared to oseltamivir in the treatment of influenza A virus infections.

Example 35 Toxicity Study of Conjugate 6

The safety of conjugate 6 was assessed in a 14-day dose-ranging toxicity study in rats. Rats were administered conjugate 6 at a dose of 5, 20 or 50 mg/kg body weight intravenously on days 0 and 7 of the study. Compared to vehicle controls, no significant effects on body weight gain (FIG. 33), organ weight, or food intake were observed at any dose tested. Plasma concentrations (measured by AUC) increased in a dose-proportional manner. These preclinical safety results are consistent with a high therapeutic index. A summary of the observations is presented in Table 14.

Example 36: Efficacy of Conjugate 6 versus Dose, by Three Different Routes, in a Lethal Mouse Influenza Model: Study #5

Conjugate 6 was evaluated against lethal IAV H1N1 influenza virus infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks). Viral Elimination Assessment and Characterization Virus (A/Texas/36/91) is a mouse-adapted isolate capable of causing lethal infections in mice. The experiment involved 15 groups of 5 mice. On day 0, all mice were infected with virus at 1x LD90 by intranasal inoculation in a volume of 50 μl after light anesthesia with isoflurane. Groups 1-14 received a single treatment 4 hours before infection (Table 15). To determine the effectiveness of the conjugate, appropriate concentrations of the conjugate (10, 2, 0.4 and 0.1 mg/kg) were administered intravenously (IV), intramuscularly (IM) or subcutaneously (SC) in 6 different ways. In addition to the vehicle-only group (PBS), human IgG1 (Fc alone) was included as an additional negative control (group 2). Group 15 received oseltamivir phosphate orally starting 8 hours after infection twice daily for 5 days. All mice were observed for survival (Table 15) for 14 days.

Mice treated with conjugate 6 against influenza virus (H1N1) challenge with single doses of 10, 2 or 0.4 mg/kg showed 100% survival at day 14, regardless of route of administration. Only at the lowest concentration of test drug were differences observed in individual mice between intravenous (IV), intramuscular (IM), and subcutaneous (SC) doses (Table 16; 60, 80, and 100% survival, respectively). These results strongly suggest that protective concentrations of conjugate 6 in the lungs can be achieved by several routes of administration.

Example 37: Efficacy of Conjugate 6 against Oseltamivir-Resistant Isolate in a Lethal Influenza Model: Study #6

Conjugate 6 was evaluated against lethal IAV H1N1 influenza virus infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Perth/261/2009) is a mouse-adapted isolate carrying the H275Y mutation, resulting in resistance to the neuraminidase inhibitor oseltamivir. The experiment involved 10 groups of 5 mice. On day 0, all mice were infected with A/Perth/261/2009 (H1N1) at 1x LD90 by intranasal inoculation in a volume of 50 μl to mice lightly anesthetized with isoflurane. Groups 1-9 received a single treatment by intravenous administration 4 hours before infection (Table 17). In addition to the vehicle-only (PBS) group, human IgG1 (Fc alone) was included as an additional negative control. Group 10 received oseltamivir phosphate orally starting 8 hours after infection twice daily for 5 days. All mice were monitored for survival (FIGS. 34A-34D, Table 18) and weight loss (FIG. 35, Table 19) for 15 days post-challenge.

Mice treated with conjugate 6 at single doses of 50, 10 and 2 mg/kg showed 100% survival after influenza virus infection (A/Perth/261/2009). Additionally, despite the small group size (n=5), these results were statistically significant compared to the vehicle control group (Table 18). At conjugate 6 concentrations of 0.4, 0.2, or 0.1 mg/kg, survival was 60%, with no mice surviving to the end of the study when administered vehicle alone (PBS) or hIgG1 (Fc alone). ). In the oseltamivir group, only one animal survived (20% efficacy) despite treatment with a dose previously shown to protect against oseltamivir-susceptible isolates (20 mg/kg, twice daily x 5 days). These results confirm that the challenge virus is resistant to oseltamivir and sensitive to conjugate 6.

The activity of conjugate 6 against mutants containing the H275Y mutation is further supported by body weight data (Fig. 35, Table 19). Groups receiving a single dose of Conjugate 6 at concentrations of 2 mg/kg or more demonstrated short-term weight loss of 5% or less.

Example 38. 7-day pharmacokinetic study (PK) in rats after intravenous administration of the study drug.

Pharmacokinetic (PK) studies in rats were performed at Seventh Wave Laboratories (Maryland Heights, MO) using male Sprague Dawley rats aged 46-49 days. Rats were administered intravenously through the tail vein 75 mg/kg of the test drug (dose volume 5 ml/kg). Animals were maintained under standard housing conditions approved by the IACUC. At appropriate time points, animals were induced to bleed non-fatally (retro-orbital, buccal or tail vein) and the blood was collected in tubes containing K ₂ EDTA to prevent coagulation. Collected blood was centrifuged (2000 x g for 10 minutes) and plasma was collected for analysis of study drug concentrations over time.

Plasma concentrations of conjugate 6 or hIgG1 Fc at each time point were measured by sandwich ELISA as follows: conjugate 6 molecules were captured on either neuraminidase-coated plates or anti-hIgG1 antibody-coated plates and then detected with an HRP-conjugated antibody against human IgG-Fc. hIgG1 was captured using an anti-hIgG1 Fc antibody. Protein concentration was calculated in GraphPad Prism using 4PL nonlinear regression of conjugate 6 (or Fc hIgG1) standard curves. A more detailed description of the method is given below.

Nunc Maxisorp 96-well plates (cat. no. 12-565-136, ThermoFisher) were coated with either A/California/04/2009 (H1N1) neuraminidase (11058-VNAHC, Sino Biological) or anti-Fc IgG1 antibody in IX KPL coating buffer (5150-0041, SeraCare). In cases where an anti-IgG1 Fc antibody was used to capture the test drug, anti-IgG1 Fc capture and detection antibodies that bind different epitopes were selected. The plates were incubated at room temperature for 1 h on an orbital plate shaker (500 rpm). Serial dilutions of plasma samples were added to plates and incubated at room temperature for 2 h (sample diluent: 1% BSA in PBS 0.05% Tween 20 + final plasma concentration in unexposed rats 1:900). Standard curves of conjugate 6 or hIgG1 Fc in the range of 500-0.230 ng/ml were reproduced in duplicate on each plate.

After a 2-hour incubation, the plates were washed 5 times in 300 μl PBS with 0.05% Tween 20. Neuraminidase conjugate 6 on the plates (or anti-hIgG1-Fc antibody) was then labeled with HRP-conjugated anti-human IgG Fc antibody F( ab')2 (709-036-098, Jackson) diluted 1:1000 in sample diluent for 1 h at room temperature. The plates were then washed 8 times in 300 µl PBS with 0.05% Tween 20 and developed with TMB substrate for 7-8 minutes. The reaction was stopped with 1N H ₂ SO ₄ . Absorbance was read at 450 nm. A similar protocol was used for hIgG1-Fc, where only the anti-hIgG1-Fc antibody was used for capture. Amounts of conjugate 6 measured at different time points using either neuraminidase or anti-hIgG1-Fc capture were similar within experimental error, suggesting that the intact conjugate is stable in vivo.

The total conjugate 6 (or hIgG1-Fc) in the test samples was interpolated using Graphpad Prism Version 6 software after non-linear regression analysis (sigmoid curve analysis using a four-parameter nonlinear regression (4PL) model) of the standard conjugate 6 (or hIgG1-Fc) curves. Curves comparing conjugate 6 and hIgG1-Fc are shown in Fig. 36. Amounts of conjugate 6 measured at different time points using either neuraminidase or anti-hIgG1-Fc capture were similar within experimental error, suggesting that the intact conjugate is stable in vivo.

Example 39. 14-day pharmacokinetic (PK) study in rats after intravenous administration of the test drug

Pharmacokinetic (PK) studies in rats were performed at Seventh Wave Laboratories (Maryland Heights, MO) using male Sprague Dawley rats aged 46-49 days. Rats were administered intravenously via the tail vein 5, 20, or 50 mg/kg of test drug (dose volume 5 ml/kg) on days 1 and 8. Animals were maintained under standard IACUC-approved housing conditions. At certain time points, animals were induced to bleed non-fatally (retro-orbital, buccal or tail vein) and the blood was collected in tubes containing K ₂ EDTA to prevent coagulation. Collected blood was centrifuged (2000 x g for 10 minutes) and plasma was collected for analysis of test drug concentrations over time.

Plasma concentrations of conjugate 6 at each time point were measured by sandwich ELISA as follows: conjugate 6 molecules were captured on neuraminidase-coated plates and then detected using an HRP-conjugated anti-human IgG-Fc antibody. hIgG1 was captured using anti-hIgG1-Fc antibody. Protein concentration was calculated in GraphPad Prism using 4PL nonlinear regression of conjugate 6 standard curves. A more detailed description of the method is provided below.

Nunc Maxisorp 96-well plates (cat. no. 12-565-136, ThermoFisher) were coated with A/California/04/2009 (H1N1) neuraminidase (11058-VNAHC, Sino Biological) in IX KPL coating buffer (5150-0041, SeraCare ). The plates were incubated at room temperature for 1 h on an orbital plate shaker (500 rpm). Serial dilutions of plasma samples were added to plates and incubated at room temperature for 2 h (sample diluent: 1% BSA in PBS 0.05% Tween 20 + final plasma concentration in unexposed rats 1:900). 6 standard curves of the conjugate in the range of 500-0.230 ng/ml, in duplicate, were reproduced on each plate.

After 2 h of incubation, the plates were washed 5 times in 300 μl PBS with 0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates was then labeled with HRP-conjugated anti-human IgG Fc antibody F(ab')2 (709-036- 098, Jackson), diluted 1:1000 in sample diluent for 1 hour at room temperature. The plates were then washed 8 times in 300 µl PBS with 0.05% Tween 20 and developed with TMB substrate for 7-8 minutes. The reaction was stopped with 1N H ₂ SO ₄ . Absorbance was read at 450 nm.

Total conjugate 6 (or hIgG1-Fc) in test samples was interpolated using Graphpad Prism Version 6 after nonlinear regression analysis (sigmoidal curve analysis using a four-parameter nonlinear regression (4PL) model) of the standard conjugate 6 curves. Curves comparing conjugate 6 shown in Fig. 37 demonstrate linear proportionality to dose.

Example 40: 28-day pharmacokinetic (PK) study in rats comparing intravenous (IV) and subcutaneous (SC) administration of test drug

Pharmacokinetic (PK) studies in rats were performed at Seventh Wave Laboratories (Maryland Heights, MO) using male Sprague Dawley rats aged 46-49 days. Rats were administered intravenously or subcutaneously the test drug at a dose of 5 mg/kg (dose volume 5 ml/kg). Animals were maintained under standard housing conditions approved by the IACUC. At certain time points, animals were subjected to non-lethal bleeding (retro-orbital, buccal or tail vein), with blood collected in tubes containing _K2EDTA to prevent coagulation. Collected blood was centrifuged (2000 x g for 10 minutes) and plasma was collected for analysis of test drug concentrations over time.

Plasma concentrations of conjugate 6 or hIgG1-Fc at each time point were measured by sandwich ELISA as follows: conjugate 6 molecules were captured on neuraminidase-coated plates and then detected with HRP-conjugated anti-human IgG-Fc antibody. Protein concentration was calculated in GraphPad Prism using 4PL nonlinear regression of conjugate 6 standard curves. A more detailed description of the method is provided below.

Nunc Maxisorp 96-well plates (cat. no. 12-565-136, ThermoFisher) were coated with A/California/04/2009 (H1N1) neuraminidase (11058-VNAHC, Sino Biological) in 1X KPL coating buffer (5150-0041, SeraCare) . The plates were incubated at room temperature for 1 h on an orbital plate shaker (500 rpm). Serial dilutions of plasma samples were added to plates and incubated at room temperature for 2 h (sample diluent: 1% BSA in PBS 0.05% Tween 20 + final plasma concentration in unexposed rats 1:900). Standard curves of conjugate 6 in the range of 500-0.230 ng/ml, in duplicate, were reproduced on each plate.

After a 2-hour incubation, the plates were washed 5 times in 300 µl PBS with 0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates was then labeled with HRP-conjugated anti-human IgG antibody Fc F(ab')2 (709 -036-098, Jackson), diluted 1:1000 in sample diluent for 1 h at room temperature. The plates were then washed 8 times in 300 µl PBS with 0.05% Tween 20 and developed with TMB substrate for 7-8 minutes. The reaction was stopped with 1N H ₂ SO ₄ . Absorbance was read at 450 nm.

Total conjugate 6 (or hIgG1-Fc) in test samples was interpolated using Graphpad Prism Version 6 after nonlinear regression analysis (sigmoidal curve analysis using a four-parameter nonlinear regression (4PL) model) of the standard conjugate 6 curves. Curves comparing conjugate 6 in FIG. .38 show that plasma levels for SC and IV administration converged at 24 hours and were similar within experimental error up to 336 hours.

Example 41: 28-Day Pharmacokinetic (PK) Study in Non-Human Primates Following Intravenous Administration of Study Drug

Pharmacokinetic (PK) studies in non-human primates (NHPs) were performed by BTS Research (San Diego, CA) using male and female cynomolgus monkeys aged 4.5-8 years with a body weight of 2.5-6.5 kg. NHP primates were administered intravenously via the saphenous vein of the leg or the saphenous vein of the arm with 5 or 20 mg/kg of the test drug (dose volume 5 ml/kg). Animals were maintained under standard housing conditions approved by the IACUC. At certain time points, animals were subjected to non-lethal bleeding (from the femoral or saphenous vein of the arm), with the blood collected in tubes containing K ₂ EDTA to prevent coagulation. Collected blood was centrifuged (2000 x g for 10 minutes) and plasma was collected for analysis of test drug concentrations over time.

Plasma concentrations of conjugate 6 at each time point were measured by sandwich ELISA as follows: conjugate 6 molecules were captured on neuraminidase-coated plates and then detected with HRP-conjugated anti-human IgG-Fc antibody. Protein concentration was calculated in GraphPad Prism using 4PL nonlinear regression of conjugate 6 standard curves. A more detailed description of the method is provided below.

Nunc Maxisorp 96-well plates (cat. no. 12-565-136, ThermoFisher) were coated with A/California/04/2009 (H1N1) neuraminidase (11058-VNAHC, Sino Biological) in IX KPL coating buffer (5150-0041, SeraCare) . The plates were incubated at room temperature for 1 h on an orbital plate shaker (500 rpm). Serial dilutions of plasma samples were added to plates and incubated at room temperature for 2 h (sample diluent: 1% BSA in PBS 0.05% Tween 20 + final plasma concentration in unexposed cynomolgus monkeys 1:2,500). Standard curves of conjugate 6 in the range of 500-0.230 ng/ml, in duplicate, were reproduced on each plate.

After a 2-hour incubation, the plates were washed 5 times in 300 μl PBS with 0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates was then labeled with HRP-conjugated anti-human IgG Fc antibody F(ab')2 (709-036 -098, Jackson), diluted 1:1000 in sample diluent for 1 h at room temperature. The plates were then washed 8 times in 300 µl PBS with 0.05% Tween 20 and developed with TMB substrate for 7-8 minutes. The reaction was stopped with 1N H ₂ SO ₄ . Absorbance was read at 450 nm.

Total conjugate 6 in test samples was interpolated using Graphpad Prism Version 6 after nonlinear regression analysis (sigmoidal curve analysis using a four-parameter nonlinear regression (4PL) model) of conjugate 6 (or hIgG1-Fc) standard curves. RK parameters were calculated using the WinNonlin program. Curves comparing conjugate 6 are shown in FIG. 39, and a summary of key PK parameters is presented in Table 20. The dose response is linear in the range of 5-20 IV IV, resulting in a half-life of approximately 9 days for both doses ( compared to mouse/rat).

Example 42. Pharmacokinetic (PK) study of distribution in the lungs in mice after intravenous administration of the test drug

Mouse pharmacokinetic (PK) studies were performed on 6-week-old male CD-I mice. Mice were administered intravenously through the tail vein with 10 mg/kg of the test drug (dose volume 5 ml/kg). Animals were maintained under standard housing conditions approved by the IACUC. At certain time points, animals were sacrificed to collect blood (by cardiac puncture) into K ₂ EDTA tubes and lungs. The blood was centrifuged (2000 x g, for 10 minutes) to obtain plasma. Lungs were weighed and homogenized in 1.5 ml tubes using a sterile disposable pestle (Z359947, Sigma) in 100 μl homogenization buffer consisting of 11.64 ml tissue protein extraction reagent (78510, Thermo Scientific), 0.24 ml protease inhibitor cocktail (78410, Thermo Scientific) and 0.12 ml EDTA. After 1–2 min of homogenization, the volume was adjusted to 1 ml with homogenization buffer and the samples were incubated on ice for 20 min with occasional mixing by gentle shaking. Homogenates were centrifuged at 8000 x g for 10 min, and the supernatant was retained for analysis of test drug concentrations over time.

Plasma and lung concentrations of conjugate 6 at each time point were measured by sandwich ELISA as follows: conjugate 6 molecules were captured on neuraminidase-coated plates and detected with HRP-conjugated anti-human IgG-Fc antibody. Protein concentration was calculated in GraphPad Prism using 4PL nonlinear regression of conjugate 6 standard curves. A more detailed description of the method is provided below.

Nunc Maxisorp 96-well plates (cat. no. 12-565-136, ThermoFisher) were coated with A/California/04/2009 (H1N1) neuraminidase (11058-VNAHC, Sino Biological) in IX KPL coating buffer (5150-0041, SeraCare) . The plates were incubated at room temperature for 1 h on an orbital plate shaker (500 rpm). Serial dilutions of plasma samples were plated and incubated at room temperature for 2 h (sample diluent: 1% BSA in PBS 0.05% Tween 20 + final concentration in plasma of naïve mice 1:100). Standard curves of conjugate 6 in the range of 500-0.230 ng/ml, in duplicate, were reproduced on each plate.

After a 2-hour incubation, the plates were washed 5 times in 300 μl PBS with 0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates was then labeled with HRP-conjugated anti-human IgG Fc antibody F(ab')2 (709-036 -098, Jackson), diluted 1:1000 in sample diluent for 1 hour at room temperature. The plates were then washed 8 times in 300 µl PBS with 0.05% Tween 20 and developed with TMB substrate for 7-8 minutes. The reaction was stopped with 1N H ₂ SO ₄ . Absorbance was read at 450 nm.

Total conjugate 6 in test samples was interpolated using Graphpad Prism Version 6 after nonlinear regression analysis (sigmoidal curve analysis using a four-parameter nonlinear regression (4PL) model) of conjugate 6 standard curves. Figure 40 shows curves comparing conjugate 6 concentrations. in plasma and lungs. Surprisingly, _Cmax in the lungs was reached at t=1 h (19.5 μg/g lung tissue, ~310 nM). Lung exposure to conjugate 6 was approximately 10% compared to plasma.

Example 43: 5-Day Pharmacokinetic (PK) Study in Mice Comparing Intravenous (IV), Subcutaneous (SC), and Intramuscular (IM) Administration of Test Drug

Pharmacokinetic (PK) studies were performed on male CD-I mice at 6 weeks of age. Mice were administered intravenously through the tail vein with 5 mg/kg of the test drug (dose volume 5 ml/kg). Animals were maintained under standard housing conditions approved by the IACUC. At specified times, animals were subjected to non-lethal bleeding (retro-orbital, buccal or tail vein), with blood collected in tubes containing K ₂ EDTA to prevent coagulation. Collected blood was centrifuged (2000 x g for 10 minutes) and plasma was collected for analysis of test drug concentrations over time.

Plasma concentrations of conjugate 6 at each time point were measured by sandwich ELISA as follows: conjugate 6 molecules were captured on neuraminidase-coated plates and then detected with HRP-conjugated anti-human IgG-Fc antibody. Protein concentration was calculated in GraphPad Prism using 4PL nonlinear regression of conjugate 6 (or hIgG1-Fc) standard curves. A more detailed description of the method is given below.

Nunc Maxisorp 96-well plates (cat. no. 12-565-136, ThermoFisher) were coated with A/California/04/2009 (H1N1) neuraminidase (11058-VNAHC, Sino Biological) in 1X KPL coating buffer (5150-0041, SeraCare) . The plates were incubated at room temperature for 1 h on an orbital plate shaker (500 rpm). Serial dilutions of plasma samples were added to plates and incubated at room temperature for 2 h (sample diluent: 1% BSA in PBS 0.05% Tween 20 + final concentration in plasma of naïve mice 1:100). Standard curves of conjugate 6 in the range of 500-0.230 ng/ml, in duplicate, were reproduced on each plate.

After a 2-hour incubation, the plates were washed 5 times in 300 µl PBS with 0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates was then labeled with HRP-conjugated anti-human IgG antibody Fc F(ab')2 (709 -036-098, Jackson), diluted 1:1000 in sample diluent for 1 hour at room temperature. The plates were then washed 8 times in 300 µl PBS with 0.05% Tween 20 and developed with TMB substrate for 7-8 minutes. The reaction was stopped with 1N H ₂ SO ₄ . Absorbance was read at 450 nm.

Total conjugate 6 in test samples was interpolated using Graphpad Prism Version 6 after nonlinear regression analysis (sigmoidal curve analysis using a four-parameter nonlinear regression (4PL) model) of the conjugate 6 standard curves. Curves comparing conjugate 6 are shown in Figure 41 . Exposure levels for IV, IM, and SC were similar with AUCs of 2082, 1944, and 2500, respectively.

Example 44: Efficacy in Mice and Many Species of RK Enables Infrequent Prophylactic Dosing in Humans

Allometric scaling of effective dosing in mice based on PK studies in mice, rats, and primates has been used to predict dosing and PK parameters in human subjects. Allometric scaling was based on the area under the curve (AUC) at an effective dose of 2.5 mg/kg in a 28-day prophylaxis study in mice (see Example 33).

For allometric scaling of cynomolgus only, human clearance (CL) was calculated from cynomolgus-only PK data (Example 41) using a simple allometric equation: CL (human)=CL (macaque) ⋅ [BW(human)/BW(macaque )] ^w , where BW = body weight and w is a scaling factor fixed at 0.85. Scaling results for cynomolgus monkeys only are presented in Table 21.

For mouse-rat-cynomolgus allometric scaling, human clearance (CL) based on animal species was plotted against body weight (BW) of the animal on a log-log scale according to the following allometric equation: CL=a ⋅ BW ^x where a represents the coefficient and x represents the exponent of the allometric equation. The coefficient a and the exponent x were calculated from the intercept and slope of the linear regression line, respectively. The mouse-rat-cynomolgus scaling results are presented in Table 22.

The human clearance values calculated using the appropriate algorithms described above were then used to calculate the appropriate human dose required to achieve a target effective AUC value of 3700 μg-h/ml (from a mouse dose of 2.5 mg/kg, Example 33). using the following equation: Dose=CL ⋅ AUC.

Example 45 Synthesis of Intermediate 11

Stage a.

A solution of tert-butyl (4-bromobutyl)carbamate (11.2 g, 60 mmol) and tert-butyl (4-aminobutyl)carbamate (10 g, 40 mmol) dissolved in DMF (150 ml) was treated with potassium carbonate (16 .4 g, 120 mmol), then stirred at 80°C for 6 hours. The reaction mixture was partitioned between DCM (500 ml) and brine (100 ml). The organic layer was separated and washed with brine, then dried with sodium sulfate. The solution was filtered, concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10-100% acetonitrile and water with 0.1% TFA as modifier. Product yield 11.0 g, 77%. Ion(s) found using LCMS: M+H=360.4

Stage b.

The product obtained in the previous step (0.4 g, 1.11 mmol) and Fmoc-OSu (0.45 mg, 1.3 mmol) were dissolved in DCM (10 ml), then treated with N-methylmorpholine (0.22 ml , 2 mmol) and stirred for 1 hour at room temperature. The reaction mixture was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10%-100% acetonitrile and water without 0.1% TFA as modifier. The product yield was 450 mg, 69%. Ion(s) found using LCMS: M/2+H=582.4.

Stage c.

The product obtained in the previous step (0.4 g, 0.7 mmol) was treated with TFA (5 ml) at room temperature for 0.5 h, then concentrated to dryness and used in the next step without further purification. The output at this stage was quantitative. Ion(s) found using LCMS: M/2+H=382.3.

Stage d.

Fmoc diamine (24 mg, 0.063 mmol) obtained in the previous step was added to a solution of carboxylic acid (80 mg, 0.126 mmol, described in the synthesis of intermediate 10, step f) in DMF (5 ml), then treated with HATU (50 mg , 0.13 mmol), then N-methyl morpholin (0.06 ml, 0.50 mmol). After stirring for 1 hour, the resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10-100% acetonitrile and water without TFA as modifier. The product yield was 80 mg, 79%. Ion(s) found using LCMS: M/2+H=803.9.

Stage e.

The product obtained in the previous step (80 mg, 0.050 mmol) was treated with 1% DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in DMF (2 ml) and stirred at room temperature. When the reaction was complete by LCMS (15 minutes), the reaction mixture was concentrated, then treated with TFA (2 ml) and stirred for 30 minutes at room temperature. The reaction solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10-100% acetonitrile and water, using 0.1% TFA as a modifier. The product yield as TFA salt was 52 mg. Ion(s) found using LCMS: M/2+H=492.7.

Stage f.

The product obtained in the previous step was dissolved in water (2 ml), then treated with a solution of lithium hydroxide (12 mg, 0.50 mmol, in 1 ml of water). The progress of the reaction was monitored using LCMS. After stirring for 10 min, the reaction was quenched with 0.1 ml of acetic acid. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0%-30% acetonitrile and water, using 0.1% TFA as a modifier. Product yield: 30 mg as TFA salt, 66%. Ion(s) found using LCMS: M/2+H=452.7. M/3+H=302.1.

Example 46 Synthesis of Intermediate 12

Stage a.

To a solution of tert-butyl (4-bromobutyl)carbamate (4.8 g, 19 mmol) and propargyl-PEG4 amine (2 g, 8.6 mmol) in DMF (50 ml) was added potassium carbonate (3.6 g, 26 mmol). The solution was stirred at 80°C for 6 hours, then distributed between DCM (200 ml) and brine (50 ml). The organic layer was separated, washed with brine, dried with sodium sulfate, filtered and concentrated, then purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10%-100% acetonitrile and water with 0.1% TFA as modifier. The product yield was 3.5 g, 70%. Ion(s) found using LCMS: M/2+H=574.4.

Stage b.

The product obtained in the previous step (3.5 g, 8.6 mmol) was treated with TFA (20 ml) at room temperature for 0.5 h, then concentrated to dryness, dissolved in water, frozen, lyophilized and used for next stage without additional purification. At this stage the output was quantitative. Ion(s) found using LCMS: M/2+H=374.3.

Stage c.

The diamine TFA salt obtained in the previous step (37 mg, 0.1 mmol) was added to a solution of the carboxylic acid (130 mg, 0.2 mmol, described in the synthesis of intermediate 10, step f) in 10 ml DMF, then treated with HATU (80 mg, 0.2 mmol) and N-methylmorpholine (0.25 ml, 2 mmol). The resulting solution was stirred for 1 hour, then concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10%-100% acetonitrile and water without 0.1% TFA as modifier. The product yield was 120 mg, 75%. Ion(s) found using LCMS: M/2+H=799.9.

Stage d.

The product obtained in the previous step (120 mg, 0.075 mmol) was treated with 2 ml of trifluoroacetic acid and stirred for 30 min at room temperature. The resulting solution was concentrated, dissolved in water (2 ml), then treated with a solution of lithium hydroxide (12 mg, 0.5 mmol) in water (1 ml). The resulting solution was stirred for 10 min, then slightly acidified with 0.1 ml acetic acid, concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0%-30% acetonitrile and water using 0.1% TFA as modifier. The product yield was 48 mg as TFA salt, 57%. Ion(s) found using LCMS: M/2+H=559.757. M/3+H=373.5.

Example 47 Synthesis of conjugate 8

To a solution of h-IgG1 Pc-PEG4-azide in PBSx1 buffer solution (100 mg, 1.71 μmol, 7.011 ml, MW = 58200 Da, DAR = 3.7) was added a small molecule cerivatized with alkyne (TFA salt of intermediate-12 , 45 mg, 0.031 mmol), and freshly prepared solutions in PBS at pH 7.4 CuSO ₄ (0.7 ml, 50.0 mmol, 20 liquid), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.7 ml, 50.0 mmol, 20 equiv.) and sodium ascorbate (1.05 ml, 50.0 mmol, 30 equiv.). The resulting homogeneous solution was stirred on a shaker for 12 hours. The crude solutions were diluted with PBS at pH 7.4 to a final concentration of 1 mg/ml and ultrafiltered twice (10,000 MWCO) to a volume of 1 ml. The crude mixtures were then diluted 1:10 in PBS at pH 7.4 and purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA), followed by size exclusion chromatography. The purified product was quantified using a Nanodrop™ UV-vis spectrophotometer (using the calculated extinction coefficient based on the amino acid sequence of the Fc used for conjugation, and concentrated to approximately 10 mg/ml using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule onto the gel and staining using Flash Blue.Each gel included a molecular weight bar with the molecular weight standards indicated (Figure 42).Yields are typically 40-60%. MALDI MS analysis showed a mass range (60000-90000) with an average mass of 62358. Average DAR=3.

Example 48 Comparison of in vitro and in vivo activity of selected inhibitors with their Fc conjugates in CPE assays and in a lethal murine influenza model

To demonstrate that conjugation of the neuraminidase inhibitors described herein to Fc enhances their activities in viral replication assays and in vivo efficacy models, we compared the activities of selected unconjugated inhibitors with their Fc conjugates in cytopathic effect (CPE) assays and in lethal mouse models. infection caused by the influenza virus. For the CPE microneutralization assay, ten two-fold serial dilutions of each test drug (TA), starting at 160 nM or 400 nM (9600 nM for zanamivir and oseltamivir controls), were prepared in duplicate for a one-hour inoculation of INF V A (A/C A/ 09 H IN 1) at a multiplicity of infection (MOI) of 0.001 and INFV B at an MOI of 0.01 for a one-hour incubation. The TA-virus mixture was then added to MDCK cells seeded in 96-well plates and incubated for one hour. On day 3 post-infection with INFV A and on day 5 post-infection with INFV B (B/Brisbane), cells were fixed and stained with crystal violet, and the absorbance was read to calculate the 50% effective concentration (EC50) of each TA using XLfit dose-response models. The intrinsic cytotoxicity of each TA was also assessed using ten 2-fold serial dilutions of each TA, starting at 160 nM or 400 nM, prepared in duplicate for inoculation with MDCK cells in 96-well culture plates. Cell viability was determined 3 and 5 days after treatment using the CellTiter-Glo kit. The 50% cytotoxic concentration (CC50) value was calculated using the XLfit dose-response model. No cytotoxicity was observed for any of the TAs at the highest concentrations tested. A summary of the CPE-based micro neutralization assay data is shown in Table 23.

The data summarized in Table 23 demonstrate that Fc-conjugated forms of neuraminidase dimers have higher activity in the CPE microneutralization assay than their unconjugated counterparts. When comparing conjugate 6 with intermediate-7, a 166-fold and 2-fold increase was observed against INFV A and INFV B, respectively. When comparing conjugate 8 with intermediate-12, a 26-fold and 2-fold increase was observed against INFV A and INFV B, respectively.

Conjugate 6 was compared to the most potent neuraminidase inhibitor dimer from the study summarized in Table 23 (intermediate-11, only the dimer corresponding to intermediate-12 without the trimeric linker allowing Fc conjugation) in a model of lethal viral infection influenza IAV HIN 1 using female BALB/c mice (Charles River Laboratories, 6–8 weeks). The challenge control virus (A/Puerto Rico/08/1934, also known as PR8) is a mouse-adapted isolate with an LD ₉₀ value of approximately 30 plaque-forming units (PFU) per mouse.

The experiment involved 8 groups of 5 mice. On day 0, all mice were challenged with PR8 at 10-fold LD90 by intranasal inoculation in a volume of 50 μl into mice anesthetized with ketamine/xylazine (150 and 10 mg/kg, respectively). Groups received a single treatment with vehicle, hIgG1-Fc alone, conjugate 6, or intermediate-11 intravenously 2 hours after infection (Table 24; groups 1-6). Group 7 received intermediate-11 (15 mg/kg) twice daily (bid) for 5 days, also intravenously. Group 8 received oseltamivir (15 mg/kg) orally (PO) twice daily for 5 days. All mice were observed for survival (Table 25) and body weight loss (data not shown) for 10 days after virus infection.

As expected, mice treated with vehicle or hIgG1-Fc alone died of infection on day 7 (Table 25). Mice treated with 10 doses (total 150 mg/mouse) of oseltamivir a showed a statistically significant delay in death, but only one animal survived to day 10. All mice that received a single dose of 0.3 or 3 mg/kg Conjugate 6 survived to the end of the study and showed a net weight gain over the course of the experiment. It is important to note that all mice treated with intermediate-11 at 0.3 or 3 mg/kg died during the study with only a minimal (2 days or less) delay in death compared to vehicle and hIgG1-Fc alone as control . Since hIgG1-Fc does not have intrinsic antiviral activity (group 2), this suggests that the significantly improved activity of conjugate 6 is the result of improved avidity as a result of multivalent display on Fc, as supported by the results shown in Table 23, as well as improved pharmacokinetics and contribution of Fc-mediated immune interaction. The difference in activity between the inhibitor dimer alone and conjugate 6 was statistically significant at both dose concentrations (compare groups 3 and 6 and groups 4 and 5; Table 25). On a mass basis, a 500-fold greater cumulative dose of intermediate-11 was required to observe potency equivalent to conjugate 6.

Example 49 Synthesis of Intermediate-13 ((5R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-E/Z-[3-(propargyl-PEG4)- propenyl]pyrrolidine-(2R)-carboxylic acid)

Stage a. (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxy-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester

To a stirred mixture of (5R)-((1R)-acetylamino(2S)-methoxy-(2S)-methylpentyl)-(4S)-Z-propenylpyrrolidine-(2R)-carboxylic acid salt HCl (prepared according to JACS, 2002 , 124, 4716-4721; 1.0 mmol) trimethylammonium hydroxide (1.5 mmol) was added to acetonitrile (10 ml). After stirring for 3 hours at room temperature, di-tert-butyl dicarbonate (4 eq.) was added. After completion of the reaction, all volatile components were evaporated using vacuum technology. The residue was diluted with water (10 ml). Ethyl acetate (10 mL) was added and 1 M aqueous sulfuric acid was added until the aqueous layer reached pH ~3. The aqueous layer was washed with two additional aliquots of ethyl acetate (10 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated. The residue is purified by chromatographic methods to obtain the desired product.

Stage b. (2R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester

To a mixture of (2R)-((1R)-acetylamino(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxy-(3S)-7-propenylpyrrolidine-1-carboxylic acid tert- butyl ether (1.0 mmol) in dichloromethane (5.0 ml) and methanol (1.0 ml), (trimethylyl yl)diazo methane (1.1 mmol) was slowly added. The mixture was stirred until the reaction was complete, with the temperature gradually reaching room temperature. All volatile substances were evaporated using vacuum technology. If necessary, the residue was purified by chromatography to obtain the desired product.

Stage c. (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-formylpyrrolidine-1-carboxylic acid tert-butyl ester

Room temperature mixture of (2R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-7-propenylpyrrolidine-1-carboxylic acid tert-butyl complex ether (1 mmol) in dichloromethane (15 ml) was cooled to -78°C. Ozone was bubbled through the solution until the dissolved ozone turned a faint blue color. Nitrogen was bubbled through the solution until the blue color disappeared, then dimethyl sulfide (4.0 mmol) was added, the flask was transferred to a freezer (-20°C) and left for 1 hour. The solution was concentrated and the residue was purified by chromatography to obtain the desired product.

Stage d. (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-E/Z-[3-(propargyl-PEG4)-propenyl]pyrrolidine -carboxylic acid tert-butyl ester

Sodium hydride (1.1 mmol) was added to a mixture of propargyl-PEG4-phosphonium bromide (1.0 mmol) in DMF (5.0 ml) stirred at 0° C. and after 10 minutes the temperature was raised to room temperature. Stirring was continued for 1 hour, then (2R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-formylpyrrolidine-1-carboxylic acid tert -butyl ether (1.0 mmol) in DMF (1.0 ml). Upon completion, the reaction was quenched with a saturated ammonium chloride solution. The aqueous solution was extracted several times with ethyl acetate and the combined organic phases were washed with brine, dried and evaporated. The residue was purified by chromatography to obtain the desired product.

Step f. (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxy-(3S) E/Z-[3-(propargyl-PEG4)-propenyl ]pyrrolidine-1-carboxylic acid tert-butyl ester

To a mixture of (2R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-E/Z-[3-(propargyl) stirred at 0°C -PEG4)-propenyl]-pyrrolidine-1-carboxylic acid tert-butyl ester (1.0 mmol) in tetrahydrofuran (12.0 ml) and water (3.0 ml) added lithium hydroxide (1.1 mmol) . Stirring was continued and after 15 minutes the temperature was raised to room temperature. Once complete, the pH of the solution was adjusted to acidic by excess addition of AMBERLITE® IRN-77 resin. The mixture was filtered and the filtrate was concentrated in vacuo using vacuum technology to obtain the title compound. If necessary, the residue was purified by chromatography to obtain the desired product.

Stage f. (5R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-E/Z-[3-(propargyl-PEG4)-propenyl]pyrrolidine-(2R)-carboxylic acid

To a mixture of tert-butyl ether (2R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxy-(3S)-E/Z-[, stirred at 0°C Trifluoroacetic acid (4, 0 ml). After 10 minutes the temperature rose to room temperature. Upon completion, all volatile components were evaporated using vacuum technology. The residue was purified by chromatography to obtain the desired product.

Example 50 Synthesis of conjugate 9

A solution of hIgG1 Fc-PEG4 azide in PBS × 1 buffer solution at pH 7.4 (100 mg, 1.71 μmol, 7.011 ml, MW = 58.200 Da, DAR = 3.7) was added to PBS × 1 buffer solution at pH 7.4 (2.45 ml) intermediate-13 ((5R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-E/Z-[3-( propargyl-PEG4)-propenyl]-pyrrolidine-(2R)-carboxylic acid) (0.031 mmol), copper sulfate (0.62 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (0.62 mmol) and sodium ascorbate (0.62 mmol) 93 mmol). The resulting homogeneous solution was shaken gently on a shaker for 12 hours. The crude solution was diluted with PBS at pH 7.4 to a final concentration of 1 mg/ml and ultrafiltered twice (10,000 MWCO) to a volume of 1 ml. The crude mixture was then diluted 1:10 in PBS at pH 7.4 and purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA), followed by size exclusion chromatography, followed by size exclusion chromatography. The purified product was quantified using a Nanodrop™ UV-visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of the Fc used for conjugation, and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule onto the gel and staining using flash blue staining Each gel included a molecular weight bar with the molecular weight standards indicated MALDI MS analysis was used to determine the mean DAR.

Example 51. Synthesis of propargyl-PEG4-phosphonium bromide

A mixture of propargyl-PEG4 bromide (1.0 mmol) and triphenylphosphine (1.2 mmol) in toluene (10 ml) was refluxed. Upon completion, the mixture was cooled to room temperature. The solids were filtered and used in the next step without further purification.

Example 52 Synthesis of Intermediate-14 ((5R)-[(1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl]-(4S)-Z-propenylpyrollidine-(2R) -carboxylic acid, HCl salt)

Stage a. N-{(2S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrollidin-(2R)-yl]-(2S)-methylpentyl}-( propargyl-PEG4)-carboxyamide

To a mixture of tert-butyl ether {(2S)-methoxy-(1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-7-propenylpyrrolidin-(2R)-yl, stirred at 0°C ]-(2S)-methylpentyl}-carbamic acid (prepared according to JACS, 2002, 124, 4716-4721; 1.0 mmol) in dry dichloromethane (5 ml), trifluoroacetic acid (10 mmol) was added and the temperature was raised to room temperature. Upon completion, the solvent was removed under reduced pressure. The resulting residue was dissolved in dichloromethane (20 ml) and extracted with saturated aqueous sodium bicarbonate solution. The organic layer was separated, dried (sodium sulfate), filtered and evaporated. The crude amine was dissolved in DMF (5 ml) and treated at 0°C with steam stirring with gyl-PEG4 acid (1.1 mmol), diisopropylethylamine (3.0 mmol) and HATU (1.1 mmol). Upon completion of the reaction, all volatile components were evaporated using vacuum technology. The residue was dissolved in ethyl acetate (15 ml) and washed with saturated aqueous sodium bicarbonate (10 ml), then with 1 M aqueous sulfuric acid (10 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated. The residue was purified by chromatography to obtain the desired product.

Stage b. Tert-butyl ester (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-oxo-(3S)-Z-propenylpyrrolidine-1- carboxylic acid

To a stirred solution of N-{(2.8)-methoxy-(1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-7-propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl} -(propargyl-PEG4)-carboxyamide (1.0 mmol) in a mixture of acetonitrile and water (10:1, 5 ml) was added in small portions of cerium ammonium nitrate (2.0 mmol) at 45°C for 1 hour, and stirring was continued until the reaction was complete. The reaction was quenched with saturated aqueous sodium bicarbonate (5 ml). The aqueous layer was extracted with EtOAc (3×10 ml), the combined organic layers were dried (sodium sulfate) and evaporated to obtain the crude product, which was used in the next step without further purification. The product was dissolved in acetonitrile (5 ml), di-tert-butyl carbonate (1.5 mmol) was added, followed by triethylamine (2.0 mmol) and DM AP (catalytic amount). Upon completion, the reaction was quenched with saturated ammonium chloride solution (5 ml). The aqueous layer was extracted with EtOAc (3×10 ml) and the combined organic layers were dried (sodium sulfate). All volatile substances were removed by vacuum methods. If necessary, the residue was purified by chromatographic methods to obtain the desired product.

Stage c. Tert-butyl ether (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R/S)-methoxy-(3S)-Z-propenylpyrrolidine- 1-carboxylic acid

To a solution of tert-butyl ether (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-oxo- (3S)-7-propenylpyrrolidine-1-carboxylic acid (1.0 mmol) in THF (8 ml) was added SUPER-HYDRIDE® (1 M in THF, 2.2 mmol). After 30 minutes, the reaction mixture was quenched with a saturated aqueous solution of sodium bicarbonate (4 ml) and 30% hydrogen peroxide (5 drops). The mixture was warmed to room temperature and stirred for another 30 minutes, and the aqueous layer was extracted with EtOAc (3×10 ml). The combined organic layers were dried (sodium sulfate) and the solvent was evaporated to give hemiaminal, which was used without further purification. To a solution of the above product in methanol (16 ml) was added p-toluenesulfonic acid hydrate (0.1 mmol) at room temperature. The reaction mixture was stirred overnight and quenched with saturated aqueous sodium bicarbonate (10 ml). Methanol was removed under reduced pressure, and the resulting residue was added with water (10 ml) and extracted with EtOAc (3×10 ml). The organic layers were separated and dried with brine and sodium sulfate, filtered and concentrated. If necessary, the residue was purified by chromatographic methods to obtain the desired product.

Stage d. Tert-butyl ester (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-cyano-(3S)-Z-propenylpyrrolidine-1- carboxylic acid

To a solution of tert-butyl ether stirred at -78°C (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R/S)- methoxy-(3S)-7-propenylpyrrolidine-1-carboxylic acid (1.0 mmol) in dichloromethane (20 ml) was added trimethylsilyl cyanide (2.0 mmol), followed by boron trifluoride diethyl etherate (1.2 mmol). The reaction mixture was stirred at a temperature ranging from -78°C to -50°C for 3 hours. Saturated aqueous sodium bicarbonate (40 ml) was added and the aqueous layer was extracted with EtOAc (3×15 ml). The combined organic layers were dried (sodium sulfate), filtered and concentrated under reduced pressure. The resulting residue, consisting of a mixture of epimeric cyano derivatives, was purified by chromatographic methods to obtain the desired product.

Step f. (5R)-[(1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl]-(4S)-Z-propenylpyrrolidine-(2R)-carboxylic acid, HCl salt

To a solution of tert-butyl ether (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-cyano-(3S)-Z-propenylpyrrolidine- 1-Carboxylic acid (1.0 mmol) in AcOH (10 ml) was added 12N HCl (10 ml) at room temperature. The solution was stirred at room temperature until the reaction was complete, and the solvent was evaporated under reduced pressure. If necessary, the residue was purified by chromatographic methods to obtain the desired product.

Example 53 Synthesis of conjugate 10

A solution of hIgG1 Ec-PEG4 azide in PBS×1 buffer solution at pH 7.4 (100 mg, 1.71 µmol, 7.011 ml, MW=58.200 Da, DAR=3.7) was added to PBS×1 buffer solution at pH 7.4 (2.45 ml) intermediate-14 ((5R)-[(1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl]-(4S)-7- propenylpyrrolidine-(2R)-carboxylic acid, HCl salt) (0.031 mmol), copper sulfate (0.62 mmol), tris(3-hydroxypropyltriazolylmethyl)-amine (0.62 mmol) and sodium ascorbate (0.93 mmol). The resulting homogeneous solution was shaken gently on a shaker for 12 hours. The crude solution was diluted with PBS at pH 7.4 to a final concentration of 1 mg/ml and ultrafiltered twice (10,000 MWCO) to a volume of 1 ml. The crude mixture was then diluted 1:10 in PBS at pH 7.4 and purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA), followed by size exclusion chromatography. The purified product was quantified using a Nanodrop™ UV-visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of the Fc used for conjugation, and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule onto the gel and staining using flash blue staining Each gel included a molecular weight bar with the molecular weight standards indicated MALDI MS analysis was used to determine the mean DAR.

Example 54 Synthesis of Intermediate-15, HCl Salt

Stage a. N-{(2.S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl} -(3-butynyl)carboxamide

To a mixture of tert-butyl ether {(2S)-methoxy-(1R)-[l-(4-methoxybenzyl)-5-oxo-(3S)-7-propenylpyrrolidin-(2R)-yl, stirred at 0°C ]-(2S)-methylpentyl}-carbamic acid (prepared according to JACS, 2002, 124, 4716-4721; 1.0 mmol) in dry dichloromethane (5 ml), trifluoroacetic acid (10 mmol) was added and the temperature was raised to room temperature. Upon completion, the solvent was removed under reduced pressure. The resulting residue was dissolved in dichloromethane (20 ml) and extracted with saturated aqueous sodium bicarbonate solution. The organic layer was separated, dried (sodium sulfate), filtered and evaporated. The crude amine was dissolved in DMF (5 ml) and treated at 0° C. with stirring with 4-pentinoic acid (1.1 mmol), diisopropylethylamine (3.0 mmol) and HATU (1.1 mmol). Upon completion of the reaction, all volatile components were evaporated using vacuum technology. The residue was dissolved in ethyl acetate (15 ml) and washed with saturated aqueous sodium bicarbonate (10 ml), then 1 M aqueous sulfuric acid (10 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated. The residue was purified by chromatographic methods to obtain the desired product.

Stage b. (2R)-((1R)-(4-pentinoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-oxo-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester

To a stirred solution of N-{(2.S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo(3S)-7-propenylpyrrolidin-(2R)-yl]-(2S)- methylpentyl}-(3-butynyl)-carboxyamide (1.0 mmol) in a mixture of acetonitrile and water (10:1, 5 ml), cerium ammonium nitrate (2.0 mmol) was added in small portions at 45°C for 1 hour , and stirring was continued until the reaction was complete. The reaction was quenched with saturated aqueous sodium bicarbonate (5 ml). The aqueous layer was extracted with EtOAc (3 x 10 ml), the combined organic layers were dried (sodium sulfate) and evaporated to obtain the crude product, which was used next step without further purification. The product was dissolved in acetonitrile (5 ml), di-tert-butyl carbonate (1.5 mmol) was added, then triethylamine (2.0 mmol) and DM AP (catalytic amount). Once complete, the reaction was quenched with a saturated solution ammonium chloride (5 ml).The aqueous layer was extracted with EtOAc (3×10 ml), and the combined organic layers were dried (sodium sulfate).All volatiles were removed by vacuum methods. If necessary, the residue was purified by chromatographic methods to obtain the desired product.

Stage c. Tert-butyl ester (2R)-((1R)-(4-pentinoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R/S)-methoxy-(3S)-Z-propenylpyrrolidine-1- carboxylic acid

To a solution of tert-butyl ether (2R)-((1R)-(2R)-((1R)-(4-pentinoyl)-(2S)-methoxy-(2S)-methylpentyl) stirred at -78°C -(5R)-oxo-(3S)-Z-propenylpyrrolidine-1-carboxylic acid (1.0 mmol) in THF (8 ml) was added SUPER-HYDRIDE® (1Mb THF, 2.2 mmol).After 30 min the reaction the mixture was quenched with saturated aqueous sodium bicarbonate (4 ml) and 30% hydrogen peroxide (5 drops).The mixture was warmed to room temperature and stirred for an additional 30 minutes, and the aqueous layer was extracted with EtOAc (3x10 ml).The combined organic layers were dried. (sodium sulfate) and the solvent was evaporated to give hemiaminal, which was used without further purification. To a solution of the above product in methanol (16 ml) was added p-toluenesulfonic acid hydrate (0.1 mmol) at room temperature. The reaction mixture was stirred overnight and quenched with saturated aqueous sodium bicarbonate (10 ml).Methanol was removed under reduced pressure, and the resulting residue was added with water (10 ml) and extracted with EtOAc (3×10 ml). The organic layers were separated and dried with brine and sodium sulfate, filtered and concentrated. If necessary, the residue was purified by chromatographic methods to obtain the desired product.

Stage d. (2R)-((1R)-(4-pentinoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-cyano-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester

To a solution of tert-butyl ether (2R)-((1R)-(4-pentinoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R/8)-methoxy- (3S)-7-propenylpyrrolidine-1-carboxylic acid (1.0 mmol) in dichloromethane (20 ml) was added trimethylsilyl cyanide (2.0 mmol), followed by boron trifluoride diethyl etherate (1.2 mmol). The reaction mixture was stirred at -78°C to -50°C for 3 hours. Saturated aqueous sodium bicarbonate (40 mL) was added and the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic layers were dried (sodium sulfate), filtered and concentrated under reduced pressure. The resulting residue, consisting of a mixture of epimeric cyano derivatives, was purified by chromatographic methods to obtain the desired product.

Stage e.

To a stirred mixture of N-{(2.8)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl }-(3-butynyl)carboxyamide (1.0 mmol), bis-[N'-(2-azidoethyl)]iminodiacetic amide (0.5 mmol), 1H-1,2,3-triazole-4-methanamine, 1-(phenylmethyl)-N,N-bis[1-(phenylmethyl)-1H-1,2,3-triazol-4-yl]methyl] (0.1 mmol) and sodium ascorbate (1.0 mmol) in copper sulfate (0.1 mmol) was added to ethanol (5 ml) and water (5 ml). Once complete, the reaction was treated with SiliaMetS TAAcONa (0.3 mmol) for 30 minutes. The reaction mixture was filtered and all volatiles were evaporated by vacuum methods. The residue purified by chromatographic methods to obtain the desired product.

Stage f.

HATU (2 .0 mmol). All volatile substances were evaporated using vacuum methods and the residue was purified by chromatographic methods to obtain the desired product.

Stage g. Intermediate-15, HCl salt

To a solution of the intermediate obtained in step f (1.0 mmol) in AcOH (10 ml) was added 12N HCl (10 ml) added at room temperature. The solution was stirred at room temperature until the reaction was complete, and the solvent was evaporated under reduced pressure. If necessary, the residue was purified by chromatographic methods to obtain the desired product.

Example 55 Synthesis of conjugate 11

A solution of hIgG1 Ec-PEG4 azide in PBS×1 buffer solution at pH 7.4 (100 mg, 1.71 µmol, 7.011 ml, MW=58.200 Da, DAR=3.7) was added to PBS×1 buffer solution at pH 7.4 (2.45 ml) intermediate-15, HCI salt (0.031 mmol), copper sulfate (0.62 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (0.62 mmol) and sodium ascorbate (0.93 mmol). The resulting homogeneous solution was gently shaken on a shaker for 12 hours. The crude solution was diluted with PBS at pH 7.4 to a final concentration of 1 mg/mL and ultrafiltered twice (10,000 MWCO) to a volume of 1 mL. The crude mixture was then diluted 1:10 in PBS at pH 7.4 and purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA) followed by size exclusion chromatography. The purified product was quantified using a Nanodrop™ UV-visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of the Fc used for conjugation, and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule onto the gel and staining using flash blue staining Each gel included a molecular weight bar with the molecular weight standards indicated MALDI MS analysis was used to determine the mean DAR.

Example 56 Synthesis of bis-[N'-(2-azidoethyl)]-iminodiacetic amide

Stage a. Bis-[N'-(2-azidoethyl)]-N-Boc-iminodiacetic amide

HATU was added to a stirred solution of N-Boc-iminodiacetic acid (1.0 mmol), 2-azidoethane-1-amine hydrochloride (2.0 mmol) and DIPEA (6.0 mmol) in DMF (10 ml) at 0°C. (2.0 mmol). All volatile components were evaporated by vacuum methods and the residue was purified by chromatographic methods to obtain the desired product.

Stage b. Bis-[N'-(2-azidoethyl)]-iminodiacetic amide

To a mixture of bis-[N'-(2-azidoethyl)]-]N-Boc-iminodiacetic amide (1.0 mmol) and 2-methyl-2-butene (0.5 ml) in dichloromethane, stirred at 0°C 8.0 ml) trifluoroacetic acid (4.0 ml) was added. After 10 minutes the temperature rose to room temperature. Upon completion, all volatile substances were evaporated using vacuum methods. The residue was purified by chromatographic methods to obtain the desired product.

Example 57 Synthesis of Intermediate-16

A sulfosanamivir monomer conjugated to a PEG4-alkyne linker and which can be further conjugated to an Fc domain or albumin protein was prepared according to the following synthesis scheme. The starting product sulfosanamivir was prepared according to Hadhazi et al. A sulfozanamivir analogue has potent anti-influenza virus activity. ChemMedChem Comm. 13:785-789 (2018).

Example 58 Synthesis of Intermediate-17

A sulfosanamivir dimer conjugated to a PEG4-alkyne linker and which can be further conjugated to an Fc domain or albumin protein was prepared according to the following synthesis scheme. The starting product sulfosanamivir was prepared according to Hadhazi et al. A sulfozanamivir analogue has potent anti-influenza virus activity. ChemMedChem Comm. 13:785-789 (2018).

Example 59. Synthesis of conjugate 12

A solution of azido-functionalized aglycosylated Fc (70 mg, 4.7 mL, 1.3709 μmol) was added to a 15 mL centrifuge tube containing the alkyne-derivatized small molecule (29.8 mg, 0.0216 mmol, intermediate-7). After gentle shaking to dissolve all solids, 206 µl of a mixed solution of sodium L-ascorbic acid (59.4 mg, 0.3 mmol), copper(II) sulfate (15.9 mg, 0.1 mmol) and THPTA (43.5 mg, 0.1 mmol) in PBS buffer at pH 7.4 (1 ml). The resulting mixture was shaken gently overnight. It was purified by affinity chromatography on a Protein A column followed by size exclusion chromatography as described in Example 8. Maldi TOF analysis of the purified final product gave an average mass of 56,177 Da (DAR=3.6). The yield was 10.1 mg, yield 14%. In fig. 54 shows SDS-PAGE analysis under non-reducing conditions of conjugate 12.

Example 60 Synthesis of Intermediate-18

Stage a. Synthesis of tert-butyl (17-{4-[(tert-butoxycarbonyl)amino]butyl}-16-oxo-4,7D0D3-tetraoxa-17-azachenicos-1-in-21-yl)carbamate

To a solution of di-tert-butyl[azandiyldi(butan-4,1-diyl)]biscarbamate (1.5 g, 4.17 mmol from example 45, step a) and pro pargyl-PEG4 acid (1.08 g, 4.17 mmol) in DCM (40 ml) was added EDC (1.0 g, 5 mmol), HOBt (650 mg, 5 mmol) and DIE A (1.4 ml, 10 mmol) at room temperature, then stirred overnight at room temperature. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10%-100% acetonitrile and unmodified water. Yield 1.9 g, 76%. Ion(s) found by LCMS: M+H=602.4.

Stage b. Synthesis of N,N-bis(4-aminobutyl)-4,7,10,13-tetraoxahexadec-15-yne-1-amide

Tert-butyl (17-{4-[(tert-butoxycarbonyl)amino]butyl}-16-oxo-4,7,10,13-tetraoxa-17-azachenicos-1-yn-21-yl)carbamate (1, 90, 3.1 mmol) were treated with 20 ml TFA at room temperature for 0.5 h, then concentrated to dryness and used in the next step without further purification. The yield is quantitative for this stage. Ion(s) found using LCMS: M/2+H=402.3.

Stage c. Synthesis of fully protected intermediate-18

N,N-bis(4-aminobutyl)-4,7,10,13-tetraoxahexadec-15-yne-1-amide (0.150 g, 0.32 mmol) was added to a solution of zanamivir acid ester (0.400 g 0.63 mmol described in example 31, step f) in DCM (10 ml), then treated with EDC (0.200 g, 1.0 mmol), HOBt (0.135 g, 1.00 mmol) and DIE A (0.14 ml, 1 .00 mmol) at room temperature overnight. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10%-100% acetonitrile and water without 0.1% TFA as modifier. The product yield was 310 mg, 60.3%. Ion(s) detected by LCMS: M/2+H=813.9.

Stage d. Synthesis of intermediate-18

The product obtained in the previous step (300 mg, 0.18 mmol) was treated with trifluoroacetic acid (2 ml) acid and stirred for 30 min at room temperature. The resulting solution was concentrated, redissolved in water (2 ml), then treated with a solution of lithium hydroxide (24 mg, 1 mmol) in water (1 ml). The reaction mixture was stirred for 10 min, then quenched with 0.1 ml acetic acid, concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0%-50% acetonitrile and water using 0.1% TFA as a modifier. The product yield was 140 mg, 52%. Ion(s) found using LCMS: M/2+H=573.8, M/3+H=382.9.

Example 61. Synthesis of PEG4-azido Fc for conjugate 13a

Preparation of a 0.05 M solution of PEG4-azidoNHS ester in DMF/PBS

PEG4-azido NHS ester (80.5 mg) was dissolved in DMF (0.50 ml) at 0°C and diluted to 4.063 ml by adding 3.50 ml PBS 1x buffer at 0°C. This solution was used to prepare another PEG4-azido Fc with different drug o-antibody ratio (DAR) values by adjusting the equivalents of this PBS x1 PEG4-azido NHS ester solution.

Preparation of PEG4-azido Fc

A 0.05M solution of PEG4-azidoNHS ester in PBS x1 buffer (0.0984 ml, 4.92 µmol, 2.5 equivalents) was added to a solution of h-IgG1-Fc (105 mg in 5.031 ml PBS at pH 7.4, MW ~ 53360 Da, 1.968 µmol) and the mixture was shaken gently for 12 hours at ambient temperature. The solution was concentrated using a centrifugal concentrator (30,000 MWCO) to a volume of ~1.5 mL. The crude mixture was diluted 1:10 in PBS pH 7.4 and concentrated again. This washing procedure was repeated a total of three times. Unreacted azido reagent was removed using this procedure. Concentrated Ec-PEG4 azide was diluted to 5.03 ml with PBS 1x buffer at pH 7.4 and ready for click conjugation. The purified product was quantified using a Nanodrop™ UV-visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1). Yield was quantitative after buffer exchange/purification.

Example 62. Synthesis of conjugate 13a

Preparation of a click reagent solution: 0.0050M CuSO ₄ in a PBS buffer solution: 20.0 mg of CuSO ₄ was dissolved in 25.0 ml of PBS xl, then 22.0 ml of the above CuSO ₄ solution was taken and 189.4 mg of BTTAA and 1090 mg of sodium ascorbate to obtain a clear solution (0.0050 M CuSO ₄ , 0.020 M BTTAA and 0.25 M sodium ascorbate). A solution of azide-functionalized Fc (100 mg, 4.79 mL, 1.87 μmol, STP-161-4A-Linker-1-Azide) was added to a 15 mL centrifuge tube containing an alkyne-derivatized small molecule, intermediate-18 ( 11.2 mg, 0.00750 mmol, obtained in example 60). After gentle shaking to dissolve all solids, the solution was treated with 3.00 mL of the above click reagent solution. The resulting colorless homogeneous solution was gently shaken overnight. It was purified by affinity chromatography on a Protein A column followed by size exclusion chromatography (see general conjugate purification protocol in Example 10). Maldi TOF analysis of the purified final product gave an average mass of 55913 Da (DAR=1.7). The yield was 54.0 mg, yield 54%.

Example 63 Synthesis of conjugate 13b, conjugate 13c, conjugate 13d, conjugate 13e, conjugate 13f and conjugate 13g

PEG4-azido Fc for conjugate 13b, conjugate 13c, conjugate 13d, conjugate 13e, conjugate 13f and conjugate 13g were prepared similarly to PEG4-azido Fc conjugate 13a (Example 61) by adjusting the amount of NHS PEG4-azido ester equivalents as described in the table below. Conjugate 13b, conjugate 13c, conjugate 13d, conjugate 13e, conjugate 13f, and conjugate 13g were prepared similarly to conjugate 13a in Example 62, where the number of equivalents of the targeting moiety (intermediate-18) was adjusted based on the desired DAR value (Table 26), and the volume The click reagent solution used was the same as that used in the procedure for Example 62. The table below lists the DAR values, molecular weights and yields. The product conjugates were purified by affinity chromatography on a Protein A column followed by size exclusion chromatography as described in Example 8.

SDS-PAGE analysis under non-stop conditions of conjugates 13a-13g is presented in FIG. 55.

Example 64 In vitro stability of conjugate 6

To demonstrate the in vitro stability of conjugate 6, mouse and human plasma and liver microsomes treated with freshly prepared K2EDTA were used. In vitro stability in mouse and human plasma was determined by comparing the drug to antibody ratio (DAR) after 24 hours of incubation in plasma at 37°C using MALDI-TOF mass spectrometric detection. Microsomal stability in the liver using mouse and human microsomes was also assessed after incubation for 24 hours at 37°C using MALDI-TOF mass spectrometric detection. This was done to identify potential metabolically labile sites on the Fc protein, linker, or target moiety.

64.1 Sample preparation for plasma stability determination

First, 60 μl of conjugate 6 at a concentration of 3 mg/ml was mixed with 120 μl of plasma. Aliquots of each type of plasma were added to 2 tubes. One aliquot of each plasma type was immediately frozen. The remaining aliquot was placed in a water bath (37°C) for 24 hours. MAGNE® Protien A beads (Promega) were equilibrated by gently shaking the beads in suspension. In duplicate for both plasma types, 50 μl of the bead suspension was added to a 1.5 ml microcentrifuge tube and placed on a magnetic rack for 10 seconds. After 10 seconds, the storage buffer was removed and discarded. 500 μl of binding/wash buffer (0.1% BSA in 1X PBS, pH 7.4) was added to a 1.5 ml microcentrifuge tube containing the beads. The granules were mixed (shaking) and placed on a magnetic stand for 10 seconds. After 10 seconds, the binding/wash buffer was removed and discarded. 50 μl of buffer (1x PBS, pH 7.4) was added to the microcentrifuge tube containing the beads. 50 μl of the plasma mixture was added to the beads and gently shaken to mix. Using a tube shaker, the sample was mixed at room temperature for 60 minutes to keep the beads in suspension. After mixing, the tube was placed on a magnetic stand for 10 seconds and the supernatant was removed. Add 500 μl of buffer (1x PBS, pH 7.4) and shake gently to mix. After mixing, the tube was placed on a magnetic rack for 10 seconds before the wash buffer was removed and discarded. The washing step was repeated only 2 times. After 2 washes with 500 μL buffer (lx PBS, pH 7.4), 3 washes were performed with 500 μL, 200 μL, and 100 μL water, respectively. An appropriate volume of water was added to the tube and gently shaken to mix well, then placed on a magnetic rack for 10 seconds before the water was removed and discarded. After the third wash with water, 30 μL of elution buffer (90:10:0.4 water:acetonitrile:TGA) was added to the beads. Using a tube shaker, the elution buffer and sample were mixed for 30 minutes at room temperature. After mixing, the tube was placed on a magnetic rack for 10 seconds, and the elution buffer containing the sample was removed and stored. 2 μL of sample was mixed with 2 μL of MALDI matrix (20 mg/mL sinapic acid in a 70:30:0.1 water:acetonitrile:TGA ratio) and applied to a MALDI target plate using a two-layer technique. The sample was then analyzed by MALDI-TOF mass spectrometry.

64.2 Preparation of samples with liver macros

10x buffer was prepared with 500 mM Tris-HCl at pH 7.5 and 50 mM magnesium chloride hexahydrate. ACV-006 was diluted to 50 μM in 1x PBS, pH 7.4. Liver microsomes were thawed and vortexed. An aliquot of liver microsomes from each species (human and mouse) was heat inactivated at 70°C for 15 minutes to serve as a control. Reaction mixtures were prepared for both species according to Table 27. The tubes were incubated in a water bath (37°C) for 24 hours. Samples were extracted for analysis using MAGNE® Protein A beads (Promega) according to the protocol in 59.1.

64.3 Analysis of samples and data obtained

Samples were acquired using Bruker Compass Flex Control version 3.4 to obtain MALDI-TOF mass spectra over the full scan range (Table 28). BSA was used as an internal calibrator for the range of masses measured. Data were further analyzed using Bruker Compass Flex Analysis version 3.4 software. In addition, the DAR pattern of the control was compared with the DAR pattern of the test sample.

64.4 Results

The test compound, conjugate 6 (FIG. 43), was tested for in vitro stability in fresh K2EDTA-treated plasma and mouse and human liver microsomes. Conjugate 6 was added to mouse and human plasma treated with freshly prepared K2EDTA to a final concentration of 1 mg/ml. The plasma was divided into 2 aliquots, one was immediately frozen, and the other was incubated in a water bath at 37°C for 24 hours. At the end of incubation, samples were extracted from the plasma matrix using MAGNE® Protein A magnetic beads. After plasma incubation, samples were analyzed using MALDI-TOF mass spectrometry for changes in DAR. It was found that conjugate 6 in mouse (Fig. 44) or human (Fig. 45) plasma upon incubation did not cause any changes in DAR.

The stability of conjugate 6 in liver microsomes was tested at a final concentration of 5 μM in a 50 mM Tris-HCl buffer solution, pH 7.5, which contained either active or heat-inactivated liver microsomes at a final concentration of 0.5 mg/ml and MgCl ₂ at a final concentration concentration 5 mM. All samples were incubated at a constant temperature of 37°C and nicotinamide adenine dinucleotide phosphate (NADPH) regenerating solution was used to ensure constant cofactor availability during incubation. Incubations were carried out for 24 hours. At the end of incubation, samples were extracted from the microsomal matrix using Protein A magnetic beads. After incubation with liver microsomes, samples were analyzed by MALDI-TOF mass spectrometry for changes in DAR. Conjugate 6 was not found to cause any changes in DAR in mouse (FIG. 46) or human (FIG. 47) liver microsome incubations.

Stability in plasma in vitro after incubation at 37°C for 24 hours suggests no degradation of Fc-conjugate 6, linker, or targeting moiety in either mouse or human. Similarly, no degradation was observed after incubation in mouse and human liver microsomes, indicating the absence of metabolites. The results of these in vitro stability studies confirm that it is a stable compound with degradation products that may have biological properties.

Example 65 Efficacy of Conjugate 13 at Various Drug-Antibody Ratios (DAR) against Influenza A (H1N1) in a Lethal Mouse Model

Conjugate 13 was evaluated against lethal influenza IAV H1N1 infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/1934) is a mouse-adapted isolate capable of causing lethal infections in mice. The experiment involved 13 groups of 5 mice. On day 0, all mice were infected with the virus at 3x LD95 by intranasal inoculation in a volume of 30 μl after anesthesia with a mixture of ketamine/xylazine (150 and 10 mg/kg, respectively).

Groups 1-13 received a single IV treatment with test drug or vehicle (PBS) 2 hours after viral infection. The study evaluated conjugate 13 constructs with 4 different DAR values corresponding to conjugate 13a, conjugate 13c, conjugate 13d, and conjugate 13g (DAR 1.7, 3.8, 5.8, and 10.3, respectively). The synthesis of conjugate 13a, conjugate 13c, conjugate 13d and conjugate 13g is described in Example 61-Example 63. Each construct was evaluated at 0.03, 0.1 and 0.3 mg/kg. The general study design for each conjugate is summarized in Table 29.

All constructs were fully protective at 0.3 mg/kg, in contrast, no construct was active at 0.03 mg/kg (0% survival for all groups), indicating that the low dose was below the effective dose threshold . However, groups receiving 0.1 mg/kg of conjugates can be distinguished (Table 30). At this dose level, conjugates with DAR values of 1.7, 3.8, and 5.8 were significantly more protective than mice treated with vehicle alone (p=0.0027). The high DAR construct (10.3), however, was not significantly more protective than vehicle-only treated mice (p=0.091). The underlying mechanism by which a high DAR construct loses its activity is currently unknown but may be caused by several factors, including interference with antibody recycling, resulting in a shorter half-life.

Example 66: Efficacy of Conjugate 6 and Conjugate 12 in Vivo

Conjugate 6 and Conjugate 12, an analog with the Fc mutation at N297A, were evaluated against lethal influenza IAV H1N1 infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/34) is a mouse-adapted isolate. The experiment involved 5 mice per group. Mice were anesthetized with ketamine/xylazine (150/10 mg/kg) and infected with influenza virus at 3-5 LD95 by intranasal inoculation in a volume of 30 μl. A single 1 dose of treatment was administered IV 2 hours after infection. PBS was used as a negative control. All mice were observed for % body weight loss (FIG. 48) and survival (FIG. 49) for 15 days post-challenge.

Example 67 In vitro binding of conjugate 6 and conjugate 12 to Fcγ receptor IIIA

Binding of conjugate 6 and conjugate 12, an analogue with the Fc mutation in N297A, was assessed against FcγRIIIA by ELISA. The plate was coated with 1 μg/ml recombinant human FcγRIIIA overnight. The next day, the plate was blocked with 1% BSA for 1 hour. Conjugates were added to the plate in doses ranging from 0.01-1000 nM and incubated for 2 hours. Binding was detected by incubation with peroxidase-conjugated anti-human Fc for 1 hour and followed by incubation with TMB substrate for 10-15 minutes. Binding was determined by reading the absorbance at 450 nm (FIG. 50 and FIG. 51).

Example 68 In Vivo Analysis of Conjugate 6 in Plasma Samples

Conjugate 6 in plasma samples was quantified by detecting neuraminidase uptake using ELISA. Briefly, molecules were captured on neuraminidase-coated plates and then detected using an HRP-conjugated anti-human IgG-Fc antibody. Protein concentration was calculated in GraphPad Prism using 4PL nonlinear regression of conjugate 6 standard curves. A more detailed description of the method is provided below.

Nunc Maxisorp 96-well plates (cat. no. 12-565-136, ThermoFisher) were coated with 0.1 U/well of neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Biological) in IX KPL coating buffer (5150-0041, SeraCare). The plates were incubated at room temperature for 1 h on an orbital plate shaker (500 rpm). Serial dilutions of plasma samples were added to plates and incubated at room temperature for 2 hours (sample diluent: 0.5% BSA in PBS, 0.025% Tween 20 + final plasma concentration in unexposed cynomolgus monkeys 1:2500). Standard curves of conjugate 6 ranging from 0.230 to 500 ng/ml, in duplicate, were reproduced on each plate. After a 2-hour incubation, the plates were washed 5 times in 300 μl PBS with 0.05% Tween 20. The neuraminidase conjugate on the plates was then probed with HRP-conjugated anti-human IgG antibody Fc F(ab')2 (709- 036-098, Jackson), diluted 1:1000 in sample diluent for 1 hour at room temperature. The plates were then washed 8 times in 300 µl PBS with 0.05% Tween 20 and developed with TMB substrate for 7-8 minutes. The reaction was stopped IN H ₂ SO ₄ . Absorbance was read at 450 nm. Conjugate 6 in plasma samples was interpolated using GraphPad Prism Version 6 after nonlinear regression analysis (sigmoidal curve analysis using a four-parameter nonlinear regression (4PL) model) of the standard curves.

The resulting mean plasma concentrations were then used to calculate pharmacokinetic parameters by noncompartmental analysis using Phoenix WinNonlin 7.0 software.

Toxicokinetics (TR), groups 2 (IV, 5 mg/kg) and 3 (IV, 20 mg/kg)

Concentrations were comparable between male and female animals in the same dose group on day 1 (Table 31) and day 8 (Table 32) after administration. Mean plasma concentrations increased approximately proportionally to dose on both days. After the 2nd dose, there was a slight accumulation of about 30% in the different dose groups. A plot of mean plasma concentrations on days 1 and 8 for the different dose groups is shown in FIG. 52.

Pharmacokinetics (PR), groups 4 (IV, 10 mg/kg) and 5 (SC, 10 mg/kg)

After intravenous administration, plasma concentrations in males and females were comparable. Following intravenous administration, very low clearance was observed, resulting in a long terminal half-life (Table 33A and Table 33B).

Following subcutaneous administration, the time to reach maximum concentrations was 72 hours post-dose, but concentrations could be measured 672 hours post-dose (Table 34A and Table 34B). Bioavailability after subcutaneous dosing was high and was approximately 139%. In fig. Figure 53 shows a comparison of plasma concentrations over time for intravenous and subcutaneous administration of 10 mg/kg.

Example 69: Efficacy of Conjugate 6 against Influenza A/Puerto Rico/8/1934 (H1N1) in a Lethal Mouse Model

Conjugate 6 was evaluated against lethal influenza IAV H1N1 infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/1934) is a mouse-adapted isolate capable of causing lethal infections in mice. The experiment involved 7 groups of 5 mice. On day 0, all mice were infected with the virus at 3x LD95 by intranasal inoculation in a volume of 30 μl after anesthesia with a mixture of ketamine/xylazine (150 and 10 mg/kg, respectively). Mortality and body weight were recorded daily, and any animal with a 20% body weight loss was scored as dead. Test groups were treated 2 hours after viral challenge with a single intravenous injection of conjugate 6, hIgG1-Fc control, or vehicle (PBS). Oseltamivir-treated animals were dosed orally twice daily for 5 days, starting 2 hours after viral challenge. Table 35 shows the study design.

As expected, mice treated with vehicle or hIgG1-Fc alone died of infection by day 6. Similarly, mice treated with low dose oseltamivir (5 mg/kg; twice daily for 5 days) died by day 8 (Table 36). However, mice treated with oseltamivir at a dose of 20 mg/kg using the same dosing regimen were completely protected (p=0.0027). In contrast to oseltamivir a, mice treated with conjugate 6 were fully protected at all dose levels (10, 2 and 0.4 mg/kg) following a single IV dose (p=0.0027).

The effectiveness of conjugate 6 was further confirmed by daily body weight measurements. As expected, mice treated with vehicle or hIgG1-Fc showed a sustained decrease in body weight until it exceeded 20%, at which point they were considered dead (Table 37). The group treated with oseltamivir 5 mg/kg also showed consistent weight loss until death on day 8. Mice treated with high dose oseltamivir (20 mg/kg) showed sustained but reduced body weight loss, which reached 14% on day 8, before recovery.

In contrast to control and oseltamivir-treated mice, those groups receiving Conjugate 6 maintained healthy body weight throughout the study, even at the lowest dose concentration (0.4 mg/kg) (Table 37). The greatest transient weight loss among mice treated with conjugate 6 was only 2% on day 14 in the 2 mg/kg dose group. Based on survival and body weight measurements, Conjugate 6 demonstrated robust protection against influenza A/Puerto Rico/8/1934 with a single IV dose of as little as 0.4 mg/kg.

Example 70 Synthesis of conjugate 14

The title conjugate was prepared analogously to conjugate 13a (Example 61) using PEG-azido-Ec (SEQ ID NO: 35) and intermediate-7 (Example 19). Maldi TOF analysis of the purified final product showed an average mass of 56,502 Da (DAR=2.1). The yield was 43.4 mg, 43.4%.

Example 71 Synthesis of conjugate 15

This conjugate was prepared analogously to Example 62 (Conjugate 13a) using PEG4-azido-Ec (SEQ ID NO: 35, Example 61) and intermediate-12 (Example 46). Maldi TOF analysis of the purified final product showed an average mass of 56,528 Da (DAR=2.2). The yield was 40.0 mg, 40.0%.

Example 72 Synthesis of conjugate 16

This conjugate was prepared analogously to Example 62 (Conjugate 13a) using PEG4-azido-Ec (SEQ ID NO: 35, Example 61) and intermediate-10 (Example 31). Maldi TOF analysis of the purified final product showed an average mass of 56,507 Da (DAR=2.1). The yield was 41.7 mg, 42%.

Example 73 Synthesis of Intermediate-19

Stage a.

Carbonate was added to a solution of tert-butyl [2-(2-bromoethoxy)ethyl]carbamate (1.8 g, 6.6 mmol) and propargyl-PEG4-amine (0.7 g, 3.0 mmol) in 30 ml DMF potassium (1.2 g, 9 mmol). The reaction mixture was stirred at 80°C for 6 hours, and then partitioned between DCM (200 ml) and brine (50 ml). The organic layer was separated, washed with brine and dried with anhydrous sodium sulfate. After filtration, the resulting filtrate was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10% to 100% acetonitrile and water with 0.1% TFA as modifier. The product yield was 1.0 g, 65%. Ion(s) found using LCMS: M/2+H=606.4.

Stage b.

The product obtained in the previous step (1.0 g, 1.6 mmol) was treated with TFA (10 ml) at room temperature for 0.5 h, then concentrated to dryness and used in the next step without further purification. The output at this stage was quantitative. Ion(s) found using LCMS: M/2+H=406.3.

Stage c.

The product obtained in the previous step (120 mg, 0.17 mmol) was treated with a solution of zanamivir acid ester (230 mg, 0.38 mmol, example 31) in 10 ml DMF. EDC (100 mg, 0.5 mmol), HOBt (65 mg, 0.5 mmol) and DIE A (0.14 ml, 1 mmol) were added to this solution. The resulting solution was stirred at room temperature overnight, then purified and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10% to 100% acetonitrile and water without TFA as a modifier. The product yield was 180 mg, 60.2%. Ion(s) found using LCMS: M/2+H=816.9.

Stage d.

The product obtained in the previous step (180 mg, 0.11 mmol) was treated with trifluoroacetic acid (2 ml) for 30 min at room temperature. The resulting solution was concentrated and dissolved in water (2 ml), then treated with a solution of lithium hydroxide (24 mg, 1 mmol) dissolved in H ₂ O (1 ml). The resulting reaction mixture was stirred for 10 minutes, then quenched with 0.1 ml of acetic acid. The solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0% to 50% acetonitrile and water, using 0.1% TFA as a modifier. The product yield was 140 mg, 52%. Ion(s) found using LCMS: M/2+H=575.8, M/3+H=384.2.

Example 74 Synthesis of conjugate 17

This conjugate was prepared analogously to Example 62 (Conjugate 13a) using PEG4-azido-Pc (SEQ ID NO: 35, Example 61) and intermediate-19 (Example 73). Maldi TOF analysis of the purified final product showed an average mass of 56,672 Da (DAR=2.2). Yield 36.7 mg, 36.7%.

Example 75 Synthesis of Intermediate-20

Stage a.

To a solution of diethyliminodiacetate (3.1 g, 16.06 mmol) in anhydrous DMF (36 mL) was added benzyl bromide (2.38 mL, 19.64 mmol) and potassium carbonate (6.44 g, 46.46 mmol). The resulting mixture was heated at 70°C for 16 hours. After cooling to room temperature, the reaction mixture was diluted with water and extracted with tert-butyl methyl ether (3×100 ml). The organic layers were washed with brine, dried over Na ₂ SO ₄ and filtered, then concentrated. The residue was purified by normal phase silica gel chromatography (Isco, 0 to 10% ethyl acetate and hexane). Yield 3.13 g, 69.8%. Ion found using LCMS: [M+H]+=280.2.

Stage b.

A solution of the diethyl ether obtained in step a (3.2 g, 11.2 mmol) in anhydrous THF (5 ml) was slowly added to the round bottom flask containing LiALH ₄ (425 mg, 14.2 mmol) in THF (2 ml) in an atmosphere of nitrogen gas at 0°C. The syringe was flushed with THF (2×5 ml). The resulting mixture was slowly warmed to room temperature overnight. Methanol (2 ml) was added slowly to quench the reaction, after which aqueous NaOH (1 ml) was added. The resulting mixture was stirred for 1 hour, then filtered under vacuum. The filtrate was concentrated and used in the next step without purification. Yield 2.4 g, 109%. Ion found using LCMS: [M+H]+=196.2.

Stage c.

The product obtained in the previous step (1.02 g, 5.24 mmol) in anhydrous THF (4 ml) was slowly added to a flask containing NaH (60% purity, 2.09 g, 52.4 mmol) and THF (5 ml), at 0°C in a nitrogen gas atmosphere. The resulting mixture was stirred for 1 hour and then 3-(Boc-amino)propyl bromide (3.8 g, 15.7 mmol) in THF (20 ml) was added dropwise. The reaction mixture was slowly warmed to room temperature and stirred for 3 days. The reaction mixture was cooled to 0°C, then quenched with water (6 ml) and stirred for 1 hour. The reaction mixture was extracted with ethyl acetate (2×100 ml). The combined organic layers were washed with aqueous IN HCl and brine. They were dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by normal phase chromatography (Isco, 0 to 5% methanol and dichloromethane). Yield 984 mg, 37%. Ion found using LCMS [M+H]+=510.0.

Stage d.

Palladium hydroxide (543 mg, 0.77 mmol) was added to the flask containing the product obtained from step c (985.3 mg, 1.93 mmol) in anhydrous methanol (19.5 ml), under H ₂ atmosphere. The resulting mixture was stirred at room temperature for 16 hours. It was then filtered through a pad of celite, washed with methanol and concentrated. The residue was used in the next step without purification. Yield 828.6 mg, 102%. Ion found using LCMS [M+H]+=420.0.

Stage e.

The product from step d (829 mg, 1.97 mmol) in H ₂ O:THF (1:1, 16 ml) was cooled to 0°C. To this solution was added Na ₂ CO ₃ (314 mg, 2.96 mmol) followed by the addition of Fmoc N-hydroxysuccinimide ester (826 mg, 2.37 mmol). The resulting mixture was warmed to room temperature and stirred until the reaction was complete according to LCMS, then extracted with ethyl acetate. The organic layer was washed with brine and dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by normal phase chromatography (Isco, 0 to 60% ethyl acetate and hexane). Yield 784 mg, 62%. Ion found using LCMS [M+H-Boc]+=542.0.

Stage f.

The product from step e (1.01 g, 1.57 mmol) was stirred in TFA (5 ml) and CH ₂ Cl ₂ (9 ml) at room temperature for 1 hour, then concentrated under reduced pressure. The residue was purified using RPLP (Isco, 5-100% methanol in water without modifier). Yield 903 mg, 86%. Ion found using LCMS [M+H]+=442.2.

Stage g.

To a mixture of zanamivir acid ether (340 mg, 0.49 mmol), the product obtained from step f (111 mg, 0.25 mmol, example 31) and HATU (206 mg, 0.53 mmol) in anhydrous DMF (3 ml) DIE A (162 mg, 1.23 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, then purified directly using RPLC (Isco, 30-100% methanol in water without modifier). Yield 249 mg, 61%. Ion found using LCMS [(M+2H)/2]+=833.8.

Stage h.

To a solution of the product obtained in step g (249 mg, 0.15 mmol) in anhydrous DMF (0.5 ml) was added SilaMetS Thiol (1.2 g, 1.47 mmol) and 1,8-diazabicyclo[5.4. 0]-undec-7-ene (12 mg, 0.07 mmol). The resulting mixture was stirred for 1.5 hours, then filtered directly into a mixture containing HATU (69 mg, 0.18 mmol), PEG-4 propargylic acid (43 mg, 0.16 mmol) and DIEA (43 mg, 0.18 mmol). 33 mmol). The reaction mixture was stirred for 1 hour, then purified directly using RPLC (Isco, 30-100% methanol in water without modifier). Yield 298 mg, 118%. Ion found using LCMS [(M+2H-Boc)/2]+=843.9.

Stage i.

The product from step h (298 mg, 0.18 mmol) was dissolved in TFA (3 ml) and CH2CI2 (3 ml) and the solution was stirred at room temperature for 16 hours. It was then concentrated under reduced pressure and purified by HPLC (ACCQ Isco, 0-25% acetonitrile in water, using 0.1% TFA as modifier). Yield 112 mg, 44%. Ions found using LCMS [(M+2H)/2]+=643.8 and [(M+3H)/3]+=429.6.

Stage j.

To a solution of the product obtained in step-i (112 mg, 0.072 mmol) in MeOH (4 ml) and water (2 ml) was added LiOH (10.6 mg, 0.43 mmol). The resulting solution was stirred at room temperature for 1 hour, then acidified with TFA and concentrated under reduced pressure. The residue was purified by HPLC (ACCQ Isco, 0-25% acetonitrile in water, using 0.1% TFA as modifier). Yield 37 mg, 36%. Ions found using LCMS [(M+2H)/2]+=603.8, [(M+3H)/3]+=402.9.

Example 76 Synthesis of conjugate 18

A solution of azide-functionalized aglycosylated Fc (100 mg, 5.4 mL, 1.87 µmol, SEQ ID NO: 35) was added to a 15 mL centrifuge tube containing the alkyne-derived small molecule (16.1 mg , 0.011 mmol, intermediate-20). After gentle shaking to dissolve all solids, the mixture was added to 3 ml of premixed solution of sodium L-ascorbic acid (149 mg, 0.75 mmol, 0.25 M), copper(II) sulfate (2.4 mg, 0.015 mmol , 0.005 M) and BTTAA (25.8 mg, 0.6 mmol, 0.02 M) in PBS buffer at pH 7.4. The resulting solution was shaken gently overnight. It was purified by affinity chromatography on a Protein A column followed by size exclusion chromatography (see General Conjugate Purification Protocol in Example 10). Maldi TOF analysis of the purified final product showed an average mass of 56,826 Da (DAR 2.2). Yield 36.64 mg, 37%.

Example 77 Synthesis of Intermediate-21

Stage a.

DIPEA (7.8 g, 60 mmol) and DMAP (366.6 mg, 3 mmol) were added to a solution of 2-(2-Boc-aminoethoxy)ethanol (6.15 g, 30 mmol) in anhydrous DCM (60 mL). . p-Toluenesulfonyl chloride (6.86 g, 36 mmol) was then added in portions over 30 minutes. After stirring the resulting mixture for 3 days, it was concentrated by rotary evaporation and purified by RPLC (20% to 70% acetonitrile/water without modifier). Yield 3.71 g, 34.4%. Ion found using LCMS: [M-Boc+H]+=260.

Stage b.

To a solution of the product obtained in step-a (2.1 g, 5.83 mmol) in anhydrous THF (10 ml) was added sodium carbonate (1.24 g, 11.7 mmol) and mono-N-Boc-1, 4-diaminobutane (1.32 g, 7 mmol). The resulting mixture was heated at 60°C for 1 day. The salt was then filtered off and the filtrate was concentrated on a rotary evaporator. The residue was purified by RPLC (100 g, 5-50% acetonitrile in water, using 0.1% TFA as modifier). Yield 1.94 g, 88.6%. Ion found using LCMS: [M+H]+=376.0.

Stage c.

To a solution of propargyl-PEG-4-acid (781 mg, 3 mmol) and HATU (1.14 g, 3 mmol) in anhydrous DMF (3 ml) was added DIPEA (390 mg, 3 mmol) followed by addition of a solution of the product obtained in step-b (940 mg, 2.5 mmol) and DIPEA (390 mg, 3 mmol) in anhydrous DMF (3 ml). The reaction mixture was stirred for 30 minutes, then purified directly by RPLC (100 g, 5-80% acetonitrile in water, using 0.1% TFA as modifier). Yield 960.2 mg, 65.3%. Ion found using LCMS: [M+H]+=618.3, [M-Boc+H]+=518.3.

Stage d.

The product obtained in step-c (960.2 mg, 1.63 mmol) was dissolved in anhydrous THF (6 ml). A solution of 4N HCl in dioxane (4 ml) was added and the reaction mixture was stirred overnight. It was then concentrated on a rotary evaporator. The residue was extracted with water (3 ml×3) and ethyl acetate (10 ml). The combined aqueous layers were lyophilized. Yield 760 mg, 95.1%. Ion found using LCMS: [M+H]+=418.0.

Stage e.

To a mixture of zanamivir acid ester (315 mg, 0.5 mmol) and HATU (190 mg, 0.5 mmol) in anhydrous DMF (1 ml), a solution of the diamine product obtained in step-d (148 mg, 0.3 mmol), and DIPEA (165 mg, 1.5 mmol) in anhydrous DMF (1 ml) for 20 minutes. After addition, the reaction mixture was stirred for an additional 30 minutes and purified directly by RPLC (50 g, 30-90% acetonitrile in water, using 0.1% TFA as modifier). Yield 233 mg, 56.7%. Ion found using LCMS: [(M+2H)/2]+=821.3.

Stage f.

The product obtained in step-e (233 mg, 0.142 mmol) was dissolved in TFA (1.5 ml) and the solution was heated at 30°C for 30 minutes. It was then concentrated and directly purified by RPLC (0-30% acetonitrile in water, using 0.1% TFA as modifier). Yield 120 mg, 57.4%. Ions found using LCMS: [(M+2H)/2]+=621.4, [(M+3H)/3]+=414.7.

Stage g.

To a solution of the product obtained in step-f (120 mg, 0.0816 mmol) in MeOH (2 ml) was added a solution of LiOH monohydrate (63 mg, 1.5 mmol) in water (2 ml). The resulting mixture was stirred for 1.5 hours and then concentrated on a rotary evaporator. The residue was acidified with 4N HCl in dioxane (0.5 ml) and purified by HPLC (0-15% acetonitrile in water, using 0.1% TFA as modifier). Yield 98.2 mg, 86.6%. Ions found using LCMS: [(M+2H)/2]+=581.8,[(M+3H)/3]+=388.2.

Example 78 Synthesis of conjugate 19

This conjugate was prepared analogously to Example 62 (Conjugate 13a) using PEG4-azido-Fc (SEQ ID NO: 35, Example 61) and intermediate-21 (Example 78). Maldi TOF analysis of the purified final product showed an average mass of 56,548 Da (DAR=2.1). Yield 39.7 mg, 39.7%.

Example 79 Synthesis of Intermediate-22

Stage a.

EDC was added to a mixture of zanamivir acid ester (1.8 g, 2.8 mmol, Example 31) and propargyl-PEG4-amine (0.82 g, 3.5 mmol, 1.2 eq.) in dichloromethane (50 ml). (1.0 g, 5 mmol), HOBt (0.65 g, 5 mmol) and DIEA (1.4 ml, 10 mmol). The resulting solution was stirred at room temperature overnight, concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10%-100% acetonitrile and water without 0.1% TFA as modifier. Product yield 1.35 g, 50.2%. Ion(s) found using LCMS: M+H=830.4.

Stage b.

The product obtained in the previous step (1.35 g, 1.6 mmol) was treated with trifluoroacetic acid (20 ml) for 30 minutes at room temperature. The resulting solution was concentrated, dissolved in water (10 ml) and MeOH (10 ml), then treated with a solution of lithium hydroxide (120 mg, 5 mmol) in water (10 ml). The reaction mixture was stirred for 10 min, then quenched with 0.5 ml of acetic acid. The reaction mixture was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 0%-50% acetonitrile and water, using 0.1% TFA as a modifier. Product yield 510 mg, 52.8%. Ions found using LCMS: M+H=604.3.

Example 80 Synthesis of conjugate 20

This conjugate was prepared analogously to Example 62 (Conjugate 13a) using PEG4-azido-Pc (SEQ ID NO: 35, Example 61) and intermediate-22 (Example 79). Maldi TOF analysis of the purified final product showed an average mass of 55508 Da (DAR=2.3). Yield 37.0 mg, 37.0%.

Example 81 Synthesis of Intermediate-23

Stage a.

To a solution of 1N-Boc-1,4-diaminobutane (941.5 mg, 5 mmol) in anhydrous THF (10 ml) was added sodium carbonate (1.06 g, 10 mmol) and 3-(Boc-amino)propyl bromide (1 .43 g, 6 mmol). The resulting mixture was heated at 50°C for 1 day. The salt was then filtered and concentrated on a rotary evaporator. The residue was purified by RPLC (100 g, 5-50% acetonitrile in water, using 0.1% TFA as modifier). Yield 1.35 g, 58.8%. Ion found using LCMS: [M+H]+=346.0.

Stage b.

To a solution of propargyl-PEG-4-acid (781 mg, 3 mmol) and HATU (1.14 g, 3 mmol) in anhydrous DMF (3 ml) was added DIPEA (390 mg, 3 mmol) followed by addition of a solution of the product obtained in step-a (863.8 mg, 2.5 mmol), and DIPEA (390 mg, 3 mmol) in anhydrous DMF (3 ml). The reaction mixture was stirred for 30 minutes, then purified directly by RPLC (100 g, 5-80% acetonitrile in water, using 0.1% TFA as modifier). Yield 1.19 g, 81%. Ion found using LCMS: [M+H]+=588.3.

Stage c.

The product obtained in step-b (1.19 g, 2.02 mmol) was dissolved in anhydrous THF (6 ml). A solution of 4N HCl in dioxane (4.5 ml) was added and the reaction mixture was stirred for 1 day. It was then concentrated on a rotary evaporator. The residue was extracted with water (3 ml×3) and ethyl acetate (15 ml). The combined aqueous layers were lyophilized. Yield 940 mg, quantitative yield. Ion found using LCMS: [M+H]+=388.3.

Stage d.

To a mixture of zanamivir acid ester (315 mg, 0.5 mmol, Example 31) and HATU (209.1 mg, 0.55 mmol) in anhydrous DMF (1 ml) was added DIPEA (65 mg, 0.5 mmol). After 5 minutes, a solution of the product obtained in step-c (170 mg, 0.439 mmol) and DIPEA (130 mg, 1 mmol) in anhydrous DMF (1 ml) were added portionwise over 20 minutes. The reaction mixture was stirred for an additional 30 minutes, then purified directly by RPLC (50 g, 30-90% acetonitrile in water, using 0.1% TFA as modifier). Yield 208 mg, 51.6%. Ion found using LCMS: [(M+2H)/2] ⁺ =806.7.

Stage e.

The product obtained in step-d (208 mg, 0.129 mmol) was dissolved in TFA (1.5 ml) and the solution was heated at 30°C for 30 minutes. It was then directly purified by RPLC (100 g, 0-30% acetonitrile in water, using 0.1% TFA as modifier). Yield 134 mg, 72%. Ions found using LCMS: [(M+2H)/2] ⁺ =606.8, [(M+3H)/3] ⁺ =405.0.

Stage f.

To a solution of the product obtained in step-e (134 mg, 0.093 mmol) in MeOH (2 ml) was added a solution of LiOH monohydrate (63 mg, 1.5 mmol) in water (2 ml). The resulting mixture was stirred for 1.5 hours and then concentrated on a rotary evaporator. The residue was acidified with a solution of 4N HCl in dioxane (0.5 ml) and purified by HPLC (0-15% acetonitrile in water, using 0.1% TFA as modifier). Yield 78.4 mg, 62%. Ions found using LCMS: [(M+2H)/2] ⁺ =566.4, [(M+3H)/3] ⁺ =378.4.

Example 82 Synthesis of conjugate 21

This conjugate was prepared analogously to Example 62 (Conjugate 13a) using PEG4-azido-Fc (SEQ ID NO: 35, Example 61) and intermediate-23 (Example 81). Maldi TOF analysis of the purified final product showed an average mass of 56,503 Da (DAR=2.2). Yield 34.4 mg, 34%.

Example 83. Activity of conjugates 13-21 against highly pathogenic influenza A H7N9 virus and two influenza B isolates in a cytopathic effect assay (CPE)

A total of 9 conjugates (Table 38) were used at concentrations of 100, 10, 1 and 0.1 nM, and were compared with ribavirin in the CPE assay. The CPE assay followed standard methodology but briefly used 80–100% confluent MDCK cell monolayers in a 96-well plate. Test drugs were added to them in triplicate and incubated at 37°C (+5% CO ₂ ) until the effects of CPE became visually obvious. Once CPE was noted, cell layers were stained with 0.011% neutral red for approximately 2 hours. Thereafter, a 50:50 mixture of Sorensen's citrate buffer/ethanol was added and left to incubate for 30 minutes, then the A ₅₄₀ was read on a spectrophotometer and the EC ₅₀ /CC ₅₀ values were calculated using regression analysis.

All conjugates had significant activity against influenza isolate B/Florida/4/2006 with EC ₅₀ values ranging from 3.05 to 33.5 nM (Table 39). On average, the conjugates demonstrated a 275-fold potency advantage over ribavirin (EC ₅₀ 3250 nM). The activity of the conjugates against influenza B/Brisbane/60/2008 was very similar to the activity observed against B/Florida, with the exception of conjugate 14, which had an EC ₅₀ of more than 100 nM.

Importantly, all conjugates were also highly active against the highly pathogenic influenza virus isolate A/Anhui/1/2013 (H7N9). The average EC ₅₀ value for all conjugates was 21.2 nM against this isolate (range 12 to 28.5 nM) compared to 14,000 nM for ribavirin. Finally, no direct cytotoxic effects of the conjugates on MDCK monolayers were detected at the concentrations tested.

Method 39: Activity of conjugates in the CPE assay against influenza subtypes

Example 84 Synthesis of conjugate 22

Stage a. Synthesis of PEG4-azido IVIG

Preparation of a 0.05 M solution of PEG4 azido-NHS ester in DMF/PBS: 6.05 mg of PEG4 azido-NHS ester was dissolved in 0.050 ml DMF at 0°C and diluted to 0.305 ml by adding 0.250 ml 1x buffer PBS at 0°C. This solution was used to prepare another PEG4-azido IVIG with different DAR values by adjusting equivalents of this PEG4-azido-NHS-ester solution in PBS.

Preparation of PEG4-azido IVIG: 0.05 M PEG4-azido-NHS-c false ester PBS buffer solution (0.301 ml, 15.0 µmol, 5.5 equivalents) was added to the IVIG (intravenous immune globulin, Baxter) solution (407 mg in 9.25 ml, pH 7.4, PBS, MW ~ 148863 Da, 1.968 µmol), and the mixture was shaken gently for 12 hours at ambient temperature. The solution was concentrated using a centrifugal concentrator (100,000 MWCO) to a volume of ~1.5 mL. The crude mixture was diluted 1:10 in PBS, pH 7.4, and concentrated again. This washing procedure was repeated a total of three times. This washing procedure removed the low molecular weight reagent. Concentrated IVIG-PEG4-azide was diluted to 9.25 ml with 1x PBS pH 7.4 buffer and prepared for click conjugation. The purified product was quantified using a Nanodrop™ UV-visible spectrophotometer (using a calculated extinction coefficient based on the IVIG amino acid sequence). The yield is quantitative after purification.

Stage b. Synthesis of conjugates

A solution of the “click” reagent was obtained: 0.0050 M CuSO ₄ in a PBS buffer solution: 10.0 mg of CuSO ₄ was dissolved in 12.53 ml of PBS x1, then 6.00 ml of 0.0050 M CuSO ₄ solution was taken and 57 was added. 7 mg BTTAA (CAS number 1334179-85-9) and 297.6 mg sodium ascorbate to obtain a click reagent solution (0.0050 M CuSO ₄ , 0.020 M BTTAA and 0.25 M sodium ascorbate).

A solution of azide-functionalized IVIG (140 mg, 3.17 mL, 0.936 μmol, IVIG-linker-1-azide) was added to a 15 mL centrifuge tube containing the alkyne-derivatized small molecule (8.4 mg, 0.00618 mmol, 6 .6 eq., described in example 60). After gentle shaking to dissolve all solids, add 1.50 ml of the above click reagent solution (sodium L-ascorbic acid, 0.25 M, 74.2 mg, 0.374 mmol, copper(II) sulfate 0.0050 M, 1.2 mg, 0.0075 mmol, and BTTAA 0.020 M, 12.9 mg, 0.0300 mmol). The resulting mixture was shaken gently overnight. It was purified by affinity chromatography on a protein A column followed by size exclusion chromatography (see conjugate purification protocol in example 10). Maldi TOF analysis of the purified final product showed an average mass of 151,873 Da (DAR=2.7). Yield 51.0 mg, yield 36%.

Example 85 Synthesis of conjugate 23

Conjugate 23 was prepared similarly to conjugate 22 by replacing the corresponding alkyne-functionalized small molecule (intermediate-23 described in Example 81) in the click conjugation step.

Example 86 Synthesis of conjugate 24

Conjugate 24 was prepared similarly to conjugate 22 by replacing the corresponding alkyne-functionalized small molecule (described in Example 19) in the click conjugation step.

Example 87 Efficacy of conjugates 13, 14 and 21 against influenza B in a lethal mouse model

The conjugates were evaluated against lethal influenza B infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks). The challenge virus (B/Malaysia/2506/04) is a mouse-adapted isolate capable of causing lethal infections in mice. The experiment involved 11 groups of 5 mice. On day 0, all mice were infected with virus at 3xLD95 by intranasal inoculation in a volume of 30 μl (approximately 1E4 per mouse) after anesthesia with a mixture of ketamine/xylazine (150 and 10 mg/kg, respectively).

All groups were treated 2 hours after viral challenge with a single IV dose of test drug, vehicle (PBS), or Fc alone (hIgG1-Fc) as a control. The study evaluated intermediate-18, intermediate-7, and intermediate-23 conjugated to identical Fc monomers (conjugates 13, 14, and 21, respectively), tested at 3.0, 1.0, and 0.3 mg /kg. Mice were monitored for 2 weeks, and animals that showed more than 20% body weight loss or were considered moribund were scored as dead.

All mice treated with vehicle or Fc alone as a control died on day 7. In contrast, mice treated with conjugates 13, 14 and 21 were completely protected after receiving a single IV dose of 0.3 mg/kg (Table 40). As expected, groups receiving 1.0 or 3.0 mg/kg conjugates were also fully protected. The effectiveness of all conjugates against influenza B was further supported by daily body weight measurements (Table 41), which showed a transient drop of less than 5% throughout the study for any conjugate-treated group. The activity of conjugates 13, 14 and 21 is comparable in dose to that of conjugate 6 against the H1N1 and H3N2 subtypes of influenza A. Because conjugates 6 and 14 have identical targeting moieties (corresponding to intermediate-7), a single conjugate can be active against the dominant types of seasonal influenza ( influenza A (H1N1), influenza A (H3N2) and influenza B).

Example 88 Synthesis of PEG4-azido Fc for conjugate 25, conjugate 26, conjugate 27 and conjugate 28

Preparation of 0.05 M PEG4-azido-NHS-ester in DMF/PBS solution x1: 27.40 mg PEG4-azido-NHS-ester was dissolved in 0.155 ml DMF at 0°C and diluted by adding 1,200 ml of 1x PBS buffer at 0°C. This solution was used to prepare another PEG4-azido-Ec with different DAR values by adjusting the equivalents of this solution of PEG4-azido-NHS ester in x1 PBS.

Preparation of PEG4-azido-Ec (SEQ ID NO: 48): 0.05 M PEG4-azido-NHS ester in PBS buffer x1 (0.0984 ml, 4.92 µmol, 2.5 equivalents) was added to the h- solution IgG1-Fc (SEQ ID NO: 48) (234 mg in 13.605 ml PBS pH 7.4, MW -57976 Da, 4.036 µmol) and the mixture was shaken gently for 2 hours at ambient temperature. The solution was concentrated using a centrifugal concentrator (30,000 MWCO) to a volume of ~2 mL. The crude mixture was diluted 1:7 in PBS pH 7.4 and concentrated again. This washing procedure was repeated a total of three times. This washing procedure removed the low molecular weight reagent. Concentrated Fc(SEQ ID NO: 48)-PEG4-azide was diluted to 13.60 ml with 1x PBS buffer pH 7.4 and prepared for click conjugation. The purified product was quantified using a Nanodrop™ UV-visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1).

The preparation of PEG4-azido-Ec (SEQ ID NO: 50) is similar to the above PEG4-azido-Fc (SEQ ID NO: 48).

Example 89. Synthesis of conjugate 25

Preparation of a “click” reagent solution: 0.0050M CuSO ₄ in a buffer solution x1 PBS: 10.0 mg of CuSO ₄ was dissolved in 12.53 ml x1 PBS, then 10.00 ml of this solution of CuSO ₄ was taken and 86.1 mg was added BTTAA and 495.3 mg of sodium ascorbate to obtain a “click” reagent solution (0.0050 M CuSO ₄ , 0.020 M BTTAA and 0.25 M sodium ascorbate). This Click reagent solution is used for conjugate 25 and conjugate 26.

A solution of azide-functionalized Fc (78.0 mg, 4.535 mL, 1.35 μmol, SEQ ID NO: 48-PEG4-azide) was added to a 15 mL centrifuge tube containing the alkyne-derivatized small molecule (13.2 mg, 8. 88 µmol, intermediate-23). After gentle shaking to dissolve all solids, 2.153 mL of the above click reagent solution (L-ascorbic acid sodium salt, 0.25 M, 106.6 mg, 0.538 mmol, copper(II) sulfate, 0. 0050M, 1.72 mg, 0.0107 mmol, and BTTAA 0.020 M, 18.5 mg, 0.0431 mmol). The resulting mixture was shaken gently for 6 hours at ambient temperature. It was purified by affinity chromatography on a protein A column followed by size exclusion chromatography (see conjugate purification protocol in example 10). Maldi TOF analysis of the purified final product showed an average mass of 60973 Da (DAR=2.1). Yield 50.3 mg, yield 64%.

Example 90 Synthesis of conjugate 26

The preparation of conjugate 26 is similar to conjugate 25 using the same batch of PEG4-azido-Ec (SEQ ID NO: 48) and an alkyne-derivatized small molecule (intermediate-7). Maldi TOF analysis of the purified final product showed an average mass of 61068 Da (DAR=2.2). Yield 49.5 mg, yield 63%.

Example 91 Synthesis of conjugate 27

Preparation of a “click” reagent solution: 0.0050 M CuSO ₄ in a buffer solution x1 PBS: 10.0 mg CuSO ₄ was dissolved in 12.53 ml x1 PBS, then 12.00 ml of this CuSO ₄ solution was taken and 103.3 was added mg BTTAA and 594.3 mg sodium ascorbate to obtain a “click” reagent solution (0.0050 M CuSO ₄ , 0.020 M BTTAA and 0.25 M sodium ascorbate). This click reagent solution will be used for conjugate 28.

A solution of azide-functionalized Fc (80.0 mg, 4.535 mL, 1.38 μmol, SEQ ID NO: 50-PEG4-azide) was added to a 15 mL centrifuge tube containing the alkyne-derivatized small molecule (13.5 mg, 9. 10 µmol, intermediate-23). After gentle shaking to dissolve all solids, 2.21 ml of the above click reagent solution (sodium L-ascorbic acid, 0.25 M, 109.3 mg, 0.552 mmol, copper(II) sulfate, 0 .0050 M, 1.76 mg, 0.0110 mmol, and BTTAA, 0.020 M, 19.0 mg, 0.0441 mmol). The resulting mixture was shaken gently for 6 hours at ambient temperature. It was purified by affinity chromatography on a protein A column followed by size exclusion chromatography (see conjugate purification protocol in example 10). Maldi TOF analysis of the purified final product showed an average mass of 61,447 Da (DAR=2.5). Yield 37.1 mg, yield 46%.

Example 92 Synthesis of conjugate 28

Conjugate 28 was prepared similarly to conjugate 27 using the same batch of PEG4-azido-Ec (SEQ ID NO: 50) and an alkyne-derivatized small molecule (intermediate-7). Maldi TOF analysis of the purified final product gave an average mass of 61,388 Da (DAR=2.4). Yield 44.6 mg, yield 56%.

Example 93. Activity of conjugate 6 and conjugate 21 against highly pathogenic influenza A (H5N1, H7N9) in the cytopathic effect assay (CPE)

In vitro assays to determine the activity of the conjugates of the present invention were performed against BSL-3 (highly pathogenic) influenza A and generally followed standard procedures. Briefly, various concentrations of conjugates were mixed with virus (approximately 250 TC _ID50 ) and left to incubate at 35°C for one hour. After incubation, the mixture was added to an 80-90% confluent monolayer of MDCK cells. After a 90-minute incubation, the cells were washed and the conjugates were reapplied. The monolayer was then coated with carboxymethylcellulose to minimize viral spread and left to incubate for two days. After two days of culture, the cells were washed with PBS and fixed with 10% formaldehyde. After fixation, the MDCK monolayer was permeabilized with Triton X-100 and immunostained with mouse anti-influenza nucleoprotein mAb. Monolayers were read and the area of staining per well was calculated to determine EC _50/100 values.

The results of the study are summarized in Table 42 and demonstrate the effectiveness of Conjugate 6 and Conjugate 21 against highly pathogenic strains with pandemic potential. Importantly, both conjugates generated EC ₁₀₀ values of 15 nM or lower against four H5N1 and one H7N9 isolates. In contrast, oseltamivir had an EC ₁₀₀ of approximately 15 nM against only one isolate (A/Vietnam/1194/2004) and values ranging from 125 to >1000 nM against other highly pathogenic strains. These results suggest that the potential of conjugate 6 and conjugate 21 for the treatment of highly virulent influenza pandemics is superior to that of oseltamivir.

Example 94: Activity of Conjugate 6 and Conjugate 21 against Influenza A (H1N1) at Various Multiplicities of Infection (MOI) in a Cytopathic Effect Assay (CPE)

MDCK cells were seeded at a density of 4×10 ⁴ cells/well in MEM medium in a 96-well plate (treated with TC) and incubated at 37°C, 5% CO ₂ for 18-24 hours. Test drugs (zanamivir, oseltamivir, baloxavir , conjugate 6 and conjugate 21) in the dose range of 1.93-10000 nM were incubated with influenza A/WSN/1933 virus at a multiplicity of infection (MOI) virus: cell from 0.001 to 1 for 1 h at room temperature (RT). After 1 hour, the preincubated virus and test drug were added to a 90-100% confluent monolayer of MDCK cells and incubated for 1 hour at room temperature.

After 1 h, MEM medium supplemented with L-glutamine and penicillin/streptomycin was added to the wells. Infected cells were incubated at 37°C, 5% CO ₂ for 72 hours. CPE was determined after fixation and staining of cells with crystal violet. The EC ₅₀ value was calculated by nonlinear regression analysis using Graphpad Prism 6 software. The results of the CPE analysis presented in Table 43 show that conjugate 6 and conjugate 21 are superior to standard in vitro treatment agents, especially at high MOI values.

Example 95: Efficacy of Conjugate 6 against influenza A (H1N1) in a mouse model of lethal severe combined immunodeficiency

The conjugates were evaluated against lethal influenza A infection in male BALB/c severe combined immunodeficiency (SCID) mice (Stock #001803; Jackson Laboratories, 6-8 weeks old). The challenge virus (A/Puerto Rico/08/1934) is a mouse-adapted isolate capable of causing lethal infections in mice. The experiment involved 5 groups of 5 mice each. On day 0, all mice were infected with virus at 3xLD ₉₅ by intranasal inoculation in a volume of 30 μl (approximately 1U per mouse) after anesthesia with a mixture of ketamine and xylazine (150 and 10 mg/kg, respectively).

All groups were treated 2 hours after viral infection with a single IV dose of test drug, vehicle (PBS), or Fc alone (hIgG1-Fc) as a control. The study evaluated 3 different dose concentrations of Conjugate 6 (0.3, 1.0, or 3.0 mg/kg). Mice were monitored for 5 weeks, and animals that had greater than 20% weight loss or were considered moribund were scored as dead. Body weight was also recorded to monitor the general health of the animals.

All mice treated with vehicle or Fc alone as a control died by 2 weeks. In contrast, mice treated with conjugate 6 were completely protected after receiving single intravenous doses of 1 or 3 mg/kg throughout the study (Fig. 56, Table 44). When the dose of conjugate 6 was reduced to 0.3 mg/kg, survival dropped to 20% at the end of the study. A dose of 0.3 mg/kg was completely protective over 3 weeks. The effectiveness of conjugate 6 in this model of severe immunodeficiency is further supported by body weight data (Fig. 57, Table 45). Groups receiving conjugate 6 at doses of 1 or 3 mg/kg demonstrated no more than transient body weight loss of less than 3% over the course of the study. Additionally, by the end of the study, the groups taking both doses showed a net weight gain (7.5% and 2.2%, respectively). The group receiving the lowest concentration of conjugate 6 (0.3 mg/kg) had less than 4% temporary weight loss during the first 3 weeks of the study before the onset of signs of infection at week 4, which ultimately resulted in death for four of the patients. five animals.

Taken together, these data demonstrate the effectiveness of conjugate 6 in protecting lethally infected mice using single IV doses of conjugate as low as 1 mg/kg. Moreover, this protection was long lasting, lasting for 5 weeks of the study. This was achieved in an extreme mouse model of immunodeficiency completely lacking the immune T and B cells needed to clear influenza infections. These data support the use of conjugate 6 for the treatment of both immunocompetent and immunodeficient patient populations.

Example 96. Dose-dependent clearance of virus in the lungs of conjugate 6

Efficacy studies were performed on female BALB/c mice aged 6-8 weeks (Charles River), which were intranasally injected with 3x10 ² PFU/mouse (3x LD ₉₅ ) of mouse-adapted influenza A/Puerto Rico/8/1934 (H1N1) virus ). Conjugate 6 or human IgG1-Fc as a control was administered as a single intravenous (IV) dose 2 hours after infection at a dose of 0.1-3 mg/kg. Oseltamivir was administered orally twice daily for 4 days, starting 2 hours after infection at a dose of 5 or 15 mg/kg. Body weight (BW) was recorded for 4 days. 4 days after infection, mice were sacrificed using CO ₂ and both lobes of the lungs were collected. Lungs were homogenized with 1 mm silica beads in 1 ml PBS using a MagNA Lyser (Roche). Homogenization was performed at 6000 rpm for 60 sec and cooled on ice for 5 min between cycles. After homogenization of the lungs, the tubes were centrifuged for 10 min at 600 × g and the supernatant was transferred to a new tube.

To determine viral load in the lungs (measured in plaque-forming units (PFU)), lung homogenate supernatants were diluted in infection buffer ranging from 10-1 to 10-6. 100 μl of virus dilutions were added to a confluent monolayer of MDCK cells in 24-well plates and incubated for 1 h at room temperature with shaking every 15 min. After virus removal, liquid medium containing Avicel was added to MDCK cells. Cells were incubated at 37°C, 5% CO ₂ for 40 hours. After incubation, the medium was removed and cells were stained with crystal violet for plaque counting. PFU was calculated relative to lung weight (PFU/g lung).

The results of this study demonstrate that low doses of conjugate 6 rapidly reduce viral load by several orders of magnitude better than oseltamivir (TAMIFLU®) (Fig. 58, Table 46). This observation has clinical significance because severe influenza infections are caused by viral transfer from the initial upper respiratory tract infection to the lungs.

Example 97 Conjugate 6 Dose-Dependent Reduction of Inflammatory Cytokines in the Lungs

Efficacy studies were performed on female BALB/c mice aged 6-8 weeks (Charles River), which were intranasally injected with 3x10 ² PFU/mouse (3x LD ₉₅ ) of mouse-adapted influenza A/Puerto Rico/8/1934 (H1N1) virus ). Conjugate 6 or human IgG1-Fc as a control was administered as a single intravenous (IV) dose 2 hours postinfection at 0.1-3 mg/kg. Oseltamivir was administered orally twice daily for 4 days, starting 2 hours after infection, at a dose of 5 or 15 mg/kg. Body weight (BW) was recorded for 4 days. 4 days after infection, mice were sacrificed using CO ₂ and both lobes of the lungs were collected. Lungs were homogenized with 1 mm silica beads in 1 ml PBS using a MagNA Lyser (Roche). Homogenization was performed at 6000 rpm for 60 sec and cooled on ice for 5 min between cycles. After homogenization of the lungs, the tubes were centrifuged for 10 min at 600 × g and the supernatant was transferred to a new tube.

For cytokine analysis, lung homogenate supernatants were serially diluted 2-fold in a 96-well plate. Cytokine levels for INF-γ, TNF-α, IL-6, MIP-1α, and MCP-1 were determined by ELISA according to the manufacturer's instructions (R&D Systems).

The morbidity and mortality of severe influenza is ultimately caused by the virus-induced influx of proinflammatory cytokines into the lungs. One potential concern with the use of an Fc conjugate to treat influenza is whether the Fc moiety may exacerbate cytokine-induced inflammation. Results from a lethal H1N1 infection model show just the opposite: a dose-dependent reduction of conjugate 6 in proinflammatory cytokines (eg, TNFα and IL-6) in infected lung tissues (Fig. 59, Table 47).

Table 47: Cytokine response on day 4 postinfection

Example 98 In Vivo Analysis of Conjugate 6 Plasma Sample Comparison of PK in CD-1 and BALB/c Mice with Severe Combined Immunodeficiency

Conjugate 6 in plasma samples was quantified by detecting neuraminidase uptake using ELISA. Briefly, molecules were captured on neuraminidase-coated plates and then detected using an HRP-conjugated anti-human IgG-Fc antibody. Protein concentration was calculated in GraphPad Prism using 4PL nonlinear regression of conjugate 6 standard curves. A more detailed description of the method is provided below.

Nunc Maxisorp 96-well plates (cat. no. 12-565-136, ThermoFisher) were coated with 0.1 U/well of neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Biological) in IX KPL coating buffer (5150-0041, SeraCare). The plates were incubated at room temperature for 1 h on an orbital plate shaker (500 rpm). Serial dilutions of plasma samples were added to plates and incubated at room temperature for 2 hours (sample diluent: 0.5% BSA in PBS, 0.025% Tween 20 + final plasma concentration in naïve mice 1:2500). Standard curves of conjugate 6 ranging from 0.230 to 500 ng/ml, in duplicate, were reproduced on each plate. After a 2-hour incubation, the plates were washed 5 times in 300 μl PBS with 0.05% Tween 20. The neuraminidase conjugate on the plates was then probed with HRP-conjugated anti-human IgG Fc F(ab') ₂ antibody. (709-036-098, Jackson), diluted 1:1000 in sample diluent for 1 hour at room temperature. The plates were then washed 8 times in 300 µl PBS with 0.05% Tween 20 and developed with TMB substrate for 7-8 minutes. The reaction was stopped IN H ₂ SO ₄ . Absorbance was read at 450 nm. Conjugate 6 in plasma samples was interpolated using GraphPad Prism Version 6 after nonlinear regression analysis (sigmoidal curve analysis using a four-parameter nonlinear regression (4PL) model) of the standard curves.

PK profiles, CD-I mice compared to SCID BALB/c

Conjugate 6 administered intravenously to SCID and CD-I (immunocompetent) mice at a dose of 5 mg/kg exhibited similar PK profiles (FIG. 60). Concentrations were comparable at the selected time points. Biphasic DO profiles contain 24-hour distribution phases followed by a gentle removal phase. Plasma levels of conjugate 6 remained high (~10 μg/mL) relative to _Cmax levels during the one-week course of the study.

Example 99 Synthesis of the propargyldiamine central linker.

Stage a.

A solution of 2-(2-Boc-aminoethoxy)ethanol (16.0 g, 78.0 mmol) and CBr ₄ (31.0 g, 93.5 mmol) in DCM (100 ml) at 0°C was slowly treated with PPh ₃ (24.5 g, 93.5 mmol) for 15 minutes (exothermic reaction). During addition, the internal temperature was maintained below 30°C. After adding PPh ₃ the reaction mixture was stirred overnight at room temperature. The crude reaction mixture was concentrated to an oil, then purified by normal phase chromatography, eluting with 10% ethyl acetate/hexanes to 80% ethyl acetate/hexanes. Fractions containing oil droplets inside collection tubes were pooled and concentrated to a colorless oil. Yield 18.1 g, 86%.

Stage b.

A solution of the product from step-a (10 g, 37.3 mmol), benzylamine (1.60 g, 14.9 mmol) and K ₂ CO ₃ (6.19 g, 44.8 mmol) in DMF (20 ml) was heated in an oil bath at 75°C for 8 hours. The mixture was filtered, concentrated and purified by RPLC (5% ACN/water to 100% ACN). Yield 6.8 g, 95%.

Stage c.

To a solution of the product obtained in step-b (5.35 g, 8.98 mmol) in CHCl ₃ /EtOH (1:20, 100 ml) was added 20% Pd(OH) ₂ /C (1.26 g, 1 /80 mmol). The reaction mixture was stirred overnight under a hydrogen cylinder at ambient temperature. The reaction mixture was filtered through a pad of celite. Solvents were removed and carried to the next step without purification.

Stage d.

The product obtained in step-c was re-dissolved in 20 ml of DMF/dichloromethane (1:5). To this free amine solution was added propargyl-PEG4 acid (2.36 g, 8.98 mmol), EDCI (2.57 g, 13.5 mmol), HOAt (1.83 g, 13.5 mmol) and base Hunig (3.13 ml, 18.0 mmol). The reaction mixture was stirred for four hours, then concentrated and purified by RPLC (10% ACN/water to 60% ACN/water). Yield 4.00 g, 70% in two stages. Ions found using LCMS: [M-Boc+H] ⁺ =534.2, [M+H] ⁺ =634.2.

Stage e.

The product obtained from step-d (4.00 g, 6.31 mmol) was treated with 4N HCl in dioxane (30 ml) for 2 hours. Excess HCl and dioxane were removed using a rotary evaporator and the residue was further dried under high vacuum to give Intermediate-10 as the 2 HCl salt. Yield 3.15 g, 99%. Ion found using LCMS: [M+H] ⁺ =434.2.

Example 100 Synthesis of intermediate-7a (C7-C7 isomer)

Stage a.

Methyl 5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate (30 g, 65.7 mmol) was dissolved in methanol (100 ml) and combined with Lindlar catalyst (15 g). The resulting mixture was purged with hydrogen and stirred for 5 hours, purging hydrogen through the headspace every 30 minutes. After completion of the reaction as determined by HPLC, the catalyst was filtered through celite. The filtrate was used in the next step.

The crude amine obtained in the previous step (18.9 g, 43.8 mmol) was treated with N,N'-bis-boc-1-guanylpyrazole (14.3 g, 46.0 mmol) and DIEA (9.9 ml , 57.0 mmol) in methanol (100 ml). The resulting solution was stirred at room temperature until complete consumption of the starting material, as determined by LCMS (-30 min). The solution was concentrated to a foam and stored under high vacuum overnight, then used without further purification in the next step.

The crude triacetate obtained in the previous step (43.8 mmol) was dissolved in 100 ml of dry methanol, then treated with sodium methoxide in methanol (1.9 ml, 25% solution in methanol, 8.76 mmol) at room temperature. The reaction progress was monitored using LCMS, which was completed after 10 minutes. The reaction was quenched with IN HCl to pH ~7. The resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 10-100% acetonitrile and water. No TFA modifier was used for this purification. Product yield 15.6 g, 65%.

Stage b.

A mixture of the product obtained in step-a (5.47 g, 10 mmol) and DMAP (1.222 g, 10 mmol) was dissolved in anhydrous THF (30 ml). After cooling in an ice-water bath, the solution was slowly treated with 1,1'-carbonyldiimidazole (2.6 g, 16 mmol), then stirred for 30 minutes at 0°C followed by heating at 60°C for 2 hours. It was then cooled to room temperature and extracted with water (50 ml) and EtOAc/hexane (1:1, 100 ml). The organic layer was washed with water (50 ml x 3); dried over Na ₂ SO ₄ and concentrated by rotary evaporation. The white foam product was further dried under high vacuum and carried to the next step without further purification. Ion found using LCMS: [M+H] ⁺ =573.2.

Stage c.

The reaction flask containing the product obtained in step-b was purged with nitrogen under vacuum and dissolved in anhydrous DCM (50 ml), then cooled in an ice water bath. DMAP (4.89 g, 40 mmol) was added to the cooled solution, and then 4-nitrophenyl chloroformate (6.05 g, 30 mmol) was added in portions over 20 minutes. The solution was stirred at 0°C, then warmed to room temperature for 1 hour. After 1 hour, LCMS analysis indicated the presence of starting material, so additional DMAP (1.22 g, 10 mmol) and 4-nitrophenylchloroformate (1 g, 5 mmol) were added. The reaction was continued for 4 hours, then purified on two silica gel columns (220 g, pre-wet with a mixture of 20% EtOAc and hexane) and eluted with 20-80% EtOAc and hexane. Yield 4.42 g, 59.9% in two stages. Ion found using LCMS: [M+H] ⁺ =738.2

Stage d.

To a solution of the product obtained in step-c (3.2 g, 4.34 mmol) in anhydrous DCM (3 ml), a mixture of the central linker propargyldiamine (1.26 g, 2.5 mmol, described in example 99) and DIPEA (1.68 g, 13 mmol) in anhydrous DMF (5 ml). The reaction mixture was stirred at room temperature for 2 hours. It was then concentrated and purified by RPLC (30-90% acetonitrile in water, no TFA modifier). Ions found using LCMS: [(M+2H)/2] ⁺ =815.8, [(M-Boc+2H)/2] ⁺ =765.8, [(M-2Boc+2H)/2] ⁺ =716. Yield 3.33 g, 94.2%.

Stage e.

The product obtained in step-d (3.33 g, 2.04 mmol) was dissolved in DCM (5 ml) and TFA (5 ml), then stirred at 35°C for ~6 hours. The reaction was monitored using LCMS. After completion of the reaction, the solution was concentrated and purified by RPLC (5-30% acetonitrile in water, no TFA). Ions found using LCMS: [(M+2H)/2] ⁺ =615.8, [(M+3H)/3] ⁺ =411. Yield 2.29 g, 91.3%.

Stage f.

The product obtained in step-e (61.5 mg, 0.05 mmol) was dissolved in MeOH/water (1:1, 0.6 ml). After cooling the solution to -6°C (salt/ice bath), 1.0 M LiOH (0.3 mL, 0.3 mmol) was added dropwise and the reaction mixture was stirred for 30 minutes. It was then quenched to pH ~ 7.0 with 4N HCl in dioxane solution (75 µl) and directly purified using preparative HPLC (Isco ACCQ prep, Luna 5 µm C18(2) 100 Å LC column 100 mm x 30 mm gradient; : 0% acetonitrile/water for 2 minutes, then 0%-15% acetonitrile/water for 12 minutes, then isocratic mode at 15% acetonitrile for 10 minutes using 0.1% TFA). Yield 45 mg, 65.3%. Ions found using LCMS: [(M+2H)/2] ⁺ =575.8, [(M+3H)/3] ⁺ =384.2. Analytical retention time: 6.013 min. Conditions: Phenomemex Gemini HPLC column, 3 µm WX-C18 110 Å, 100 mm × 3 mm, eluting over 25 minutes with a gradient of 5-95% acetonitrile and water using 0.1% TFA.

Example 101 Synthesis of Intermediate-7b (C7-C9 Isomer)

The C7-C9 heterodimer (intermediate-7b) was prepared similarly to intermediate-7a (C7-C7 isomer, Example 100), except that the reaction was carried out at 0°C and monitored by HPLC (retention time: 6.112 min. Conditions : See Example 100, synthesis of intermediate-7a and stopping when C7-C9 isomer predominates (~3 h).This isomer was isolated under the same conditions used to isolate intermediate-7a. Ions found by LCMS: [(M+2H)/2] ⁺ =575.8, [(M+3H)/3] ⁺ =384.2.

Example 102 Synthesis of intermediate-7c (C9-C9 isomer)

Intermediate-7c (C9-C9 isomer) was prepared similarly to Intermediate-7a (C7-C7 isomer, Example 100), except that the reaction was carried out at 0°C and monitored by HPLC (retention time: 6.232 min. Conditions : See Example 100, synthesis of intermediate-7a and stopped when the C9-C9 isomer predominates (~6 h). This isomer was isolated under the same conditions used to isolate intermediate-7a. Ions found by LCMS: [(M+2N)/2] ⁺ =575.8, [(M+ZN)/3] ⁺ =384.2.

Example 103 Synthesis of Intermediate-7 (Acetonide Pathway)

Stage a.

Methyl 5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate (10.0 g, 22 mmol) was dissolved in 60 ml of dry methanol, then treated with 20 ml of sodium methoxide in methanol (0.5 M in methanol, 10 mmol) while cooling in an ice water bath. The reaction progress was monitored by LCMS and was completed after 2 hours. The pH of the reaction solution was then adjusted to 5 to 6 using Amberlite IRN-77 ion exchange resin. The mixture was filtered to remove the resin and evaporated to dryness in vacuo. The resulting oil was used in the next step without further purification. Ions found using LCMS: M+H=331.1.

Stage b.

To a solution of the products obtained in the previous step in 70 ml of acetone was added 30 ml of 2,2-dimethoxypropane and p-toluenesulfonic acid 1 hydrate (400 mg, 2.0 mmol), the resulting solution was stirred at room temperature overnight. After this time, sodium bicarbonate (170 mg, 2.0 mmol) was added and the mixture was concentrated to dryness. The resulting residue was used in the next step without purification. Ion(s) found by LCMS: M+H=371.2.

Stage c.

5.0 g of Lindlar catalyst was added to a solution of the product obtained in the previous step in 60 ml of methanol. The resulting mixture was purged with hydrogen every 30 minutes and stirred for 5 hours. After completion of the reaction as determined by HPLC, the catalyst was filtered through celite. The filtrate was concentrated and used in the next step without purification.

The crude product obtained in the previous step in 60 ml THF was treated with N,N'-bis-boc-1-guanylpyrazole (9.3 g, 30.0 mmol) and DIEA (9.9 ml, 57.0 mmol). The resulting solution was stirred at room temperature until all starting material was consumed as determined by LCMS (4 h). The solution was concentrated and purified by flash chromatography, eluting with 20%-80% ethyl acetate/dichloromethane. Yield 8.8 g, 59.0% in four stages. Ion(s) found by LCMS: M+H 587.3.

Stage d.

The reaction flask containing the product obtained in the previous step (6.5 g, 11 mmol) was purged with nitrogen in vacuo and dissolved in anhydrous dichloromethane (100 ml). After cooling the solution in an ice water bath, DMAP (4.89 g, 40 mmol) was added and stirred until dissolved, then 4-nitrophenyl chloroformate (5.58 g, 28 mmol) was added portionwise with stirring at 0° C. to room temperature for 1 hour. After 1 hour, LCMS analysis indicated the presence of starting material, so additional DMAP (1.22 g, 10 mmol) and 4-nitrophenylchloroformate (1.0 g, 5 mmol) were added. The reaction was continued for an additional 4 hours, then concentrated and purified by flash chromatography, eluting with 20%-80% ethyl acetate/dichloromethane. Yield 5.1 g, 59.9%. Ion found using LCMS: [M+H] ⁺ =752.2

Stage e.

To a solution of the nitrophenyl carbonate obtained in the previous step (1.8 g, 2.3 mmol) in anhydrous dichloromethane (20 ml) was added a mixture of the central linker (0.51 g, 1.0 mmol, added in portions over 30 minutes) and DIPEA (1.4 ml, 10 mmol) in anhydrous DMF (20 ml). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash chromatography, eluting with 0%-10% methanol/dichloromethane. Yield 1.35 g, 80%. Ions found using LCMS: [(M+2H)/2] ⁺ =830.4, [(M-Boc+2H)/2] ⁺ =780.4, [(M -2 Boc+2H)/2] ⁺ =730.4 .

Stage f.

The product obtained in the previous step (200 mg, 0.2 mmol) was dissolved in 2 ml MeOH and 2 ml THF, then treated with a solution of lithium hydroxide (24 mg, 1 mmol) dissolved in 2 ml water. The reaction mixture was stirred for 10 minutes at room temperature, after which HPLC analysis indicated completion of the reaction. The pH of the reaction solution was adjusted to 5-6 using Amberlite IRN-77 ion exchange resin, and filtered to remove the resin. The crude product was evaporated to dryness under vacuum and used in the next purification step. Ion(s) found using LCMS: [(M+2H)/2] ⁺ =815.4, [(M-Boc+2H)/2] ⁺ =765.4, [(M-2 Boc+2H)/2] ⁺ =715.4.

Stage g.

The product obtained in step g (400 mg, 0.25 mmol) was dissolved in 5 ml of dichloromethane and 5 ml of TFA, and the resulting reaction solution was stirred at room temperature. The progress of the reaction was monitored using LCMS. After completion of the reaction (6 h), the solution was evaporated to dryness and then dissolved in 4 ml of water and 4 ml of acetonitrile. The resulting solution was stirred for an additional 2 hours at room temperature, at which point LCMS analysis showed complete deprotection of the acetonide protecting groups. This mixture was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph, eluting with 5-40% acetonitrile/water with 0.1% TFA as modifier. Yield 270 mg, 68.0%. Ions found using LCMS: [(M+2H)/2] ⁺ =575.8, [(M+3H)/3] ⁺ =384.2.

Example 104 NMR Results for Intermediate-7a, Intermediate-7b, and Intermediate-7c

¹ H NMR (500 MHz, Methanol-d ₄ ) δ 5.91-5.89 (m, 2H), 5.00-4.96 (m, 2H), 4.58-4.53 (m, 2H),4.42-4.37 (m, 2H), 4.20 -4.17 (m, 6H), 4.02-3.97 (m, 2H), 3.78-3.49 (m, 28H), 3.29-3.18 (m, 4H), 2.86 (t,J=2.6 Hz, 1H), 2.85-2.72 (m, 2H), 1.96 (s, 3H), 1.95 (s, 3H).

¹³ C NMR (125 MHz, MeOD) δ 171.73, 170.80, 170.29, 161.95, 156.06, 155.08, 154.99, 144.07, 143.93, 116.41, 114.10, 106.03, 105.86, 77.71, 74.46, 73.22, 68.62, 68.54, 68.50, 68.38, 68.12, 68.00, 67.94, 67.67, 67.64, 67.53, 67.20, 66.96, 65.44, 61.53, 56.17, 49.74, 49.64, 47.37, 45.17, 39.16, 39.06, 31.7 3, 19.96, 19.92.

NMR of intermediate-7b

¹ H NMR (500 MHz, Methanol-d ₄ ) δ 5.91-5.87 (m, 2H), 5.04-4.95 (m, 1H), 4.58-4.48 (m, 2H), 4.45-4.37 (m, 3H), 4.20 -4.10 (m, 5H), 4.08-3.98 (m, 2H), 3.79-3.44 (m, 29H), 3.29-3.18 (m, 4H), 2.88-2.70 (m, 3H), 2.02 (s, 3H) , 1.96-1.94 (m, 3H).

¹ H NMR (500 MHz, DMSO-d ₆ ) δ: 8.28 (d, J=8.4 Hz, 2H, -NH), 7.98 (d, J=11.5 Hz, 2H, -NH), 7.78 (d, J= 8.7 Hz, 2H, -NH), 7.60 (t, J=8.5 Hz, 2H, -NH), 7.20 (bs, 2H, -NH), 7.06 (bs, 2H, -NH), 5.69 (bs, 1H) , 5.67 (d, J=2.4 Hz, 1H), 4.83 (dd, J=9.2, 2.2 Hz, 1H), 4.45 (dt, J=9, 2.5 Hz, 1H), 4.37 (d, J=2.2 Hz, 1H), 4.33-4.24 (m, 2H), 4.13 (d, J=2.5 Hz, 2H), 4.05-4.32 (m, 41H), 3.23 (m, 1H), 3.15-3.09 (m, 2H), 3.06 -3.02 (m, 2H), 2.59 (t, J=6.7 Hz, 2H), 1.91 (s, 3H), 1.78 (s, 3H).

¹³ C NMR (125 MHz, MeOD) δ:173.19, 172.98, 172.25, 171.75, 164.26, 163.82, 157.88, 157.55, 156.51, 146.09, 107.21, 106.93, 106.59, 79.22, 76.19, 75.92, 74.72, 74.68, 70.12, 70.08 , 70.03, 69.99, 69.92, 69.88, 69.66, 69.48, 69.12, 69.01, 68.94, 68.73, 68.69, 68.50, 68.19, 67.08, 66.93, 66.79, 63.04, 57 .68, 51.24, 51.15, 50.13, 48.87, 48.72, 46.72, 46.58 , 46.23, 40.64, 40.41, 33.21, 21.49, 21.45, 21.40.

NMR of intermediate -7c

¹ H NMR (500 MHz, Methanol-d ₄ ) δ: 5.88 (d, J=2.6 Hz, 2H), 4.50 (dt, J=8.5, 2.7 Hz, 2H), 4.43 (ddd, J=9.7, 3.9, 1.5 Hz, 2H), 4.39 (dd, J=11.5, 2.4 Hz, 2H), 4.25-4.16 (m, 2H), 4.19 (d, J=2.4 Hz, 2H), 4.13 (dt, J=11.5, 5.9 Hz, 2H), 4.05 (m, 2H), 3.77 (t, J=6.2 Hz, 2H), 3.72-3.55 (m, 22H), 3.51 (m, 4H), 3.35-3.23 (m, 4H), 2.86 (t, J=2.4 Hz, 1H), 2.74 (t, J=6.2 Hz, 2H), 2.02 (s, 6H).

¹³ C NMR (125 MHz, MeOD) δ:173.03, 163.76, 157.89, 157.54, 145.60, 107.21, 79.28, 76.29, 74.70, 70.11, 70.07, 70.03, 69.92, 69.70, 69.47, 68.96, 68.89, 68.72, 68.54, 68.19 , 67.05, 66.75, 57.69, 50.09, 48.69, 48.08, 46.10, 40.44, 40.38, 33.23, 21.42.

Example 105 Synthesis of Intermediate-60

Stage a.

To a stirred solution at 0°C of a pre-prepared zanamivir acid ester (1.00 g, 1.586 mmol, example 31), 2-azidoethylamine hydrochloride (213 mg, 1.744 mmol) and DIPEA (1.105 ml, 6.343 mmol) in DMF (8 .0 ml) HATU (615 mg, 1.618 mmol) was added. The temperature was raised to ambient temperature and stirring was continued until the reaction was complete. All volatile substances were removed by vacuum methods. The residue was taken up in ethyl acetate, washed with 1 M aqueous sulfuric acid (1 x 50 ml), then saturated aqueous sodium bicarbonate (3 x 20 ml) and brine (1 x 50 ml). The resulting organic layers were dried with magnesium sulfate, filtered, and all volatile substances were removed by vacuum methods. 816 mg of the desired azide intermediate was thus obtained in high purity and used in the next step without any further purification. (Ion found by LCMS: [M+H] ⁺ =699.2). To a stirred solution of the described crude product (816 mg, 1.168 mmol), dipropargylamine (54 mg, 0.584 mmol), tris((1-benzyl-4-triazolyl)-methyl)amine (31 mg, 0.058 mmol) and sodium ascorbate (58 mg, 0.292 mmol) in ethanol (10 ml) and water (5 ml) was added copper sulfate (10 mg, 0.061 mmol). Upon completion, the copper scavenger SiliaMetS TAAcONa (300 mg, load 0.45 mmol/g) was added and stirring was continued for 1 hour. The mixture was filtered with dichloromethane. The filtrate was washed with saturated sodium bicarbonate solution. The aqueous layer was further washed with dichloromethane (3 times). The combined organic layers were dried with magnesium sulfate, filtered and concentrated. The residue was purified on a silica column using liquid chromatography with Isco COMBIFLASH®, eluting with 0%-100% hexane in ethyl acetate followed by 0%-30% dichloromethane. Yield 817 mg, yield 94%. Ions found using LCMS: [(M+2H)/2] ⁺ =745.8, [(M+3H)/3] ⁺ =497.5.

Stage b.

To a stirred solution at 0°C of the product obtained in step a (817 mg, 0.548 mmol), propargyl-PEG4 acid (185 mg, 0.712 mmol) and DIPEA (286 μl, 1.644 mmol) in DMF (7.0 ml ) HATU (212 mg, 0.544 mmol) was added. The temperature was raised to room temperature and stirring was continued until complete. All volatile substances were removed by vacuum methods. The residue was purified by HPLC (0-90% methanol in water). Yield 520 mg, 56%. Ions found using LCMS: [(M+2H)/2] ⁺ =866.8, [(M+3H)/3] ⁺ =578.4.

Stage c.

A stirred solution of the compound obtained in step-b (520 mg, 0.300 mmol) in 2-methyl-2-butene (0.25 ml), dichloromethane (4.0 ml) and TFA (2.0 ml) was stirred until evolution ceased gas All volatile substances were removed by vacuum methods. The residue was purified by HPLC (0-30% methanol in water, using 0.1% TFA as modifier). Yield 221 mg, 47%. Ions found using LCMS: [(M+2H)/2] ⁺ =666.8, [(M+3H)/3] ⁺ =444.8.

Stage d.

Lithium hydroxide (20 mg, 0.850 mmol) was added to a stirred solution of the product obtained in step-c (221 mg, 0.142 mmol) in water (3.0 ml) at 0°C. The reaction was quenched with acetic acid (120 μl) and all volatiles were removed by vacuum methods. The residue was purified by HPLC (0-20% methanol in water, using 0.1% TFA as modifier). Yield 130 mg, 62%. Ions found using LCMS: [(M+2H)/2] ⁺ =626.8, [(M+3H)/3] ⁺ =418.2.

Example 106. Synthesis of conjugate 29

A solution of azido-functionalized aglycosylated Fc (SEQ ID NO: 35) in x1 PBS buffer at pH 7.4 (100 mg, 10 ml, 1.874 μmol) was added to a centrifuge tube containing the alkyne-derivatized small molecule (17 mg, 0 .0112 mmol; Example 105, Intermediate-60), copper sulfate (4 mg, 0.0225 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (39 mg, 0.0900 mmol) and sodium ascorbate (7.4 mg, 0.375 mmol) in x1 PBS buffer solution at pH 7.4 (10.50 ml). The resulting mixture was shaken gently overnight. It was purified by affinity chromatography on a protein A column followed by size exclusion chromatography (see example 10). Maldi TOF analysis of the purified final product showed an average mass of 56938 Da (DAR=2.3). Yield 49.9 mg, yield 49%.

Example 107 Synthesis of Intermediate-65

Stage a.

Isobutyl chloroformate (0.617 ml, 4 .7574 mmol). After 10 minutes, the temperature was raised to room temperature and stirring was continued for 20 minutes. The temperature was again reduced to 0°C and sodium borohydride (360 mg, 9.515 mmol) was added in one portion, followed by dropwise addition (10 ml) over 5 minutes. Once complete, the reaction was quenched with acetic acid (2.860 mL, 50 mmol) and after 5 minutes the temperature was raised to room temperature while stirring was continued until gas evolution ceased. All volatile substances were evaporated, the residue was suspended in dichloromethane and filtered. The filtrate was concentrated and the residue was purified on a silica column using liquid chromatography using Isco COMBIFLASH®, eluting with 20%-100% hexanes and ethyl acetate using 3% methanol as a modifier. Yield 1.302 g, yield 66%. Ions found using LCMS: [(M+H)] ⁺ =617.2.

Stage b.

To a stirred solution of the product from step-a (1.25 g, 2.027 mmol) and DIPEA (1.095 mL, 6.284 mmol) in dichloromethane (15 mL) at 0° C. was added methanesulfonyl chloride (0.314 mmol, 4.054 mmol). Once complete, the reaction mixture was treated with water (15 ml). The layers were separated and the dichloromethane layer was dried with brine, then magnesium sulfate and filtered. The solution was concentrated, the residue was dissolved in DMF (10 ml) and sodium azide (264 mg, 4.054 mmol) was added while raising the temperature to 50°C. Completion was observed after 18 hours and all volatiles were evaporated by vacuum. The residue was purified on a silica column using liquid chromatography using Isco COMBIFLASH®, eluting with a gradient of 20% to 100% hexanes in ethyl acetate using 3% methanol as modifier. Yield 767 mg, yield 59%. Ions found using LCMS: [(M+H)] ⁺ =642.2.

Stage c.

To a stirred solution of the product obtained in step-b (496 mg, 0.773 mmol), dipropargylamine (36 mg, 0.386 mmol), tris((1-benzyl-4-triazolyl)methyl)-amine (41 mg, 0.077 mmol) and sodium ascorbate (115 mg, 0.580 mmol) in ethanol (16 ml) and water (8 ml), copper sulfate (13 mg, 0.081 mmol) was added. Once complete, the copper scavenger SiliaMetS TAAcONa (600 mg, load 0.45 mmol/g) was added and stirring was continued for 1 hour. The mixture was filtered with dichloromethane. The filtrate was washed with saturated sodium bicarbonate solution. The aqueous layer was further washed with dichloromethane (3 times). The combined organic layers were dried with magnesium sulfate, filtered and all volatiles were evaporated in vacuo. HATU (220 mg, 0.580 mmol) was added to a stirred solution of the residue, propargyl-PEG4 acid (151 mg, 0.580 mmol) and DIPEA (337 μL, 1.933 mmol) in DMF (10.0 mL) at 0°C. The temperature was raised to room temperature and stirring was continued until complete. All volatile substances were removed by vacuum methods. The residue was purified by HPLC (0 to 90% methanol in water). Yield 457 mg, 73%. Ions found using LCMS: [(M+2H)/2] ⁺ =809.8, [(M+2H-Boc)/2] ⁺ =759.8.

Stage d.

A stirred solution of the compound obtained in step-c (451 mg, 0.279 mmol) in 2-methyl-2-butene (0.25 ml), dichloromethane (4.0 ml) and TFA (2.0 ml) was stirred until evolution ceased gas All volatile substances were removed by vacuum methods. Ions found using LCMS: [(M+2H)/2] ⁺ =609.8, [(M+3H)/3] ⁺ =407.0. Lithium hydroxide (240 mg, 10.03 mmol) was added to a stirred solution of the residue in tetrahydrofuran (6 ml) and water (6 ml) at 0°C. Once complete, the reaction was quenched with acetic acid (0.638 mL, 11.14 mmol) and all volatiles were removed by vacuum. The residue was purified by HPLC (0 to 90% methanol in water, using 0.1% TFA as modifier). Yield 209 mg, 67%. Ions found using LCMS: [(M+2H)/2] ⁺ =569.8, [(M+2H-Boc)/2] ⁺ =380.3.

Example 108 Synthesis of conjugate 30

A solution of azido-functionalized aglycosylated Fc (Example 7, SEQ ID NO: 35) in x1 PBS buffer at pH 7.4 (50 mg, 5 ml, 1.874 μmol) was added to a centrifuge tube containing an alkyne-derivatized small molecule (8.7 mg, 0.0064 mmol, intermediate-65), copper sulfate (2 mg, 0.013 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (22 mg, 0.0508 mmol) and sodium ascorbate (25 mg, 0.127 mmol) in buffer solution x1 PBS at pH 7.4 (9.50 ml). The resulting mixture was shaken gently overnight. It was purified by affinity chromatography on a protein A column followed by size exclusion chromatography (see example 10). Maldi TOF analysis of the purified final product showed an average mass of 61548 Da (DAR=2.4). Yield 32.33 mg, yield 67%.

Example 109: Synthesis of Zanaminnra p-nitrophenyl carbonate intermediate

The zanamivir triacetoxyazido intermediate (10.0 g, 22 mmol) was dissolved in 60 ml dry methanol, then treated with 20 ml sodium methoxide in methanol (0.5 M in methanol, 10 mmol) while cooling in an ice-water bath. The reaction progress was monitored by LCMS and was completed after 2 hours. The pH of the reaction solution was then adjusted to 5-6 using Amberlite IRN-77 ion exchange resin. The mixture was filtered to remove resin and evaporated to dryness under vacuum. The resulting oil was used in the next step without further purification. Ion(s) found by LCMS: M+H=331.1.

Stage b.

To a solution of the products obtained in the previous stage in 70 ml of acetone was added 30 ml of 2,2-dimethoxypropane and p-toluenesulfonic acid 1 hydrate (400 mg, 2.0 mmol), the resulting solution was stirred at room temperature overnight. After this time, sodium bicarbonate (170 mg, 2.0 mmol) was added and the mixture was concentrated to dryness. The resulting residue was used in the next step without purification. Ion(s) found by LCMS: M+H=371.2.

Stages c and d

5.0 g of Lindlar catalyst was added to a solution of the product obtained at the previous stage in 60 ml of methanol. The resulting mixture was purged with hydrogen every 30 minutes and stirred for 5 hours. After completion of the reaction as determined by HPLC, the catalyst was filtered through celite. The filtrate was concentrated and used in the next step without purification.

The crude product obtained in the previous step in 60 ml THF was treated with N,N'-bis-boc-1-guanylpyrazole (9.3 g, 30.0 mmol) and DIEA (9.9 ml, 57.0 mmol). The resulting solution was stirred at room temperature until all the starting material was completely consumed according to LCMS data (4 h). The solution was concentrated and purified by flash chromatography, eluting with a gradient of 20% to 80% ethyl acetate in dichloromethane. Yield 8.8 g, 59.0% in four stages. Ion(s) found by LCMS: M+H 587.3.

Stage e.

The reaction flask containing the product obtained in the previous step (6.5 g, 11 mmol) was purged with nitrogen in vacuo and dissolved in anhydrous dichloromethane (100 ml). After cooling the solution in an ice water bath, DMAP (4.89 g, 40 mmol) was added and stirred until dissolved, then 4-nitrophenyl chloroformate (5.58 g, 28 mmol) was added portionwise with stirring at 0°C to room temperature. temperature for 1 hour. LCMS analysis indicated the presence of starting product after 1 hour, so additional DMAP (1.22 g, 10 mmol) and 4-nitrophenylchloroformate (1.0 g, 5 mmol) were added. The reaction was continued for an additional 4 hours, then the reaction mixture was concentrated and purified by flash chromatography, eluting with a gradient of 20% to 80% ethyl acetate in dichloromethane. Yield 5.1 g, 59.9%. Ion found using LCMS: [M+H] ⁺ =752.2.

Example 110 Synthesis of Intermediate-71

Stage a.

DIPEA (7.8 g, 60 mmol) and DMAP (366.6 mg, 3 mmol) were added to a solution of 2-(2-Boc-aminoethoxy)ethanol (6.15 g, 30 mmol) in anhydrous DCM (60 mL). . p-Toluenesulfonyl chloride (6.86 g, 36 mmol) was then added in portions over 30 minutes. After stirring the resulting mixture for 3 days, it was concentrated by rotary evaporation and purified by RPLC (20-70% acetonitrile/water). Yield 3.71 g, 34.4%. Ion found using LCMS: [M -Boc+H] ⁺ =260.

Stage b.

To a solution of the product obtained in step-a (2.1 g, 5.83 mmol) in anhydrous THF (10 ml) was added sodium carbonate (1.24 g, 11.7 mmol) and 1H-Boc-1,4- diaminobutane (1.32 g, 7 mmol). The resulting mixture was heated at 60°C for 1 day. The salt was then filtered off and the filtrate was concentrated on a rotary evaporator. The residue was purified by RPLC (100 g, 5-50% acetonitrile in water). Yield 1.94 g, 88.6%. Ion found using LCMS: [M+H] ⁺ =376.0.

Stage c.

To a solution of propargyl-PEG-4-acid (781 mg, 3 mmol) and HATU (1.14 g, 3 mmol) in anhydrous DMF (3 ml) was added DIPEA (390 mg, 3 mmol), followed by the addition of that obtained in step b a solution of product (940 mg, 2.5 mmol) and DIPEA (390 mg, 3 mmol) in anhydrous DMF (3 ml). The reaction mixture was stirred for 30 minutes, then purified directly by RPLC (5-80% acetonitrile in water, using 0.1% TFA as modifier). Yield 960.2 mg, 65.3%. Ion found using LCMS: [M+H] ⁺ =618.3, [M-Boc+H] ⁺ =518.3.

Stage d.

The product obtained in step-c (960.2 mg, 1.63 mmol) was dissolved in anhydrous THF (6 ml). A 4N solution of HCl in dioxane (4 ml) was added and the reaction mixture was stirred overnight. It was then concentrated on a rotary evaporator. The residue was extracted with water (3x3 ml) and ethyl acetate (10 ml). The combined aqueous layers were lyophilized. Yield 760 mg, 95.1%. Ion found using LCMS: [M+H] ⁺ =418.0.

Stage e.

To a mixture of the product obtained in step d (556.2 mg, 1.13 mmol) and DIPEA (741 mg, 5.7 mmol) in anhydrous DMF (3 ml), zanamivir p-nitrophenyl carbonate (1.67 g, 2. 26 mmol, as described in example 109) for 20 minutes. The reaction mixture was stirred for 1 hour, then purified directly by RPLC (30-90% acetonitrile/water, using 0.1% TF A as modifier). Yield 1.35 g, 74%. Ion found using LCMS: [(M+2H)/2] ⁺ =807.9.

Stage f.

The product obtained in step-e (1.35 g, 0.836 mmol) was dissolved in TFA (5 ml). The reaction mixture was heated at 30°C for 1 hour and purified directly by RPLC (0-35% acetonitrile/water using 0.1% TFA as modifier). Yield 1.00 g, 82.9%. Ions found using LCMS: [(M+2H)/2] ⁺ =607.8.

Stage g.

The product from step f (1.00 g, 0.693 mmol) was dissolved in MeOH (12 mL), then cooled in an ice water bath. It was then treated with a solution of LiOH monohydrate (286 mg, 6.6 mmol) in water (9 ml). The resulting mixture was stirred overnight and then acidified with a 4N solution of HCl in dioxane (2 ml). After removal of organic solvents on a rotary evaporator, the residue was purified by preparative HPLC (Isco ACCQ Prep, Luna 5 μm C18(2), 100 Å, LC column 100 mm × 30 mm; gradient: 0% acetonitrile/water for 2 min, then 0- 15% acetonitrile/water for 12 minutes, then isocratic mode at 15% acetonitrile for 10 minutes using 0.1% TFA). Yield 275.9 mg, 29.2%. Ions found using LCMS: [(M+2H)/2] ⁺ =567.8, [(M+3H)/3] ⁺ =378.9.

Example 111. Synthesis of conjugate 31

A sterile 15 mL centrifuge tube was loaded with sodium ascorbate (68.1 mg, 0.344 mmol), THPTA (14.9 mg, 0.0344 mmol), alkyne-derivatized small molecule (17.5 mg, 0.00953 mmol, described in example 110) and PBS 7.4 buffer (1 ml). After vortexing to dissolve all components, Peg-4-azido-Pc (50 mg, 0.0008588 mmol, described in Example 7 SEQ ID No. 18) was added, followed by a solution of CuSO ₄ (2.05 mg, 0.0129 mmol) in PBS (0.5 ml). The mixture was gently rotated for 20 hours, then purified by affinity chromatography on a protein A column followed by size exclusion chromatography. Maldi TOF analysis of the purified final product showed an average mass of 63,586 Da (DAR=3.8). Yield 32.8 mg, yield 66%.

Example 112 Synthesis of Intermediate-72

Stage a.

To a solution of N-Boc-1,4-diaminobutane (1.56 g, 8.28 mmol) in anhydrous DMF (7 ml) was added sodium carbonate (742 mg, 7 mmol) and 5-(Boc-amino)-1- pentyl bromide (1.73 g, 6.5 mmol). The resulting mixture was heated at 50°C for 24 hours. The salt was then filtered off and the filtrate was concentrated on a rotary evaporator. The residue was purified by RPLC (5-50% acetonitrile/water, using 0.1% TFA as modifier). Yield 2 g, 63.2%. Ion found using LCMS: [M+H] ⁺ =374.4.

Stage b.

To a solution of propargyl-PEG-4-acid (1.3 g, 5 mmol) and HATU (2.1 g, 5.5 mmol) in anhydrous DMF (6 ml) was added DIPEA (1.3 g, 10 mmol). After 5 minutes, the reaction mixture was added to the product obtained in step a (2 g, 4.1 mmol) and stirred for 1 hour. It was then directly purified by RPLC (5-80% acetonitrile/water using 0.1% TFA as modifier). Yield 2.34 g, 95%. Ion found using LCMS: [M+H] ⁺ =616.4, [M-Boc+H] ⁺ =516.4.

Stage c.

The product obtained in step-b (2.34 g, 3.8 mmol) was dissolved in anhydrous THF (12 ml). A 4N solution of HCl in dioxane (10 ml) was added and the reaction mixture was stirred overnight. It was then concentrated on a rotary evaporator. The residue was redissolved in acetonitrile/water (1:1, -16 ml) and the solution was lyophilized. The crude product was transferred to the next stage without further purification. Yield 1.91 g, quantitative yield. Ion found using LCMS: [M+H] ⁺ =416.4.

Stage d.

To a solution of zanamivir p-nitropenylcarbonate (2.25 g, 3.05 mmol, described in Example 109) in anhydrous DCM (3 ml), a mixture of the product obtained in step-c (610 mg, 1.249 mmol) and DIPEA ( 1.05 g, 8.1 mmol) in anhydrous DMF (4 ml) for 20 minutes. After stirring for 1 hour, the reaction mixture was concentrated and purified by RPLC (30-80% acetonitrile/water, using 0.1% TFA as modifier). Yield 1.73 g, 85.9%. Ion found using LCMS: [(M+2H)/2] ⁺ =806.8, [(M-Boc+2H)/2] ⁺ =756.8.

Stage e.

The product obtained in step-d (1.73 g, 1.073 mmol) was dissolved in TFA (5 ml). After heating the solution at 30°C for 3 hours, it was purified directly by RPLC (0-35% acetonitrile/water, using 0.1% TFA as modifier). Yield 1.176 g, 76.1%. Ions found using LCMS: [(M+2H)/2] ⁺ =606.8, [(M+3H)/3] ⁺ =405.

Stage f.

The product obtained in step-e (1.176 g, 0.817 mmol) was dissolved in MeOH (12 ml) and the solution was cooled in an ice water bath. It was treated by adding dropwise a solution of LiOH monohydrate (344.4 mg, 8.2 mmol) in water (9 ml). The resulting mixture was stirred overnight and then acidified with a 4N solution of HCl in dioxane (2 ml). After removal of organic solvents using a rotary evaporator, the residue was purified by preparative HPLC (Isco ACCQ prepare, Luna 5 μm C18 (2) 100 Å LC column 100 mm × 30 mm; gradient: 0% acetonitrile/water for 2 minutes, then 0-15 % acetonitrile/water for 12 minutes, then isocratic mode at 15% acetonitrile for 10 minutes using 0.1% TFA). Yield 108 mg, 9.7%. Ions found using LCMS: [(M+2H)/2] ⁺ =566.8, [(M+3H)/3] ⁺ =378.2.

Example 113 Synthesis of conjugate 32

A sterile 15 mL centrifuge tube was charged with sodium ascorbate (68.1 mg, 0.344 mmol), THPTA (14.9 mg, 0.0344 mmol), product from Example 112, intermediate-72 (17.5 mg, 0.0344 mmol). 00953 mmol) and PBS 7.4 (1 ml). After vortexing to dissolve all components, azido-Fc (50 mg, 0.0008588 mmol, described in Example 7 SEQ ID NO: 4) was added, followed by a solution of CuSO ₄ (2.05 mg, 0.0129 mmol) in PBS (0.5 ml). The mixture was rotated for 20 hours. It was purified by affinity chromatography on a protein A column followed by size exclusion chromatography. Maldi TOF analysis of the purified final product showed an average mass of 63588. Yes (DAR=3.8). Yield 30.9 mg, yield 62%.

Example 114 Synthesis of Intermediate-73

Stage a.

To a solution of zanamivir p-nitrophenylcarbonate (698.4 mg, 0.95 mmol, described in Example 109) in anhydrous DCM (2 ml), a mixture of the central linker propargyldiamine (209 mg, 0.426 mmol, described in Example 110) was added in portions over 10 minutes ) and DIPEA (330.8 mg, 2.56 mmol) in anhydrous DMF (2 ml). The reaction mixture was stirred at room temperature for 1 hour. It was then concentrated and purified by RPLC (30-85% acetonitrile/water, no TFA modifier). Yield 531 mg, 69.2%. Ions found using LCMS: [(M+2H)/2] ⁺ =807.8, [(M-Boc+2H)/2] ⁺ =757.8.

Stage b.

The product obtained in step-b (531 mg, 0.329 mmol) was dissolved in DCM (1.5 ml) and TFA (1.5 ml), then stirred at 35°C for 3 hours. It was concentrated and purified by RPLC (5-30% acetonitrile/water, no TFA modifier). Yield 387 mg, 97.1%. Ions found using LCMS: [(M+2H)/2] ⁺ =607.6, [(M+3H)/3] ⁺ =405.4.

Stage c.

The product obtained in step-b (121.4 mg, 0.1 mmol) was dissolved in 1.0 M NaCl (3 ml) and acetonitrile (1 ml). After cooling the solution to -8°C (ice salt bath), 1.0 M NaOH (0.4 mL, 0.4 mmol) was added dropwise and the reaction mixture was stirred at -14°C to -8°C in within 6 hours. It was then neutralized with 4N HCl in dioxane (100 µl) and directly purified by preparative HPLC (Isco ACCQ Prep, Luna 5 µm C18(2) 100 Å LC column 100 mm x 30 mm; gradient: 0% acetonitrile in water over 2 min , then 0% to 17.8% acetonitrile in water for 12 minutes, then isocratically at 17.8% acetonitrile for 10 minutes using 0.1% TFA). Yield 85.5 mg, 62.8%. Ions found using LCMS: [(M+2H)/2] ⁺ =567.8, [(M+3H)/3] ⁺ =378.9.

Example 115 Synthesis of Intermediate-74

Stage a.

To a solution of N-Boc-2-(2-aminoethoxy)ethylamine (1.2 g, 5 mmol) in anhydrous DMF (5 ml) was added sodium carbonate (691 mg, 5 mmol) and N-Boc-6-bromo-hexylamine (1.4 g, 5 mmol). The resulting mixture was heated at 70°C for 24 hours. The salt was then filtered off and the filtrate was concentrated on a rotary evaporator. The residue was purified by RPLC (gradient from 5% to 50% acetonitrile in water, using 0.1% TFA as modifier). Yield 444 mg, 22%. Ion found using LCMS: [M+H] ⁺ =404.3.

Stage b.

DIPEA (258 mg, 2 mmol) was added to a solution of propargyl-PEG-4-acid (342.6 mg, 1.32 mmol) and HATU (601.5 mg, 1.58 mmol) in anhydrous DMF (2 mL). After 5 minutes, the reaction mixture was added to the product obtained in step a (444.3 mg, 0.858 mmol) and stirred for 1 hour. It was then directly purified by RPLC (gradient from 5% to 80% acetonitrile in water, using 0.1% TFA as modifier). Yield 297.1 mg, 53.6%. Ion found using LCMS: [M+H] ⁺ =646.2, [M-Boc+H] ⁺ =546.2.

Stage c.

The product obtained in step-b (297.1 mg, 0.46 mmol) was dissolved in anhydrous THF (2 ml). A 4N solution of HCl in dioxane (4 ml) was added and the reaction mixture was stirred overnight. It was then concentrated by rotary evaporation and purified by preparative HPLC (5% to 50% acetonitrile in water, using 0.1% TFA as modifier). Yield 239.6 mg, 77.3%. Ion found using LCMS: [M+H] ⁺ =446.2.

Stage d.

To a solution of zanamivir p-nitrophenylcarbonate (523.8 mg, 0.71 mmol, described in Example 2) in anhydrous DCM (2 ml), the mixture of the product obtained in step-c (239.6 mg, 0.356 mmol) and DIPEA (245.5 mg, 1.9 mmol) in anhydrous DMF (2 ml). The reaction mixture was stirred at room temperature for 1 hour. It was then concentrated and purified by RPLC (30% to 85% acetonitrile in water, no TFA modifier). Yield 553 mg, 96.5%. Ions found using LCMS: [(M+2H)/2] ⁺ =821.8, [(M-Boc+2H)/2] ⁺ =771.8.

Stage e.

The product from step d (553 mg, 0.343 mmol) was dissolved in DCM (1.5 ml) and TFA (1.5 ml), then stirred at 35°C for 3 hours. It was concentrated and purified by RPLC (5% to 30% acetonitrile in water, no TFA modifier). Yield 424.6 mg, 99.6%. Ions found using LCMS: [(M+2H)/2] ⁺ =621.6, [(M+3H)/3] ⁺ =415.0.

Stage f.

The product obtained in step-e (424.6 mg, 0342 mmol) was dissolved in 1.0 M NaCl (4 ml) and acetonitrile (4 ml). After cooling the solution to -11°C (ice salt bath), 1.0 M NaOH (1.53 mL, 1.53 mmol) was added dropwise and the reaction mixture was stirred at -14°C to -8°C in within 6 hours. The reaction mixture was then quenched with 4N HCl in dioxane solution (375 µl) and directly purified by preparative HPLC (Isco ACCQ Prep, Luna 5 µm C 18(2) 100 Å LC column 100 mm × 30 mm; gradient: 0% acetonitrile in water for 2 min, then 0% to 20.9% acetonitrile in water for 13.6 min, then isocratic at 20.9% acetonitrile for 10 min using 0.1% TFA). Yield 439 mg, 92.3%. Ions found using LCMS: [(M+2H)/2] ⁺ =581.8, [(M+3H)/3] ⁺ =388.2.

Example 116 Synthesis of Intermediate-75

Stage a.

Mixture of tert-butyl (4-oxobutyl)carbamate (850 mg, 4.54 mg) and tert-butyl N-{2-[2-(2-aminoethoxy)ethoxy]ethyl}carbamate (1.99 g, 8 mmol) dissolved in DCM (30 ml) and dried over Na ₂ SO ₄ . After filtration and concentration, the residue was redissolved in anhydrous DCM (20 ml). Acetic acid (641 mg, 11 mmol) was added followed by sodium triacetoxyborohydride (3.4 g, 16 mmol) in portions over 1 hour. The reaction mixture was stirred overnight, then quenched with AcOH (3 ml) and MeOH (10 ml). The mixture was filtered and the filtrate was concentrated by rotary evaporation and purified by RPLC (5% to 45% acetonitrile in water). Yield 618 mg, 32.5%. Ion found using LCMS: [M+H] ⁺ =420.4.

Stage b.

This compound was prepared similarly to the product obtained in step-b of Example 115. Ions found by LCMS: [M+H] ⁺ =662.4, [M-Boc+H] ⁺ =562.4.

Stage c.

This compound was prepared analogously to the product obtained in step-c of Example 115. Ions found by LCMS: [M+H] ⁺ =462.4.

Stage d.

This compound was prepared similarly to the product obtained in step-d of Example 115. Ions found by LCMS: [(M+2H)/2] ⁺ =829.8, [(M-Boc+2H)/2] ⁺ =779.8.

Stage e.

This compound was prepared similarly to the product obtained in step e of Example 115. Ions found by LCMS: [(M+2H)/2] ⁺ =629.8, [(M+3H)/3] ⁺ =420.2.

Stage f.

This compound was prepared similarly to the product obtained in step-f of Example 115. Ions found by LCMS: [(M+2H)/2] ⁺ =598.8, [(M+3H)/3] ⁺ =393.6.

Example 117 Synthesis of Intermediate-76

Stage a.

To a solution of N-Boc-peg-1 tosylate (1.54 g, 4.28 mmol, described in Example 110) in anhydrous THF (8 ml) was added tert-butyl N-2{2-[2-(2-aminoethoxy )ethoxy]ethyl}-carbamate (1.59 g, 6.42 mmol) and sodium carbonate (453.7 mg, 4.28 mmol). The resulting mixture was heated at 50°C for 24 hours. The solid was filtered and washed with acetonitrile. The filtrate was concentrated and purified by RPLC (100 g, 5% to 90% acetonitrile in water). Yield 1.15 g, 61.7%. Ion found using LCMS: [M+H] ⁺ =436.4.

Stage b.

This compound was prepared similarly to the product obtained in step-b of Example 115. Ions found by LCMS: [M+H] ⁺ =678.2, [M-Boc+H] ⁺ =578.2.

Stage c.

This compound was prepared analogously to the product obtained in step-c of Example 115. Ions found by LCMS: [M+H] ⁺ =478.2.

Stage d.

This compound was prepared similarly to the product obtained in step-d of Example 115. Ions found by LCMS: [(M+2H)/2] ⁺ =837.6, [(M-Boc+2H)/2] ⁺ =767.8.

Stage e.

This compound was prepared similarly to the product obtained in step e of Example 115. Ions found by LCMS: [(M+2H)/2] ⁺ =637.5, [(M+3H)/3] ⁺ =425.6.

Stage f.

This compound was prepared similarly to the product obtained in step-f of Example 115. Ions found by LCMS: [(M+2H)/2] ⁺ =597.8, [(M+3H)/3] ⁺ =399.0.

Example 118 Synthesis of Intermediate-67

Stage a.

The previously obtained starting product ester-zanamivir-acid (0.90 g, 1.43 mmol, described in example 22) and N-methylmorpholine (0.23 ml, 2.14 mmol) were dissolved in THF (35 ml) and cooled to 0°C (ice water bath) under nitrogen atmosphere.

Isobutyl chloroformate (0.24 ml, 1.85 mmol, in 2 ml DCM) was added dropwise using a syringe over a period of 5 minutes. The mixture was stirred at 0°C for 30 minutes, then 15 minutes at ambient temperature, and then cooled to 0°C and sodium borohydride (540 mg, 14.3 mmol, dissolved in 5 ml methanol) was added dropwise over 5 minutes. The reaction mixture was stirred for 15 minutes, after which all the starting product was consumed (as determined by LC/MS). A few drops (~1 ml) of glacial acetic acid were added to acidify the mixture (pH ~5). The mixture was diluted with ethyl acetate and water, and extracted with ethyl acetate (3x). The organic layer was washed with brine, the organic extracts were dried over sodium sulfate and concentrated on a rotary evaporator. The crude material was purified by silica gel chromatography, first dried on Celite, and then eluted with 0-10% methanol in DCM for 30 minutes. Yield 0.66 g, 75%. Ion found using LCMS: [M+H] ⁺ =617.2.

Stage b.

Triethylamine (0.30 mL, 1.3 mmol) was added to a stirred mixture of the product obtained in the previous step (0.66 g, 1.10 mmol) in 20 ml DCM. The mixture was cooled to 0°C (ice water bath) under nitrogen, then treated with mesyl chloride (0.15 g, 1.3 mmol) added dropwise over 5 minutes using a syringe. The ice bath was removed and the reaction mixture was stirred for 45 minutes. The reaction was quenched with saturated aqueous sodium bicarbonate, then extracted with DCM (3x). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated on a rotary evaporator. Yield 0.74 g, 99%. Ion found using LCMS: [M+H] ⁺ =695.2. The intermediate product was used in the next step without purification.

Stage c.

The mesylate from the previous step (0.74 g, 1.1 mmol) was stirred in DMF (5 ml) at 80°C with 3 eq. sodium azide (0.21 g, 3.3 mmol) for 5 hours. The mixture was diluted with water, extracted with DCM (3x). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated. Yield 0.67 g, 95%. Ion found using LCMS: [M+H]+ = 642.4. The azide was used in the next step without purification.

Stage d.

The azide from the previous step (0.67 g, 1.04 mmol) was stirred in methanol (20 ml) in the presence of Lindlar catalyst (300 mg) under hydrogen gas for 12 hours. The mixture was filtered through celite and concentrated to give the title compound as a clear oil. The amine was used in the next step without purification. Yield 0.37 g, 54%, 3 stages. Ion found using LCMS: [M+H]+ = 616.2.

Stage e.

EDC (150 mg, 0.78 mmol) was added to a stirred solution of the amine obtained in the previous step (370 mg, 0.60 mmol), propargyl-peg4-carboxylic acid (188 mg, 0.72 mmol) and triethylamine (0.100 ml , 0.72 mmol) in DMF (4 ml). The reaction mixture was stirred for 2 hours at ambient temperature, then purified directly by RPLC (10% to 95% acetonitrile in water, no modifier, 30 minute gradient). Pure fractions were pooled, lyophilized and sent directly to the next step. Yield 490 mg, 80%. Ion found by LC/MS: [M+H]+ = 858.2

Stage f.

The product from the previous step (490 mg, 0.57 mmol) was stirred in TFA (4 ml) for 45 minutes, then concentrated and azeotroped with methanol (3x) on a rotary evaporator. Ion found by LC/MS: [M+H]+ = 658.2. The residue was stirred in 1/1 methanol/water containing LiOH (43 mg, 1.8 mmol) for 30 minutes. The reaction was neutralized with a few drops of glacial acetic acid and the volume was halved on a rotary evaporator. The crude product was purified by semipreparative HPLC (0% to 75% acetonitrile in water, 0.1% TFA, 30 min gradient). The pure fractions were combined and lyophilized to give the title compound. Yield 105 mg, 29%, 3 stages. Ion found by LC/MS: [M+H]+ = 618.2.

Example 119 Synthesis of Intermediate-68

Stage a.

p-Nitrophenyl chloroformate (0.23 g, 1.14 mmol) was added to a stirred mixture of primary alcohol (0.47 g, 0.76 mmol, described in Example 118) and triethylamine (0.21 ml, 1.52 mmol), dissolved in DCM (15 ml, anhydrous). The reaction mixture was stirred for 1 hour, then additional trimethylamine (0.21 ml) and p-nitrophenyl chloroformate (230 mg) was added and stirring was continued for another hour, after which the mixture was diluted with water and extracted with DCM (3x). The combined organic extracts were washed with brine and dried over sodium sulfate. The solvent was removed by rotary evaporation. The crude residue was dissolved in DCM (3 ml), applied to celite and purified by silica gel chromatography (0% to 70% ethyl acetate in hexanes, 30 min gradient). The pure fractions were combined and concentrated to give the title compound as a white solid. Yield 525 mg, 88%. Ion found using LCMS: [M+H]+ = 782.2.

Stage b.

Triethylamine (0.14 ml, 0.97 mmol) was added to a stirred solution of propargyl-peg4-amine (181 mg, 0.78 mmol) in 1 ml acetonitrile. The triethylamine/propargyl-peg4-amine mixture was added to the product obtained in the previous step (510 mg, 0.65 mmol, in 2 ml acetonitrile) and stirred for 1 hour, after which all starting materials were consumed. The solvent was removed using a rotary evaporator. The crude residue was purified by RPLC (20% to 95% acetonitrile in water, 30 min gradient, no modifier). Yield 475 mg, 83%. Ion found by LC/MS: [M+H]+ = 874.2.

Stage c.

The product from the previous step (475 mg, 0.54 mmol) was stirred in TFA (5 ml) for 2 hours and then concentrated and azeotroped with methanol (3x) on a rotary evaporator. Ion found by LC/MS: [M+H]+ = 674.2. The residue was stirred in 1:1 methanol/water containing LiOH (49 mg, 1.6 mmol) for 30 minutes. The reaction was neutralized with glacial acetic acid and then concentrated by rotary evaporation. The product was purified by semipreparative HPLC (0% to 75% acetonitrile in water, 0.1% TFA, 30 min gradient). Pure fractions were pooled and lyophilized. Yield 204 mg, 59%. Ion found using LCMS: [M+H]+ = 634.2.

Example 120 Synthesis of Intermediate-77

Stage a.

ΗATU (0.44 g, 1.16 mmol, in 1.5 ml DMF) was added dropwise to a stirred solution of propargyl-peg4-amine (0.24 g, 1.05 mmol), bis-Boc-D-ornithine ( 0.35 g, 1.05 mmol) and triethylamine (0.59 ml, 4.21 mmol) in DMF (2 ml). The reaction mixture was stirred for 1 hour at ambient temperature, then purified directly by RPLC (5% to 90% acetonitrile in water, 0.1% TFA, 35 minute gradient). The pure fractions were combined and lyophilized to give the product as a viscous clear oil. Ion found by LC/MS: [M+H]+ = 546.2.

The Boe-protected intermediate was stirred in 4N HCl in dioxane (10 ml) for 45 minutes at ambient temperature. The solvent was removed by rotary evaporation, then the resulting residue was dissolved in deionized water (20 ml), frozen and lyophilized to give the product as a clear oil. Yield 310 mg, 70%, 2 stages. Ion found using LCMS: [M+H]+ = 346.2.

Stage b.

The product obtained in the previous step (96 mg, 0.28 mmol) and triethylamine (0.15 ml, 1.11 mmol) in acetonitrile (2 ml) were added to a stirred solution of zanamivir p-nitrophenyl carbonate (435 mg, 0.56 mmol , described in example 118, in 6 ml of acetonitrile) and stirred for 12 hours at ambient temperature, then for 2 hours at 80°C. The solvent was removed and the crude residue was purified by RPLC (10% to 95% acetonitrile in water, no modifier, 35 min gradient). Pure fractions were combined and concentrated on a rotary evaporator. Ion found using LC/MS: [(M+2H)/2] ⁺ = 815.8

This intermediate was stirred in TFA (5 ml) for 45 minutes, then concentrated and dried under high vacuum. Ion found using LC/MS[(M+2H)/2] ⁺ = 615.8.

This TFA salt was stirred in a mixture (1/1) MeOH/DI water (5 ml) containing LiOH (27 mg, 1.11 mmol) for 30 minutes at 0°C. The mixture was acidified (~pH ~5) with glacial acetic acid. Methanol was removed by rotary evaporation and the resulting residue was purified by semipreparative HPLC (5% to 70% acetonitrile in water, 0.1% TFA, 35 min gradient). Pure fractions were pooled and lyophilized. Yield 35 mg, 11%, 3 stages. Ion found using LC/MS: [(M+2H)/2] ⁺ = 575.8.

Example 121 Synthesis of Intermediate-78

Stage a.

Propargyl-Ρeg4-acid (640 mg, 2.46 mmol), diol-HCl salt (350 mg, 2.46 mmol), EDC (471 mg, 2.46 mmol), HOBt (377 mg, 2.46 mmol) and triethylamine (249 mg, 2.46 mmol) were stirred in DMF (3 ml) at ambient temperature for 4 hours. The mixture was purified directly by RPLC (0% to 80% acetonitrile in water, no modifier, 35 min gradient). The pure fractions were combined and concentrated to give the product as a clear oil. Yield 580 mg, 68%. Ion found using LCMS: [M+H] ⁺ = 348.4.

Stage b.

p-Nitrophenyl chloroformate (1.23 g, 1.25 mmol) was added to a stirred solution of the product obtained in the previous step (530 mg, 6.10 mmol) and triethylamine (925 mg, 9.15 mmol) in DCM (25 ml ), then cooled to 0°C in a nitrogen atmosphere. The mixture was stirred for 15 minutes at 0°C and then at room temperature for 2 hours. The reaction mixture was diluted with deionized water and extracted with DCM (3 x 20 ml). The combined organic extracts were washed with brine and dried over sodium sulfate. The crude residue was purified by silica gel chromatography (10% to 100% ethyl acetate in hexanes, 25 minute gradient) to give the product as a white solid. Yield 250 mg, 24%. Ion found using LCMS: [M+H] ⁺ = 678.2.

Stage c.

Amine-functionalized zanamivir (505 mg, 0.82 mmol, described in Example 118) and triethylamine (0.15 ml, 1.11 mmol) in acetonitrile (2 ml) were added to a stirred solution of the product obtained in the previous step (220 mg, 0.37 mmol), in acetonitrile (10 ml) and stirred for 4 hours at ambient temperature. The reaction mixture was concentrated on a rotary evaporator. The resulting residue was purified by RPLC (15% to 95% acetonitrile in water, no modifier, 35 min gradient). Pure fractions were combined and concentrated on a rotary evaporator. Ion found using LC/MS: [(M+2H)/2] ⁺ = 815.2.

This intermediate was stirred in TFA (5 ml) for 45 minutes, then concentrated and dried under high vacuum. Ion found using LC/MS: [(M+2H)/2] ⁺ = 615.2.

The resulting TFA salt was stirred in a mixture (1/1) MeOH/DI water (5 ml) containing LiOH (71 mg, 2.98 mmol) for 30 minutes at 0°C. The mixture was acidified (~ ρΗ ~ 5) with glacial acetic acid. Methanol was removed by rotary evaporation and purified by semipreparative HPLC (5% to 70% acetonitrile in water, 0.1% TFA, 35 min gradient). Pure fractions were pooled and lyophilized. Yield 125 mg, 29% in 3 stages. Ion found using LC/MS: [(M+2H)/2] ⁺ = 575.8.

Example 122 Synthesis of Intermediate-4a

Stage a.

Propargyl-Ρeg4-amine (165 mg, 0.71 mmol) was added to a stirred solution of zanamivir p-nitrophenylcarbonate (350 mg, 0.47 mmol, described in Example 109) in acetonitrile (20 ml). The reaction mixture was stirred for 1 hour at ambient temperature, then the solvent was removed using a rotary evaporator. The residue was purified by RPLC (10% to 95% acetonitrile in water, no modifier, 30 min gradient). The pure fractions were combined and lyophilized to obtain the boc-protected intermediate as a white solid. Ion found using LCMS: [M+H] ⁺ = 830.2.

This intermediate was stirred in TFA (5 ml) at ambient temperature for 30 minutes. The solvent was removed by rotary evaporation and the residue was purified by RPLC (10% to 95% acetonitrile in water, 0.1% TFA, 30 min gradient). The pure fractions were combined and lyophilized to give the product as a white solid. Yield 155 mg, 52%, 2 stages. Ion found using LCMS: [M+H] ⁺ = 630.2.

Stage b.

The product obtained in the previous step (140 mg, 0.22 mmol) was stirred in a 1:3 methanol:water (8 ml) mixture containing LiOH (21 mg, 0.89 mmol) at 0°C for 20 minutes . The mixture was acidified with a few drops of glacial acetic acid and concentrated on a rotary evaporator. The crude material was purified by semipreparative HPLC (5% to 95% acetonitrile in water, 0.1% TFA, 30 min gradient). The pure fractions were combined and lyophilized to give the product as a white solid. Yield 60 mg, 45%. Ion found using LCMS: [M+H] ⁺ = 590.2.

¹ H NMR (500 MHz, Methanol-d ₄ ) δ 5.86 (d, J=2.6 Hz, 1H), 4.99 (dd, J=9.0, 2.7 Hz, 1H), 4.55 (dd, J=9.7, 2.7 Hz, 1H), 4.38 (dd, J=8.6, 2.6 Hz, 1H), 4.24-4.14 (m, 1H), 4.19 (d, J=2.6 Hz, 2H), 4.02-3.98 (m, 1H), 3.74-3.59 (m, 13H), 3.54 (t, J=5.6 Hz, 2H), 3.52-3.48 (m, 1H), 3.29-3.22 (m, 2H), 2.84 (t, J=2.4 Hz, 1H), 1.96 ( s, 3H).

¹³ C NMR (126 MHz, MeOD) δ 172.33, 163.68, 157.54, 156.58, 106.80, 79.23, 75.91, 74.56, 70.18, 70.13, 69.96, 69.87, 69.59, 69.51, 6 9.14, 68.74, 62.98, 57.67, 51.11, 40.54, 21.37.

Example 123 Synthesis of Intermediate-4b

The Peg-functionalized intermediate (100 mg, 0.13 mmol, described in Example 122) was stirred in a 1:3 methanol:water (5 ml) mixture containing LiOH (12 mg, 0.52 mmol), at ambient temperature within 2 hours. The mixture was acidified with glacial acetic acid and concentrated on a rotary evaporator. The crude material was purified by RPLC (5-95% acetonitrile in deionized water, 0.1% TFA, 30 min gradient). The pure fractions were combined and lyophilized to give the product as a white solid. Yield 21 mg, 31%. Ion found using LCMS: [M+H] ⁺ = 590.2.

¹ H NMR (Methanol-d ₄ ) δ: 5.86 (d, J=2.6 Hz, 1H), 4.48 (dd, J=8.6, 2.7 Hz, 1H), 4.42 (dd, J=9.6, 1.4 Hz, 1H) , 4.38 (d, J=12 Hz, 1H), 4.22-4.19 (m, 1H), 4.19 (d, J=2.4 Hz, 2H), 4.16-4.12 (dd, J=11.5, 6 Hz, 1H), 4.09-4.02 (m, 1H), 3.75-3.58 (m, 13H), 3.55 (t, J=5.5 Hz, 2H), 3.33-3.29 (m, 2H), 2.84 (t, J=2.4 Hz, 1H) , 2.02 (s, 3H).

¹³ C NMR (126 MHz, MeOD) δ 172.94, 163.82, 157.90, 157.53, 106.80, 79.23, 76.18, 74.55, 70.19, 70.13, 69.96, 69.90, 69.61, 68.96, 6 8.74, 68.17, 66.69, 57.67, 50.11, 40.43, 21.33.

Example 124. General procedure for the synthesis of azido-Fc.

Preparation of a solution of PEG4-azido-NHS ester (0.050M) in DMF/PBS: 16.75 mg of PEG4-azido-NHS ester was dissolved in 0.100 ml DMF at 0°C and diluted to 0.837 ml by adding 1x PBS buffer at 0°C. This solution was used to prepare another PEG4-azido-Fc with different DAR values by adjusting equivalents of this PEG4-azido-NHS ester solution in PBS.

Pretreatment of h-lgG1-Fc, SEQ ID NO: 48 (107.2 mg in 8.800 ml PBS at pH 7.4, MW ~57891 Da, 1.852 µmol): Fc solution was transferred to four centrifugal concentrators (30000 MWCO, 15 ml ) and diluted to 15 ml with PBS buffer x1, and concentrated to ~1.5 ml. The residue was diluted 1:10 in PBS pH 7.4 and concentrated again. This washing procedure was repeated a total of four times followed by dilution to 8.80 mL.

Preparation of PEG4-azido-Fc: 0.05M PEG4-azido-NHS ester in PBS buffer x1 (0.593 ml, 29.6 µmol, 16 equivalents) was added to the h-IgG1-Fc solution (SEQ ID NO: 48) and the mixture was shaken for 2 hours at ambient temperature. The solution was concentrated using four 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 washing procedure was repeated a total of three times. Concentrated Ec-PEC4-azide was diluted to 8.80 ml with 1x PBS pH 7.4 buffer and prepared for click conjugation. The purified product was quantified using a Nanodrop™ UV-visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1). The yield was quantitative after purification.

Example 125 Synthesis of conjugate 34

A solution of azido-functionalized Fc (40 mg, 2.5 mL, 0.69 μmol, described in General Preparation of Azido-Fc, Example 124, SEQ ID No. 18) was added to a 15 mL centrifuge tube containing the alkyne-derivatized small molecule ( 6.0 mg, 8.23 µmol, intermediate-67, example 118). After stirring gently to dissolve all solids, the mixture was added to a solution of sodium L-ascorbic acid (54 mg, 0.27 mmol), copper(II) sulfate (0.88 mg, 5.5 μmol) and BTTA (9.4 mg, 22 mmol) in PBS buffer at pH 7.4 (1.09 ml). The resulting mixture was gently rotated overnight. It was then purified by affinity chromatography on a Protein A column followed by size exclusion chromatography (see General Conjugate Purification Protocol). Maldi TOF analysis of the purified final product showed an average mass of 64,678 Da (DAR=7.2). Yield 24 mg, yield 54%.

Example 126 Synthesis of conjugate 35

A solution of azido-functionalized Fc (40 mg, 2.5 ml, 0.69 μmol, described in General Preparation of Azido-Fc, Example 124, SEQ ID No. 18) was added to a 15 ml centrifuge tube containing the alkyne-derivatized small molecule ( 6.1 mg, 8.23 µmol, intermediate-68, example 119). After stirring gently to dissolve all solids, the mixture was added to a solution of sodium L-ascorbic acid (54 mg, 0.27 mmol), copper(II) sulfate (0.88 mg, 5.5 μmol) and BTTA (9.4 mg, 22 µmol) in PBS buffer at 7.4 (1.09 ml). The resulting solution was gently rotated overnight. It was then purified by affinity chromatography on a Protein A column followed by size exclusion chromatography (see General Conjugate Purification Protocol). Maldi TOF analysis of the purified final product showed an average mass of 64,830 Da (DAR=7.3). Yield 23 mg, yield 52%.

Example 127 Synthesis of conjugate 36

A solution of azido-functionalized Fc (50 mg, 2.5 mL, 0.86 μmol, described in General Preparation of Azido-Fc, Example 124, SEQ ID No. 18) was added to a 15 mL centrifuge tube containing the alkyne-derivatized small molecule ( 7.1 mg, 5.2 µmol, intermediate-77, example 120). After stirring gently to dissolve all solids, the mixture was added to a solution of sodium L-ascorbic acid (68 mg, 0.34 mmol), copper(II) sulfate (1.1 mg, 6.9 μmol) and BTTA (12 mg, 27 µmol) in PBS buffer at 7.4 (1.37 ml). The resulting mixture was gently rotated overnight. It was then purified by affinity chromatography on a Protein A column followed by size exclusion chromatography (see General Conjugate Purification Protocol). Maldi TOF analysis of the purified final product showed an average mass of 63,300 Da (DAR=3.6). Yield 37 mg, yield 73%.

Example 128. Synthesis of conjugate 37

A solution of azido-functionalized Fc (70 mg, 2.5 mL, 1.2 μmol, described in General Preparation of Azido-Fc, Example 124, SEQ ID No. 18) was added to a 15 mL centrifuge tube containing the alkyne-derivatized small molecule ( 13.3 mg, 9.6 µmol, intermediate 78, example 121). After stirring gently to dissolve all solids, the mixture was added to a solution of sodium L-ascorbic acid (96 mg, 0.48 mmol), copper(II) sulfate (1.6 mg, 10 µmol) and BTTA (17 mg, 38 mmol ) in PBS buffer at pH 7.4 (1.92 ml). The resulting mixture was shaken gently overnight. It was then purified by affinity chromatography on a Protein A column followed by size exclusion chromatography (see General Conjugate Purification Protocol). Maldi TOF analysis of the purified final product showed an average mass of 63,574 Da (DAR=3.6). Yield 42 mg, yield 61%.

Example 129. Synthesis of conjugate 33

Preparation of a “click” reagent solution: 0.0050 M CuSO ₄ in a PBS buffer solution: 10.0 mg of CuSO ₄ was dissolved in 12.53 ml of PBS, then 5.00 ml of this CuSO ₄ solution was taken and 43.1 mg of BTTAA was added ( CAS# 1334179-85-9) and 247.5 mg of sodium ascorbate to obtain a click reagent solution (0.0050M CuSO ₄ , 0.020M BTTAA and 0.25M sodium ascorbate).

An alkyne-derivatized small molecule (29.7 mg, 19. 9 µmol described in Example 100, 2.5 equivalents for each azido on Fc). After gentle stirring to dissolve all solids, the mixture was treated with click reagent solution (4.34 ml) (sodium L-ascorbic acid, 0.25 M, 1086 µmol, copper(II) sulfate, 0.0050 M, 21.7 µmol, and BTTAA 0.020M, 86.9 µmol). The resulting mixture was gently rotated for 6 hours at ambient temperature. It was purified by affinity chromatography on a protein A column followed by size exclusion chromatography (see General Conjugate Purification Protocol). Maldi TOF analysis of the purified final product showed an average mass of 64,550 Da (DAR=4.6). Yield 90.7 mg with 98% purity.

Example 130 Efficacy of conjugate 33 against influenza B/Brisbane/60/2008 in a lethal mouse model

Test drugs were evaluated against lethal influenza B infection in female BALB/c mice (Jackson Laboratories, 6-8 weeks old). The challenge virus (B/Brisbane/60/2008) is a mouse-adapted isolate capable of causing lethal infections in mice. The experiment involved 10 groups of 5 mice in each group. On day 0, all mice were infected with virus at 3x LD95 by intranasal inoculation in a volume of 50 μl (approximately 1E5 virus per mouse) after isoflurane anesthesia.

All groups received a single intravenous injection of conjugate 33 (Example 129), vehicle (PBS), or Fc alone (hIgG1 Fc) as a control two hours after virus challenge. An additional group was treated with oral oseltamivir (20 mg/kg, twice daily for 5 days), starting 8 hours after viral challenge.

The study evaluated 7 different dose concentrations of Conjugate 33 (10, 3, 1, 0.3, 0.1, 0.03, and 0.01 mg/kg). Mice were monitored for 2 weeks, and animals that had a weight loss greater than 20% or were considered moribund were scored as mortality. Body weight was also recorded to monitor the general health of the animals.

All mice treated with vehicle or Fc alone as a control achieved mortality by day 8, as expected. In contrast, all mice treated with conjugate 33 were completely protected after receiving single intravenous doses of 10 to 0.3 mg/kg throughout the study (Table 48). In contrast, the group receiving oseltamivir resulted in only a 40% survival rate, although the total dose these mice received was 200 mg/kg over the course of the experiment. The effectiveness of conjugate 33 was further confirmed by daily body weight measurements (Table 49; data shown only up to the first death in the group). Mice treated with conjugate 33 at a dose of 0.3 mg/kg showed only a transient loss of body weight, reaching a maximum of 7%, for one day. Taken together, this study demonstrates the effectiveness of conjugate 33, as measured by two parameters indicative of influenza B infection (survival and body weight), against the Yamagata strain of influenza.

Example 131. Characteristics of regioisomers

Intermediates of monomers and dimers can be obtained as a specific regioisomer or a mixture of regioisomers. Examples 100-102 show how to prepare regioisomers of intermediate-7 (C7-C7 example 100; C7-C9 example 101; C9-C9 example 102; optimized C7-C7 example 103). The methods described herein can be used to separate mixtures of regioisomers of any intermediate described herein. Table 50 provides a characterization of the relative percentage (%) amounts of bound C7 and C9 monomers, and bound C7-C7, C7-C9 and C9-C9 dimers in previously described methods for synthesizing the intermediate prior to conjugation.

Example 132 Efficacy of subcutaneous conjugate 33 against influenza A/Puerto Rico/8/34 (H1N1) in a lethal mouse model

Conjugate 33 was evaluated against lethal influenza IAV H1N1 infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/1934) is a mouse-adapted isolate capable of causing lethal infections in mice. The experiment involved 6 groups of 5 mice. On day 0, all mice were infected with the virus at 3x LD95 by intranasal inoculation in a volume of 30 μl after anesthesia with a mixture of ketamine/xylazine (150 and 10 mg/kg, respectively). Mortality and body weight were recorded daily, and any animal with a 20% body weight loss was scored as dead. Test groups received a single subcutaneous (SC) treatment with conjugate 33, hIgG1-Fc control, or vehicle (PBS) 2 hours after viral challenge. Table 51 shows the study design.

As expected, mice treated with vehicle or hIgG1-Fc as a control died of infection on days 6-7 (Table 52). However, mice treated with conjugate 33 were fully protected at dose levels of 1, 0.3 and 0.1 mg/kg. Mortality for conjugate 33 was observed only at the lowest dose concentration of 0.03 mg/kg.

The effectiveness of conjugate 33 was further confirmed by daily body weight measurements. As expected, mice treated with vehicle or hIgG1 Fc showed a sustained decrease in body weight until it exceeded 20%, at which point they were assessed as mortality (Table 53).

In contrast to control mice, those groups that received conjugate 33 at doses of 1, 0.3 and 0.1 mg/kg maintained a healthy body weight throughout the study and never showed more than a transient decrease in body weight of less than 8 % (0.1 mcg/kg group, day 8; Table 53). Based on survival and body weight measurements, conjugate 33 demonstrated robust protection against lethal influenza A/Puerto Rico/8/1934 challenge with a single subcutaneous dose of as little as 0.1 mg/kg.

Example 133 Conjugate 33 Efficacy Against A/PR/8/1934 (H1N1), Pulmonary PFU Load and Cytokine Levels

Studies of the effectiveness of conjugate 33 were carried out on female BALB/c mice aged 6-8 weeks (Charles River), which were intranasally injected with 3E2 PFU/mouse (3x LD95) of the mouse-adapted influenza A/PR/8/1934 (H1N1) virus. Conjugate 33 or human IgG1-Fc as a control was administered as a single subcutaneous (SC) dose 2 h postinfection at 0.1–3 mg/kg. Oseltamivir was administered orally twice daily for 4 days, starting 2 hours after infection, at a dose of 5 or 50 mg/kg. Baloxavir was resuspended in 0.5% methyl cellulose and administered orally twice daily for 4 days, starting 2 hours after infection, at a dose of 30 mg/kg (Table 54). Body weight (BW) was recorded for 4 days (FIGS. 62A-62B). 4 days after infection, mice were sacrificed using CO ₂ and both lobes of the lungs were collected. Lungs were homogenized with 1 mm silica beads in 1 ml PBS using a MagNA Lyser (Roche). Homogenization was performed at 6000 rpm for 60 sec and cooled on ice for 5 min between cycles. After homogenization of the lungs, the tubes were centrifuged for 10 min at 600 x g and the supernatant was transferred to a new tube.

To determine PFU, lung homogenate supernatants were diluted with infection buffer in the range from 10 ^-1 to 10 ^-6 . 100 μl of virus dilutions were added to a confluent monolayer of MDCK cells in 24-well plates and incubated for 1 h at room temperature with shaking every 15 min. After virus removal, liquid medium containing Avicel was added to MDCK cells. Cells were incubated at 37°C, 5% CO ₂ for 40 hours. After incubation, the medium was removed and cells were stained with crystal violet to count plaques and the number of plaque forming units (PFU) relative to lung weight (PFU/g lung) was calculated.

For cytokine analysis, lung homogenate supernatants were serially diluted 2-fold in a 96-well plate. Cytokine levels for INF-γ, TNF-α, IL-6, MIP-1α, and MCP-1 were determined by ELISA according to the manufacturer's instructions (R&D Systems). Following lethal influenza challenge in a murine model, lung PFU load (FIGS. 63A-63B) and lung cytokine levels were determined at day 4 postinfection (Table 55 and Table 56, respectively). Conjugate 33 demonstrated dose-dependent log reductions in viral load that were 0.7 at 0.1 mg/kg, 1.88 at 0.3 mg/kg, 3 at 1 mg/kg, and 3.8 at 3 mg/kg. Oseltamivir at a dose of 5 mg/kg and 50 mg/kg had a moderate effect on viral load, resulting in a reduction of 0.86 and 2 log, respectively. Baloxavir reduced viral load below the detection limit of 1e2 PFU/mL, thereby reducing viral load by >5.99 log compared to PBS control.

As expected, no biologically significant difference was observed between the negative controls, PBS, and hIgG1-Fc.

Conjugate 33 reduced viral load in a dose-dependent manner on day 4 post-influenza A infection in a mouse model (Table 55, Figures 64A-64B). Similarly, conjugate 33 demonstrated a dose-dependent fold reduction in cytokine levels for TNF-α, IL-6, INF-γ, MCP-1 and MIP-1α (Figs. 65A-65E, respectively) compared to uninfected controls on day 4 after infection with influenza A virus in a mouse model (Table 56).

The highest concentration of conjugate 33 tested, 3 mg/kg, demonstrated no weight loss throughout the infection period, similar to uninfected control mice (Table 57).

Example 134 Efficacy of Conjugate 33 against influenza A (H1N1) in a mouse model of lethal severe combined immunodeficiency (SCID)

Test drugs were evaluated against lethal influenza A infection in female BALB/c scid mice (Jackson Laboratories, 6-8 weeks old). The challenge virus (A/Puerto Rico/8/1934) is a mouse-adapted isolate capable of causing lethal infections in mice. The experiment involved 11 groups of 5 mice each. On day 0, all mice were infected with virus at 3xLD95 by intranasal inoculation in a volume of 30 μl (approximately 1U virus per mouse) after anesthesia with a mixture of ketamine/xylazine (150 and 10 mg/kg, respectively).

Groups received a single subcutaneous injection of vehicle (PBS), hIgG1-Fc control, or conjugate 33 (3, 1, 0.3, 0.1, 0.03 mg/kg) two hours after virus challenge. A separate study group consisted of 3 groups of mice treated with baloxavir marboxil (DC Chemicals, Shanghai, China) orally twice daily for 1 day; also, starting 2 hours after viral infection. Mice were monitored for 2 weeks, and animals that had a weight loss greater than 20% or were considered moribund were scored as mortality.

At the end of the study (day 14), mice treated with conjugate 33 were fully protected at all dose concentrations from 3 to 0.1 mg/kg (Table 58). Conjugate 33 failed to protect against lethal virus challenge only at the lowest concentration tested, 0.03 mg/kg. As expected, groups receiving vehicle or hIgG1-Fc were not protected. Mice treated with baloxavir were also protected, but at significantly higher total doses of 60 and 20 mg/kg, with only 40% of mice surviving to day 14 at a total dose of 6 mg/kg.

The effectiveness of conjugate 33 in this model of severe immunodeficiency was also evident based on body weight (Table 59). The lowest conjugate concentration that provided complete protection based on mortality was 0.1 mg/kg. At this dose level, the greatest mean weight loss for the group was transient and resulted in a decrease of less than 5% (occurring on day 2). In addition, the difference in body weight for the 1 and 3 mg/kg dose groups was less than 2% at day 14 compared to uninfected mice.

Taken together, these data demonstrate the effectiveness of conjugate 33 in protecting lethally challenged mice using single SC doses of conjugate as low as 0.1 mg/kg. This was achieved in a severe immunodeficiency mouse model completely lacking the immune T and B cells needed to clear influenza infections. These data support the use of conjugate 33 for the treatment of immunocompromised patient populations.

Example 135 Efficacy of subcutaneously administered conjugate 33 against influenza A/California/07/2009 (H1N1) pdm in a lethal mouse model

Conjugate 33 was evaluated against lethal influenza IAV H1N1 infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks). The challenge challenge virus (A/California/07/2009 (H1N1) pdm) is a pandemic isolate capable of causing lethal infections in mice. The experiment involved 6 groups of 5 mice. On day 0, all mice were infected with the virus at 3x LD95 by intranasal inoculation in a volume of 30 μl after anesthesia with a mixture of ketamine/xylazine (150 and 10 mg/kg, respectively). Mortality and body weight were recorded daily, and any animal with a 20% body weight loss was scored as dead.

Test groups received a single subcutaneous (SC) injection of conjugate 33, hIgG1-Fc control, or vehicle (PBS) 2 hours after viral challenge. Table 60 shows the study design and dose levels.

As expected, mice treated with vehicle or control hIgG1-Fc died of infection on days 6-7 (Table 61). However, mice treated with conjugate 33 were completely protected at 1 mg/kg and almost the same at 0.3 (80% survival). Significant mortality for conjugate 33 was observed only at the lower dose concentrations of 0.1 and 0.03 mg/kg.

The effectiveness of conjugate 33 was further confirmed by daily body weight measurements. As expected, mice treated with vehicle or hIgG1 Fc showed a sustained decrease in body weight until it exceeded 20%, at which point they were assessed as mortality (Table 62).

In contrast to control mice, mice treated with Conjugate 33 at a dose of 1 mg/kg showed only a transient decrease in body weight of approximately 10% with a peak on day 3 (Table 62). When measuring survival and body weight, conjugate 33 demonstrated robust protection against lethal challenge with influenza A/California/07/2009 (H1N1) pdm virus using a single subcutaneous dose of 1 mg/kg. Activity against the clinically relevant pandemic strain used in this study , confirms the utility of conjugate 33 in the treatment of serious influenza infections.

Example 136 Efficacy of intravenous (IV) conjugate 33 against influenza A/Puerto Rico/8/1934 (H1N1) in a lethal delayed treatment mouse model

Conjugate 33 was evaluated against lethal influenza A (H1N1) infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/1934) is a mouse-adapted isolate capable of causing lethal infections in mice. On day 0, all mice were infected with the virus at 3x LD95 by intranasal inoculation in a volume of 30 μl after anesthesia with a mixture of ketamine/xylazine (150 and 10 mg/kg, respectively). Mortality and body weight were recorded daily, and any animal with a 20% body weight loss was scored as dead.

The study design is detailed in Table 63 and consists of several groups. The control group included the vehicle-only (PBS) and hIgG1-Fc groups dosed 24 hours after viral challenge (the uninfected group was also included in this group). The second group received oseltamivir, which was administered at a dose 4 times higher than the humanized dose, with a delay in the start of treatment by 24, 48 or 72 hours. The last 3 groups received conjugate 33 administered as single intravenous doses of 10, 3, or 1 mg/kg; each of them was administered on the same schedule as the oseltamivir group described above.

As expected, vehicle and hIgG1-Fc were not protective when dosed 24 hours after viral challenge and resulted in complete mortality by day 7. In our hands, oseltamivir, even at 4 times the humanized dose (total dose of 200 mg/kg), was only partially effective when dosing was delayed by 24 hours (Table 64; 40% survival). However, conjugate 33 was fully protective at all concentrations (10, 3, and 1 mg/kg) using the same 24-hour dosing schedule.

When dosing was escalated to a full 48 hours after viral challenge, oseltamivir was no longer effective (0% survival), while conjugate 33 had 80% protection at 10 and 3 mg/kg doses. When dosing was delayed to 72 hours, only the 10 mg/kg dose of conjugate 33 demonstrated partial protection (40%). The effectiveness of conjugate 33 was also evident based on daily body weight measurements (Table 65). This was particularly significant in the T+24 hour groups, where for any group receiving conjugate 33 there was a reduction of less than 3% that was transient and occurred on day 1. In this study, conjugate 33 was more effective than oseltamivir, an approved treatment for influenza.

Example 137 Synthesis of Conjugate 38 (Intermediate-73), Conjugate 39 (Intermediate-74), Conjugate 40 (Intermediate-75) and Conjugate 41 (Intermediate-76)

A sterile 15 mL centrifuge tube was charged with sodium ascorbate (68.3 mg, 0.345 mmol), BTTAA (11.9 mg, 0.0276 mmol), product from Examples 114 (Intermediate-73), 115 (Intermediate-74 ), 116 (intermediate-75) or 117 (intermediate-76) (0.00953 mmol) and PBS 7.4 (1 ml). The reagents were vortexed until homogeneous, then mixed with azido-Fc (50 mg, 0.0008624 mmol, described in Example 124, SEQ ID NO: 64), and then with a solution of CuSO ₄ (1.1 mg, 0.0069 mmol) in water (0.5 ml). The mixture was rotated for 12 hours, then purified by affinity chromatography on a protein A column followed by size exclusion chromatography. Conjugates were characterized using Maldi TOF analysis (DAR typically=4.5). Yields were typically 50%.

Example 138 Synthesis of Intermediate-79

Stage a.

Zanamavir ester acid (0.90 g, 1.43 mmol, example 31) and N-methylmorpholine (0.23 ml, 2.14 mmol) were dissolved in THF (35 ml) and cooled to 0°C (bath with ice water) in a nitrogen atmosphere. Isobutyl chloroformate (0.24 mL, 1.85 mmol, in 2 mL DCM) was added dropwise via syringe over 5 minutes. The mixture was stirred at 0°C for 30 minutes, then for 15 minutes at ambient temperature, and then cooled to 0°C. Sodium borohydride (540 mg, 14.3 mmol, dissolved in 5 ml methanol) was added dropwise over 5 minutes. The reaction mixture was stirred for 15 minutes, after which all starting material was consumed (as determined by LC/MS). A few drops (~1 ml) of glacial acetic acid were added to acidify the mixture (pH ~5). Dilute with ethyl acetate and water, and extract with ethyl acetate (3x). The organic layer was washed with brine, the organic extracts were dried over sodium sulfate and concentrated on a rotary evaporator. The crude product was purified by silica gel chromatography (plated on celite first) (0-10% methanol in DCM, 30 min). Yield 0.66 g, 75%. Ion found using LCMS: [M+H]+ = 617.2.

Stage b.

Triethylamine (0.30 ml, 1.3 mmol) was added to a stirred mixture of alcohol (0.66, 1.1 mol, in 20 ml CH ₂ Cl ₂ ). The mixture was cooled to 0°C (ice water bath) under 1 atmosphere of nitrogen and mesyl chloride (150 mg, 1.3 mmol) was added dropwise over 5 minutes via syringe. The ice bath was removed and the reaction mixture was stirred for 45 minutes. The mixture was diluted with saturated aqueous sodium bicarbonate, extracted with DCM (3x). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated on a rotary evaporator. Ion detected by LCMS: [M+H]+ = 695.2. The intermediate product was used in the next step without purification.

Zanamvir mesylate was stirred in DMF at 80°C with 3 eq. sodium azide for 5 hours. The mixture was diluted with water, extracted with DCM (3x). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated. Ion found using LCMS: [Μ+H]+ = 695.2. The azide was carried to the next step without purification.

Zanamavir azide (670 mg, 1.04 mmol) was stirred in methanol (20 ml) in the presence of Lindlar catalyst (300 mg) under hydrogen gas for 12 hours. The mixture was filtered through celite and concentrated to give the title compound as a clear oil. The amine was carried to the next step without purification. Yield 0.37 g, 54%, 3 stages. Ion found using LCMS: [M+H]+ = 616.2.

Stage c.

Methyl 3-(benzylamino)propanoate (1 g, 5.2 mmol), methyl 4-bromobutanoate (1.2 g, 6.2 mmol) and diisopropylethylamine (1.34 ml, 7.8 mmol) were stirred in DMF (5 ml) at 65°C for 4 hours. Most of the solvent was removed by rotary evaporation and the crude material was purified by silica gel chromatography (Isco, 0 to 9% methanol in DCM, 30 minute gradient) to give the benzyl-protected intermediate as a clear oil. The benzyl-protected intermediate was stirred in methanol (20 ml) under hydrogen gas at 1 atmosphere in the presence of 20% palladium hydroxide on carbon (300 mg) for 12 hours. The mixture was filtered through celite and concentrated to give the title compound as a clear oil. Yield 0.72 g, 69%. Ion found using LCMS: [M+H]+ = 204.2.

Stage d.

Product from step-c (0.70 g, 2.16 mmol), propargyl-PEG-4-acid (0.63 mg, 2.38 mmol), HATU (1.26 g, 3.24 mmol) in DMF (2 ml) was stirred at room temperature followed by the addition of DIEA (1.15 ml, 6.49 mmol). The reaction mixture was stirred for 2 hours, then purified by reverse phase liquid chromatography (Isco, 5% to 50% acetonitrile in water with 0.1% TFA as modifier). Yield 840 mg, 87%. Ion found using LCMS [M+H]+ = 446.2.

Stage e.

A solution of the product obtained in step-d (840 mg, 1.85 mmol) and LiOH (113.1 mg, 4.72 mmol) in H ₂ O:MeOH (1:2, 9 ml) was stirred at room temperature in within 2 hours. The reaction mixture was then acidified with TFA. The resulting solution was concentrated, then purified by reverse phase liquid chromatography (Isco, 0% to 20% acetonitrile in water). Yield 454.5 mg, 59%. Ion found using LCMS [M+H]+ = 418.2.

Stage f.

To a solution of the product obtained in step-b (334.7 mg, 0.52 mmol), the product obtained in step-e (102 mg, 0.24 mmol), and HATU (273.25 mg, 0.704 mmol) in anhydrous DMF (3 ml) was added DIEA (217 mg, 16.4 mmol) at room temperature. The resulting mixture was stirred for 1 hour, then purified using RPLC (20% to 100% methanol in water without modifier). Yield 206.5 mg, 55%. Ion found using LCMS [(M+2H)/2]+ = 806.8.

Stage g.

The product obtained from step-f (206.5 mg, 0.128 mmol) and TFA (3 ml) in CH ₂ Cl ₂ (5 ml) were stirred at room temperature overnight, then concentrated under reduced pressure. The resulting residue was purified by semipreparative HPLC (0% to 30% acetonitrile in water with 0.1% TFA as modifier). Yield 144.5 mg, 69%. Ion found using LCMS [(M+2H)/2]+ = 606.8.

Stage h.

To a solution of the product obtained in step-g (144.5 mg, 0.119 mmol) in MeOH (9 ml) and water (3 ml) was added LiOH (18 mg, 0.75 mmol). The resulting solution was stirred at room temperature for 1 hour, then acidified with TFA and concentrated under reduced pressure. The residue was purified by semipreparative HPLC (0% to 25% acetonitrile in water, using 0.1% TFA as modifier). Yield 45 mg, 28%. Ions found using LCMS [(M+2H)/2]+ = 566.8.

Example 139. Synthesis of conjugate 42

A solution of azido-functionalized Fc (50 mg, 5.4 mL, 0.859 μmol, Example 124, SEQ ID NO: 18) was added to a 10 mL centrifuge tube containing the alkyne-derived small molecule (7.87 mg, 0.057 mmol , example 138). After gentle shaking to dissolve all solids, the mixture was added to 3 ml of pre-mixed solution of sodium L-ascorbic acid (0.68 mg, 0.34 mmol, 0.25 M), copper(II) sulfate (1.1 mg, 0 .0069 mmol, 0.005 M) and BTTAA (11.8 mg, 0.027 mmol, 0.02 M) in PBS buffer at pH 7.4. The resulting solution was stirred gently overnight. It was purified by affinity chromatography on a protein A column followed by size exclusion chromatography (see General Conjugate Purification Protocol). Maldi TOF analysis of the purified final product showed an average mass of 63,623 Da (DAR 3.8). Yield 36.01 mg, 72%.

Example 140 Synthesis of Intermediate-80

Stage a.

To a solution of zanamivir p-nitrophenylcarbonate (0.3 g, 0.4 mmol, Example 103) in anhydrous dichloromethane (5 ml) was added propargyl-PEG4-methylamine (0.11 g, 0.44 mmol) and DIPEA (0.14 ml, 1.0 mmol) in anhydrous DMF (5 ml). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash chromatography, eluting with a 0% to 10% methanol/dichloromethane mixture. Yield 0.28 g, 81%. Ions found using LCMS: (M+H)+ = 858.4, (M - Boc)+H ⁺ = 758.4.

Stage b.

The product obtained in the previous step (280 mg, 0.2 mmol) was dissolved in 2 ml MeOH and 2 ml THF, then treated with a solution of lithium hydroxide (24 mg, 1 mmol) dissolved in 2 ml water. The reaction mixture was stirred for 10 minutes at room temperature, after which the reaction was complete according to HPLC. The reaction pH was adjusted to 5-6 using Amberlite IRN-77 ion exchange resin, then filtered to remove the resin. The crude filtrate was evaporated to dryness under vacuum and used in the next step without purification; the yield was quantitative. Ions found using LCMS: (M+H)+=844.4, (M - Boc+H) ⁺ = 744.4. Stage c

The product obtained in step-b was dissolved in 2 ml of dichloromethane and 2 ml of TFA, and stirred at room temperature. The progress of the reaction was monitored using LCMS. After completion of the reaction (6 h), the solution was evaporated to dryness and then dissolved in 2 ml of water and 2 ml of acetonitrile. The resulting solution was stirred for another 2 hours at room temperature, and LCMS analysis showed complete deprotection of the acetonide protecting groups. This mixture was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco CombiFlash liquid chromatograph, eluting with a gradient of 5% to 40% acetonitrile in water with 0.1% TFA as modifier. Yield 180 mg, 78.0%. Ions found using LCMS: (Μ+H) ⁺ = 604.2.

Example 141: Stability data for Intermediate-80 compared to Intermediate-4

Intermediate-80 (Example 140) and Intermediate-4 (Example 13) were solubilized to 10 mg/ml in deionized water and then diluted 1:10 in 1X PBS to a final concentration of 1 mg/ml. Samples were incubated at 37°C or 60°C for 1 week. A 25 μL aliquot was diluted in 75 μL water for HPLC analysis. Using an Acquity Η-Class UPLC with a Phenomenex Biozen PS-CIS column (150 x 2.1 mm, 1.6 µm), a gradient from 0.1% formic acid in water to 0.1% formic acid in acetonitrile was performed as follows: 5% B for 0-1 min, 5-20% B for 1-20 min. Detection was performed at 240 nm using a diode array detector. Intermediate-80 showed less than 1% degradation over a week at 60°C, while intermediate-4 showed 15% degradation over the same time period (FIG. 66).

Example 142. Synthesis of conjugate 43

Preparation of a “click” reagent solution: 0.0050M CuSO ₄ in a buffer solution x1 PBS: 10.0 mg of CuSO ₄ was dissolved in 12.53 ml x1 PBS, then 10.00 ml of this solution of CuSO ₄ was taken and 86.1 mg was added BTTAA and 495.3 mg of sodium ascorbate to obtain a “click” reagent solution (0.0050M CuSO ₄ , 0.020M BTTAA and 0.25M sodium ascorbate).

A solution of azido-functionalized Fc (78.0 mg, 4.535 mL, 1.35 μmol; Example 124, SEQ ID NO: 64) was added to a 15 mL centrifuge tube containing the alkyne-derivatized small molecule (13.2 mg, 8.88 µmol, intermediate-80, example 140). After stirring gently to dissolve all solids, 2.153 ml of the above click reagent solution (sodium L-ascorbic acid, 0.25 M, 106.6 mg, 0.538 mmol, copper(II) sulfate 0.0050) was added to the mixture M, 1.72 mg, 0.0107 mmol, and BTTAA 0.020 M, 18.5 mg, 0.0431 mmol). The resulting mixture was gently rotated for 6 hours at ambient temperature. It was purified by affinity chromatography on a protein A column followed by size exclusion chromatography (see Conjugate Purification Protocol). Maldi TOF analysis of the purified final product showed an average mass of 64,012 Da (DAR=7.0). Yield 50.3 mg, yield 62%.

Example 143 Synthesis of Intermediate-81

Stage a.

To a well stirred solution of N-Boc-N-Me-glycinol (3.5 g, 20 mmol) in DMSO (20 mL), cooled in an ice water bath, was added allyl bromide (3.6 g, 30.0 mmol) with followed by the addition of finely ground KOH powder (3.5 g, 30.0 mmol) over 15 minutes. The resulting solution was stirred overnight at room temperature. The resulting mixture was distributed between 5% aq. HOAc (50 ml) and ethyl acetate (200 ml). The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated, then purified by flash chromatography, eluting with a 10% to 80% gradient of ethyl acetate/hexane. Product yield 4.1 g, 95%. Ions found using LCMS: M+H=216.3.

Stage b.

Ozone was bubbled through a solution of the compound obtained in step-a (8.0 g, 37 mmol) in MeOH (50 ml) and DCM (50 ml) at -78° C. until a light blue color appeared. Unreacted ozone was removed by bubbling oxygen for 10 minutes before adding NaBH ₄ (1.6 g, 40 mmol) in a small amount over 10 minutes. After all of the NaBH ₄ had been added, the mixture was gradually warmed to room temperature. The resulting solution was distributed between ethyl acetate (100 ml) and brine (50 ml). The organic layer was separated, washed with brine, dried over sodium sulfate, filtered, concentrated to an oil and then purified by flash chromatography, eluting with a 10% to 80% gradient of ethyl acetate/dichloromethane. Product yield 5.0 g, 62%. Ions found using LCMS: M+H=220.2.

Stage c.

To a solution of the product (4.4 g, 20 mmol) from the previous step and CBr ₄ (10.0 g, 30.0 mmol) in DCM (50 ml) at 0° C. PPh ₃ (8.0 g, 30 mmol) for 15 minutes (exothermic reaction). During addition, the internal temperature was maintained below 30°C. After adding PPh ₃ the reaction mixture was stirred overnight at room temperature. The crude reaction mixture was concentrated to an oil, then purified by normal phase chromatography, eluting with a gradient of 10% ethyl acetate/hexanes to 80% ethyl acetate/hexanes. Fractions containing oil droplets inside collection tubes were pooled and concentrated to a colorless oil. 4.0 g, 70.5%. Ions found using LCMS: M+H=282.1.

Stage d.

A solution of the product obtained in step-c (4 g, 14 mmol), benzylamine (0.60 g, 5.7 mmol) and K ₂ CO ₃ (2.35 g, 17 mmol) in DMF (20 ml) was heated to oil bath at 75°C for 8 hours. The mixture was filtered, concentrated and purified by RPLC (5% ACN/water to 100% ACN). Yield 2.1 g, 72.7%.

Stage e.

To a solution of the product obtained in step-d (1.3 g, 2.0 mmol) dissolved in CHCl ₃ /EtOH (1:20, 20 ml), 20% Pd(OH) ₂ /C (0.50 G). The reaction mixture was stirred overnight under a hydrogen cylinder at ambient temperature. The reaction mixture was filtered through a pad of celite, then concentrated using a rotary evaporator and transferred to the next step without further purification.

Stage f.

The product obtained in step-e was dissolved in 10 ml DMF, then treated with propargyl-PEG4 acid (0.52 g, 2.0 mmol), EDCI (0.6 g, 3.0 mmol), HOAt (0. 45 g, 3 mol) and Hunig's base (0.7 ml, 5.0 mmol) at room temperature. The reaction mixture was stirred for four hours, then concentrated and purified by RPLC (10% ACN/water to 60% ACN/water). Yield 0.43 g, 65% in two stages. Ions found using LCMS: [M - Boc+H] ⁺ = 562.4, [M+H] ⁺ = 662.4.

Stage g.

The product obtained from step-d (70 mg, 0.1 mmol) was treated with TFA (2 ml) for 2 hours at room temperature. The TFA was removed by rotary evaporation and the remaining oil was further dried under high vacuum for 12 hours to obtain the desired bis-TFA salt product. The output was quantitative. Ion found using LCMS: [Μ+H] ⁺ = 462.4.

Stage h.

To a solution of the nitrophenyl carbonate described in Example 109 (0.72 g, 0.95 mmol) in anhydrous DMF (5 ml) was added a mixture of the diamine obtained in step-g (0.3 g, 0.43 mmol), added portionwise for 30 minutes) and DIPEA (0.28 ml, 2 mmol) in anhydrous DMF (20 ml). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash chromatography, eluting with a 0 to 10% methanol/dichloromethane gradient. Yield 0.63 g, 86%. Ions found using LCMS: [(M+2H)/2] ⁺ = 844.4, [(M - Boe+2H)/2] ⁺ = 794.4, [(M - 2 Boe+2H)/2] ⁺ = 744.4 .

Stage i.

The product obtained in the previous step (600 mg, 0.35 mmol) was dissolved in 5 ml MeOH and 5 ml THF, then treated with a solution of lithium hydroxide (48 mg, 2 mmol) dissolved in 2 ml water. The reaction mixture was stirred for 10 minutes at room temperature, after which HPLC analysis indicated completion of the reaction. The pH of the reaction solution was adjusted to 5-6 using Amberlite IRN-77 ion exchange resin, then filtered to remove the resin. The crude product was evaporated to dryness on a rotary evaporator and used in the next step without purification. Ions found using LCMS: [(M+2H)/2] ⁺ = 829.9, [(M - Boe+2H)/2] ⁺ = 779.4, [(M - 2 Boe+2H)/2] ⁺ = 729.4 .

Stage j.

The product obtained in step-i was dissolved in 5 ml of dichloromethane and 5 ml of TFA and stirred at room temperature. The progress of the reaction was monitored using LCMS. After completion of the reaction (6 h), the solution was evaporated to dryness and then dissolved in 4 ml of water and 4 ml of methanol. The resulting solution was stirred for another 2 hours at room temperature, after which LCMS analysis showed complete deprotection of the acetonide protecting groups. This mixture was concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco CombiFlash liquid chromatograph, eluting with a 5% to 40% acetonitrile/water gradient with 0.1% TFA as modifier. Yield 380 mg, 71.0%. Ions found using LCMS: [(Μ+2Н)/2] ⁺ = 589.8, [(Μ+3Н)/3] ⁺ = 392.5.

Example 144 Synthesis of Intermediate-82

The title compound was prepared analogously to Example 143, Intermediate-81, where N-Boc-N-Me-glycinol was replaced by N-Boc-N-ethyl-glycinol in step-a. Ions found using LCMS: [M/2]+1=603.8, [M/3]+1=402.9.

Example 145 Synthesis of Intermediate-83

Stage a.

CBZ-protected-amino-peg1-bromide (7.6 g, 25.2 mmol), benzylamine (1.1 g, 10.1 mmol) and potassium carbonate (2.8 g, 2.8 mmol) were stirred in DMF (10 ml) at 60°C for 12 hours. The solution was concentrated by rotary evaporation and purified by silica gel chromatography (0-10% methanol in DCM, 30 minute gradient) to give the product as a clear viscous oil. Yield 3.2 g, 57%. Ion found by LC/MS [M+H]+ = 550.2.

Stage b.

The product obtained in step-a (1.9 g, 3.5 mmol) was dissolved in THF (20 ml) and cooled to 0° C. in an ice water bath under nitrogen atmosphere. LAH (6.9 mL, 13.8 mmol, 2 M in THF) was added dropwise via syringe over 10 minutes. The mixture was stirred at reflux for 2 hours, then cooled to 0°C in an ice water bath. 1 ml of water was added dropwise, followed by dropwise addition of 1 ml of aqueous (15% by weight) NaOH solution. 3 ml of water was added and the mixture was stirred for 15 minutes, after which 2 g of magnesium sulfate was added. The mixture was stirred for 10 minutes, then filtered through celite, washed with 2 additional 10 ml portions of THF and the combined filtrates were concentrated on a rotary evaporator. The residue was dissolved in acetonitrile (20 ml), triethylamine (1.4 g, 13.8 mmol) and boc anhydride (3.0 g, 13.8 mmol) were added. The mixture was stirred for 45 minutes, concentrated and purified by reverse phase HPLC (5-95% acetonitrile/deionized water, 0.1% TFA modifier, 30 minute gradient). Yield 1.4 g, 79%. Ion found by LC/MS [M+H]+ = 510.2.

Stage c.

The product obtained from step-b (1 g, 1.9 mmol) of this example was stirred in methanol (25 ml) in the presence of palladium hydroxide (200 mg) under a hydrogen atmosphere for 2 hours. The mixture was filtered through celite and concentrated to give the product as a clear oil, which was used without further purification. Yield 0.73 g, 89%. Ion found by LC/MS [M+H]+ = 420.4.

Stage d.

The product from step-c (0.73 g, 1.7 mmol), pargyl-peg4-tosylate (0.91 g, 2.4 mmol) and diisopropylethylamine (0.76 g, 5.9 mmol) were stirred in DMF (5 ml) at 85°C for 4 hours. The mixture was concentrated on a rotary evaporator, then purified by reverse phase HPLC (5-95% acetonitrile/deionized water, 0.1% TFA modifier, 30 minute gradient) to give the product as a clear viscous oil. Yield 0.89 g, 82%. Ion found by LC/MS [M+H]+ = 634.4.

Stage e.

The product obtained in step-d (0.89 g, 1.4 ml) was stirred in 4N HCl (in dioxane) for 45 minutes at ambient temperature. The mixture was concentrated on a rotary evaporator and subjected to azeotropic distillation (3x) with benzene. The resulting residue was dissolved in deionized water (15 ml), frozen and lyophilized to give the product as a clear oil, bis-HCl salt. Yield 0.65 g, 91%. Ion found by LC/MS [M+H}+ = 434.4.

Stage f.

The remaining four steps in the synthesis of this compound were similar to those used in the synthesis of Example 143, Intermediate-81. Ions found using LCMS: (M+2H)/2=575.8

Example 146 Alternative Synthesis of Intermediate-83

The product described in Example 145, Intermediate-83, can be prepared alternatively using the above reaction scheme by one skilled in the art using the methods described in this patent.

Example 147 Serial passage experiments to select resistant influenza viruses

To assess the potential for the development of drug-resistant mutant viral strains under the selective pressure of viral inhibitors, serial passage studies were conducted with conjugates 6 and 33 in comparison with the comparators oseltamivir and baloxavir. Serial passage studies were performed using A549 or MDCK cells. Passages were performed as follows: 150,000 A549 or MDCK cells were seeded per well (12-well plate) in 500 μl DMEM, 10% FBS, 1% PS, 1% NaPyr and 1% HEPES, and incubated for approximately 24 hours. When cells reached approximately 80% confluence, they were washed once with PBS and incubated for 2 hours in the presence of compounds or PBS alone under normal culture conditions. The concentrations of the tested drugs were properly optimized to maximize viral inhibition while maintaining sufficient viral production for subsequent passages. The concentrations of the test samples used in the serine passage experiments are shown in FIG. 67 and 68. Cells were then infected at an MOI of 0.01 or 0.05 (MDCK and A549 cells, respectively; 150 μl diluted in infection buffer) for 1 hour at room temperature in a buffer containing PBS, bovine albumin 35% and Ca ²⁺ /Mg ²⁺ , followed by removing the inoculum and washing the cells once with DMEM 1% PS, 1% NaPyr and 1% HEPES (no FBS). Cells were then incubated for 24 hours in the presence of test drugs diluted in DMEM 1% PS, 1% NaPyr, 1% HEPES and 1 μg/ml TRSA-treated trypsin. After incubation, viral supernatants were collected, and cells and debris were removed by centrifugation (5 min, 4°C, 1400 × g). The supernatants were then used to:

i. quantification of virus titer using plaque assay;

ii performing a hemagglutination assay to determine whether the virus has escaped inhibition of the compound;

iii reinfection of newly seeded A549 or MDCK cells in the presence of compounds.

The process was repeated for 10 passages or until resistance to oseltamivir and baloxavir was observed in the control. Once elevated titers in the presence of drugs are detected in two consecutive passages, the viruses can be purified from plaques. After plaque purification, all 8 genome segments can be sequenced and compared to a PBS-treated control virus to detect escape mutations. A summary of two different serial passage experiments is shown in FIG. 67 and 68. In the experiment shown in FIG. 67, conjugate 6 was compared to oseltamivir and baloxavir using A549 cells infected with A/California/04/09/H1N1 pdm. Conjugate 6, baloxavir and oseltamivir were used at concentrations of 0.5, 0.5 and 200 nM, respectively. For conjugate 6, no increase in virus titer was observed over passage 11 (indicating the absence of emergence of resistant mutants), whereas baloxavir and oseltamivir titers increased to levels similar to those observed in the PBS control after passages 5 and 11, respectively. In the experiment presented in FIG. 68, conjugates 6 and 33 were compared to oseltamivir and baloxavir using MDCK cells infected with A/WSN/1933 H1N1. Conjugate 6, conjugate 33, baloxavir and oseltamivir were used at concentrations of 4 nM, 2 nM, 4 nM and 50 nM, respectively. There was no increase in viral titer for conjugates 6 or 33 over 10 passages (indicating no emergence of resistant mutants), while baloxavir and oseltamivir titers increased to levels similar to those observed in the PBS control after passages 5 and 10. respectively.

Claims (71)

1. A conjugate for the treatment of a viral infection caused by influenza virus or parainfluenza virus, described by formula (D-I): (D-I) where each A ₁ and each A ₂ are independently selected from any of the formulas (AI)-(A-XII): where R ₁ is selected from -OH, -NH ₂ , -NHC(=NH)NH ₂ and -NHC(=NH)NHR ₆ ; R ₂ and R ₃ are each independently selected from -H, -OH, -F, -Cl and -Br; R ₄ is selected from -CO ₂ H _, -P(=O)(OH) ₂ , -SO ₃ H; R ₅ is selected from -COCH ₃ , -COCF ₃ , -SO ₂ CH ₃ ; X is selected from -O- and -S-; Y selected from -O-, -S-, -NR ₇ -, -O(C=O)NR ₇ -, -O(C=S)NR ₇ -, -O(C=O)O-, -O (C=O)-, -NH(C=O)O-, -NH(C=O)-, -NH(C=NH)-, -NH(C=O)NR ₇ -, -NH(C =NH)NR ₇ -, -NH(C=S)NR ₇ -, -NH(C=S)-, -OCH ₂ (C=O)NR ₇ -, -NH(SO ₂ )-, -NH( SO ₂ )NR ₇ -, -OR ₈ -, -NHR ₈ - and -SR ₈ -; R ₆ selected from R ₇ is selected from H, (C ₁ -C ₂₀ )alkyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl; (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; R ₈ is selected from (C ₃ -C ₂₀ )heterocycloalkyl, (C ₅ -C ₁₅ )aryl and (C ₂ -C ₁₅ )heteroaryl; each E contains an Fc domain monomer; n is 1 or 2; T represents an integer from 1 to 20, L is a linker covalently attached to each of E, A ₁ and A ₂ , where L contains one or more of (C ₁ -C ₂₀ )alkylene, (C ₁ -C ₂₀ )heteroalkylene, (C ₂ -C ₂₀ ) alkenylene, (C ₂ -C ₂₀ )heteroalkenylene, (C ₂ -C ₂₀ )alkynylene, (C ₂ -C ₂₀ )heteroalkynylene, (C ₃ -C ₂₀ )cycloalkylene, (C ₃ -C ₂₀ )heterocycloalkylene, (C ₄ -C ₂₀ )cycloalkenylene, (C ₄ -C ₂₀ )heterocycloalkenylene, (C ₈ -C ₂₀ )cycloalkynylene, (C ₈ -C ₂₀ )heterocycloalkynylene, (C ₅ -C ₁₅ )arylene, (C ₂ -C ₁₅ )heteroarylene , O, S, NR ⁱ , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino, where R ⁱ represents H, (C ₁ -C ₂₀ )alkyl, (C ₁ -C ₂₀ )heteroalkyl, (C ₂ -C ₂₀ )alkenyl, (C ₂ -C ₂₀ )heteroalkenyl, (C ₂ -C ₂₀ )alkynyl, (C ₂ -C ₂₀ )heteroalkynyl, (C ₃ -C ₂₀ )cycloalkyl, (C ₃ -C ₂₀ )heterocycloalkyl , (C ₄ -C ₂₀ )cycloalkenyl, (C ₄ -C ₂₀ )heterocycloalkenyl, (C ₈ -C ₂₀ )cycloalkynyl, (C ₈ -C ₂₀ )heterocycloalkynyl, (C ₅ -C ₁₅ )aryl or (C ₂ - C ₁₅ )heteroaryl, or a pharmaceutically acceptable salt thereof. 2. The conjugate according to claim 1, where L is optionally substituted with (C ₁ -C ₂₀ )alkyl, (C ₂ -C ₂₀ )alkenyl, (C ₂ -C ₂₀ )alkynyl, (C ₅ -C ₁₅ )aryl, C5- C15 alkaryl, (C ₁ -C ₂₀ )acyl, (C ₂ -C ₁₅ )heteroaryl, including a total of one to ten heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur; (C ₁ -C ₂₀ )heteroalkyl containing in total from one to ten heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur; (C ₂ -C ₂₀ )heteroalkenyl containing in total from one to ten heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur; (C ₂ -C ₂₀ )heteroalkynyl containing from one to ten heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur; (C ₂ -C ₁₅ )heteroalkaryl containing from one to ten heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur; a halogen selected from the group consisting of F, Cl, Br and I; oxo, cyano, nitro, amino, (C ₁ -C ₂₀ )alkyno, hydroxy, (C ₁ -C ₂₀ )alkoxy, (C ₁ -C ₂₀ )alkanoyl, carbonyl, carbamoyl, guanidinyl, ureido or amidinyl group. 3. The conjugate according to claim 1 or 2, where each A ₁ and each A ₂ are independently selected from any of the formulas (AI) - (AV): 4. The conjugate according to claim 1, where each A ₁ and each A ₂ are independently selected from any of formulas (A-VI)-(A-IX): 5. Conjugate according to claim 1, where the conjugate is described by formula (D-II): or a pharmaceutically acceptable salt thereof. 6. Conjugate according to claim 1, where the conjugate is described by formula (D-III): or a pharmaceutically acceptable salt thereof. 7. The conjugate according to claim 1, where the conjugate is described by the formula (D-IV): or a pharmaceutically acceptable salt thereof. 8. The conjugate according to claim 1, where the conjugate is described by the formula (D-V): or a pharmaceutically acceptable salt thereof. 9. The conjugate according to claim 1, where the conjugate is described by the formula (D-VI): (D-VI) or a pharmaceutically acceptable salt thereof. 10. The conjugate according to claim 1, where the conjugate is described by the formula (D-VII): or a pharmaceutically acceptable salt thereof. 11. The conjugate according to claim 1, where the conjugate is described by the formula (D-VIII): or a pharmaceutically acceptable salt thereof. 12. The conjugate according to claim 1, where the conjugate is described by the formula (D-IX): or a pharmaceutically acceptable salt thereof. 13. The conjugate according to claim 1, where the conjugate is described by the formula (D-X): or a pharmaceutically acceptable salt thereof. 14. Conjugate according to any one of paragraphs. 1-13, where L is oxo-substituted. 15. Conjugate according to any one of paragraphs. 1-14, where L contains no more than 250 atoms. 16. Conjugate according to any one of paragraphs. 1-15, where L is covalently attached to the surface-exposed nitrogen atom of lysine in E. 17. Conjugate according to any one of paragraphs. 1-15, where L is covalently attached to the sulfur atom of the surface exposed cysteine in E. 18. The conjugate according to claim 16 or 17, where n is 2 and each E dimerizes to form an Fc domain. 19. Conjugate according to any one of paragraphs. 1-18, where T is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. 20. Use of the conjugate according to any one of paragraphs. 1-19 for the treatment of viral infection caused by influenza virus or parainfluenza virus. 21. Pharmaceutical composition for the treatment of a viral infection caused by the influenza virus or parainfluenza virus, containing a conjugate according to any one of paragraphs. 1-19 or a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient thereof. 22. A method of treating a subject having a viral infection caused by an influenza virus or parainfluenza virus, comprising administering to the subject an effective amount of a conjugate according to any one of claims. 1-19 or compositions according to claim 21. 23. A method of prophylactic treatment of a viral infection caused by an influenza virus or a parainfluenza virus in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate according to any one of claims. 1-19 or compositions according to claim 21. 24. The method according to claim 22 or 23, where the viral infection is an influenza A, B or C virus, or a parainfluenza virus. 25. Method according to any one of paragraphs. 22-24, characterized in that the conjugate or composition is administered intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesional, intracranial, intraarticular, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, locally, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, oral, topical, inhalation, injection or infusion. 26. Method according to any one of paragraphs. 22-25, wherein the subject is treated with a second therapeutic agent. 27. The method of claim 26, wherein the second therapeutic agent is an antiviral agent. 28. The method according to claim 26, where the antiviral agent is selected from oseltamivir, zanamivir, peramivir, laninamivir, amantadine or rimantadine. 29. The method of claim 26, wherein the second therapeutic agent is an antiviral vaccine. 30. The method of claim 29, wherein the antiviral vaccine induces an immune response in the subject against an influenza A, B or C virus, or a parainfluenza virus.