Fc conjugate containing a CD73 inhibitor and its use

Abstract

The present disclosure provides a conjugate comprising an Fc domain monomer or an Fc domain covalently attached to a moiety that binds to or inhibits CD73. The present disclosure also provides a pharmaceutical composition comprising such a conjugate, and the use of such a conjugate in the treatment of disorders associated with CD73 dysregulation or overexpression, such as cancer, fibrosis, or viral infections.

Description

Background Art

[0001] Cluster of Differentiation 73 (CD73) is a glycosylphosphatidylinositol-anchored membrane protein present in most tissues, which catalyzes the conversion of extracellular adenosine monophosphate (AMP) to adenosine. It functions as a homodimer, can be shed, and is active as a soluble protein in circulation. In addition to its enzymatic function, CD73 is also a cell adhesion molecule and plays a role in the regulation of leukocyte trafficking.

[0002] In cancer, CD73 is expressed by many cell subsets present in the tumor mass, including tumor cells, stromal cells, and endothelial cells, as well as infiltrating immune cells. CD73 levels are known to be upregulated by tissue injury or hypoxic conditions, and CD73 levels are elevated in multiple solid tumors. Upregulation of CD73 within the tumor contributes to an adenosine-rich tumor microenvironment with numerous tumor-promoting and immunosuppressive effects. High CD73 tumor expression has been associated with shortened overall survival and poor prognosis in certain cancers. Also, CD73 in cancer patients has been associated with resistance to anti-tumor therapies.

[0003] Furthermore, dysregulation of CD73 observed in various immune cell populations in viral infections suggests a functional role of purine nucleotides and nucleoside signaling in the context of the immune response to viral infections, including SARS-CoV-2 virus infection.

[0004] Also, CD73 dysregulation has been shown to play an important role in the etiology of pulmonary fibrosis induced by radiotherapy or other insults to the lung tissue. In a mechanism independent of its catalytic activity, multivalent ligation of the CD73 enzyme has been shown to stimulate B cell activation, clonal expansion, and the generation of memory B cells, suggesting that multivalent CD73-binding molecules could be used as adjuvants to enhance vaccine efficacy.

[0005] There is a need for novel treatments for disorders associated with CD73. The development of small molecule inhibitors of CD73 has been hampered by low metabolic stability. Given the role that CD73 plays in cancer and a wide variety of other diseases, disorders, and conditions, and the lack of CD73 inhibitors currently available to healthcare providers, there is a need for new CD73 inhibitors, as well as related compositions and methods. SUMMARY OF THE INVENTION

[0006] The present disclosure relates to conjugates comprising an Fc domain monomer or an Fc domain covalently attached to a moiety that binds to or inhibits CD73. Specifically, such conjugates comprise monomers or dimers of moieties that bind to or inhibit CD73 conjugated to an Fc monomer or Fc domain. The Fc monomer or Fc domain within the conjugate binds to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells, such as neutrophils, and activates effector functions such as phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC). The present disclosure also provides pharmaceutical compositions comprising such conjugates, and the use of such conjugates in the treatment of disorders associated with CD73 dysregulation or overexpression (e.g., cancer, fibrosis, or viral infections).

[0007] In one aspect, the present disclosure features a conjugate represented by formula (D-I) or (M-I), or a pharmaceutically acceptable salt thereof, CHEMICAL wherein A 1 and A 2 each independently has the structure of formula (A), CHEMICAL m is 0, 1, 2, 3, 4, 5, or 6, s is 0 or 1, X 1 , X2 , X 3 , X 4 , X 5 , and X 6 each of which is independently N, CR 4 or C-Y-R 5 and X 1 , X 2 , X 3 , X 4 , X 5 , and X 6 at least one of which is C-Y-R 5 wherein R 5 is a bond to L, R 1 is

Chemical formula

[0008] In some embodiments, the conjugate is represented by formula (D-I).

Chemical formula

[0009] In some embodiments, the conjugate is represented by formula (M-I).

Chemical formula

[0010] In some embodiments, A 1 and A 2 have the structure of formula (A-I).

Chemical formula

[0011] In some embodiments, A 1 and A 2 each have the structure of formula (A-Ia).

Chemical formula

[0012] In some embodiments, A 1 and A 2 each have the structure of formula (A-Ib).

Chemical formula

[0013] In some embodiments, A 1 and A 2 each have the structure of formula (A-Ib-1).

Chemical formula

[0014] In some embodiments, A 1 and A 2 each have the structure of formula (A-Ib-2).

Chemical formula

[0015] In some embodiments, A 1 and A 2 each have the structure of formula (A-II).

Chemical formula

[0016] In some embodiments, A 1 and A 2 each have the structure of formula (A-IIa).

Chemical formula

[0017] In some embodiments, A 1 and A 2 each has the structure of formula (A-IIb). [Chemical formula]

[0018] In some embodiments, A 1 and A 2 each has the structure of formula (A-IIb-1). [Chemical formula]

[0019] In some embodiments, A 1 and A 2 each has the structure of formula (A-IIb-2). [Chemical formula]

[0020] In some embodiments, s is 0. In some embodiments, s is 1.

[0021] In some embodiments, each of R 2a and R 2b is independently H, optionally substituted C1-C 20 alkyl, or optionally substituted C1-C 20 heteroalkyl. In some embodiments, each of R 2a and R 2b is H.

[0022] In some embodiments, R 4 is H, halogen, OH, SH, optionally substituted amino, optionally substituted C1-C 20 alkyl, or optionally substituted C1-C 20It is heteroalkyl. In some embodiments, R 4 is halogen. In some embodiments, R 4 is Cl.

[0023] In some embodiments, R 1 is

Chemical formula

[0024] In some embodiments, R 1 is

Chemical formula

[0025] In some embodiments, each of R 6a and R 6b is independently H, optionally substituted C1-C 20 alkyl, or optionally substituted C1-C 20 heteroalkyl. In some embodiments, each of R 6a and R 6b is independently H, -CH3, -CH2CH3, -CH2OH, -CH2OCH3, -CH2CH2OH, or -CH2CH2OCH3. In some embodiments, each of R 6a and R 6bの is H.

[0026] In some embodiments, Y is

Chemical formula

Chemical formula

[0027] In some embodiments, Y is

Chemical formula

Chemical formula

[0028] In some embodiments, Y is [Chemical formula] as follows.

[0029] In some embodiments, Y is [Chemical formula] as follows. In some embodiments, Y is [Chemical formula] as follows.

[0030] In some embodiments, Y is [Chemical formula] as follows. In some embodiments, Y is [Chemical formula] is. In some embodiments, Y is

Chemical Formula

[0031] In some embodiments, Y is

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

[0032] In some embodiments, L is one or more optionally substituted C1-C 40 alkylene, optionally substituted C1-C 40 heteroalkylene, optionally substituted C1-C 40 alkoxylene, optionally substituted C2-C 20 alkenylene, optionally substituted C2-C 20 heteroalkenylene, optionally substituted C2-C 20 alkynylene, optionally substituted C2-C 20 heteroalkynylene, optionally substituted C3-C 20 cycloalkylene, optionally substituted C2-C 20 heterocycloalkylene, optionally substituted C4-C 20 cycloalkenylene, optionally substituted C4-C 20 heterocycloalkenylene, optionally substituted C8-C 20 cycloalkynylene, optionally substituted C8-C20 Heterocycloalkynylene, optionally substituted C5-C 15 Arylene, optionally substituted C2-C 15 Heteroarylene, O, S, NR i , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino, and R i is H, optionally substituted C1-C 20 alkyl, optionally substituted C1-C 20 heteroalkyl, optionally substituted C2-C 20 alkenyl, optionally substituted C2-C 20 heteroalkenyl, optionally substituted C2-C 20 alkynyl, optionally substituted C2-C 20 heteroalkynyl, optionally substituted C3-C 20 cycloalkyl, optionally substituted C2-C 20 heterocycloalkyl, optionally substituted C4-C 20 cycloalkenyl, optionally substituted C4-C20 heteroheterocycloalkenyl, optionally substituted C8-C 20 cycloalkynyl, optionally substituted C8-C 20 heterocycloalkynyl, optionally substituted C5-C 15 aryl, or optionally substituted C2-C 15 heteroaryl.

[0033] In some embodiments, L is oxo-substituted. In some embodiments, L contains 1 to 250 atoms. In some embodiments, L can form an amide, carbamate, sulfonyl, or urea bond.

[0034] In some embodiments, L is represented by the following formula, 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 In the formula, J 1 is a bond bonded to A1, J 2 is a bond bonded to E or a functional group capable of reacting with a functional group conjugated with E, Q 1 , Q 2 , Q 3 , Q 4 , and Q 5 each independently is optionally substituted C1-C40 alkylene, optionally substituted C1-C40 heteroalkylene, optionally substituted C1-C40 alkoxylene, 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 C2-C 20 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, T 1 , T 2 , T 3 , T 4 each independently is O, S, NR i , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, imino, or oximo, R iis H, 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, C2-C 20 heterocycloalkyl, C4-C20 cycloalkenyl optionally substituted, C4-C20 heterocycloalkenyl optionally substituted, C8-C20 cycloalkynyl optionally substituted, C8-C20 heterocycloalkynyl optionally substituted, C5-C15 aryl optionally substituted, or C2-C15 heteroaryl optionally substituted, and each of g, h, i, j, k, l, m, n, and o is independently 0, 1, or 2.

[0035] In some embodiments, Q 1 is

Chemical formula

[0036] In some embodiments, Q 2 is C1-C 40 alkylene optionally substituted, C1-C 40 heteroalkylene optionally substituted, C1-C 40 alkoxylene optionally substituted, or C2-C 15 heteroarylene optionally substituted.

[0037] In some embodiments, Q 3 is C2-C 15 heteroarylene optionally substituted.

[0038] In some embodiments, Q 4 is C1-C 40 alkylene optionally substituted, C1-C40 heteroalkylene, or optionally substituted C1-C 40 alkoxylene.

[0039] In some embodiments, J 2 is

Chemical formula

[0040] The present disclosure features the intermediates (Int) of Table 1. These intermediates contain one or more inhibitors of CD73 and a linker and can be used in the synthesis of the conjugates described herein. The intermediates of Table 1 can be conjugated to, for example, an Fc domain or an Fc domain monomer (e.g., via a linker) by any suitable method known to those skilled in the art (including the methods described or exemplified herein). In some embodiments, the conjugate contains E, where E is an Fc domain monomer or an Fc domain. In a preferred embodiment, one or more nitrogen atoms of one or more surface-exposed lysine residues of E, or one or more sulfur atoms of one or more surface-exposed cysteines of E, are linked to a linker (e.g., PEG2-PEG 20is covalently conjugated with a linker). The linker conjugated with E can be functionalized to react with any of the Ints described herein (e.g., Int in Table 1) to form a covalent bond. In a preferred embodiment, E is conjugated with a linker functionalized with an azide group, and Int (e.g., Int in Table 1) is functionalized with an alkyne group. The conjugation of the linker-azide of E and the linker-alkyne of Int (e.g., by click chemistry) forms the conjugate of the present disclosure. In other embodiments, E is conjugated with a linker functionalized with an alkyne group, and Int (e.g., Int in Table 1) is functionalized with an azide group. The conjugation of the linker-alkyne of E and the linker-azide of Int (e.g., by click chemistry) forms the conjugate of the present disclosure. In still other embodiments, Int (e.g., Int in Table 2) is functionalized with a phenyl ester group (e.g., a trifluorophenyl ester group or a tetrafluorophenyl ester group). The conjugation of E and the linker-phenyl ester of Int (e.g., trifluorophenyl ester or tetrafluorophenyl ester) (e.g., by acylation) forms the conjugate of the present invention. The conjugation of E and the linker-phenyl ester of Int (e.g., trifluorophenyl ester or tetrafluorophenyl ester) (e.g., by acylation) is carried out by the methods described herein or methods known in the art.

[0041] The present disclosure further features a composition (e.g., a pre-conjugated intermediate) having the structure of Int in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

Table 1-41

Table 1-42

Table 1-43

Table 1-44

Table 1-45

Table 1-46

Table 1-47

Table 1-48

[0042] In addition, the present disclosure is also characterized by the conjugates in Table 2. Each conjugate in Table 2 corresponds to a conjugate of formula (D-I) or (M-I). The conjugates in Table 2 include conjugates formed by the covalent reaction between Int and E in Table 1. The conjugates in Table 2 further include conjugates formed by the covalent reaction between Int in Table 1 and a linker that is now conjugated to E. In some embodiments, the reactive moiety of Int (e.g., an alkyne group or an azide group) reacts with the corresponding reactive group of the linker covalently bound to E (e.g., an alkyne group or an azide group), resulting in the covalent bonding of Int in Table 1 to E. In some embodiments, the reactive moiety of Int (e.g., a phenyl ester group, e.g., a tetrafluorophenyl ester or a trifluorophenyl ester group) reacts with the corresponding reactive group of the amino acid side chain of E (e.g., a nitrogen or sulfur atom), resulting in the covalent bonding of Int in Table 1 to E.

[0043] In some embodiments of any conjugate in Table 2, n is 1 or 2. When n is 1, E includes an Fc domain monomer (e.g., an Fc domain monomer having any one of the sequences of SEQ ID NOs: 1 to 112 and 115 to 120). When n is 2, each E includes an Fc domain monomer (e.g., an Fc domain monomer having any one of the sequences of SEQ ID NOs: 1 to 112 and 115 to 120), and the Fc domain monomers dimerize to form an Fc domain.

[0044] In some embodiments of any conjugate in Table 2, T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). The present disclosure also provides any population of the conjugates described in Table 2 where the average value of T is from 1 to 20 (e.g., the average value of T is from 1 to 2, from 1 to 3, from 1 to 4, from 1 to 5, from 5 to 10, from 10 to 15, or from 15 to 20). In some embodiments, the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0045] The wavy lines within the conjugate in Table 2 indicate that each Int is covalently bonded to an amino acid side chain within E (e.g., the nitrogen atom of a surface-exposed lysine or the sulfur atom of a surface-exposed cysteine within E) or a pharmaceutically acceptable salt thereof.

[0046] This disclosure also provides the conjugate of Table 2. This conjugate is produced by conjugating the Int of Table 1 to an Fc domain or an Fc domain monomer (e.g., via a linker).

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

[0047] The present disclosure further features a method of making an Fc conjugate by conjugating an Int of Table 1 to an Fc domain monomer or Fc domain (e.g., via a linker). In some embodiments, the present disclosure provides a conjugate comprising a small molecule targeting agent described in the Int of Table 1, which is conjugated to Fc (e.g., via a linker).

[0048] In some embodiments, the wavy line connected to E indicates that the L of each A1-L or each A1-L-A2 is covalently bonded to the nitrogen atom of a solvent-exposed lysine of E. In some embodiments, the wavy line connected to E indicates that the L of each A1-L or each A1-L-A2 is covalently bonded to the sulfur atom of a solvent-exposed cysteine of E.

[0049] In some embodiments, n is 2 and each E dimerizes to form an Fc domain.

[0050] In some embodiments, each E is a human IgG1 Fc domain monomer. In some embodiments, each E contains a substitution mutation at N297 selected from N297A, N297G, or N297Q, and the amino acid numbering of each Fc domain monomer follows the Kabat EU index. In some embodiments, each E contains a C220S substitution mutation, and the amino acid numbering of each Fc domain monomer follows the Kabat EU index. In some embodiments, each E contains M252Y, S254T, and T256E substitution mutations, and the amino acid numbering of each Fc domain monomer follows the Kabat EU index.

[0051] In some embodiments, each E contains an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 112 and 115 to 120. In some embodiments, each E contains any one of the amino acid sequences of SEQ ID NOs: 1 to 112 and 115 to 120.

[0052] In some embodiments, each E contains an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18. In some embodiments, each E contains any one of the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18. In some embodiments, each E contains the amino acid sequence of SEQ ID NO: 13. In some embodiments, each E contains the amino acid sequence of SEQ ID NO: 14. In some embodiments, each E contains the amino acid sequence of SEQ ID NO: 17. In some embodiments, each E contains the amino acid sequence of SEQ ID NO: 18.

[0053] In some embodiments, each E comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. In some embodiments, each E comprises any one of the amino acid sequences of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 83.

[0054] In some embodiments, each E comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 115. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 115. In some embodiments, each E comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 116. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 116. In some embodiments, each E comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 117. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 117. In some embodiments, each E comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 118. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, each E comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, each E comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 120.

[0055] In some embodiments, n is 1, and T represents the number of A1-L or A1-L-A2 moieties attached to each E. In some embodiments, n is 2, two Es dimerize to form an Fc domain, and T represents the number of A1-L or A1-L-A2 moieties attached to the Fc domain. In some embodiments, T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0056] In addition, the present disclosure also provides a population of conjugates described herein, wherein the average value of T is 1 to 20 (for example, the average value of T is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, or 15 to 20). In some embodiments, the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0057] In another aspect, the present disclosure provides a pharmaceutical composition comprising a conjugate or a population of conjugates described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0058] In another aspect, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject a conjugate, a population of conjugates, or a pharmaceutical composition described herein.

[0059] In some embodiments, the cancer is selected from lung cancer, optionally non-small cell lung cancer or small cell lung cancer, head and neck cancer, optionally squamous cell carcinoma, renal cell carcinoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, urothelial cancer, cholangiocarcinoma, endometrial cancer, melanoma, or esophageal cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer overexpresses or is known to overexpress CD73 compared to non-cancer cells of the same tissue type.

[0060] In some embodiments, the method further comprises administering an immune checkpoint inhibitor to the subject. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 monoclonal antibody.

[0061] In another aspect, the present disclosure provides a method for treating or preventing a viral infection in a subject, the method comprising administering to the subject a conjugate, a population of conjugates, or a pharmaceutical composition described herein. In some embodiments, the viral infection is a beta-coronavirus infection. In some embodiments, the beta-coronavirus is SARS-CoV-2. In some embodiments, the SARS-CoV-2 is an alpha, delta, or omicron variant. In some embodiments, the SARS-CoV-2 is an omicron variant. In some embodiments, the omicron variant is of the BA.1, BA.2, BA.3, BA.4, or BA.5 lineage. In some embodiments, the method further comprises administering an antiviral agent or an antiviral vaccine to the subject.

[0062] In another aspect, the present disclosure provides a method for treating or preventing fibrosis in a subject, the method comprising administering to the subject a conjugate, a population of conjugates, or a pharmaceutical composition described herein. In some embodiments, the fibrosis is pulmonary fibrosis, dermal fibrosis, renal fibrosis, hepatic fibrosis, cardiac fibrosis, or systemic sclerosis. In some embodiments, the fibrosis is pulmonary fibrosis. In some embodiments, the pulmonary fibrosis is associated with a viral infection (e.g., associated with SARS-CoV-2 infection), drug-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, pneumoconiosis, interstitial lung disease, sarcoidosis, silicosis, or systemic sclerosis.

[0063] In some embodiments of any of the treatment methods described herein, the conjugate, the population of conjugates, or the pharmaceutical composition is administered intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, rectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intracystically, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, by injection, or by infusion.

[0064] Definitions To facilitate understanding of the present invention, a number of terms are defined below. The terms defined herein have the meanings commonly understood by those skilled in the art related to the present invention. Terms such as "a", "an", and "the" are not intended to refer only to singular entities and include general classifications for which specific examples may be used for illustration. The terms in this specification are used to describe particular embodiments of the present invention, but their use does not define the scope of the present invention except as outlined in the claims.

[0065] As used herein, the term "Fc domain monomer" refers to at least the hinge domain and the second and third antibody constant domains (C H 2 and C H3) or a functional fragment thereof (e.g., a fragment that (i) can dimerize with another Fc domain monomer to form an Fc domain and (ii) can bind to an Fc receptor). The Fc domain monomer can be an isotype of any immunoglobulin antibody, including IgG, IgE, IgM, IgA, or IgD (e.g., IgG). Further, the Fc domain monomer can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4) (e.g., IgG1). In some embodiments, the Fc domain monomer does not include any portion of an immunoglobulin that can function as an antigen recognition region (e.g., a variable domain or a complementarity-determining region (CDR)). The Fc domain monomer in the compositions described herein can include one or more changes (e.g., 1 to 10, 1 to 8, 1 to 6, 1 to 4 amino acid substitutions, additions, or deletions) from the wild-type Fc domain monomer sequence that modify the interaction between the Fc domain and the Fc receptor. Examples of suitable changes are known in the art. In certain embodiments, the human Fc domain monomer (e.g., an IgG heavy chain such as IgG1) includes the region extending 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 and does not affect the structure or stability of the Fc region. The C-terminal Lys447 can be proteolytically cleaved during polypeptide expression. In some embodiments of any Fc domain monomer described herein, the C-terminal Lys 447 is optionally present or absent. The N (e.g., Asn201) on the N-terminal side of the Fc region may or may not be present and does not affect the structure of the stability of the Fc region. The N-terminal Asn can be deamidated when the polypeptide is expressed. In some embodiments of any Fc domain monomer described herein, the N-terminal Asn is optionally present or absent.Unless otherwise specified herein, the numbering of amino acid residues within an IgG or Fc domain monomer follows the EU numbering system for antibodies, which is also referred to as the Kabat EU index and is 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.

[0066] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers capable of binding to an Fc receptor. In the case of the wild-type Fc domain, the two Fc domain monomers dimerize through interactions between the two C H 3 antibody constant domains, although in some embodiments, one or more disulfide bonds are formed between the hinge domains of the two dimerized Fc domain monomers.

[0067] The term "Fab" or "fragment antigen binding" is used interchangeably herein and refers to the region on an antibody that binds an antigen. Fab is a technical term and its meaning is known to those of ordinary skill in the art. The Fab region is composed of one constant domain and one variable domain of each of the heavy and light chains. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region can be composed of three domains, CH1, CH2, and / or CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), with conserved regions (referred to as "framework regions" (FR)) dispersed between these regions. Within an antibody, the heavy chain (e.g., VH and CH regions) is bound via a hinge to an Fc domain monomer. The Fc domain monomer described herein can include residues between 10 and / or 20 of the Fab domain and the hinge region (e.g., 11, 12, 13, 14, 15, 16, 17, 18, or 19 residues). In certain embodiments, the N-terminus of the Fc domain monomer is any one of amino acid residues 198-205 (corresponding to residues of the Fab domain). In some embodiments, the N-terminus of the Fc domain monomer is amino acid residue 201 (e.g., Asn201). In certain embodiments, the N-terminus of the Fc domain monomer is amino acid residue 202 (e.g., Val202).

[0068] The term "covalently bound" refers to two parts of a conjugate being bound to each other by a covalent bond formed between two atoms of the two parts of the conjugate.

[0069] As used herein, "surface-exposed amino acid" or "solvent-exposed amino acid" (e.g., surface-exposed cysteine or surface-exposed lysine) refers to an amino acid that is accessible to the solvent surrounding the protein. Surface-exposed amino acids can be natural or can be variants of the protein (e.g., substitutions or insertions). In some embodiments, surface-exposed amino acids are amino acids that, when substituted, do not substantially change the three-dimensional structure of the protein.

[0070] As used herein, the term "linker" refers to a covalent bond or connection between two or more components within a conjugate (e.g., between two CD73 inhibitors within a conjugate described herein, between a CD73 inhibitor and an Fc domain within a conjugate described herein, and between a dimer of two CD73 inhibitors and an Fc domain within a conjugate described herein). In some embodiments, the conjugate described herein can include a linker having a divalent structure (e.g., a divalent linker). A divalent linker has two arms, each arm being covalently bound to a component of the conjugate (e.g., the first arm is conjugated to a CD73 inhibitor and the second arm is conjugated to an Fc domain). In some embodiments, the conjugate described herein can include a linker having a trivalent structure (e.g., a trivalent linker). A trivalent linker has three arms, each arm being covalently bound to a component of the conjugate (e.g., the first arm is conjugated to a CD73 inhibitor, the second arm is conjugated to a second CD73 inhibitor, and the third arm is conjugated to an Fc domain). The linker of the present disclosure can be linear or branched.

[0071] In some embodiments, the molecule that can be used as a linker comprises at least two functional groups that may be the same or different, for example, two carboxylic acid groups, two amine groups, two sulfonic acid groups, a carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid group, an amine group and a maleimide group, an amine group and an alkyne group, or an amine group and a sulfonic acid group. The first functional group can form a covalent bond with the first component within the conjugate, and the second functional group can form a covalent bond with the second component within the conjugate. In some embodiments of the trivalent linker, the two arms of the linker can comprise two dicarboxylic acids, in which case the first carboxylic acid can form a covalent bond with the first CD73 inhibitor within the conjugate, the second carboxylic acid can form a covalent bond with the second CD73 inhibitor within the conjugate, and the third arm of the linker can form a covalent bond with the Fc domain within the conjugate. Examples of dicarboxylic acids are further described herein. In some embodiments, a molecule comprising one or more maleimide groups can be used as a linker, in which case the maleimide group can form a carbon-sulfur bond with a cysteine within a component (e.g., the Fc domain) within the conjugate. In some embodiments, a molecule comprising one or more alkyne groups can be used as a linker, in which case the alkyne group can form a 1,2,3-triazole bond with an azide within a component (e.g., the Fc domain) within the conjugate. In some embodiments, a molecule comprising one or more azide groups can be used as a linker, in which case the azide group can form a 1,2,3-triazole bond with an alkyne within a component (e.g., the Fc domain) within the conjugate. In some embodiments, a molecule comprising one or more bis-sulfone groups can be used as a linker, in which case the bis-sulfone group can form a bond with an amine group within a component (e.g., the Fc domain) within the conjugate.In some embodiments, a molecule containing one or more sulfonic acid groups can be used as a linker, in which case the sulfonic acid group can form a sulfonamide bond with a component within the conjugate. In some embodiments, a molecule containing one or more isocyanate groups can be used as a linker, in which case the isocyanate group can form a urea bond with a component within the conjugate. In some embodiments, a molecule containing one or more haloalkyl groups can be used as a linker, in which case the haloalkyl group can form a covalent bond (e.g., a C-N bond and a C-O bond) with a component within the conjugate. In some embodiments, a molecule containing one or more phenyl ester groups (e.g., a trifluorophenyl ester group or a tetrafluorophenyl ester group) can be used as a linker, in which case the phenyl ester group (e.g., a trifluorophenyl ester group or a tetrafluorophenyl ester group) can form an amide with an amine within a component (e.g., a fusion protein) within the conjugate.

[0072] In some embodiments, the linker provides space, rigidity, and / or flexibility between two or more components. In some embodiments, the linker can be a bond (e.g., a covalent bond). The term "bond" refers to a chemical bond, e.g., 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 resulting from a chemical reaction (e.g., chemical conjugation). In some embodiments, the linker comprises 250 or fewer atoms. In some embodiments, the linker comprises 250 or fewer non-hydrogen atoms. In some embodiments, the backbone of the linker comprises 250 or fewer atoms. The "backbone" of the linker refers to the atoms within the linker that form the shortest path (e.g., the shortest path linking a first CD73 inhibitor and a second CD73 inhibitor) from one part of the conjugate to another part of the conjugate. The atoms included in the backbone of the linker are directly involved in the bond between one part of the conjugate and another part (e.g., the bond between a first CD73 inhibitor and a second CD73 inhibitor). For example, hydrogen atoms bonded to carbon within the backbone of the linker are not considered to be directly involved in the bond between one part of the conjugate and another part.

[0073] In some embodiments, the linker can comprise a synthetic group derived from, for example, a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). In some embodiments, the linker can comprise one or more amino acid residues (e.g., D- or L-amino acid residues). In some embodiments, the linker can be a residue of an amino acid sequence (e.g., a 1-25 amino acid, 1-10 amino acid, 1-9 amino acid, 1-8 amino acid, 1-7 amino acid, 1-6 amino acid, 1-5 amino acid, 1-4 amino acid, 1-3 amino acid, 1-2 amino acid, or 1 amino acid sequence). In some embodiments, the linker is one or more (e.g., 1-100, 1-50, 1-25, 1-10, 1-5, or 1-3) optionally substituted alkylene, optionally substituted heteroalkylene (e.g., PEG unit), optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted alkynylene, optionally substituted heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted cycloalkenylene, optionally substituted heterocycloalkenylene, optionally substituted cycloalkynylene, optionally substituted heterocycloalkynylene, optionally substituted arylene, optionally substituted heteroarylene (e.g., pyridine), O, S, NR i (R iis 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 heterocycloalkenyl, 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 can be one or more optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene (e.g., PEG units), 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), optionally substituted C2-C 20 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 (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 C2-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.

[0074] As used herein, the terms "alkyl", "alkenyl", and "alkynyl" include linear and branched chain monovalent substituents, as well as combinations thereof, and when unsubstituted contain only C and H. When an alkyl group contains at least one carbon-carbon double bond or carbon-carbon triple bond, this alkyl group can be referred to as an "alkenyl" or "alkynyl" group, respectively. The monovalency of an alkyl, alkenyl, or alkynyl group does not include any optional substituents on the alkyl, alkenyl, or alkynyl group. For example, when an alkyl, alkenyl, or alkynyl group is attached to a compound, the monovalency of the alkyl, alkenyl, or alkynyl group means the 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, an alkyl or heteroalkyl group can contain, for example, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2 carbon atoms (e.g., C1-C20, C1-C18, C1-C16, 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 can contain, for example, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 carbon atoms (e.g., C2-C20, C2-C18, C2-C16, C2-C14, C2-C12, C2-C10, 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. A heterocycloalkyl group, heterocycloalkenyl group, or heterocycloalkynyl group refers to a cycloalkyl group, cycloalkenyl group, or cycloalkynyl group having one or more heteroatoms independently selected from N, O, and S. Exemplary heterocycloalkyl groups include pyrrolidine, thiophene, thiorane, tetrahydrofuran, piperidine, and tetrahydropyran.

[0075] As used herein, the term "cycloalkyl" represents a monovalent saturated or unsaturated non-aromatic cyclic alkyl group. A cycloalkyl can have, for example, from 3 to 20 carbons (e.g., C3-C7, C3-C8, C3-C9, C3-C10, C3-C11, C3-C12, C3-C14, C3-C16, C3-C18, or C3-C20 cycloalkyl). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. When a cycloalkyl group contains at least one carbon-carbon double bond, this cycloalkyl group can be referred to as a "cycloalkenyl" group. A cycloalkenyl can have, for example, from 4 to 20 carbons (e.g., C4-C7, C4-C8, C4-C9, C4-C10, C4-C11, C4-C12, C4-C14, C4-C16, C4-C18, or C4-C20 cycloalkenyl). Exemplary cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexenyl, and cycloheptenyl. When a cycloalkyl group contains at least one carbon-carbon triple bond, this cycloalkyl group can be referred to as a "cycloalkynyl" group. A cycloalkynyl can have, for example, from 8 to 20 carbons (e.g., C8-C9, C8-C10, C8-C11, C8-C12, C8-C14, C8-C16, C8-C18, or C8-C20 cycloalkynyl). Also, the term "cycloalkyl" includes cyclic compounds having a bridged polycyclic structure in which one or more carbons bridge two non-adjacent members of a monocyclic ring (e.g., bicyclo[2.2.1.]heptyl and adamantane). Also, the term "cycloalkyl" includes bicyclic, tricyclic, and tetracyclic fused ring structures, such as decalin and spirocyclic compounds.

[0076] As used herein, the term "aryl" refers to any monocyclic or fused bicyclic or tricyclic system having aromatic characteristics in terms of the electronic distribution of the entire ring system, such as phenyl, naphthyl, or phenanthrene. In some embodiments, the ring system contains 5 to 15 ring member atoms or 5 to 10 ring member atoms. An aryl group can have, for example, 5 to 15 carbons (e.g., C5-C6, C5-C7, C5-C8, C5-C9, C5-C10, C5-C11, C5-C12, C5-C13, C5-C14, or C5-C15 aryl). Also, the term "heteroaryl" refers to such monocyclic or fused bicyclic ring systems containing one or more (e.g., 1 to 4, 1 to 3, 1, 2, 3, or 4) heteroatoms selected from O, S, and N. A heteroaryl group can have, for example, 2 to 15 carbons (e.g., 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). By including heteroatoms, it becomes possible to include 5-membered rings as being considered aromatic in the same way 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. Due to the possibility of tautomers, groups such as phthalimide are also considered heteroaryl. In some embodiments, the aryl or heteroaryl group is an optionally substituted 5- or 6-membered aromatic ring system containing 1 to 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 optionally be substituted with a substituent (e.g., an aryl substituent such as biphenyl).

[0077] The term "alkaryl" refers to an aryl group attached to an alkylene, alkenylene, or alkynylene group. Generally, when a compound is attached to an alkaryl group, the alkylene, alkenylene, or alkynylene moiety of the alkaryl is attached to the compound. In some embodiments, the alkaryl is C6-C35 alkaryl (e.g., C6-C16, C6-C14, C6-C12, C6-C10, C6-C9, C6-C8, C7, or C6 alkaryl), where the number of carbons indicates the total number of carbons in both the aryl moiety and the alkylene, alkenylene, or alkynylene moiety of the alkaryl. Examples of alkaryl 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 the case of heteroalkaryl, one or more heteroatoms selected from N, O, and S may be present in the alkylene, alkenylene, or alkynylene moiety of the alkaryl group and / or in the aryl moiety of the alkaryl group. In the case of optionally substituted alkaryl, the substituent may be present in the alkylene, alkenylene, or alkynylene moiety of the alkaryl group and / or in the aryl moiety of the alkaryl group.

[0078] As used herein, the term "amino" represents -N(R x )2 or -N + (R x )3, wherein each R x is independently H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R x together form a heterocycloalkyl. In some embodiments, the amino group is -NH2.

[0079] As used herein, the term "alkamino" refers to an amino group as described herein attached to an alkylene (e.g., C1-C5 alkylene), alkenylene (e.g., C2-C5 alkenylene), or alkynylene group (e.g., C2-C5 alkynylene). Generally, when an alkanamino group is attached to a compound, the alkylene, alkenylene, or alkynylene moiety of the alkanamino is attached to the compound. The amino moiety of the alkanamino is -N(R x )2 or -N + (R x )3, where each R x is independently H, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, or two R x together form a heterocycloalkyl. In some embodiments, the amino moiety of the alkanamino is -NH2. An example of an alkanamino group is C1-C5 alkanamino, e.g., C2 alkanamino (e.g., CH2CH2NH2 or CH2CH2N(CH3)2). In the case of a heteroalkamino group, one or more (e.g., 1-4, 1-3, 1, 2, 3, or 4) heteroatoms selected from N, O, and S may be present in the alkylene, alkenylene, or alkynylene moiety of the heteroalkamino group. In some embodiments, the alkanamino group may be optionally substituted. In the case of a substituted alkanamino group, the substituent may be present in the alkylene, alkenylene, or alkynylene moiety of the alkanamino group and / or in the amino moiety of the alkanamino group.

[0080] As used herein, the term "alkanamide" refers to an amide group attached to an alkylene (e.g., C1-C5 alkylene), alkenylene (e.g., C2-C5 alkenylene), or alkynylene (e.g., C2-C5 alkynylene) group. Generally, when a compound is attached to an alkanamide group, the alkylene, alkenylene, or alkynylene moiety of the alkanamide is attached to the compound. The amide moiety of the alkanamide is -C(O)-N(R x )2, where each Rx Each independently is H, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, or two Rs x together form heterocycloalkyl. In some embodiments, the amide portion of the alkamide is -C(O)NH2. The alkamide group can be -(CH2)2-C(O)NH2 or -CH2-C(O)NH2. In the case of a heteroalkamide group, one or more (e.g., 1 to 4, 1 to 3, 1, 2, 3, or 4) heteroatoms selected from N, O, and S may be present in the alkylene, alkenylene, or alkynylene portion of the heteroalkamide group. In some embodiments, the alkamide group may be optionally substituted. In the case of a substituted alkamide group, the substituent may be present in the alkylene, alkenylene, or alkynylene portion of the alkamide group and / or in the amide portion of the alkamide group.

[0081] As used herein, the terms "alkylene", "alkenylene", and "alkynylene" refer to divalent groups having the specified size. In some embodiments, alkylene can include, for example, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2 carbon atoms (e.g., 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 can include, for example, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 carbon atoms (e.g., 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 linear and branched chain forms, as well as combinations thereof. The divalency of an alkylene, alkenylene, or alkynylene group does not include any optional substituents on the alkylene, alkenylene, or alkynylene group. For example, two CD73 inhibitors can be linked to each other via a linker (including alkylene, alkenylene, and / or alkynylene, or combinations thereof). Each of the alkylene, alkenylene, and / or alkynylene groups within the linker is considered divalent with respect to the two bonds at both ends 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 the bonds to the two alkylene groups at the ends of the linker. Any optional substituents on the alkenylene are not included in the divalency of the alkenylene. The divalent nature of an alkylene, alkenylene, or alkynylene group (e.g., an alkylene, alkenylene, or alkynylene group within a linker) refers to both ends of the group and does not include any optional substituents that may be present within the alkylene, alkenylene, or alkynylene group.Since the CD73 inhibitor is divalent, multiple (e.g., two) moieties of the conjugate, such as a first CD73 inhibitor and a second CD73 inhibitor, can be linked together. The alkylene, alkenylene, and / or alkynylene groups may be substituted by typically suitable groups as substituents for the alkyl, alkenyl, and alkynyl groups described herein. For example, C=O is a C1 alkylene substituted by oxo (=O). For example, -HCR-C≡C- can be regarded as an optionally substituted alkynylene and is regarded as a divalent group even if it has an optional substituent R. The heteroalkylene, heteroalkenylene, and / or heteroalkynylene groups refer to alkylene, alkenylene, and / or alkynylene containing one or more (e.g., 1 to 4, 1 to 3, 1, 2, 3, or 4) heteroatoms (e.g., N, O, and S). For example, a polyethylene glycol (PEG) polymer or a PEG unit -(CH2)2-O- in a PEG polymer is regarded as a heteroalkylene containing one or more oxygen atoms.

[0082] As used herein, the term "cycloalkylene" refers to a divalent cyclic group that joins two portions of a compound together. For example, one carbon within the cycloalkylene group may be bonded to one portion of the compound, while another carbon within the cycloalkylene group may be bonded to another portion of the compound. The cycloalkylene group can include a saturated or unsaturated non-aromatic cyclic group. The cycloalkylene can have, for example, 3 to 20 carbons in the cyclic portion of the cycloalkylene (e.g., 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 contains at least one carbon-carbon double bond, this cycloalkylene group can be referred to as a "cycloalkenylene" group. The cycloalkenylene can have, for example, 4 to 20 carbons in the cyclic portion of the cycloalkenylene (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 the cycloalkylene group contains at least one carbon-carbon triple bond, this cycloalkylene group can be referred to as a "cycloalkynylene" group. The cycloalkynylene can have, for example, 4 to 20 carbons in the cyclic portion of the cycloalkynylene (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 by typically suitable groups as substituents for the alkyl, alkenyl, and alkynyl groups described herein. Heterocycloalkylene refers to a cycloalkylene group containing one or more (e.g., 1 to 4, 1 to 3, 1, 2, 3, or 4) heteroatoms (e.g., N, O, and S). Examples of cycloalkylene include, but are not limited to, cyclopropylene and cyclobutylene. Tetrahydrofuran can be regarded as heterocycloalkylene.

[0083] As used herein, the term "arylene" refers to a polyvalent (e.g., divalent or trivalent) aryl group that joins together multiple (e.g., two or three) moieties of a compound. For example, one carbon within the arylene group may be bonded to one moiety of the compound, while another carbon within the arylene group may be bonded to another moiety of the compound. Arylene can have, for example, 5 to 15 carbons 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 by groups that are typically suitable as substituents for the alkyl, alkenyl, and alkynyl groups described herein. Heteroarylene refers to an aromatic group that contains one or more (e.g., 1-4, 1-3, 1, 2, 3, or 4) heteroatoms (e.g., N, O, and S). The heteroarylene group can have, for example, 2 to 15 carbons (e.g., 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).

[0084] As used herein, the term "optionally substituted" refers to having zero, one, or more substituents (e.g., 0 to 25, 0 to 20, 0 to 10, or 0 to 5 substituents). Alkyl, heteroalkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl may be substituted with alkyl, halogen, alkenyl, alkynyl, aryl, aralkyl, acyl, heteroaryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaralkyl, halogen, oxo, cyano, nitro, amino, alkylamino, hydroxy, alkoxy, alkanoyl, carbonyl, carbamoyl, guanidinyl, ureido, amidinyl, oximo, benzyl, OR, NR2, SR, SOR, SO2R, OCOR, NRCOR, NRCONR2, NRCOOR, OCONR2, RCO, COOR, alkyl - OOCR, SO3R, CONR2, SO2NR2, NRSO2NR2, CN, CF3, OCF3, SiR3, and NO2 (wherein each R is independently H, alkyl, cycloalkyl, heterocyclyl, alkenyl, aryl, heteroalkyl, heteroalkenyl, or heteroaryl). In some embodiments, the substituents are further substituted as described herein. For example, a C1 alkyl group, i.e., methyl, can be substituted with oxo to form a formyl group, and further substituted with -OH or -NHR to form a carboxyl group or an amide group.

[0085] An optionally substituted group or moiety refers to a group or moiety in which one of the atoms (e.g., a hydrogen atom) is optionally replaced with another substituent (e.g., any one of the groups or moieties described above). For example, an optionally substituted alkyl may be an optionally substituted methyl, in which case the hydrogen atom of the methyl group is replaced, for example, with OH. As another example, a substituent on a heteroalkyl or its divalent counterpart, heteroalkylene, can replace a hydrogen on carbon or a hydrogen on a heteroatom (e.g., N). For example, the hydrogen atom in the group -R-NH-R- may be replaced with an alkylamide substituent (e.g., -R-N[(CH2C(O)N(CH3)2]-R).

[0086] Generally, optional substituents are non-interfering substituents. A "non-interfering substituent" refers to a substituent that retains the ability to bind to CD73 of the conjugate described herein. Thus, in some embodiments, the substituent may modify the degree of such activity. However, as long as the conjugate retains the ability to bind to CD73 or inhibit tumor growth, the substituent is classified as "non-interfering". For example, a non-interfering substituent is considered to retain the ability of the compound to provide antiviral efficacy based on an IC50 value of 10 μM or less in a viral plaque reduction assay. Thus, the substituent may modify the degree of inhibition based on plaque reduction or CD73 inhibition. However, as long as the compounds herein retain the ability to inhibit CD73, the substituent is classified as "non-interfering". A plurality of assays are available in the art for quantifying viral plaque reduction or tumor growth inhibition, or the ability of a compound to inhibit CD73, some of which are exemplified in the examples below.

[0087] The term "hetero", when used to describe a chemical group or moiety, refers to having at least one heteroatom (e.g., N, O, and S) that is neither carbon nor hydrogen. Any one of the above groups or moieties may be termed hetero if it contains at least one heteroatom. For example, a heterocycloalkyl, heterocycloalkenyl, or heterocycloalkynyl group refers to a cycloalkyl, cycloalkenyl, or cycloalkynyl group having one or more heteroatoms independently selected from, for example, N, O, and S. An example of a heterocycloalkenyl group is maleimide. For example, a heteroaryl group refers to an aromatic group having one or more heteroatoms independently selected from, for example, N, O, and S. Also, one or more heteroatoms may be included in a substituent that replaces a hydrogen atom of a group or moiety as described herein. For example, in an optionally substituted heteroaryl group, if one of the hydrogen atoms of the heteroaryl group is replaced by a substituent (e.g., methyl), this substituent may also contain one or more heteroatoms (e.g., methanol).

[0088] As used herein, the term "acyl" refers to a group having the structure: [Chemical formula] wherein R z is optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, alkamino, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaryl, heteroalkaryl, or heteroalkamino.

[0089] As used herein, the term "halo" or "halogen" refers to any halogen atom, e.g., F, Cl, Br, or I. Any one of the groups or moieties described herein may be referred to as a "halo moiety" when it contains at least one halogen atom (e.g., haloalkyl).

[0090] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, or more) halo groups. Haloalkyl groups include, but are not limited to, fluoroalkyl (e.g., trifluoromethyl and pentafluoroethyl) and chloroalkyl.

[0091] As used herein, the term "hydroxyl" represents an -OH group.

[0092] As used herein, the term "oxo" refers to a substituent having the structure =O (a double bond exists between the atom and the oxygen atom).

[0093] As used herein, the term "carbonyl" refers to a group having the following structure. [Chemical formula] As used herein, the term "thiocarbonyl" refers to a group having the following structure. [Chemical formula] As used herein, the term "phosphate" represents a group having the following structure. [Chemical formula] As used herein, the term "phosphoryl" represents a group having the following structure. [Chemical formula] As used herein, the term "sulfonyl" represents a group having the following structure. [Chemical formula] As used herein, the term "imino" represents a group containing C=N (for example, the following structures: [Chemical formula] are included). For example, the imino group can be any one of the following structures: [Chemical formula] wherein each of R i1 and R i2 is each H or any one of the substituents described herein (for example, C1-C 20 alkyl), and each of R i3 and R i4 is unsubstituted or methylene substituted with one or more of the substituents described herein (for example, C1-C 20 alkyl), and each of i1 and i2 is independently 0, 1, 2, or 3.

[0094] As used herein, the term "oxime" represents a group containing C=N-O (for example, the following structure [Chemical formula] including).

[0095] As used herein, the term “N-protecting group” refers to a group that is intended to protect an 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) (incorporated herein by reference).Examples of N-protecting groups include acyl, allyloyl, and carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-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-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (BOC), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, and phenylthiocarbonyl; aralkyl groups such as benzyl, triphenylmethyl, and benzyloxymethyl; and silyl groups such as trimethylsilyl.

[0096] As used herein, the term "amino acid" means natural and unnatural amino acids.

[0097] As used herein, the term "natural amino acid" means an amino acid including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

[0098] As used herein, the term "non-natural amino acid" means an alpha amino acid that is not naturally produced or found in the body of a mammal. Examples of non-natural amino acids include D-amino acids; amino acids having an acetylaminomethyl group bonded to the sulfur atom of cysteine; pegylated amino acids; the formula NNH2(CH2) nOmega amino acids of COOH (where n is from 2 to 6), neutral non-polar amino acids, such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, and norleucine; oxy-methionine, 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 include α-aminobutyric acid, α-amino-α-methylbutyric acid, aminocyclopropanecarboxylate, aminoisobutyric acid, aminonorbornylcarboxylate, L-cyclohexylalanine, cyclopentylalanine, L-N-methylleucine, L-N-methylmethionine, L-N-methylnorvaline, L-N-methylphenylalanine, L-N-methylproline, L-N-methylserine, L-N-methyltryptophan, D-ornithine, L-N-methylethylglycine, L-norleucine, α-methylaminoisobutyric acid, α-methylcyclohexylalanine, D-α-methylalanine, D-α-methylarginine, D-α-methylasparagine, D-α-methylaspartic acid, 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, D-N-methylserine, D-α-methylthreonine, D-α-methyltryptophan, D-α-methyltyrosine, D-α-methylvaline, D-N-methylalanine, D-N-methylarginine, D-N-methylasparagine, D-N-methylaspartic acid, D-N-methylcysteine, D-N-methylglutamine, D-N-methylglutamic acid, D-N-methylhistidine, D-N-methylisoleucine, D-N-methylleucine, D-N-methyllysine, N-methylcyclohexylalanine, D-N-methylornithine, N-methylglycine, N-methylaminoisobutyric acid,N-(1-methylpropyl)glycine, N-(2-methylpropyl)glycine, D-N-methyltryptophan, D-N-methyltyrosine, D-N-methylvaline, gamma-aminobutyric acid, L-t-butylglycine, L-ethylglycine, L-homophenylalanine, L-alpha-methylarginine, L-alpha-methylaspartic acid, L-alpha-methylcysteine, L-alpha-methylglutamine, L-alpha-methylhistidine, L-alpha-methylisoleucine, L-alpha-methyll leucine, L-alpha-methylmethionine, L-alpha-methylnorvaline, L-alpha-methylphenylalanine, L-alpha-methylserine, L-alpha-methyltryptophan, L-alpha-methylvaline, N-(N-(2,2-diphenylethyl)carbamoylmethyl)glycine, 1-carboxy-1-(2,2-diphenylethylamino)cyclopropane, 4-hydroxyproline, ornithine, 2-aminobenzoyl (anthraniloyl), D-cyclohexylalanine, 4-phenyl-phenylalanine, L-citrulline, alpha-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, meta-tyrosine, nor-tyrosine, L-N,N',N''-trimethyllysine, homolysine, norlysine, N-glycanasparagine, 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-amino-cyclohexaneacetic acid, D / L-allylglycine, 4-aminobenzoic acid, 1-amino-cyclobutanecarboxylic acid, 2 or 3 or 4-aminocyclohexanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-aminoindan-1-carboxylic acid, 4-amino-pyrrolidine-2-carboxylic acid, 2-aminotetralin-2-carboxylic acid, azetidine-3-carboxylic acid,4-Benzyl-pyrrolidine-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-phenoxy-pyrrolidine-2-carboxylic acid, (2R,5S) and (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid, (2S,4S)-4-amino-1-benzoyl-pyrrolidine-2-carboxylic acid, t-butylalanine, (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid, 1-aminomethyl-cyclohexaneacetic acid, 3,5-bis-(2-amino)ethoxybenzoic acid, 3,5-diaminobenzoic acid, 2-methylaminobenzoic acid, N-methylanthranilic acid, L-N-methylalanine, L-N-methylarginine, L-N-methylasparagine, L-N-methylaspartic acid, L-N-methylcysteine, L-N-methylglutamine, L-N-methylglutamic acid, L-N-methylhistidine, L-N-methylisoleucine, L-N-methyllysine, L-N-methylnorleucine, L-N-methylo r nithine, L-N-methylthreonine, L-N-methyltyrosine, L-N-methylvaline, L-N-methyl-t-butylglycine, L-norvaline, a-methyl-g-aminobutyric acid, 4,4’-biphenylalanine, a-methylsylcopentylalanine, a-methyl-a-naphthylalanine, a-methylpenicillamine, N-(4-aminobutyl)glycine, N-(2-aminoethyl)glycine, N-(3-aminopropyl)glycine, N-amino-a-methylbutyric acid, a-naphthylalanine, N-benzylglycine, N-(2-carbamoylethyl)glycine, N-(carbamoylmethyl)glycine, N-(2-carboxyethyl)glycine, N-(carboxymethyl)glycine, N-cyclobutylglycine, N-cyclodecylglycine, N-cycloheptylglycine, N-cyclohexylglycine, N-cyclodecylglycine, N-cyclododecylglycine, 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-aminobutyric acid, D-N-methylmethionine, N-methylcyclopentylalanine, D-N-methylphenylalanine, D-N-methylproline, D-N-methylthreonine, N-(1-methylethyl)glycine, N-methylnaphthylalanine, N-methylpenicillamine, N-(p-hydroxyphenyl)glycine, N-(thiomethyl)glycine, penicillamine, L-α-methylalanine, L-α-methylasparagine, L-α-methyl-t-butylglycine, L-methylethylglycine, L-α-methylglutamic acid, L-α-methylhomophenylalanine, N-(2-methylthioethyl)glycine, L-α-methyllysine, L-α-methylnorleucine, L-α-methylo r nithine, L-α-methylproline, L-α-methylthreonine, L-α-methyltyrosine, L-N-methyl-homophenylalanine, N-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine, L-pyroglutamic acid, D-pyroglutamic acid, O-methyl-L-serine, O-methyl-L-homoserine, 5-hydroxylysine, α-carboxyglutamic acid, phenylglycine, L-pipecolic acid (homoproline), L-homoleucine, L-lysine (dimethyl), L-2-naphthylalanine, L-dimethyldopa or L-dimethoxy-phenylalanine, L-3-pyridylalanine, L-histidine (benzoyloxymethyl), N-cycloheptylglycine, L-diphenylalanine, O-methyl-L-homotyrosine, L-β-homolysine, O-glycantreoin, ortho-tyrosine, L-N,N’-dimethyllysine, L-homoarginine, neotryptophan, 3-benzothienylalanine, isoquinoline-3-carboxylic acid, diaminopropionic acid, homocysteine, 3,4-dimethoxyphenylalanine, 4-chlorophenylalanine, L-1,2,3,4-tetrahydronormann-3-carboxylic acid, adamantylalanine, symmetric dimethylarginine, 3-carboxythiomorpholine,D-1,2,3,4-Tetrahydronorvaline, 3-aminobenzoic acid, 3-amino-1-carboxymethyl-pyridin-2-one, 1-aminocyclohexanecarboxylic acid, 2-aminocyclopentanecarboxylic acid, 1-aminocyclopropanecarboxylic acid, 2-aminoindane-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-(2-naphthyl), pyrrolidine-2-carboxylic acid, nipecotic acid, (2S,4R) and (2S,4S)-4-(4-phenylbenzyl)pyrrolidine-2-carboxylic acid, (3S)-1-pyrrolidine-3-carboxylic acid, (2S,4S)-4-tritylmercapto-pyrrolidine-2-carboxylic acid, (2S,4S)-4-mercaptoproline, t-butylglycine, N,N-bis(3-aminopropyl)glycine, 1-aminocyclohexane-1-carboxylic acid, N-mercaptoethylglycine, and selenocysteine. In some embodiments, the amino acid residue may be charged or polar. Charged amino acids include alanine, lysine, aspartic acid, or glutamic acid, or analogs of these of non-natural origin. Polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, or tryptophan, or analogs of these of non-natural origin. In some embodiments, it is particularly contemplated that the terminal amino group within the amino acid can be an amide group or a carbamate group.,

[0099] As used herein, the term "percent identity (%)" refers to the percentage of amino acid residues of a candidate sequence (e.g., Fc-IgG) or a fragment thereof that are identical to the amino acid residues of a reference sequence, after aligning the sequences to achieve maximum identity and introducing gaps if necessary (i.e., gaps can be introduced into one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for the purpose of comparison). Alignments for the purpose of quantifying percent identity can be achieved in a variety of ways within the skill of the art, e.g., using publicly available computer software (e.g., BLAST, ALIGN, or Megalign (DNASTAR) software). One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid sequence identity of a given candidate sequence to, with respect to, or for a given reference sequence (alternatively, can be expressed as a given candidate sequence having or comprising a certain percent amino acid sequence identity to, with respect to, or for a given reference sequence) is calculated as follows. 100×(ratio of A / B) Wherein, A is the number of amino acid residues scored as identical in the alignment of the candidate and reference sequences, and 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 percent amino acid sequence identity of the candidate sequence to the reference sequence is considered not to be equal to the percent amino acid sequence identity of the reference sequence to the candidate sequence.

[0100] Two polynucleotide sequences or polypeptide sequences are "identical" when the nucleotide or amino acid sequences in the two sequences are the same when aligned to maximize correspondence as described above. The comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions where the sequences are similar. As used herein, a "comparison window" refers to a segment of at least about 15 contiguous positions, about 20 contiguous positions, about 25 contiguous positions, or more (e.g., about 30 to about 75 contiguous positions, or about 40 to about 50 contiguous positions), where the sequences can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0101] As used herein, the term "treating" or "treatment" refers to therapeutic treatment of a disease (e.g., cancer, fibrosis, or an infectious disease) in a subject. In some embodiments, the therapeutic treatment can slow the progression of the disease, improve the outcome of the subject, and / or eliminate a tumor. In some embodiments, the therapeutic treatment of a disease in a subject alleviates or improves one or more symptoms or conditions associated with the disease, reduces the degree of the symptoms, stabilizes (i.e., does not worsen) the condition of the disease, prevents the spread of the disease, and / or delays or slows the progression of the disease compared to the condition and / or the state of the disease in the absence of the therapeutic treatment.

[0102] As used herein, the term "average value of T" refers to the average number of Fc domain monomers or monomers of CD73 conjugated to the Fc domain or dimers of a CD73 inhibitor within a population of conjugates. In some embodiments, within the population of conjugates, the average number of monomers of a CD73 inhibitor conjugated to an Fc domain monomer or dimers of a CD73 inhibitor can be from 1 to 20 (e.g., the average value of T can be from 1 to 2, from 1 to 3, from 1 to 4, from 1 to 5, from 5 to 10, from 10 to 15, or from 15 to 20). In some embodiments, the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0103] As used herein, the term "subject" can be a human or non-human primate.

[0104] As used herein, the term "therapeutically effective amount" refers to an amount effective to induce a desired effect in a subject or to treat a subject having a condition or disorder described herein, e.g., a pharmaceutical dosage. Also as used herein, "therapeutically effective amount" can be understood to mean an amount that provides a desired therapeutic and / or prophylactic effect when administered in one or more dosages, or by any dosage or route, and / or alone or in combination with other therapeutic agents. For example, in the context of administering a conjugate described herein for the treatment of a disease described herein, an effective amount of the conjugate is, for example, an amount sufficient to prevent, slow, or reverse the progression of the disease as compared to the response obtained without administration of the conjugate.

[0105] As used herein, the term "pharmaceutical composition" refers to a medical or pharmaceutical preparation comprising at least one active ingredient and one or more excipients and diluents for rendering the active ingredient suitable for a method of administration. The pharmaceutical compositions of the present disclosure include pharmaceutically acceptable components compatible with the conjugates described herein.

[0106] As used herein, the term "pharmaceutically acceptable carrier" refers to an excipient or diluent in a pharmaceutical composition. For example, a pharmaceutically acceptable carrier can be a vehicle capable of suspending or dissolving the active conjugate. A pharmaceutically acceptable carrier must be compatible with the other components of the formulation and must not be harmful to the recipient. In the present disclosure, a pharmaceutically acceptable carrier must provide reasonable pharmaceutical stability for the conjugates described herein. The nature of the carrier will vary with the mode of administration. For example, for oral administration, a solid carrier is preferred, and for intravenous administration, an aqueous solution carrier (e.g., WFI, and / or buffer solution) is commonly used.

[0107] As used herein, the term "pharmaceutically acceptable salt" refers to salts of the conjugates described herein that are suitable for use in the methods described herein without undue toxicity, irritation, and / or allergic response within the scope of reasonable medical judgment. 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. The salts can be prepared in situ during the final isolation and purification of the conjugates described herein or separately by reacting the free base moiety with a suitable organic acid.

[0108] As used herein, the term "about" indicates a deviation of ±5%. For example, about 10% refers to 9.5% to 10.5%.

[0109] Any value shown in a range of values includes both the upper and lower limits, as well as any value included between the upper and lower limits.

[0110] Other features and advantages of the conjugates described herein will become apparent from the following detailed description of the invention and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0111]

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Figure 50A

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Mode for Carrying Out the Invention

[0112] The present disclosure relates to a conjugate comprising an Fc domain monomer or an Fc domain covalently bound to a moiety that binds to or inhibits CD73. Specifically, such a conjugate comprises a monomer or dimer of a moiety that binds to or inhibits CD73 conjugated to an Fc monomer or an Fc domain. The CD73 inhibitor (e.g., adenosine monophosphate, adenosine diphosphate, or an analog thereof) in the conjugate targets CD73. The Fc monomer or Fc domain in the conjugate binds to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells, such as neutrophils, and activates effector functions such as phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC). The conjugate exhibits a desirable tissue distribution (e.g., lung distribution).

[0113] The present disclosure also provides a pharmaceutical composition comprising such a conjugate, and the use of such a conjugate in the treatment of disorders associated with dysregulation or overexpression of CD73 (e.g., cancer, fibrosis, or viral infections).

[0114] I. Conjugate This specification provides a synthetic conjugate comprising one or more CD73 inhibitors (e.g., a CD73 inhibitor of formula (A), (A-I), (A-Ia), (A-Ib), (A-Ib-1), (A-Ib-2), (A-II), (A-IIa), (A-IIb), (A-IIb-1), or (A-IIb-2)) or an Fc domain conjugated to one or more dimers of two CD73 inhibitors. The dimer of two CD73 inhibitors comprises a first CD73 inhibitor (e.g., a first CD73 inhibitor of formula (A), (A-I), (A-Ia), (A-Ib), (A-Ib-1), (A-Ib-2), (A-II), (A-IIa), (A-IIb), (A-IIb-1), or (A-IIb-2)) and a second CD73 inhibitor (e.g., a second CD73 inhibitor of formula (A), (A-I), (A-Ia), (A-Ib), (A-Ib-1), (A-Ib-2), (A-II), (A-IIa), (A-IIb), (A-IIb-1), or (A-IIb-2)). In the context of a dimer (e.g., a dimer of formula (D-I)), the first and second CD73 inhibitors are linked to each other via a linker.

[0115] The conjugates of the present disclosure include monomers and dimers of CD73 inhibitors conjugated to an Fc domain, an Fc monomer, or an Fc-binding peptide. The Fc domain in the conjugates described herein binds to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells. Binding of the Fc domain in the conjugates described herein to FcγRs on immune cells activates phagocytosis and effector functions (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC)).

[0116] The conjugate provided in this specification is described by any one of formula (D-I) or (M-I). In some embodiments, the conjugate described herein comprises a monomer of the Fc domain or one or more monomers of a CD73 inhibitor conjugated to the Fc domain. In some embodiments, the conjugate described herein comprises a dimer of the Fc domain monomer or a CD73 inhibitor conjugated to the Fc domain. In some embodiments, when n is 2, E (Fc domain monomer) dimerizes to form an Fc domain.

[0117] The conjugates described herein can be synthesized using chemical synthesis techniques available in the art. When functional groups are not available for conjugation, the molecule can be derivatized using conventional chemical synthesis techniques well known in the art. In some embodiments, the conjugates described herein contain one or more chiral centers. The conjugate includes each of the isolated stereoisomeric forms, as well as mixtures of stereoisomers with different chiral purities (including racemic mixtures). The conjugate also includes various diastereomers, enantiomers, and tautomers.

[0118] Conjugate of a monomer of a CD73 inhibitor bound to an Fc domain In some embodiments, the conjugates described herein include an Fc domain monomer or an Fc domain covalently bonded to one or more monomers of a CD73 inhibitor, for example, a conjugate represented by formula (M-I). The conjugate of the Fc domain monomer or Fc domain and one or more monomers of the CD73 inhibitor can be formed by binding the Fc domain monomer or Fc domain to each of the monomers of the CD73 inhibitor via a linker (e.g., any linker described herein).

[0119] In a conjugate having an Fc domain monomer or an Fc domain covalently bound to one or more monomers of the CD73 inhibitor described herein, the wavy line leading to E indicates that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) monomers of the CD73 inhibitor can bind to the Fc domain monomer or the Fc domain. In some embodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) monomers of the CD73 inhibitor can bind to the Fc domain monomer. 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) monomers of the CD73 inhibitor can bind to the Fc domain. The wavy line in the conjugate described herein should not be construed as a single bond between one or more monomers of the CD73 inhibitor and an atom in the Fc domain monomer or the Fc domain. In some embodiments, when T is 1, one monomer of the CD73 inhibitor can bind to an atom in the Fc domain monomer or the Fc domain. In some embodiments, when T is 2, two monomers of the CD73 inhibitor can bind to an atom in the Fc domain monomer or the Fc domain.

[0120] 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 A1-L can be independently selected (e.g., independently selected from any of the A1-L structures described herein). In some embodiments, E can conjugate with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different A1-L moieties. In some embodiments, E conjugates with a first A1-L moiety and a second A1-L moiety. In some embodiments, each A1 of the first A1-L moiety and the second A1-L moiety is independently selected from any one of formulas (A), (A-I), (A-Ia), (A-Ib), (A-Ib-1), (A-Ib-2), (A-II), (A-IIa), (A-IIb), (A-IIb-1), and (A-IIb-2).

[0121] In some embodiments, the first A1-L moiety specifically conjugates with a lysine residue of E (e.g., the nitrogen atom of a surface-exposed lysine residue of E), and the second A1-L moiety specifically conjugates with a cysteine residue of E (e.g., the sulfur atom of a surface-exposed cysteine residue of E). In some embodiments, the first A1-L moiety specifically conjugates with a cysteine residue of E (e.g., the sulfur atom of a surface-exposed cysteine residue of E), and the second A1-L moiety specifically conjugates with a lysine residue of E (e.g., the nitrogen atom of a surface-exposed lysine residue of E).

[0122] As further described herein, the linker in a conjugate having an Fc domain monomer or an Fc domain covalently bound to one or more monomers of a CD73 inhibitor (e.g., L) described herein can have a divalent structure with two arms. One arm in the divalent linker can bind to a monomer of the CD73 inhibitor, and the other arm can bind to the Fc domain monomer or the Fc domain.

[0123] As represented by the above formula, in a conjugate having an Fc domain covalently bound to one or more monomers of a CD73 inhibitor, when n is 2, two Fc domain monomers (each Fc domain monomer is represented by E) dimerize to form an Fc domain.

[0124] Conjugate of a dimer of a CD73 inhibitor bound to an Fc domain The conjugates described herein include Fc domain monomers or Fc domains covalently bound to one or more dimers of a CD73 inhibitor, for example, conjugates represented by formula (D-I). The two dimers of the CD73 inhibitor include a first CD73 inhibitor (e.g., a first CD73 inhibitor of formula (A), (A-I), (A-Ia), (A-Ib), (A-Ib-1), (A-Ib-2), (A-II), (A-IIa), (A-IIb), (A-IIb-1), or (A-IIb-2)), and a second CD73 inhibitor (e.g., a second CD73 inhibitor of formula (A), (A-I), (A-Ia), (A-Ib), (A-Ib-1), (A-Ib-2), (A-II), (A-IIa), (A-IIb), (A-IIb-1), or (A-IIb-2)). The first and second CD73 inhibitors are bound to each other via a linker (e.g., a linker described herein). In some embodiments of the dimer of the CD73 inhibitor, the first and second CD73 inhibitors are the same. In some embodiments, the first and second CD73 inhibitors are different.

[0125] 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 A1-L-A2 can be independently selected (e.g., independently selected from any of the A1-L-A2 structures described herein). In some embodiments, E can conjugate with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different A1-L-A2 moieties. In some embodiments, E conjugates with a first A1-L-A2 moiety and a second A1-L-A2 moiety. In some embodiments, each of A1 and A2 of the first A1-L-A2 moiety and the second A1-L-A2 moiety is independently selected from any one of formula (A), (A-I), (A-Ia), (A-Ib), (A-Ib-1), (A-Ib-2), (A-II), (A-IIa), (A-Iib), (A-Iib-1), or (A-Iib-2).

[0126] In some embodiments, the first A1-L-A2 moiety specifically conjugates with a lysine residue of E (e.g., the nitrogen atom of a surface-exposed lysine residue of E), and the second A1-L-A2 moiety specifically conjugates with a cysteine residue of E (e.g., the sulfur atom of a surface-exposed cysteine residue of E). In some embodiments, the first A1-L-A2 moiety specifically conjugates with a cysteine residue of E (e.g., the sulfur atom of a surface-exposed cysteine residue of E), and the second A1-L-A2 moiety specifically conjugates with a lysine residue of E (e.g., the nitrogen atom of a surface-exposed lysine residue of E).

[0127] In the conjugate described herein, the 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) dimers of the CD73 inhibitor can bind to the Fc domain monomer or the Fc domain. In some embodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) dimers of the CD73 inhibitor can bind to the Fc domain monomer. 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) dimers of the CD73 inhibitor can bind to the Fc domain. The wavy line in the conjugate described herein should not be construed as a single bond between one or more dimers of the CD73 inhibitor and the Fc domain monomer or an atom within the Fc domain. In some embodiments, when T is 1, one dimer of the CD73 inhibitor can bind to the Fc domain monomer or an atom within the Fc domain. In some embodiments, when T is 2, two dimers of the CD73 inhibitor can bind to the Fc domain monomer or an atom within the Fc domain.

[0128] As further described herein, the linker within the conjugate described herein may have a branched structure. As further described herein, the linker within the conjugate described herein can be a multivalent structure, e.g., a divalent or trivalent structure having two or three arms, respectively. In some embodiments where the linker has three arms, two of the arms can bind to the first and second CD73 inhibitors, and the third arm can bind to the Fc domain monomer or the Fc domain.

[0129] As represented by the above formula, in a conjugate having an Fc domain covalently bound to one or more dimers of a CD73 inhibitor, when n is 2, two Fc domain monomers (each Fc domain monomer is represented by E) dimerize to form an Fc domain.

[0130] II. Fc domain monomer and Fc domain The present disclosure features a composition (e.g., conjugate) that includes one or more Fc domain monomers. When two compositions containing Fc domain monomers dimerize, the resulting conjugate contains an Fc domain. The Fc domain monomer includes a hinge domain, C H 2 antibody constant domains, and C H 3 antibody constant domains. The Fc domain monomer can be an isotype of an immunoglobulin antibody, such as IgG, IgE, IgM, IgA, or IgD. The Fc domain monomer can be any isotype of an immunoglobulin antibody (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain monomer can be any immunoglobulin antibody allotype (e.g., IGHG1*01 (i.e., G1m(za)), IGHG1*07 (i.e., G1m(zax)), IGHG1*04 (i.e., G1m(zav)), IGHG1*03 (G1m(f)), IGHG1*08 (i.e., G1m(fa)), IGHG2*01, IGHG2*06, IGHG2*02, IGHG3*01, IGHG3*05, IGHG3*10, IGHG3*04, IGHG3*09, IGHG3*11, IGHG3*12, IGHG3*06, IGHG3*07, 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) (e.g., as described in Vidarsson et al. IgG subclasses and allotypes: from structure to effector function. Frontiers in Immunology. 5(520):1-17(2014)). Also, the Fc domain monomer can be of any species (e.g., human, mouse, or rat). The dimer of the Fc domain monomer is an Fc domain that can bind to an Fc receptor (a receptor on the surface of leukocytes).

[0131] In some embodiments, the Fc domain monomer described herein may contain one or more amino acid substitutions, additions, and / or deletions with respect to the Fc domain monomer having any one of the sequences of SEQ ID NOs: 1-112 and 115-120. In some embodiments, Asn (e.g., N297) in the Fc domain monomer within the conjugate described herein can be replaced with Ala (e.g., N297A), Gly (e.g., N297G), or Gln (e.g., N297Q) to prevent N-linked glycosylation. In some embodiments, the amino acid corresponding to N297 is replaced with Ala, Gly, or Gln.

[0132] In some embodiments, the Fc domain monomer within the conjugate described herein contains an additional moiety for purification (e.g., a hexa-histidine peptide), or a signal sequence (e.g., an IL2 signal sequence) attached to the N- or C-terminus of the Fc domain monomer. In some embodiments, the additional moiety for purification (e.g., a hexa-histidine peptide), or the signal sequence (e.g., an IL2 signal sequence) attached to the N- or C-terminus of the Fc domain monomer is cleaved after expression of the polypeptide. In some embodiments, the Fc domain monomer in the composition does not contain any type of antibody variable region (e.g., V H 、V L , complementarity determining region (CDR), or hypervariable region (HVR)).

[0133] In some embodiments, the Fc domain monomer within the conjugate described herein can have a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to any one of the sequences of SEQ ID NOs: 1-112 and 115-120 shown below. In some embodiments, the Fc domain monomer within the fusion protein or conjugate described herein can contain any one of the sequences of SEQ ID NOs: 1-112 and 115-120 shown below.

[0134] Sequence number 1: Mature human Fc IgG1, Z1 is Cys or Ser, X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, X7 is Asn or Ser TIFF2025521815000116.tif31170

[0135] Sequence number 2: Mature human Fc IgG1, Cys→Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, X7 is Asn or Ser TIFF2025521815000117.tif37170

[0136] Sequence number 3: Mature human IgG1 Fc, Cys→Ser substitution (#), X4 is Asp or Glu, X5 is Leu or Met TIFF2025521815000118.tif31170

[0137] Sequence number 4: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(f) (bold italic) TIFF2025521815000119.tif31170

[0138] Sequence number 5: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(fa) (bold italic) TIFF2025521815000120.tif31170

[0139] Sequence number 6: Mature human IgG1 Fc, Cys→Ser substitution (#), M428L, N434S mutations (bold / underline), allotype G1m(fa) (bold italic) TIFF2025521815000121.tif31170

[0140] Accession number 7: Mature human IgG1 Fc, Cys→Ser substitution (#), M428L, N434S mutations (bold / underline), allotype G1m(f) (bold italic) TIFF2025521815000122.tif31170

[0141] Accession number 8: Mature human IgG1 Fc, Cys→Ser substitution (#), YTE triple mutations (bold and underline), allotype G1m(fa) (bold italic) TIFF2025521815000123.tif31170

[0142] Accession number 9: Mature human IgG1 Fc, Cys→Ser substitution (#), YTE triple mutations (bold and underline), allotype G1m(f) (bold italic) TIFF2025521815000124.tif31170

[0143] Accession number 10: Mature human Fc IgG1, Z1 is Cys or Ser, X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, X7 is Asn or Ser TIFF2025521815000125.tif31170

[0144] Accession number 11: Mature human Fc IgG1, Cys→Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, X7 is Asn or Ser TIFF2025521815000126.tif31170

[0145] Accession number 12: Mature human IgG1 Fc, Cys→Ser substitution (#), X4 is Asp or Glu, X5 is Leu or Met TIFF2025521815000127.tif31170

[0146] Accession number 13: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(f) (bold italic) TIFF2025521815000128.tif31170

[0147] Accession number 14: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(fa) (bold italic) TIFF2025521815000129.tif31170

[0148] Accession number 15: Mature human IgG1 Fc, Cys→Ser substitution (#), M428L, N434S mutations (bold / underline), allotype G1m(fa) (bold italic) TIFF2025521815000130.tif31170

[0149] Accession number 16: Mature human IgG1 Fc, Cys→Ser substitution (#), M428L, N434S mutations (bold / underline), allotype G1m(f) (bold italic) TIFF2025521815000131.tif31170

[0150] Accession number 17: Mature human IgG1 Fc, Cys→Ser substitution (#), YTE triple mutation (bold and underline), allotype G1m(fa) (bold italic) TIFF2025521815000132.tif31170

[0151] Accession number 18: Mature human IgG1 Fc, Cys→Ser substitution (#), YTE triple mutation (bold and underline), allotype G1m(f) (bold italic) TIFF2025521815000133.tif31170

[0152] SEQ ID NO: 19: Mature human Fc IgG1, Z1 is Cys or Ser, X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, X7 is Asn or Ser TIFF2025521815000134.tif31170

[0153] SEQ ID NO: 20: Mature human Fc IgG1, Cys→Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, X7 is Asn or Ser TIFF2025521815000135.tif31170

[0154] SEQ ID NO: 21: Mature human IgG1 Fc, Cys→Ser substitution (#), X4 is Asp or Glu, X5 is Leu or Met TIFF2025521815000136.tif31170

[0155] SEQ ID NO: 22: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(f) (bold italic) TIFF2025521815000137.tif31170

[0156] SEQ ID NO: 23: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(fa) (bold italic) TIFF2025521815000138.tif31170

[0157] SEQ ID NO: 24: Mature human IgG1 Fc, Cys→Ser substitution (#), M428L, N434S mutations (bold / underline), allotype G1m(fa) (bold italic) TIFF2025521815000139.tif31170

[0158] Accession No. 25: Mature human IgG1 Fc, Cys→Ser substitution (#), M428L, N434S mutations (bold / underline), allotype G1m(f) (bold italic) TIFF2025521815000140.tif31170

[0159] Accession No. 26: Mature human IgG1 Fc, Cys→Ser substitution (#), YTE triple mutation (bold and underline), allotype G1m(fa) (bold italic) TIFF2025521815000141.tif31170

[0160] Accession No. 27: Mature human IgG1 Fc, Cys→Ser substitution (#), YTE triple mutation (bold and underline), allotype G1m(f) (bold italic) TIFF2025521815000142.tif31170

[0161] Accession No. 28: Mature human Fc IgG1, Z1 is Cys or Ser, X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, X7 is Asn or Ser TIFF2025521815000143.tif31170

[0162] Accession No. 29: Mature human Fc IgG1, Cys→Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, X7 is Asn or Ser TIFF2025521815000144.tif31170

[0163] Accession No. 30: Mature human IgG1 Fc, Cys→Ser substitution (#), X4 is Asp or Glu, X5 is Leu or Met TIFF2025521815000145.tif31170

[0164] SEQ ID No. 31: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(f) (bold italic) TIFF2025521815000146.tif31170

[0165] SEQ ID No. 32: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(fa) (bold italic) TIFF2025521815000147.tif31170

[0166] SEQ ID No. 33: Mature human IgG1 Fc, Cys→Ser substitution (#), M428L, N434S mutations (bold / underline), allotype G1m(fa) (bold italic) TIFF2025521815000148.tif31170

[0167] SEQ ID No. 34: Mature human IgG1 Fc, Cys→Ser substitution (#), M428L, N434S mutations (bold / underline), allotype G1m(f) (bold italic) TIFF2025521815000149.tif31170

[0168] SEQ ID No. 35: Mature human IgG1 Fc, Cys→Ser substitution (#), YTE triple mutation (bold and underline), allotype G1m(fa) (bold italic) TIFF2025521815000150.tif31170

[0169] SEQ ID No. 36: Mature human IgG1 Fc, Cys→Ser substitution (#), YTE triple mutation (bold and underline), allotype G1m(f) (bold italic) TIFF2025521815000151.tif31170

[0170] Sequence number 37: Mature human Fc IgG1, J1 is Asn or absent, J2 is Lys or absent, Z1 is Cys or Ser, X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asn or Ala, X5 is Leu or Asp, X6 is Gln or His, X7 is Asp or Glu, X8 is Leu or Met, X 10 is Asn or Ser TIFF2025521815000152.tif31170

[0171] Sequence number 38: Mature human Fc IgG1, Cys→Ser substitution (#), J1 is Asn or absent, J2 is Lys or absent, X4 is Asn or Ala, X5 is Leu or Asp, X6 is Gln or His, X7 is Asp or Glu, X8 is Leu or Met, X 10 is Asn or Ser TIFF2025521815000153.tif37170

[0172] Sequence number 39: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), J1 is Asn or absent, J2 is Lys or absent, X4 Asn or Ala, X7 is Asp or Glu, X8 is Leu or Met TIFF2025521815000154.tif31170

[0173] Sequence number 40: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), X4 is Asn or Ala, X7 is Asp or Glu, X8 is Leu or Met TIFF2025521815000155.tif31170

[0174] Sequence number 41: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), X7 is Asp or Glu, X8 is Leu or Met TIFF2025521815000156.tif31170

[0175] Sequence number 42: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000157.tif31170

[0176] Sequence number 43: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000158.tif31170

[0177] Sequence number 44: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000159.tif31170

[0178] Sequence number 45: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000160.tif31170

[0179] Sequence number 46: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000161.tif31170

[0180] Sequence number 47: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000162.tif31170

[0181] Accession number 48: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000163.tif31170

[0182] Accession number 49: Mature human Fc IgG1, Cys→Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000164.tif31170

[0183] Accession number 50: Mature human Fc IgG1, Cys→Ser substitution (#), Asn→Ala substitution (*), DHS triple mutation (bold and underlined), X7 is Asp or Glu, X8 is Leu or Met TIFF2025521815000165.tif31170

[0184] Accession number 51: Mature human Fc IgG1, Cys→Ser substitution (#), Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000166.tif31170

[0185] Accession number 52: Mature human Fc IgG1, Cys→Ser substitution (#), Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000167.tif31170

[0186] Accession number 53: Mature human Fc IgG1, Cys→Ser substitution (#), Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000168.tif31170

[0187] Accession number 54: Mature human Fc IgG1, Cys→Ser substitution (#), Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000169.tif31170

[0188] Accession number 55: Mature human Fc IgG1, Cys→Ser substitution (#), Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000170.tif31170

[0189] Accession number 56: Mature human Fc IgG1, Cys→Ser substitution (#), Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000171.tif31170

[0190] Accession number 57: Mature human Fc IgG1, Cys→Ser substitution (#), Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000172.tif31170

[0191] Accession number 58: Mature human Fc IgG1, Cys→Ser substitution (#), Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000173.tif31170

[0192] Accession number 59: Mature human Fc IgG1, J1 is Asn or absent, J2 is Lys or absent, X4 is Asn or Ala, X5 is Leu or Asp, X6 is Gln or His, X7 is Asp or Glu, X8 is Leu or Met, X 10 is Asn or Ser TIFF2025521815000174.tif31170

[0193] Accession No. 60: Mature human Fc IgG1, DHS triple mutation (bold and underlined), J1 is Asn or absent, J2 is Lys or absent, X4 is Asn or Ala, X7 is Asp or Glu, X8 is Leu or Met TIFF2025521815000175.tif31170

[0194] Accession No. 61: Mature human Fc IgG1, DHS triple mutation (bold and underlined), X4 is Asn or Ala, X7 is Asp or Glu, X8 is Leu or Met TIFF2025521815000176.tif31170

[0195] Accession No. 62: Mature human Fc IgG1, DHS triple mutation (bold and underlined), X7 is Asp or Glu, X8 is Leu or Met TIFF2025521815000177.tif31170

[0196] Accession No. 63: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000178.tif31170

[0197] Accession No. 64: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000179.tif31170

[0198] Accession No. 65: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000180.tif31170

[0199] Accession No. 66: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000181.tif31170

[0200] Sequence number 67: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000182.tif31170

[0201] Sequence number 68: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000183.tif31170

[0202] Sequence number 69: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000184.tif31170

[0203] Sequence number 70: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000185.tif31170

[0204] Sequence number 71: Mature human Fc IgG1, Asn→Ala substitution (*), DHS triple mutation (bold and underlined), X7 is Asp or Glu, X8 is Leu or Met TIFF2025521815000186.tif31170

[0205] Sequence number 72: Mature human Fc IgG1, Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000187.tif31170

[0206] Accession No. 73: Mature human Fc IgG1, Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000188.tif31170

[0207] Accession No. 74: Mature human Fc IgG1, Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000189.tif31170

[0208] Accession No. 75: Mature human Fc IgG1, Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000190.tif31170

[0209] Accession No. 76: Mature human Fc IgG1, Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000191.tif31170

[0210] Accession No. 77: Mature human Fc IgG1, Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000192.tif31170

[0211] Accession No. 78: Mature human Fc IgG1, Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italic) TIFF2025521815000193.tif31170

[0212] Accession No. 79: Mature human Fc IgG1, Asn→Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic) TIFF2025521815000194.tif31170

[0213] Sequence number 80: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(f) (bold italic), Asn→Ala substitution (*) TIFF2025521815000195.tif31170

[0214] Sequence number 81: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(fa) (bold italic), Asn→Ala substitution (*) TIFF2025521815000196.tif31170

[0215] Sequence number 82: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(f) (bold italic), YTE triple mutation (bold and underlined), Asn→Ala substitution (*) TIFF2025521815000197.tif31170

[0216] Sequence number 83: Mature human IgG1 Fc, Cys→Ser substitution (#), allotype G1m(fa) (bold italic), YTE triple mutation (bold and underlined), Asn→Ala substitution (*) TIFF2025521815000198.tif31170

[0217] In some embodiments, the variant Fc domain comprises an amino acid substitution at position 246 (e.g., K246X, where X is any amino acid other than Lys, e.g., K246S, K246G, K246A, K246T, K246N, K246Q, K246R, K246H, K246E, or K246DC220S). In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, M252Y, S254T, and T256E (where X is not Lys). In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, V309D, Q311H, and N434S (where X is not Lys). In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, M428L, and N434S (where X is not Lys). In some embodiments, the variant Fc domain further comprises a mutation at position 220 (e.g., the C220S mutation). The amino acid substitutions are substitutions relative to the wild-type Fc monomer amino acid sequence, e.g., wild-type human IgG1 or IgG2.

[0218] In some embodiments, the variant Fc domain monomer comprises a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to any one of the sequences of SEQ ID NOs: 84-112 and 115-120 shown below. In some embodiments, the variant Fc domain monomer comprises any one of the sequences of SEQ ID NOs: 84-112 and 115-120 shown below.

[0219] In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, M252Y, S254T, and T256E (where X is not Lys). In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, V309D, Q311H, and N434S (where X is not Lys). In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, M428L, and N434S (where X is not Lys). In some embodiments, the substitution at K246X is selected from Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp. In some embodiments, the substitution at K246X is Ser.

[0220] SEQ ID NO: 84: mature human IgG1 Fc; X1 (position 201) is Asn or absent; X2 (position 220) is Cys or Ser; X3 (position 246) is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X4 (position 252) is Met or Tyr; X5 (position 254) is Ser or Thr; X6 (position 256) is Thr or Glu; X7 (position 297) is Asn or Ala; X8 (position 309) is Leu or Asp; X9 (position 311) is Gln or His; X 10 (position 356) is Asp or Glu; X 11 (position 358) is Leu or Met; X 12 (position 428) is Met or Leu; X 13 (position 434) is Asn or Ser; X 14 (position 447) is Lys or absent; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000199.tif37170

[0221] SEQ ID NO: 85: mature human IgG1 Fc; Cys→Ser substitution (#); X1 is Asn or absent; X2 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X3 is Asn or Ala; X4 is Asp or Glu; X5 is Leu or Met; X6 is Lys or absent; N-terminal Fab residue is underlined; hinge residue is italicized TIFF2025521815000200.tif31170

[0222] SEQ ID NO: 86: mature human IgG1 Fc; Cys→Ser substitution (#); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asn or Ala; X3 is Asp or Glu; X4 is Leu or Met; N-terminal Fab residue is underlined; hinge residue is italicized TIFF2025521815000201.tif31170

[0223] SEQ ID NO: 87: mature human IgG1 Fc; Cys→Ser substitution (#); Lys→Ser substitution (*); X1 is Asp or Glu; X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residue is italicized TIFF2025521815000202.tif31170

[0224] SEQ ID NO: 88: mature human IgG1 Fc, Cys→Ser substitution (#), Lys→Ser substitution (*), allotype G1m(fa) (bold italic), N-terminal Fab residue: underlined, hinge residue: italicized TIFF2025521815000203.tif31170

[0225] SEQ ID NO: 89: mature human IgG1 Fc, Cys→Ser substitution (#), Lys→Ser substitution (*), allotype G1m(f) (bold italic), N-terminal Fab residue: underlined, hinge residue: italicized TIFF2025521815000204.tif31170

[0226] Accession No. 90: Mature human IgG1 Fc; Cys→Ser substitution (#); Asn→Ala substitution (^); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asp or Glu; X3 is Leu or Met; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000205.tif31170

[0227] Accession No. 91: Mature human IgG1 Fc; Cys→Ser substitution (#); Lys→Ser substitution (*); Asn→Ala substitution (^); X1 is Asp or Glu; X2 is Leu or Met; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000206.tif31170

[0228] Accession No. 92: Mature human IgG1 Fc; Cys→Ser substitution (#); YTE triple mutation (bold and underlined); X 1 is Asn or absent; X2 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X3 is Asn or Ala; X4 is Asp or Glu; X5 is Leu or Met; X6 is Lys or absent; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000207.tif31170

[0229] Accession No. 93: Mature human IgG1 Fc; Cys→Ser substitution (#); YTE triple mutation (bold and underlined); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asn or Ala; X3 is Asp or Glu; X4 is Leu or Met; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000208.tif31170

[0230] Accession number 94: Mature human IgG1 Fc; Cys→Ser substitution (#); Lys→Ser substitution (*); YTE triple mutation (bold and underlined); X1 is Asp or Glu; X2 is Leu or Met; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000209.tif31170

[0231] Accession number 95: Mature human IgG1 Fc, Cys→Ser substitution (#), Lys→Ser substitution (*), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italicized), N-terminal Fab residues: underlined, hinge residues: italicized TIFF2025521815000210.tif31170

[0232] Accession number 96: Mature human IgG1 Fc, Cys→Ser substitution (#), Lys→Ser substitution (*), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italicized), N-terminal Fab residues: underlined, hinge residues: italicized TIFF2025521815000211.tif31170

[0233] Accession number 97: Mature human IgG1 Fc; Cys→Ser substitution (#); Asn→Ala substitution (^); YTE triple mutation (bold and underlined); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asp or Glu; X3 is Leu or Met; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000212.tif31170

[0234] Accession number 98: Mature human IgG1 Fc; Cys→Ser substitution (#); Lys→Ser substitution (*); Asn→Ala substitution (^); YTE triple mutation (bold and underlined); X1 is Asp or Glu; X2 is Leu or Met; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000213.tif31170

[0235] Accession number 99: Mature human IgG1 Fc; Cys→Ser substitution (#); DHS triple mutation (bold and underlined); X 1 is Asn or absent; X2 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X3 is Asn or Ala; X4 is Asp or Glu; X5 is Leu or Met; X6 is Lys or absent; N-terminal Fab residue is underlined; hinge residue is italicized TIFF2025521815000214.tif31170

[0236] Accession number 100: Mature human IgG1 Fc; Cys→Ser substitution (#); DHS triple mutation (bold and underlined); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asn or Ala; X3 is Asp or Glu; X4 is Leu or Met; N-terminal Fab residue is underlined; hinge residue is italicized TIFF2025521815000215.tif31170

[0237] Accession number 101: Mature human IgG1 Fc; Cys→Ser substitution (#); Lys→Ser substitution (*); DHS triple mutation (bold and underlined); X1 is Asp or Glu; X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residue is italicized TIFF2025521815000216.tif31170

[0238] Accession number 102: Mature human IgG1 Fc, Cys→Ser substitution (#), Lys→Ser substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italicized), N-terminal Fab residue: underlined, hinge residue: italicized TIFF2025521815000217.tif31170

[0239] Accession number 103: Mature human IgG1 Fc, Cys→Ser substitution (#), Lys→Ser substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italic), N-terminal Fab residue: underlined, hinge residue: italic TIFF2025521815000218.tif31170

[0240] Accession number 104: Mature human IgG1 Fc; Cys→Ser substitution (#); Asn→Ala substitution (^); DHS triple mutation (bold and underlined); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asp or Glu; X3 is Leu or Met; N-terminal Fab residue is underlined; hinge residue is italic TIFF2025521815000219.tif31170

[0241] Accession number 105: Mature human IgG1 Fc; Cys→Ser substitution (#); Lys→Ser substitution (*); Asn→Ala substitution (^); DHS triple mutation (bold and underlined); X1 is Asp or Glu; X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residue is italic TIFF2025521815000220.tif37170

[0242] Accession number 106: Mature human IgG1 Fc; Cys→Ser substitution (#); LS double mutation (bold and underlined); X 1 is Asn or absent; X2 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X3 is Asn or Ala; X4 is Asp or Glu; X5 is Leu or Met; X6 is Lys or absent; N-terminal Fab residue is underlined; hinge residue is italic TIFF2025521815000221.tif31170

[0243] Accession No. 107: Mature human IgG1 Fc; Cys→Ser substitution (#); LS double mutation (bold and underlined); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asn or Ala; X3 is Asp or Glu; X4 is Leu or Met; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000222.tif31170

[0244] Accession No. 108: Mature human IgG1 Fc; Cys→Ser substitution (#); Lys→Ser substitution (*); LS double mutation (bold and underlined); X1 is Asp or Glu; X2 is Leu or Met; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000223.tif31170

[0245] Accession No. 109: Mature human IgG1 Fc, Cys→Ser substitution (#), Lys→Ser substitution (*), LS double mutation (bold and underlined), allotype G1m(fa) (bold italicized), N-terminal Fab residues: underlined, hinge residues: italicized TIFF2025521815000224.tif31170

[0246] Accession No. 110: Mature human IgG1 Fc, Cys→Ser substitution (#), Lys→Ser substitution (*), LS double mutation (bold and underlined), allotype G1m(f) (bold italicized), N-terminal Fab residues: underlined, hinge residues: italicized TIFF2025521815000225.tif31170

[0247] Accession No. 111: Mature human IgG1 Fc; Cys→Ser substitution (#); Asn→Ala substitution (^); LS double mutation (bold and underlined); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asp or Glu; X3 is Leu or Met; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000226.tif31170

[0248] Accession No. 112: Mature human IgG1 Fc; Cys→Ser substitution (#); Lys→Ser substitution (*); Asn→Ala substitution (^); LS double mutation (bold and underlined); X1 is Asp or Glu; X2 is Leu or Met; N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000227.tif31170

[0249] Accession No. 113: Palivizumab full-length antibody; anti-RSV IgG; underlined is the leader sequence Heavy chain: TIFF2025521815000228.tif56170

[0250] Accession No. 114: Palivizumab full-length antibody; anti-RSV IgG; underlined is the leader sequence Light chain: TIFF2025521815000229.tif31170

[0251] Accession No. 115: Mature human IgG1 Fc; Cys→Ser substitution (#); Asn→Gln substitution (^); allotype G1m(fa) (bold italic); N-terminal Fab residues are underlined; hinge residues are italicized TIFF2025521815000230.tif31170

[0252] Accession No. 116: Mature human IgG1 Fc, Cys→Ser substitution (#), Asn→Gln substitution (^), allotype G1m(f) (bold italic), N-terminal Fab residues: underlined, hinge residues: italicized TIFF2025521815000231.tif31170

[0253] Accession number 117: Mature human IgG1 Fc, Cys→Ser substitution (#); Lys→Ser substitution (*); LS double mutation (bold and underlined); Asn→Gln substitution (^); allotype G1m(f) (bold italic); N-terminal Fab residue: underlined, hinge residue: italic TIFF2025521815000232.tif31170

[0254] Accession number 118: Mature human IgG1 Fc; Cys→Ser substitution (#); LS double mutation (bold and underlined); Asn→Gln substitution (^); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asp or Glu; X3 is Leu or Met; N-terminal Fab residue is underlined; hinge residue is italic TIFF2025521815000233.tif31170

[0255] Accession number 119: Mature human IgG1 Fc; Cys→Ser substitution (#); Lys→Ser substitution (*); Asn→Gln substitution (^); LS double mutation (bold and underlined); X1 is Asp or Glu; X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residue is italic TIFF2025521815000234.tif31170

[0256] Accession number 120: Mature human IgG1 Fc, Cys→Ser substitution (#), Asn→Gln substitution (^), allotype G1m(f) (bold italic), N-terminal Fab residue: underlined, hinge residue: italic TIFF2025521815000235.tif31170

[0257] As defined herein, the Fc domain is C HIt includes two Fc domain monomers dimerized by the interaction between the constant domains of 3 antibodies, and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domain monomers. The Fc domain forms the minimum structure that binds to Fc receptors, such as Fc-gamma receptors (i.e., Fcγ receptors (FcγR)), Fc-alpha receptors (i.e., Fcα receptors (FcαR)), Fc-epsilon receptors (i.e., Fcε receptors (FcεR)), and / or neonatal Fc receptor (FcRn). In some embodiments, the Fc domain of the present disclosure binds to Fcγ receptors (e.g., FcRn, FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b)), and / or FcγRIV and / or neonatal Fc receptor (FcRn).

[0258] In some embodiments, the Fc domain monomer or Fc domain of the present disclosure is an aglycosylated Fc domain monomer or Fc domain (e.g., an Fc domain monomer or Fc domain that maintains engagement with an Fc receptor (e.g., FcRn)). For example, the Fc domain is an aglycosylated IgG1 variant that maintains engagement with an Fc receptor (e.g., IgG1 having amino acid substitutions at N297 and / or T299 of the glycosylation motif). Exemplary aglycosylated Fc domains and methods for making aglycosylated Fc domains are known in the art, as described, for example, in Sazinsky S.L. et al., Aglycosylated immunoglobulin G1 variants productively engage activating Fc receptors, PNAS, 2008, 105(51):20167-20172 (incorporated herein by reference in its entirety).

[0259] In some embodiments, the Fc domain or Fc domain monomer of the present disclosure is engineered to enhance binding to the neonatal Fc receptor (FcRn). For example, the Fc domain can include a triple mutation corresponding to M252Y / S254T / T256E (YTE) (e.g., IgG1 having the YTE mutation (e.g., human or humanized IgG1)). The Fc domain can include a single variant corresponding to N434H (e.g., IgG1 having the N434H mutation (e.g., human or humanized IgG1)). The Fc domain can include a single variant corresponding to C220S (e.g., IgG1 having the C220S mutation (e.g., human or humanized IgG1)). The Fc domain can include a quadruple variant corresponding to C220S / L309D / Q311H / N434S (CDHS) (e.g., IgG1 having the DHS mutation (e.g., human or humanized IgG1)). The Fc domain can include a triple variant corresponding to L309D / Q311H / N434S (DHS) (e.g., IgG1 having the DHS mutation (e.g., human or humanized IgG1)). The Fc domain can include one or more combinations of the above-described mutations that enhance binding to FcRn. By enhancing binding to FcRn, the half-life of the Fc domain-containing conjugate can be increased. For example, by incorporating one or more amino acid mutations that increase binding to FcRn (e.g., the YTE mutation, the LS mutation, or the N434H mutation), the half-life of the conjugate can be increased by 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to a conjugate having a corresponding Fc domain without the mutation that enhances FcRn binding.Exemplary Fc domains with enhanced binding to FcRN, and methods for making Fc domains with enhanced binding to FcRN are known in the art, for example, as described in Maeda, A. et al., Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody, MABS, 2017, 9(5):844-853 (incorporated herein by reference in its entirety). As used herein, an amino acid "corresponding" to a particular amino acid residue (e.g., of a particular SEQ ID NO.) is to be understood to include any amino acid residue that would be understood by one of ordinary skill in the art to align with the particular residue (e.g., of a particular sequence). For example, any one of SEQ ID NOs: 1-112 and 115-120 can be mutated to include the YTE mutation, the LS mutation, and / or the N434H mutation by mutating the "corresponding residue" of the amino acid sequence.

[0260] In some embodiments, the Fc domain or Fc domain monomer of the present disclosure has the sequence of any one of SEQ ID NOs: 1-112 and 115-120, and may further include additional amino acids (Xaa)x at the N-terminus and / or additional amino acids (Xaa)z at the C-terminus, where Xaa is any amino acid and x and z are integers of zero or more, generally less than 100, preferably less than 10, more preferably 0, 1, 2, 3, 4, or 5. For example, the additional amino acid can be a single amino acid on the C-terminus corresponding to Lys330 of IgG1.

[0261] In some embodiments, the Fc domain monomer comprises less than about 300 amino acid residues (e.g., less than about 300, less than about 295, less than about 290, less than about 285, less than about 280, less than about 275, less than about 270, less than about 265, less than about 260, less than about 255, less than about 250, less than about 245, less than about 240, less than about 235, less than about 230, less than about 225, or less than about 220 amino acid residues). In some embodiments, the Fc domain monomer is less than about 40 kDa (e.g., less than about 35 kDa, less than about 30 kDa, less than about 25 kDa).

[0262] In some embodiments, the Fc domain monomer comprises at least 200 amino acid residues (e.g., at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 amino acid residues). In some embodiments, the Fc domain monomer is at least 20 kDa (e.g., at least 25 kDa, at least 30 kDa, or at least 35 kDa).

[0263] In some embodiments, the Fc domain monomer comprises 200 - 400 amino acid residues (e.g., 200 - 250, 250 - 300, 300 - 350, 350 - 400, 200 - 300, 250 - 350, or 300 - 400 amino acid residues). In some embodiments, the Fc domain monomer is 20 - 40 kDa (e.g., 20 - 25 kDa, 25 - 30 kDa, 35 - 40 kDa, 20 - 30 kDa, 25 - 35 kDa, or 30 - 40 kDa).

[0264] In some embodiments, the Fc domain monomer comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 98%) identical to any one of the sequences of SEQ ID NOs: 1 - 112 and 115 - 120 or a region thereof. In some embodiments, the Fc domain monomer comprises any one of the amino acid sequences of SEQ ID NOs: 1 - 112 and 115 - 120, or a region thereof.

[0265] In some embodiments, the Fc domain monomer comprises any one of the regions of SEQ ID NOs: 1-112 and 115-120, which regions include positions 220, 252, 254, and 256. In some embodiments, the region comprises at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 110 amino acid residues, at least 120 amino acid residues, at least 130 amino acid residues, at least 140 amino acid residues, at least 150 amino acid residues, at least 160 amino acid residues, at least 170 amino acid residues, at least 180 amino acid residues, at least 190 amino acid residues, or at least 200 amino acid residues.

[0266] Activation of immune cells Fcγ receptors (FcγR) bind to the Fc portion of immunoglobulin G (IgG) and play important roles in the activation and regulation of immunity. For example, the IgG Fc domain within an immune complex (Ic) engages with FcγR with high avidity, inducing a signaling cascade that regulates the activation of immune cells. The human FcγR family includes several activating receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) and one inhibitory receptor (FcγRIIb). FcγR signaling is mediated by intracellular domains that include immunoreceptor tyrosine-based activation motifs (ITAM) for activating FcγR and immunoreceptor tyrosine-based inhibitory motifs (ITIM) for the inhibitory receptor FcγRIIb. In some embodiments, FcγR binding by the Fc domain results in ITAM phosphorylation by Src family kinases, which activates Syk family kinases and induces downstream signaling networks including the PI3K and Ras pathways.

[0267] In the fusion protein or conjugate described in this specification, the Fc domain portion of the fusion protein or conjugate binds to FcγRs (e.g., FcRn, FcγRI, FcγIIa, FcγIIc, FcγIIIa, and FcγIIIb) on immune cells and activates phagocytosis and effector functions (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC)). Examples of immune cells that can be activated by the conjugates described in this specification include, but are not limited to, macrophages, neutrophils, eosinophils, basophils, lymphocytes, follicular dendritic cells, natural killer cells, and mast cells.

[0268] Tissue distribution After entering the systemic circulation, the therapeutic agent is distributed to the tissues of the body. Distribution is generally non-uniform because of differences in blood perfusion, tissue binding, local pH, and cell membrane permeability. The rate at which a drug enters a tissue depends on the rate of blood flow to the tissue, tissue mass, and partitioning characteristics between blood and tissue. The distribution equilibrium between blood and tissue (when the rates of entry and exit are the same) is reached more rapidly in highly vascularized regions, unless diffusion across the cell membrane is the rate-limiting step. Size, shape, charge, target binding, FcRn and target binding mechanisms, route of administration, and formulation affect tissue distribution.

[0269] In some cases, the fusion proteins described in this specification can be optimized to distribute to lung tissue. In some cases, the fusion protein has a ratio of distribution concentration in epithelial lining fluid to the concentration of the fusion protein in plasma of at least 30% within 2 hours after administration. In certain embodiments, the ratio of concentrations is at least 45% within 2 hours after administration. In some embodiments, the ratio of concentrations is at least 55% within 2 hours after administration. Specifically, the ratio of concentrations within 2 hours after administration is at least 60%.

[0270] III. Linker "Linker" refers to a covalent bond or connection between two or more components within the conjugate described herein (e.g., between two CD73 inhibitors within the conjugate described herein, between a CD73 inhibitor and an Fc domain within the conjugate described herein, and between a dimer of two CD73 inhibitors and an Fc domain within the conjugate described herein).

[0271] Linker within a conjugate having an Fc domain covalently bound to a monomer of a CD73 inhibitor In a conjugate comprising an Fc domain monomer or an Fc domain covalently bound to one or more monomers of a CD73 inhibitor as described herein, the linker within the conjugate can be a bivalent structure having two arms. One arm in the bivalent linker can bind to a monomer of the CD73 inhibitor, and the other arm can bind to the Fc domain monomer or the Fc domain. In some embodiments, one or more monomers of the CD73 inhibitor within the conjugate described herein can each independently bind to an atom within the Fc domain monomer or the Fc domain.

[0272] In some embodiments, the linker is represented by the following formula. J 1 -(Q 1 ) g -(T 1 ) h -(Q 2 ) i -(T 2 ) j -(Q 3 ) k -(T 3 ) l -(Q 4 ) m -(T 4 ) n -(Q 5 ) o -J 2 Wherein J 1 is a bond bound to A1, J 2is a bond attached to E or a functional group capable of reacting with a functional group conjugated with E (e.g., maleimide and cysteine, amine and activated carboxylic acid (e.g., carboxylic acid activated by tetrafluorophenyl or trifluorophenol), thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrazine), Q 1 , Q 2 , Q 3 , Q 4 , and Q 5 each of which is independently optionally substituted C1-C40 alkylene, optionally substituted C1-C40 heteroalkylene, optionally substituted C1-C40 alkoxylene, 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 C2-C 20 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, T 1 , T 2 , T 3 , T 4 each of which is independently O, S, NR i , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, imino, or oximo, 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 C2-C20 A heterocycloalkyl, an optionally substituted C4-C20 cycloalkenyl, an optionally substituted C4-C20 heterocycloalkenyl, an optionally substituted C8-C20 cycloalkynyl, an optionally substituted C8-C20 heterocycloalkynyl, an optionally substituted C5-C15 aryl, or an optionally substituted C2-C15 heteroaryl, wherein each of g, h, i, j, k, l, m, n, and o is independently 0, 1, or 2. In some embodiments, each of g, h, i, j, k, l, m, n, and o is independently 0 or 1.

[0273] In some embodiments, being optionally substituted includes substitution with polyethylene glycol (PEG). PEG has a repeating unit structure (-CH2CH2O-) n wherein n is an integer from 2 to 100. Polyethylene glycol is PEG2 to PEG 100 (e.g., PEG2, PEG3, PEG4, PEG5, PEG5-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 ) can be selected.

[0274] In some embodiments, J 2 can have an Fc domain monomer or two binding sites to the Fc domain (e.g., two J 2 ).

[0275] The linker in a conjugate having an Fc domain covalently bound to a dimer of a CD73 inhibitor In a conjugate comprising an Fc domain monomer or an Fc domain covalently bound to one or more dimers of a CD73 inhibitor as described herein, the linker within the conjugate can have a branched structure. As further described herein, the linker within the conjugate described herein can have a multivalent structure, e.g., a divalent or trivalent structure having two or three arms, respectively. In some embodiments where the linker has three arms, two of the arms can bind to the first and second CD73 inhibitors, and the third arm can bind to the Fc domain monomer or the Fc domain. In some embodiments where the linker has two arms, one arm can bind to the Fc domain and the other arm can bind to one of the two CD73 inhibitors. In other embodiments, a linker having two arms can be used to bind two CD73 inhibitors on a conjugate comprising an Fc domain covalently bound to one or more dimers of a CD73 inhibitor.

[0276] In some embodiments, the linker within a conjugate having an Fc domain covalently bound to one or more dimers of a CD73 inhibitor is represented by formula (D-L-I).

Chemical formula

[0277] In some embodiments, the optional substitution includes substitution with PEG. PEG has a repeating unit structure (-CH2CH2O-) n wherein n is an integer from 2 to 100. Polyethylene glycol is PEG2 to PEG100 (e.g., PEG2, PEG3, PEG4, PEG5, PEG5-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 ) may be selected.

[0278] In some embodiments, L C can have two binding points with the Fc domain (e.g., two G C2 ).

[0279] In some embodiments, L comprises a polyethylene glycol (PEG) linker. The PEG linker comprises a linker having a repeating unit structure (-CH2CH2O-) n wherein n is an integer from 2 to 100. The polyethylene glycol linker can covalently bond the CD73 inhibitor and E (e.g., in the case of the conjugate of formula (M-I)). The polyethylene glycol linker can covalently bond the first CD73 inhibitor and the second CD73 inhibitor (e.g., in the case of the conjugate of formula (D-I)). The polyethylene glycol linker can covalently bond the CD73 inhibitor dimer and E (e.g., in the case of the conjugate of formula (D-I)). The polyethylene glycol linker is PEG2 to PEG 100 (e.g., PEG2, PEG3, PEG4, PEG5, PEG5-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 ) can be selected from. In some embodiments, L c includes a PEG linker, and L C is covalently bonded to each of Q i and E.

[0280] Linker In some embodiments, the linker provides space, rigidity, and / or flexibility between the CD73 inhibitor and the Fc domain monomer or Fc domain in the conjugate described herein, or between two CD73 inhibitors in the conjugate described herein. In some embodiments, the linker can be a bond resulting from a linkage, e.g., a covalent bond, e.g., 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 resulting from a chemical reaction (e.g., chemical conjugation). In some embodiments, the linker (e.g., L as shown in formula (D-I) or (M-I)) comprises 250 atoms or fewer (e.g., 1 to 2, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 110, 1 to 120, 1 to 130, 1 to 140, 1 to 150, 1 to 160, 1 to 170, 1 to 180, 1 to 190, 1 to 200, 1 to 210, 1 to 220, 1 to 230, 1 to 240, or 1 to 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)).In some embodiments, the linker (L) comprises 250 or fewer non-hydrogen atoms (e.g., 1 to 2, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 110, 1 to 120, 1 to 130, 1 to 140, 1 to 150, 1 to 160, 1 to 170, 1 to 180, 1 to 190, 1 to 200, 1 to 210, 1 to 220, 1 to 230, 1 to 240, or 1 to 250 non-hydrogen atoms (plural possible); 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 (plural possible)). In some embodiments, the backbone of the linker (L) comprises 250 or fewer atoms (e.g., 1 to 2, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 110, 1 to 120, 1 to 130, 1 to 140, 1 to 150, 1 to 160, 1 to 170, 1 to 180, 1 to 190, 1 to 200, 1 to 210, 1 to 220, 1 to 230, 1 to 240, or 1 to 250 atoms (plural possible); 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 (plural possible)). The "backbone" of the linker refers to the atoms within the linker that together form the shortest path from one part of the conjugate to another. The atoms within the backbone of the linker are directly involved in the connection between one part and another part of the conjugate.For example, a hydrogen atom bonded to a carbon within the backbone of a linker is not considered to be directly involved in the bonding between one part and another part of the conjugate.

[0281] Molecules that can be used to make a linker (L) include at least two functional groups (e.g., two carboxylic acid groups). In some embodiments of a trivalent linker, two arms of the linker can include two dicarboxylic acids, in which case the first carboxylic acid can form a covalent bond with a first CD73 inhibitor within the conjugate, the second carboxylic acid can form a covalent bond with a second CD73 inhibitor within the conjugate, and the third arm of the linker can form a covalent bond (e.g., a C—O bond) with an Fc domain monomer or an Fc domain within the conjugate. In some embodiments of a bivalent linker, the bivalent linker can include two carboxylic acids, in which case the first carboxylic acid can form a covalent bond with one component (e.g., a CD73 inhibitor) within the conjugate, and the second carboxylic acid can form a covalent bond (e.g., a C—S bond or a C—N bond) with another component (e.g., an Fc domain monomer or an Fc domain) within the conjugate.

[0282] In some embodiments, a dicarboxylic acid molecule can be used as a linker (e.g., a dicarboxylic acid linker). For example, in a conjugate comprising an Fc domain monomer covalently bound to one or more dimers of a CD73 inhibitor, the first carboxylic acid of the dicarboxylic acid molecule can form a covalent bond with a hydroxyl or amine group of the first CD73 inhibitor, and the second carboxylic acid can form a covalent bond with a hydroxyl or amine group of the second CD73 inhibitor.

[0283] In some embodiments, a dicarboxylic acid molecule (e.g., those described herein) may be further functionalized to include one or more additional functional groups. The dicarboxylic acid can be further functionalized to provide a point of attachment to an Fc domain monomer or an Fc domain, for example, via a linker (such as a PEG linker).

[0284] In some embodiments, when the CD73 inhibitor is bound to the Fc domain monomer or the Fc domain, the linker can include a moiety comprising a carboxylic acid moiety and an amino moiety separated by 1 to 25 atoms.

[0285] In some embodiments, the linker can include a diamino moiety (e.g., as described herein) and can be further functionalized to include one or more additional functional groups. Such a diamino linker can be further functionalized to provide a point of attachment to the Fc domain monomer or the Fc domain, for example, via a linker such as a PEG linker.

[0286] In some embodiments, a linker can be formed using a molecule comprising an azide group, in which case the azide group can undergo an addition cyclization reaction with an alkyne to form a 1,2,3-triazole bond. In some embodiments, a linker can be formed using a molecule comprising an alkyne group, in which case the alkyne group can undergo an addition cyclization reaction with an azide to form a 1,2,3-triazole bond. In some embodiments, a linker can be formed using a molecule comprising a maleimide group, in which case the maleimide group can react with a cysteine to form a C-S bond. In some embodiments, a linker can be formed using a molecule comprising one or more haloalkyl groups, in which case the haloalkyl group can form a covalent bond (e.g., C-N and C-O bonds) with the CD73 inhibitor.

[0287] In some embodiments, the linker (L) can include a synthetic group derived from, for example, a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). In some embodiments, the linker can include one or more amino acid residues. In some embodiments, the linker can be an amino acid sequence (e.g., a 1-25 amino acid, 1-10 amino acid, 1-9 amino acid, 1-8 amino acid, 1-7 amino acid, 1-6 amino acid, 1-5 amino acid, 1-4 amino acid, 1-3 amino acid, 1-2 amino acid, or 1 amino acid sequence). In some embodiments, the linker (L) is one or more optionally substituted C1-C40 alkylene, optionally substituted C1-C40 heteroalkylene (e.g., PEG units), optionally substituted C1-C40 alkoxylene, 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), optionally substituted C2-C 20 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 (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 C2-C 20heterocycloalkyl, 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.

[0288] Conjugation chemistry The CD73 inhibitor monomer or dimer can be conjugated to the Fc domain monomer or Fc domain by any standard conjugation chemistry known to those skilled in the art, for example, via a linker. For example, the following conjugation chemistries are particularly contemplated for conjugating a PEG linker (e.g., a functionalized PEG linker) to the Fc domain monomer or Fc domain.

[0289] The covalent bond of two or more components within a conjugate using a linker can be achieved using well-known organic chemical synthesis techniques and methods. Complementary functional groups on two components can react with each other to form a covalent bond. Examples of complementary reactive functional groups include, but are not limited to, for example, 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 with a polypeptide can be achieved using techniques known in the art. Exemplary techniques for site-specific conjugation of a small molecule to the Fc domain are shown in Agarwall.P., et al. Bioconjugate Chem. 26:176-192 (2015).

[0290] Other examples of functional groups capable of reacting with an amino group include, for example, alkylating agents and acylating agents. Representative alkylating agents include: (i) an α-haloacetyl group (e.g., XCH2CO- where X = Br, Cl, or I); (ii) an N-maleimide group (capable of reacting with an amino group by a Michael-type reaction or by acylation through addition to a cyclic carbonyl group); (iii) an aryl halide (e.g., a nitrohaloaromatic group); (iv) an alkyl halide; (v) an aldehyde or ketone capable of forming a Schiff base with an amino group; (vi) an epoxide (e.g., epichlorohydrin and bisoxirane capable of reacting with an amino group, a sulfhydryl group, or a phenolic hydroxyl group); (vii) a chlorine-containing s-triazine (reactive towards nucleophilic groups such as amino groups, sulfhydryl groups, and hydroxyl groups); (viii) an aziridine (reactive towards nucleophilic groups such as amino groups upon ring opening); (ix) diethyl squarate; and (x) an α-haloalkyl ether.

[0291] Examples of amino-reactive acylating groups include, for example: (i) isocyanates and isothiocyanates; (ii) sulfonyl chlorides; (iii) acid halides; (iv) active esters (e.g., nitrophenyl esters or N-hydroxysuccinimidyl esters); (v) acid anhydrides (e.g., mixed, symmetric, or N-carboxy anhydrides); (vi) acyl azides; and (vii) imido esters. Aldehydes and ketones can react with amines to form Schiff bases, which can be stabilized by reductive amination.

[0292] It will be appreciated that a particular functional group can be converted to another functional group prior to the reaction, for example, to impart additional reactivity or selectivity. Examples of methods useful for this purpose include the conversion of an amine to a carboxyl using a reagent such as a dicarboxylic acid anhydride; the conversion of an amine to a thiol using a reagent such as N-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, or a thiol-containing succinimidyl derivative; the conversion of a thiol to a carboxyl using a reagent such as an α-haloacetate; the conversion of a thiol to an amine using a reagent such as ethyleneimine or 2-bromoethylamine; the conversion of a carboxyl to an amine using a reagent such as a carbodiimide, followed by conversion to a diamine; the conversion of an alcohol to a thiol using a reagent such as tosyl chloride, followed by transesterification with thioacetate and hydrolysis to a thiol with sodium acetate.

[0293] In some embodiments, the linker of the present disclosure (e.g., L, such as the L of D-L-I) Cbinds to E (e.g., the Fc domain) by (e.g., by any method described herein). In a preferred embodiment of the present disclosure, the linker is (a) a thiourea bond with a lysine of E (i.e., -NH(C=S)NH-); (b) a carbamate bond with a lysine of E (i.e., -NH(C=O)-O); (c) an amine bond by reductive amination between lysine and E (i.e., -NHCH2 between lysine and E); (d) an amide with a lysine of E (i.e., -NH-(C=O)CH2); I a cysteine-maleimide conjugate between a maleimide of the linker and a cysteine of E; (f) an amine bond by reductive amination between the linker and a carbohydrate of E (e.g., an Fc domain monomer or a glycosyl group of the Fc domain) (i.e., -NHCH2); (g) a re-crosslinked cysteine conjugate in which the linker is conjugated to two cysteines of E; (h) an oxime bond between the linker and a carbohydrate of E (e.g., an Fc domain monomer or a glycosyl group of the Fc domain); (i) an oxime bond between the linker and an amino acid residue of E; (j) an azide bond between the linker and E; (k) direct acylation of the linker with E; or (l) a thioether bond between the linker and E.

[0294] In some embodiments, the linker binds to E and the bond comprises the structure -NH(C=NH)X-, where X is O, HN, or a bond. In some embodiments, the linker binds to E and the bond between the remainder of the linker and E comprises the structure -NH(C=O)NH-.

[0295] In some embodiments, the linker binds to E and the bond comprises the structure -R9OR9C(=O)NH-, where R9 is H, optionally substituted C1-C 20 alkyl, optionally substituted C3-C 20 cycloalkyl, optionally substituted C2-C 20 heterocycloalkyl, optionally substituted C5-C 15 aryl, or optionally substituted C2-C 15It is heteroaryl. In some embodiments, the linker conjugates with E, and the bond between the remainder of the linker and E contains the structure -CH2OCH2C(=O)NH-.

[0296] Exemplary conjugation strategies (e.g., methods for conjugating monomers or dimers of CD73 inhibitors to E (e.g., via a linker)) are further described in the examples.

[0297] In some embodiments, the 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., azido-PEG2-PEG 40 -NHS ester))) conjugates with E, and T (e.g., the drug-antibody ratio or DAR) 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 these cases, E-(PEG2-PEG 40 )-azide can react with Int having a terminal alkyne linker (e.g., L, e.g., L of D-L-I C ) by click conjugation. During click conjugation, the azide (e.g., Fc-(PEG2-PEG 40 )-azide) and the alkyne (e.g., the terminal alkyne linker (e.g., L, e.g., L of D-L-I CA five-membered heteroatomic ring is formed by a copper-catalyzed reaction with Int having ( ) and Int having a terminal azide. In some embodiments, the linker that conjugates with E is a terminal alkyne and conjugates with Int having a terminal azide. E-(PEG2-PEG 40 Exemplary preparations in the preparation of )-azide are described in the examples. Those skilled in the art will readily understand the final product from click chemistry conjugation.

[0298] Exemplary binding strategies are further described herein.

[0299] IV. Treatment Methods The present disclosure provides for the use of the conjugates and pharmaceutical compositions described herein in the treatment of diseases associated with CD73 dysregulation or overexpression (e.g., cancer, fibrosis, or viral infections).

[0300] Cancer The conjugates and pharmaceutical compositions described herein can be used for the treatment of cancer in a subject. In some embodiments, the cancer overexpresses or is known to overexpress CD73 compared to non-cancer cells of the same tissue type. In some embodiments, the subject is determined to have a cancer that overexpresses CD73 compared to non-cancer cells of the same tissue type. In some embodiments, the method further comprises determining whether the cancer overexpresses CD73 compared to non-cancer cells of the same tissue type and administering the conjugate only if the cancer overexpresses CD73.

[0301] In some embodiments, the cancer is selected from lung cancer, optionally non-small cell lung cancer or small cell lung cancer, head and neck cancer, optionally squamous cell carcinoma, renal cell carcinoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, urothelial cancer, cholangiocarcinoma, endometrial cancer, melanoma, or esophageal cancer. In some embodiments, the cancer is a solid tumor.

[0302] In some embodiments, the method further comprises administering an immune checkpoint inhibitor to the subject. In some embodiments, the immune checkpoint inhibitor is an inhibitor of any one or more of the following immune checkpoint targets: CTLA-4, PD-1, PD-L1, LAG-3, B7.1, B7-H3, B7-H4, TIM3, VISTA, CD137, OX-40, CD40, CD27, CCR4, GITR, NKG2D, and KIR. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody selected from one or more of the following: anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG3 antibody, anti-B7.1 antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-TIM3 antibody, anti-VISTA antibody, anti-CD137 antibody, anti-OX40 antibody, anti-CD40 antibody, anti-CD27 antibody, anti-CCR4 antibody, anti-GITR antibody, anti-NKG2D antibody, and anti-KIR antibody. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 monoclonal antibody.

[0303] Approved or investigational immune checkpoint inhibitors include, but are not limited to, YERVOY® (ipilimumab), OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab), tremelimumab, galiximab, MDX-1106, BMS-936558, MEDI4736, MPDL3280A, MEDI6469, BMS-986016, BMS-663513, PF-05082566, IPH2101, KW-0761, CDX-1127, CP-870, CP-893, GSK2831781, MSB0010718C, MK3475, CT-011, AMP-224, MDX-1105, IMP321, and MGA271, as well as numerous other antibodies or fusion proteins directed to the immune checkpoint proteins described herein.

[0304] In some embodiments, the method comprises administering to the subject (1) the conjugate described herein and (2) an immune checkpoint inhibitor. In some embodiments, the conjugate described herein is administered first, and then the immune checkpoint inhibitor is administered alone. In some embodiments, the immune checkpoint inhibitor is administered first, and then the conjugate described herein is administered alone. In some embodiments, the conjugate described herein and the immune checkpoint inhibitor are administered substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, when the conjugate described herein and the immune checkpoint inhibitor are administered together (e.g., substantially simultaneously in the same or separate pharmaceutical compositions or separately in the same treatment regimen), the tumor growth inhibition of each of the conjugate and the immune checkpoint inhibitor can be greater (e.g., can occur at lower concentrations) than the tumor growth inhibition of each of the conjugate and the immune checkpoint inhibitor when each is used alone in a treatment regimen.

[0305] Viral infection The conjugates and pharmaceutical compositions described herein can be used for the treatment of viral infections in a subject. Also, the conjugates and pharmaceutical compositions described herein can be used to prevent viral infections in subjects that are susceptible to viral infections or have an increased risk of contracting a viral infection (e.g., subjects who are hospitalized, immunocompromised, preparing for surgery, recently had surgery, or are receiving medications (e.g., chemoradiotherapy) that affect the immune system).

[0306] In some embodiments, the viral infection is a beta coronavirus infection. In some embodiments, the beta coronavirus is SARS-CoV-2. In some embodiments, SARS-CoV-2 is an alpha, delta, or omicron variant. In some embodiments, SARS-CoV-2 is an omicron variant. In some embodiments, the omicron variant is a BA.1, BA.2, BA.3, BA.4, or BA.5 lineage.

[0307] In some embodiments, the method further comprises administering an antiviral agent or an antiviral vaccine to the subject. In some embodiments, the method comprises administering (1) the conjugate described herein, and (2) an antiviral agent or an antiviral vaccine to the subject. In some embodiments, the conjugate described herein is administered first, and then the antiviral agent or the antiviral vaccine is administered alone. In some embodiments, the antiviral agent or the antiviral vaccine is administered first, and then the conjugate described herein is administered alone. In some embodiments, the conjugate described herein and the antiviral agent or the antiviral vaccine are administered substantially simultaneously (e.g., in the same pharmaceutical composition, or in separate pharmaceutical compositions). In some embodiments, when the conjugate described herein and the antiviral agent or the antiviral vaccine are administered together (e.g., substantially simultaneously in the same or separate pharmaceutical compositions, or separately in the same treatment regimen), the inhibition of viral replication of each of the conjugate and the antiviral agent or the antiviral vaccine can be greater (e.g., can occur at a lower concentration) than the inhibition of viral replication of each of the conjugate and the antiviral agent or the antiviral vaccine when each is used alone in the treatment regimen.

[0308] Fibrosis The conjugate and pharmaceutical composition described herein can be used for the treatment or prevention of fibrosis in a subject.

[0309] In some embodiments, the fibrosis is pulmonary fibrosis, dermal fibrosis, renal fibrosis, hepatic fibrosis, cardiac fibrosis, or systemic sclerosis. In some embodiments, the fibrosis is pulmonary fibrosis. In some embodiments, the pulmonary fibrosis is associated with a viral infection (e.g., associated with SARS-CoV-2 infection), drug-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, pneumoconiosis, interstitial lung disease, sarcoidosis, silicosis, or systemic sclerosis.

[0310] In some embodiments, the fibrosis is selected from the group consisting of scleroderma, cystic fibrosis, cirrhosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, Dupuytren's contracture, keloid, chronic kidney disease, chronic graft rejection, scar, wound healing, postoperative adhesions, reactive fibrosis, polymyositis, ANCA vasculitis, Behçet's disease, antiphospholipid syndrome, relapsing polychondritis, familial Mediterranean fever, giant cell arteritis, Graves ophthalmopathy, discoid lupus erythematosus, pemphigoid, bullous pemphigoid, hidradenitis suppurativa, sarcoidosis, bronchiolitis obliterans, primary sclerosing cholangitis, primary biliary cirrhosis, and organ fibrosis (e.g., dermal fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, or cardiac fibrosis). In some embodiments, the fibrosis is scleroderma (e.g., systemic sclerosis, localized scleroderma, or sine scleroderma). In some embodiments, the fibrosis is organ fibrosis (e.g., dermal fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, or cardiac fibrosis). In some embodiments, the fibrosis is cystic fibrosis.

[0311] Treatment of fibrosis can be evaluated by suitable methods known to those skilled in the art, including improvement, alleviation, or delay in progression of one or more symptoms associated with the specific fibrosis being treated.

[0312] V. Pharmaceutical Compositions The conjugates described herein can be formulated into pharmaceutical compositions for use in the methods described herein. In some embodiments, the conjugates described herein can be formulated into pharmaceutical compositions alone. In some embodiments, the conjugates described herein can be formulated in combination with a second therapeutic agent in a pharmaceutical composition. In some embodiments, the conjugates described herein can be administered in combination with a second therapeutic agent (e.g., sequentially or simultaneously) as part of a dosing regimen. In some embodiments, the pharmaceutical composition comprises a conjugate described herein and a pharmaceutically acceptable carrier and excipient.

[0313] The acceptable carriers and excipients in the pharmaceutical composition are non-toxic to the recipient at the dosages and concentrations used. Acceptable carriers and excipients include buffers (e.g., phosphate, citrate, HEPES, and TAE), antioxidants (e.g., ascorbic acid and methionine), preservatives (e.g., hexamethonium chloride, octadecyl dimethyl benzyl ammonium chloride, resorcinol, and benzalkonium chloride), proteins (e.g., human serum albumin, gelatin, dextran, and immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), amino acid residues (e.g., glycine, glutamine, histidine, and lysine), and carbohydrates (e.g., glucose, mannose, sucrose, and sorbitol).

[0314] Examples of other excipients include, but are not limited to, antiadherents, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers (diluents), film formers or coatings, flavorants, fragrances, fluidizing agents (flow enhancers), lubricants, adsorbents, suspending or dispersing agents, or sweeteners. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0315] The conjugates herein can have ionizable groups such that they can be prepared as pharmaceutically acceptable salts. Such salts can be acid addition salts with inorganic or organic acids, or, in the case of the acidic forms of the conjugates herein, salts can be prepared from inorganic or organic bases. In many cases, the conjugates are prepared or used as pharmaceutically acceptable salts that are prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art and include, for example, hydrochloric acid, sulfuric acid, hydrobromic acid, acetic acid, lactic acid, citric acid, or tartaric acid for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, etc. for forming basic salts. Methods for preparing appropriate salts are well established in the art.

[0316] Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate. Representative alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, 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).

[0317] Depending on the route of administration and dosage, the conjugates or pharmaceutical compositions thereof described herein used in the methods described herein are formulated into suitable pharmaceutical compositions to enable easy delivery. The conjugate or its pharmaceutical composition can be administered intramuscularly, intravenously (e.g., as a sterile solution suitable for intravenous use and in a solvent system), intradermally, intraarterially, intraperitoneally, intralesionally, intracerebrally, intraarticularly, intraprostatically, intrasclerally, intratracheally, intranasally, intravitreally, intravaginally, rectally, topically, intratumorally, transperitoneally, subcutaneously, subconjunctivally, intracellularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally (e.g., as tablets, capsules, caplets, gelcaps, or syrups), topically (e.g., as creams, gels, lotions, or ointments), locally, by inhalation, injection, or infusion (e.g., sustained infusion, local perfusion directly immersing target cells, catheters, washes, creams, or lipid compositions). Depending on the route of administration, the conjugate or its pharmaceutical composition herein can be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels (including hydrogels), pastes, ointments, creams, plasters, lotions, osmotic delivery devices, suppositories, enemas, injection solutions, implants, sprays, formulations suitable for iontophoresis delivery, or aerosols. The compositions can be formulated according to conventional pharmaceutical practices.

[0318] The conjugates described herein can be formulated by a variety of methods known in the art. For use in the treatment of human and animal subjects, the conjugates described herein can be formulated as a pharmaceutical composition or a veterinary composition. Depending on the subject to be treated (e.g., human), the mode of administration, and the type of treatment desired (e.g., prophylactic or therapeutic), the conjugates described herein are formulated in a manner consistent with these parameters. An overview of such techniques 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.

[0319] Formulations can be prepared in a manner suitable for systemic or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous, or subcutaneous injection), or those prepared for transdermal, transmucosal, or oral administration. Formulations generally include a diluent and, optionally, adjuvants, buffers, and preservatives. The conjugates can also be administered as liposomal compositions or microemulsions. Systemic administration can also include relatively non-invasive methods (e.g., 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 will be understood by those skilled in the art.

[0320] The pharmaceutical composition can be administered parenterally in the form of a preparation for injection. The pharmaceutical composition for injection can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. The preparation can be prepared in a solid form suitable for dissolving or suspending in a liquid before injection, or as an emulsion. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagle's Medium (α-MEM), F-12 medium). Such injection compositions can also include non-toxic auxiliary substances (e.g., wetting agents or emulsifiers, pH buffering agents, e.g., sodium acetate and sorbitan monolaurate). Methods of formulation are known in the art, see, for example, Pharmaceutical Preformulation and Formulation, 2nd Edition, M. Gibson, Taylor & Francis Group, CRC Press (2009).

[0321] The pharmaceutical composition can be prepared in the form of an oral preparation. Examples of preparations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (such as sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch (including potato starch), calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (such as cellulose derivatives including microcrystalline cellulose, starch including potato starch, croscarmellose sodium, alginate, or alginic acid); binders (such as sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethyl cellulose, ethyl cellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, fluidizing agents, and anti-adhesion agents (such as magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Also, the preparation for oral use can be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (such as potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with a water or oil medium (such as peanut oil, liquid paraffin, or olive oil). Powders, granules, and pellets can be prepared in the usual manner using the above-described components under tablets and capsules, for example, using a mixer, a fluidized bed apparatus, or a spray dryer.

[0322] Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colorants, flavorants, plasticizers, humectants, and buffering agents. Also, formulations for oral use can be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with a medium of water or oil (e.g., peanut oil, liquid paraffin, or olive oil). Powders, granules, and pellets can be prepared in the usual manner using the ingredients described above under tablets and capsules, for example using a mixer, a fluid bed apparatus, or a spray dryer.

[0323] Dissolution or controlled release of the conjugate or its pharmaceutical composition described herein can be achieved by appropriate coating of tablets, capsules, pellets, or granules of the conjugate, or by incorporating the conjugate into an appropriate matrix. Controlled release coatings can include one or more of the coating substances described above, and / or, for example, shellac, beeswax, glyceryl wax, hydrogenated castor oil, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glyceryl palmitostearate, ethyl cellulose, acrylic resin, dipolylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxy methacrylate, methacrylate hydrogel, 1,3 butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. In controlled release matrix formulations, matrix materials can include, for example, hydrous methyl cellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbon.

[0324] The pharmaceutical composition can be formed in unit dosage form as required. The amount of the active component (e.g., the conjugate described herein) contained in the pharmaceutical composition is an amount such that a suitable dosage within the specified range is provided (e.g., a dosage within the range of 0.01 to 100 mg / kg body weight).

[0325] VI. Route of Administration and Dosage In any of the methods described herein, the conjugates herein can be administered by any suitable route for treating or preventing the disorders described herein (e.g., cancer, viral infections, or fibrotic conditions). The conjugates described herein can be administered to humans, household pets, livestock, or other animals together with a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, administration includes administering any of the conjugates or compositions herein intramuscularly, intravenously (e.g., as a sterile solution suitable for intravenous use and in a solvent system), intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intravitreally, intravaginally, rectally, topically, intratumorally, transperitoneally, subcutaneously, subconjunctivally, intracystically, transmucosally, intrapericardially, intraumbilically, intraocularly, orally (e.g., as tablets, capsules, caplets, gelcaps, or syrups), topically (e.g., as creams, gels, lotions, or ointments), locally, by inhalation, injection, or infusion (e.g., continuous infusion, local perfusion directly immersing target cells, catheters, washes, creams, or lipid compositions). In some embodiments, when a second therapeutic agent is also administered in addition to the conjugates described herein, the second therapeutic agent or its pharmaceutical composition can also be administered by any of the routes of administration described herein.

[0326] The dosage of the conjugate or its pharmaceutical composition described herein depends on factors including the route of administration, the disease to be treated, and the physical characteristics of the subject (e.g., age, weight, general health status). Typically, the amount of the conjugate or its pharmaceutical composition included within a single dosage can be an amount that effectively prevents, delays, or treats the disorder without inducing significant toxicity. The pharmaceutical composition can include a dosage of the conjugate described herein in the range of 0.01 - 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), in more specific embodiments about 0.1 - about 30 mg / kg, and in more specific embodiments about 1 - about 30 mg / kg. In some embodiments, when the conjugate described herein and a second therapeutic agent are administered in combination (e.g., substantially simultaneously in the same or separate pharmaceutical compositions, or separately in the same treatment regimen), the required dosage of the conjugate described herein can be lower than the required dosage of the conjugate when the conjugate is used alone in the treatment regimen.

[0327] The conjugate or its pharmaceutical composition described herein can be administered to a subject in need thereof, e.g., one or more times (e.g., 1 - 10 times or more; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), daily, weekly, monthly, twice a year, annually, or when medically necessary. The dosage can be provided in either a single or multiple dosage regimens. The timing between administrations can decrease as the medical condition improves or increase as the health of the patient deteriorates. The dosage and frequency of administration can be adapted by a physician according to conventional factors (e.g., the degree of infection and various parameters of the subject).

Example

[0328] The following examples are provided to illustrate to those skilled in the art how the compositions and methods described herein can be used, made, and evaluated, and are intended to be a pure exemplification of the present disclosure and are not intended to limit the scope of what the inventors regard as the present disclosure.

[0329] General procedure for the preparation of Fc constructs The reverse translation of amino acids containing the protein construct was synthesized by solid-phase synthesis. The oligonucleotide template was cloned in pcDNA3.1 (Life Technologies, Carlsbad, CA, USA) at the cloning sites BamHI and XhoI (New England Biolabs, Ipswich, MA, USA), including the signal sequence derived from human interleukin-2 or human albumin. The pcDNA3.1 plasmid was transformed into Top10 E. coli cells (LifeTech). DNA amplification, extraction, and purification were performed using the PURELINK® HiPure Plasmid Filter Maxiprep Kit (LifeTech). The plasmid DNA was delivered to HEK-293 cells using the EXPIFECTAMINE™ 293 Transfection Kit (LifeTech) according to the manufacturer's protocol. The cells were centrifuged, filtered, and the supernatant was purified using MabSelect Sure Resin (GE Healthcare (Chicago, IL, USA)). The purified molecules were analyzed using 4-12% Bis Tris SDS PAGE.

[0330] General procedure for the conjugation of intermediate (int) and Fc A solution of the trifluorophenyl ester dissolved in DMF (1 mL) was added to a solution of 50 mg of Fc in PBS (pH 7.4, 19.5 mg / mL) at ambient temperature. The pH of the resulting solution was adjusted to approximately 8.5 with boric acid buffer (300 μL, 1 M, pH 8.5) or carbonate buffer (300 μL, 1 M, pH 8.5). The homogeneous colorless reaction product was gently rocked for 3 hours and then subjected to purification according to general procedures. The average mass was obtained by Maldi TOF analysis of the purified final product, from which the DAR was calculated.

[0331] General procedure for purification of the conjugate Protein A, dialysis, and SEC: The conjugate was purified using Mabselect PrismA (Protein A purification) resin (eluted with TBS pH 7.4), then dialyzed using Slide-d-lyzer G2 dialysis cassettes (30,000 MWCO) in 150 mM histidine (twice), then in 150 mM NaCl pH 8.5 buffer, and then size exclusion chromatography was performed using TBS pH 7.4 buffer. The final product was in TBS (25 mM Tris, 150 mM NaCl) pH 7.4 buffer. The purified material was quantified using a UV-visible spectrophotometer (Protein Bradford assay) and concentrated to approximately 10 mg / ml using a centrifugal concentrator (30,000 MWCO).

[0332] Synthesis of Intermediate A [Chemical formula] EDC (1.6 g, 8.2 mmol) was added in four portions to a stirred mixture of azido-peg4-carboxylic acid (2 g, 6.9 mmol) and 2,4,6-trifluorophenol (1.2 g, 8.2 mmol) in DCM (20 mL). The mixture was stirred at ambient temperature for 2 h. The organic phase was washed with DI water (30 mL) and the aqueous phase was back-extracted with DCM (2 × 20 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated. The mixture of crude products was purified by silica gel chromatography (0 - 80% ethyl acetate in hexane, 25 min gradient) to give the product as a clear oil. Yield 2.15 g, 79%. Ion measured by LC / MS: [M+Na] + = 443.8.

[0333] Synthesis of Intermediate B

Chemical Structure

[0334] Synthesis of Intermediate C [Chem.] 4,6-dichloro-1H-pyrazolo[3,4-d]pyridine (2.5 g, 13.3 mmol) and ammonium sulfate (45 mg) were dissolved in 100 mL of hexamethyldisilazane. The mixture was then heated to 130 °C and stirred for 3 hours. The mixture was then concentrated on a rotary evaporator and dried under high vacuum for 12 hours. The solid residue was then taken up in 100 mL of acetonitrile and b-D-ribofuranose 1,2,3,5-tetraacetate (3.8 g, 13.3 mmol) was added. The mixture was cooled to 0 °C and TMSOTf (3.6 mL, 19.9 mmol) was added dropwise over 5 minutes. The reaction mixture was slowly warmed to ambient temperature and stirred for 3 hours. The mixture was cooled to 0 °C and saturated sodium bicarbonate was carefully added to neutralize the TMS triflate. The mixture was extracted with ethyl acetate (3 x 40 mL). The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The crude residue was purified by silica gel column chromatography (hexane / EtOAc) to give the desired compound as a white foam. Yield 1.85 g, 31%. Ion measured by LCMS: + [M+H]

[0335] Synthesis of Intermediate D [Chem.] 2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (5 g, 26.6 mmol) and ammonium sulfate (38 mg, 0.291 mmol) were dissolved in 30 mL of hexamethyldisilazane. The mixture was then warmed to reflux and stirred for 3 hours. The mixture was then concentrated on a rotary evaporator and dried under high vacuum for 12 hours. The solid residue was then taken up in 100 mL of acetonitrile and b-D-ribofuranose 1,2,3,5-tetraacetate (10.2 g, 31.9 mmol) was added. The mixture was cooled to 0 °C and TMSOTf (5.28 mL, 29.1 mmol) was added dropwise. The reaction mixture was gradually warmed to room temperature and stirred overnight. The mixture was then concentrated and taken up in ethyl acetate. The organic layer was washed with saturated sodium bicarbonate and brine, dried over sodium sulfate, filtered, and concentrated. The crude residue was purified by silica gel column chromatography (hexane / EtOAc) to give the desired compound as a white foam. Yield 2.35 g, 19.8%. Ions measured by LCMS: [M+H] + = 446.2.

[0336] Synthesis of Int-132

Chemical formula

[0337] Step a

Chemical formula

[0338] Step b.

Chem.

[0339] Step c.

Chem.

[0340] Step d.

Chem.

[0341] Step e.

Chemical formula

[0342] Synthesis of conjugate 70 To a solution of Array No. 13 (3.40 mL, 100 mg, 0.0017 mmol) in PBS 7.4, Int-132 (21 mg, 0.017 mmol) in DMF (0.200 mL) was added. The pH of the reaction mixture was slowly adjusted to approximately 8.5 by adding 2 mL of 1 M potassium carbonate buffer (pH 9). The reaction was then gently rocked for 4 hours. The reaction was quenched by stirring in 150 mM His / 100 mM ammonium hydroxide buffer (pH 8.5) for 12 hours and then subjected to purification according to the general procedure. Maldi TOF analysis of the purified final product gave an average mass of 62,472 Da (DAR = 3.8). Yield: 52 mg, 50%.

[0343] Synthesis of Int-110

Chemical Structure

[0344] Synthesis of Conjugate 58 The title compound was prepared in the same manner as Conjugate 70, except that the starting material used in Int-132 was replaced with Int-110. Maldi TOF analysis of the purified final product gave an average mass of 63,530 Da (DAR = 4.8).

[0345] Synthesis of Int-6

Chemical Structure

[0346] Synthesis of Conjugate 4b The title compound was prepared in the same manner as conjugate 70, except that the starting material used in Int-132 was replaced with Int-6. Maldi TOF analysis of the purified final product gave an average mass of 61,147 Da (DAR = 2.8).

[0347] Synthesis of conjugate 4a To a solution of SEQ ID NO: 17 (5.4 mL, 100 mg, 0.0017 mmol) in PBS 7.4 was added the product described in Int-6 (14 mg, 0.016 mmol) in DMF (0.200 mL). The pH of the reaction mixture was slowly adjusted to approximately 8.5 by adding 2 mL of 1 M potassium carbonate buffer (pH 9). The reaction was then gently rocked for 4 h and then subjected to purification according to the general procedure. Maldi TOF analysis of the purified final product gave an average mass of 63,505 Da (DAR = 5.3). Yield: 76 mg, 75%.

[0348] Synthesis of Int-7

Chemical formula

[0349] Synthesis of conjugate 5b The title compound was prepared in the same manner as conjugate 70, except that the starting material described in Int-110 was replaced with Int-7. Maldi TOF analysis of the purified final product gave an average mass of 62,224 (DAR = 3.9).

[0350] Synthesis of conjugate 5a The title compound was prepared in the same manner as conjugate 4a, except that the starting material described in Int-110 was replaced with Int-7. Maldi TOF analysis of the purified final product gave an average mass of 62,404 Da (DAR = 4.2).

[0351] Synthesis of Int-74

Chem.

[0352] Synthesis of Conjugate 40 The title compound was prepared in the same manner as Conjugate 70, except that the starting material used in Int-110 was replaced with Int-74. Maldi TOF analysis of the purified final product gave an average mass of 66,569 (DAR = 7.6).

[0353] Synthesis of Int-75

Chem.

[0354] Synthesis of Conjugate 41 The title compound was prepared in the same manner as Conjugate 70, except that the starting material used in Int-110 was replaced with Int-75. Maldi TOF analysis of the purified final product gave an average mass of 63,771 (DAR = 5.0).

[0355] Synthesis of Int-111

Chem.

[0356] Synthesis of Conjugate 59 The title compound was prepared in the same manner as conjugate 70, except that the starting material used in Int-110 was replaced with Int-111. Maldi TOF analysis of the purified final product gave an average mass of 63,411 (DAR = 4.7).

[0357] Synthesis of Int-16

Chem.

[0358] Step a.

Chem.

[0359] Step b.

Chem.

[0360] Step c.

Chemical formula

[0361] Step d.

Chemical formula

[0362] Step e.

Chemical formula

[0363] Step f.

Chemical formula

[0364] Synthesis of conjugate 11b The title compound was prepared in the same manner as conjugate 70, except that the starting material used in Int-110 was replaced with Int-16. Maldi TOF analysis of the purified final product gave an average mass of 62,471 Da (DAR = 4.0).

[0365] Synthesis of conjugate 11a The title compound was prepared in the same manner as conjugate 4a, except that the starting material used in Int-110 was replaced with Int-16. Maldi TOF analysis of the purified final product gave an average mass of 62,404 Da (DAR = 4.2).

[0366] Synthesis of Int-27

Chemical Structure

[0367] Synthesis of conjugate 16 The title compound was prepared in the same manner as conjugate 70, except that the starting material used in Int-110 was replaced with Int-27. Maldi TOF analysis of the purified final product gave an average mass of 60,745 (DAR = 2.4).

[0368] Synthesis of Int-106 [Chemical formula]

[0369] Step a. [Chemical formula] HATU (411 mg, 1.08 mmol) was added to a mixture of (racemic)-cis 1-tert-butoxycarbonylaminoindane-2-carboxylic acid (250 mg, 0.90 mmol), propargyl-peg4 amine (312 mg, 1.35 mmol), and DIEA (349 mg, 2.70 mmol) in DMF (2 mL). The reaction was stirred at ambient temperature for 4 h and applied directly to reverse phase HPLC (5 - 85% acetonitrile in DI water, 0.1% TTFA modifier, 25 min gradient). The pure fractions were pooled and lyophilized. The boc-protected amine was stirred in a 1 / 1 mixture of TFA / DCM (10 mL) at ambient temperature for 30 min. The solvent was removed on a rotary evaporator and dried under high vacuum to give the TFA salt of the intermediate (racemate) as a clear viscous oil. Yield 71%, 2 steps, 325 mg. Ion measured by LC / MS: [M + H]+ = 391.2.

[0370] Step b. [Chemical formula] Intermediate B (200 mg, 44 mmol), the intermediate described in the step before this example (175 mg, 0.45 mmol), and triethylamine (90 mg, 0.89 mmol) were stirred in ethanol (25 mL) at 50 °C for 4 hours. The mixture was cooled to ambient temperature and concentrated. The crude residue was purified by silica gel chromatography (0 - 10% methanol in DCM, 25 minutes), and the product was obtained as a pair of diastereomers. Yield 70%, 250 mg. Ion measured by LC / MS: [M+H]+ = 801.2.

[0371] Step c.

Chemical formula

[0372] Step d.

Chemical formula

[0373] Step e.

Chemical formula

[0374] Synthesis of conjugate 56 The title compound was prepared in the same manner as conjugate 70, except that the starting material used in Int-110 was replaced with Int-106. Maldi TOF analysis of the purified final product gave an average mass of 63,241 Da (DAR = 4.5).

[0375] Synthesis of Int-61

Chemical formula

[0376] Synthesis of conjugate 38 The title compound was prepared in the same manner as conjugate 70, except that the starting material from Int-110 was replaced with Int-61. Maldi TOF analysis of the purified final product gave an average mass of 63,348 Da (DAR = 4.8).

[0377] Synthesis of Int-36

Chemical formula

[0378] Synthesis of conjugate 19 The title compound was prepared in the same manner as conjugate 70, except that the starting material used in Int-110 was replaced with Int-36. Maldi TOF analysis of the purified final product gave an average mass of 63,363 Da (DAR = 4.8).

[0379] Synthesis of Int-10 [Chemical formula]

[0380] Step a. [Chemical formula] 1-[(tert-Butyl)oxycarbonyl]azetidine-3-carboxylic acid (500 mg, 2.48 mmol), HATU (1.42 g, 3.72 mmol), and DIEA (0.88 mL, 2.48 mmol) (0.88 mL, 2.48 mmol) in DMF (6 mL) were stirred together at ambient temperature for 10 minutes. To this was added propargyl-peg4-amine (862 mg, 3.72 mmol) in DMF (1 mL), and the resulting mixture was stirred for 1 hour. The reaction was concentrated and purified by reverse-phase HPLC (5% - 100% ACN / water) to give the boc-protected intermediate as a yellow viscous liquid. The boc-protected intermediate was taken up in DCM (10 mL) and treated with 4 M aqueous HCl in dioxane (8 mL) for 3 hours. The solvent was removed under reduced pressure and then dried under high vacuum to give the amine-HCl salt as a yellowish viscous oil. Yield 627 mg, 99%. Ion measured by LCMS: [M+H] + = 315.9.

[0381] Step b. [Chemical formula] A mixture of intermediate B (447 mg, 1.118 mmol), the product from the previous step (456 mg, 1.34 mmol), and triethylamine (0.31 mL) in methanol (6 mL) was heated at 50 °C for 2 hours. After complete consumption of the starting materials, the reaction mixture was cooled to room temperature and the volatiles were removed by rotary evaporation. The crude residue was redissolved in ethyl acetate (50 mL) and washed with water and brine. The combined organic layers were dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure to give the compound, which was used in the next step without further purification. Ion measured by LCMS: [M+H]+ = 724.6.

[0382] Step c.

Chem.

[0383] Step d.

Chem.

[0384] Step e.

Chemical formula

[0385] Synthesis of conjugate 8a Trifluorophenol ester (13 mg, 0.011 mmol, described for the synthesis of Int-10) in DMF (0.5 mL) was added to the Fc carrier SEQ ID NO: 17 (80 mg in 4 mL in PBS at pH 7.4), and then the pH was adjusted to approximately 8 with boric acid buffer (0.200 mL, pH 8.5, 1.0 M). The mixture was stirred at ambient temperature for 2 hours and then subjected to purification according to the general procedure. Maldi TOF analysis of the purified final product gave an average mass of 64,894 (DAR: 6.7). This conjugate was purified by buffer dialysis (PBS pH 7.4) and SEC chromatography. Yield 54 mg, 77.1%.

[0386] Synthesis of conjugate 8b Trifluorophenol ester (17 mg, 0.014481 mmol) described in Int-10 was conjugated with the Fc carrier SEQ ID NO: 13 (100 mg, 0.00181 mmol, in 5.13 mL in PBS at pH 7.4) as described for the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 65,370 Da (DAR = 6.9). Yield 80 mg, 80.6%.

[0387] Synthesis of Int-9

Chemical Structure

[0388] Step a.

Chemical Structure

[0389] Step b.

Chemical Structure

[0390] Step c. [Chemistry] To the trihydroxy product (497 mg, 0.75 mmol) from the previous step and 2,2-dimethoxypropane (0.46 mL, 3.75 mmol) in acetone (20 mL) at room temperature was added p-TsOH (13 mg, 0.075 mmol). The reaction mixture was stirred at ambient temperature for 2 h and then concentrated under reduced pressure. The crude product was dissolved in ethyl acetate (10 mL) and washed with saturated sodium bicarbonate. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated in vacuo to give an off-white solid, which was used in the next step without further purification. Yield 497 mg, 100%. Ion measured by LCMS: [M+H] + = 702.8.

[0391] Step d. [Chemistry] To a suspension of methylenebis(phosphonic dichloride) (562 mg, 2.25 mmol) in THF (5 mL) at 0 °C was added DIPEA (0.14 mL, 0.83 mmol). To the resulting mixture was added dropwise a solution of the acetonide (497 mg, 0.75 mmol) from the previous step in THF (2 mL) over 1 h. After the addition, the resulting mixture was stirred at 0 °C for an additional 15 min and then the solution was transferred to a pre-cooled (0 °C) flask containing 0.2 M aqueous HCl (2.25 mL). The reaction mixture was warmed to ambient temperature and stirred for 4 h. When complete (monitored by LCMS), the reaction mixture was concentrated and purified by reverse-phase HPLC (5% ACN / water → 100% ACN / water, using 0.1% TFA modifier). White solid, yield 454 mg, 74%. Ion measured by LCMS: [M+H] + = 820.6.

[0392] Step e. [Chemistry] A solution of the product from step c (0.1 g, 0.122 mmol) and intermediate A (0.037 g, 0.088 mmol) dissolved in DMF:H2O (1:3, 1.5 mL) was cooled to 0 °C. To this, a premixed solution of copper(II) sulfate (0.0020 g, 0.0121 mmol), sodium ascorbate (0.072 g, 0.365 mmol), and BTTA (0.010 g, 0.0244 mmol) dissolved in water (0.5 mL) was added, and the mixture was stirred at the same temperature for 5 minutes, gradually warmed to room temperature, and stirred at room temperature for 15 minutes. After completion of the reaction, the reaction mixture was quenched by adding a few drops of AcOH and EDTA to adjust the pH to 6, and the product was purified by reverse-phase HPLC (10% - 100% ACN / water, 0.1% TFA). White solid (0.142 g, 93.84%). LCMS[(M + 2H) / 2] + = 621.2.

[0393] Synthesis of conjugate 7a The trifluorophenol ester (described in the synthesis of Int-9) was conjugated with the Fc carrier SEQ ID NO: 17 as described in the general conjugation procedure. Maldi TOF analysis of the purified final product gave an average mass of 63,727 Da (DAR = 5.2). Yield 87.5 mg, 87.5%.

[0394] Synthesis of conjugate 7b The trifluorophenol ester (9 mg, 0.0072 mmol) (described in the synthesis of Int-9) was conjugated with the Fc carrier SEQ ID NO: 13 (50 mg, 2.56 mL in PBS at pH 7.4, 0.00091 mmol) as described in the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 63,528 Da (DAR = 4.8). Yield 34.3 mg, 68.6%.

[0395] Synthesis of Int-21

Chemical formula

[0396] Step a. [Chemical formula] Propargyl-PEG-4 mesylate (931 mg, 3 mmol), N-Boc piperazine (558 mg, 3 mmol), and potassium carbonate (828 mg, 6 mmol) in acetonitrile (50 mL) were heated to reflux for 15 h. The reaction mixture was cooled to ambient temperature and the solvent was removed under reduced pressure. The crude material was partitioned between water and ethyl acetate, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2 x 10 mL). The combined organic extracts were washed with brine, water, and dried over sodium sulfate. The solvent was concentrated to give the crude N-Boc protected product as a yellow viscous liquid. Ion measured by LCMS: [M+H] + = 401.9. The N-Boc protected amine was redissolved in DCM and cooled to 0 °C in an ice bath. HCl (4 mL, 10 eq) in dioxane was added thereto, and the reaction mixture was gradually warmed to ambient temperature and stirred until LCMS analysis showed that the starting material had been completely converted to the product. The solvent was removed under reduced pressure, and the crude material was dried under reduced pressure to give the product as a white solid (HCl salt). Yield: 672 mg, 85%, 2 steps. Ion measured by LCMS: [M+H] + = 301.2.

[0397] Step b. [Chemical formula] A mixture of Intermediate B (250 mg, 0.56 mmol), the amine from the previous step (201 mg, 0.67 mmol), and triethylamine (0.12 mL) in EtOH (6 mL) was heated at 50 °C for 2 h. After the starting material was completely consumed (by LCMS), the reaction mixture was cooled to ambient temperature and the volatiles were removed on a rotary evaporator to give a crude material, which was purified by normal phase column chromatography (using hexane:ethyl acetate). The product was a white solid. Yield 200 mg, 50%. Ion measured by LCMS: [M+H] + = 710.8.

[0398] Step c. [Chemical formula] The triacetate product (220 mg, 0.31 mmol) from the previous step was dissolved in methanol (10 mL), treated with potassium carbonate (149 mg, 1.08 mmol), and then stirred at ambient temperature for 1.5 h. The reaction mixture was filtered through celite and the filter cake was washed with methanol (3 × 20 mL). The solution was concentrated in vacuo to remove volatiles to afford the crude product, which was purified by reverse phase HPLC to give the product. The product was a white solid. Yield 139 mg, 77%. Ion measured by LCMS: [M+H] + = 585.2.

[0399] Step d. [Chemical formula] To a solution of the product (139 mg, 0.24 mmol) from the previous step and 2,2-dimethoxypropane (0.145 mL, 1.188 mmol) in acetone (10 mL) at ambient temperature was added p-TsOH (23 mg, 0.005 mmol). The reaction was stirred for 2 h and then concentrated under reduced pressure to afford the acetonide intermediate. The crude material was dissolved in ethyl acetate (20 mL) and washed with saturated sodium bicarbonate. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated in vacuo to give the acetonide derivative as a white solid, which was used in the next step without further purification. Ion measured by LCMS: [M+H] +=624.8. A suspension of methylenebis(phosphonic dichloride) (178 mg, 0.71 mmol) in THF (5 mL) at 0 °C was added with DIEA (0.046 mL, 0.26135 mmol). To the resulting mixture, a solution of acetonide (148 mg, 0.24 mmol) in THF (2 mL) was added dropwise over 10 minutes. After the addition, the resulting mixture was stirred at 0 °C for an additional 15 minutes and then the solution was transferred to a pre-cooled (0 °C) flask containing 0.2 M aqueous HCl (1.5 mL). The reaction mixture was warmed to ambient temperature and stirred for 4 hours. Upon completion (by LCMS), the reaction mixture was concentrated and purified by reverse-phase HPLC. The product was a white solid. Yield 100 mg, 57%. Ion measured by LCMS: [M+H] + =743.6.

[0400] Step e.

Chemical formula

[0401] Synthesis of Conjugate 13 Trifluorophenol ester (described in the synthesis of Int-21) (0.017 g, 0.01448 mmol) was conjugated with the Fc carrier SEQ ID NO: 13 (0.1 g, 0.0018 mmol) as described in the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 62149 Da (DAR = 3.9). Yield 42.3 mg, 42.3%.

[0402] Synthesis of Int-55 [Chemical formula]

[0403] Step a. [Chemical formula] A mixture of intermediate B (1 g, 2.24 mmol), cyclopentylamine (228 mg, 2.68 mmol), triethylamine (0.46 mL), and ethanol (20 mL) was heated at 50 °C for 1 hour. The mixture was cooled to ambient temperature and concentrated. The residue was purified by column chromatography (petroleum ether / ethyl acetate: 5:1) to give the product. The product was a white solid. Yield 1.1 g, 98%. Ion measured by LCMS: [M+H] + = 495.8.

[0404] Step b. [Chemical formula] The triacetate product from the previous step (1.1 g, 2.21 mmol) was dissolved in THF (30 mL) and treated with 2 M aqueous LiOH (9 mL) at ambient temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and then acidified with 1 N aqueous HCl. The crude material was purified by reverse-phase HPLC (10% - 100% ACN / water, 0.1% TFA). The product was a white solid. Yield 735 mg, 89%. Ion measured by LCMS: [M+H] + = 369.8.

[0405] Step c.

Chem.

[0406] Step d.

Chem.

[0407] Step e.

Chem.

[0408] Step f.

Chemical Structure

[0409] Synthesis of Conjugate 31 The trifluorophenol ester (described in the synthesis of int-55) was conjugated with the Fc carrier SEQ ID NO: 13 as described in the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 61,874 Da (DAR = 3.7). Yield 61.9 mg, 77.4%.

[0410] Synthesis of Int-28

Chemical formula

[0411] Step a.

Chemical formula

[0412] Step b.

Chemical formula

[0413] Step c.

Chem.

[0414] Step d.

Chem.

[0415] Step e.

Chem.

[0416] Synthesis of conjugate 17 The trifluorophenol ester (9 mg, 0.00724 mmol) described in the synthesis of Int-28 was conjugated with the Fc carrier SEQ ID NO: 13 (50 mg, 2.58 mL in PBS at pH 7.4, 0.0009 mmol) as described in the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 60,273 Da (DAR = 1.9). Yield 43.9 mg, 87.7%.

[0417] Synthesis of Int-49

Chemical Structure

[0418] Step a.

Chemical Structure

[0419] Step b.

Chemical formula

[0420] Step c.

Chemical formula

[0421] Step d.

Chemical formula

[0422] Step e.

Chemical formula

[0423] Synthesis of conjugate 28 The trifluorophenol ester (7 mg, 0.0058 mmol) (described in the synthesis of Int-49) was conjugated with the Fc carrier SEQ ID NO: 13 (40 mg, 2 mL in PBS at pH 7.4, 0.000724 mmol) as described in the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 64,083 Da (DAR = 5.5). Yield 30.3 mg, 75.6%.

[0424] Synthesis of Int-81

Chemical Structure

[0425] Step a.

Chemical Structure

[0426] Step b.

Chemical Structure

[0427] Step c.

Chemical Structure

[0428] Step d.

Chemical Structure

[0429] Synthesis of conjugate 44 Trifluorophenol ester (14 mg, 0.011584 mmol) (described in the synthesis of Int-81) was conjugated with the Fc carrier SEQ ID NO: 13 (80 mg, 4.1 mL in PBS at pH 7.4, 0.00145 mmol)5 as described in the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 63,709 Da (DAR = 5.3). Yield 63 mg, 78.9%.

[0430] Synthesis of Int-90

Chemical formula

[0431] Synthesis of conjugate 48 Trifluorophenol ester (17 mg, 0.01448 mmol) (described in the synthesis of Int-90) was conjugated with the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL, 0.0018 mmol) in PBS at pH 7.4 as described in the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 63,468 Da (DAR = 5). Yield 53.2 mg, 53.2%.

[0432] Synthesis of Int-91

Chemical Structure

[0433] Synthesis of conjugate 49 The trifluorophenol ester (17.5 mg, 0.01448 mmol) described in the synthesis of Int-91 was conjugated with the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL in PBS at pH 7.4, 0.0018 mmol) as described in the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 64,352 Da (DAR = 5.8). Yield 70 mg, 70%.

[0434] Synthesis of Int-97

Chemical Structure

[0435] Synthesis of conjugate 52 The trifluorophenol ester (17 mg, 0.01448 mmol) described in the synthesis of Int-97 was conjugated with the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL, 0.0018 mmol) in PBS at pH 7.4 as described in the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 63,232 Da (DAR = 4.9). Yield 73 mg, 73%.

[0436] Synthesis of Int-99

Chemical Structure

[0437] Synthesis of conjugate 53 The trifluorophenol ester (17 mg, 0.01448 mmol) described in the synthesis of Int-99 was conjugated with the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL, 0.0018 mmol) in PBS at pH 7.4 as described in the synthesis of conjugate 8a. Maldi TOF analysis of the purified final product gave an average mass of 61,730 Da (DAR 3.4). Yield 70 mg, 70%.

[0438] Synthesis of Int-113

Chemical Structure

[0439] Step a.

Chemical Structure

[0440] Step b. The triacetate (324 mg, 0.45 mmol) from the previous step in methanol (5 mL) was treated with potassium carbonate (217 mg, 1.57 mmol) at ambient temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, dissolved in methanol:water (1:1, 2 mL), and purified by reverse phase HPLC (10% - 100% ACN / water, 0.1% TFA). White foam. Yield 210 mg, 78%. Ion measured by LCMS: [M+H] + = 598.2.

[0441] Step c.

Chemical Structure

[0442] Step d.

Chemical Structure

[0443] Synthesis of Conjugate 60 Trifluorophenol ester (18 mg, 0.01448 mmol) (described in the synthesis of Int-113) in DMF (0.5 mL) was added to the Fc carrier SEQ ID NO: 13 (100 mg in 5.15 mL of PBS at pH 7.4), and then the pH was adjusted to about 7.4 with sodium carbonate buffer (0.200 mL, pH 9.2 - 10.6, 0.1 M). The mixture was stirred at room temperature for 4 hours. The reaction mixture was quenched by adding 150 mM histidine / 100 mM ammonium hydroxide buffer, pH 8.5 (about 0.5 mL of buffer mixture / 10 mg of protein), stirred for 12 hours, and then purified according to the general procedure. Maldi TOF analysis of the purified final product gave an average mass of 61,118 Da (DAR = 2.9). Yield 54.9 mg, 61%.

[0444] Synthesis of Int-115

Chemical formula

[0445] Step a.

Chemical formula

[0446] Step b.

Chemical formula

[0447] Step c.

Chemical Structure

[0448] Step d.

Chemical Structure

[0449] Synthesis of conjugate 61 The trifluorophenol ester (18 mg, 0.01448 mmol) (described in the synthesis of Int-115) in DMF (0.5 mL) was added to Fc carrier SEQ ID NO: 13 (100 mg in 5.15 mL of PBS at pH 7.4) 205, and then adjusted to pH ~7.4 with sodium carbonate buffer (0.200 mL, pH 9.2 - 10.6, 0.1 M). The mixture was stirred at ambient temperature for 4 hours. The reaction mixture was quenched by adding 150 mM histidine / 100 mM ammonium hydroxide buffer, pH 8.5 (about 0.5 mL of buffer mixture / 10 mg of protein), stirred for 12 hours, and then subjected to purification according to the general procedure. Maldi TOF analysis of the purified final product gave an average mass of 61,737 Da (DAR: 3.4). Yield 59.9 mg, 59.9%.

[0450] Synthesis of Int-117

Chemical formula

[0451] Step a.

Chem.

[0452] Step b.

Chem.

[0453] Step c.

Chem.

[0454] Step d.

Chem.

[0455] Step e.

Chem.

[0456] Step f.

Chemical Structure

[0457] Synthesis of conjugate 62 Trifluorophenol ester (19 mg, 0.01448 mmol) (described in the synthesis of Int-117) was conjugated with the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL in PBS at pH 7.4, 0.0018 mmol) as described in the conjugation procedure of conjugate 60. Maldi TOF analysis of the purified final product gave an average mass of 62,537 Da (DAR: 3.8). Yield 66.9 mg, 66.9%.

[0458] Synthesis of Int-119

Chemical Structure

[0459] Step a.

Chemical Structure

[0460] Step b.

Chemical formula

[0461] Step c.

Chemical formula

[0462] Step d.

Chemical Structure

[0463] Synthesis of Conjugate 63a The trifluorophenol ester (16 mg, 0.01448 mmol) described in the synthesis of Int-119 was conjugated with the Fc carrier SEQ ID NO: 13 (100 mg, 3.47 mL in PBS at pH 7.4, 0.0018 mmol) described in the conjugation procedure of conjugate 61. Maldi TOF analysis of the purified final product gave an average mass of 62259 Da (DAR = 3.8). Yield 59.2 mg, 59.2%.

[0464] Synthesis of Int-130

Chemical Structure

[0465] Synthesis of conjugate 69 The trifluorophenol ester (18 mg, 0.01448 mmol) described in the synthesis of Int-130 was conjugated with the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL in PBS at pH 7.4, 0.0018 mmol) described in the conjugation procedure of conjugate 61. MALDI TOF analysis of the purified final product gave an average mass of 62259 Da (DAR: 3.8). Yield 59.2 mg, 59.2%.

[0466] Synthesis of Int-134

Chemical Structure

[0467] Step a.

Chemical Structure

[0468] Step b.

Chemical Structure

[0469] Step c.

Chemical Structure

[0470] Step d.

Chem.

[0471] Synthesis of conjugate 71 The trifluorophenol ester (17 mg, 0.0144 mmol) (described in the synthesis of Int-134) in DMF (0.5 mL) was added to the Fc carrier SEQ ID NO: 13 (100 mg in 5.15 mL of PBS at pH 7.4), and then adjusted to pH ~7.4 with sodium carbonate buffer. The mixture was stirred at ambient temperature for 4 hours. The reaction was quenched by stirring in 150 mM His / 100 mM ammonium hydroxide buffer (pH 8.5) for 12 hours and purified by dialysis into 150 mM His pH ~8.5 buffer, Protein A, and SEC column. Maldi TOF analysis of the purified final product gave an average mass of 62,647 Da (DAR = 4.4). Yield 43.7 mg, 43.7%.

[0472] Synthesis of Int-136

Chem.

[0473] Synthesis of conjugate 72 The trifluorophenol ester described in the synthesis of Int-136 (17 mg, 0.01448 mmol) was conjugated with the Fc carrier SEQ ID NO: 13 described in the conjugation procedure of conjugate 71 (100 mg, 3.4 mL in PBS at pH 7.4, 0.0018 mmol). Maldi TOF analysis of the purified final product gave an average mass of 64,461 Da (DAR = 5.6). Yield 47.9 mg, 47.9%.

[0474] Synthesis of Int-2

Chemical Structure

[0475] Step a.

Chemical Structure

[0476] Step b.

Chemical Structure

[0477] Step c.

Chemical formula

[0478] Step d.

Chemical formula

[0479] Step e.

Chemical formula

[0480] Step f.

Chemical formula

[0481] Step g.

Chem.

[0482] Step h.

Chem.

[0483] Synthesis of Conjugate 2 A solution of trifluorophenyl ester (10.6 mg, 0.0086 mmol, Int-2) dissolved in DMF (1 mL) was added to a solution of Fc (50 mg, 0.00086 mmol, SEQ ID NO: 17 in PBS at pH 7.4, 17.3 mg / mL) at ambient temperature. The pH of the resulting solution was adjusted to approximately 8.5 with boric acid buffer (300 uL, 1 M, pH 8.5). The homogeneous colorless reaction mixture was gently agitated for 3 hours and then subjected to purification according to the general procedure. Maldi TOF analysis of the purified final product gave an average mass of 62,957 Da (DAR = 4.3). Yield: 41 mg, 83%.

[0484] Synthesis of Conjugate 146 The title compound was prepared in the same manner as Conjugate 2, except that the Fc SEQ ID NO: 17 was replaced with Fc SEQ ID NO: 13.

[0485] Synthesis of Int-1

Chemical Structure

[0486] Synthesis of Conjugate 1 The title conjugate was prepared in the same manner as Conjugate 2, except that the trifluorophenyl ester from Int-2 was replaced with the trifluorophenyl ester from Int-1.

[0487] Synthesis of Conjugate 144 The title compound was prepared in the same manner as Conjugate 1, except that the Fc SEQ ID NO: 17 was replaced with Fc SEQ ID NO: 13.

[0488] Synthesis of Int-14

Chemical Structure

[0489] Step a.

Chemical Structure

[0490] Step b.

Chemical Structure

[0491] Synthesis of Conjugate 10 The title conjugate was prepared in the same manner as conjugate 2, except that the trifluorophenyl ester from Int-2 was replaced with the trifluorophenyl ester from Int-14.

[0492] Synthesis of Int-13

Chemical Structure

[0493] Synthesis of Conjugate 9 The title conjugate was prepared in the same manner as Conjugate 2, except that the trifluorophenyl ester from Int-2 was replaced with the trifluorophenyl ester from Int-13.

[0494] Synthesis of Int-33

Chem.

[0495] Step a.

Chem.

[0496] Step b.

Chem.

[0497] Step c.

Chemical formula

[0498] Step d.

Chemical formula

[0499] Step e.

Chemical formula

Claims

1. A conjugate represented by formula (D-I) or (M-I) or a pharmaceutically acceptable salt thereof, wherein 【Chemical 1】 wherein, A １ and A ２ each independently has the structure of formula (A), 【Chemical 2】 m is 0, 1, 2, 3, 4, 5, or 6; s is 0 or 1; X １ , X ２ , X ３ , X ４ , X ５ , and X ６ each of which is independently N, CR ４ , or C−Y−R ５ and X １ , X ２ , X ３ , X ４ , X ５ , and X ６ at least one of which is C−Y−R ５ where R ５ is a bond to L R １ is 【Chemical 3】 and Z is O, S, or sulfonyl; R ２ａ and R ２ｂ each independently is H, optionally substituted C １ -C ２０ alkyl, optionally substituted C ２ -C ２０ alkenyl, optionally substituted C ２ -C ２０ alkynyl, optionally substituted C １ -C ２０ heteroalkyl, optionally substituted C ２ -C ２０ heteroalkenyl, optionally substituted C ２ -C ２０ heteroalkynyl, optionally substituted C ３ -C ２０ cycloalkyl, optionally substituted C ３ -C ２０ cycloalkenyl, optionally substituted C ２ -C ２０ heterocycloalkyl, optionally substituted C ２ -C ２０ heterocycloalkenyl, optionally substituted C ６ -C １８ aryl, or optionally substituted C ２ -C １９ heteroaryl, and Each R ３ is independently OH, SH, halogen, optionally substituted amino, optionally substituted C １ -C ２０ -C ２ -alkyl, optionally substituted C ２０ -C ２ -alkenyl, optionally substituted C ２０ -C １ -alkynyl, optionally substituted C ２０ -heteroalkyl, optionally substituted C ２ -C ２０ -heteroalkenyl, optionally substituted C ２ -C ２０ -heteroalkynyl, optionally substituted C ３ -C ２０ -cycloalkyl, optionally substituted C ３ -C ２０ -cycloalkenyl, optionally substituted C ２ -C ２０ -heterocycloalkyl, optionally substituted C ２ -C ２０ -heterocycloalkenyl, optionally substituted C ６ -C １８ -aryl, or optionally substituted C ２ -C １９ -heteroaryl, and R ４ is H, halogen, OH, SH, optionally substituted amino, optionally substituted C １ ~C ２０ alkyl, optionally substituted C ２ ~C ２０ alkenyl, optionally substituted C ２ ~C ２０ alkynyl, optionally substituted C １ ~C ２０ heteroalkyl, optionally substituted C ２ ~C ２０ heteroalkenyl, optionally substituted C ２ ~C ２０ heteroalkynyl, optionally substituted optionally substituted C ３ ~C ２０ cycloalkyl, optionally substituted C ３ ~C ２０ cycloalkenyl, optionally substituted C ２ ~C ２０ heterocycloalkyl, optionally substituted C ２ ~C ２０ heterocycloalkenyl, optionally substituted C ６ ~C １８ aryl, or optionally substituted C ２ ~C １９ heteroaryl, and R ６ａ and R ６ｂ each independently is H, optionally substituted C １ to C ２０ alkyl, optionally substituted C ２ to C ２０ alkenyl, optionally substituted C ２ to C ２０ alkynyl, optionally substituted C １ to C ２０ heteroalkyl, optionally substituted C ２ to C ２０ heteroalkenyl, optionally substituted C ２ to C ２０ heteroalkynyl, optionally substituted C ３ to C ２０ cycloalkyl, optionally substituted C ３ to C ２０ cycloalkenyl, optionally substituted C ２ to C ２０ heterocycloalkyl, optionally substituted C ２ to C ２０ heterocycloalkenyl, optionally substituted C ６ to C １８ aryl, or optionally substituted C ２ to C １９ heteroaryl, and Y is a first linker; L is a second linker; n is 1 or 2; each E contains an Fc domain monomer; T is an integer from 1 to 20; The wavy line indicates that L is covalently bonded to E, said conjugate, or a pharmaceutically acceptable salt thereof.

2. The conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the conjugate is represented by formula (D-I). 【Chemical Formula 4】

3. The conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the conjugate is represented by formula (M-I). 【Chemical Formula 5】

4. A １ and A ２ The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, having the structure of formula (A-I). 【Chemical Formula 6】

5. A １ and A ２ The conjugate according to claim 4, or a pharmaceutically acceptable salt thereof, wherein each of A and A has the structure of formula (A-Ia). [Chemical Formula 7]

6. A １ and A ２ The conjugate according to claim 5, or a pharmaceutically acceptable salt thereof, wherein each of them has the structure of formula (A-Ib). 【Chemical 8】

7. A １ and A ２ The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, each having the structure of formula (A-I). 【Chemical Formula 9】

8. A １ and A ２ The conjugate according to claim 7, or a pharmaceutically acceptable salt thereof, wherein each of them has the structure of formula (A-IIa). 【Chemical Formula 10】

9. A １ and A ２ The conjugate according to claim 8, or a pharmaceutically acceptable salt thereof, wherein each of them has the structure of formula (A-IIb). 【Chemical Formula 11】

10. The conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein s is 0.

11. The conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein s is 1.

12. R ２ａ and R ２ｂ each independently is H, C １ to C ２０ alkyl, or optionally substituted C １ to C ２０ heteroalkyl, the conjugate according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.

13. R ２ａ and R ２ｂ each of which is H, the conjugate according to claim 12, or a pharmaceutically acceptable salt thereof.

14. R ４ is H, halogen, OH, SH, optionally substituted amino, optionally substituted C １ to C ２０ alkyl, or optionally substituted C １ to C ２０ heteroalkyl, the conjugate according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.

15. R ４ The conjugate according to claim 14, or a pharmaceutically acceptable salt thereof, wherein R is halogen.

16. R ４ The conjugate according to claim 15, or a pharmaceutically acceptable salt thereof, wherein R is Cl.

17. R １ is 【Chemical Formula 12】 The conjugate according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein is.

18. R １ is 【Chemical 13】 The conjugate according to any one of claims 1 to 17, or a pharmaceutically acceptable salt thereof, wherein is.

19. R ６ａ and R ６ｂ each independently is H, optionally substituted C １ to C ２０ alkyl, or optionally substituted C １ to C ２０ heteroalkyl, the conjugate according to claim 18, or a pharmaceutically acceptable salt thereof.

20. R ６ａ and R ６ｂ each independently is H, -CH ３ , -CH ２ CH ３ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, or -CH ２ CH ２ OCH ３ The conjugate according to claim 19, or a pharmaceutically acceptable salt thereof.

21. R ６ａ and R ６ｂ each of which is H, the conjugate according to claim 20, or a pharmaceutically acceptable salt thereof.

22. Y is 【Chemical 14】 wherein in the formula each of p1, p2, p3, and p4 is independently 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4. Each X ７ is independently N or CH, Each X ８ is independently O, NH, CH ２ or C(=O), and R Ｎ１ is H, C optionally substituted with １ - C ２０ alkyl, C optionally substituted with ２ - C ２０ alkenyl, C optionally substituted with ２ - C ２０ alkynyl, C optionally substituted with １ - C ２０ heteroalkyl, C optionally substituted with ２ - C ２０ heteroalkenyl, C optionally substituted with ２ - C ２０ heteroalkynyl, C optionally substituted with ３ - C ２０ cycloalkyl, C optionally substituted with ３ - C ２０ cycloalkenyl, C optionally substituted with ２ - C ２０ heterocycloalkyl, C optionally substituted with ２ - C ２０ heterocycloalkenyl, C optionally substituted with ６ - C １８ aryl, C optionally substituted with ２ - C １９ heteroaryl, C optionally substituted with １ - C ２０ alkaryl, or C optionally substituted with １ - C ２０ alkylcycloalkyl, and Each R ７ is independently 【Chemical Formula 15】 Optionally substituted C ３ ~C ２０ Cycloalkyl, optionally substituted C ３ ~C ２０ Cycloalkenyl, optionally substituted C ２ ~C ２０ Heterocycloalkyl, or optionally substituted C ２ ~C ２０ Is heterocycloalkenyl, R ７ａ and R ７ｂ each independently is H, optionally substituted C １ -C ２０ alkyl, optionally substituted C ２ -C ２０ alkenyl, optionally substituted C ２ -C ２０ alkynyl, optionally substituted C １ -C ２０ heteroalkyl, optionally substituted C ２ -C ２０ heteroalkenyl, optionally substituted C ２ -C ２０ heteroalkynyl, optionally substituted C ３ -C ２０ cycloalkyl, optionally substituted C ３ -C ２０ cycloalkenyl, optionally substituted C ２ -C ２０ heterocycloalkyl, optionally substituted C ２ -C ２０ heterocycloalkenyl, optionally substituted C ６ -C １８ aryl, or optionally substituted C ２ -C １９ heteroaryl, and Each R ８ is independently halogen, OH, SH, optionally substituted amino, optionally substituted C １ to C ２０ alkyl, optionally substituted C ２ to C ２０ alkenyl, optionally substituted C ２ to C ２０ alkynyl, optionally substituted C １ to C ２０ heteroalkyl, optionally substituted C ２ to C ２０ heteroalkenyl, optionally substituted C ２ to C ２０ heteroalkynyl, optionally substituted C ３ to C ２０ cycloalkyl, optionally substituted C ３ to C ２０ cycloalkenyl, optionally substituted C ２ to C ２０ heterocycloalkyl, optionally substituted C ２ to C ２０ heterocycloalkenyl, optionally substituted C ６ to C １８ aryl, or optionally substituted C ２ to C １９ heteroaryl, the conjugate according to any one of claims 1 to 21.

23. Y is 【Chemical 16】 The conjugate according to claim 22, or a pharmaceutically acceptable salt thereof.

24. q is 1 and Y is 【Chemical 17】 The conjugate according to claim 23, or a pharmaceutically acceptable salt thereof.

25. R ７ is 【Chemical 18】 and Y is 【Chemical Formula 19】 The conjugate according to claim 24, or a pharmaceutically acceptable salt thereof.

26. Y is the following for the conjugate according to claim 24, or a pharmaceutically acceptable salt thereof. 【Chemical 20】

27. q is 0 and Y is 【Chemical 21】 The conjugate according to claim 23, or a pharmaceutically acceptable salt thereof.

28. R Ｎ１ is H, 【Chemical 22】 The conjugate according to any one of claims 23 to 27, or a pharmaceutically acceptable salt thereof.

29. Y is 【Chemical 23】 The conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof.

30. Y is 【Chemical Formula 24】 The conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof.

31. Y is 【Chemical 25】 The conjugate according to claim 30, or a pharmaceutically acceptable salt thereof.

32. Y is 【Chemical 26】 The conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof.

33. Y is 【Chemical 27】 The conjugate according to claim 32, or a pharmaceutically acceptable salt thereof.

34. Y is ​ The conjugate according to claim 33, or a pharmaceutically acceptable salt thereof.

35. Y is 【Chemical 29】 The conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof.

36. Y is 【Chemical Formula 30】 The conjugate according to claim 35, or a pharmaceutically acceptable salt thereof.

37. Y is as follows, the conjugate according to claim 36, or a pharmaceutically acceptable salt thereof. 【Chemical 31】

38. L is C which is optionally substituted with one or more １ ~C ４０ alkylene, C which is optionally substituted １ ~C ４０ heteroalkylene, C which is optionally substituted １ ~C ４０ alkoxylene, C which is optionally substituted ２ ~C ２０ alkenylene, C which is optionally substituted ２ ~C ２０ heteroalkenylene, C which is optionally substituted ２ ~C ２０ alkynylene, C which is optionally substituted ２ ~C ２０ heteroalkynylene, C which is optionally substituted ３ ~C ２０ cycloalkylene, C which is optionally substituted ２ ~C ２０ heterocycloalkylene, C which is optionally substituted ４ ~C ２０ cycloalkenylene, C which is optionally substituted ４ ~C ２０ heterocycloalkenylene, C which is optionally substituted ８ ~C ２０ cycloalkynylene, C which is optionally substituted ８ ~C ２０ heterocycloalkynylene, C which is optionally substituted ５ ~C １５ arylene, C which is optionally substituted ２ ~C １５ heteroarylene, O, S, NR ｉ , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino, and R ｉ 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 ２ ~C ２０ heterocycloalkyl, optionally substituted C ４ ~C ２０ cycloalkenyl, optionally substituted C ４ ~C20 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, the conjugate according to any one of claims 1 to 37.

39. The conjugate according to any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof, wherein L is oxo-substituted.

40. The conjugate according to any one of claims 1 to 39, or a pharmaceutically acceptable salt thereof, wherein the skeleton of L contains 1 to 250 atoms.

41. The conjugate according to any one of claims 1 to 40, or a pharmaceutically acceptable salt thereof, wherein L is capable of forming an amide, carbamate, sulfonyl, or urea bond.

42. The conjugate according to any one of claims 3 to 41, or a pharmaceutically acceptable salt thereof, wherein L is represented by the following formula, 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 is a bond bonded to A １ and J ２ is a bond bonded to E or a functional group capable of reacting with a functional group conjugated with E, Q １ , Q ２ , Q ３ , Q ４ , and Q ５ each of which is independently, optionally substituted C1-C40 alkylene, optionally substituted C1-C40 heteroalkylene, optionally substituted C1-C40 alkoxylene, 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 C ２ -C ２０ 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, and T １ 、T ２ 、T ３ 、T ４ Each of T, T, T, and T is independently O, S, NR ｉ , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, imino, or oximo, 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 C ２ -C ２０ 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, and each of g, h, i, j, k, l, m, n, and o is independently 0, 1, or 2, the conjugate, or a pharmaceutically acceptable salt thereof.

43. Q １ The conjugate according to claim 42, or a pharmaceutically acceptable salt thereof, wherein Q is as follows. 【Chemical 32】

44. Q ２ is optionally substituted C １ -C ４０ alkylene, optionally substituted C １ -C ４０ heteroalkylene, optionally substituted C １ -C ４０ alkoxylene, or optionally substituted C ２ -C １５ heteroarylene, the conjugate according to claim 42 or 43, or a pharmaceutically acceptable salt thereof.

45. Q ３ is optionally substituted C ２ to C １５ is a heteroarylene, the conjugate according to any one of claims 42 to 44, or a pharmaceutically acceptable salt thereof.

46. Q ４ is C optionally substituted １ -C ４０ alkylene, C optionally substituted １ -C ４０ heteroalkylene, or C optionally substituted １ -C ４０ alkoxylene, the conjugate according to any one of claims 42 to 45, or a pharmaceutically acceptable salt thereof.

47. J ２ is 【Chemical 33】 The conjugate according to any one of claims 42 to 46, or a pharmaceutically acceptable salt thereof.

48. The conjugate represented by the formula in Table 2, or a pharmaceutically acceptable salt thereof.

49. The wavy line connected to E is each A １ -L or each A １ -L-A ２ The conjugate according to any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the L of is covalently bonded to the nitrogen atom of the solvent-exposed lysine of E.

50. The wavy line connected to E is each A １ -L or each A １ -L-A ２ The conjugate according to any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the L of is covalently bonded to the sulfur atom of the solvent-exposed cysteine of E.

51. The conjugate according to any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein n is 2 and each E dimerizes to form an Fc domain.

52. The conjugate according to any one of claims 1 to 51, or a pharmaceutically acceptable salt thereof, wherein each E is a human IgG1 Fc domain monomer.

53. The conjugate according to any one of claims 1 to 52, or a pharmaceutically acceptable salt thereof, wherein each E contains a substitution mutation at N297 selected from N297A, N297G, or N297Q, and the amino acid numbering of each Fc domain monomer follows the Kabat EU index.

54. The conjugate according to any one of claims 1 to 53, or a pharmaceutically acceptable salt thereof, wherein each E contains a C220S substitution mutation, and the amino acid numbering of each Fc domain monomer follows the Kabat EU index.

55. The conjugate according to any one of claims 1 to 54, or a pharmaceutically acceptable salt thereof, wherein each E contains M252Y, S254T, and T256E substitution mutations, and the amino acid numbering of each Fc domain monomer follows the Kabat EU index.

56. The conjugate according to any one of claims 1 to 52, or a pharmaceutically acceptable salt thereof, wherein each E contains an amino acid sequence of any one of SEQ ID NOs: 1 to 112 or 115 to 120.

57. The conjugate according to claim 56, or a pharmaceutically acceptable salt thereof, wherein each E contains the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO:

18.

58. The conjugate according to claim 56, or a pharmaceutically acceptable salt thereof, wherein each E contains the sequence of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 116, or SEQ ID NO:

120.

59. The conjugate according to any one of claims 1 to 58, or a pharmaceutically acceptable salt thereof, wherein T is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

60. A population of conjugates according to any one of claims 1 to 59, or a pharmaceutically acceptable salt thereof, wherein the average value of T is 1 to 10.

61. A population of conjugates according to claim 60, or a pharmaceutically acceptable salt thereof, wherein the average value of T is 1 to 5.

62. A pharmaceutical composition comprising the conjugate or population of conjugates according to any one of claims 1 to 61, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

63. A method for treating cancer in a subject, the method comprising administering to the subject the conjugate, population of conjugates, or pharmaceutical composition according to any one of claims 1 to 62.

64. The method according to claim 63, wherein the cancer is selected from lung cancer, optionally non-small cell lung cancer or small cell lung cancer, head and neck cancer, optionally squamous cell carcinoma, renal cell carcinoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, urothelial cancer, cholangiocarcinoma, endometrial cancer, melanoma, or esophageal cancer.

65. The method according to claim 63 or 64, wherein the cancer is a solid tumor.

66. The method according to any one of claims 63 to 65, wherein the cancer overexpresses, or is known to overexpress, CD73 compared to non-cancerous cells of the same tissue type.

67. The method according to any one of claims 63 to 66, further comprising administering an immune checkpoint inhibitor to the subject.

68. The method according to claim 67, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

69. A method for treating or preventing a viral infection in a subject, the method comprising administering to the subject the conjugate, population of conjugates, or pharmaceutical composition according to any one of claims 1 to 62.

70. The method according to claim 69, wherein the viral infection is a beta coronavirus infection.

71. The method according to claim 70, wherein the beta coronavirus is SARS-CoV-2.

72. The method according to claim 71, wherein the SARS-CoV-2 is an alpha, delta, or omicron variant.

73. The method according to claim 72, wherein the SARS-CoV-2 is an omicron variant.

74. The method according to claim 73, wherein the omicron variant is of the BA.1, BA.2, BA.3, BA.4, or BA.5 lineage.

75. The method according to any one of claims 69 to 74, further comprising administering an antiviral agent or antiviral vaccine to the subject.

76. A method for treating or preventing fibrosis in a subject, the method comprising administering to the subject the conjugate, population of conjugates, or pharmaceutical composition according to any one of claims 1 to 62.

77. The method according to claim 76, wherein the fibrosis is pulmonary fibrosis, skin fibrosis, renal fibrosis, hepatic fibrosis, cardiac fibrosis, or systemic sclerosis.

78. The method according to claim 77, wherein the fibrosis is pulmonary fibrosis.

79. The method according to claim 78, wherein the pulmonary fibrosis is associated with a viral infection, drug-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, pneumoconiosis, interstitial lung disease, sarcoidosis, silicosis, or systemic sclerosis.

80. The method according to any one of claims 63 to 79, wherein the conjugate, the population of conjugates, or the pharmaceutical composition is administered by intramuscular, intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrathoracic, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topical, intratumoral, intraperitoneal, subcutaneous, subconjunctival, intracavitary, transmucosal, epicardial, intraumbilical, intraocular, oral, topical, inhalation, injection, or infusion.

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