FC conjugates comprising CD73 inhibitors and uses thereof

Abstract

The present disclosure provides conjugates comprising an Fc domain monomer or an Fc domain covalently linked to a moiety that binds or inhibits CD73. The disclosure also provides pharmaceutical compositions comprising such conjugates and the use of such conjugates in the treatment of conditions associated with disorder or overexpression of CD73 (e.g., cancer, fibrosis or viral infection).

Description

Background Art

[0001] Cluster of differentiation 73 (CD73) is a glycosylphosphatidylinositol-linked membrane protein found in most tissues that catalyzes the conversion of extracellular adenosine monophosphate (AMP) to adenosine. It functions as a homodimer and can be shed and circulate as a soluble protein. 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 that occupy tumor lesions, including tumor cells, stromal cells, and endothelial cells, as well as infiltrating immune cells. CD73 levels are known to be upregulated due to tissue damage or hypoxic conditions, and many solid tumors have elevated CD73 levels. Upregulation of CD73 within tumors contributes to an adenosine-rich tumor microenvironment that has multiple pro-tumor and immunosuppressive effects. High CD73 tumor expression has been associated with shorter overall survival and poorer prognosis in certain cancers. CD73 in cancer patients has also been associated with resistance to anti-tumor therapy.

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

[0004] CD73 dysregulation has also been shown to play a key role in the pathogenesis of pulmonary fibrosis induced by radiation therapy or other insults to lung tissue. In mechanisms independent of its catalytic activity, multivalent ligation of the CD73 enzyme has been shown to stimulate B cell activation, clonal expansion, and the development 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 new treatments for conditions associated with CD73. The development of small molecule inhibitors of CD73 has been hampered by poor metabolic stability. Given the role that CD73 plays in cancer and a wide variety of other diseases, disorders, and conditions, and the current lack of CD73 inhibitors available to practitioners, there is a need for new CD73 inhibitors and compositions and methods related thereto. Summary of the Invention

[0006] The present disclosure relates to conjugates comprising an Fc domain monomer or an Fc domain covalently linked to a portion that binds to or inhibits CD73. Specifically, such conjugates contain a monomer or dimer that binds to or inhibits a portion of CD73 conjugated to an Fc monomer or Fc domain. The Fc monomer or Fc domain in the conjugate binds to FcγR (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells (e.g., neutrophils) to activate phagocytosis and effector functions, such as 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 conditions associated with dysregulation or overexpression of CD73 (e.g., cancer, fibrosis, or viral infection).

[0007] In one aspect, the disclosure features a conjugate described by formula (DI) or (MI), or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Among them A 1 and A 2 Each of which independently has the structure of formula (A):

[0010]

[0011] m is 0, 1, 2, 3, 4, 5 or 6;

[0012] s is 0 or 1;

[0013] X 1 、X 2 、X 3 、X 4 、X 5 and X 6 Each of which is independently N, CR 4 or CYR 5 , where X 1 、X 2 、X 3 、X 4 、X 5 and X 6 At least one of them is CYR 5 And R 5 is the bond with L;

[0014] R 1 for

[0015] Z is O, S or sulfonyl;

[0016] R2a and R 2b Each of which is independently H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 heteroaryl;

[0017] Each R 3 are independently OH, SH, halogen, optionally substituted amino, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 heteroaryl;

[0018] R 4 is H, halogen, OH, SH, optionally substituted amino, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 heteroaryl;

[0019] R 6a and R 6b Each of which is independently H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 heteroaryl;

[0020] Y is the first connector;

[0021] L is the second connector;

[0022] n is 1 or 2;

[0023] Each E comprises an Fc domain monomer;

[0024] T is an integer from 1 to 20; and

[0025] The curved line indicates that L is covalently attached to E.

[0026] In some embodiments, the conjugate is described by formula (DI):

[0027]

[0028] In some embodiments, the conjugate is described by Formula (MI):

[0029]

[0030] In some embodiments, A 1 and A 2 Having the structure of formula (AI):

[0031]

[0032] In some embodiments, A 1 and A 2 Each has the structure of formula (A-Ia):

[0033]

[0034] In some embodiments, A 1 and A 2 Each has the structure of formula (A-Ib):

[0035]

[0036] In some embodiments, A 1 and A 2 Each has the structure of formula (A-Ib-1):

[0037]

[0038] In some embodiments, A 1 and A 2 Each has the structure of formula (A-Ib-2):

[0039]

[0040] In some embodiments, A 1 and A 2 Each has the structure of formula (A-II):

[0041]

[0042] In some embodiments, A 1 and A 2 Each has the structure of formula (A-IIa):

[0043]

[0044] In some embodiments, A 1 and A 2 Each has the structure of formula (A-IIb):

[0045]

[0046] In some embodiments, A 1 and A 2 Each has the structure of formula (A-IIb-1):

[0047]

[0048] In some embodiments, A 1 and A 2Each has the structure of formula (A-IIb-2):

[0049]

[0050] In some embodiments, s is 0. In some embodiments, s is 1.

[0051] In some embodiments, R 2a and R 2b Each of which is independently H, optionally substituted C1-C 20 Alkyl or optionally substituted C1-C 20 In some embodiments, R 2a and R 2b Each of them is H.

[0052] In some embodiments, R 4 is H, halogen, OH, SH, optionally substituted amino, optionally substituted C1-C 20 Alkyl or optionally substituted C1-C 20 In some embodiments, R 4 In some embodiments, R 4 For Cl.

[0053] In some embodiments, R 1 for

[0054] In some embodiments, R 1 for

[0055] In some embodiments, R 6a and R 6b Each of which is independently H, optionally substituted C1-C 20 Alkyl or optionally substituted C1-C 20 In some embodiments, R 6a and R 6b Each of R is independently H, -CH3, -CH2CH3, -CH2OH, -CH2OCH3-CH2CH2OH or -CH2CH2OCH3. 6a and R 6b Each of them is H.

[0056] In some embodiments, Y is:

[0057]

[0058] Each of p1, p2, p3 and p4 is independently 0, 1, 2, 3 or 4;

[0059] q is 0, 1, 2, 3, or 4;

[0060] Each X 7 are independently N or CH;

[0061] Each X 8 are independently O, NH, CH2 or C(=O);

[0062] R N1 is H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl, optionally substituted C2-C 19 Heteroaryl, optionally substituted C1-C 20 Alkaryl or optionally substituted C1-C 20 alkylcycloalkyl;

[0063] Each R 7 Independently Optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl or optionally substituted C2-C 20 heterocycloalkenyl;

[0064] R 7a and R 7b Each of which is independently H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 heteroaryl; and

[0065] Each R 8 are independently halogen, OH, SH, optionally substituted amino, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 Heteroaryl.

[0066] In some embodiments, Y is In some embodiments, q is 1 and Y is In some embodiments, R 7 for And Y is In some embodiments, Y is In some embodiments, q is 0 and Y is In some embodiments, R N1 For H,

[0067] In some embodiments, Y is

[0068] In some embodiments, Y is In some embodiments, Y is

[0069] In some embodiments, Y is In some embodiments, Y is In some embodiments, Y is

[0070] In some embodiments, Y is In some embodiments, Y is In some embodiments, Y is In some embodiments, Y is

[0071] In some embodiments, L comprises one or more optionally substituted C1-C 40 Alkylene, optionally substituted C1-C 40 Heteroalkylene, optionally substituted C1-C 40 Alkyleneoxy, optionally substituted C2-C 20 Alkenylene, optionally substituted C2-C 20 Heteroalkenylene, optionally substituted C2-C 20 Alkynylidene, 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 Heterocycloalkenyl, optionally substituted C8-C 20 Cycloalkynylene, optionally substituted C8-C 20 Heterocycloalkynyl, optionally substituted C5-C 15 Arylene, optionally substituted C2-C 15 Heteroarylene, O, S, NR i , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino,

[0072] where 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 heterocycloalkenyl, 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.

[0073] In some embodiments, L is oxo-substituted. In some embodiments, L comprises 1 to 250 atoms. In some embodiments, L is capable of forming an amide, carbamate, sulfonyl, or urea bond.

[0074] In some embodiments, L is described by the formula:

[0075] 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

[0076] Among them J 1 is the key attached to A1;

[0077] J 2 is a bond attached to E or a functional group capable of reacting with a functional group conjugated to E;

[0078] Q 1 , Q 2 , Q 3 , Q 4 and Q 5 Each of the groups is independently an optionally substituted C1-C40 alkylene, an optionally substituted C1-C40 heteroalkylene, an optionally substituted C1-C40 alkyleneoxy, an optionally substituted C2-C20 alkenylene, an optionally substituted C2-C20 heteroalkenylene, an optionally substituted C2-C20 alkynylene, an optionally substituted C2-C20 heteroalkynylene, an optionally substituted C3-C20 cycloalkylene, an 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;

[0079] T 1 、T 2 、T 3 、T 4Each of which is independently O, S, NR i , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, imino or oxime;

[0080] 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 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

[0081] Each of g, h, i, j, k, l, m, n and o is independently 0, 1 or 2.

[0082] In some embodiments, Q 1 for

[0083]

[0084] In some embodiments, Q 2 is an optionally substituted C1-C 40 Alkylene, optionally substituted C1-C 40 Heteroalkylene, optionally substituted C1-C 40 Alkyleneoxy or optionally substituted C2-C 15 Heteroarylene.

[0085] In some embodiments, Q 3 is an optionally substituted C2-C 15 Heteroarylene.

[0086] In some embodiments, Q 4 is an optionally substituted C1-C 40 Alkylene, optionally substituted C1-C 40 Heteroalkylene or optionally substituted C1-C 40 Alkyleneoxy.

[0087] In some embodiments, J 2 for

[0088] The present disclosure features the intermediates (Int) of Table 1. These intermediates include one or more CD73 inhibitors and linkers and can be used to synthesize 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 any method described or exemplified herein). In some embodiments, the conjugate comprises E, wherein E is an Fc domain monomer or an Fc domain. In preferred embodiments, one or more nitrogen atoms of one or more surface-exposed lysine residues of E or one or more sulfur atoms of one or more surface-exposed cysteines in E are covalently conjugated to a linker (e.g., PEG2-PEG 20 Linker). The linker conjugated to E can be functionalized so that it can react with any Int described herein (e.g., Int of Table 1) to form a covalent bond. In a preferred embodiment, E is conjugated to a linker functionalized with an azido group, and Int (e.g., Int of Table 1) is functionalized with an alkyne group. The linker-azido of E is conjugated with the linker-alkyne of Int (e.g., by click chemistry) to form a conjugate of the present disclosure. In yet other embodiments, E is conjugated to a linker functionalized with an alkyne group, and Int (e.g., Int of Table 1) is functionalized with an azido group. The linker-alkyne of E is conjugated with the linker-azido of Int (e.g., by click chemistry) to form a conjugate of the present disclosure. In yet other embodiments, Int (e.g., Int of Table 2) is functionalized with a phenyl ester group (e.g., a trifluorophenyl ester group or a tetrafluorophenyl ester group). The conjugation of E to a linker-phenyl ester (e.g., trifluorophenyl ester or tetrafluorophenyl ester) of Int (e.g., by acylation) forms a conjugate of the invention. The conjugation of E to a linker-phenyl ester (e.g., trifluorophenyl ester or tetrafluorophenyl ester) of Int (e.g., by acylation) is performed by methods described herein or known in the art.

[0089] The disclosure also features compositions having the structure of Int of Table 1 (eg, pre-conjugated intermediates).

[0090] Table 1. Intermediates

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] The present disclosure also features the conjugates of Table 2. Each conjugate of Table 2 corresponds to a conjugate of formula (DI) or (MI). The conjugates of Table 2 include conjugates formed by the covalent reaction of Int of Table 1 with E. The conjugates of Table 2 also include conjugates formed by the covalent reaction of Int of Table 1 with a linker (which is in turn conjugated to E). In some embodiments, the reactive moiety of Int (e.g., an alkyne or azido group) reacts with a corresponding reactive group (e.g., an alkyne or azido group) of a linker covalently attached to E, such that Int of Table 1 is covalently attached to E. In some embodiments, the reactive moiety of Int (e.g., a phenyl ester group, such as a tetrafluorophenyl ester or trifluorophenyl ester group) reacts with a corresponding reactive group (e.g., a nitrogen atom or a sulfur atom) of an amino acid side chain of E, such that Int of Table 1 is covalently attached to E.

[0139] In some embodiments of any of the conjugates of Table 2, n is 1 or 2. When n is 1, E comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-112 and 115-120). When n is 2, each E comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-112 and 115-120), and the Fc domain monomers dimerize to form an Fc domain.

[0140] In some embodiments of any of the conjugates of 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 a population of any of the conjugates of Table 2, wherein 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.

[0141] The curved lines in the conjugates of Table 2 indicate that each Int is covalently attached to an amino acid side chain in E (eg, a nitrogen atom of a surface-exposed lysine or a sulfur atom of a surface-exposed cysteine ​​in E), or a pharmaceutically acceptable salt thereof.

[0142] The present disclosure also provides the conjugate of Table 2, wherein the conjugate is produced by conjugating (eg, via a linker) Int of Table 1 to an Fc domain or an Fc domain monomer.

[0143] Table 2. Conjugates corresponding to selected intermediates in Table 1

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] The present disclosure also features a method of preparing an Fc conjugate by conjugating (e.g., via a linker) an Int of Table 1 to an Fc domain monomer or an Fc domain. In some embodiments, the present disclosure provides a conjugate, wherein the conjugate comprises a small molecule targeting agent, wherein the targeting agent is described by an Int of Table 1, conjugated to Fc (e.g., via a linker).

[0163] In some embodiments, the curved line connected to E indicates that each A1-L or each L of A1-L-A2 is covalently attached to a nitrogen atom of a solvent-exposed lysine of E. In some embodiments, the curved line connected to E indicates that each A1-L or each L of A1-L-A2 is covalently attached to a sulfur atom of a solvent-exposed cysteine ​​of E.

[0164] In some embodiments, n is 2, and each E dimerizes to form an Fc domain.

[0165] In some embodiments, each E is a human IgG1 Fc domain monomer. In some embodiments, each E comprises a substitution mutation selected from N297A, N297G, or N297Q at N297, wherein the amino acid numbering of each Fc domain monomer is according to the Kabat EU index. In some embodiments, each E comprises a C220S substitution mutation, wherein the amino acid numbering of each Fc domain monomer is according to the Kabat EU index. In some embodiments, each E comprises M252Y, S254T, and T256E substitution mutations, wherein the amino acid numbering of each Fc domain monomer is according to the Kabat EU index.

[0166] 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 any one of SEQ ID NOs: 1-112 and 115-120. In some embodiments, each E comprises an amino acid sequence of any one of SEQ ID NOs: 1-112 and 115-120.

[0167] 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 any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18. In some embodiments, each E comprises an amino acid sequence of any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, each E comprises the amino acid sequence of SEQ ID NO: 18.

[0168] 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 any one of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. In some embodiments, each E comprises an amino acid sequence of any one 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.

[0169] In some embodiments, each E comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 115. In some embodiments, each E comprises an 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 with the amino acid sequence of SEQ ID NO: 116. In some embodiments, each E comprises an 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 with the amino acid sequence of SEQ ID NO: 117. In some embodiments, each E comprises an 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 with the amino acid sequence of SEQ ID NO: 118. In some embodiments, each E comprises an 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 an amino acid sequence of SEQ ID NO: 119. In some embodiments, each E comprises an 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 an amino acid sequence of SEQ ID NO: 120. In some embodiments, each E comprises an amino acid sequence of SEQ ID NO: 120.

[0170] In some embodiments, n is 1 and T represents the number of A1-L or A1-L-A2 moieties bound to each E. In some embodiments, n is 2 and two E dimerize to form an Fc domain, and T represents the number of A1-L or A1-L-A2 moieties bound 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).

[0171] The present disclosure also provides a population of conjugates described herein, wherein the average value of T is between 1 and 20 (e.g., the average value of T is between 1 and 2, 1 and 3, 1 and 4, 1 and 5, 5 and 10, 10 and 15, or 15 and 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.

[0172] 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.

[0173] In another aspect, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject a conjugate, a population of conjugates, or a pharmaceutical composition described herein.

[0174] 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 carcinoma; bile duct cancer; 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 relative to non-cancerous cells of the same tissue type.

[0175] In some embodiments, the method further comprises administering to the subject an immune checkpoint inhibitor. 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.

[0176] 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, 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. In some embodiments, the method further comprises administering to the subject an antiviral agent or an antiviral vaccine.

[0177] In another aspect, the present disclosure provides a method for treating or preventing fibrosis in a subject, the method comprising administering a conjugate as described herein, a colony of a conjugate, or a pharmaceutical composition to the subject. In some embodiments, fibrosis is pulmonary fibrosis, skin fibrosis, renal fibrosis, liver fibrosis, cardiac fibrosis, or systemic sclerosis. In some embodiments, fibrosis is pulmonary fibrosis. In some embodiments, pulmonary fibrosis is associated with 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.

[0178] In some embodiments of any of the methods of treatment described herein, the conjugate, population of conjugates, or pharmaceutical composition is administered intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, by injection, or by infusion.

[0179] definition

[0180] 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 of ordinary skill in the art to which the present invention relates. Terms such as "a," "an," and "the" are not intended to refer only to a singular entity, but also to general categories for which specific examples may be used for illustration. The terms herein are used to describe specific embodiments of the present invention, but their use does not limit the present invention, except as outlined in the claims.

[0181] As used herein, the term "Fc domain monomer" refers to an Fc domain monomer comprising at least a hinge domain and second and third antibody constant domains (C H 2 and C H3) or its functional fragment (for example, a fragment capable of the following operations: (i) dimerization with another Fc domain monomer to form an Fc domain, and (ii) binding to an Fc receptor) polypeptide chain. The Fc domain monomer can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA or IgD (for example, IgG). In addition, the Fc domain monomer can be an IgG subtype (for example, IgG1, IgG2a, IgG2b, IgG3 or IgG4) (for example, IgG1). In some embodiments, the Fc domain monomer does not include any part of an immunoglobulin that can serve as an antigen recognition region, such as a variable domain or a complementary determining region (CDR). The Fc domain monomer in the compositions as described herein can contain one or more changes (for example, 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions or deletions) relative to the wild-type Fc domain monomer sequence, which changes the interaction between the Fc domain and the Fc receptor. Examples of suitable variations are known in the art. In certain embodiments, human Fc domain monomers (e.g., IgG heavy chains, such as IgG1) comprise a region extending from any one of Asn208, Glu216, Asp221, Lys222, or Cys226 to the carboxyl-terminal Lys447 of the heavy chain. The C-terminal Lys447 of the Fc region may be present or absent without affecting the structure or stability of the Fc region. After polypeptide expression, the C-terminal Lys447 may be proteolytically cleaved. In some embodiments of any Fc domain monomer described herein, the C-terminal Lys447 is optionally present or absent. The N-terminal N (e.g., Asn201) of the Fc region may be present or absent without affecting the structure or stability of the Fc region. The N-terminal Asn may be deamidated during polypeptide expression. In some embodiments of any Fc domain monomer described herein, the N-terminal Asn is optionally present or absent. Unless otherwise indicated herein, the numbering of amino acid residues in an IgG or Fc domain monomer is according to the EU numbering system for antibodies (also known as the Kabat EU index), as described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0182] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers capable of binding to an Fc receptor. In a wild-type Fc domain, the two Fc domain monomers are bound by two C H3. Interactions between antibody constant domains Dimerization. In some embodiments, one or more disulfide bonds are formed between the hinge domains of two dimerized Fc domain monomers.

[0183] The terms "Fab" or "antigen-binding fragment," as used interchangeably herein, refer to the region of an antibody that binds to an antigen. Fab is a term of art, and its meaning is known to those skilled in the art. The Fab region is composed of one constant region and one variable region of each heavy and light chain. 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), interspersed with more conserved regions, called "framework regions" (FRs). In antibodies, the heavy chains (e.g., VH and CH regions) are connected to the Fc domain monomer by a hinge. The Fc domain monomers described herein can comprise 10 and / or 20 residues (e.g., 11, 12, 13, 14, 15, 16, 17, 18, or 19 residues) of the Fab domain and the hinge region. In certain embodiments, the N-terminus of an Fc domain monomer is any one of amino acid residues 198-205 (corresponding to residues of a Fab domain). In some embodiments, the N-terminus of an Fc domain monomer is amino acid residue 201 (e.g., Asn 201). In certain embodiments, the N-terminus of an Fc domain monomer is amino acid residue 202 (e.g., Val 202).

[0184] The term "covalently linked" means that the two parts of the conjugate are linked to each other via a covalent bond formed between two atoms in the two parts of the conjugate.

[0185] As used herein, "surface-exposed amino acids" or "solvent-exposed amino acids," such as surface-exposed cysteine ​​or surface-exposed lysine, refer to amino acids that are accessible to the solvent surrounding a protein. Surface-exposed amino acids can be naturally occurring or engineered variants (e.g., substitutions or insertions) of a protein. In some embodiments, surface-exposed amino acids are amino acids that, when substituted, do not substantially alter the three-dimensional structure of the protein.

[0186] As used herein, the term "linker" refers to a covalent bond or connection between two or more components in a conjugate (e.g., between two CD73 inhibitors in a conjugate described herein, between a CD73 inhibitor and an Fc domain in a conjugate described herein, and between a dimer of two CD73 inhibitors and an Fc domain in a conjugate described herein). In some embodiments, the conjugates described herein may comprise a linker having a bivalent structure (e.g., a bivalent linker). A bivalent linker has two arms, wherein each arm is covalently linked to a component of the conjugate (e.g., a first arm is conjugated to a CD73 inhibitor and a second arm is conjugated to an Fc domain). In some embodiments, the conjugates described herein may comprise a linker having a trivalent structure (e.g., a trivalent linker). A trivalent linker has three arms, wherein each arm is covalently linked to a component of the conjugate (e.g., a first arm is conjugated to a CD73 inhibitor, a second arm is conjugated to a second CD73 inhibitor, and a third arm is conjugated to an Fc domain). The linkers of the present disclosure may be linear or branched.

[0187] In some embodiments, a molecule useful as a linker comprises at least two functional groups, which may be the same or different, such as 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 may form a covalent bond with the first component of the conjugate, and the second functional group may form a covalent bond with the second component of the conjugate. In some embodiments of a trivalent linker, two arms of the linker may contain two dicarboxylic acids, wherein the first carboxylic acid may form a covalent bond with the first CD73 inhibitor in the conjugate and the second carboxylic acid may form a covalent bond with the second CD73 inhibitor in the conjugate, and the third arm of the linker may form a covalent bond with the Fc domain in the conjugate. Examples of dicarboxylic acids are further described herein. In some embodiments, molecules containing one or more maleimide groups can be used as linkers, wherein the maleimide group can form a carbon-sulfur bond with a cysteine ​​in a component (e.g., an Fc domain) in the conjugate. In some embodiments, molecules containing one or more alkyne groups can be used as linkers, wherein the alkyne group can form a 1,2,3-triazole bond with an azide group in a component (e.g., an Fc domain) in the conjugate. In some embodiments, molecules containing one or more azide groups can be used as linkers, wherein the azide group can form a 1,2,3-triazole bond with an alkyne in a component (e.g., an Fc domain) in the conjugate. In some embodiments, molecules containing one or more bissulfone groups can be used as linkers, wherein the bissulfone group can form a bond with an amine group of a component (e.g., an Fc domain) in the conjugate. In some embodiments, molecules containing one or more sulfonic acid groups can be used as linkers, wherein the sulfonic acid group can form a sulfonamide bond with a component in the conjugate. In some embodiments, molecules containing one or more isocyanate groups can be used as linkers, wherein the isocyanate group can form a urea bond with a component in the conjugate. In some embodiments, molecules containing one or more haloalkyl groups can be used as linkers, wherein the haloalkyl groups can form covalent bonds, such as C-N bonds and C-O bonds, with components in the conjugate. In some embodiments, molecules containing one or more phenyl ester groups (e.g., trifluorophenyl ester groups or tetrafluorophenyl ester groups) can be used as linkers, wherein the phenyl ester groups (e.g., trifluorophenyl ester groups or tetrafluorophenyl ester groups) can form amides with amines in components in the conjugate (e.g., fusion proteins).

[0188] 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, such as a covalent bond. The term "bond" refers to a chemical bond, such as an amide bond, a disulfide bond, a CO bond, a CN bond, a NN bond, a CS bond, or any type of bond formed by a chemical reaction, such as a chemical conjugation. In some embodiments, the linker contains no more than 250 atoms. In some embodiments, the linker contains no more than 250 non-hydrogen atoms. In some embodiments, the backbone of the linker contains no more than 250 atoms. The "backbone" of the linker refers to the atoms in the linker that together form the shortest path from one part of the conjugate to another part of the conjugate (e.g., the shortest path connecting the first CD73 inhibitor and the second CD73 inhibitor). The atoms in the linker backbone are directly involved in connecting one part of the conjugate to another part of the conjugate (e.g., connecting the first CD73 inhibitor and the second CD73 inhibitor). For example, a hydrogen atom attached to a carbon in the linker backbone is not considered to be directly involved in connecting one part of the conjugate to another part of the conjugate.

[0189] In some embodiments, the linker can contain 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, such as D-amino acid residues or L-amino acid residues. In some embodiments, the linker can be a residue of an amino acid sequence (e.g., 1-25 amino acids, 1-10 amino acids, 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, 1-2 amino acids, or 1 amino acid sequence). In some embodiments, the linker can comprise one or more (e.g., 1-100, 1-50, 1-25, 1-10, 1-5, or 1-3) optionally substituted alkylene, optionally substituted heteroalkylene (e.g., PEG units), 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 iwherein R is 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 comprise 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-C 20 C-C15 aryl or optionally substituted C2-C15 heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino.

[0190] As used herein, the terms "alkyl," "alkenyl," and "alkynyl" include linear and branched monovalent substituents, as well as combinations of these substituents, containing only C and H when unsubstituted. When an alkyl group contains at least one carbon-carbon double bond or carbon-carbon triple bond, the alkyl group may be referred to as an "alkenyl" or "alkynyl" group, respectively. The monovalence of an alkyl, alkenyl, or alkynyl group does not include optional substituents on the alkyl, alkenyl, or alkynyl group. For example, if an alkyl, alkenyl, or alkynyl group is attached to a compound, the monovalence of the alkyl, alkenyl, or alkynyl group refers to its attachment to the compound and does not include any additional substituents that may be present on the alkyl, alkenyl, or alkynyl group. In some embodiments, an alkyl or heteroalkyl group can contain, for example, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2 carbon atoms (e.g., C1-C20, C1-C18, C1-C16, C1-C14, C1-C12, C1-C10, C1-C8, C1-C6, C1-C4, or C1-C2). In some embodiments, alkenyl, heteroalkenyl, alkynyl or heteroalkynyl groups can contain, for example, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6 or 2-4 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. Heterocycloalkyl, heterocycloalkenyl or heterocycloalkynyl groups refer to cycloalkyl, cycloalkenyl or cycloalkynyl groups having one or more heteroatoms independently selected from, for example, N, O and S. Exemplary heterocycloalkyls include pyrrolidine, thiophene, thiolane, tetrahydrofuran, piperidine and tetrahydropyran.

[0191] As used herein, term " cycloalkyl " represents monovalent saturated or unsaturated non-aromatic cyclic alkyl group.Cycloalkyl can have, for example, three to twenty carbons (for example, C3-C7, C3-C8, C3-C9, C3-C10, C3-C11, C3-C12, C3-C14, C3-C16, C3-C18 or C3-C20 cycloalkyl).The example of cycloalkyl includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.When cycloalkyl group includes at least one carbon-carbon double bond, cycloalkyl group is referred to as " cycloalkenyl " group.Cycloalkenyl can have, for example, four to twenty carbons (for example, 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 cycloalkyl group includes at least one carbon-carbon triple bond, cycloalkyl group is called " cycloalkynyl " group.Cycloalkynyl can have for example eight to twenty carbons (for example, C8-C9, C8-C10, C8-C11, C8-C12, C8-C14, C8-C16, C8-C18 or C8-C20 cycloalkynyl).Term " cycloalkyl " also includes the cyclic compound with bridged polycyclic structure, wherein two non-adjacent members of one or more carbon bridged monocycles, for example bicyclo [2.2.1.] heptyl and adamantane.Term " cycloalkyl " also includes bicyclo, tricyclo and tetracyclic condensed ring structure, for example decalin and spirocyclic compound.

[0192] As used herein, the term "aryl" refers to any monocyclic or fused bicyclic or tricyclic ring system with aromatic characteristics in terms of the electron distribution of the entire ring system, such as phenyl, naphthyl or phenanthrene. In some embodiments, the ring system contains 5-15 ring member atoms or 5-10 ring member atoms. The aryl group can have, for example, five to fifteen carbons (for example, C5-C6, C5-C7, C5-C8, C5-C9, C5-C10, C5-C11, C5-C12, C5-C13, C5-C14 or C5-C15 aryl). The term "heteroaryl" also refers to such monocyclic or fused bicyclic ring systems containing one or more (for example, 1-4, 1-3, 1, 2, 3 or 4) heteroatoms selected from O, S and N. Heteroaryl groups can have, for example, two to fifteen carbons (for example, C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C2-C9, C2-C10, C2-C11, C2-C12, C2-C13, C2-C14 or C2-C15 heteroaryl). Comprise heteroatoms and allow to comprise 5-membered rings and 6-membered rings that are considered to be aromatic. Therefore, typical heteroaryl systems include, for example, pyridyl, pyrimidinyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, benzoxazolyl, benzisoxazolyl and imidazolyl. Because tautomers are possible, groups such as phthalimido are also considered to be heteroaryl. In some embodiments, aryl or heteroaryl groups are 5-yuan or 6-yuan aromatic ring systems optionally containing 1-2 nitrogen atoms. In some embodiments, aryl or heteroaryl groups are optionally substituted phenyl, pyridyl, indolyl, pyrimidinyl, pyridazinyl, benzothiazolyl, benzimidazolyl, pyrazolyl, imidazolyl, isoxazolyl, thiazolyl or imidazopyridinyl. In some embodiments, aryl groups are phenyl. In some embodiments, aryl groups can be substituted by substituents such as aryl substituents, for example, biphenyl is optionally substituted.

[0193] The term "alkaryl" refers to an aryl group connected to an alkylene, alkenylene or alkynylene group. Generally speaking, if 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 a C6-C35 alkaryl (e.g., C6-C16, C6-C14, C6-C12, C6-C10, C6-C9, C6-C8, C7 or C6 alkaryl), wherein the number of carbons indicates the total number of carbons in 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 a benzyl or phenethyl group. In heteroalkaryl groups, one or more heteroatoms selected from N, O and S may be present in the alkylene, alkenylene or alkynylene portion of the alkaryl group and / or may be present in the aryl portion of the alkaryl group. In optionally substituted alkaryl groups, substituents may be present in the alkylene, alkenylene or alkynylene portion of the alkaryl group and / or may be present in the aryl portion of the alkaryl group.

[0194] As used herein, the term "amino" refers to -N(R x )2 or –N + (R x )3, where each R x are independently H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R x In some embodiments, the amino group is -NH2.

[0195] As used herein, the term "alkylamino" refers to an amino group as described herein that is attached to an alkylene (e.g., C1-C5 alkylene), alkenylene (e.g., C2-C5 alkenylene), or alkynylene group (e.g., C2-C5 alkenylene). Generally, if a compound is attached to an alkylamino group, the alkylene, alkenylene, or alkynylene portion of the alkylamino group is attached to the compound. The amino portion of an alkylamino group refers to a -N(R x )2 or –N + (R x )3, where each R x are independently H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R xIn some embodiments, the amino group of the alkylamino group is -NH2. Examples of alkylamino groups are C1-C5 alkylamino groups, such as C2 alkylamino groups (e.g., CH2CH2NH2 or CH2CH2N(CH3)2). In heteroalkylamino groups, 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 heteroalkylamino group. In some embodiments, the alkylamino group may be optionally substituted. In substituted alkylamino groups, substituents may be present in the alkylene, alkenylene, or alkynylene moiety of the alkylamino group and / or may be present in the amino group of the alkylamino group.

[0196] As used herein, the term "alkanoamide" 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 alkenylene) group. Generally, if a compound is attached to an alkanoamide group, the alkylene, alkenylene, or alkynylene portion of the alkanoamide is attached to the compound. The amide portion of an alkanoamide refers to -C(O)-N(R x )2, where each R x are independently H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R x Combine to form a heterocycloalkyl. In some embodiments, the amide portion of the alkaneamide is -C(O)NH2. The alkaneamide group can be (CH2)2-C(O)NH2 or -CH2-C(O)NH2. In a heteroalkaneamide group, one or more (e.g., 1-4, 1-3, 1, 2, 3, or 4) heteroatoms selected from N, O, and S may be present in the alkylene, alkenylene, or alkynylene portion of the heteroalkaneamide group. In some embodiments, the alkaneamide group may be optionally substituted. In a substituted alkaneamide group, substituents may be present in the alkylene, alkenylene, or alkynylene portion of the alkaneamide group and / or may be present in the amide portion of the alkaneamide group.

[0197] As used herein, the terms "alkylene," "alkenylene," and "alkynylene" refer to divalent groups of a specified size. In some embodiments, the alkylene group may contain, for example, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2 carbon atoms (e.g., C1-C20, C1-C18, C1-C16, C1-C14, C1-C12, C1-C10, C1-C8, C1-C6, C1-C4, or C1-C2). In some embodiments, the alkenylene or alkynylene group may contain, for example, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, or 2-4 carbon atoms (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 groups include straight and branched forms, as well as combinations of these forms. The divalency of an alkylene, alkenylene, or alkynylene group does not include optional substituents on the alkylene, alkenylene, or alkynylene group. For example, two CD73 inhibitors can be attached to each other via a linker comprising an alkylene, alkenylene, and / or alkynylene group, or a combination thereof. Each of the alkylene, alkenylene, and / or alkynylene groups in the linker is considered divalent with respect to two attachments at either terminus of the alkylene, alkenylene, and / or alkynylene group. For example, if the linker comprises -(optionally substituted alkylene)-(optionally substituted alkenylene)-(optionally substituted alkylene)-, the alkenylene group is considered divalent with respect to its attachment to two alkylene groups at the termini of the linker. The optional substituents on the alkenylene group are not included in the divalency of the alkenylene group. The divalent nature of an alkylene, alkenylene, or alkynylene group (e.g., an alkylene, alkenylene, or alkynylene group in the linker) refers to both termini of the group and does not include optional substituents that may be present in the alkylene, alkenylene, or alkynylene group. Because they are divalent, they can link together multiple (e.g., two) moieties of the conjugate (e.g., a first CD73 inhibitor and a second CD73 inhibitor). Alkylene, alkenylene and / or alkynylene groups may be substituted with groups that are generally suitable as substituents for alkyl, alkenyl and alkynyl groups as described herein. For example, C=O is a C1 alkylene substituted with oxo (=O). For example, -HCR-C≡C- can be considered an optionally substituted alkynylene and is considered a divalent group, even if it has an optional substituent R. Heteroalkylene, heteroalkenylene and / or heteroalkynylene groups refer to alkylene, alkenylene and / or alkynylene groups that include one or more (e.g., 1-4, 1-3, 1, 2, 3 or 4) heteroatoms (e.g., N, O and S). For example, a polyethylene glycol (PEG) polymer or a PEG unit in a PEG polymer, -(CH2)2-O-, is considered a heteroalkylene containing one or more oxygen atoms.

[0198] As used herein, the term "cycloalkylene" refers to a divalent cyclic group that connects two parts of a compound together. For example, a carbon in a cycloalkylene group can be connected to a part of the compound, and another carbon in a cycloalkylene group can be connected to another part of the compound. A cycloalkylene group can include saturated or unsaturated non-aromatic cyclic groups. Cycloalkylene can have, for example, three to twenty carbons (for example, C3-C7, C3-C8, C3-C9, C3-C10, C3-C11, C3-C12, C3-C14, C3-C16, C3-C18 or C3-C20 cycloalkylene) in the cyclic portion of cycloalkylene. When a cycloalkylene group includes at least one carbon-carbon double bond, the cycloalkylene group is referred to as a "cycloalkenylene" group. Cycloalkenylene can have, for example, four to twenty carbons (for example, C4-C7, C4-C8, C4-C9, C4-C10, C4-C11, C4-C12, C4-C14, C4-C16, C4-C18 or C4-C20 cycloalkenylene) in the cyclic portion of cycloalkenylene. When the cycloalkylene group includes at least one carbon-carbon triple bond, the cycloalkylene group is referred to as a "cycloalkynylene" group. The cycloalkynylene group can have, for example, four to twenty carbons (for example, C4-C7, C4-C8, C4-C9, C4-C10, C4-C11, C4-C12, C4-C14, C4-C16, C4-C18 or C8-C20 cycloalkynylene) in the cyclic portion of the cycloalkynylene. The cycloalkylene group can be substituted by a group that is generally suitable as a substituent of an alkyl, alkenyl and alkynyl group as described herein. Heterocycloalkylene refers to a cycloalkylene group containing one or more (e.g., 1-4, 1-3, 1, 2, 3, or 4) heteroatoms (e.g., N, O, and S). Examples of cycloalkylene groups include, but are not limited to, cyclopropylene and cyclobutylene. Tetrahydrofuran can be considered a heterocycloalkylene group.

[0199] As used herein, the term "arylene" refers to a polyvalent (e.g., divalent or trivalent) aryl group by which multiple (e.g., two or three) parts of a compound are connected together. For example, a carbon in the arylene group can be connected to a part of the compound, and another carbon in the arylene group can be connected to another part of the compound. Arylene can have, for example, five to fifteen carbons (e.g., C5-C6, C5-C7, C5-C8, C5-C9, C5-C10, C5-C11, C5-C12, C5-C13, C5-C14 or C5-C15 arylene) in the aryl moiety of arylene. Arylene groups can be substituted by groups that are generally suitable as substituents of alkyl, alkenyl and alkynyl groups as described herein. Heteroarylene refers to an aromatic group comprising one or more (e.g., 1-4, 1-3, 1, 2, 3 or 4) heteroatoms (e.g., N, O and S). A heteroarylene group can have, for example, two to fifteen 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).

[0200] As used herein, the term "optionally substituted" means having 0, 1 or more substituents, such as 0-25, 0-20, 0-10 or 0-5 substituents. Alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl groups can be substituted with alkyl, halogen, alkenyl, alkynyl, aryl, alkaryl, acyl, heteroaryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroalkaryl, halogen, oxo, cyano, nitro, amino, alkylamino, hydroxy, alkoxy, alkanoyl, carbonyl, carbamoyl, guanidino, urea, amidino, oxime, benzyl, OR, NR2, SR, SOR, S02R, OCOR, NRCOR, NRCONR2, NRCOOR, OCONR2, RCO, COOR, alkyl-OOCR, S03R, 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, ie, a methyl group, can be substituted with oxo to form a formyl group, and further substituted with -OH or -NHR to form a carboxyl group or an amido group.

[0201] The optionally substituted group or part refers to a group or part (for example, any one of the above-mentioned groups or parts) in which an atom (for example, a hydrogen atom) is optionally substituted by another substituent. For example, the optionally substituted alkyl can be an optionally substituted methyl, in which the hydrogen atom of the methyl group is replaced by, for example, OH. As another example, the substituent on heteroalkyl or its divalent counterpart (heteroalkylene) can replace the hydrogen on the carbon or the hydrogen on a heteroatom (such as N). For example, the hydrogen atom in the group -R-NH-R- can be replaced by an alkaneamide substituent such as -RN[(CH2C(O)N(CH3)2]-R.

[0202] Typically, optional substituents are non-interfering substituents. A "non-interfering substituent" refers to a substituent that preserves the ability of the conjugates described herein to bind to CD73. Thus, in some embodiments, a substituent may alter the extent of such activity. However, as long as the conjugate retains the ability to bind to CD73 or inhibit tumor growth, the substituent will be classified as "non-interfering." For example, based on an IC50 value of 10 μM or less in a viral plaque reduction assay, a non-interfering substituent will preserve the ability of the compound to provide antiviral efficacy. Thus, a substituent may alter the extent of inhibition based on plaque reduction or CD73 inhibition. However, as long as the compound herein retains the ability to inhibit CD73, the substituent will be classified as "non-interfering." Many assays for determining viral plaque reduction or tumor growth inhibition, or the ability of any compound to inhibit CD73, are available in the art, and some assays are exemplified in the Examples below.

[0203] When used to describe a chemical group or part, the term "hetero" refers to having at least one heteroatom that is not carbon or hydrogen, such as N, O and S. If any of the above groups or parts contains at least one heteroatom, it can be referred to as hetero. 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 a maleimido group. For example, a heteroaryl group refers to an aromatic group having one or more heteroatoms independently selected from, for example, N, O and S. One or more heteroatoms can also be included in the substituent replacing the hydrogen atom in a group or part as described herein. For example, in an optionally substituted heteroaryl group, if a hydrogen atom in the heteroaryl group is replaced by a substituent (e.g., methyl), the substituent can also contain one or more heteroatoms (e.g., methanol).

[0204] As used herein, the term "acyl" refers to a group having the following structure: where R zis optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, alkylamino, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaryl, heteroalkaryl, or heteroalkylamino.

[0205] As used herein, the term "halo" or "halogen" refers to any halogen atom, such as F, Cl, Br, or I. If any of the groups or moieties described herein contains at least one halogen atom, such as a haloalkyl group, it can be referred to as a "halo moiety."

[0206] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more (e.g., one, two, three, four, five, six or more) halo groups. Haloalkyl includes, but is not limited to, fluoroalkyl (e.g., trifluoromethyl and pentafluoroethyl) and chloroalkyl.

[0207] The term "hydroxy" as used herein refers to an -OH group.

[0208] As used herein, the term "oxo" refers to a substituent having the structure =0 where a double bond exists between the atom and the oxygen atom.

[0209] As used herein, the term "carbonyl" refers to a radical of the structure:

[0210] As used herein, the term "thiocarbonyl" refers to a group having the following structure:

[0211] As used herein, the term "phosphate" refers to a group having the following structure:

[0212] As used herein, the term "phosphoryl" refers to a group having the following structure:

[0213] As used herein, the term "sulfonyl" refers to a group having the following structure:

[0214] As used herein, the term "imino" refers to a group comprising C=N (e.g., comprising the structure: ). For example, the imino group can have any of the following structures: where R i1 and R i2 Each of is H or any of the substituents described herein (e.g., C1-C 20 alkyl); R i3 and R i4Each of the alkylene groups is a methylene group, which is unsubstituted or substituted with one or more substituents described herein (e.g., C1-C 20 alkyl); and each of i1 and i2 is independently 0, 1, 2 or 3.

[0215] As used herein, the term "oxime" refers to an oxime comprising C=NO (e.g., comprising the structure ) group.

[0216] As used herein, the term "N-protecting group" refers to those groups intended to protect amino groups from undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis," 5th Edition (John Wiley & Sons, New York, 2014), which is incorporated herein by reference. N-protecting groups include, for example, acyl, aroyl and carbamoyl groups such as formyl, acetyl, propionyl, pivaloyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthaloyl, 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 benzylsulfonyl and p-toluenesulfonyl; carbamate-forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxy ... ,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzylhydroxycarbonyl, tert-butoxycarbonyl (BOC), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl and phenylthiocarbonyl; alkaryl groups such as benzyl, triphenylmethyl and benzyloxymethyl; and silyl groups such as trimethylsilyl.

[0217] The term "amino acid" as used herein means naturally occurring amino acids and non-naturally occurring amino acids.

[0218] As used herein, the term "naturally occurring amino acids" refers to amino acids including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

[0219] As used herein, the term "non-naturally occurring amino acid" means an alpha amino acid that is not naturally produced or present in mammals. Examples of non-naturally occurring amino acids include D-amino acids; amino acids having an acetamidomethyl group attached to the sulfur atom of cysteine; PEGylated amino acids; amino acids of the formula NH2(CH2) nCOOH ω amino acids, wherein n is 2-6, neutral non-polar amino acids such as sarcosine, tert-butylalanine, tert-butylglycine, N-methylisoleucine and norleucine; oxymethionine; phenylglycine; citrulline; methionine sulfoxide; cysteic acid; ornithine; diaminobutyric acid; 3-aminoalanine; 3-hydroxy-D-proline; 2,4-diaminobutyric acid; 2-aminopentanoic acid; 2-aminooctanoic acid, 2-carboxypiperazine; piperazine-2-carboxylic acid, 2-amino-4-phenylbutyric acid; 3-(2-naphthyl)alanine and hydroxyproline. The other amino acids are α-aminobutyric acid, α-amino-α-methylbutyric acid, aminocyclopropane-carboxylic acid, aminoisobutyric acid, aminonorbornyl-carboxylic acid, L-cyclohexylalanine, cyclopentylalanine, LN-methylleucine, LN-methylmethionine, LN-methylnorvaline, LN-methylphenylalanine, LN-methylproline, LN-methylserine, LN-methyltryptophan, D-ornithine, LN-methylethylglycine, L-norleucine, α-methyl-aminoisobutyrate, α-methylcyclohexylalanine, D-α-methylalanine, and D-α-methylsilanine. amino acid, 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, DN-methylserine, D-α-methylthreonine, D-α-methyltryptophan, D-α-methyltyrosine, D-α-methylvaline, DN-methylalanine , DN-methylarginine, DN-methylasparagine, DN-methylaspartic acid, DN-methylcysteine, DN-methylglutamine, DN-methylglutamic acid, DN-methylhistidine, DN-methylisoleucine, DN-methylleucine, DN-methyllysine, N-methylcyclohexylalanine, DN-methylornithine, N-methylglycine, N-methylaminoisobutyric acid, N-(1-methylpropyl)glycine, N-(2-methylpropyl)glycine, DN-methyltryptophan, DN-methyltyrosine, DN-methylvaline, γ-aminobutyric acid, L -tert-Butylglycine, L-Ethylglycine, L-Homophenylalanine, L-α-Methylarginine, L-α-Methylaspartic acid, L-α-Methylcysteine, L-α-Methylglutamine, L-α-Methylhistidine, L-α-Methylisoleucine, L-α-Methylleucine, L-α-Methylmethionine, L-α-Methylnorvaline, L-α-Methylphenylalanine, L-α-Methylserine, L-α-Methyltryptophan, L-α-Methylvaline, N-(N-(2,2-diphenylethyl))carbamoylmethylglycine, 1-Carboxy-1-(2,2-Diphenyl-ethylamino)cyclopropane, 4-hydroxyproline, ornithine, 2-aminobenzoyl (o-aminobenzoyl), D-cyclohexylalanine, 4-phenyl-phenylalanine, L-citrulline, α-cyclohexylglycine, L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, L-thiazolidine-4-carboxylic acid, L-homotyrosine, L-2-furanylalanine, L-histidine (3-methyl), N-(3-guanidinopropyl)glycine, O-methyl-L-tyrosine, O-glycan-serine, m-tyrosine, n-tyrosine, LN,N′,N″-trimethyllysine, homolysine, n-lysine, N-glycan-asparagine, 7-hydroxy-1,2,3,4-tetrahydro -4-Fluorophenylalanine, 4-methylphenylalanine, bis-(2-pyridyl)amine, pentafluorophenylalanine, indoline-2-carboxylic acid, 2-aminobenzoic acid, 3-amino-2-naphthoic acid, asymmetric dimethylarginine, L-tetrahydroisoquinoline-1-carboxylic acid, D-tetrahydroisoquinoline-1-carboxylic acid, 1-aminocyclohexaneacetic acid, D / L-allylglycine, 4-aminobenzoic acid, 1-aminocyclobutanecarboxylic acid, 2- or 3- or 4-aminocyclohexanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-aminoindan-1-carboxylic acid, 4-amino-pyrrolidine-2-carboxylic acid, 2-aminotetralin-2-carboxylic acid, azetidine-3-carboxylic acid, 4-benzyl-pyrrolidine-2-carboxylic acid, tert-butyl Glycine, b-(benzothiazol-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, tert-butylalanine, (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid, 1-aminomethyl-cyclohexane -acetic acid, 3,5-bis-(2-amino)ethoxy-benzoic acid, 3,5-diamino-benzoic acid, 2-methylamino-benzoic acid, N-methylanthranilic acid, LN-methylalanine, LN-methylarginine, LN-methylasparagine, LN-methylaspartic acid, LN-methylcysteine, LN-methylglutamine, LN-methylglutamic acid, LN-methylhistidine, LN-methylisoleucine, LN-methyllysine, LN-methylnorleucine, LN-methylornithine, LN-methylthreonine, LN-methyltyrosine, LN-methylvaline, LN-methyl-tert-butylglycine, L-norvaline, α-methyl-γ-aminobutyrate, 4,4′-biphenylalanine, α-methylcyclopentylalanine, α-methyl-α-naphthylalanine, α-methylpenicillamine, N-(4-aminobutyl)glycine, N-(2-aminoethyl)glycine, N-(3-aminopropyl)glycine, N-amino-α-methylbutyrate, α-naphthylalanine, N-benzylglycine, N-(2-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-cyclodecanylglycine Monoalkylglycine, N-(2,2-diphenylethyl)glycine, N-(3,3-diphenylpropyl)glycine, N-(3-guanidinopropyl)glycine, N-(1-hydroxyethyl)glycine, N-(hydroxyethyl)glycine, N-(imidazoethyl)glycine, N-(3-indolethyl)glycine, N-methyl-γ-aminobutyric acid, DN-methylmethionine, N-methylcyclopentylalanine, DN-methylphenylalanine, DN-methylproline, DN-methylthreonine, N-(1-methylethyl)glycine, N-methyl-naphthylalanine, N-methylpenicillamine, N-(p-hydroxyphenyl)glycine, N-(thiomethyl)glycine, penicillamine, L-α-methylalanine, L-α-methylasparagine , L-α-methyl-tert-butylglycine, L-methylethylglycine, L-α-methylglutamic acid, L-α-methylhomophenylalanine, N-(2-methylthioethyl)glycine, L-α-methyllysine, L-α-methylnorleucine, L-α-methylornithine, L-α-methylproline, L-α-methylthreonine, L-α-methyltyrosine, LN-methyl-homophenylalanine, N-(N-(3,3-diphenylpropyl))carbamoylmethylglycine, 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 yl), 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-polysaccharide-threonine, o-tyrosine, LN,N′-dimethyllysine, L-homoarginine, neotryptophan, 3-benzothienylalanine, isoquinoline-3-carboxylic acid, diaminopropionic acid, homocysteine, 3,4-dimethoxyphenylalanine, 4-chlorophenylalanine, L-1,2,3,4-tetrahydronorharman-3-carboxylic acid, adamantane alanine, symmetrical dimethylarginine, 3-carboxythiomorpholine, D-1,2,3,4-Tetrahydronorharman-3-carboxylic acid, 3-aminobenzoic acid, 3-amino-1-carboxymethyl-pyridin-2-one, 1-amino-1-cyclohexanecarboxylic acid, 2-aminocyclopentanecarboxylic acid, 1-amino-1-cyclopropanecarboxylic acid, 2-aminoindan-2-carboxylic acid, 4-amino-tetrahydrothiopyran-4-carboxylic acid, azetidine-2-carboxylic acid, b-(benzothiazol-2-yl)-alanine, neopentylglycine, 2-carboxymethylpiperidine, b-cyclobutylalanine, allylglycine, diaminopropionic acid, homo-cyclohexylalanine, (2S,4R)-4-hydroxypiperidine-2-carboxylic acid, Octahydroindole-2-carboxylic acid, (2S,4R) and (2S,4R)-4-(2-naphthyl), pyrrolidine-2-carboxylic acid, hexahydronicotinic 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, tert-butylglycine, N,N-bis(3-aminopropyl)glycine, 1-amino-cyclohexane-1-carboxylic acid, N-mercaptoethylglycine and selenocysteine. In some embodiments, the amino acid residues can be charged or polar. Charged amino acids include alanine, lysine, aspartic acid or glutamic acid, or non-naturally occurring analogs thereof. Polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine or tryptophan, or non-naturally occurring analogs thereof. It is specifically contemplated that in some embodiments, the terminal amino group in the amino acid may be an acylamino group or a carbamate group.

[0220] As used herein, the term "percent identity (%)" refers to the percentage of amino acid residues in 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 and, if necessary, introducing gaps to achieve maximum percent identity (i.e., gaps can be introduced in one or both of the candidate sequence and the reference sequence for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment for determining the purpose of percent identity can be achieved in various ways known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. In some embodiments, the amino acid sequence identity percentage of a given candidate sequence relative to a given reference sequence, with a given reference sequence, or for a given reference sequence (which can alternatively be expressed as a given candidate sequence having or comprising a certain amino acid sequence identity percentage relative to a given reference sequence, with a given reference sequence, or for a given reference sequence) is calculated as follows:

[0221] 100x(A / B fraction)

[0222] wherein A is the number of amino acid residues scored as identical in an alignment of the candidate sequence and the reference sequence, and wherein 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 between the candidate sequence and the reference sequence will not be equal to the percent amino acid sequence identity between the reference sequence and the candidate sequence.

[0223] Two polynucleotides or polypeptide sequences are considered to be "identical" if, when the maximum correspondence comparison is performed as described above, the nucleotides or amino acid sequences in the two sequences are identical. The comparison between the two sequences is typically performed by comparing the sequences over a comparison window to identify and compare the local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 15 continuous positions, about 20 continuous positions, about 25 continuous positions or more (e.g., about 30 to about 75 continuous positions or about 40 to about 50 continuous positions), wherein after the two sequences are optimally aligned, the sequence can be compared to a reference sequence with the same number of continuous positions.

[0224] As used herein, the term "treating" or "to treat" refers to the therapeutic treatment of a disease (e.g., cancer, fibrosis, or infection) in a subject. In some embodiments, therapeutic treatment can slow the progression of the disease, improve the subject's outcome, and / or eliminate the tumor. In some embodiments, therapeutic treatment of a disease in a subject can alleviate or improve one or more symptoms or conditions associated with the disease, reduce the extent of the symptoms, stabilize (i.e., not worsen) the state of the disease, prevent the spread of the disease, and / or delay or slow the progression of the disease, as compared to the state and / or condition of the disease in the absence of therapeutic treatment.

[0225] As used herein, the term "average value of T" refers to the average number of CD73 inhibitor monomers or CD73 inhibitor dimers conjugated to Fc domain monomers or Fc domains within a population of conjugates. In some embodiments, within a population of conjugates, the average number of CD73 inhibitor monomers or CD73 inhibitor dimers conjugated to Fc domain monomers can be 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.

[0226] As used herein, the term "subject" can be a human or a non-human primate.

[0227] As used herein, the term "therapeutically effective amount" refers to an amount, such as a drug dosage, that is effective to induce a desired effect in a subject or to effectively treat a subject suffering from a condition or disorder described herein. It should also be understood herein that a "therapeutically effective amount" can be interpreted as an amount that, when taken in one or more doses or by any dose or route, and / or taken alone or in combination with other therapeutic agents, gives the desired therapeutic and / or preventive effect. For example, in the context of administering a conjugate described herein for treating a disease described herein, an effective amount of the conjugate is an amount sufficient to prevent, slow, or reverse the progression of the disease, for example, as compared to the response obtained without administering the conjugate.

[0228] As used herein, the term "pharmaceutical composition" refers to a medicament or pharmaceutical preparation comprising at least one active ingredient and one or more excipients and diluents to render the active ingredient suitable for the method of administration. The pharmaceutical compositions of the present disclosure comprise pharmaceutically acceptable components that are compatible with the conjugates described herein.

[0229] 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 that can suspend or dissolve the active conjugate. A pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not harmful to the recipient. In the present disclosure, a pharmaceutically acceptable carrier must provide sufficient pharmaceutical stability for the conjugates described herein. The nature of the carrier varies depending on the mode of administration. For example, for oral administration, solid carriers are preferred; for intravenous administration, aqueous carriers (e.g., WFI and / or buffered solutions) are typically used.

[0230] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of the conjugates described herein that is suitable for use in the methods described herein without undue toxicity, irritation, and / or allergic response, within the scope of sound 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 (edited by 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 group with a suitable organic acid.

[0231] As used herein, the term "about" indicates a deviation of ±5%. For example, about 10% refers to 9.5% to 10.5%.

[0232] Any number provided in a numerical range includes the upper and lower limits, and any number included within the upper and lower limits.

[0233] Other features and advantages of the conjugates described herein will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0234] Figure 1A Figure 2 is a graph showing that conjugates 133a and 133b exhibit single-digit nM potency in a cell-free CD73 inhibition assay. AB680 is a known CD73 inhibitor. Int-258 corresponds to the CD73 inhibitor portion of conjugates 133a and 133b without the Fc domain. Figure 1B is a graph showing that conjugate 172a exhibits single-digit nM potency in a cell-free CD73 inhibition assay. AB680 is a known CD73 inhibitor. Figure 1C is a graph showing the percentage of CD73 inhibition by oleclumab and mupadolimab in a cell-free CD73 inhibition assay.

[0235] Figure 2 Figure 2 is a graph showing that conjugates 133a and 133b exhibit single-digit nM potency in a cell-based CD73 inhibition assay (MDA-MB231 cells, human breast cancer). AB680 is a known CD73 inhibitor. Int-258 corresponds to the CD73 inhibitor portion of conjugates 133a and 133b without the Fc domain.

[0236] Figure 3 is a graph showing that conjugate 172a exhibits subnanomolar potency in a cell-based CD73 inhibition assay (MDA-MB231 cells, human breast cancer).

[0237] Figure 4 is a graph showing PBMC rescue assay on AMP-repressed cells using conjugate 172a.

[0238] Figure 5 is a series of graphs showing that conjugate 172a exhibits potent and complete CD73 inhibition in human PBMC cells similar to the small molecule inhibitors AB680, OP5244, and SHR170008.

[0239] Figure 6 is a series of graphs showing that conjugate 172a exhibits potent and complete CD73 inhibition in 4T1 cancer cells similar to the small molecule inhibitors AB680, OP5244, and SHR170008.

[0240] Figure 7 It was shown that conjugate 172a showed binding to CD73-expressing B cells (CD3 - CD19 + ) and CD8 +T cells (CD3 + CD8 + ), showing subnanomolar and single-digit nanomolar binding, respectively. SEQ ID NO: 80 (conjugate 172a lacking Int) was used as a negative control.

[0241] Figure 8 It was shown that conjugate 172a exhibited B cell (CD3 - CD19 + SEQ ID NO: 80 (conjugate 172a lacking Int) was used as a negative control.

[0242] Figure 9A and Figure 9B is a series of graphs showing binding of conjugate 172a to MDA-MB-231 cells at low concentrations. Figure 9A It was shown that the binding of conjugate 172a in the presence of AMP was reduced in an AMP-dependent manner, demonstrating that conjugate 172a is an AMP-competitive CD73 inhibitor. Figure 9B It was shown that in the presence of increasing concentrations of the small molecule CD73 inhibitor AB680, the binding of conjugate 172a decreased in a dose-dependent manner, demonstrating that conjugate 172a is a catalytic site CD73 inhibitor.

[0243] Figure 10A and Figure 10B is a series of graphs showing that conjugate 133b reactivates adenosine-inhibited T cells via inhibition of CD73. Conjugate 133b has an EC of 175.3 nM in the presence of 30 μM adenosine monophosphate (AMP). 50 ( Figure 10A ) or with an EC of 976 nM in the presence of 100 μM AMP 50 ( Figure 10B ).

[0244] Figure 11 is a graph showing that conjugate 172a and anti-CD73 monoclonal antibody exhibit potent subnanomolar CD73 internalization activity in MDA-MB-231 cells.

[0245] Figure 12 It is shown that conjugate 172a exhibits binding to MDA-MB-231 cells (IC 50 is 0.39 nM) is mopalimumab (IC 50 SEQ ID NO: 80 (hIgG1 Fc vector) was used as a negative control.

[0246] Figure 13Figure 1 is a graph showing a 7-day mouse pharmacokinetic study of conjugate 133a administered intramuscularly at 10 mg / kg. After 168 hours, similar plasma exposure levels were observed for both the CD73 (28.6 μg / mL) and Fc (21.5 μg / mL) capture assays. The AUCs for the CD73 (3565) and Fc (7538) captures were within approximately 2-fold of each other, indicating minimal loss of Int over time.

[0247] Figure 14 Graph showing a 7-day mouse pharmacokinetic study of conjugate 172a administered intramuscularly at 10 mg / kg. After 168 hours, similar plasma exposure levels were observed for CD73 and Fc capture assays. These results highlight the long half-life, high plasma exposure, and overall stability of conjugate 172a in vivo.

[0248] Figure 15 is a graph showing the efficacy of conjugate 133a in a mouse syngeneic model with a colon tumor cell line.

[0249] Figure 16 Figure 2 is a graph showing the efficacy of conjugate 133b in a mouse syngeneic model with a colon tumor cell line. Conjugate 133b was administered alone (5 mg / kg or 20 mg / kg) or in combination with an anti-PD-1 antibody (RMP1-14).

[0250] Figure 17 Graph showing the efficacy of conjugate 133b over time (12 days) in a mouse syngeneic model with a colon tumor cell line. Conjugate 133b was administered alone (5 mg / kg or 20 mg / kg) or in combination with an anti-PD-1 antibody (RMP1-14).

[0251] Figure 18 is a graph showing the efficacy of conjugate 133b and conjugate 161 against CT26 (colon) tumors after 10 days of growth in mice.

[0252] Figure 19A and Figure 19B is a series of graphs showing the efficacy of Conjugate 133b, Conjugate 161, Conjugate 169, Conjugate 165, Conjugate 172a, and Conjugate 175 against a colon tumor cell line (CT26) in a syngeneic mouse model.

[0253] Figure 20A is a graph showing tumor growth over 8 days in animals treated with vehicle control and conjugate 172a. Figure 20B is a bar graph showing the percent inhibition of tumor growth in animals treated with conjugate 172a relative to vehicle control. Figure 20C are box plots showing tumor volumes for individual animals on study day 8.

[0254] Figure 21 is a graph of plasma levels of conjugate 172a in mice over time.

[0255] Figure 22A and Figure 22B is a series of graphs showing the effect of different dosing regimens on the efficacy of conjugate 133b on CT26 (colon) tumor growth in mice and the effect of the conjugate on this efficacy.

[0256] Figure 23 is a graph showing IV tolerability of conjugate 172a in mice.

[0257] Figure 24 is a graph showing that conjugate 172a and conjugate 172c exhibit nM potency in a cell-free CD73 inhibition assay.

[0258] Figure 25 is a graph showing that various batches of conjugate 172c exhibited single-digit nM or sub-nM potency in a cell-based CD73 inhibition assay (MDA-MB231 cells, human breast cancer).

[0259] Figure 26 is a series of graphs showing the binding of conjugate 172c (batch 9), olerumumab, and mopalimumab to human MDA-MB-231 cancer cells in the presence and absence of AMP as determined by flow cytometry.

[0260] Figure 27 is a series of graphs showing the binding of conjugate 172c (batch 9), olerumumab, and mopalimumab to human MDA-MB-231 cancer cells in the presence and absence of the small molecule CD73 inhibitor AB680, as determined by flow cytometry.

[0261] Figure 28 is a graph showing the activity of conjugate 172c (batch 9) and CD73-targeting comparators in a CD73 inhibition assay using human PBMCs at 3 hours.

[0262] Figure 29 is a graph showing the activity of conjugate 172c (batch 9) and CD73-targeting comparators in a CD73 inhibition assay using human PBMCs at 24 hours.

[0263] Figure 30 is a graph showing the activity of conjugate 172c (batch 9) and CD73-targeting comparators in a CD73 inhibition assay using mouse EMT6 cancer cells at 3 hours.

[0264] Figure 31is a graph showing the activity of conjugate 172c (batch 9) and CD73-targeting comparators in a CD73 inhibition assay using mouse EMT6 cancer cells at 24 hours.

[0265] Figure 32 is a series of graphs showing the activity of conjugate 172c (batch 9) and other experimental articles targeting CD73 as determined by flow cytometry in a human PBMC activation assay.

[0266] Figure 33 is a graph showing the activity of conjugate 172c (batch 9) and other test articles targeting CD73 in a human PBMC activation assay measuring the percent inhibition of adenosine production by CellTiter-Glo.

[0267] Figure 34 is a graph showing the percentage of CD73 internalization dose response curves for conjugate 172c (batch 9), olerumab, mopalimab, and hIgG Fc into MDA-MB-231 human breast adenocarcinoma cells.

[0268] Figure 35 is a graph showing the 14-day plasma concentration-time curve obtained by CD73 capture / Fc detection after administration of dose-linear concentrations of conjugate 172a.

[0269] Figure 36 is a graph showing the 14-day plasma concentration-time profile obtained by Fc capture / Fc detection following administration of dose-linear concentrations of conjugate 172a.

[0270] Figure 37 is a graph showing the 14-day plasma concentration-time curve obtained by CD73 capture / Fc detection after administration of dose-linear concentrations of conjugate 172a.

[0271] Figure 38 is a graph showing the 14-day plasma concentration-time profile obtained by Fc capture / Fc detection following administration of dose-linear concentrations of conjugate 172a.

[0272] Figure 39 is a graph showing the 7-day plasma concentration-time profile obtained by CD73 capture / Fc detection following intraperitoneal (IP) administration of conjugate 172c DAR scan.

[0273] Figure 40 is a graph showing the 7-day plasma concentration-time profile obtained by Fc capture / Fc detection following IP administration of conjugate 172c DAR scan.

[0274] Figure 41is a graph showing 14-day plasma concentration-time profiles of conjugate 172a following intravenous (IV), IP, and subcutaneous (SC) administration as determined by CD73 capture / Fc detection.

[0275] Figure 42 is a graph showing 14-day plasma concentration-time profiles obtained by Fc capture / Fc detection following IV, IP, and SC administration of conjugate 172a.

[0276] Figure 43 is a bar graph showing the diameter of 3D tumor spheroids in the presence of 100 nM of conjugate 201 and other test articles.

[0277] Figure 44 is a bar graph showing 3D tumor spheroid penetration in microns in the presence of 100 nM conjugate 201 and 100 nM olerumab.

[0278] Figure 45A is a graph showing the mean tumor volume (±SEM) over time in mice treated with conjugate 172a, conjugate 172a / α-PD-1, and other test articles. Figure 45B is a graph showing the mean tumor volume on day 22 (17 days after dosing) in mice treated with conjugate 172a, conjugate 172a / α-PD-1, and other test articles.

[0279] Figure 46A is a graph showing the mean tumor volume (±SEM) over time in mice treated with conjugate 172a, conjugate 172a / α-PD-1, α-PD-1, and vehicle. Figure 46B is a graph showing tumor growth over time in mice treated with the conjugate 172a / α-PD-1 with complete tumor regression.

[0280] Figure 47 Timeline for rechallenge studies in mice to determine if animals that achieve complete remission with the conjugate 172a / anti-PD-1 combination also acquire immunity to the EMT-6 cancer cell line.

[0281] Figure 48A are box plots showing tumor inhibition against the EMT-6 breast cancer cell line in mice treated with conjugate 172a or vehicle. Figure 48B is a line graph showing tumor inhibition against the EMT-6 breast cancer cell line in mice treated with conjugate 172a or vehicle.

[0282] Figure 49 Is displayed when using CD8 + CD25 on T cells +Graph of the activity of conjugates with different linker lengths targeting CD73 in a PBMC activation assay as readout.

[0283] Figure 50A is a graph showing the inhibition of purified recombinant human CD73 in the presence of conjugate 172c (batch 9) or a CD73 small molecule inhibitor using a cell-free CD73 enzyme inhibition assay. Figure 50B is a graph showing the inhibition of purified recombinant human CD73 in the presence of conjugate 172c (batch 9) or CD73 monoclonal antibody using a cell-free CD73 enzyme inhibition assay.

[0284] Figure 51 is a graph showing the inhibition of surface-expressed CD73 on MDA-MB231 (human breast cancer) cells in the presence of conjugate 172c (batch 9) and other test articles using a cell-based CD73 enzyme inhibition assay.

[0285] Figure 52 is a graph showing the inhibition of surface-expressed CD73 on MDA-MB231 (human breast cancer) cells in the presence of various conjugates containing different linker lengths using a cell-based CD73 enzyme inhibition assay.

[0286] Figure 53A is a graph showing tumor volume over time in a mouse colon tumor model treated with conjugate 172c (batch 9) and in combination with α-PD-1 mAb. Figure 53B is a graph showing tumor volume at day 20 for individual mice treated with conjugate 172c (batch 9) and in combination with α-PD-1 mAb. DETAILED DESCRIPTION

[0287] The present disclosure relates to conjugates comprising an Fc domain monomer or an Fc domain covalently linked to a portion that binds to or inhibits CD73. Specifically, such conjugates contain monomers or dimers that bind to or inhibit a portion of CD73 conjugated to an Fc monomer or Fc domain. The CD73 inhibitor (e.g., adenosine monophosphate, adenosine diphosphate, or its analogues) in the conjugate targets CD73. The Fc monomer or Fc domain in the conjugate binds to FcγR (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells (e.g., neutrophils) to activate phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The characteristic conjugate exhibits desired tissue distribution (e.g., lung distribution).

[0288] The present disclosure also provides pharmaceutical compositions comprising such conjugates and uses of such conjugates in treating disorders associated with dysregulation or overexpression of CD73 (e.g., cancer, fibrosis, or viral infection).

[0289] I. Conjugates

[0290] Provided herein are synthetic conjugates comprising an Fc domain conjugated to one or more CD73 inhibitors (e.g., a CD73 inhibitor of Formula (A), (AI), (A-Ia), (A-Ib), (A-Ib-1), (A-Ib-2), (A-II), (A-IIa), (A-IIb), (A-IIb-1), or (A-IIb-2)) or to one or more dimers of two CD73 inhibitors. A dimer of two CD73 inhibitors comprises a CD73 inhibitor (e.g., a first CD73 inhibitor of Formula (A), (AI), (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), (AI), (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 case of a dimer (e.g., a dimer of Formula (DI)), the first and second CD73 inhibitors are linked to each other via a linker.

[0291] The conjugates of the present disclosure include CD73 inhibitor monomers and dimers conjugated to Fc domains, Fc monomers, or Fc binding peptides. The Fc domains in the conjugates described herein bind to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells. Binding of the Fc domains in the conjugates described herein to FcγRs on immune cells activates phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC).

[0292] The conjugates provided herein are described by any one of formula (DI) or (MI). In some embodiments, the conjugates described herein comprise one or more CD73 inhibitor monomers conjugated to an Fc domain monomer or an Fc domain. In some embodiments, the conjugates described herein comprise one or more CD73 inhibitor dimers conjugated to an Fc domain monomer or an Fc domain. In some embodiments, when n is 2, E (Fc domain monomer) dimerizes to form an Fc domain.

[0293] The conjugates described herein can be synthesized using chemical synthesis techniques available in the art. Where functional groups are not available for conjugation, conventional chemical synthesis techniques well known in the art can be used to derivatize the molecule. In some embodiments, the conjugates described herein contain one or more chiral centers. The conjugates include each isolated stereoisomeric form and mixtures of stereoisomers of varying degrees of chiral purity, including racemic mixtures. It also encompasses the various diastereomers, enantiomers, and tautomers that may be formed.

[0294] Conjugate of CD73 inhibitor monomer linked to Fc domain

[0295] In some embodiments, the conjugates described herein comprise an Fc domain monomer or Fc domain covalently linked to one or more CD73 inhibitor monomers, such as a conjugate described by formula (MI). A conjugate of an Fc domain monomer or Fc domain with one or more CD73 inhibitor monomers can be formed by linking the Fc domain monomer or Fc domain to each CD73 inhibitor monomer via a linker, such as any linker described herein.

[0296] In conjugates having an Fc domain monomer or Fc domain covalently linked to one or more CD73 inhibitor monomers described herein, the curve 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) CD73 inhibitor monomers can be attached to the Fc domain monomer or 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) CD73 inhibitor monomers can be attached 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) CD73 inhibitor monomers can be attached to the Fc domain. The curved lines in the conjugates described herein should not be interpreted as single bonds between one or more CD73 inhibitor monomers and an Fc domain monomer or an atom in the Fc domain. In some embodiments, when T is 1, one CD73 inhibitor monomer may be attached to an Fc domain monomer or an atom in the Fc domain. In some embodiments, when T is 2, two CD73 inhibitor monomers may be attached to an Fc domain monomer or an atom in the Fc domain.

[0297] 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 A1-L structure described herein). In some embodiments, E can be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different A1-L moieties. In some embodiments, E is conjugated to a first A1-L moiety and a second A1-L moiety. In some embodiments, A1 of each of the first A1-L moiety and the second A1-L moiety is independently selected from any of Formulas (A), (AI), (A-Ia), (A-Ib), (A-Ib-1), (A-Ib-2), (A-II), (A-IIa), (A-IIb), (A-IIb-1), and (A-IIb-2).

[0298] In some embodiments, the first A1-L moiety is specifically conjugated to a lysine residue of E (e.g., a nitrogen atom of a surface-exposed lysine residue of E), and the second A1-L moiety is specifically conjugated to a cysteine ​​residue of E (e.g., a sulfur atom of a surface-exposed cysteine ​​residue of E). In some embodiments, the first A1-L moiety is specifically conjugated to a cysteine ​​residue of E (e.g., a sulfur atom of a surface-exposed cysteine ​​residue of E), and the second A1-L moiety is specifically conjugated to a lysine residue of E (e.g., a nitrogen atom of a surface-exposed lysine residue of E).

[0299] As further described herein, the linker (e.g., L) in a conjugate having an Fc domain monomer or Fc domain covalently linked to one or more CD73 inhibitor monomers described herein can be a bivalent structure having two arms. One arm of the bivalent linker can be attached to the CD73 inhibitor monomer and the other arm can be attached to the Fc domain monomer or Fc domain.

[0300] In the conjugate having an Fc domain covalently linked to one or more CD73 inhibitor monomers, as shown in the above formula, when n is 2, two Fc domain monomers (each Fc domain monomer is represented by E) dimerize to form an Fc domain.

[0301] Conjugate of CD73 inhibitor dimer linked to Fc domain

[0302] The conjugates described herein comprise an Fc domain monomer or Fc domain covalently linked to one or more CD73 inhibitor dimers, such as a conjugate described by formula (DI). The dimer of two CD73 inhibitors comprises a first CD73 inhibitor (e.g., a first CD73 inhibitor of Formula (A), (AI), (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), (AI), (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 linked to each other by a linker, such as a linker described herein. In some embodiments of the dimer of CD73 inhibitors, the first and second CD73 inhibitors are the same. In some embodiments, the first and second CD73 inhibitors are different.

[0303] 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 A1-L-A2 structure described herein). In some embodiments, E can be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different A1-L-A2 moieties. In some embodiments, E is conjugated to 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), (AI), (A-la), (A-lb), (A-Ib-1), (A-Ib-2), (A-II), (A-lla), (A-Iib), (A-Iib-1), or (A-Iib-2).

[0304] In some embodiments, the first A1-L-A2 moiety is specifically conjugated to a lysine residue of E (e.g., a nitrogen atom of a surface-exposed lysine residue of E), and the second A1-L-A2 moiety is specifically conjugated to a cysteine ​​residue of E (e.g., a sulfur atom of a surface-exposed cysteine ​​residue of E). In some embodiments, the first A1-L-A2 moiety is specifically conjugated to a cysteine ​​residue of E (e.g., a sulfur atom of a surface-exposed cysteine ​​residue of E), and the second A1-L-A2 moiety is specifically conjugated to a lysine residue of E (e.g., a nitrogen atom of a surface-exposed lysine residue of E).

[0305] In the conjugates described herein, the curve 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) CD73 inhibitor dimers can be attached to an Fc domain monomer or an 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) CD73 inhibitor dimers can be attached to an 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) CD73 inhibitor dimers can be attached to an Fc domain. The curved lines in the conjugates described herein should not be interpreted as single bonds between one or more CD73 inhibitor dimers and an Fc domain monomer or an atom in the Fc domain. In some embodiments, when T is 1, one CD73 inhibitor dimer may be attached to an Fc domain monomer or an atom in the Fc domain. In some embodiments, when T is 2, two CD73 inhibitor dimers may be attached to an Fc domain monomer or an atom in the Fc domain.

[0306] As further described herein, the linker in the conjugates described herein can be a branched structure. As further described herein, the linker in the conjugates described herein can be a multivalent structure, such as a bivalent or trivalent structure having two or three arms, respectively. In some embodiments, when the linker has three arms, two arms can be attached to the first and second CD73 inhibitors and the third arm can be attached to an Fc domain monomer or an Fc domain.

[0307] In the conjugate having an Fc domain covalently linked to one or more CD73 inhibitor dimers, as shown in the above formula, when n is 2, two Fc domain monomers (each Fc domain monomer is represented by E) dimerize to form an Fc domain.

[0308] II. Fc Domain Monomers and Fc Domains

[0309] The present disclosure features compositions (e.g., conjugates) comprising one or more Fc domain monomers. When two compositions comprising Fc domain monomers are dimerized, the resulting conjugate comprises an Fc domain. The Fc domain monomer comprises a hinge domain, a C H 2 Antibody constant domain and C H3 antibody constant domains. The Fc domain monomer can be an immunoglobulin antibody isotype IgG, IgE, IgM, IgA or IgD. The Fc domain monomer can also be any immunoglobulin antibody isotype (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, IGHG 3*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) (for example, as described in Vidarsson et al. IgG subclasses and allotypes: from structure to effector function. Frontiers in Immunology. 5(520): 1-17 (2014)). The Fc domain monomer can also be of any species, such as human, rat or mouse. A dimer of Fc domain monomers is an Fc domain that can bind to an Fc receptor, which is a receptor located on the surface of leukocytes.

[0310] In some embodiments, the Fc domain monomers described herein contain one or more amino acid substitutions, additions, and / or deletions relative to an Fc domain monomer having a sequence of any one of SEQ ID NOs: 1-112 and 115-120. In some embodiments, the Asn (e.g., N297) in the Fc domain monomers in the conjugates as described herein can be replaced by 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 substituted by Ala, Gly, or Gln.

[0311] In some embodiments, the Fc domain monomer in the conjugates described herein comprises an additional moiety for purification (e.g., a six-histidine peptide), or a signal sequence (e.g., an IL2 signal sequence) attached to the N-terminus or C-terminus of the Fc domain monomer. In some embodiments, the additional moiety for purification (e.g., a six-histidine peptide) or the signal sequence (e.g., an IL2 signal sequence) attached to the N-terminus or C-terminus of the Fc domain monomer is cleaved after polypeptide expression. In some embodiments, the Fc domain monomer in the composition does not contain any type of antibody variable region, such as V H 、V L , complementarity determining regions (CDRs) or hypervariable regions (HVRs).

[0312] In some embodiments, the Fc domain monomer in the conjugates described herein may have a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to the sequence of any one of SEQ ID NOs: 1-112 and 115-120 shown below. In some embodiments, the Fc domain monomer in the fusion proteins or conjugates as described herein may comprise the sequence of any one of SEQ ID NOs: 1-112 and 115-120 shown below.

[0313] SEQ ID NO: 1: Mature human Fc IgG1, Z1 is Cys or Ser, and wherein X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser

[0314]

[0315] SEQ ID NO: 2: Mature human Fc IgG1, Cys to Ser substitution (#), and wherein X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser

[0316]

[0317] SEQ ID NO: 3: Mature human IgG1 Fc, Cys to Ser substitution (#), X4 is Asp or Glu, and X5 is Leu or Met

[0318]

[0319] SEQ ID NO: 4: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics)

[0320]

[0321] SEQ ID NO: 5: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(fa) (bold italics)

[0322]

[0323] SEQ ID NO:6: Mature human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations (bold / underlined), allotype G1m(fa) (bold italics)

[0324]

[0325]

[0326] SEQ ID NO:7: Mature human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations (bold / underlined), allotype G1m(f) (bold italics)

[0327]

[0328] SEQ ID NO: 8: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0329]

[0330] SEQ ID NO:9: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0331]

[0332]

[0333] SEQ ID NO: 10: Mature human Fc IgG1, Z1 is Cys or Ser, and wherein X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser

[0334]

[0335] SEQ ID NO: 11: Mature human Fc IgG1, Cys to Ser substitution (#), and wherein X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser

[0336]

[0337] SEQ ID NO: 12: Mature human IgG1 Fc, Cys to Ser substitution (#), X4 is Asp or Glu, and X5 is Leu or Met

[0338]

[0339]

[0340] SEQ ID NO: 13: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics)

[0341]

[0342] SEQ ID NO: 14: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(fa) (bold italics)

[0343]

[0344] SEQ ID NO: 15: Mature human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations (bold / underlined), allotype G1m(fa) (bold italics)

[0345]

[0346] SEQ ID NO: 16: Mature human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations (bold / underlined), allotype G1m(f) (bold italics)

[0347]

[0348] SEQ ID NO: 17: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0349]

[0350] SEQ ID NO: 18: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0351]

[0352] SEQ ID NO: 19: Mature human Fc IgG1, Z1 is Cys or Ser, and wherein X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser

[0353]

[0354] SEQ ID NO:20: Mature human Fc IgG1, Cys to Ser substitution (#), and wherein X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser

[0355]

[0356] SEQ ID NO: 21: Mature human IgG1 Fc, Cys to Ser substitution (#), X4 is Asp or Glu, and X5 is Leu or Met

[0357]

[0358] SEQ ID NO: 22: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics)

[0359]

[0360] SEQ ID NO: 23: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(fa) (bold italics)

[0361]

[0362] SEQ ID NO:24: Mature human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations (bold / underlined), allotype G1m(fa) (bold italics)

[0363]

[0364] SEQ ID NO: 25: Mature human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations (bold / underlined), allotype G1m(f) (bold italics)

[0365]

[0366]

[0367] SEQ ID NO: 26: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0368]

[0369] SEQ ID NO: 27: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0370]

[0371] SEQ ID NO: 28: Mature human Fc IgG1, Z1 is Cys or Ser, and wherein X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser

[0372]

[0373]

[0374] SEQ ID NO:29: Mature human Fc IgG1, Cys to Ser substitution (#), and wherein X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser

[0375]

[0376] SEQ ID NO:30: Mature human IgG1 Fc, Cys to Ser substitution (#), X4 is Asp or Glu, and X5 is Leu or Met

[0377]

[0378] SEQ ID NO:31: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics)

[0379]

[0380]

[0381] SEQ ID NO:32: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(fa) (bold italics)

[0382]

[0383] SEQ ID NO:33: Mature human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations (bold / underlined), allotype G1m(fa) (bold italics)

[0384]

[0385] SEQ ID NO:34: Mature human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations (bold / underlined), allotype G1m(f) (bold italics)

[0386]

[0387] SEQ ID NO:35: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0388]

[0389] SEQ ID NO:36: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0390]

[0391] SEQ ID NO:37: Mature human Fc IgG1, J1 is Asn or absent, J2 is Lys or absent, Z1 is Cys or Ser, and wherein 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, and X8 is Leu or Met, X9 is Met or Leu, and X 10 Asn or Ser

[0392]

[0393] SEQ ID NO: 38: Mature human Fc IgG1, Cys to Ser substitution (#), J1 is Asn or absent, J2 is Lys or absent, and wherein X4 is Asn or Ala, X5 is Leu or Asp, X6 is Gln or His, X7 is Asp or Glu, and X8 is Leu or Met, and X 10 Asn or Ser

[0394]

[0395] SEQ ID NO: 39: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), J1 is Asn or absent, J2 is Lys or absent, wherein X4 is Asn or Ala, X7 is Asp or Glu, and X8 is Leu or Met

[0396]

[0397] SEQ ID NO: 40: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), wherein X4 is Asn or Ala, X7 is Asp or Glu, and X8 is Leu or Met

[0398]

[0399]

[0400] SEQ ID NO:41: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), wherein X7 is Asp or Glu and X8 is Leu or Met

[0401]

[0402] SEQ ID NO:42: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0403]

[0404] SEQ ID NO:43: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0405]

[0406] SEQ ID NO:44: Mature human Fc IgG1, Cys substituted with Ser (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0407]

[0408] SEQ ID NO:45: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0409]

[0410] SEQ ID NO:46: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0411]

[0412] SEQ ID NO:47: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0413]

[0414]

[0415] SEQ ID NO:48: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0416]

[0417] SEQ ID NO:49: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0418]

[0419] SEQ ID NO:50: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), wherein X7 is Asp or Glu and X8 is Leu or Met

[0420]

[0421]

[0422] SEQ ID NO:51: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0423]

[0424] SEQ ID NO:52: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0425]

[0426] SEQ ID NO:53: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0427]

[0428]

[0429] SEQ ID NO:54: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0430]

[0431] SEQ ID NO:55: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0432]

[0433] SEQ ID NO:56: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0434]

[0435] SEQ ID NO:57: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0436]

[0437] SEQ ID NO:58: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0438]

[0439] SEQ ID NO: 59: Mature human Fc IgG1, J1 is Asn or absent, J2 is Lys or absent, and wherein X4 is Asn or Ala, X5 is Leu or Asp, X6 is Gln or His, X7 is Asp or Glu, and X8 is Leu or Met, and X 10 Asn or Ser

[0440]

[0441] SEQ ID NO:60: Mature human Fc IgG1, DHS triple mutation (bold and underlined), J1 is Asn or absent, J2 is Lys or absent, and wherein X4 is Asn or Ala, X7 is Asp or Glu, and X8 is Leu or Met

[0442]

[0443] SEQ ID NO:61: Mature human Fc IgG1, DHS triple mutation (bold and underlined), wherein X4 is Asn or Ala, and X7 is Asp or Glu, and X8 is Leu or Met

[0444]

[0445] SEQ ID NO:62: Mature human Fc IgG1, DHS triple mutation (bold and underlined), wherein X7 is Asp or Glu and X8 is Leu or Met

[0446]

[0447] SEQ ID NO:63: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0448]

[0449] SEQ ID NO:64: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0450]

[0451] SEQ ID NO:65: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0452]

[0453]

[0454] SEQ ID NO:66: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0455]

[0456] SEQ ID NO:67: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0457]

[0458] SEQ ID NO:68: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0459]

[0460]

[0461] SEQ ID NO:69: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0462]

[0463] SEQ ID NO:70: Mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0464]

[0465] SEQ ID NO:71: Mature human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined), wherein X7 is Asp or Glu and X8 is Leu or Met

[0466]

[0467] SEQ ID NO:72: Mature human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0468]

[0469] SEQ ID NO:73: Mature human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0470]

[0471] SEQ ID NO:74: Mature human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0472]

[0473] SEQ ID NO:75: Mature human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0474]

[0475] SEQ ID NO:76: Mature human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0476]

[0477] SEQ ID NO:77: Mature human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0478]

[0479] SEQ ID NO:78: Mature human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)

[0480]

[0481]

[0482] SEQ ID NO:79: Mature human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)

[0483]

[0484] SEQ ID NO:80: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics), Asn to Ala substitution (*)

[0485]

[0486] SEQ ID NO:81: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(fa) (bold italics), Asn to Ala substitution (*)

[0487]

[0488]

[0489] SEQ ID NO:82: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics), YTE triple mutation (bold and underlined), Asn to Ala substitution (*)

[0490]

[0491] SEQ ID NO:83: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(fa) (bold italics), YTE triple mutation (bold and underlined), Asn to Ala substitution (*)

[0492]

[0493] In some embodiments, the variant Fc domain comprises an amino acid substitution at position 246 (e.g., K246X, wherein X is any amino acid other than Lys, such as 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, wherein X is not Lys. In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, V309D, Q311H, and N434S, wherein X is not Lys. In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, M428L, and N434S, wherein X is not Lys. In some embodiments, the variant Fc domain further comprises a mutation, eg, a C220S mutation, at position 220. The amino acid substitutions are relative to the amino acid sequence of a wild-type Fc monomer, eg, wild-type human IgG1 or IgG2.

[0494] In some embodiments, the variant Fc domain monomer comprises a sequence having at least 95% identity (e.g., 97%, 99%, or 99.5% identity) to the sequence of any one of SEQ ID NOs: 84-112 and 115-120 set forth below. In some embodiments, the variant Fc domain monomer comprises a sequence of any one of SEQ ID NOs: 84-112 and 115-120 set forth below.

[0495] In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, M252Y, S254T, and T256E, wherein X is not Lys. In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, V309D, Q311H, and N434S, wherein X is not Lys. In some embodiments, the variant Fc domain monomer comprises at least the following mutations: K246X, M428L, and N434S, wherein 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.

[0496] 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; and X 11 (position 358) is Leu or Met; X 12 (Position 428) is Met or Leu; X 13 (position 434) is Asn or Ser; X 14 (Position 447) is Lys or absent; N-terminal Fab residue is underlined; hinge residues are italicized

[0497]

[0498]

[0499] SEQ ID NO:85: Mature human IgG1 Fc; Cys to 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; and X5 is Leu or Met; X6 is Lys or absent; N-terminal Fab residue is underlined; hinge residues are italicized

[0500]

[0501] SEQ ID NO:86: Mature human IgG1 Fc; Cys to 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; and X4 is Leu or Met; the N-terminal Fab residue is underlined; hinge residues are italicized

[0502]

[0503] SEQ ID NO:87: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); X1 is Asp or Glu; and X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0504]

[0505] SEQ ID NO:88: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); allotype G1m(fa) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0506]

[0507] SEQ ID NO:89: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); allotype G1m(f) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0508]

[0509] SEQ ID NO:90: ​​Mature human IgG1 Fc; Cys to Ser substitution (#); Asn to Ala substitution (^); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asp or Glu; and X3 is Leu or Met; the N-terminal Fab residue is underlined; hinge residues are italicized

[0510]

[0511] SEQ ID NO:91: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); Asn to Ala substitution (^); X1 is Asp or Glu; and X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0512]

[0513] SEQ ID NO:92: Mature human IgG1 Fc; Cys to Ser substitution (#); YTE triple mutation (bold and underlined); 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; and X5 is Leu or Met; X6 is Lys or absent; N-terminal Fab residue is underlined; hinge residues are italicized

[0514]

[0515]

[0516] SEQ ID NO:93: Mature human IgG1 Fc; Cys to 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; and X4 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0517]

[0518] SEQ ID NO:94: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); YTE triple mutation (bold and underlined); X1 is Asp or Glu; and X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0519]

[0520] SEQ ID NO:95: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); YTE triple mutation (bold and underlined); allotype G1m(fa) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0521]

[0522] SEQ ID NO:96: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); YTE triple mutation (bold and underlined); allotype G1m(f) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0523]

[0524] SEQ ID NO:97: Mature human IgG1 Fc; Cys to Ser substitution (#); Asn to 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; and X3 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0525]

[0526] SEQ ID NO:98: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); Asn to Ala substitution (^); YTE triple mutation (bold and underlined); X1 is Asp or Glu; and X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0527]

[0528] SEQ ID NO:99: Mature human IgG1 Fc; Cys to Ser substitution (#); DHS triple mutation (bold and underlined); 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; and X5 is Leu or Met; X6 is Lys or absent; N-terminal Fab residue is underlined; hinge residues are italicized

[0529]

[0530] SEQ ID NO: 100: Mature human IgG1 Fc; Cys to 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; and X4 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0531]

[0532] SEQ ID NO: 101: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); DHS triple mutation (bold and underlined); X1 is Asp or Glu; and X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0533]

[0534] SEQ ID NO: 102: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); DHS triple mutation (bold and underlined); allotype G1m(fa) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0535]

[0536] SEQ ID NO: 103: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); DHS triple mutation (bold and underlined); allotype G1m(f) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0537]

[0538] SEQ ID NO: 104: Mature human IgG1 Fc; Cys to Ser substitution (#); Asn to 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; and X3 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0539]

[0540] SEQ ID NO: 105: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); Asn to Ala substitution (^); DHS triple mutation (bold and underlined); X1 is Asp or Glu; and X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0541]

[0542] SEQ ID NO: 106: Mature human IgG1 Fc; Cys to Ser substitution (#); LS double mutation (bold and underlined); 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; and X5 is Leu or Met; X6 is Lys or absent; N-terminal Fab residue is underlined; hinge residues are italicized

[0543]

[0544] SEQ ID NO: 107: Mature human IgG1 Fc; Cys to 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; and X4 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0545]

[0546] SEQ ID NO: 108: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); LS double mutation (bold and underlined); X1 is Asp or Glu; and X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0547]

[0548] SEQ ID NO: 109: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); LS double mutation (bold and underlined); allotype G1m(fa) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0549]

[0550] SEQ ID NO: 110: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); LS double mutation (bold and underlined); allotype G1m(f) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0551]

[0552] SEQ ID NO: 111: Mature human IgG1 Fc; Cys to Ser substitution (#); Asn to 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; and X3 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0553]

[0554] SEQ ID NO: 112: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); Asn to Ala substitution (^); LS double mutation (bold and underlined); X1 is Asp or Glu; and X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0555]

[0556] SEQ ID NO: 113: Palivizumab full length antibody; anti-RSV IgG; leader sequence underlined

[0557] Heavy chain:

[0558] MGWSCIILFLVATATGVHSQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGA GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0559] SEQ ID NO: 114: Palivizumab full length antibody; anti-RSV IgG; leader sequence underlined

[0560] Light chain:

[0561] MGWSCIILFLVATATGVHS DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0562] SEQ ID NO: 115: Mature human IgG1 Fc; Cys to Ser substitution (#); Asn to Gln substitution (^); allotype G1m(fa) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0563]

[0564] SEQ ID NO: 116: Mature human IgG1 Fc; Cys to Ser substitution (#); Asn to Gln substitution (^); allotype G1m(f) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0565]

[0566] SEQ ID NO: 117: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); LS double mutation (bold and underlined); Asn to Gln substitution (^); allotype G1m (f) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0567]

[0568] SEQ ID NO: 118: Mature human IgG1 Fc; Cys to Ser substitution (#); LS double mutation (bold and underlined); Asn to Gln substitution (^); X1 is Ser, Gly, Ala, Thr, Asn, Gln, Arg, His, Glu, or Asp; X2 is Asp or Glu; and X3 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0569]

[0570] SEQ ID NO: 119: Mature human IgG1 Fc; Cys to Ser substitution (#); Lys to Ser substitution (*); Asn to Gln substitution (^); LS double mutation (bold and underlined); X1 is Asp or Glu; and X2 is Leu or Met; N-terminal Fab residue is underlined; hinge residues are italicized

[0571]

[0572]

[0573] SEQ ID NO: 120: Mature human IgG1 Fc; Cys to Ser substitution (#); Asn to Gln substitution (^); allotype G1m(f) (bold italics); N-terminal Fab residue is underlined; hinge residues are italicized

[0574]

[0575] As defined herein, an Fc domain comprises H3 Interactions between antibody constant domains Two Fc domain monomers that dimerize, and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domain monomers. The Fc domain forms a minimal structure that binds to an Fc receptor, such as an Fc-γ receptor (ie, Fcγ receptor (FcγR)), an Fc-α receptor (ie, Fcα receptor (FcαR)), an Fc-ε receptor (ie, Fcε receptor (FcεR)), and / or a neonatal Fc receptor (FcRn). In some embodiments, the Fc domain of the present disclosure binds to an Fcγ receptor (e.g., FcRn, FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b)) and / or FcγRIV and / or a neonatal Fc receptor (FcRn).

[0576] In some embodiments, the Fc domain monomer or Fc domain of the present disclosure is a non-glycosylated 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 a non-glycosylated IgG1 variant that maintains engagement with an Fc receptor (e.g., an IgG1 having an amino acid substitution at N297 and / or T299 of a glycosylation motif). Exemplary non-glycosylated Fc domains and methods for preparing non-glycosylated Fc domains are known in the art, for example, as described in Sazinsky SL et al., Aglycosylated immunoglobulin G1 variants productively engage activating Fc receptors, PNAS, 2008, 105(51):20167-20172, which is incorporated herein in its entirety.

[0577] In some embodiments, the Fc domains or Fc domain monomers of the present disclosure are engineered to enhance binding to neonatal Fc receptors (FcRn). For example, the Fc domains may include a triple mutation corresponding to M252Y / S254T / T256E (YTE) (e.g., IgG1, such as a human or humanized IgG1 with a YTE mutation). The Fc domains may include a single mutant corresponding to N434H (e.g., IgG1, such as a human or humanized IgG1 with a N434H mutation). The Fc domains may include a single mutant corresponding to C220S (e.g., IgG1, such as a human or humanized IgG1 with a C220S mutation). The Fc domains may include a quadruple mutant corresponding to C220S / L309D / Q311H / N434S (CDHS) (e.g., IgG1, such as a human or humanized IgG1 with a DHS mutation). The Fc domain may include a triple mutant corresponding to L309D / Q311H / N434S (DHS) (e.g., IgG1, such as a human or humanized IgG1 with a DHS mutation). The Fc domain may include a combination of one or more of the above mutations that enhance binding to FcRn. Enhanced binding to FcRn may increase the half-life of an Fc domain-containing conjugate. For example, incorporation of one or more amino acid mutations that enhance FcRn binding (e.g., a YTE mutation, an LS mutation, or an N434H mutation) may increase the half-life of the conjugate by 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more relative to a conjugate having a corresponding Fc domain without mutations that enhance FcRn binding. Exemplary Fc domains with enhanced binding to FcRN and methods for preparing 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, which is incorporated herein by reference. As used herein, an amino acid "corresponding to" a particular amino acid residue (e.g., an amino acid residue of a particular SEQ ID NO.) is understood to include any amino acid residue that would be understood by one skilled in the art to be aligned with a particular residue (e.g., a residue of a particular sequence). For example, any of SEQ ID Nos: 1-112 and 115-120 can be mutated to include a YTE mutation, an LS mutation, and / or an N434H mutation by mutating the "corresponding residue" of the amino acid sequence.

[0578] 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 comprise an additional amino acid at the N-terminus (Xaa)x and / or an additional amino acid at the C-terminus (Xaa)z, wherein Xaa is any amino acid, and x and z are integers greater than or equal to zero, typically less than 100, preferably less than 10, and more preferably 0, 1, 2, 3, 4, or 5. For example, the additional amino acid may be a single amino acid at the C-terminus corresponding to Lys330 of IgG1.

[0579] In some embodiments, an 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, an 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).

[0580] In some embodiments, an 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, an Fc domain monomer is at least 20 kDa (e.g., at least 25 kDa, at least 30 kDa, or at least 35 kDa).

[0581] In some embodiments, an Fc domain monomer comprises 200 to 400 amino acid residues (e.g., 200 to 250, 250 to 300, 300 to 350, 350 to 400, 200 to 300, 250 to 350, or 300 to 400 amino acid residues). In some embodiments, an Fc domain monomer is 20 to 40 kDa (e.g., 20 to 25 kDa, 25 to 30 kDa, 35 to 40 kDa, 20 to 30 kDa, 25 to 35 kDa, or 30 to 40 kDa).

[0582] In some embodiments, the Fc domain monomer comprises an amino acid sequence that is at least 90% identical (e.g., at least 95%, at least 98% identical) to the sequence of any one of SEQ ID NOs: 1-112 and 115-120, or a region thereof. In some embodiments, the Fc domain monomer comprises the amino acid sequence of any one of SEQ ID NOs: 1-112 and 115-120, or a region thereof.

[0583] In some embodiments, an Fc domain monomer comprises a region of any one of SEQ ID NOs: 1-112 and 115-120, wherein the region comprises 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.

[0584] Activation of immune cells

[0585] Fc-γ receptors (FcγR) bind to the Fc portion of immunoglobulin G (IgG) and play an important role in immune activation and regulation. For example, the IgG Fc domain in the immune complex (Ic) engages FcγR with high affinity, thereby triggering the signal transduction cascade that regulates immune cell activation. The human FcγR family contains several activating receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb) and an inhibitory receptor (FcγRIIb). FcγR signaling is mediated by an intracellular domain containing an immunotyrosine-based activation motif (ITAM) for activating FcγR and an immunotyrosine-based inhibitory motif (ITIM) for inhibitory receptor FcγRIIb. In some embodiments, the Fc domain binds to FcγR, resulting in ITAM phosphorylation of Src family kinases; this activates Syk family kinases and induces downstream signal transduction networks including PI3K and Ras pathways.

[0586] In the fusion proteins or conjugates described herein, the Fc domain portion of the fusion protein or conjugate binds to FcγR (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells and activates phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Examples of immune cells that can be activated by the conjugates described herein include, but are not limited to, macrophages, neutrophils, eosinophils, basophils, lymphocytes, follicular dendritic cells, natural killer cells, and mast cells.

[0587] Tissue distribution

[0588] After the therapeutic agent enters the systemic circulation, it is distributed to the tissues of the body. Distribution is usually uneven due to differences in blood perfusion, tissue binding, regional pH, and cell membrane permeability. The rate at which the drug enters the tissue depends on the blood flow rate to the tissue, tissue mass, and the distribution characteristics between the blood and tissue. Distribution equilibrium between the blood and tissue (when the inflow and outflow rates are the same) is reached more rapidly in areas with rich blood vessels, unless diffusion across the cell membrane is the rate-limiting step. Size, shape, charge, target binding, FcRn and target binding mechanism, route of administration, and formulation affect tissue distribution.

[0589] In some cases, the fusion protein described herein can be optimized to be distributed to lung tissue. In some cases, the distribution concentration ratio of the fusion protein in the epithelial fluid is at least 30% of the fusion protein concentration in the plasma within 2 hours after administration. In certain embodiments, the concentration ratio is at least 45% within 2 hours after administration. In some embodiments, the concentration ratio is at least 55% within 2 hours after administration. Specifically, the concentration ratio is at least 60% within 2 hours after administration.

[0590] III. Connectors

[0591] A linker refers to a bond or connection between two or more components in a conjugate described herein (e.g., between two CD73 inhibitors in a conjugate described herein, between a CD73 inhibitor and an Fc domain in a conjugate described herein, and between a dimer of two CD73 inhibitors and an Fc domain in a conjugate described herein).

[0592] Linkers in conjugates having an Fc domain covalently linked to a monomer of a CD73 inhibitor

[0593] In conjugates containing an Fc domain monomer or Fc domain covalently linked to one or more CD73 inhibitor monomers described herein, the linker in the conjugate can be a bivalent structure having two arms. One arm of the bivalent linker can be attached to the monomer of the CD73 inhibitor and the other arm can be attached to the Fc domain monomer or Fc domain. In some embodiments, one or more CD73 inhibitor monomers in the conjugates described herein can each independently be linked to an atom in the Fc domain monomer or Fc domain.

[0594] In some embodiments, the linker is described by the formula:

[0595] 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

[0596] Among them J 1 is the key attached to A1; J 2 is a bond attached to E or a functional group capable of reacting with a functional group conjugated to E (e.g., maleimide and cysteine, amine and activated carboxylic acid (e.g., carboxylic acid activated with 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 the groups is independently an optionally substituted C1-C40 alkylene, an optionally substituted C1-C40 heteroalkylene, an optionally substituted C1-C40 alkyleneoxy, an optionally substituted C2-C20 alkenylene, an optionally substituted C2-C20 heteroalkenylene, an optionally substituted C2-C20 alkynylene, an optionally substituted C2-C20 heteroalkynylene, an optionally substituted C3-C20 cycloalkylene, an optionally substituted C2-C 20T is an optionally substituted C4-C20 heterocycloalkylene group, an optionally substituted C4-C20 heterocycloalkylene group, an optionally substituted C8-C20 cycloalkynylene group, an optionally substituted C8-C20 heterocycloalkynylene group, an optionally substituted C5-C15 arylene group, or an optionally substituted C2-C15 heteroarylene group; 1 、T 2 、T 3 、T 4 Each of which is independently O, S, NR i , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, imino or oxime; 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 20 wherein g, h, i, j, k, l, m, n, and o are independently 0, 1, or 2. In some embodiments, g, h, i, j, k, l, m, n, and o are independently 0 or 1.

[0597] In some embodiments, the optional substitution comprises substitution with polyethylene glycol (PEG). PEG has a repeating unit structure (-CH2CH2O-) n , wherein n is an integer from 2 to 100. Polyethylene glycol can be selected from PEG2 to PEG 100 Any of (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 ).

[0598] In some embodiments, J 2 Can have two points of attachment to an Fc domain monomer or Fc domain (e.g., two J 2 ).

[0599] Linkers in conjugates having an Fc domain covalently linked to a dimer of a CD73 inhibitor

[0600] In conjugates containing an Fc domain monomer or Fc domain covalently linked to one or more CD73 inhibitor dimers described herein, the linker in the conjugate can be a branched structure. As further described herein, the linker in the conjugates described herein can be a multivalent structure, such as a bivalent or trivalent structure having two or three arms, respectively. In some embodiments, when the linker has three arms, two arms can be attached to the first and second CD73 inhibitors and the third arm can be attached to the Fc domain monomer or Fc domain. In some embodiments, when the linker has two arms, one arm can be attached to the Fc domain and the other arm can be attached to one of the two CD73 inhibitors. In other embodiments, a linker with two arms can be used to attach two CD73 inhibitors to a conjugate containing an Fc domain covalently linked to one or more CD73 inhibitor dimers.

[0601] In some embodiments, the linker in the conjugate having an Fc domain covalently attached to one or more CD73 inhibitor dimers is described by formula (DLI):

[0602]

[0603] Among them L A From formula G A1 -(Z A1 ) g1 -(Y A1 ) h1 -(Z A2 ) i1 -(Y A2 ) j1 -(Z A3 ) k1 -(Y A3 ) l1 -(Z A4 ) m1 -(Y A4 ) n1 -(Z A5 )o1-G A2 Description; L B From formula G B1 -(Z B1 ) g2 -(Y B1 )h2 -(Z B2 ) i2 -(Y B2 ) j2 -(Z B3 ) k2 -(Y B3 ) l2 -(Z B4 ) m2 -(Y B4 ) n2 -(Z B5 )o2-G B2 Description; L C From formula G C1 -(Z C1 ) g3 -(Y C1 ) h3 -(Z C2 ) i3 -(Y C2 ) j3 -(Z C3 ) k3 -(Y C3 ) l3 -(Z C4 ) m3 -(Y C4 ) n3 -(Z C5 )o3-G C2 Description; G A1 is attached to Q in the formula (DLI) i Key; G A2 is a bond attached to the first CD73 inhibitor (e.g., A1); G B1 is attached to Q in the formula (DLI) i Key; G B2 is a bond attached to a second CD73 inhibitor (e.g., A2); G C1 is attached to Q in the formula (DLI) i Key; G C2 is a bond attached to an Fc domain monomer or an Fc domain, or is a functional group capable of reacting with a functional group conjugated to E (e.g., maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and olefin and tetrazine); Z A1 , Z A2 , Z A3 , Z A4 , Z A5 , Z B1 , Z B2 , Z B3 , Z B4 , ZB5 , Z C1 , Z C2 , Z C3 , Z C4 and Z C5 Each of the groups is independently an optionally substituted C1-C40 alkylene, an optionally substituted C1-C40 heteroalkylene, an optionally substituted C1-C40 alkyleneoxy, an optionally substituted C2-C20 alkenylene, an optionally substituted C2-C20 heteroalkenylene, an optionally substituted C2-C20 alkynylene, an optionally substituted C2-C20 heteroalkynylene, an optionally substituted C3-C20 cycloalkylene, an optionally substituted C2-C 20 Y is an optionally substituted C4-C20 heterocycloalkylene group, an optionally substituted C4-C20 heterocycloalkenylene group, an optionally substituted C8-C20 cycloalkynylene group, an optionally substituted C8-C20 heterocycloalkynylene group, an optionally substituted C5-C15 arylene group, or an optionally substituted C2-C15 heteroarylene group; A1 、Y A2 、Y A3 、Y A4 、Y B1 、Y B2 、Y B3 、Y B4 、Y C1 、Y C2 、Y C3 and Y C4 Each of which is independently O, S, NR i , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, imino or oxime; 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; g1, h1, i1, j1, k1, l1, m1, n1, o1, g2, h2, i2, j2, k2, l2, m2, n2, o2, g3, h3, Each of i3, j3, k3, l3, m3, n3 and o3 is independently 0 or 1; Q is a nitrogen atom, an optionally substituted C1-C20 alkylene group, an optionally substituted C1-C20 heteroalkylene group, an optionally substituted C2-C20 alkenylene group, an optionally substituted C2-C20 heteroalkenylene group, an optionally substituted C2-C20 alkynylene group, an optionally substituted C2-C20 heteroalkynylene group, an optionally substituted C3-C20 cycloalkylene group, an 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.

[0604] In some embodiments, the optional substitution comprises substitution with PEG. PEG has a repeating unit structure (-CH2CH2O-) n , wherein n is an integer from 2 to 100. Polyethylene glycol can be selected from PEG2 to PEG 100 Any of (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 ).

[0605] In some embodiments, L C There may be two points of attachment to the Fc domain (e.g., two G C2 ).

[0606] In some embodiments, L comprises a polyethylene glycol (PEG) linker. The PEG linker comprises a repeating unit structure (-CH2CH2O-) n wherein n is an integer from 2 to 100. The polyethylene glycol linker can covalently link the CD73 inhibitor and E (e.g., in a conjugate of formula (MI)). The polyethylene glycol linker can covalently link the first CD73 inhibitor and the second CD73 inhibitor (e.g., in a conjugate of formula (DI)). The polyethylene glycol linker can covalently link the CD73 inhibitor dimer and E (e.g., in a conjugate of formula (DI)). The polyethylene glycol linker can be selected from PEG2 to PEG 100 Any of (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 ). In some embodiments, L c Including PEG linker, where L C Covalently attached to Q i and each of E.

[0607] connector

[0608] 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 conjugates described herein, or between two CD73 inhibitors in the conjugates described herein. In some embodiments, the linker can be a bond, e.g., a covalent bond, e.g., an amide bond, a disulfide bond, a CO bond, a CN bond, a N-N bond, a C-S bond, or any type of bond resulting from a chemical reaction, e.g., a chemical conjugation. In some embodiments, a linker (e.g., L as shown in Formula (DI) or (MI)) comprises no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240 or 1-250 atoms; 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 10 0, 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).In some embodiments, the linker (L) comprises no more than 250 non-hydrogen atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-2 00, 1-210, 1-220, 1-230, 1-240 or 1-250 non-hydrogen atoms; 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95 In some embodiments, the present invention comprises at least one non-hydrogen atom (of the present invention). In some embodiments, the present invention comprises at least one non-hydrogen atom (of the present invention), at least one non-hydrogen atom (of the present invention), at least one non-hydrogen atom (of the present invention), at least one non-hydrogen atom (of the present invention), at least one non-hydrogen atom (of the present invention), at least one non-hydrogen atom (of the present invention), at least one non-hydrogen atom (of the present invention), In some embodiments, the backbone of the Linker (L) comprises no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1 -200, 1-210, 1-220, 1-230, 1-240 or 1-250 atoms; 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 9 5, 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).The "backbone" of a linker refers to the atoms in the linker that together form the shortest path from one part of the conjugate to another part of the conjugate. The atoms in the linker backbone are directly involved in connecting one part of the conjugate to another part of the conjugate. For example, a hydrogen atom attached to a carbon in the linker backbone is not considered to be directly involved in connecting one part of the conjugate to another part of the conjugate.

[0609] Molecules that can be used to prepare linkers (L) contain at least two functional groups, such as two carboxylic acid groups. In some embodiments of trivalent linkers, two arms of the linker may contain two dicarboxylic acids, wherein the first carboxylic acid can form a covalent bond with the first CD73 inhibitor in the conjugate and the second carboxylic acid can form a covalent bond with the second CD73 inhibitor in the conjugate, and the third arm of the linker can form a covalent bond (e.g., a C-O bond) with the Fc domain monomer or Fc domain in the conjugate. In some embodiments of bivalent linkers, the bivalent linker may contain two carboxylic acids, wherein the first carboxylic acid can form a covalent bond with one component in the conjugate (e.g., a CD73 inhibitor) and the second carboxylic acid can form a covalent bond (e.g., a C-S bond or a C-N bond) with another component in the conjugate (e.g., an Fc domain monomer or an Fc domain).

[0610] In some embodiments, a dicarboxylic acid molecule can be used as a linker (e.g., a dicarboxylic acid linker). For example, in a conjugate containing an Fc domain monomer or Fc domain covalently linked to one or more CD73 inhibitor dimers, the first carboxylic acid in 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.

[0611] In some embodiments, dicarboxylic acid molecules such as those described herein can be further functionalized to include one or more additional functional groups. The dicarboxylic acid can be further functionalized, for example, to provide an attachment point to an Fc domain monomer or Fc domain (e.g., via a linker, such as a PEG linker).

[0612] In some embodiments, when the CD73 inhibitor is attached to an Fc domain monomer or Fc domain, the linker may comprise a moiety comprising a carboxylic acid moiety and an amino moiety separated by 1 to 25 atoms.

[0613] In some embodiments, the linker may comprise a diamino moiety, such as a diamino moiety described herein, which may be further functionalized to comprise one or more additional functional groups. Such diamino linkers may be further functionalized, for example, to provide a point of attachment to an Fc domain monomer or Fc domain (e.g., via a linker, such as a PEG linker).

[0614] In some embodiments, molecules containing an azide group can be used to form a linker, wherein the azide group can undergo a cycloaddition with an alkyne to form a 1,2,3-triazole bond. In some embodiments, molecules containing an alkyne group can be used to form a linker, wherein the alkyne group can undergo a cycloaddition with an azide group to form a 1,2,3-triazole bond. In some embodiments, molecules containing a maleimide group can be used to form a linker, wherein the maleimide group can react with cysteine ​​to form a C-S bond. In some embodiments, molecules containing one or more haloalkyl groups can be used to form a linker, wherein the haloalkyl group can form a covalent bond, such as a C-N bond and a C-O bond, with a CD73 inhibitor.

[0615] In some embodiments, the linker (L) may contain a synthetic group derived from, for example, a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). In some embodiments, the linker may comprise one or more amino acid residues. In some embodiments, the linker may be an amino acid sequence (e.g., 1-25 amino acids, 1-10 amino acids, 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, 1-2 amino acids, or 1 amino acid sequence). In some embodiments, the linker (L) may comprise one or more optionally substituted C1-C40 alkylene, optionally substituted C1-C40 heteroalkylene (e.g., PEG units), optionally substituted C1-C40 alkyleneoxy, 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 20C-C15 aryl or optionally substituted C2-C15 heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino.

[0616] Conjugation chemistry

[0617] 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, such as via a linker. The following conjugation chemistry is particularly contemplated, for example, for conjugating a PEG linker (e.g., a functionalized PEG linker) to an Fc domain monomer or Fc domain.

[0618] The covalent conjugation of two or more components in the conjugate using a linker can be achieved using well-known organic chemical synthesis techniques and methods. The complementary functional groups on the two components can react with each other to form a covalent bond. Examples of complementary reactive functional groups include, but are not limited to, maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and olefin and tetrazine. Site-specific conjugation with polypeptides can be accomplished using techniques known in the art. Exemplary techniques for site-specific conjugation of small molecules to Fc domains are provided in Agarwall.P. et al., Bioconjugate Chem.26:176-192 (2015).

[0619] Other examples of functional groups capable of reacting with amino groups include, for example, alkylating agents and acylating agents. Representative alkylating agents include: (i) α-haloacetyl groups, such as XCH2CO- (wherein X = Br, Cl, or I); (ii) N-maleimido groups, which can react with amino groups via a Michael-type reaction or via an acylation reaction with a ring carbonyl addition; (iii) aryl halides, such as nitrohaloaryl groups; (iv) alkyl halides; (v) aldehydes or ketones capable of forming Schiff bases with amino groups; (vi) epoxides, such as epichlorohydrin and dioxirane, which can react with amino groups, sulfhydryl groups, or phenolic hydroxyl groups; (vii) chlorine-containing s-triazines, which are reactive toward nucleophiles such as amino groups, sulfhydryl groups, and hydroxyl groups; (viii) aziridines, which are reactive toward nucleophiles such as amino groups via ring opening; (ix) diethyl squarate; and (x) α-haloalkyl ethers.

[0620] Examples of amino-reactive acylating groups include, for example, (i) isocyanates and isothiocyanates; (ii) sulfonyl chlorides; (iii) acyl halides; (iv) active esters, such as nitrophenyl esters or N-hydroxysuccinimidyl esters; (v) anhydrides, such as mixed, symmetrical, or N-carboxylic anhydrides; (vi) acyl azides; and (vii) imidates. Aldehydes and ketones can react with amines to form Schiff bases, which can be stabilized by reductive amination.

[0621] It should be understood that certain functional groups can be converted to other functional groups prior to the reaction, for example to impart additional reactivity or selectivity. Examples of methods that can be used for this purpose include converting amines to carboxyl groups using reagents such as dicarboxylic anhydrides; converting amines to thiols using reagents such as N-acetylhomocysteine ​​thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives; converting thiols to carboxyl groups using reagents such as α-haloacetates; converting thiols to amines using reagents such as ethyleneimine or 2-bromoethylamine; converting carboxyl groups to amines using reagents such as carbodiimides followed by diamines; and converting alcohols to thiols using reagents such as p-toluenesulfonyl chloride, followed by transesterification with thioacetates and hydrolysis to thiols with sodium acetate.

[0622] In some embodiments, a linker of the present disclosure (e.g., L, such as L of DLI) C ) is conjugated (e.g., by any of the methods described herein) to E (e.g., an Fc domain). In preferred embodiments of the present disclosure, the linker is conjugated by: (a) a thiourea bond to a lysine of E (i.e., -NH(C=S)NH-); (b) a carbamate bond to a lysine of E (i.e., -NH(C=O)-O); (c) an amine bond between a lysine and E (i.e., -NHCH2) by reductive amination; (d) an amide bond to a lysine of E (i.e., -NH-(C=O)CH2); a cysteine-maleimide conjugate between a maleimide of the linker I and a cysteine ​​of E; (f) a linker to (i) an oxime bond between a linker and an amino acid residue of E; (j) an azide bond between a linker and E; (k) direct acylation of the linker to E; or (l) a thioether bond between a linker and E.

[0623] In some embodiments, the linker is conjugated to E, wherein the bond comprises the structure -NH(C=NH)X-, wherein X is O, HN, or a bond. In some embodiments, the linker is conjugated to E, wherein the bond between the remainder of the linker and E comprises the structure -NH(C=O)NH-.

[0624] In some embodiments, the linker is conjugated to E, wherein the bond comprises the structure -R9OR9C(=O)NH-, wherein 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 15 In some embodiments, the linker is conjugated to E, wherein the bond between the remainder of the linker and E comprises the structure -CH2OCH2C(=O)NH-.

[0625] Exemplary linking strategies (e.g., methods for linking a monomer or dimer of a CD73 inhibitor to E, such as via a linker) are further described in the Examples.

[0626] In some embodiments, a linker (e.g., an active ester such as a nitrophenyl ester or an N-hydroxysuccinimidyl ester, or a derivative thereof) (e.g., a functionalized PEG linker (e.g., an azido-PEG2-PEG 40 -NHS ester)) is conjugated to E, wherein T (e.g., drug-antibody ratio or DAR) is between 0.5 and 10.0, for example, 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, , 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-(PEG-PEG) 40)-azido groups can be click-conjugated to oligonucleotides with terminal alkyne linkers (e.g., L, such as DLI) C During click conjugation, the azido group (e.g., Fc–(PEG2-PEG 40 )-azido) with an alkyne (e.g., with a terminal alkyne linker (e.g., L, such as DLI) C ) of Int) to form a 5-membered heteroatom ring. In some embodiments, the linker conjugated to E is a terminal alkyne and is conjugated to Int having a terminal azide group. The examples describe E-(PEG2-PEG 40 One skilled in the art will readily understand the final product from click chemistry conjugation.

[0627] Exemplary linking strategies are described further herein.

[0628] IV. Treatment Methods

[0629] The present disclosure provides for the use of the conjugates and pharmaceutical compositions described herein for treating disorders associated with dysregulation or overexpression of CD73 (e.g., cancer, fibrosis, or viral infection).

[0630] cancer

[0631] The conjugates and pharmaceutical compositions described herein can be used to treat cancer in a subject. In some embodiments, the cancer overexpresses or is known to overexpress CD73 relative to non-cancerous cells of the same tissue type. In some embodiments, the subject has been determined to have a cancer that overexpresses CD73 relative to non-cancerous cells of the same tissue type. In some embodiments, the method further comprises the step of determining whether the cancer overexpresses CD73 relative to non-cancerous cells of the same tissue type and administering the conjugate only if the cancer overexpresses CD73.

[0632] 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 carcinoma; bile duct cancer; endometrial cancer; melanoma; or esophageal cancer. In some embodiments, the cancer is a solid tumor.

[0633] 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 anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-LAG3 antibodies, anti-B7.1 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-TIM3 antibodies, anti-VISTA antibodies, anti-CD137 antibodies, anti-OX40 antibodies, anti-CD40 antibodies, anti-CD27 antibodies, anti-CCR4 antibodies, anti-GITR antibodies, anti-NKG2D antibodies and anti-KIR antibodies. 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.

[0634] Immune checkpoints approved or under development include but are not limited to (ipilimumab), (nivolumab), KEY (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, IMP 321, and MGA271, as well as many other antibodies or fusion proteins to the immune checkpoint proteins described herein.

[0635] In some embodiments, the method includes administering (1) a conjugate as described herein and (2) an immune checkpoint inhibitor to the subject. In some embodiments, the conjugate as 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 as described herein is administered alone. In some embodiments, the conjugate as 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 as described herein and the immune checkpoint inhibitor are administered together (e.g., administered substantially simultaneously in the same or separate pharmaceutical compositions, or administered separately in the same treatment regimen), the tumor growth inhibitory effect of each of the conjugate and the immune checkpoint inhibitor may be greater (e.g., occurring at a lower concentration) than when each is used alone in a treatment regimen.

[0636] Viral infection

[0637] The conjugates and pharmaceutical compositions described herein can be used to treat viral infections in subjects. The conjugates and pharmaceutical compositions described herein can also be used to prevent viral infections in subjects susceptible to or at increased risk of viral infections (e.g., subjects who are hospitalized, immunocompromised, preparing for surgery, recently had surgery, or are taking drugs that affect the immune system, such as radiochemotherapy).

[0638] In some embodiments, the viral infection is a betacoronavirus infection. In some embodiments, the betacoronavirus 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.

[0639] In some embodiments, the method further comprises administering an antiviral agent or antiviral vaccine to the subject. In some embodiments, the method comprises administering to the subject (1) a conjugate described herein and (2) an antiviral agent or antiviral vaccine. In some embodiments, the conjugate described herein is administered first, followed by the antiviral agent or antiviral vaccine alone. In some embodiments, the antiviral agent or antiviral vaccine is administered first, followed by the conjugate described herein alone. In some embodiments, the conjugate described herein and the antiviral agent or antiviral vaccine are administered substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, when the conjugate described herein and the antiviral agent or antiviral vaccine are administered together (e.g., administered substantially simultaneously in the same or separate pharmaceutical compositions, or administered separately in the same treatment regimen), the inhibitory effect of the conjugate and the antiviral agent or antiviral vaccine on viral replication may be greater (e.g., occurring at lower concentrations) than when each is used alone in a treatment regimen.

[0640] fibrosis

[0641] The conjugates and pharmaceutical compositions described herein can be used to treat or prevent fibrosis in a subject.

[0642] In some embodiments, the fibrosis is pulmonary fibrosis, skin fibrosis, renal fibrosis, liver fibrosis, cardiac fibrosis, or systemic sclerosis. In some embodiments, the fibrosis is pulmonary fibrosis. In some embodiments, the pulmonary fibrosis is associated with 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.

[0643] 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, keloids, chronic kidney disease, chronic transplant rejection, scarring, wound healing, postoperative adhesions, reactive fibrosis, polymyositis, ANCA vasculitis, Behcet's disease, antiphospholipid syndrome, relapsing polychondritis, familial Mediterranean fever, giant cell arteritis, Graves' ophthalmopathy, discoid lupus, pemphigus, bullous pemphigoid, hidradenitis suppurativa, sarcoidosis, bronchiolitis obliterans, primary sclerosing cholangitis, primary biliary cirrhosis, and organ fibrosis (e.g., skin fibrosis, lung fibrosis, liver fibrosis, kidney fibrosis, or cardiac fibrosis). In some embodiments, the fibrosis is scleroderma (e.g., systemic sclerosis, localized scleroderma, or scleroderma without cutaneous sclerosis). In some embodiments, the fibrosis is organ fibrosis (e.g., skin fibrosis, lung fibrosis, liver fibrosis, kidney fibrosis, or cardiac fibrosis). In some embodiments, the fibrosis is cystic fibrosis.

[0644] Treatment of fibrosis can be assessed by appropriate methods known to those skilled in the art, including improvement, amelioration, or slowing of progression of one or more symptoms associated with the particular fibrotic disease being treated.

[0645] V. Pharmaceutical Compositions

[0646] The conjugates herein can be formulated in pharmaceutical compositions for use in the methods described herein. In some embodiments, the conjugates described herein can be formulated alone in a pharmaceutical composition. 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 as part of a dosing regimen (e.g., sequentially or simultaneously). In some embodiments, the pharmaceutical composition comprises a conjugate described herein and a pharmaceutically acceptable carrier and excipient.

[0647] Acceptable carriers and excipients in the pharmaceutical composition are nontoxic to the recipient at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acid residues such as glycine, glutamine, histidine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol.

[0648] Examples of other excipients include, but are not limited to, anti-adherents, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, adsorbents, suspending or dispersing agents, or sweeteners. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium hydrogen phosphate, calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0649] The conjugates described herein have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. These salts can be acid addition salts involving inorganic or organic acids, or, in the case of the acidic forms of the conjugates herein, the salts can be prepared from inorganic or organic bases. Typically, the conjugates are prepared or used as pharmaceutically acceptable salts, which are prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art, such as hydrochloric 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 suitable salts are well known in the art.

[0650] Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, 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 salts. Representative alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0651] According to route of administration and dosage, the conjugate herein or its pharmaceutical composition for use in the methods described herein will be formulated into a suitable pharmaceutical composition to allow easy delivery. The conjugate or its pharmaceutical composition can be formulated for intramuscular, intravenous (e.g., as a sterile solution and in a solvent system suitable for intravenous use), intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, local, intratumoral, intraperitoneal, subcutaneous, subconjunctival, intravesical, mucosal, pericardial, intraumbilical, intraocular, oral (e.g., tablet, capsule, microcapsule, capsule or syrup), external (e.g., as a cream, gel, lotion or ointment), local, by inhalation, by injection or by infusion (e.g., continuous infusion, local perfusion directly soaking target cells, catheter, lavage, in cream or lipid composition) use. Depending on the route of administration, the conjugates herein or pharmaceutical compositions thereof can be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels (including hydrogels), pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injections, implants, sprays, formulations suitable for iontophoretic delivery, or aerosols. The compositions can be formulated according to conventional pharmaceutical practice.

[0652] The conjugates described herein can be formulated in a variety of ways known in the art. In order to be used as treatment for human and animal subjects, the conjugates described herein can be formulated as pharmaceutical or veterinary compositions. According to the subject to be treated (e.g., mankind), the mode of administration, and the desired type of treatment (e.g., prevention or treatment), the conjugates described herein are formulated in a manner consistent with these parameters. An overview of such technology 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 JC Boylan, Marcel Dekker, New York (2013), each of which is incorporated herein by reference.

[0653] The preparation can be prepared in a manner suitable for systemic administration or topical administration. Systemic preparations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection), or can be prepared for transdermal, transmucosal or oral administration. The preparation will typically comprise a diluent, and in some cases adjuvants, buffers and preservatives. The conjugate can also be administered in a liposome composition or as a microemulsion. Systemic administration can also include relatively non-invasive methods, such as using suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also applicable to the conjugates herein. Suitable forms include syrups, capsules and tablets, as understood in the art.

[0654] The pharmaceutical composition can be administered parenterally in the form of an injectable preparation. The pharmaceutical composition for injection can be prepared using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. The preparation can be prepared as 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, normal saline, and cell culture medium (e.g., Dulbecco's modified Eagle's medium (DMEM), α-modified Eagle's medium (α-MEM), F-12 culture medium). Such injectable compositions may also contain a certain amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, such as sodium acetate and sorbitan monolaurate. Preparation methods are known in the art, see, for example, Pharmaceutical Preformulation and Formulation, 2nd edition, M.Gibson, Taylor & Francis Group, CRC Press (2009).

[0655] Pharmaceutical compositions can be prepared in the form of oral preparations. Preparations for oral use include tablets containing active ingredients mixed with non-toxic pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch (including potato starch), calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate); granulating agents and disintegrants (e.g., cellulose derivatives (including microcrystalline cellulose), starch (including potato starch), cross-linked sodium carboxymethyl cellulose, alginate or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinyl pyrrolidone or polyethylene glycol); and lubricants, glidants and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil or talc). Formulations for oral use may also 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 water or an oil medium (e.g., peanut oil, liquid paraffin or olive oil). Powders, granules and pills may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, for example, a mixer, fluidized bed equipment or spray drying equipment.

[0656] Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colorants, flavorings, plasticizers, wetting agents, and buffers. Preparations for oral use may also be provided as chewable tablets, or as hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules, wherein the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin, or olive oil). Powders, granules, and pills may be prepared in a conventional manner using, for example, a mixer, fluidized bed apparatus, or spray drying equipment, using the ingredients mentioned above under tablets and capsules.

[0657] The dissolution or diffusion controlled release of the conjugates described herein or their pharmaceutical compositions can be achieved by appropriate coating of tablets, capsules, pills or granular formulations of the conjugate or by incorporating the conjugate into an appropriate matrix. The controlled release coating can include the coating materials mentioned above and / or one or more of the following, for example: shellac, beeswax, sugar wax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glyceryl palmitostearate, ethyl cellulose, acrylic resin, dl-polylactic 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, the matrix material may also include, for example, hydrated methylcellulose, Kahnawa wax and stearyl alcohol, Carbopol 934, silicone, tristearin, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene and / or halogenated fluorocarbons.

[0658] The pharmaceutical composition may be formed into a unit dosage form as desired. The amount of active ingredient, such as a conjugate described herein, contained in the pharmaceutical composition is such as to provide a suitable dosage within the specified range (eg, a dosage within the range of 0.01-100 mg / kg body weight).

[0659] VI. Route of Administration and Dosage

[0660] In any of the methods described herein, the conjugates herein can be administered by any appropriate route for treating or preventing a condition described herein (e.g., cancer, viral infection, or fibrotic condition). The conjugates described herein can be administered to humans, domestic pets, livestock, or other animals together with a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, administration includes intramuscular, intravenous (e.g., as a sterile solution and in a solvent system suitable for intravenous use), intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topical, intratumoral, intraperitoneal, subcutaneous, subconjunctival, intravesical, intramucosal, intrapericardial, intraumbilical, intraocular, oral (e.g., tablets, capsules, microcapsules, caplets or syrups), external (e.g., as a cream, gel, lotion or ointment), topical, by inhalation, by injection or by infusion (e.g., continuous infusion, local perfusion directly soaking target cells, catheter, lavage, in a cream or lipid composition) administration of any conjugate or composition described herein. In some embodiments, if a second therapeutic agent is administered in addition to a conjugate described herein, the second therapeutic agent or its pharmaceutical composition can also be administered by any route of administration described herein.

[0661] The dosage of the conjugates described herein or their pharmaceutical compositions thereof depends on a variety of factors, including route of administration, disease to be treated, and physical characteristics of the subject, such as age, weight, and overall health. Typically, the amount of the conjugate or its pharmaceutical composition contained in a single dose can be an amount that can effectively prevent, delay, or treat a condition without inducing significant toxicity. The pharmaceutical composition may comprise 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg), and in more specific embodiments, about 0.1 to about 30 mg / kg, and in more specific embodiments, about 1 to about 30 mg / kg of the conjugates described herein. In some embodiments, when a conjugate described herein and a second therapeutic agent are administered in combination (e.g., administered substantially simultaneously in the same or separate pharmaceutical compositions, or administered separately in the same treatment regimen), the required dosage of the conjugate described herein may be lower than the dosage of the conjugate required when the conjugate is used alone in the treatment regimen.

[0662] The conjugates described herein, or pharmaceutical compositions thereof, can be administered to a subject in need thereof, for example, daily, weekly, monthly, semi-annually, annually, or as medically necessary, one or more times (e.g., 1-10 times or more; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). Dosages can be provided in single or multiple dosing regimens. The time between administrations can be reduced as the medical condition improves or increased as the patient's health declines. The dosage and frequency of administration can be adjusted by the physician based on conventional factors such as the extent of infection and the different parameters of the subject.

[0663] Example

[0664] The following examples are put forth so as to provide one of ordinary skill in the art with a description of how the compositions and methods described herein might be used, prepared, and evaluated and are intended solely as exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

[0665] General procedure for preparing Fc constructs

[0666] The reverse translation of the amino acids, including the protein construct, was synthesized by solid phase synthesis. The oligonucleotide template was cloned into the cloning sites BamHI and XhoI (New England Biolabs, Ipswich, MA, USA) in pcDNA3.1 (Life Technologies, Carlsbad, CA, USA) and included a signal sequence derived from human interleukin-2 or human albumin. The pcDNA3.1 plasmid was transformed into Top10 Escherichia coli (E. coli) cells (LifeTech). DNA was amplified, extracted, and purified using the HiPure Plasmid Filter Maxiprep Kit (LifeTech). 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 purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA). The purified molecules were analyzed using 4-12% Bis Tris SDS PAGE.

[0667] General procedure for conjugation of intermediate (int) to Fc

[0668] At ambient temperature, a solution of the trifluorophenyl ester dissolved in DMF (1 mL) was added to a solution of 50 mg Fc in PBS at pH 7.4 (19.5 mg / mL). The resulting solution was adjusted to pH 8.5 with borate buffer (300 uL, 1 M, pH 8.5) or carbonate buffer (300 uL, 1 M, pH 8.5). The uniform colorless reaction mixture was gently shaken for 3 hours and then purified according to the general procedure. The Maldi TOF analysis of the purified final product gave an average mass from which the DAR was calculated.

[0669] General procedure for purification of conjugates

[0670] Protein A, dialysis and SEC: Using Mabselect PrismA (Protein A purification) resin, eluted with TBS pH 7.4, then dialyzed into 150mM histidine (2x) using a Slide-d-lyzer G2 dialysis cassette (30,000 MWCO), then dialyzed into 150mM NaCl pH 8.5 buffer, and then size exclusion chromatography was performed using TBS pH 7.4 buffer to purify the conjugate. The final product was in TBS (25mM Tris, 150mM NaCl) pH 7.4 buffer. The purified material was quantified using a UV-visible spectrophotometer (Bradford protein assay) and concentrated to approximately 10 mg / ml using a centrifugal concentrator (30,000 MWCO).

[0671] Synthesis of intermediate A

[0672]

[0673] EDC (1.6 g, 8.2 mmol) was added 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) in 4 portions. The mixture was stirred at ambient temperature for 2 hours. The organic phase was washed with DI water (30 mL), and the aqueous phase was back extracted with DCM (2x, 20 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated. The crude product mixture was purified by silica gel chromatography (0-80% ethyl acetate in hexane, 25 minute gradient) to obtain the product as a clear oil. Yield 2.15 g, 79%. Measured ions [M+Na] by LC / MS + =443.8.

[0674] Synthesis of intermediate B

[0675]

[0676] 4,6-Dichloro-1H-pyrazolo[3,4,d]pyrimidine (5 g, 26.5 mmol) and ammonium sulfate (62 mg) were dissolved in 150 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 bD-ribofuranose 1,2,3,5-tetraacetate (9.3 g, 29.1 mmol) was added, and the mixture was stirred until all the solids dissolved. The mixture was cooled to 0 ° C, and TMSOTf (6.2 mL, 34.4 mmol) was added dropwise over 5 minutes. The reaction mixture was gradually warmed to ambient temperature and stirred for 3 hours. The mixture was concentrated and taken up in ethyl acetate (100 mL). The organic extract was washed with saturated sodium bicarbonate, then 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 7.8 g, 66%. LCMS found ions: [M+Na] + =469.2.

[0677] Synthesis of intermediate C

[0678]

[0679] 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 in 100 mL of acetonitrile and bD-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 gradually warmed to ambient temperature and stirred for 3 hours. The mixture was then cooled to 0 ° C and saturated sodium bicarbonate was carefully added to neutralize TMS triflate. The mixture was extracted with ethyl acetate (3x, 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%. LCMS ion: [M+H] + =446.0.

[0680] Synthesis of intermediate D

[0681]

[0682] 2,4-dichloro-7H-pyrrolo[2,3,d]pyrimidine (5g, 26.6mmol) and ammonium sulfate (38mg, 0.291mmol) are dissolved in 30mL hexamethyldisilazane. The mixture is then warmed to reflux and stirred for 3 hours. The mixture is then concentrated on a rotary evaporator and dried under high vacuum for 12 hours. The solid residue is then taken in 100mL acetonitrile and bD-ribofuranose 1,2,3,5-tetraacetate (10.2g, 31.9mmol) is added. The mixture is cooled to 0°C and TMSOTf (5.28mL, 29.1mmol) is added dropwise. The reaction mixture is gradually warmed to room temperature and stirred overnight. The mixture is then concentrated and taken in ethyl acetate. The organic layer is washed with saturated sodium bicarbonate and brine, and dried over sodium sulfate, filtered and concentrated. The crude residue is purified by silica gel column chromatography (hexane / EtOAc) to obtain the desired compound as white foam. Yield: 2.35 g, 19.8%. LCMS ion: [M+H] + =446.2.

[0683] Synthesis of Int-132

[0684]

[0685] Step a

[0686]

[0687] Sodium triacetoxyborohydride (412 mg, 1.95 mmol) is added to a stirred mixture of 2-trifluoromethylbenzaldehyde (226 mg, 1.3 mmol) and propargyl-peg4-amine (300 mg, 1.3 mmol) in DCM (25 mL), and the reactant is stirred at ambient temperature for 16 hours. Methanol (3 mL) is added and the mixture is concentrated and purified by silica gel chromatography (0-10% methanol in DCM, 25 minutes) to give the product as a clear oil. Yield 73%, 375 mg. Measured ion by LC / MS: [M+H] + = 390.0.

[0688] Step b.

[0689]

[0690] Intermediate B (430 mg, 0.96 mmol), the amine product from the previous step (430 mg, 0.96 mmol) and triethylamine (194 mg, 1.93 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 minute gradient) to give the product as a clear oil. Yield 79%, 610 mg. Measured ion by LC / MS: [M+H] + = 800.2.

[0691] Step c.

[0692]

[0693] The triacetate product (610 mg, 0.76 mmol) and potassium carbonate (30 mg) from the previous step were stirred in methanol (30 mL) at ambient temperature for 2 hours. The mixture was filtered and neutralized with glacial acetic acid (0.5 mL) and concentrated. The crude residue was purified by silica gel chromatography (0-10% methanol in DCM, 25 minutes). The triol was taken in DMF (5 mL) and 2,2-dimethoxypropane (10 mL), p-toluenesulfonic acid hydrate (15 mg) was added and the mixture was stirred at 70 ° C for 1 hour. Triethylamine (0.5 mL) was added to the reactant, and the mixture was concentrated on a rotary evaporator. The crude residue was purified by silica gel chromatography (0-10% methanol in DCM, 25 minutes) to obtain a product in the form of a clear oil. Yield 89%, 2 steps, 420 mg. Measured ion by LC / MS: [M+H] + = 714.2.

[0694] Step d.

[0695]

[0696] The acetone compound (460 mg, 0.64 mmol) from the previous step in THF (5 mL) was added dropwise to a mixture of methylene (phosphorus dichloride) (633 mg, 2.68 mmol) in THF (5 mL) cooled to 0 ° C via an ice bath. When the addition was complete, the ice bath was removed and the reactant was stirred at ambient temperature for 3 hours. The mixture was then cooled to 0 ° C via an ice bath and a 0.5 N HCl aqueous solution (10 mL) was added. The mixture was stirred at 0 ° C for 15 minutes and then stirred at ambient temperature for 3 hours (monitored by LC / MS). The solvent was reduced to about 5 mL on a rotary evaporator and the crude mixture was purified by reverse phase HPLC (5-85% acetonitrile in DI water, 0.1% TFA modifier, 25 minute gradient). The pure fractions were combined and lyophilized to obtain the product as a white hygroscopic solid. Yield 55%, 295 mg. Measured ion by LC / MS: [M+H] + =832.2.

[0697] Step e.

[0698]

[0699] The product (70 mg, 0.84 mmol) and intermediate A (35 mg, 0.84 mmol) from the previous step were dissolved in DMF (1 mL) and cooled to 0 ° C via an ice-water bath. Copper sulfate (2 mg, 0.013 mmol) was added to a mixture of BTTA (9 mg, 0.021 mmol) and sodium ascorbate (50 mg, 0.25 mmol) in DI water (2 mL), and the mixture was gently shaken for 15-20 seconds until the solution was clear. The copper mixture was added to the alkyne / azide mixture, and the reactants were stirred at 0 ° C for 10 minutes, then at ambient temperature for 20 minutes. The crude reaction mixture was directly applied to reverse phase HPLC (5%-85% acetonitrile in DI water, 0.1% TFA modifier, 25 minute gradient). Pure fractions were combined and lyophilized to give the product as a white hygroscopic solid. Yield 43%, 45 mg. Measured ion [(M / 2)+H] + = 627.2 by LC / MS.

[0700] Synthesis of conjugate 70

[0701] To a solution of SEQ ID NO: 13 (3.40 mL, 100 mg, 0.0017 mmol) in PBS 7.4 was added Int-132 (21 mg, 0.017 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 reactants were then gently shaken for 4 hours. The reaction was quenched by stirring for 12 hours in 150 mM His / 100 mM ammonium hydroxide buffer (pH 8.5) and then purified 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%.

[0702] Synthesis of Int-110

[0703]

[0704] The title compound was prepared as described for Int-132, where 2-trifluoromethylbenzaldehyde was replaced with 3-trifluoromethylbenzaldehyde. Yield: 45 mg, 43%. LC / MS found ion [(M / 2)+H]+ = 627.2.

[0705] Synthesis of conjugate 58

[0706] The title compound was prepared analogously to conjugate 70, wherein the starting material used in Int-132 was replaced by Int-110. Maldi TOF analysis of the purified final product gave an average mass of 63,530 Da (DAR = 4.8).

[0707] Synthesis of Int-6

[0708]

[0709] The title compound was prepared as described for Int-132, where 2-trifluoromethylbenzaldehyde was replaced with benzaldehyde. Found by LC / MS: ion [(M / 2)+H]+ = 593.2.

[0710] Synthesis of conjugate 4b

[0711] The title compound was prepared analogously to conjugate 70, wherein the starting material used in Int-132 was replaced by Int-6. Maldi TOF analysis of the purified final product gave an average mass of 61,147 Da (DAR = 2.8).

[0712] Synthesis of conjugate 4a

[0713] To a solution of SEQ ID NO: 17 (5.4 mL, 100 mg, 0.0017 mmol) in PBS 7.4 was added the product (14 mg, 0.016 mmol) described in Int-6 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 reactant was then gently shaken for 4 hours and then purified 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%.

[0714] Synthesis of Int-7

[0715]

[0716] The title compound was prepared as described for Int-132, where 2-trifluoromethylbenzaldehyde was replaced with cyclopentanone. Found by LC / MS: ion [(M / 2)+H]+ = 582.2.

[0717] Synthesis of conjugate 5b

[0718] The title compound was prepared analogously to conjugate 70, wherein the starting material described in Int-110 was replaced by Int-7. Maldi TOF analysis of the purified final product gave an average mass of 62,224 (DAR=3.9).

[0719] Synthesis of conjugate 5a

[0720] The title compound was prepared analogously to conjugate 4a, wherein the starting material described in Int-110 was replaced by Int-7. Maldi TOF analysis of the purified final product gave an average mass of 62,404 Da (DAR = 4.2).

[0721] Synthesis of Int-74

[0722]

[0723] The title compound was prepared as described for Int-132, where 2-trifluoromethylbenzaldehyde was replaced with 3,4-dichlorobenzaldehyde. Found by LC / MS: ion [(M / 2)+H]+ = 627.2.

[0724] Synthesis of conjugate 40

[0725] The title compound was prepared analogously to conjugate 70, wherein the starting material used in Int-110 was replaced by Int-74. Maldi TOF analysis of the purified final product gave an average mass of 66,569 (DAR = 7.6).

[0726] Synthesis of Int-75

[0727]

[0728] The title compound was prepared as described for Int-132, where 2-trifluoromethylbenzaldehyde was replaced with 3,4-dichlorobenzaldehyde. Found by LC / MS: ion [(M / 2)+H]+ = 627.2.

[0729] Synthesis of conjugate 41

[0730] The title compound was prepared analogously to conjugate 70, wherein 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).

[0731] Synthesis of Int-111

[0732]

[0733] The title compound was prepared as described for Int-132, where 2-trifluoromethylbenzaldehyde was replaced with 1-formylbenzofuran. Found by LC / MS: ion [(M / 2)+H]+ = 613.2.

[0734] Synthesis of conjugate 59

[0735] The title compound was prepared analogously to conjugate 70, wherein the starting material used in Int-110 was replaced by Int-111. Maldi TOF analysis of the purified final product gave an average mass of 63,411 (DAR = 4.7).

[0736] Synthesis of Int-16

[0737]

[0738] Step a.

[0739]

[0740] Intermediate B (350 mg, 0.78 mmol), R-2-piperidinecarboxylic acid methyl ester (112 mg, 0.78 mmol) and triethylamine (194 mg, 1.93 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) to obtain the product as a clear oil. Yield 375 mg, 85%. Measured ion by LC / MS: [M+H] + = 554.2.

[0741] Step b.

[0742]

[0743] The triacetate product (375 mg, 0.68 mmol) and potassium carbonate (30 mg) from the previous step were stirred in methanol (30 mL) at ambient temperature for 2 hours. The mixture was filtered, neutralized with glacial acetic acid (0.5 mL) and concentrated. The crude residue was purified by silica gel chromatography (0-10% methanol in DCM, 25 minute gradient). The triol was taken in DMF (5 mL) and 2,2-dimethoxypropane (10 mL). p-Toluenesulfonic acid hydrate (15 mg) was added to the reaction and the mixture was stirred at 70 ° C for 1 hour. Triethylamine (0.5 mL) was added to the reactant, and the mixture was concentrated on a rotary evaporator. The crude residue was purified by silica gel chromatography (0-10% methanol in DCM, 25 minutes) to obtain a product as a clear oil. Yield 175 mg, 54%, 2 steps. Measured ion [M+H] + = 468.2 by LC / MS.

[0744] Step c.

[0745]

[0746] The acetonide (175 mg, 0.37 mmol) from the previous step in THF (5 mL) is added dropwise to a stirred mixture of methylene (phosphorus dichloride) (467 mg, 1.87 mmol) and DIEA (53 mg, 0.41 mmol) in THF (5 mL) cooled to 0 ° C via an ice bath. When the addition is complete, the ice bath is removed and the reactant is stirred at ambient temperature for 3 hours. The mixture is then cooled to 0 ° C via an ice bath, and a 0.5 N HCl aqueous solution (10 mL) is added. The mixture is stirred at 0 ° C for 15 minutes, then stirred at ambient temperature for 3 hours (monitored by LC / MS). The solvent is reduced to approximately 0.5 mL on a rotary evaporator, and the crude mixture is purified by reversed-phase HPLC (5-85% acetonitrile in DI water, 0.1% TFA modifier, 25 minutes gradient). Pure fractions are combined and lyophilized to obtain the product as a white hygroscopic solid. Yield 57%, 125 mg. LC / MS [M+H]+=586.2

[0747] Step d.

[0748]

[0749] The intermediate from the previous step of this example (125 mg, 0.21 mmol) was stirred in a 1 / 2 mixture of methanol / DI water containing lithium hydroxide (30 mg, 1.3 mmol) at ambient temperature for 4 hours. The mixture was acidified with glacial acetic acid (1 mL), concentrated on a rotary evaporator and purified by reverse phase HPLC (5% to 80% acetonitrile in DI water, 0.1% TTFA modifier, 25 minute gradient). Pure fractions were combined and lyophilized to give the carboxylic acid as a hygroscopic white solid. Yield 84%, 106 mg. Found by LC / MS: [M+H] + = 572.2.

[0750] Step e.

[0751]

[0752] EDC (50 mg, 0.26 mmol) was added to a mixture of the carboxylic acid intermediate (106 mg, 0.19 mmol), propargyl-peg4 amine (60 mg, 0.26 mmol) and triethylamine (44 mg, 0.43 mmol) in DMF (2 mL) from the previous step. The reactant was stirred at ambient temperature for 4 hours and applied directly to reverse phase HPLC (5% to 85% acetonitrile in DI water, 0.1% TTFA modifier, 25 minute gradient). Pure fractions were combined and lyophilized to give the carboxylic acid as a hygroscopic white solid. Yield 54%, 93 mg. Measured ion by LC / MS: [M+H] + = 786.2.

[0753] Step f.

[0754]

[0755] The product (250mg, 0.31mmol) and intermediate A (161mg, 0.38mmol) from the previous step are dissolved in DMF (1mL) and cooled to 0°C via an ice-water bath. Copper sulfate (8mg, 0.048mmol) is added to a mixture of BTTA (34mg, 0.08mmol) and sodium ascorbate (189mg, 0.96mmol) in DI water (2mL), and the mixture is gently shaken for 15-20 seconds until the solution is clear. The copper mixture is added to the alkyne / azide mixture, and the reactant is stirred at 0°C for 10 minutes, then stirred at ambient temperature for 20 minutes. The crude reaction mixture is directly applied to reversed-phase HPLC (5%-85% acetonitrile in DI water, 0.1% TFA modifier, 25 minute gradient). Pure fractions are combined and lyophilized to obtain the product as a white hygroscopic solid. Yield 215mg, 56%. LC / MS found ion [(M / 2)+H]+=603.8.

[0756] Synthesis of conjugate 11b

[0757] The title compound was prepared analogously to conjugate 70, wherein the starting material used in Int-110 was replaced by Int- 16. Maldi TOF analysis of the purified final product gave an average mass of 62,471 Da (DAR = 4.0).

[0758] Synthesis of conjugate 11a

[0759] The title compound was prepared analogously to conjugate 4a, wherein the starting material used in Int-110 was replaced by Int- 16. Maldi TOF analysis of the purified final product gave an average mass of 62,404 Da (DAR = 4.2).

[0760] Synthesis of Int-27

[0761]

[0762] The title compound was prepared as described for Int-15. Found by LC / MS: [M+H]+ = 603.8.

[0763] Synthesis of conjugate 16

[0764] The title compound was prepared analogously to conjugate 70, wherein the starting material used in Int-110 was replaced by Int-27. Maldi TOF analysis of the purified final product gave an average mass of 60,745 (DAR = 2.4).

[0765] Synthesis of Int-106

[0766]

[0767] Step a.

[0768]

[0769] HATU (411 mg, 1.08 mmol) is added to a mixture of (racemic)-cis 1-tert-butyloxycarbonyl aminoindan-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 reactant is stirred at ambient temperature for 4 hours and directly applied to reverse phase HPLC (5% to 85% acetonitrile in DI water, 0.1% TTFA modifier, 25 minute gradient). Pure fractions are merged and lyophilized. The boc-protected amine is stirred at ambient temperature for 30 minutes in a 1 / 1 mixture of TFA / DCM (10 mL). The solvent is removed on a rotary evaporator and dried under high vacuum to obtain the intermediate TFA salt (racemic) of a clear viscous oil. Yield 71%, 2 steps, 325 mg. Found by LC / MS: [M+H] + = 391.2.

[0770] Step b.

[0771]

[0772] Intermediate B (200 mg, 44 mmol), the intermediate described in the previous step of this embodiment (175 mg, 0.45 mmol) and triethylamine (90 mg, 0.89 mmol) were stirred at 50 ° C in ethanol (25 mL) 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) to obtain a pair of diastereomers. Yield 70%, 250 mg. Measured ion by LC / MS: [M+H] + = 801.2.

[0773] Step c.

[0774]

[0775] The triacetate product (250 mg, 0.31 mmol) and potassium carbonate (30 mg) from the previous step were stirred in methanol (30 mL) at ambient temperature for 2 hours. The mixture was filtered and neutralized with glacial acetic acid (0.5 mL) and concentrated. The crude residue was purified by silica gel chromatography (0-10% methanol in DCM, 25 minutes). The triol was taken in DMF (2 mL) and 2,2-dimethoxypropane (5 mL), p-toluenesulfonic acid hydrate (15 mg) was added to the reaction and the mixture was stirred at 70 ° C for 1 hour. Triethylamine (0.5 mL) was added to the reactant, and the mixture was concentrated on a rotary evaporator. The crude residue was purified by silica gel chromatography (0-10% methanol in DCM, 25 minutes) to obtain a pair of diastereomers. Yield 60%, 135 mg for 2 steps. Measured ion by LC / MS: [M+H] + = 715.2.

[0776] Step d.

[0777]

[0778] The acetonide (200mg, 0.27mmol) from the previous step in THF (5mL) is added dropwise to a stirred mixture of methylene (phosphorus dichloride) (197mg, 1.12mmol) and DIEA (36mg, 0.28mmol) in THF (5mL) cooled to 0°C via an ice bath. When the addition is complete, the ice bath is removed and the reactant is stirred at ambient temperature for 3 hours. The mixture is then cooled to 0°C via an ice bath, and a 0.5N HCl aqueous solution (10mL) is added. The mixture is stirred at 0°C for 15 minutes, then stirred at ambient temperature for 3 hours (monitored by LC / MS). The solvent is reduced to approximately 5mL on a rotary evaporator, and the crude mixture is purified by reversed-phase HPLC (5-85% acetonitrile in DI water, 0.1% TFA modifier, 25 minutes gradient). Pure fractions are combined and lyophilized to obtain the product (diastereoisomer pair) as a white hygroscopic solid. Yield 45%, 107mg. Found ion by LC / MS: [M+H]+=833.2.

[0779] Step e.

[0780]

[0781] The product (90mg, 0.11mmol) and intermediate A (46mg, 0.11mmol) from the previous step are dissolved in DMF (1mL) and cooled to 0 DEG C via an ice-water bath. Copper sulfate (3mg, 0.016mmol) is added to a mixture of BTTA (12mg, 0.027mmol) and sodium ascorbate (64mg, 0.32mmol) in DI water (2mL), and the mixture is gently shaken for 15-20 seconds until the solution is clear. The copper mixture is added to the alkyne / azide mixture, and the reactant is stirred at 0 DEG C for 10 minutes, then stirred at ambient temperature for 20 minutes. The crude reaction mixture is directly applied to reverse phase HPLC (5%-85% acetonitrile in DI water, 0.1% TFA modifier, 25 minutes gradient). Pure fractions are combined and lyophilized to obtain a diastereoisomer mixture as a white hygroscopic solid. Yield 104mg, 76%. The ion [(M / 2)+H]+ was found to be 627.8 by LC / MS.

[0782] Synthesis of conjugate 56

[0783] The title compound was prepared analogously to conjugate 70, wherein the starting material used in Int-110 was replaced by Int- 106. Maldi TOF analysis of the purified final product gave an average mass of 63,241 Da (DAR = 4.5).

[0784] Synthesis of Int-61

[0785]

[0786] The title compound was prepared as described for Intermediate 106, where (rac)-cis 1-tert-butoxycarbonylamino-indan-2-carboxylic acid was replaced with (rac) trans-boc-amino-cyclopentanecarboxylic acid. Found by LC / MS: [(M / 2)+H]+ = 603.8.

[0787] Synthesis of conjugate 38

[0788] The title compound was prepared analogously to conjugate 70, wherein the starting material from Int-110 was replaced by Int-61. Maldi TOF analysis of the purified final product gave an average mass of 63,348 Da (DAR = 4.8).

[0789] Synthesis of Int-36

[0790]

[0791] The title compound was prepared as described for Intermediate 106, where (rac)-cis 1-tert-butoxycarbonylamino-indan-2-carboxylic acid was replaced with (rac) trans-Boc-amino-cyclopentanecarboxylic acid. Found by LC / MS: [(M / 2)+H]+ = 603.8.

[0792] Synthesis of conjugate 19

[0793] The title compound was prepared analogously to conjugate 70, wherein the starting material used in Int-110 was replaced by Int-36. Maldi TOF analysis of the purified final product gave an average mass of 63,363 Da (DAR = 4.8).

[0794] Synthesis of Int-10

[0795]

[0796] Step a.

[0797]

[0798] A solution of 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) in DMF (6 mL) (0.88 mL, 2.48 mmol) was stirred together at ambient temperature for 10 minutes. Propargyl-peg4-amine (862 mg, 3.72 mmol) in DMF (1 mL) was added thereto, and the resulting mixture was stirred for 1 hour. The reactant was concentrated and purified by reverse phase HPLC (5% to 100% ACN / water) to obtain a boc-protected intermediate in a yellow viscous liquid. The boc-protected intermediate was placed in DCM (10 mL) and treated with a 4M HCl aqueous solution (8 mL) in dioxane for 3 hours. The solvent was removed under reduced pressure and then dried under high vacuum to obtain an amine-HCl salt in a light yellow viscous oil. Yield 627 mg, 99%. Detected ions by LCMS: [M+H] + =315.9.

[0799] Step b.

[0800]

[0801] 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 the starting material was completely consumed, the reaction mixture was cooled to room temperature and 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 layer was dried over sodium sulfate, filtered and the solvent was removed under reduced pressure to obtain a compound, which was used in the next step without further purification. Measured ion by LCMS: [M+H] + =724.6.

[0802] Step c.

[0803]

[0804] The triacetate (800mg, 1.118mmol) from the previous step is dissolved in methanol (6mL) again and potassium carbonate (540mg, 3.91mmol) is added, and the reactant is stirred 2 hours at ambient temperature. The reaction mixture is filtered through diatomite, and the filter cake is washed with methanol (2x 20mL). The solution is concentrated in a vacuum to remove volatiles, and the thick material thus obtained is purified to obtain compound by reversed-phase HPLC (5% to 100% ACN / water, 0.1% TFA modifier). White foam. Yield 300mg, 45%. By LCMS measured ion: [M+H] + =598.8.

[0805] Step d.

[0806]

[0807] To a solution of triol (300 mg, 0.5 mmol) and 2,2-dimethoxypropane (0.31 mL, 2.5 mmol) from the previous step in acetone (20 mL) was added p-TsOH (8 mg, 0.05 mmol) at room temperature. The reactant was stirred for two hours and then concentrated under reduced pressure. The crude oil was redissolved in ethyl acetate (10 mL) and washed with saturated sodium bicarbonate. The organic layer was separated and dried over sodium sulfate, filtered and concentrated in vacuo to provide an acetonide derivative as a white solid, which was used in the next step without further purification. Measured ions by LCMS:

[0808] [M+H] +=639.8. DIEA (0.09 mL, 0.55 mmol) was added to a suspension of methylenebis(phosphorus chloride) (375 mg, 1.5 mmol) in THF (5 mL) at 0 ° C. A solution of acetonide in THF (2 mL) was added dropwise to the resulting mixture over a 10-minute period. After addition, the resulting mixture was stirred at ambient temperature for another hour, and the solution was then transferred to a pre-cooled (0 ° C.) flask containing a 0.2 M HCl aqueous solution (8 mL). The reaction mixture was warmed to ambient temperature and stirred for 2 hours. After completion, the reaction mixture was concentrated under reduced pressure and the crude material was purified by reverse phase HPLC using ACN: water (0.1% TFA modifier). The product was a white solid. Yield 243 mg, 81%. Measured ion by LCMS: [M+H] + =757.8.

[0809] Step e.

[0810]

[0811] The product (50mg, 0.066mmol) and intermediate A (27mg, 0.066mmol) from the previous step are dissolved in DMF:H The solution in O (1:3, 1.5mL) is cooled to 0 DEG C. The premixed solution of the solution in which copper sulfate (II) (1mg g, 0.007mmol), sodium ascorbate (57mg, 0.198mmol) and BTTA (6mg, 0.013mmol) are added into water (0.5mL) is stirred at the same temperature for 5 minutes, and is gradually warmed to room temperature, and is stirred at room temperature for 15 minutes. When the reaction is complete (monitored by HPLC), the mixture is quenched by adding a few drops of glacial acetic acid and 125mM EDTA (pH 6), and the product is purified by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA).White solid.Yield 142mg, 93.8%.By LCMS measured ion [M+H] + =1179.4.

[0812] Synthesis of conjugate 8a

[0813] Trifluorophenol ester (13 mg, 0.011 mmol, described in the synthesis of Int-10) in DMF (0.5 mL) was added to the Fc carrier SEQ ID NO: 17 (80 mg, 4 mL, in PBS at pH 7.4), and then adjusted to pH ~ 8 with borate buffer (0.200 mL, pH 8.5, 1.0 M). The mixture was stirred at ambient temperature for 2 hours and then purified according to the general procedure. Maldi TOF analysis of the purified final product gave an average mass of 64,894 (DAR of 6.7). The conjugate was purified by buffer dialysis (PBS pH 7.4) and SEC chromatography. Yield 54 mg, 77.1%.

[0814] Synthesis of conjugate 8b

[0815] As described in the synthesis of conjugate 8a, the trifluorophenol ester described in Int-10 (17 mg, 0.014481 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (100 mg, 0.00181 mmol, 5.13 mL in PBS at pH 7.4). Maldi TOF analysis of the purified final product gave an average mass of 65,370 Da (DAR = 6.9). Yield 80 mg, 80.6%.

[0816] Synthesis of Int-9

[0817]

[0818] Step a.

[0819]

[0820] A mixture of intermediate B (447 mg, 1.112 mmol), 2-[benzylamino] methyl acetate (240 mg, 1.34 mmol), triethylamine (0.23 mL) and ethanol (6 mL) was heated at 50 ° C for 1 hour. The mixture was cooled to ambient temperature and concentrated. The crude residue was redissolved in ethyl acetate (50 mL) and washed with water and brine. The organic layer was dried over sodium sulfate and the solvent was removed under reduced pressure to give a compound (646 mg, 98%) as a white solid, which was used in the next step without further purification. Ions were measured by LCMS: [M+H] +=589.8. Crude ester (646 mg, 0.98 mmol) is dissolved in THF (5 mL), and 1M LiOH aqueous solution (11 mL) is added thereto, and stirred at room temperature until TLC shows that the reaction is complete. The reaction mixture is concentrated and diluted with water, and acidified with 1M HCl, and extracted with ethyl acetate (3x20 mL). The combined organic extracts are washed with water, brine and dried over sodium sulfate. Remove the solvent to obtain a crude product, which is purified by reverse phase HPLC (5% to 100% ACN / water) to obtain the desired compound as white foam. Yield 392 mg, 88.8%. Measured ion by LCMS: [M+H] + =449.8.

[0821] Step b.

[0822]

[0823] The carboxylic acid (392 mg, 0.87 mmol), HATU (497 mg, 1.31 mmol) and DIEA (0.23 mL, 1.31 mmol) from the previous step in DMF (5 mL) were stirred together at ambient temperature for 10 minutes. Propargyl-PEG4-amine (241 mg, 1.04 mmol) added in DMF (1 mL) was stirred at room temperature for 1 hour, then concentrated under reduced pressure. The crude material was purified by reversed-phase HPLC (5% ACN: water, 100% ACN) to obtain the desired compound. The yield of white solid was 466 mg, 81%. Measured ion by LCMS: [M+H] + =662.8.

[0824] Step c.

[0825]

[0826] p-TsOH (13 mg, 0.075 mmol) was added to the tri-hydroxy product (497 mg, 0.75 mmol) and 2,2-dimethoxypropane (0.46 mL, 3.75 mmol) from the previous step in acetone (20 mL) at room temperature. The reactant was stirred at ambient temperature for two hours 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 and dried over sodium sulfate, filtered and concentrated in vacuo to provide an off-white solid, which was used in the next step without further purification. Yield 497 mg, 100%. Measured ion by LCMS: [M+H] + =702.8.

[0827] Step d.

[0828]

[0829] At 0 ℃, DIPEA (0.14mL, 0.83mmol) is added to a suspension of methylenebis(phosphorus chloride) (562mg, 2.25mmol) in THF (5mL). In the process of 1 hour, a solution of acetonide (497mg, 0.75mmol) in THF (2mL) from the previous step is added dropwise to the obtained mixture. After addition, the obtained mixture is stirred for another 15 minutes at 0 ℃, and the solution is then transferred to a pre-cooled (0 ℃) flask containing a 0.2M HCl aqueous solution (2.25mL). The reaction mixture is warmed to ambient temperature and stirred for 4 hours. After completion (monitored by LCMS), the reaction mixture is concentrated and purified by reversed-phase HPLC using 5% ACN / water to 100% ACN / water and 0.1% TFA modifier. White solid yield 454mg, 74%. Measured ion by LCMS: [M+H] + =820.6.

[0830] Step e.

[0831]

[0832] The product (0.1g, 0.122mmol) and intermediate A (0.037g, 0.088mmol) from step c are dissolved in DMF:H The solution in O (1:3, 1.5mL) is cooled to 0 DEG C. To which copper sulfate (II) (0.0020g, 0.0121mmol), sodium ascorbate (0.072g, 0.365mmol) and BTTA (0.010g, 0.0244mmol) are added are dissolved in water (0.5mL) in a premixed solution and stirred at the same temperature for 5 minutes, gradually warmed to room temperature and stirred at room temperature for 15 minutes. After the reaction is complete, it is quenched by adding a few drops of AcOH and EDTA to pH 6 by the reaction mixture, and by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA) purified product. White solid (0.142g, 93.84%). LCMS[(M+2H) / 2] + =621.2.

[0833] Synthesis of conjugate 7a

[0834] Trifluorophenol ester (described in the synthesis of Int-9) was conjugated to 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%.

[0835] Synthesis of conjugate 7b

[0836] As described in the synthesis of conjugate 8a, trifluorophenol ester (9 mg, 0.0072 mmol) (described in the synthesis of Int-9) was conjugated to the Fc carrier SEQ ID NO: 13 (50 mg, 2.56 mL, in PBS at pH 7.4, 0.0009 1 mmol). 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%.

[0837] Synthesis of Int-21

[0838]

[0839] Step a.

[0840]

[0841] Propargyl-PEG-4 methanesulfonate (931 mg, 3 mmol), N-Boc piperazine (558 mg, 3 mmol) and potassium carbonate (828 mg, 6 mmol) were refluxed in acetonitrile (50 mL) for 15 hours. The reaction mixture was cooled to ambient temperature and the solvent was removed under reduced pressure. The crude material was distributed between water and ethyl acetate, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2 × 10 mL). The combined organic extracts were washed with brine, water, and dried over sodium sulfate. The solvent was concentrated to obtain the product of the crude N-Boc protection as a yellow viscous liquid. Measured ion 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 in dioxane (4 mL, 10 equivalents) was added thereto, and the reaction mixture was gradually warmed to ambient temperature and stirred until LCMS analysis indicated complete conversion of the starting material to the product. The solvent was removed under reduced pressure and the crude material was dried under high vacuum to give the product (HCl salt) as a white solid. Yield: 672 mg, 85% in 2 steps. Measured ion by LCMS: [M+H] + =301.2.

[0842] Step b.

[0843]

[0844] By intermediate B (250mg, 0.56mmol), the amine (201mg, 0.67mmol) from the previous step and a mixture of triethylamine (0.12mL) in EtOH (6mL) was heated at 50°C for 2 hours. After the complete consumption of starting material (monitored by LCMS), the reaction mixture was cooled to ambient temperature and volatiles were removed on a rotary evaporator to obtain a crude material, which was purified by normal phase column chromatography using hexane: ethyl acetate. The product was a white solid. Yield 200mg, 50%. Measured ion by LCMS: [M+H] + =710.8.

[0845] Step c.

[0846]

[0847] The triacetate product (220mg, 0.31mmol) from the previous step is dissolved in methanol (10mL) and processed with potassium carbonate (149mg, 1.08mmol), then stirred at ambient temperature for 1.5 hours. The reaction mixture is filtered through diatomite, and the filter cake is washed with methanol (3x20mL). The solution is concentrated in vacuo to remove volatiles, and crude product is obtained. The crude product is purified by reversed-phase HPLC to obtain product. The product is a white solid. Yield 139mg, 77%. By LCMS measured ion: [M+H] + =585.2.

[0848] Step d.

[0849]

[0850] To a solution of the product (139 mg, 0.24 mmol) and 2,2-dimethoxypropane (0.145 mL, 1.188 mmol) from the previous step in acetone (10 mL) was added p-TsOH (23 mg, 0.005 mmol) at ambient temperature. The reactant was stirred for two hours and then concentrated under reduced pressure to give an acetonide intermediate. The crude material was dissolved in ethyl acetate (20 mL) and washed with saturated sodium bicarbonate. The organic layer was separated and dried over sodium sulfate, filtered and concentrated in vacuo to provide an acetonide derivative as a white solid, which was used in the next step without further purification. Measured ions by LCMS: [M+H] +=624.8. To a suspension of methylenebis(phosphorus chloride) (178 mg, 0.71 mmol) in THF (5 mL) was added DIEA (0.046 mL, 0.26135 mmol) at 0 ° C. A solution of acetonide (148 mg, 0.24 mmol) in THF (2 mL) was added dropwise over a period of 10 minutes. After addition, the mixture was stirred at 0 ° C for another 15 minutes, and the solution was then transferred to a pre-cooled (0 ° C) flask containing a 0.2M HCl aqueous solution (1.5 mL). The reaction mixture was warmed to ambient temperature and stirred for 4 hours. After completion (monitored by LCMS), the reaction mixture was concentrated and purified by reverse phase HPLC. The product was a white solid. Yield 100 mg, 57%. Measured ion by LCMS: [M+H] + =743.6.

[0851] Step e.

[0852]

[0853] The product (0.07g, 0.0942mmol) and intermediate A (0.04g, 0.0942mmol) from step d are dissolved in DMF:H The solution in O (1:3, 1.5mL) is cooled to 0 ° C. A premixed solution of a solution in which copper sulfate (II) (0.0015g, 0.00942mmol), sodium ascorbate (0.056g, 0.2826mmol) and BTTA (0.019g, 0.0188mmol) are added and stirred at the same temperature for 5 minutes, gradually warmed to room temperature and stirred at room temperature for 15 minutes. After the reaction is complete, the reaction mixture is quenched by adding a few drops of AcOH and EDTA to pH 6, and the product is purified by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA). White solid (0.07g, 63.81%). LCMS[M+H] + =1165.4.

[0854] Synthesis of conjugate 13

[0855] As described in the synthesis of conjugate 8a, trifluorophenol ester (described in the synthesis of Int-21) (0.017 g, 0.01448 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (0.1 g, 0.0018 mmol). MaldiTOF analysis of the purified final product gave an average mass of 62149 Da (DAR = 3.9). Yield 42.3 mg, 42.3%.

[0856] Synthesis of Int-55

[0857]

[0858] Step a.

[0859]

[0860] 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 provide the product. The product was a white solid. Yield 1.1 g, 98%. Measured by LCMS ion: [M+H] + =495.8.

[0861] Step b.

[0862]

[0863] The triacetate product (1.1 g, 2.21 mmol) from the previous step was dissolved in THF (30 mL) and treated with a 2M LiOH aqueous solution (9 mL) at ambient temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and then acidified with a 1N HCl aqueous solution. The crude material was purified by reverse phase HPLC (10% to 100% ACN / water, 0.1% TFA). The product was a white solid. Yield 735 mg, 89%. Measured ion by LCMS: [M+H] + =369.8.

[0864] Step c.

[0865]

[0866] To a solution of the product (735 mg, 1.99 mmol) and 2,2-dimethoxypropane (1.22 mL, 9.94 mmol) from the previous step in acetone (25 mL) was added p-TsOH (34 mg, 0.198 mmol) at room temperature. The reactant was stirred for two hours and then concentrated under reduced pressure. The crude product was dissolved in ethyl acetate (30 mL) and washed with saturated sodium bicarbonate. The organic layer was separated and dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain a pale solid, which was purified by silica gel chromatography using hexane / ethyl acetate. The product was a white solid. Yield 690 mg, 85%. Measured ion by LCMS: [M+H] + =410.9.

[0867] Step d.

[0868]

[0869] The acetonide from the previous step (300 mg, 0.731 mmol), propargyl-peg 4- A mixture of amine (203 mg, 0.88 mmol) and triethylamine (0.15 mL, 1.09 mmol) in ethanol (5 mL) was heated at 80 ° C for 24 hours. The mixture was cooled to ambient temperature and concentrated. The residue was purified by reverse phase HPLC (10% to 100% ACN / water, 0.1% TFA) to obtain the title compound as a light brown viscous liquid. Yield 100 mg, 23%. Measured ion by LCMS: [M+H] + =604.9.

[0870] Step e.

[0871]

[0872] At 0 ℃, DIEA (0.032mL, 0.18mmol) was added to a suspension of methylenebis(phosphorus chloride) (124mg, 0.496mmol) in THF (5mL). A solution of acetonide (100mg, 0.17mmol) in THF (1mL) from the previous step was added dropwise to the obtained mixture over a period of 10 minutes. After addition, the obtained mixture was stirred at 0 ℃ for another 15 minutes, and the solution was then added dropwise to a pre-cooled (0 ℃) flask containing a 0.2M HCl aqueous solution (4.5mL). The reaction mixture was warmed to ambient temperature and stirred for 2 hours. After completion (monitored by LCMS), the reaction mixture was concentrated under reduced pressure and the crude material was purified by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA). The product was a white solid. Yield 72mg, 60%. Measured ion by LCMS: [M+H] + =722.6.

[0873] Step f.

[0874]

[0875] The product (0.06g, 0.03mmol) and intermediate A (0.035g, 0.083mmol) from the previous step are dissolved in DMF:H The solution in O (1:3, 1.5mL) is cooled to 0 ° C. A premixed solution of a solution of copper sulfate (II) (0.0013g, 0.0083mmol), sodium ascorbate (0.049g, 0.249mmol) and BTTA (0.007g, 0.0167mmol) dissolved in water (0.5mL) is added thereto and stirred at the same temperature for 5 minutes, gradually warmed to room temperature and stirred at room temperature for 15 minutes. After the reaction is complete, the reaction mixture is quenched by adding a few drops of AcOH and EDTA to pH 6, and the product is purified by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA). White solid (0.06g, 63.17%). LCMS[(M+2H) / 2] + =572.2.

[0876] Synthesis of conjugate 31

[0877] As described in the synthesis of conjugate 8a, trifluorophenol ester (described in the synthesis of int-55) was conjugated to the Fc carrier SEQ ID NO: 13. 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%.

[0878] Synthesis of Int-28

[0879]

[0880] Step a.

[0881]

[0882] A mixture of the pyrazolo-pyrimidine derivative (880 mg, 1.76 mmol) described in step a of the synthesis of Int-55 and the azetidine intermediate (668 mg, 2.12 mmol) and triethylamine (0.17 mL) in ethanol (25 mL) was heated at 80 ° C for 12 hours. The mixture was cooled to ambient temperature and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate: 5: 1) to provide the title compound. The product was a white solid. Yield 508 mg, 37%. Measured ion by LCMS: [M+H] + =774.8.

[0883] Step b.

[0884]

[0885] The triacetate (412mg, 0.53mmol) from the previous step was dissolved in methanol (10mL). Potassium carbonate (257mg, 1.86mmol) was added and the reaction mixture was stirred at ambient temperature for 1.5 hours. The reaction mixture was filtered through diatomaceous earth and the filter cake was washed with methanol (3x20mL). The solution was concentrated under reduced pressure to obtain a crude triol, which was purified by reverse phase HPLC to obtain the desired compound. The product was a white solid. Yield: 310mg, 89%. Measured ion by LCMS: [M+H] + =648.8.

[0886] Step c.

[0887]

[0888] To a solution of the triol (310 mg, 0.48 mmol) and 2,2-dimethoxypropane (0.29 mL, 2.39 mmol) from the previous step in acetone (25 mL) was added p-TsOH (8 mg, 0.048 mmol) at ambient temperature. The reactant was stirred for two hours and then concentrated under reduced pressure. The crude product was dissolved in ethyl acetate (30 mL) and washed with saturated sodium bicarbonate. The organic layer was separated and dried over sodium sulfate, filtered and concentrated in vacuo to provide a crude material. The product was a white solid. Yield 329 mg, 99%. Measured ion by LCMS: [M+H] + =688.6.

[0889] Step d.

[0890]

[0891] At 0 ℃, DIEA (0.092mL, 0.182mmol) is added to a suspension of methylenebis(phosphorus chloride) (359mg, 1.44mmol) in THF (5mL). Over a 10-minute period, the acetonide intermediate (328mg, 0.48mmol) from the previous step in THF (2mL) was added dropwise to the mixture. After addition, the resulting mixture was stirred for another 15 minutes at 0 ℃, and the solution was then added dropwise to a pre-cooled (0 ℃) flask containing a 0.2M HCl aqueous solution. The reaction mixture was warmed to ambient temperature and stirred for 4 hours. After completion (monitored by LCMS), the reaction mixture was concentrated under reduced pressure and the crude material was purified by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA). The product was a white solid. Yield 246mg, 64%. Measured ion by LCMS: [M+H] + =806.6.

[0892] Step e.

[0893]

[0894] The product (0.06g, 0.0745mmol) and intermediate A (0.031g, 0.0745mmol) from step d are dissolved in DMF:H the solution in O (1:3, 1.5mL) is cooled to 0 DEG C.Add copper sulfate (II) (0.0012g, 0.007446mmol), sodium ascorbate (0.044g, 0.2234mmol) and BTTA (0.006g, 0.0148mmol) to the premixed solution of the solution soluble in water (0.5mL) and stirred 5 minutes at the same temperature, gradually warmed to room temperature and stirred at room temperature 15 minutes.After the reaction is completed, by adding a few drops of AcOH and EDTA to pH be 6 by reaction mixture cancellation, and by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA) purified product.Product is white solid.Yield 70mg, 63.8%. Found by LCMS: [(M+2H) / 2] + =613.6.

[0895] Synthesis of conjugate 17

[0896] As described in the synthesis of conjugate 8a, the trifluorophenol ester described in the synthesis of Int-28 (9 mg, 0.00724 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (50 mg, 2.58 mL, in PBS at pH 7.4, 0.0009 mmol). 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%.

[0897] Synthesis of Int-49

[0898]

[0899] Step a.

[0900]

[0901] A mixture of 1-indanone (132 mg, 1 mmol), propargyl PEG4-amine (277 mg, 1.2 mmol) and acetic acid (0.003 mL, 0.06 mol) in benzene (6.0 mL) was stirred under reflux for 4 hours. The reaction mixture was cooled to 20 ° C under a nitrogen atmosphere and concentrated under reduced pressure to half the volume. At ambient temperature, the residue was added to a stirred solution of sodium borohydride (19 mg, 0.5 mmol) in ethanol (5 mL). The mixture was stirred for 24 hours and then quenched by adding water (about 3 mL). The solvent was evaporated under reduced pressure. The residue was extracted with ethyl acetate (3 × 10 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain an amine intermediate as a dark brown viscous liquid. Yield 111 mg, 32%. Measured ion by LCMS: [M+H] + =348.0.

[0902] Step b.

[0903]

[0904] A mixture of intermediate B (100 mg, 0.222 mmol), the crude amine from the previous step (93 mg, 0.268 mmol) and triethylamine (0.046 mL, 0.3354) in ethanol (5 mL) was heated at 50 ° C for 1 hour. The mixture was cooled to ambient temperature and concentrated to give the crude product as a light yellow viscous oil. The crude material was used in the next step without any purification. LCMS [M+H] + =758.6. The crude product was dissolved in THF (5 mL) and 1M LiOH aqueous solution (1.34 mL, 1.34 mmol) was added, and the resulting mixture was stirred at ambient temperature for 30 minutes. The mixture was acidified with 1N HCl aqueous solution (to pH about 4) and the solvent was removed under reduced pressure. The crude material was purified by reverse phase HPLC to obtain the product as a light yellow solid. Yield 141 mg, 84%. Measured by LCMS ion: 631.8 [M+H] + .

[0905] Step c.

[0906]

[0907] To a solution of the product (119 mg, 0.188 mmol) and 2,2-dimethoxypropane (0.11 mL, 0.94 mmol) from the previous step in acetone (10 mL) was added p-TsOH (3 mg) at room temperature. The reactant was stirred for two hours and then concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (30 mL) and washed with saturated sodium bicarbonate. The organic layer was separated and dried over sodium sulfate, filtered and concentrated in vacuo to provide an off-white solid, which was used in the next step without further purification. Yield 127 mg, 99%. Measured ion by LCMS: [M+H] + =674.6.

[0908] Step d.

[0909]

[0910] At 0 ℃, to the suspension of methylenebis(phosphorus chloride) (141mg, 0.56mmol) in THF (5mL), DIEA (0.036mL, 0.21mmol) was added. In the process of 10 minutes, a solution of the product (128mg, 0.19mmol) from the previous step in THF (2mL) was added dropwise to the obtained mixture. After addition, the obtained mixture was stirred for another 15 minutes at 0 ℃, and the solution was then added dropwise to a pre-cooling (0 ℃) flask containing the 0.2M HCl aqueous solution. The reaction mixture was warmed to ambient temperature and stirred for 4 hours. After completion (monitored by LCMS), the reaction mixture was concentrated under reduced pressure and the crude material was purified by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA). The product was a yellow solid 34mg, 23%. Measured ion by LCMS: [M+H] + =790.2.

[0911] Step e.

[0912]

[0913] The product (0.03g, 0.03797mmol) and intermediate A (0.016g, 0.03797mmol) from previous step d are dissolved in DMF:H the solution in O (1:3, 1.5mL) is cooled to 0 DEG C.Add copper sulfate (II) (0.0006g, 0.003797mmol), sodium ascorbate (0.032g, 0.1139mmol) and BTTA (0.003g, 0.007mmol) to the premixed solution of the solution dissolved in water (0.5mL) and stirred at the same temperature for 5 minutes, gradually warmed to room temperature and stirred at room temperature for 15 minutes.After the reaction is completed, it is 6 that the reaction mixture is quenched by adding a few drops of AcOH and EDTA to pH, and by reversed-phase HPLC (10% to 100% ACN / water, 0.1%TFA) purified product.White solid (0.016g, 34.78%). LCMS [(M+H) / 2] + =605.8.

[0914] Synthesis of conjugate 28

[0915] As described in the synthesis of conjugate 8a, the trifluorophenol ester described in the synthesis of Int-49 (7 mg, 0.0058 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (40 mg, 2 mL, in PBS at pH 7.4, 0.000724 mmol). 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%.

[0916] Synthesis of Int-81

[0917]

[0918] Step a.

[0919]

[0920] The propargyl-PEG4-acid (544mg, 2.09mmol), HATU (662mg, 1.74mmol) and DIEA (0.62mL, 3.48mmol) in DMF (6mL) are stirred together for 10 minutes at ambient temperature. 3-amino-azetidine carboxylic acid tert-butyl esters (300mg, 1.74mmol) added in DMF (1mL) are stirred for 1 hour at ambient temperature. The reaction mixture is concentrated under reduced pressure, and the crude residue is then purified by reversed-phase HPLC (5% to 100% ACN / water) to obtain the azetidine intermediate of the boc- protection as a yellow viscous oil. The intermediate (578mg, 1.39mmol) of boc- protection is dissolved in dioxanes (10mL), and is treated with the 4M HCl aqueous solution in dioxanes (7mL) for 3 hours at ambient temperature. The solvent is removed under reduced pressure and dried under high vacuum to obtain the azetidine as an HCl salt. The product is a yellow viscous oil. Yield 480 mg, 98%. LCMS ion: [M+H] + =315.0.

[0921] Step b.

[0922]

[0923] A mixture of intermediate B (250 mg, 0.56 mmol), azetidine (235 mg, 0.67 mmol) from the previous step and triethylamine (0.15 mL, 1.12 mmol) in ethanol (6 mL, 1.12 mmol) was heated at 50 ° C for 1 hour. The mixture was cooled to ambient temperature and concentrated on a rotary evaporator. The crude residue was dissolved in ethyl acetate (50 mL) and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give the triacetate intermediate as a colorless viscous oil. Measured ion by LCMS: [M+H] + =724.6. The crude triacetate (380 mg, 0.94 mmol) was dissolved in methanol (5 mL), potassium carbonate (253 mg, 1.83 mmol) was added, and the mixture was stirred at ambient temperature for 2 hours. The mixture was concentrated under reduced pressure and purified by silica gel chromatography using 0% to 10% MeOH in DCM. The product was a white solid. Yield 280 mg, 89%. Measured by LCMS ion: [M+H] + =598.8.

[0924] Step c.

[0925]

[0926] To the product (130 mg, 0.472 mmol) from the previous step and 2,2-dimethoxypropane (0.28 mL, 2.34 mmol) in acetone (20 mL) at room temperature was added p-TsOH (8 mg, 0.005 mmol). The reactant was stirred for two hours and then concentrated under reduced pressure. The crude material was dissolved in ethyl acetate (20 mL) and washed with saturated sodium bicarbonate. The organic layer was separated and dried over sodium sulfate, filtered and concentrated under reduced pressure to provide an acetonide derivative. The product was a white solid. Measured ions by LCMS: [M+H] + =639.8. DIEA (0.089 mL, 0.51 mmol) was added to a suspension of methylenebis(phosphorus chloride) (350 mg, 1.40 mmol) in THF (5 mL) at 0 ° C. A solution of an acetonide derivative (298 g, 0.47 mmol) in THF (2 mL) was added dropwise to the resulting mixture over a 10-minute period. After addition, the resulting mixture was stirred at 0 ° C. for another 15 minutes, and the solution was then transferred to a pre-cooled (0 ° C.) flask containing a 0.2 M HCl aqueous solution (2.5 mL). The reaction mixture was warmed to ambient temperature and stirred for 4 hours. After completion (monitored by LCMS), the mixture was concentrated under reduced pressure and purified by reverse phase HPLC. The product was a white solid. Yield 220 mg, 62%. Measured ion by LCMS: [M+H] + =757.8.

[0927] Step d.

[0928]

[0929] The product (0.05g, 0.066mmol) and intermediate A (0.027g, 0.066mmol) from the previous step are dissolved in DMF:H The solution in O (1:3, 1.5mL) is cooled to 0 DEG C. To which copper sulfate (II) (0.0001g, 0.006mmol), sodium ascorbate (0.039g, 0.198mmol) and BTTA (0.006g, 0.013mmol) are added are dissolved in water (0.5mL) premixed solution and stirred at the same temperature for 5 minutes, gradually warmed to room temperature and stirred at room temperature for 15 minutes. After the reaction is complete, it is quenched by adding a few drops of AcOH and EDTA to pH 6 by the reaction mixture, and by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA) purified product. The product is a white solid. Yield 48mg, 62%. By LCMS measured ion: [M+H] + =1179.4.

[0930] Synthesis of conjugate 44

[0931] As described in the synthesis of conjugate 8a, the trifluorophenol ester described in the synthesis of Int-81 (14 mg, 0.011584 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (80 mg, 4.1 mL, in PBS at pH 7.4, 0.00145 mmol) 5. Maldi TOF analysis of the purified final product gave an average mass of 63,709 Da (DAR = 5.3). Yield 63 mg, 78.9%.

[0932] Synthesis of Int-90

[0933]

[0934] The title compound was prepared similarly to Int-10, replacing N-Boc azetidine-3-carboxylic acid with 1-[(tert-butyl)oxycarbonyl]pyrrolidine-3-carboxylic acid. The product was a white solid. Yield 52 mg, 67%. LCMS measured ion: [(M+2H) / 2] + =596.8.

[0935] Synthesis of conjugate 48

[0936] As described in the synthesis of conjugate 8a, the trifluorophenol ester described in the synthesis of Int-90 (17 mg, 0.01448 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL, 0.0018 mmol) in PBS at pH 7.4. Maldi TOF analysis of the purified final product gave an average mass of 63,468 Da (DAR=5). Yield 53.2 mg, 53.2%.

[0937] Synthesis of Int-91

[0938]

[0939] The title compound was prepared similarly to Int-10, replacing N-Boc azetidine-3-carboxylic acid with 4-[(tert-butyl)oxycarbonyl]morpholine-3-carboxylic acid. The product was a white solid. Yield: 68 mg, 55%. LCMS measured ion: [(M+2H) / 2] + =605.2.

[0940] Synthesis of conjugate 49

[0941] As described in the synthesis of conjugate 8a, the trifluorophenol ester described in the synthesis of Int-91 (17.5 mg, 0.01448 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL, in PBS at pH 7.4, 0.0018 mmol). Maldi TOF analysis of the purified final product gave an average mass of 64,352 Da (DAR = 5.8). Yield 70 mg, 70%.

[0942] Synthesis of Int-97

[0943]

[0944] The title compound was prepared similarly to step e of the synthesis of Int-10, wherein N-Boc azetidine-2-carboxylic acid was substituted for N-Boc azetidine-3-carboxylic acid. The product was a white solid. Yield 56 mg, 72%. LCMS measured ion: [M+H] + =1179.4.

[0945] Synthesis of conjugate 52

[0946] As described in the synthesis of conjugate 8a, the trifluorophenol ester described in the synthesis of Int-97 (17 mg, 0.01448 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL, 0.0018 mmol) in PBS at pH 7.4. Maldi TOF analysis of the purified final product gave an average mass of 63,232 Da (DAR = 4.9). Yield 73 mg, 73%.

[0947] Synthesis of Int-99

[0948]

[0949] The title compound was prepared similarly to Int-10, where N-Boc-proline was substituted for N-Boc azetidine-3-carboxylic acid. The product was a white solid. Yield: 31 mg, 40.1%. LCMS analysis revealed: [(M+2H) / 2] + =596.8.

[0950] Synthesis of conjugate 53

[0951] As described in the synthesis of conjugate 8a, trifluorophenol ester (17 mg, 0.01448 mmol) described in the synthesis of Int-99 was conjugated to the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL, 0.0018 mmol) in PBS at pH 7.4. Maldi TOF analysis of the purified final product gave an average mass of 61,730 Da (DAR 3.4). Yield 70 mg, 70%.

[0952] Synthesis of Int-113

[0953]

[0954] Step a.

[0955]

[0956] A mixture of intermediate C (200 mg, 0.448 mmol) and the azetidine intermediate (188 mg, 0.54 mmol) described in step a of the synthesis of Int-10, triethylamine (0.13 mL) in ethanol (6 mL) was heated at 110 ° C for 12 hours. After the starting material was consumed (monitored by LCMS), the reaction mixture was cooled to ambient temperature and volatiles were removed under reduced pressure to obtain a crude material, which was used in the next step without further purification. Yellow viscous oil. Yield 324 mg, 100%. Measured ion by LCMS: [M+H] + =724.6.

[0957] Step b.

[0958] 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% to 100% ACN / water, 0.1% TFA). White foam. Yield 210 mg, 78%. Measured ion by LCMS: [M+H] + =598.2.

[0959] Step c.

[0960]

[0961] To a solution of the product from the previous step (190 mg, 0.32 mmol) and 2,2-dimethoxypropane (0.19 mL, 1.58 mmol) in acetone (5 mL) was added p-TsOH (6 mg, 0.032 mmol) at ambient temperature. The reactants were stirred for two hours and then concentrated under reduced pressure. The crude material was dissolved in ethyl acetate (20 mL) and washed with saturated sodium bicarbonate. The organic layer was separated and dried over sodium sulfate, filtered and concentrated under reduced pressure to provide an acetonide derivative. The product was a white solid. LCMS [M+H] + =638.2. DIEA (0.061 mL, 0.35 mmol) was added to a suspension of methylenebis(phosphorus chloride) (238 mg, 0.95 mmol) in THF (5 mL) at 0 ° C. A solution of an acetonide derivative in THF (2 mL) was added dropwise to the resulting mixture over a 10-minute period. After addition, the resulting mixture was stirred at 0 ° C. for 15 minutes, and the solution was then transferred to a pre-cooled (0 ° C.) flask containing a 0.2 M HCl aqueous solution (7 mL). The reaction mixture was warmed to ambient temperature and stirred for 4 hours. After completion (monitored by LCMS), the reaction mixture was concentrated and purified by reverse phase HPLC. The product was a white solid. Yield 130 mg, 54%. Measured ion by LCMS: [M+H] + =756.2.

[0962] Step d.

[0963]

[0964] The product (0.03g, 0.039mmol) and intermediate A (0.017g, 0.0.039mmol) from the previous step are dissolved in DMF:H the solution in O (1:3, 1.5mL) is cooled to 0 DEG C.Add copper sulfate (II) (0.00006g, 0.0.0004mmol), sodium ascorbate (0.024g, 0.118mmol) and BTTA (0.0064g, 0.008mmol) to the premixed solution of the solution dissolved in water (0.5mL) and stirred 5 minutes at the same temperature, gradually warmed to room temperature and stirred at room temperature 15 minutes.After the reaction is completed, it is 6 that the reaction mixture is quenched by adding a few drops of AcOH and EDTA to pH, and by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA) purified product.White solid (0.016g, 34.25%). LCMS [(M+2H)) / 2] + =589.6

[0965] Synthesis of conjugate 60

[0966] 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 adjusted to pH ~ 7.4 with sodium carbonate buffer (0.200 mL, pH 9.2 to 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) (approximately 0.5 mL of buffer mixture / 10 mg of protein) and 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 was 2.9). Yield 54.9 mg, 61%.

[0967] Synthesis of Int-115

[0968]

[0969] Step a.

[0970]

[0971] A mixture of intermediate D (250 mg, 0.56 mmol) and the azetidine intermediate (235 mg, 0.67 mmol) of the step a of the example synthesis of Int-10 and triethylamine (0.16 mL) in ethanol (6 mL) was heated at 50 ° C for 1 hour. When the reaction was complete (monitored by LCMS), the mixture was cooled to ambient temperature and the solvent was removed under reduced pressure to give a crude material, which was purified by silica gel column chromatography using hexane: ethyl acetate. The product was a white solid. Yield 336 mg, 83%. Measured ion by LCMS: [M+H] + =724.2.

[0972] Step b.

[0973]

[0974] The triacetate (336 mg, 0.47 mmol) from the previous step in methanol (5 mL) was treated with potassium carbonate (224 mg, 1.56 mmol) at room temperature for 2 hours, and the reaction mixture was concentrated in a vacuum to remove volatiles, and then dissolved in methanol: water (1: 1, 2 mL). The crude mixture was purified by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA). The product was a white foam. Yield 190 mg, 69%. Measured ion by LCMS: [M+H] + =598.2.

[0975] Step c.

[0976]

[0977] To a solution of the product (160 mg, 0.27 mmol) from the previous step and 2,2-dimethoxypropane (0.163 mL, 1.34 mmol) in acetone (5 mL) was added p-TsOH (5 mg, 0.027 mmol) at room temperature. The reactant was stirred for two hours and then concentrated under reduced pressure. The crude material was dissolved in ethyl acetate (20 mL) and washed with saturated sodium bicarbonate. The organic layer was separated and dried over sodium sulfate, filtered and concentrated under reduced pressure to provide an acetonide intermediate. The product was a white solid. Measured ions by LCMS: [M+H] + =638.2. DIEA (0.051 mL, 0.29 mmol) was added to a suspension of methylenebis(phosphorus chloride) (200 mg, 0.80 mmol) in THF (5 mL) at 0 ° C. A solution of an acetonide derivative in THF (2 mL) was added dropwise to the resulting mixture over a 10-minute period. After addition, the resulting mixture was stirred at 0 ° C. for another 15 minutes, and the solution was then transferred to a pre-cooled (0 ° C.) flask containing a 0.2 M aqueous HCl solution (6 mL). The reaction mixture was warmed to ambient temperature and stirred for 4 hours. After completion (monitored by LCMS), the reaction mixture was concentrated and purified by reverse phase HPLC. The product was a white solid. Yield 164 mg, 91%. Measured ion by LCMS: [M+H] + =756.2.

[0978] Step d.

[0979]

[0980] The product (0.06g, 0.079mmol) and intermediate A (0.033g, 0.079mmol) from the previous step are dissolved in DMF:H The solution in O (1:3, 1.5mL) is cooled to 0 DEG C. To which copper sulfate (II) (0.001g, 0.0079mmol), sodium ascorbate (0.047g, 0.238mmol) and BTTA (0.007g, 0.016mmol) are added are dissolved in water (0.5mL) premixed solution and stirred at the same temperature for 5 minutes, gradually warmed to room temperature and stirred at room temperature for 15 minutes. After the reaction is complete, it is quenched by adding a few drops of AcOH and EDTA to pH 6 by the reaction mixture, and by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA) purified product. The product is a white solid. Yield 50mg, 53.5%. By LCMS measured ion: [M+H] + =1177.2.

[0981] Synthesis of conjugate 61

[0982] Trifluorophenol ester (18 mg, 0.01448 mmol) (described in the synthesis of Int-115) 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), 205 and then adjusted to pH ~ 7.4 with sodium carbonate buffer (0.200 mL, pH 9.2 to 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) (approximately 0.5 mL of buffer mixture / 10 mg of protein) and 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 61,737 Da (DAR of 3.4). Yield 59.9 mg, 59.9%.

[0983] Synthesis of Int-117

[0984]

[0985] Step a.

[0986]

[0987] A solution of 1-[(tert-butyl)oxycarbonyl]azetidine-3-carboxylic acid (500 mg, 2.48 mmol), HATU (1.42 g, 3.73 mmol) and DIEA in DMF (0.19 mL, 2.48 mmol) was stirred together at ambient temperature for 10 minutes. 4-hydroxyaniline (406 mg, 3.72 mmol) in DMF (1 mL) was added to the mixture, and the resulting mixture was stirred at ambient temperature for 1 hour. The reactant was concentrated under reduced pressure and purified by reverse phase HPLC (5% to 100% ACN / water). The product was a white solid. Yield 540 mg, 74.3%. Measured ion by LCMS: [MC(CH3)3+H] + =237.2.

[0988] Step b.

[0989]

[0990] To a solution of the compound from the previous step (540 mg, 1.85 mmol) in acetonitrile (30 mL) was added potassium carbonate (510 mg, 3.69 mmol) and propargyl-PEG 4- Methanesulfonate (867 mg, 2.21 mmol). The resulting mixture was heated at reflux for 16 hours. The mixture was cooled and the excess solvent was removed under reduced pressure. Ethyl acetate (50 mL) was added to the crude material and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain the N-Boc protected derivative of the crude title compound, which was purified by reverse phase HPLC. Pale yellow viscous liquid. Yield 710 mg, 76%. Measured ion by LCMS: [M-Boc+H] + =407.2. To a solution of the N-Boc protected compound (710 mg, 1.40 mmol) in dioxane (10 mL) was added a 4 M HCl solution in dioxane (7 mL, 28 mmol). The resulting mixture was stirred at ambient temperature for 2 hours, concentrated and dried under high vacuum to give the amine intermediate as an HCl salt. Pale yellow solid. Yield 602 mg, 97%. Measured by LCMS ion: [M+H] + =407.2.

[0991] Step c.

[0992]

[0993] A mixture of intermediate B (250 mg, 0.56 mmol), the amine (296 mg, 0.67 mmol) from the previous step and triethylamine (0.16 mL, 1.12 mmol) in ethanol (6 mL, 1.12 mmol) was heated at 50 ° C for 1 hour. The mixture was cooled to ambient temperature and concentrated under reduced pressure. The crude residue was redissolved in ethyl acetate (50 mL) and washed with water and brine. The combined organic layer was dried over sodium sulfate, filtered and concentrated to give the product as a light yellow solid, which was used in the next step without further purification. Yield 437 mg, 96%. Measured ion by LCMS: [M+H] + =817.2.

[0994] Step d.

[0995]

[0996] The product from the previous step (0.437 g, 0.535 mmol) in methanol (5 mL) was treated with potassium carbonate (0.258 g, 1.87 mmol) at ambient temperature for 2 hours. The mixture was concentrated under reduced pressure, dissolved in a minimum of 1:1 MeOH:H2O, and then purified by reverse phase HPLC (10% to 100% ACN / water, 0.1% TFA). The product was a white solid. Yield 0.330 g, 89%. Measured ion by LCMS: [M+H] + =691.2.

[0997] Step e.

[0998]

[0999] To a solution of the product from the previous step (50 mg, 0.07 mmol) and 2,2-dimethoxypropane (0.044 mL, 0.36 mmol) in acetone (3 mL) was added p-TsOH (2 mg, 0.007 mmol) at ambient temperature. The reactant was stirred for two hours and then concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (10 mL) and washed with saturated sodium bicarbonate. The organic layer was collected and dried over anhydrous Na2SO4, filtered and concentrated in vacuo to provide an acetonide derivative. The product was a white solid. LCMS [M+H] +=731.2. DIEA (0.014 mL, 0.08 mmol) was added to a suspension of methylenebis(phosphorus chloride) (54 mg, 0.22 mmol) in THF (2 mL) at 0 ° C. A solution of the acetonide derivative in THF (2 mL) was added dropwise to the resulting mixture over a period of 5 minutes. After addition, the resulting mixture was stirred at 0 ° C for a further 15 minutes, and the solution was then transferred to a pre-cooled (0 ° C) flask containing a 0.2M HCl aqueous solution. The reaction mixture was warmed to ambient temperature and stirred for another 4 hours. After completion (monitored by LCMS), the reaction mixture was concentrated and purified by reverse phase HPLC using ACN: water (0.1% TFA modifier). The product was a white solid. Yield 37 mg, 60%. Measured ion by LCMS: [M+H] + =849.2.

[1000] Step f.

[1001]

[1002] The product (0.03g, 0.035mmol) and intermediate A (0.015g, 0.035mmol) from the previous step are dissolved in DMF:H The solution in O (1:3, 1.5mL) is cooled to 0 DEG C. To which copper sulfate (II) (0.0006g, 0.0035mmol), sodium ascorbate (0.021g, 0.106mmol) and BTTA (0.003g, 0.007mmol) are added are dissolved in the premixed solution of the solution in water (0.5mL) and stirred at the same temperature for 5 minutes, gradually warmed to room temperature and stirred at room temperature for 15 minutes. After the reaction is completed, it is quenched by adding a few drops of AcOH and EDTA to pH 6 by the reaction mixture, and by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA) purified product. The product is a white solid. Yield 50mg, 54%. By LCMS measured ion: [(M+2H) / 2] + =635.8.

[1003] Synthesis of conjugate 62

[1004] As described in the conjugation procedure for conjugate 60, the trifluorophenol ester described in the synthesis of Int-117 (19 mg, 0.01448 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL, in PBS at pH 7.4, 0.0018 mmol). 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%.

[1005] Synthesis of Int-119

[1006]

[1007] Step a.

[1008]

[1009] Intermediate B (250 mg, 0.56 mmol), propargyl-PEG 4- A mixture of amine (155 mg, 0.67 mmol) and triethylamine (0.16 mL, 1.12 mmol) in ethanol (6 mL) was heated at 50 ° C for 1 hour. The mixture was cooled to ambient temperature and concentrated under reduced pressure. The crude residue was redissolved in ethyl acetate (50 mL) and washed with water and brine. The organic layer was dried over sodium sulfate and the solvent was removed under reduced pressure to give the product as a colorless viscous oil, which was used in the next step without further purification. Yield 340 mg, 95%. Measured ion by LCMS: [M+H] + =642.2.

[1010] Step b.

[1011]

[1012] The product (340 mg, 0.53 mmol) from the previous step in methanol (5 mL) was treated with potassium carbonate (255 mg, 1.85 mmol) at ambient temperature for 2 hours. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography using DCM to 10% MeOH in DCM. The product was a white solid. Yield: 240 mg, 88%. Measured ion by LCMS: [M+H] + =516.2.

[1013] Step c.

[1014]

[1015] To a solution of the product from the previous step (240 mg, 0.47 mmol) and 2,2-dimethoxypropane (0.28 mL, 2.33 mmol) in acetone (4 mL) was added p-TsOH (8 mg, 0.046 mmol) at room temperature. The reactants were stirred for 2 hours and then concentrated under reduced pressure. The crude residue was redissolved in ethyl acetate (20 mL) and washed with saturated sodium bicarbonate. The organic layer was collected and dried over sodium sulfate, filtered and concentrated under reduced pressure to provide an acetonide derivative. The product was a white solid. LCMS [M+H] +=556.2. DIEA (0.089 mL, 0.51 mmol) was added to a suspension of methylenebis(phosphorus chloride) (348 mg, 1.4 mmol) in THF (5 mL) at 0 ° C. Over a 5-minute period, a solution of an acetonide derivative in THF (2 mL) was added dropwise to the resulting mixture. After addition, the resulting mixture was stirred at 0 ° C for another 15 minutes, and the solution was then transferred to a pre-cooled (0 ° C) flask containing a 0.2 M HCl aqueous solution (3 mL). The reaction mixture was warmed to ambient temperature and stirred for another 4 hours. After completion (monitored by LCMS), the reaction mixture was concentrated and purified by reversed-phase HPLC using ACN: water (0.1% TFA modifier). The product was a white solid. Yield 100 mg, 32%. Measured ion by LCMS: [M+H] + =674.2.

[1016] Step d.

[1017]

[1018] To be dissolved in DMF:H o (1: 3,1.5mL) from the product (0.06g, 0.076mmol) and intermediate A (0.032g, 0.076mmol) of the previous step are cooled to 0 DEG C. To which copper sulfate (II) (0.0012g, 0.0076mmol), sodium ascorbate (0.045g, 0.23mmol) and BTTA (0.007g, 0.015mmol) are added and stirred 5 minutes at the same temperature, gradually warmed to room temperature and stirred at room temperature 15 minutes. After the completion of the reaction, it is quenched by adding a few drops of AcOH and EDTA to pH 6 by reaction mixture, and by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA) purified product. Product is a white solid. Yield 104mg, 53.5%. Found by LCMS: [(M+2H) / 2] + =548.2.

[1019] Synthesis of conjugate 63a

[1020] As described in the conjugation procedure for conjugate 61, the trifluorophenol ester described in the synthesis of Int-119 (16 mg, 0.01448 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (100 mg, 3.47 mL, in PBS at pH 7.4, 0.0018 mmol). Maldi TOF analysis of the purified final product gave an average mass of 62259 Da (DAR = 3.8). Yield 59.2 mg, 59.2%.

[1021] Synthesis of Int-130

[1022]

[1023] The title compound was prepared similarly to Int-10, where N-Boc-piperidine-4-carboxylic acid was substituted for N-Boc azetidine-3-carboxylic acid. The product was a white solid. Yield: 72 mg, 78.1%. LCMS measured ion: [(M+2H) / 2] + =548.2.

[1024] Synthesis of conjugate 69

[1025] As described in the conjugation procedure for conjugate 61, the trifluorophenol ester described in the synthesis of Int-130 (18 mg, 0.01448 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (100 mg, 5.15 mL, in PBS at pH 7.4, 0.0018 mmol). MALDI TOF analysis of the purified final product gave an average mass of 62,259 Da (DAR 3.8). Yield 59.2 mg, 59.2%.

[1026] Synthesis of Int-134

[1027]

[1028] Step a.

[1029]

[1030] To a solution of N-Boc-3-hydroxyazetidine (346 mg, 2 mmol) in DMF (10 mL) was added sodium hydride (60% in mineral oil, 119 mg, 3 mmol) in portions at 0 ° C., and the mixture was stirred at ambient temperature for 1 hour. Propargyl-PEG4- methanesulfonate (620 mg, 2 mmol) was added to a sodium alcohol suspension, and the reaction mixture was stirred at ambient temperature for 16 hours. The resulting solution was quenched with saturated ammonium chloride solution (20 mL), extracted with ethyl acetate (3 × 20 mL), then washed with water and brine. The combined organic extracts were dried over sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (hexane: EtOAc) to obtain an N-Boc-protected intermediate as a colorless viscous oil. Measured ion by LCMS: [M+H] +=388.2. The N-Boc protected intermediate (0.640 g) was dissolved in 4N HCl in dioxane (10 mL) and stirred at ambient temperature for 4 hours. The solvent was removed under reduced pressure and dried under high vacuum to give the compound as a viscous liquid. Yield 530 mg, 83% over 2 steps. LCMS ion: [M+H] + =288.2.

[1031] Step b.

[1032]

[1033] A mixture of intermediate B (250mg, 0.56mmol), amine hydrochloride (217mg, 0.68mmol) from the previous step and triethylamine (0.16mL) in methanol (6mL) was heated at 50°C for 2 hours. After the raw material was completely consumed (monitored by LCMS), the reaction mixture was cooled to ambient temperature and the mixture was concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (50mL) and washed with water and brine. The combined organic extracts were dried over sodium sulfate. The solvent was removed under reduced pressure to obtain a triacetate derivative in the form of a yellow viscous oil, which was used in the next step without further purification. Ion [M+H] was measured by LCMS. + =668.2. The triacetate intermediate (370 mg, 0.56 mmol) was dissolved in methanol (6 mL), potassium carbonate (270 mg, 1.95 mmol) was added, and the reactants were stirred at ambient temperature for 2 hours. The mixture was concentrated under reduced pressure to give a crude product. The crude material was dissolved in a minimum of 1:1 MeOH:H2O and purified by reverse phase HPLC (5% to 100% ACN / water, 0.1% TFA modifier) ​​to give the compound. The product was a white foam. Yield 280 mg, 88%. Measured ion by LCMS: [M+H] + =572.2.

[1034] Step c.

[1035]

[1036] To a solution of the triol product (280 mg, 0.49 mmol) and 2,2-dimethoxypropane (0.3 mL, 2.45 mmol) from the previous step in acetone (4 mL) was added p-TsOH (8 mg, 0.0.048 mmol) at ambient temperature. The reactants were stirred for 2 hours and then concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (20 mL) and washed with saturated sodium bicarbonate. The organic layer was collected and dried over sodium sulfate, filtered and concentrated to give an acetonide derivative. The product was a white solid. Measured ions by LCMS: [M+H] +=612.2. DIEA (0.093 mL, 0.54 mmol) was added to a suspension of methylenebis(phosphorus chloride) (367 mg, 1.4 mmol) in THF (5 mL) at 0 ° C. A solution of an acetonide derivative in THF (2 mL) was added dropwise to the resulting mixture over a period of 5 minutes. After addition, the resulting mixture was stirred at 0 ° C for a further 15 minutes, and the solution was then transferred to a pre-cooled (0 ° C) flask containing a 0.2N HCl aqueous solution. The reaction mixture was warmed to ambient temperature and stirred for another 4 hours. After completion (monitored by LCMS), the reaction mixture was concentrated and purified by reverse phase HPLC using ACN: water (0.1% TFA modifier). The product was a white solid. Yield 280 mg, 78%. Measured ion by LCMS: 730.2 [M+H] + .

[1037] Step d.

[1038]

[1039] The product (0.06g, 0.082mmol) and intermediate A (0.035g, 0.082mmol) from the previous step are dissolved in DMF:H The solution in O (1:3, 1.5mL) is cooled to 0 DEG C.Add copper sulfate (II) (0.002g, 0.0082mmol), sodium ascorbate (0.048g, 0.246mmol) and BTTA (0.007g, 0.016mmol) to the premixed solution of the solution soluble in water (0.5mL) and stirred at the same temperature for 5 minutes, gradually warmed to room temperature and stirred at room temperature for 15 minutes.After the reaction is completed, it is 6 that the reaction mixture is quenched by adding a few drops of AcOH and EDTA to pH, and by reversed-phase HPLC (10% to 100% ACN / water, 0.1% TFA modifier) ​​purified product.Product is a white solid.Yield 52mg, 55%. Found by LCMS: [(M+2H) / 2] + =576.2.

[1040] Synthesis of conjugate 71

[1041] 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 for 12 hours in 150 mM His / 100 mM ammonium hydroxide buffer (pH 8.5) and purified by dialysis into 150 mM His pH `8.5 buffer, protein A and SEC columns. Maldi TOF analysis of the purified final product gave an average mass of 62,647 Da. (DAR was 4.4). Yield 43.7 mg, 43.7%.

[1042] Synthesis of Int-136

[1043]

[1044] The title compound was prepared similarly to step e of Int-117, where 4-hydroxyaniline was replaced with 3-hydroxyaniline. The product was a white solid. Yield: 32.8 mg, 73.1%. LCMS analysis revealed: [(M+2H) / 2] + =635.8.

[1045] Synthesis of conjugate 72

[1046] As described in the conjugation procedure for conjugate 71, the trifluorophenol ester described in the synthesis of Int-136 (17 mg, 0.01448 mmol) was conjugated to the Fc carrier SEQ ID NO: 13 (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%.

[1047] Synthesis of Int-2

[1048]

[1049] Step a.

[1050]

[1051] A solution of propargyl-Peg4-methanesulfonate (1.00 g, 3.22 mmol) and potassium thioacetate (0.74 g, 6.44 mmol) was dissolved in DMF (12 mL) and then flushed with nitrogen vacuum. After stirring for 5 minutes, the reactant became sticky, so more DMF (6 mL) was added. The reactant was stirred at room temperature overnight, and the color was now dark red. Work-up: the crude reactant was diluted with water and extracted several times with DCM. The DCM extract was dried over sodium sulfate, filtered, concentrated, and purified by RPLC (10% acetonitrile / water to 100% acetonitrile). Yield 927 mg, 99%. Measured ion by LCMS: [M+H] + =291.2

[1052] Step b.

[1053]

[1054] (R)-phenylglycinol (1.372g, 10.0mmol) dissolved in THF (20mL) is processed with Boc anhydride (2.40g, 11.0mmol) at room temperature. After 15 minutes, LCMS shows that Boc protection is complete. Then reactant is processed with DIEA (2.09mL, 12.0mmol) and mesyl chloride (0.85mL, 11.0mmol), and stirred 1 hour, when LCMS shows that it is completely converted into mesylate. The crude reaction is diluted with ethyl acetate, extracted with water, 1N HCl, salt water, and dried over sodium sulfate and concentrated. After concentration, crystals are formed. The filtrate is processed with hexane to improve the recovery of the product. The product is a crystalline white solid, yield 2.21g, 70%. By LCMS measured ion: [M-Boc+H] + =216.2

[1055] Step c.

[1056]

[1057] The Peg-thioacetic acid ester (0.45g, 1.55mmol) from the step a of the present embodiment is dissolved in DMF (4.5mL), and vacuum rinsed several times, then processed with sodium methoxide (0.39mL, 1.70mmol, 25% methanol solution).After stirring at room temperature for 20 minutes, the disposable addition mesylate (0.489g, 1.55mmol) from the previous step is added. Reactant is further stirred for 30 minutes, and now LCMS shows complete conversion. Make reactant slightly acidic with acetic acid, be condensed into oily matter, and pass through RPLC (10% acetonitrile / water to 100% acetonitrile) purification. Yield 435mg, 60%.By LCMS actual measurement ion: [M+Na] + =490.2

[1058] Step d

[1059]

[1060] The product from the previous step (435 mg, 0.93 mmol) was treated with a 1:1 solution of DCM and TFA (4 mL) and then stirred at room temperature for 30 minutes. The crude reaction was concentrated to an oil and used in the next step without further purification.

[1061] Step e.

[1062]

[1063] At room temperature, the crude product (342mg, 0.93mmol), intermediate B (416mg, 0.93mmol) and DIEA (0.648mL, 3.72mmol) from the previous step were mixed in methanol (5mL). LCMS after 5 minutes showed that approximately 90% was converted into product. LCMS after 2 hours showed that the starting material was completely consumed. Sodium methoxide (0.851mL, 3.72mmol, 25% sodium methoxide in methanol) was added at room temperature. LCMS after 10 minutes showed that acetate was completely deprotected. The reactant was neutralized with acetic acid, concentrated, and purified by RPLC (10% acetonitrile / water to 100% acetonitrile, containing 0.1% TFA). Yield 432mg, 71%. Measured ion by LCMS: [M+H] + =652.2

[1064] Step f.

[1065]

[1066] The product from the previous step (432 mg, 0.662 mmol) dissolved in acetone (3 mL) was treated with dimethoxypropane (3 mL) and toluenesulfonic acid (12.6 mg, 0.066 mmol). LCMS after 1 hour showed complete conversion to acetonide. Post-treatment: the reactant was diluted with ethyl acetate, washed with saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, concentrated, and purified by flash chromatography (10% EtOAc / DCM to 100% EtOAc). Yield 214 mg, 47%. Measured ion by LCMS: [M+H] + =692.2

[1067] Step g.

[1068]

[1069] At ambient temperature, the product (214mg, 0.309mmol) from the previous step that is dissolved in trimethoxy phosphate (6mL) is processed with methylene (phosphorus dichloride) (386mg, 1.55mmol). After 2 hours, LCMS shows that all raw materials have been consumed. The reactant is cooled with an ice bath, then processed and stirred at room temperature with 2M HCl (6mL). LCMS after 2 hours shows that the acetonide is deprotected completely. The reactant is concentrated by rotary evaporation, then purified by RPLC (10% acetonitrile / water to 100% acetonitrile, containing 0.1% TFA). Yield 160mg, 64%. Measured ion by LCMS: [M+H] + =810.2

[1070] Step h.

[1071]

[1072] To a solution of the product (50 mg, 0.062 mmol) and azide intermediate A (34 mg, 0.080 mmol) from the previous step dissolved in DMF (1 mL), copper sulfate (II) (2.5 mg, 0.0154 mmol), sodium ascorbate (12.2 mg, 0.062 mmol) and THPTA (10.7 mg, 0.025 mmol) were added in water (0.5 mL). After stirring at room temperature for 20 minutes, LCMS showed complete conversion to product. The reactant was directly purified by RPLC (10% acetonitrile / water to 100% acetonitrile, containing 0.1% TFA). Yield 49 mg, 64%. Measured ion by LCMS: [(M+2H) / 2] + =616.2

[1073] Synthesis of conjugate 2

[1074] At ambient temperature, a solution of trifluorophenyl ester (10.6 mg, 0.0086 mmol, Int-2) in DMF (1 mL) was added to Fc (50 mg, 0.00086 mmol, SEQ ID NO: 17 in PBS at pH 7.4, 17.3 mg / mL). The pH of the resulting solution was adjusted to approximately 8.5 with borate buffer (300 uL, 1 M, pH 8.5). The uniform colorless reaction was gently shaken for 3 hours and then purified 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%.

[1075] Synthesis of conjugate 146

[1076] The title compound was prepared similarly to Conjugate 2, wherein Fc SEQ ID NO: 17 was replaced with Fc SEQ ID NO: 13.

[1077] Synthesis of Int-1

[1078]

[1079] The title compound was prepared analogously to Int-2, whereby (R)-phenylglycinol was replaced by (S)-phenylglycinol in step a of the Example.

[1080] Synthesis of conjugate 1

[1081] The title conjugate was prepared analogously to conjugate 2, wherein the trifluorophenyl ester from Int-2 was replaced by the trifluorophenyl ester from Int-1.

[1082] Synthesis of conjugate 144

[1083] The title compound was prepared analogously to Conjugate 1, wherein Fc SEQ ID NO: 17 was replaced with Fc SEQ ID NO: 13.

[1084] Synthesis of Int-14

[1085]

[1086] Step a.

[1087]

[1088] A solution of the thioether (56 mg, 0.069 mmol, described in Int-2) in THF (2 mL) was treated with MCPBA (12.5 mg, 0.073 mmol). LCMS after 5 minutes showed clear conversion to sulfoxide. The product was directly purified by RPLC (5% acetonitrile / water to 100% acetonitrile, containing 0.1% TFA). Yield 55 mg, 96%. Measured ion by LCMS: [M+H] + =826.2.

[1089] Step b.

[1090]

[1091] To a solution of the product (53 mg, 0.064 mmol) and azide intermediate A (35 mg, 0.083 mmol) from the previous step dissolved in DMF (1 mL), copper sulfate (II) (2.6 mg, 0.0160 mmol), sodium ascorbate (12.7 mg, 0.064 mmol) and THPTA (11.1 mg, 0.026 mmol) were added in water (0.5 mL). After stirring at room temperature for 20 minutes, LCMS showed complete conversion to product. The reactant was directly purified by RPLC (10% acetonitrile / water to 100% acetonitrile, containing 0.1% TFA). Yield 49 mg, 64%. Measured ion by LCMS: [(M+2H) / 2] + =624.3

[1092] Synthesis of conjugate 10

[1093] The title conjugate was prepared analogously to conjugate 2, where the trifluorophenyl ester from Int-2 was replaced with the trifluorophenyl ester from Int-14.

[1094] Synthesis of Int-13

[1095]

[1096] The title compound was prepared analogously to Int-14, where the alkynyl thioether intermediate was replaced with the analogous alkynyl thioether intermediate from Int-1.

[1097] Synthesis of conjugate 9

[1098] The title conjugate was prepared analogously to conjugate 2, where the trifluorophenyl ester from Int-2 was replaced with the trifluorophenyl ester from Int-13.

[1099] Synthesis of Int-33

[1100]

[1101] Step a.

[1102]

[1103] R-phenylglycinol (411mg, 3.00mmol) is dissolved in the solution sodium hydride (156mg, 3.89mmol, 60% in mineral oil) in DMF (5mL) and is at room temperature processed 10 minutes.In this solution, propargyl-Peg4-methanesulfonate (1.12g, 3.60mmol) is added once.LCMS after 30 minutes shows to be converted into product completely.Reactant is neutralized with glacial acetic acid, concentrated, and passed through RPLC (10% acetonitrile / water to 100% acetonitrile, containing 0.1% TFA) purification.The yield of single TFA salt is 1.18g, 85%.By LCMS actual measurement ion: [M+H] + =352.2

[1104] Step b.

[1105]

[1106] At ambient temperature, the product (364mg, 0.78mmol), intermediate B (350mg, 0.78mmol) and DIEA (0.545mL, 3.13mmol) from the previous step were mixed in methanol (4mL). LCMS after 5 minutes showed that approximately 90% was converted into product. LCMS after 1 hour showed that the starting material was completely consumed. Sodium methoxide (1.07mL, 4.70mmol, 25% sodium methoxide in methanol) was added at ambient temperature. LCMS after 10 minutes showed that acetate was completely deprotected. The reactant was neutralized with glacial acetic acid, concentrated, and purified by RPLC (10% acetonitrile / water to 100% acetonitrile, containing 0.1% TFA). Yield 330mg, 66%. Measured ion by LCMS: [M+H] + =635.8

[1107] Step c.

[1108]

[1109] The product from the previous step (330 mg, 0.519 mmol) dissolved in acetone (3 mL) was treated with dimethoxypropane (3 mL) and toluenesulfonic acid (18 mg, 0.104 mmol). LCMS after 1 hour showed complete conversion to acetonide. Post-treatment: the reactant was neutralized with DIEA, concentrated, and purified by flash chromatography (0% MeOH / DCM to 10% MeOH / DCM). Yield 262 mg, 75%. Measured ion by LCMS: [M+H] + =676.2

[1110] Step d.

[1111]

[1112] At ambient temperature under nitrogen atmosphere, the product (262mg, 0.387mmol) from the previous step that is dissolved in THF (3mL) is processed with methylene (phosphorus dichloride) (387mg, 1.55mmol). After 1.5 hours, LCMS shows that all raw materials have been consumed. The reactant is cooled with an ice bath, then processed and stirred at room temperature with 2M HCl (4mL). LCMS after 1.5 hours shows that the acetonide is deprotected completely. The reactant is concentrated by rotary evaporation, then purified by RPLC (10% acetonitrile / water to 100% acetonitrile, containing 0.1% TFA). Yield 161mg, 52%. Measured ion by LCMS: [M+H] + =794.2

[1113] Step e.

[1114]

[1115] To a solution of the product (161 mg, 0.203 mmol) and azide intermediate A (111 mg, 0.264 mmol) from the previous step dissolved in DMF (1 mL), copper sulfate (II) (8.1 mg, 0.0507 mmol), sodium ascorbate (40.2 mg, 0.203 mmol) and THPTA (35.2 mg, 0.0811 mmol) were added in water (0.5 mL). After stirring at ambient temperature for 20 minutes, LCMS showed complete conversion to product. The reactant was directly purified by RPLC (10% acetonitrile / water to 100% acetonitrile, containing 0.1% TFA). Yield 224 mg, 91%. Measured ion by LCMS: [(M+2H) / 2] + =608.2

[1116] Synthesis of conjugate 20

[1117] At room temperature, a solution of trifluorophenyl ester (8.4 mg, 0.0084 mmol, Int-33) in DMF (1 mL) was added to Fc (50 mg, 0.00086 mmol, SEQ ID NO: 13 in PBS at pH 7.4, 19.5 mg / mL). The pH of the resulting solution was adjusted to approximately 8.5 with borate buffer (300 uL, 1 M, pH 8.5). The homogeneous colorless reaction was gently shaken for 3 hours and then purified according to the general procedure. Maldi TOF analysis of the purified final product gave an average mass of 62,315 Da (DAR = 3.8). Yield: 69.6 mg, 79%.

[1118] Synthesis of Int-34

[1119]

[1120] The title compound was prepared analogously to Int-33, wherein in step a of that example (R)phenylglycinol was replaced with (S)phenylglycinol.

[1121] Synthesis of conjugate 29

[1122] The title conjugate was prepared analogously to conjugate 20, where the trifluorophenyl ester from Int-33 was replaced with the trifluorophenyl ester from Int-34.

[1123] Synthesis of Int-62

[1124]

[1125] The title compound was prepared analogously to Int-33, wherein in step a of that example (R)phenylglycinol was replaced with (3S)-3-amino-3-phenylpropan-1-ol.

[1126] Synthesis of conjugate 34

[1127] At ambient temperature, a solution of trifluorophenyl ester (10.4 mg, 0.0077 mmol, Int-62) in DMF (1 mL) was added to Fc (50 mg, 0.00086 mmol, SEQ ID NO: 13 in PBS at pH 7.4, 19.5 mg / mL). The pH of the resulting solution was adjusted to approximately 8.5 with borate buffer (300 uL, 1 M, pH 8.5). The uniform colorless reaction was gently shaken for 3 hours and then purified according to the general procedure. Maldi TOF analysis of the purified final product gave an average mass of 63,518 Da (DAR=4.9). Yield: 41 mg, 82%.

[1128] Synthesis of Int-22

[1129]

[1130] Step a.

[1131]

[1132] At ambient temperature, propargyl-Peg4-amine (450mg, 1.95mmol) and 2-chloro-benzaldehyde (274mg, 1.95mmol) are stirred in methanol (10mL) for 16 hours. Sodium borohydride (296mg, 7.8mmol) is added thereto, and the solution is stirred for another hour. Excess methanol is removed by rotary evaporator, and the solution is distributed between 1N NaOH (50mL) and ethyl acetate (50mL). Abandon the water layer, remove excess ethyl acetate, and the crude mixture is passed through reverse phase flash chromatography (0% to 100% CH3CN / H2O) purification. Yield: 346mg, 50%. Measured ion by LCMS: [M+H] + =356.2.

[1133] Step b.

[1134]

[1135] Intermediate B (437 mg, 0.98 mmol), the product from the previous step (346 mg, 0.98 mmol) and DIEA (405 mg, 2.94 mmol) were stirred in ethanol (25 mL) at 50° C. for 4 hours. The mixture was cooled to ambient temperature and concentrated. The crude mixture was purified by reverse phase flash chromatography (0% to 100% CH 3 CN / H 2 O). Yield 204 mg, 27%. LC / MS [M+H] + = 753.2.

[1136] Step c.

[1137]

[1138] To a solution of triacetate (204 mg, 0.27 mmol) in methanol (20 mL) was added 25% sodium methoxide in methanol (2 mL). The solution was stirred at ambient temperature for 2 hours. The crude mixture was neutralized with glacial acetic acid (4 mL), then concentrated and purified by semi-preparative HPLC (0% to 100% CH 3 CN / H 2 O, 0.1% TFA modifier). Yield 154 mg, 89%. Measured ion by LC / MS: [M+H] + = 640.2.

[1139] Step d.

[1140]

[1141] To a solution of the triol (154 mg, 0.24 mmol) from the previous step in acetone (20 mL) was added 2,2-dimethoxypropane (1.69 g, 16.29 mmol) and TsOH (10 mg, 0.05 mmol). The solution was stirred at ambient temperature for 16 hours. Excess TsOH was neutralized with DIEA (1 mL) and the crude mixture was concentrated and purified by semi-preparative HPLC (0% to 100% CH3CN / H2O, 0.1% TFA modifier). Yield 110 mg, 67%. Found by LC / MS: [M+H]+=680.2.

[1142] Step e.

[1143]

[1144] The acetone compound (110 mg, 0.161 mmol) from the previous step in THF (6 mL) was added dropwise to a mixture of methylene (phosphorus dichloride) (121 mg, 0.485 mmol) and DIEA (31 mg, 241 mmol) in THF (10 mL) cooled to 0 ° C via an ice bath. When the addition was complete, the ice bath was removed and the reactant was stirred at ambient temperature for 3 hours. The mixture was then cooled to 0 ° C via an ice bath and a 0.1N HCl aqueous solution (30 mL) was added. The mixture was stirred at 0 ° C for 15 minutes and then stirred at room temperature for 16 hours (monitored by LC / MS). The solvent was removed on a rotary evaporator and the crude mixture was purified by semi-preparative HPLC (0% to 100% CH CN / H O, 0.1% TFA modifier). The pure fractions were combined and lyophilized to obtain the product as a white hygroscopic solid. Yield 44 mg, 34%. Measured ion by LC / MS: [M+H] = 798.1.

[1145] Step f.

[1146]

[1147] The alkyne (20 mg, 0.025 mmol) and intermediate A (18 mg, 0.042 mmol) from step e were dissolved in DMF (1 mL) and cooled to 0 ° C via an ice-water bath. Premixed copper (II) sulfate (1 mg, 0.006 mmol) and sodium ascorbate (5 mg, 0.025 mmol) in DI water (1 mL) were then added and the mixture was stirred at ambient temperature for 4 hours. The crude reaction mixture was directly purified via semi-preparative HPLC (0% to 100% CH CN / H O, 0.1% TFA modifier). Pure fractions were combined and lyophilized to give the product as a white hygroscopic solid. Yield 20 mg, 64%. Measured ion [(M / 2)+H] + = 610.3 by LC / MS.

[1148] Synthesis of conjugate 14a

[1149] To the solution of SEQ ID NO:13 (3.40mL, 100mg, 0.0017mmol) in PBS 7.4, add Int-132 (21mg, 0.017mmol) in DMF (0.200mL). The pH of the reaction mixture is slowly adjusted to about 8.5 by adding 2mL of 1M potassium carbonate buffer (pH 9). The reactant is then gently shaken for 4 hours. The reaction is quenched by stirring in 150mM His / 100mM ammonium hydroxide buffer (pH 8.5) for 12 hours and then purified according to general procedure.

[1150] As describe...

Claims

1. A conjugate described by formula (DI) or (MI) or a pharmaceutically acceptable salt thereof: Among them A 1 and A 2 Each of which independently has the structure of formula (A): 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 CYR 5 , where X 1 , X 2 , X 3 , X 4 , X 5 and X 6 At least one of them is CYR 5 And R 5 is the bond with L; R 1 for Z is O, S or sulfonyl; R 2a and R 2b Each of the following is independently H, an optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 heteroaryl; Each R 3 are independently OH, SH, halogen, optionally substituted amino, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 heteroaryl; R 4 is H, halogen, OH, SH, optionally substituted amino, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 heteroaryl; R 6a and R 6b Each of the following is independently H, an optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 heteroaryl; Y is the first connector; L is the second connector; n is 1 or 2; Each E contains an Fc domain monomer; T is an integer from 1 to 20; and The curved line indicates that L is covalently attached to E.

2. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the conjugate is described by formula (DI):

3. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the conjugate is described by formula (MI):

4. The conjugate according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein A 1 and A 2 Having the structure of formula (AI):

5. The conjugate according to claim 4 or a pharmaceutically acceptable salt thereof, wherein A 1 and A 2 Each has the structure of formula (A-Ia):

6. The conjugate according to claim 5 or a pharmaceutically acceptable salt thereof, wherein A 1 and A 2 Each has the structure of formula (A-Ib):

7. The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein A 1 and A 2 Each has the structure of formula (A-II):

8. The conjugate according to claim 7 or a pharmaceutically acceptable salt thereof, wherein A 1 and A 2 Each has the structure of formula (A-IIa):

9. The conjugate of claim 8 or a pharmaceutically acceptable salt thereof, wherein A 1 and A 2 Each has the structure of formula (A-IIb):

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. The conjugate of any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, wherein R 2a and R 2b Each of the following is independently H, an optionally substituted C1-C 20 Alkyl or optionally substituted C1-C 20 Heteroalkyl.

13. The conjugate according to claim 12 or a pharmaceutically acceptable salt thereof, wherein R 2a and R 2b Each of is H.

14. The conjugate of any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, wherein R 4 is H, halogen, OH, SH, optionally substituted amino, optionally substituted C1-C 20 Alkyl or optionally substituted C1-C 20 Heteroalkyl.

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

16. The conjugate of claim 15 or a pharmaceutically acceptable salt thereof, wherein R 4 For Cl.

17. The conjugate of any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof, wherein R 1 for 18. The conjugate of any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof, wherein R 1 for 19. The conjugate of claim 18 or a pharmaceutically acceptable salt thereof, wherein R 6a and R 6b Each of the following is independently H, an optionally substituted C1-C 20 Alkyl or optionally substituted C1-C 20 Heteroalkyl.

20. The conjugate of claim 19 or a pharmaceutically acceptable salt thereof, wherein R 6a and R 6b Each of which is independently H, -CH3, -CH2CH3, -CH2OH, -CH2OCH3, -CH2CH2OH or -CH2CH2OCH3.

21. The conjugate of claim 20 or a pharmaceutically acceptable salt thereof, wherein R 6a and R 6b Each of is H.

22. The conjugate of any one of claims 1 to 21, wherein Y is: 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 are independently N or CH; Each X 8 are independently O, NH, CH2 or C(=O); R N1 is H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl, optionally substituted C2-C 19 Heteroaryl, optionally substituted C1-C 20 Alkaryl or optionally substituted C1-C 20 alkylcycloalkyl; Each R 7 Independently Optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl or optionally substituted C2-C 20 Heterocycloalkenyl; R 7a and R 7b Each of the following is independently H, an optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 heteroaryl; and Each R 8 are independently halogen, OH, SH, optionally substituted amino, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Heteroalkenyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C3-C 20 Cycloalkenyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C2-C 20 Heterocycloalkenyl, optionally substituted C6-C 18 Aryl or optionally substituted C2-C 19 Heteroaryl.

23. The conjugate of claim 22 or a pharmaceutically acceptable salt thereof, wherein Y is 24. The conjugate of claim 23 or a pharmaceutically acceptable salt thereof, wherein q is 1 and Y is 25. The conjugate of claim 24 or a pharmaceutically acceptable salt thereof, wherein R 7 for And Y is 26. The conjugate of claim 24 or a pharmaceutically acceptable salt thereof, wherein Y is 27. The conjugate of claim 23 or a pharmaceutically acceptable salt thereof, wherein q is 0 and Y is 28. The conjugate of any one of claims 23 to 27, or a pharmaceutically acceptable salt thereof, wherein R N1 For H.

29. The conjugate of any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, wherein Y is 30. The conjugate of any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, wherein Y is 31. The conjugate of claim 30 or a pharmaceutically acceptable salt thereof, wherein Y is 32. The conjugate of any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, wherein Y is 33. The conjugate of claim 32 or a pharmaceutically acceptable salt thereof, wherein Y is 34. The conjugate of claim 33 or a pharmaceutically acceptable salt thereof, wherein Y is 35. The conjugate of any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, wherein Y is 36. The conjugate of claim 35 or a pharmaceutically acceptable salt thereof, wherein Y is 37. The conjugate of claim 36 or a pharmaceutically acceptable salt thereof, wherein Y is 38. The conjugate of any one of claims 1 to 37, or a pharmaceutically acceptable salt thereof, wherein L comprises one or more optionally substituted C1-C 40 Alkylene, optionally substituted C1-C 40 Heteroalkylene, optionally substituted C1-C 40 Alkyleneoxy, optionally substituted C2-C 20 Alkenylene, optionally substituted C2-C 20 Heteroalkenylene, optionally substituted C2-C 20 Alkyne, 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 Heterocycloalkenyl, optionally substituted C8-C 20 Cycloalkynylene, optionally substituted C8-C 20 Heterocycloalkynyl, optionally substituted C5-C 15 Arylene, optionally substituted C2-C 15 Heteroarylene, O, S, NR i , P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl or imino, Where 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 heterocycloalkenyl, 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.

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

40. The conjugate of any one of claims 1-39, or a pharmaceutically acceptable salt thereof, wherein the backbone of L comprises 1 to 250 atoms.

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

42. The conjugate of any one of claims 3-41 or a pharmaceutically acceptable salt thereof, wherein L is described by the 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 Among them J 1 is the key attached to A1; J 2 is a bond attached to E or a functional group capable of reacting with a functional group conjugated to E; Q 1 , Q 2 , Q 3 , Q 4 and Q 5 each of which is independently an optionally substituted C1-C40 alkylene, an optionally substituted C1-C40 heteroalkylene, an optionally substituted C1-C40 alkyleneoxy, an optionally substituted C2-C20 alkenylene, an optionally substituted C2-C20 heteroalkenylene, an optionally substituted C2-C20 alkynylene, an optionally substituted C2-C20 heteroalkynylene, an optionally substituted C3-C20 cycloalkylene, an 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 oxime; 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 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.

43. The conjugate of claim 42 or a pharmaceutically acceptable salt thereof, wherein Q 1 for 44. The conjugate of claim 42 or 43, or a pharmaceutically acceptable salt thereof, wherein Q 2 is an optionally substituted C1-C 40 Alkylene, optionally substituted C1-C 40 Heteroalkylene, optionally substituted C1-C 40 Alkyleneoxy or optionally substituted C2-C 15 Heteroarylene.

45. The conjugate of any one of claims 42-44, or a pharmaceutically acceptable salt thereof, wherein Q 3 is an optionally substituted C2-C 15 Heteroarylene.

46. ​​The conjugate of any one of claims 42-45, or a pharmaceutically acceptable salt thereof, wherein Q 4 is an optionally substituted C1-C 40 Alkylene, optionally substituted C1-C 40 Heteroalkylene or optionally substituted C1-C 40 Alkylene oxide.

47. The conjugate of any one of claims 42-46, or a pharmaceutically acceptable salt thereof, wherein J 2 for 48. A conjugate described by the formula of Table 2 or a pharmaceutically acceptable salt thereof.

49. The conjugate of any one of claims 1-48, or a pharmaceutically acceptable salt thereof, wherein the curved line connected to E indicates that L of each A1-L or each A1-L-A2 is covalently attached to a nitrogen atom of a lysine of E that is exposed to the solvent.

50. The conjugate of any one of claims 1-48, or a pharmaceutically acceptable salt thereof, wherein the curved line attached to E indicates that L of each A1-L or each A1-L-A2 is covalently attached to the sulfur atom of the solvent-exposed cysteine ​​of E.

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

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

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

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

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

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

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

18.

58. The conjugate of claim 56, or a pharmaceutically acceptable salt thereof, wherein each E comprises 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 of any one of claims 1-58, 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 or pharmaceutically acceptable salts thereof as described in any one of claims 1-59, wherein the average value of T is 1 to 10.

61. A population of conjugates or pharmaceutically acceptable salts thereof as claimed in claim 60, wherein the average value of T is 1 to 5.

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

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

64. The method of 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 carcinoma; bile duct cancer; endometrial cancer; melanoma; or esophageal cancer.

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

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

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

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

69. A method of 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 as described in any one of claims 1-62.

70. The method of claim 69, wherein the viral infection is a betacoronavirus infection.

71. The method of claim 70, wherein the betacoronavirus is SARS-CoV-2.

72. The method of claim 71, wherein the SARS-CoV-2 is an Alpha, Delta, or Omicron variant.

73. The method of claim 72, wherein the SARS-CoV-2 is an Omicron variant.

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

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

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

77. The method of claim 76, wherein the fibrosis is pulmonary fibrosis, skin fibrosis, kidney fibrosis, liver fibrosis, cardiac fibrosis, or systemic sclerosis.

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

79. The method of claim 78, wherein the pulmonary fibrosis is associated with 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 of any one of claims 63-79, wherein the conjugate, population of conjugates, or pharmaceutical composition is administered intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, by injection, or by infusion.

Images