Attenuated interferon proteins and fragments and multifunctional polypeptides and conjugates

Abstract

Provided herein are modified IFN [alpha] 2b proteins and functional fragments thereof. Also provided herein are polypeptides, such as bifunctional and multifunctional polypeptides, which further comprise biological moieties, such as antibodies or antigen binding fragments thereof, and conjugates of such polypeptides.

Description

Technical Field

[0001] The present disclosure relates to modified IFNα2b proteins and functional fragments thereof, as well as multifunctional polypeptides and immunoconjugates comprising IFNα2b or fragments thereof. Background Art

[0002] Human IFNα2b belongs to type I interferons (IFN-I), which are clustered on chromosome 9p. It transmits signals through the heterodimeric complex of interferon α / β receptor 1 (IFNAR1) and interferon α / β receptor 2 (IFNAR2) to trigger TYK2 and JAK1 activation, thereby triggering a cascade of immune-related interferon-stimulated genes (ISGs), which is crucial for immune activation.

[0003] There is evidence that endogenous type I IFN, including IFNα2b, can enhance the anti-tumor activity of chemotherapy, radiotherapy, and some targeted therapies. Although recombinant IFNα2b has demonstrated efficacy in cancer treatment (such as in melanoma, renal cell carcinoma, and various hematological malignancies), patients receiving this therapy usually suffer from adverse reactions, which pose a major challenge to the development of IFN-mediated therapies. Summary of the Invention

[0004] This application is to meet clinical needs. Certain embodiments provide modified IFNα2b, which has reduced adverse reactions and improved efficacy for clinical use.

[0005] In a first aspect, the present disclosure provides an IFNα2b or a functional fragment thereof, wherein the IFNα2b or the functional fragment thereof is modified to attenuate the binding affinity of the IFNα2b or the functional fragment thereof for the interferon α / β receptor. In some embodiments, the interferon α / β receptor is selected from IFNAR1, IFNAR2, and combinations thereof.

[0006] In some embodiments, the binding affinity of the IFNα2b or the functional fragment thereof for the interferon α / β receptor is attenuated by at least 10-fold, 10 2 -fold, 10 3 -fold, 10 4 -fold, or 10 5 -fold. In some embodiments, the IFNα2b or the functional fragment thereof is modified to attenuate the activation of type I interferon signaling induced by the IFNα2b or the functional fragment thereof. In some embodiments, the activation of IFNα signaling is attenuated by at least 10-fold, 10 2 -fold, 10 3 -fold, 10 4 -fold, or 10 5 -fold.

[0007] In some embodiments, IFNα2b or a functional fragment thereof is modified by amino acid substitution at one or more residues corresponding to residues R12, L15, M16, R22, L26, F27, L30, R33, H34, D35, A145, M148, R149, S152, L153, and N156 selected from SEQ ID NO:1. In some embodiments, the modification includes an amino acid substitution at the residue corresponding to S152 of SEQ ID NO:1. In some embodiments, the modification includes an amino acid substitution with cysteine at the residue corresponding to S152 of SEQ ID NO:1.

[0008] In some embodiments, IFNα2b comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0009] In some embodiments, IFNα2b or a functional fragment thereof is modified with a chemical moiety.

[0010] In some embodiments, the chemical moiety comprises a structure of formula (I):

[0011] R1-R2-R3-R4 (I),

[0012] wherein,

[0013] R1 is optionally further substituted with 1, 2, or 3 R* groups;

[0014] L1 is selected from CH or N;

[0015] R 1a is absent, or is selected from -(CH2) 0-3 -C(O)-, -(CH2) 0-3 -OC(O)-, and -(CH2) 0-3 -NHC(O)-;

[0016] each R 1b is independently absent, or is independently selected from -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)NH-, -NH-C 1-6 alkyl-, -O-C 1-6 alkyl-, -C(O)-C 1-6 alkyl-, -OC(O)-C 1-6 alkyl-, -C(O)O-C 1-6 alkyl-, and -C(O)NH-C 1-6 alkyl-;

[0017] Each R 1c is independently selected from H, C 1-6 alkyl, and C 1-6 haloalkyl;

[0018] m is an integer in the range of 1 - 30000;

[0019] R* is selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 2-6 alkenyl, or C 2-6 alkynyl;

[0020] R2 is a linker cleavable in the tumor environment;

[0021] R3 is

[0022] X is selected from -NH- or -O-;

[0023] Y is selected from -NH-, -O-, -C(O)-, -OC(O)-, -C(O)-C(O)-, or -NHC(O)-;

[0024] Each R x and R y are independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 2-6 alkenyl, or C 2-6 alkynyl;

[0025] n is 0, 1, 2, 3, 4, or 5;

[0026] R4 is

[0027] R a and R b are each independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, and C 1-6 alkoxy;

[0028] R 4a is absent, or is selected from -(CH2) 1-12 -, -(CH2) 1-12 -O-, -O-(CH2) 1-12 -, -O-(CH2) 1-12 -O-, -(CH2) 1-12 -O-(CH2) 1-12 -, (C 1-6 alkylene - O) p-(CH2) 1-12 、-(CH2) 1-12 -C 3-8 -subcycloalkyl-、-(CH2) 1-12 -C 3-8 -subcycloalkyl-(CH2) 1-12 、-(CH2) 1-12 -C 6-10 -subaryl- and -(CH2) 1-12 -C 6-10 -subaryl-(CH2) 1-12 ;

[0029] R 4a is optionally further substituted by 1, 2, 3, 4 or 5 R# groups;

[0030] R 4b is selected from -NH-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-C(O)-, -C(O)NH- or -NHC(O)-;

[0031] R# is selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 2-6 alkenyl or C 2-6 alkynyl;

[0032] p is 1, 2, 3, 4 or 5;

[0033] wherein R3 is connected to R2 through X of R3 and to R4 through Y of R3.

[0034] In some embodiments, R1 is which is optionally further substituted by 1, 2 or 3 R* groups;

[0035] L1 is selected from CH or N;

[0036] R 1a is absent, or is selected from -C(O)-, -OC(O)- and -NHC(O)-;

[0037] each R 1b is independently absent, or independently selected from -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)NH-, -NH-C 1-4 alkyl-, -O-C 1-4 alkyl-, -C(O)-C 1-4 alkyl-, -OC(O)-C 1-4 alkyl-, -C(O)O-C 1-4 alkyl- and -C(O)NH-C1-4 alkyl-;

[0038] each R 1c is independently selected from H, C 1-4 alkyl, and C 1-4 haloalkyl;

[0039] each m is independently an integer in the range of 1 - 5000;

[0040] R* is selected from H, halogen, C 1-6 alkyl, or C 1-6 haloalkyl;

[0041] alternatively, R1 is which is optionally further substituted with 1, 2, or 3 R* groups;

[0042] R 1a is absent, or is -C(O)-;

[0043] R 1c is selected from H, C 1-4 alkyl, and C 1-4 haloalkyl;

[0044] m is an integer in the range of 1 - 3000;

[0045] R* is selected from H, halogen, or C 1-4 alkyl;

[0046] alternatively, R1 is R 1c is selected from H, C 1-4 alkyl, and C 1-4 haloalkyl, and m is an integer in the range of 1 - 1000, 100 - 500, or 400 - 500;

[0047] alternatively, R1 is a linear polyethylene glycol having a molecular weight of about 20000.

[0048] In some embodiments, R2 is selected from matrix metalloproteinase (MMP)-cleavable linkers, a disintegrin and metalloprotease (ADAM)-domain-containing metalloprotease-cleavable linkers, prostate-specific antigen (PSA) protease-cleavable linkers, urokinase-type plasminogen activator (uPA) protease-cleavable linkers, membrane-type serine protease 1 (MT-SP1) protease-cleavable linkers, matriptase (ST14) protease-cleavable linkers, and legumain protease-cleavable linkers.

[0049] In some embodiments, R2 is a matrix metalloproteinase (MMP)-cleavable linker and comprises an amino acid sequence selected from any one of SEQ ID NOs: 21 - 35. In some embodiments, R2 is a prostate-specific antigen (PSA)-cleavable linker and comprises an amino acid sequence selected from any one of SEQ ID NOs: 14 - 16. In some embodiments, R2 is a urokinase-type plasminogen activator (uPA)-cleavable linker and comprises an amino acid sequence selected from any one of SEQ ID NOs: 11 - 13. In some embodiments, R2 is a legumain-cleavable linker and comprises an amino acid sequence selected from any one of SEQ ID NOs: 18 - 19 or AAN or Cbz-AAN-AMC. "Cbz" and "AMC" in Cbz-AAN-AMC represent benzyloxycarbonyl and 7-amino-4-methylcoumarin, respectively.

[0050] In some embodiments, the chemical moiety comprises the structure of formula (II):

[0051]

[0052] wherein R1, R3, and R4 are as defined above.

[0053] In some embodiments, R3 is

[0054] wherein,

[0055] X is selected from -NH- or -O-;

[0056] Y is selected from -NH-, -O-, -C(O)-, -OC(O)-, or -NHC(O)-;

[0057] each R x and R y is independently selected from H, halogen, C 1-4 alkyl, or C 1-4 haloalkyl;

[0058] n is 0, 1, 2, or 3;

[0059] Or,

[0060] R3 is

[0061] X is selected from NH or O;

[0062] Y is selected from -NH-, -O-, -C(O)-, -OC(O)-, or -NHC(O)-.

[0063] In some embodiments, the chemical moiety comprises the structure of formula (III):

[0064]

[0065] Wherein,

[0066] X is selected from NH or O;

[0067] Y is selected from -NH-, -O-, -C(O)-, -OC(O)- or -NHC(O)-;

[0068] wherein R1, R2 and R4 are as defined above.

[0069] In some embodiments, the chemical moiety comprises a structure of formula (IV):

[0070]

[0071] wherein R1 and R4 are as defined above.

[0072] In some embodiments,

[0073] R4 is

[0074] R a and R b are each independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl and C 1-6 alkoxy;

[0075] R 4a is absent, or is selected from -(CH2) 1-12 -, -(CH2) 1-12 -O-, -O-(CH2) 1-12 -, -O-(CH2) 1-12 -O-, -(CH2) 1-12 -O-(CH2) 1-12 -, (C 1-6 alkylene-O) p -(CH2) 1-12 -, -(CH2) 1-12 -C 3-8 cycloalkylene and -(CH2) 1-12 -C 3-8 cycloalkylene-(CH2) 1-12 ;

[0076] R 4a is optionally further substituted by 1, 2, 3 or 4 R# groups;

[0077] R 4bis -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)NH- or -NHC(O)-;

[0078] R# is selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl;

[0079] p is 1, 2, 3 or 4;

[0080] Or,

[0081] R a and R b are each independently selected from H and C 1-4 alkyl;

[0082] R 4a is selected from -(CH2) 1-12 -, -(CH2) 1-12 -O-, -O-(CH2) 1-12 -, -O-(CH2) 1-12 -O-, -(CH2) 1-12 -O-(CH2) 1-12 - and (C 1-4 alkylene -O) p -(CH2) 1-12 ;

[0083] R 4a is optionally further substituted by 1, 2 or 3 R# groups;

[0084] R 4b is -NH-, -O- or -C(O)-;

[0085] R# is selected from H, halogen, C 1-6 alkyl and C 1-6 haloalkyl;

[0086] p is 1, 2 or 3;

[0087] Or alternatively,

[0088] R a and R b are H;

[0089] R 4a is -(CH2) 2-12 -;

[0090] R 4b is -NH-;

[0091] R 4aOptionally further substituted by 1 or 2 R# groups;

[0092] R# is selected from H, halogen, and C 1-4 alkyl.

[0093] In some embodiments, the chemical moiety comprises a structure of formula (V):

[0094]

[0095] wherein R1, R2, and R3 are as defined above.

[0096] In some embodiments, the chemical moiety comprises a structure of formula (VI):

[0097]

[0098] wherein, PEG m is R 1c is selected from H, C 1-4 alkyl, and C 1-4 haloalkyl, and m is an integer ranging from 1 - 1000, 100 - 500, or 400 - 500.

[0099] In some embodiments, PEG m is a linear polyethylene glycol with a molecular weight of about 20000.

[0100] In some embodiments, IFNα2b or a functional fragment thereof is chemically modified at a residue corresponding to a residue selected from R12, L15, M16, R22, L26, F27, L30, R33, H34, D35, A145, M148, R149, S152, L153, and N156 of SEQ ID NO:1.

[0101] In some embodiments, IFNα2b or a functional fragment thereof comprises an amino acid substitution with cysteine at a position corresponding to a position selected from R12, L15, M16, R22, L26, F27, L30, R33, H34, D35, A145, M148, R149, S152, L153, and N156 of SEQ ID NO:1, and the chemical moiety is linked to IFNα2b or the functional fragment through cysteine.

[0102] In some embodiments, R4 of the chemical moiety is linked to the S atom of a cysteine residue through maleimide, acetylene, vinyl, monosubstituted maleic acid, or disubstituted maleimide.

[0103] In some embodiments, IFNα2b or a functional fragment thereof is conjugated to a biomolecule that specifically binds to a target. In some embodiments, IFNα2b or a functional fragment thereof is directly conjugated to the biomolecule. In some embodiments, IFNα2b or a functional fragment thereof is conjugated to the biomolecule via a linker.

[0104] In some embodiments, the biomolecule comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or an antigen-binding fragment thereof is selected from a full-length antibody, Fab, Fab’, F(ab’)2, Fd, Fd’, Fv, scFv, ds-scFv, sdAb, and nanobody.

[0105] In some embodiments, the target is PD-L1 or PD-1. In some embodiments, the biomolecule comprises an sdAb that comprises CDR1, CDR2, and CDR3 of SEQ ID NO:6, and CDR1, CDR2, and CDR3 are according to the Kabat numbering scheme. In some embodiments, the biomolecule comprises an sdAb that comprises:

[0106] (i) CDR1, which comprises the amino acid sequence of FRHYVMG (SEQ ID NO:3),

[0107] (ii) CDR2, which comprises the amino acid sequence of AISWSGSGSYYADSVKG (SEQ ID NO:4), and

[0108] (iii) CDR3, which comprises the amino acid sequence of DMTTRMSQASREYDY (SEQ ID NO:5).

[0109] In some embodiments, the biomolecule comprises an sdAb, and the sdAb comprises a sequence that has at least 80% identity to SEQ ID NO:6.

[0110] In some embodiments, the target is PD-L1 or PD-1. In some embodiments, the biomolecule comprises an sdAb that comprises CDR1, CDR2, and CDR3 of SEQ ID NO:6, and CDR1, CDR2, and CDR3 are according to the Kabat numbering scheme. In some embodiments, the biomolecule comprises an sdAb that comprises:

[0111] (i) CDR1, which comprises the amino acid sequence of SGTQFSDSKID (SEQ ID NO:50),

[0112] (ii) CDR2, which comprises the amino acid sequence of GIFQTGSTIYEDSVKG (SEQ ID NO:51), and

[0113] (iii) CDR3, which comprises the amino acid sequence of IGRGTLA (SEQ ID NO:52).

[0114] In some embodiments, the biomolecule comprises an sdAb, which comprises a sequence having at least 80% identity to SEQ ID NO:49.

[0115] In some embodiments, the biomolecule further comprises an Fc region.

[0116] In some embodiments, IFNα2b comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1 or SEQ ID NO:2.

[0117] In some embodiments, provided herein is also a multifunctional polypeptide, which comprises the IFNα2b or a functional fragment thereof of the present disclosure, and an antibody or an antigen-binding fragment thereof. In some embodiments, the multifunctional polypeptide comprises a single copy of IFNα2b or a functional fragment thereof. In some embodiments, the multifunctional polypeptide comprises an Fc fragment.

[0118] In some embodiments, IFNα2b or a functional fragment thereof is placed on the C-terminal side of the Fc fragment. In some embodiments, the antibody or an antigen-binding fragment thereof is placed on the N-terminal side of the Fc fragment. In some embodiments, the Fc fragment comprises a knobs-into-holes substitution compared to the corresponding wild-type Fc fragment.

[0119] In some embodiments, the antibody or an antigen-binding fragment thereof is specific for a tumor-associated antigen or an immune checkpoint protein. In some embodiments, the tumor-associated antigen is selected from EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, and tight junction protein 18.2.

[0120] In some embodiments, the immune checkpoint protein is selected from PD-1, PD-L1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOS-L, GITR, GITR-L, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR, and CD47. In some embodiments, the antibody or antigen-binding fragment thereof is an anti-PD-L1 or anti-PD-1 single domain antibody (sdAb).

[0121] In one embodiment, provided herein is also an immunoconjugate comprising the multifunctional polypeptide of the present disclosure, wherein the multifunctional polypeptide is conjugated to a chemical moiety.

[0122] In some embodiments, the chemical moiety comprises a structure of formula (VI):

[0123]

[0124] wherein PEG m is R 1c is selected from H, C 1-4 alkyl, and C 1-4 haloalkyl, and m is an integer ranging from 1 - 1000, 100 - 500, or 400 - 500; or, PEG m is a linear polyethylene glycol having a molecular weight of about 20,000.

[0125] In some embodiments, the immunoconjugate further comprises an Fc region.

[0126] In a third aspect, provided herein is a polynucleotide encoding the polypeptide of any one of the present application or the immunoconjugate of the present application.

[0127] In a fourth aspect, provided herein is a vector comprising the polynucleotide of the present application.

[0128] In a fifth aspect, provided herein is a host cell comprising the vector of the present application.

[0129] In a sixth aspect, provided herein is a pharmaceutical composition comprising the polypeptide, immunoconjugate, or polynucleotide of the present application.

[0130] In a seventh aspect, provided herein is a method for treating a disorder in a subject in need thereof, comprising administering to the subject an effective amount of the polypeptide, immunoconjugate, or nucleic acid of the present application. In some embodiments, the disorder is cancer.

[0131] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 Exemplary immunoconjugates of different forms of the present application are shown.

[0133] Figures 2A-2B Exemplary structures of the immunoconjugates of the present application (which contain IFNα2b modified by a chemical moiety), as well as SDS-PAGE of the immunoconjugates, are shown.

[0134] Figures 3A-3B The binding affinity of the immunoconjugates of the present application with PD-L1 is shown.

[0135] Figure 4 The activation of PD-1 / PD-L1 signaling by the immunoconjugates of the present application is shown.

[0136] Figures 5A-5B The binding affinity of the immunoconjugates of the present application with IFN receptor is shown.

[0137] Figures 6A-6B The activation of type I interferon signaling by the immunoconjugates of the present application is shown.

[0138] Figures 7A-7B A system for evaluating in vitro anti-tumor efficacy, as well as the in vitro tumor killing efficacy of the immunoconjugates of the present application, are shown.

[0139] Figure 8 The in vivo tumor suppression of the immunoconjugates of the present application is shown.

[0140] Figure 9 The toxicological analysis of the immunoconjugates of the present application is shown.

[0141] Figure 10 The structures of two bifunctional polypeptides are shown.

[0142] Figure 11 The results of activity tests of the bifunctional polypeptide and its corresponding immunoconjugate in terms of binding to the interferon receptor are shown.

[0143] Figure 12 The results of activity tests of the bifunctional polypeptide and its corresponding immunoconjugate in terms of inhibiting PD-L1 / PD-1 signal transduction are shown.

[0144] Figure 13Demonstrated the in vivo tumor suppressive activity of these bifunctional polypeptides and their corresponding immunoconjugates in the SCID RKO animal model. Detailed Description

[0145] Definitions

[0146] Before describing the embodiments in detail, it should be understood that the present disclosure is not limited to specific compositions or biological systems, which can of course vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0147] Unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and the like.

[0148] As used herein, the term "about" refers to the usual error range of the corresponding value that is readily known to those skilled in the art. Reference to a "about" value or parameter herein includes (and describes) embodiments that refer to the value or parameter itself.

[0149] It should be understood that the various aspects and embodiments of the present disclosure include "comprising" the various aspects and embodiments, "consisting of" the various aspects and embodiments, and "consisting essentially of" the various aspects and embodiments.

[0150] As used herein, the term "antibody" is used in the broadest sense and specifically encompasses intact antibodies (e.g., full-length antibodies), antibody fragments (including but not limited to Fab, F(ab’)2, scFv, scFv-Fc, single-domain antibodies (sdAb, also known as nanobodies), single-chain antibodies, and single-light-chain antibodies), monoclonal antibodies, and polyclonal antibodies, provided they exhibit the desired biological activity (e.g., epitope binding).

[0151] As used herein, the term "isolated" antibody may refer to an antibody that is substantially free of other cellular material. In one embodiment, an isolated antibody is substantially free of other proteins from the same species. In another embodiment, an isolated antibody is expressed by cells from a different species and is substantially free of other proteins from the different species. In some embodiments, an "isolated" antibody is one that has been identified and separated and / or recovered from the components of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteins or nonproteinaceous solutes. Isolation can be accomplished by using protein purification techniques well known in the art to render the antibody substantially free of natural associated components (or components associated with the cell expression system used to produce the antibody). In some embodiments, the antibody is purified (1) to greater than 75% by weight of antibody as determined by the Lowry method, most preferably greater than 80%, 90%, 95% or 99% by weight, or (2) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or preferably silver stain. Isolated antibodies include in situ antibodies within recombinant cells because at least one component of the antibody's natural environment will be absent. However, isolated antibodies are generally prepared by at least one purification step.

[0152] As used herein, the terms "native antibodies and immunoglobulins" generally refer to an approximately 150,000 dalton heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond (also referred to as a "VH / VL pair"), and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH), followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the variable domain of the light chain and the variable domain of the heavy chain. See, e.g., Chothia et al., J. Mol. Biol., 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A., 82:4592 (1985).

[0153] As used herein, the term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely between antibodies and that they are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable domain of the light chain and the variable domain of the heavy chain. The more highly conserved portions of the variable domain are called framework (FR). The variable domains of the native heavy and light chains each contain four FR regions, mostly adopting a β-sheet conformation connected by 3 CDRs, which form loops and in some cases form part of the β-sheet structure. The CDRs in each chain are held very close together by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991). The constant domains do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as the antibody's participation in antibody-dependent cytotoxicity. Variable region sequences of interest include the humanized variable region sequences of the CD47 antibodies described in detail elsewhere herein.

[0154] The terms "hypervariable region (HVR)" or "complementary determining region (CDR)" may refer to subregions of the VH and VL domains that are characterized by enhanced sequence variability and / or the formation of defined loops. These include three CDRs (H1, H2, and H3) in the VH domain and three CDRs (L1, L2, and L3) in the VL domain. H3 is believed to be crucial in conferring fine binding specificity, and L3 and H3 exhibit the highest levels of diversity. See Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).

[0155] The delineation of several CDRs / HVRs is known. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia is based on the position of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loops and is used by Oxford Molecular’s AbM antibody modeling software. The “contact” HVR is based on the analysis of available complex crystal structures. The residues from each of these HVRs / CDRs are indicated below. “Framework” or “FR” residues are those variable domain residues other than HVR / CDR residues.

[0156]

[0157] “Extended” HVRs are also known: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH (Kabat numbering).

[0158] “According to Kabat numbering” can refer to the numbering system for the heavy chain variable domain or the light chain variable domain used for antibody compilation in Kabat et al. supra. The actual linear amino acid sequence may contain deletions or insertions of fewer or additional amino acids corresponding to the FRs or HVRs of the variable domain. For a given antibody, the Kabat numbering of residues can be determined by aligning the antibody sequence with the homologous regions of the “standard” Kabat numbering sequence. Generally, Kabat numbering is used when referring to residues of the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), while the EU numbering system or index (e.g., the EU index as in Kabat, numbered according to EU IgG1) is generally used when referring to residues in the heavy chain constant region.

[0159] As used herein, the term "antibody fragment" and all its grammatical variants are defined as a part of a whole antibody that contains the antigen-binding site or variable region of the whole antibody, which, in some cases, does not contain the constant heavy-chain domains of the Fc region of the whole antibody (i.e., CH2, CH3, and / or CH4, depending on the antibody isotype). Examples of antibody fragments include Fab, Fab’, Fab’-SH, F(ab’)2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of an uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including but not limited to (1) single-chain Fv (scFv) molecules, (2) single-chain polypeptides containing only one light-chain variable domain or a fragment thereof containing the three CDRs of the light-chain variable domain, without an associated heavy-chain portion, and (3) single-chain polypeptides containing only one heavy-chain variable region or a fragment thereof containing the three CDRs of the heavy-chain variable region, without an associated light-chain portion. In antibody fragments containing one or more heavy chains, the heavy chain may contain any constant domain sequence found in the non-Fc region of the whole antibody (e.g., CH1 in the IgG isotype), and / or may contain any hinge region sequence found in the whole antibody, and / or may contain a leucine zipper sequence fused to the hinge region sequence or constant domain sequence of the heavy chain or a leucine zipper sequence located in the hinge region sequence or constant domain sequence of the heavy chain.

[0160] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab’ fragment differs from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy-chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab’-SH is the name given herein to a Fab’ in which the cysteine residue of the constant domain bears a free sulfhydryl group. The F(ab’)2 antibody fragment was originally generated as a pair of Fab’ fragments with an intervening hinge cysteine. Other chemical conjugations of antibody fragments are also known.

[0161] Single-domain antibodies (SdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. Like whole antibodies, it is capable of selectively binding a specific antigen. The molecular weight of a single-domain antibody is only 12 - 15 kDa, which is much smaller than common antibodies consisting of two heavy-chain proteins and two light chains (150 - 160 kDa), and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from the light chain and one from the heavy chain). The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids; these are called V H H fragments.

[0162] As used herein, the term "specifically binds" means binding that is selective for an antigen / receptor and can be distinguished from unwanted or non-specific interactions. The ability of a binding pair can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques known in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIACORE instrument) and other binding assays. In some embodiments, the degree of binding of an antibody / cytokine to an unrelated protein is less than about 10% of its degree of binding to an antigen / receptor, e.g., as measured by SPR. In some embodiments, the immunoglobulin / cytokine that binds to an antigen / receptor has a dissociation constant (K -8 ) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or lower, e.g., 10 -13 M - 10 -9 M, e.g., 10 -13 M - 10 D M).

[0163] As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or cytokine) and its binding partner (e.g., an antigen or receptor). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between binding pair members (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (k off and k on ). Thus, equivalent affinities can involve different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein.

[0164] "Reduced binding", e.g., reduced binding to the IFN receptor, means a decrease in the affinity of the corresponding interaction, e.g., as measured by SPR or ELISA. For clarity, the term also includes a decrease in affinity to zero (or below the limit of detection of the assay method), i.e., complete elimination of the interaction. In contrast, "increased binding" means an increase in the binding affinity of the corresponding interaction.

[0165] As used herein, the term "treatment" refers to a clinical intervention during a clinical pathologic process that is intended to alter the natural course of an individual or cell being treated. Desirable effects of treatment include slowing the rate of disease progression, ameliorating or alleviating the disease state, and relieving or improving the prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a disease, including but not limited to reducing (or destroying) the proliferation of cancer cells, reducing symptoms caused by the disease, improving the quality of life of a patient with the disease, reducing the dosage of other medications required to treat the disease, and / or prolonging the survival of the individual. In some embodiments, "treating" a disease such as cancer refers to delaying the progression of the disease, i.e., postponing, hindering, slowing, arresting, stabilizing, and / or retarding the development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay can in fact encompass prevention, i.e., the individual does not develop the disease. For example, the development of advanced cancer, such as metastasis, can be delayed.

[0166] As used herein, the term "percent amino acid sequence identity" with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific sequence after the sequences are aligned and gaps are introduced, if necessary, to achieve maximum percent sequence identity, and no conservative substitutions are considered part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved in various ways known in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared.

[0167] As used herein, the term "substitution" or "substituted amino acid" as used herein refers to the replacement of an original amino acid residue with one or more other amino acid residues.

[0168] "Effective amount" means the minimum amount required to produce a measurable improvement or preventive effect against a specific disease (such as cancer). The effective amount herein can vary depending on factors such as the patient's disease state, age, gender, and weight, as well as the ability of the therapeutic agent (or combination of therapeutic agents) to elicit a desired response in an individual. The effective amount is also the amount where the therapeutic beneficial effects exceed any toxic or detrimental effects of the treatment. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as the alleviation of one or more symptoms caused by the disease, improvement in the quality of life of a patient suffering from the disease, reduction in the dosage of other medications required to treat the disease, enhancement of the effect of additional medications (such as by targeting), delay in the progression of the disease, and / or prolongation of survival. In the case of cancer or a tumor, an effective amount of a drug can have the following effects: reduction in the number of cancer cells; reduction in tumor size; inhibition (i.e., slowing down to some extent or ideally stopping) of cancer cell invasion of surrounding organs; inhibition (i.e., slowing down to some extent or ideally stopping) of tumor metastasis; inhibition of tumor growth to some extent; and / or alleviation of one or more symptoms associated with the condition to some extent. The effective amount can be administered in one or more doses. For the purposes of this disclosure, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly effect a therapeutic treatment. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with one or more other drugs, compounds, or pharmaceutical compositions. Thus, "effective amount" can be considered in the context of administering one or more therapeutic agents, and a single substance can be considered to be administered in an effective amount if, when combined with one or more other substances, the desired outcome is achieved or accomplished.

[0169] As used herein, the term "drug antibody ratio" or "DAR" refers to the number of chemical moieties conjugated to a polypeptide of the present disclosure. For example, when the DAR is 1 (DAR1), on average, a single chemical moiety is conjugated to one chain of a bifunctional polypeptide of the present disclosure. Similarly, when the DAR is 2 (DAR2), on average, two chemical moieties are conjugated to the bifunctional polypeptide of the present disclosure. The DAR can range from 1 - 4, although higher loadings, such as 10, are also possible, depending on the number of conjugation sites in the polypeptide.

[0170] As used herein, for therapeutic purposes, the term "subject" refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo animals, sports animals, or pet animals such as dogs.

[0171] Interferons and attenuated interferons

[0172] Interferons (IFNs) belong to a large class of proteins called cytokines, which are molecules used to trigger protective defenses of the immune system through intercellular communication. IFNs are named for their ability to "interfere" with viral replication by protecting cells from viral infection. They also have a variety of other functions, including activating immune cells such as natural killer cells and macrophages, and increasing host defenses by upregulating antigen presentation through increased expression of major histocompatibility complex (MHC) antigens.

[0173] More than twenty different IFN genes and proteins have been identified in animals (including humans). They are generally divided into three classes: type I IFNs, type II IFNs, and type III IFNs. Human IFNα2b belongs to type I IFN and is clustered on chromosome 9p. It transmits signals through the heterodimeric complex of interferon α / β receptor 1 (IFNAR1) and interferon α / β receptor 2 (IFNAR2) to initiate the activation of TYK2 and JAK1, thereby triggering a cascade of immune-related interferon-stimulated genes (ISGs), which is crucial for immune activation.

[0174] Several different types of interferon therapies have been approved for humans. For example, in January 2001, the US Food and Drug Administration (FDA) approved Pegintron, a PEGylated interferon α2b. Subsequently, in October 2002, Pegasys was also approved, a PEGylated interferon α2a. PEGylated drugs can extend the in vivo half-life of interferon and can be injected once a week, rather than two or three times a week as required for conventional interferon α. When used in combination with the antiviral drug ribavirin, PEGylated interferon effectively treats hepatitis C. Interferon therapies have been developed and used in combination with chemotherapy and radiotherapy as treatments for some cancers, including hematological malignancies such as leukemia and lymphoma, chronic myeloid leukemia, nodular lymphoma, and cutaneous T-cell lymphoma, as well as melanoma. However, patients receiving this therapy usually suffer from adverse reactions. Therefore, there is a clinical need for modified IFNα2b with reduced adverse reactions and improved efficacy.

[0175] In one aspect, the present disclosure provides an IFNα2b or a functional fragment thereof that is modified to attenuate its activity. As shown in the experimental examples, this attenuated IFNα2b or its functional fragment not only significantly improves safety but also shows excellent therapeutic effects. In fact, when used in a bifunctional polypeptide that also contains a PD-L1 moiety, the resulting molecule (e.g., 112_08-IFM-knob) in combination with PEGylated interferon α (Tecentriq + PEG-IFNα) shows superior in vivo anti-tumor activity compared to Tecentriq (see, for example Figure 12 ).

[0176] Thus, in one embodiment of the present invention, provided herein is a modified IFNα2b (compared to wild-type IFNα2b protein, such as human IFNα2b protein) or a functional fragment thereof, which has a reduced binding affinity for the interferon α / β receptor. In some embodiments, the interferon α / β receptor is selected from IFNAR1, IFNAR2, and combinations thereof. In some embodiments, IFNα2b or a functional fragment thereof is modified to attenuate the binding affinity of IFNα2b or a functional fragment thereof for IFNAR1. In some embodiments, IFNα2b or a functional fragment thereof is modified to attenuate the binding affinity of IFNα2b or a functional fragment thereof for IFNAR2. In some embodiments, IFNα2b or a functional fragment thereof is modified to attenuate the binding affinity of IFNα2b or a functional fragment thereof for both IFNAR1 and IFNAR2.

[0177] In some embodiments, IFNα2b or a functional fragment thereof is attenuated such that its affinity for the interferon α / β receptor is attenuated by at least about 10-fold, at least about 10 2 -fold, at least about 10 3 -fold, at least about 10 4 -fold, or at least about 10 5 -fold. In some embodiments, the binding affinity of IFNα2b or a functional fragment thereof for IFNAR1 is reduced by at least about 10-fold, at least about 10 2 -fold, at least about 10 3 -fold, at least about 10 4 -fold, or at least about 10 5 -fold. In some embodiments, IFNα2b or a functional fragment thereof is modified to attenuate the binding affinity of IFNα2b or a functional fragment thereof for IFNAR2 by at least about 10-fold, at least about 10 2 -fold, at least about 10 3 -fold, at least about 10 4 -fold, or at least about 10 5 -fold. In some embodiments, IFNα2b or a functional fragment thereof is modified to attenuate the binding affinity of IFNα2b or a functional fragment thereof for IFNAR2 by at least about 10-fold. In some embodiments, IFNα2b or a functional fragment thereof is modified to attenuate the binding affinity of IFNα2b or a functional fragment thereof for IFNAR2 by at least about 10 2 -fold. In some embodiments, IFNα2b or a functional fragment thereof is modified to attenuate the binding affinity of IFNα2b or a functional fragment thereof for IFNAR2 by at least about 10 3-fold. In some embodiments, IFNα2b or a functional fragment thereof is modified to reduce the binding affinity of IFNα2b or a functional fragment thereof for both IFNAR1 and IFNAR2 by at least about 10-fold, at least about 10 2 -fold, at least about 10 3 -fold, at least about 10 4 -fold, or at least about 10 5 -fold.

[0178] In some embodiments, the binding affinity of IFNα2b or a functional fragment thereof for the interferon α / β receptor is reduced to reduce the adverse effects of IFNα2b or a functional fragment thereof in a subject in need thereof after administration. In some embodiments, the adverse effects include acute toxicity, subacute side effects, and chronic side effects. In some embodiments, the acute toxicity includes flu-like symptoms such as fever, chills, myalgia, headache, and nausea. In some embodiments, the subacute side effects include, but are not limited to, hematological side effects such as anemia, reduction in white blood cell and platelet counts, and reduced platelet aggregation; hepatic side effects such as elevated transaminases and inhibition of cytochrome P450 enzymes; gastrointestinal side effects such as anorexia, nausea, vomiting, and diarrhea; mental side effects such as depression, cognitive impairment, and delirium; neurological side effects such as seizures, neuropathy, multiple sclerosis-like diseases, and myasthenia gravis; renal side effects such as proteinuria; cardiovascular side effects such as arrhythmia, ischemic heart disease, cardiomyopathy, and retinal abnormalities; pulmonary side effects such as pneumonia; endocrine side effects such as thyroid diseases, diabetes, decreased sex hormone levels, and hypopituitarism; skin side effects such as hair loss, erythema, induration at the injection site, rash, pruritus, vitiligo, lichen planus, and psoriasis.

[0179] In some embodiments, the side effects include autoimmune diseases such as thyroid diseases, liver dysfunction, connective tissue diseases, skin diseases, hematological diseases, neurological diseases, pulmonary diseases, and metabolic diseases. In some embodiments, the side effects result in conditions including, but not limited to, subacute lymphocytic thyroiditis, Graves' disease, permanent hypothyroidism, autoimmune hepatitis, primary biliary cirrhosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, leukocytoclastic vasculitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, inflammatory demyelinating polyneuropathy, multiple sclerosis-like diseases, axonal neuropathy, and hearing loss, Bell palsy, multiple mononeuropathy, myasthenia gravis, abducent nerve paralysis, interstitial pneumonia, and diabetes.

[0180] In another aspect, the present disclosure provides a polypeptide that comprises IFNα2b or a functional fragment thereof, wherein the IFNα2b or the functional fragment thereof is modified to attenuate the activation of type I interferon signaling induced by the IFNα2b or the functional fragment thereof. In some embodiments, the activation of IFNα signaling is attenuated by at least 10-fold, 10 2 -fold, 10 3 -fold, 10 4 -fold or 10 5 -fold. In some embodiments, the activation of IFNα signaling is attenuated by at least 10 2 -fold. In some embodiments, the activation of IFNα signaling is attenuated by at least 10 3 -fold. The activation of IFNα signaling is attenuated by at least 10 4 -fold.

[0181] In some embodiments, the activation of type I interferon signaling is attenuated to reduce the adverse effects of IFNα2b or a functional fragment thereof following administration to a subject in need thereof. In some embodiments, the adverse effects include acute toxicity, subacute side effects, and chronic side effects. In some embodiments, the acute toxicity includes flu-like symptoms such as fever, chills, myalgia, headache, and nausea. In some embodiments, the subacute side effects include, but are not limited to, hematological side effects such as anemia, decreases in white blood cell and platelet counts, and decreased platelet aggregation; liver side effects such as elevated transaminases and inhibition of cytochrome P450 enzymes; gastrointestinal side effects such as anorexia, nausea, vomiting, and diarrhea; mental side effects such as depression, cognitive impairment, and delirium; neurological side effects such as seizures, neuropathy, multiple sclerosis-like diseases, and myasthenia gravis; renal side effects such as proteinuria; cardiovascular side effects such as arrhythmia, ischemic heart disease, cardiomyopathy, and retinal abnormalities; pulmonary side effects such as pneumonia; endocrine side effects such as thyroid diseases, diabetes, decreased sex hormone levels, and hypopituitarism; skin side effects such as hair loss, erythema, induration at the injection site, rash, pruritus, vitiligo, lichen planus, and psoriasis.

[0182] In some embodiments, side effects include autoimmune diseases such as thyroid diseases, liver dysfunction, connective tissue diseases, skin diseases, hematological diseases, neurological diseases, pulmonary diseases, and metabolic diseases. In some embodiments, the side effects result in conditions including but not limited to subacute lymphocytic thyroiditis, Graves' disease, permanent hypothyroidism, autoimmune hepatitis, primary biliary cirrhosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, leukocytoclastic vasculitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, inflammatory demyelinating polyneuropathy, multiple sclerosis-like diseases, axonal neuropathy, and hearing loss, Bell palsy, multiple mononeuropathy, myasthenia gravis, abducent nerve paralysis, interstitial pneumonia, and diabetes.

[0183] In some embodiments, IFNα2b or a functional fragment thereof is modified to improve the properties of the polypeptide following administration to a subject in need thereof. In some embodiments, the properties include but are not limited to pharmacokinetics (PK), pharmacodynamics (PD), potency, and safety. In some embodiments, potency includes anti-tumor potency. In some embodiments, anti-tumor potency includes but is not limited to inhibiting tumor growth, reducing tumor volume, increasing the survival of the subject, and inducing protection against tumor recurrence.

[0184] In some embodiments, the modification includes introducing amino acid mutations into wild-type IFNα2b (SEQ ID NO:1). As used herein, the term "amino acid mutation" is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be made to obtain the polypeptides of the present application, provided that the polypeptide has the desired characteristics, e.g., reduced binding to IFNAR1 and / or IFNAR2, or reduced activation of type I interferon signaling.

[0185] In some embodiments, the amino acid mutation is an amino acid substitution. In some embodiments, the polypeptides of the present application are obtained by replacing one amino acid with another amino acid having different structural and / or chemical properties. In some embodiments, the amino acid substitution includes replacing a hydrophobic amino acid with a hydrophilic amino acid. In some embodiments, the amino acid substitution includes replacement with a non-naturally occurring amino acid or a naturally occurring amino acid derivative of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). The standard amino acids, their abbreviations, and their properties are listed in Table 1 below.

[0186] Table 1. Standard Amino Acids

[0187] Amino acid 3-letter code 1-letter code Property Alanine Ala A Hydrophobic Arginine Arg R Basic, charged Asparagine Asn N Neutral-polar Aspartic acid Asp D Acidic, charged Cysteine Cys C Neutral-polar Glutamine Gln Q Neutral-polar Glycine Gly G Hydrophobic Glutamic acid Glu E Acidic, charged Histidine His H Basic Isoleucine Ile I Hydrophobic Leucine Leu L Hydrophobic Lysine Lys K Basic, charged Methionine Met M Neutral-polar Phenylalanine Phe F Hydrophobic Proline Pro P Conformational, cyclic Serine Ser S Neutral-polar Threonine Thr T Neutral-polar Tryptophan Trp W Aromatic Tyrosine Tyr Y Aromatic Valine Val V Aliphatic, hydrophobic

[0188] It is expected that amino acid residues at the binding interface between IFNα2b and its binding target play important roles in binding. Thus, substituting one or more such residues with non-conservative amino acids can weaken the binding affinity or biological activity of IFNα2b. The amino acid residues at the binding interface of IFNα2b are known. For example, Thomas et al., “Structural linkage between ligand discrimination and receptor activation by type I interferons,” Cell (2011) 146(4):621-32 reported that residues R12, L15, M16, R22, L26, F27, L30, R33, H34, D35, A145, M148, R149, S152, L153, and N156 are located at the binding interface.

[0189] In some embodiments, the IFNα2b or functional fragment thereof of the present disclosure includes amino acid substitutions at one or more of the residues R12, L15, M16, R22, L26, F27, L30, R33, H34, D35, A145, M148, R149, S152, L153, and N156 (positions according to SEQ ID NO:1). In some embodiments, the substitution is a non-conservative substitution. In some embodiments, the substitution is a substitution with cysteine (C), which has the additional benefit of serving as a conjugation site.

[0190] In some embodiments, it is expected that substitutions at residues C29, D35, R144, or E146 of SEQ ID NO:1 can achieve the same purpose. Thus, in some embodiments, IFNα2b or its functional fragment is modified by amino acid substitution at C29, D35, R144, or E146 of SEQ ID NO:1. In some embodiments, the substitution is a non-conservative substitution.

[0191] In some embodiments, IFNα2b or its functional fragment is modified at the residue corresponding to S152 of SEQ ID NO:1. In some embodiments, IFNα2b or its functional fragment is modified by amino acid substitution at S152 of SEQ ID NO:1. In some embodiments, the modification includes amino acid substitution with cysteine (C) at S152 of SEQ ID NO:1.

[0192] In some embodiments, IFNα2b or a fragment thereof comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the sequence of SEQ ID NO:1. In some embodiments, IFNα2b comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, IFNα2b comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the sequence of SEQ ID NO:2. In some embodiments, IFNα2b comprises the amino acid sequence of SEQ ID NO:2.

[0193] In some embodiments, IFNα2b or a fragment thereof comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the sequence of SEQ ID NO:1 while retaining any one or more of the substitutions disclosed herein. For example, amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the sequence of SEQ ID NO:1 include one or more substitutions at R12, L15, M16, R22, L26, F27, L30, R33, H34, D35, A145, M148, R149, S152, L153 and / or N156 of SEQ ID NO:1. In some embodiments, the substitution is a substitution with a cysteine (C) residue. In some embodiments, the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the sequence of SEQ ID NO:1 comprises a cysteine (C) residue at residue 152.

[0194] Multifunctional polypeptide

[0195] It is demonstrated herein that modified IFNα2b or a functional fragment thereof can be suitably incorporated into bifunctional or multifunctional polypeptides comprising one or more biological moieties such as antibodies and cytokines.

[0196] In one specific embodiment, the multifunctional polypeptide comprises one or more IFNα2b or functional fragments thereof, and one or more antibodies. In some embodiments, the multifunctional polypeptide further comprises an Fc fragment, such as a human IgG1, IgG2, IgG3 or IgG4 Fc (CH2 and CH3). Multiple forms of these multifunctional polypeptides have been tested, such as Figure 1 and 10 as shown.

[0197] In one embodiment, the multifunctional polypeptide is symmetric and comprises two identical polypeptide chains. Each chain comprises an antibody or antigen-binding fragment, an Fc portion, and IFNα2b or a functional fragment thereof. In some embodiments, in each peptide chain, the antibody / fragment and IFNα2b or a functional fragment thereof are both located on the N-terminal side of the Fc fragment (Form 1). In some embodiments, the antibody / fragment is located on the N-terminal side of the Fc fragment, while IFNα2b or a functional fragment thereof is located on the C-terminal side of the Fc fragment (Form "L1IF").

[0198] In one embodiment, the multifunctional polypeptide is asymmetric and comprises two polypeptide chains having different sequences. In some aspects of this embodiment, the multifunctional polypeptide comprises only one copy of IFNα2b or a functional fragment thereof. In other words, only one peptide chain comprises IFNα2b or a functional fragment thereof, while the other peptide chain does not. In some of these asymmetric forms, e.g., Forms 2 and 3, the single copy of IFNα2b or a functional fragment thereof is located on the C-terminal side of the Fc fragment of one of the two chains, while each chain also contains an antibody / fragment at the N-terminus. A pestle and mortar substitution can be performed to improve the correct pairing of these asymmetric peptides.

[0199] In Forms 3-5, the single copy of IFNα2b or a functional fragment thereof is placed on the N-terminal side of the Fc fragment, between one of the antibody / fragments and the Fc (Form 6), or on the most N-terminal side of both (Form 5), or on the N-terminal side of the Fc (Form 4).

[0200] The symmetric Form "L1IF" has proven to be an excellent form, exhibiting strong in vitro and in vivo activity and having an excellent safety profile (Examples 2-9). Forms 1 and 2 (asymmetric with a single IFNα2b or fragment on the C-terminal side of the Fc) exhibit even stronger anti-tumor activity (see, for example Figure 13 ), including the polypeptide alone, or the polypeptide further having a conjugated chemical moiety.

[0201] Thus, according to one embodiment of the present disclosure, there is provided a multifunctional polypeptide herein that comprises IFNα2b or a functional fragment thereof of the present disclosure and an antibody or an antigen-binding fragment thereof. In some embodiments, the multifunctional polypeptide comprises two or more IFNα2b or functional fragments thereof. In a preferred embodiment, the multifunctional polypeptide comprises a single copy of IFNα2b or a functional fragment thereof.

[0202] In some embodiments, the multifunctional polypeptide comprises an Fc fragment. In some embodiments, IFNα2b or a functional fragment thereof is placed on the C-terminal side of the Fc fragment. In some embodiments, the antibody or an antigen-binding fragment thereof is placed on the N-terminal side of the Fc fragment.

[0203] In some embodiments, the Fc fragment comprises a knobs-into-holes substitution compared to the corresponding wild-type Fc fragment. Exemplary substitutions for implementing the knobs and holes include S354C:T366W (EU numbering) in CH3 to form the knob and Y349C:T366S:Y407V (EU numbering) to form the hole.

[0204] In some embodiments, the antibody or its antigen-binding fragment is specific for a tumor-associated antigen or an immune checkpoint protein. Non-limiting examples of tumor-associated antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, and claudin 18.2.

[0205] Non-limiting examples of immune checkpoint proteins include PD-1, PD-L1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOS-L, GITR, GITR-L, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR, and CD47. In a specific embodiment, the immune checkpoint protein is PD-L1.

[0206] PD-L1 / PD-1 antibody

[0207] Programmed death receptor-1 (PD-1) is a cell surface receptor that acts as a T cell checkpoint and plays a central role in regulating T cell exhaustion. The binding of PD-1 to its ligand (programmed death ligand 1 (PD-L1)) activates downstream signaling pathways and inhibits T cell activation. In addition, the abnormally high expression of PD-L1 on tumor cells and antigen-presenting cells in the tumor microenvironment mediates tumor immune escape. Immunotherapies targeting the PD-1 / PD-L1 signaling pathway have shown unprecedented success in a variety of human cancers.

[0208] As used herein, the term "PD-1 / PD-L1 antibody" refers to any antibody that blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T cells, B cells, or NKT cells), and preferably also blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells. Other names or synonyms for PD-1 and its ligands include: for PD-1, PDCD1, PD1, CD279, and SLB2; for PD-L1, PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H; and for PD-L2, PDCD1L2, PDL2, B7-DC, and CD273. PD-1 / PD-L1 antibodies useful in the present application include, but are not limited to, monoclonal antibodies (mAbs) or antigen-binding fragments thereof that specifically bind to PD-1 or PD-L1, and preferably specifically bind to human PD-1 or human PD-L1. In some embodiments, the antibodies include, but are not limited to, polyclonal antibodies, bispecific antibodies, and multispecific antibodies. In some embodiments, the antigen-binding fragments include, but are not limited to, Fab, scFv, diabodies, triabodies, minibodies, VHH, sdAb, and nanobodies.

[0209] Any suitable "PD-1 antibody" known in the art can be used in the present application. In some embodiments, PD-1 antibodies useful in the present application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, Fabs, scFvs, diabodies, triabodies, minibodies, VHHs, sdAbs, and nanobodies. Exemplary PD-1 antibodies include, but are not limited to, pidilizumab, cemiplimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, toripalimab, dostarlimab, ABVV-181, penpulimab, pembrolizumab, genolimumab, retifanlimab, sasanlimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, nivolumab, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLA-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLA-20, SSI-361, APL-501, TJ0141H, SA-01, and antigen-binding fragments thereof.

[0210] Any suitable "PD-L1 antibody" known in the art can be used in the present application. In some embodiments, PD-L1 antibodies that can be used in the present application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, Fab, scFv, diabodies, triabodies, minibodies, VHH, sdAb, and nanobodies. Exemplary anti-PD-L1 antibodies include, but are not limited to, manelimab, atezolizumab, avelumab, cosibelimab, durvalumab, envafolimab, socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12, KY-1003, and antigen-binding fragments thereof.

[0211] In some embodiments, the "PD-L1" antibody used in the present application is a single-domain antibody (sdAb, also referred to herein as a "nanobody"). In some embodiments, the sdAb has binding specificity for the human PD-L1 protein and comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3. In some embodiments, the sdAb comprises the CDR1, CDR2, and CDR3 of SEQ ID NO:6. In some embodiments, the sdAb comprises the CDR1, CDR2, and CDR3 of SEQ ID NO:6, and the CDR1, CDR2, and CDR3 are according to the Kabat numbering scheme. In some embodiments, the sdAb comprises the CDR1, CDR2, and CDR3 of SEQ ID NO:6, and the CDR1, CDR2, and CDR3 are according to the IMGT numbering scheme. In some embodiments, the sdAb comprises the CDR1, CDR2, and CDR3 of SEQ ID NO:6, and the CDR1, CDR2, and CDR3 are according to the Chothia numbering scheme.

[0212] In some embodiments of the PD-L1 antibody of the present application, the PD-L1 antibody comprises: (1) CDR1, which comprises the amino acid sequence of FRHYVMG (SEQ ID NO:3) or an amino acid sequence having one or more substitutions as compared to SEQ ID NO:3; (2) CDR2, which comprises the amino acid sequence of AISWSGSGSYYADSVKG (SEQ ID NO:4) or an amino acid sequence having one or more substitutions as compared to SEQ ID NO:4; and (3) CDR3, which comprises the amino acid sequence of DMTTRMSQASREYDY (SEQ ID NO:5) or an amino acid sequence having one or more substitutions as compared to SEQ ID NO:5.

[0213] In some embodiments of the PD-L1 antibody of the present application, the PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6.

[0214] In some embodiments, the sdAb has binding specificity for the human PD-L1 protein and comprises complementarity determining region 1 (CDR1), CDR2, and CDR3. In some embodiments, the sdAb comprises the CDR1, CDR2, and CDR3 of SEQ ID NO:49. In some embodiments, the sdAb comprises the CDR1, CDR2, and CDR3 of SEQ ID NO:49, and the CDR1, CDR2, and CDR3 are according to the Kabat numbering scheme. In some embodiments, the sdAb comprises the CDR1, CDR2, and CDR3 of SEQ ID NO:49, and the CDR1, CDR2, and CDR3 are according to the IMGT numbering scheme. In some embodiments, the sdAb comprises the CDR1, CDR2, and CDR3 of SEQ ID NO:49, and the CDR1, CDR2, and CDR3 are according to the Chothia numbering scheme.

[0215] In some embodiments of the PD-L1 antibody of the present application, the PD-L1 antibody comprises: (1) CDR1, which comprises the amino acid sequence of SGTQFSDSKID (SEQ ID NO: 50) or an amino acid sequence having one or more substitutions compared to SEQ ID NO: 50; (2) CDR2, which comprises the amino acid sequence of GIFQTGSTIYEDSVKG (SEQ ID NO: 51) or an amino acid sequence having one or more substitutions compared to SEQ ID NO: 51; and (3) CDR3, which comprises the amino acid sequence of IGRGTLA (SEQ ID NO: 52) or an amino acid sequence having one or more substitutions compared to SEQ ID NO: 52.

[0216] In some embodiments of the PD-L1 antibody of the present application, the PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 49, or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 49.

[0217] Chemically modified with a chemical moiety

[0218] In some embodiments of the polypeptide of the present application, IFNα2b or a functional fragment is chemically modified with a chemical moiety. In some embodiments, the chemical moiety comprises a structure of formula (I): R1-R2-R3-R4 (I), wherein R1 is Optionally further substituted with 1, 2 or 3 R* groups; L1 is selected from CH or N; R 1a Is absent, or is selected from -(CH2) 0-3 -C(O)-, -(CH2) 0-3 -OC(O)- and -(CH2) 0-3 -NHC(O)-; each R 1b Independently absent, or independently selected from -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)NH-, -NH-C 1-6 Alkyl-, -O-C 1-6 Alkyl-, -C(O)-C 1-6 Alkyl-, -OC(O)-C 1-6 Alkyl-, -C(O)O-C 1-6 Alkyl- and -C(O)NH-C 1-6 Alkyl-; each R 1c Independently selected from H, C 1-6 Alkyl and C 1-6 Haloalkyl; m is an integer ranging from 1 to 30000; R* is selected from H, halogen, C1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 2-6 alkenyl or C 2-6 alkynyl; R2 is a linker cleavable in the tumor environment; R3 is X is selected from -NH- or -O-; Y is selected from -NH-, -O-, -C(O)-, -OC(O)-, -C(O)-C(O)- or -NHC(O)-; each R x and R y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl; n is 0, 1, 2, 3, 4 or 5; R4 is R a and R b are each independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl and C 1-6 alkoxy; R 4a is absent, or is selected from -(CH2) 1-12 -, -(CH2) 1-12 -O-, -O-(CH2) 1-12 -, -O-(CH2) 1-12 -O-, -(CH2) 1-12 -O-(CH2) 1-12 -, (C 1-6 alkylene -O) p -(CH2) 1-12 、-(CH2) 1-12 -C 3-8 cycloalkylene-, -(CH2) 1-12 -C 3-8 cycloalkylene-(CH2) 1-12 、-(CH2) 1-12 -C 6-10 arylene- and -(CH2) 1-12 -C 6-10 arylene-(CH2) 1-12 ; R 4a is optionally further substituted by 1, 2, 3, 4 or 5 R# groups; R 4b is selected from -NH-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-C(O)-, -C(O)NH- or -NHC(O)-, provided that it is chemically feasible; R# is selected from H, halogen, C 1-6 alkyl, C 1-6Halogenoalkyl, C 1-6 Alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl; p is 1, 2, 3, 4 or 5; wherein R3 is connected to R2 through X of R3 and to R4 through Y of R3.

[0219] R1

[0220] In one specific embodiment, R1 is Such as Optionally substituted; in another specific embodiment, R1 is In another specific embodiment, R1 is In another specific embodiment, R1 is In another specific embodiment, R1 is PEG m , which is linear polyethylene glycol with a molecular weight of about 20,000.

[0221] In specific embodiments, R1 is not further substituted by R* groups; in specific embodiments, R1 is further substituted by 1 R* group; in specific embodiments, R1 is further substituted by 2 R* groups; in specific embodiments, R1 is further substituted by 3 R* groups.

[0222] L1

[0223] In one specific embodiment, L1 is CH; in another specific embodiment, L1 is N.

[0224] R 1a

[0225] In one specific embodiment, R 1a Is absent; in another specific embodiment, R 1a Is -(CH2) 0-3 -C(O)-, such as -C(O)-; in another specific embodiment, R 1a Is -(CH2) 0-3 -OC(O)-, such as -OC(O)-; in another specific embodiment, R 1a Is -(CH2) 0-3 -NHC(O)-, such as -NHC(O)-; as long as chemically feasible.

[0226] R 1b

[0227] In one specific embodiment, R 1b Is absent; in another specific embodiment, R 1b Is NH-; in another specific embodiment, R 1bis -O-; in another specific embodiment, R 1b is -C(O)-; in another specific embodiment, R 1b is -OC(O)-; in another specific embodiment, R 1b is -C(O)O-; in another specific embodiment, R 1b is -C(O)NH-; in another specific embodiment, R 1b is -NH-C 1-6 alkyl-, preferably -NH-C 1-4 alkyl-; in another specific embodiment, R 1b is -O-C 1-6 alkyl-, preferably -O-C 1-4 alkyl-; in another specific embodiment, R 1b is -C(O)-C 1-6 alkyl-, preferably -C(O)-C 1-4 alkyl-; in another specific embodiment, R 1b is -OC(O)-C 1-6 alkyl-, preferably -OC(O)-C 1-4 alkyl-; in another specific embodiment, R 1b is -C(O)O-C 1-6 alkyl-, preferably -C(O)O-C 1-4 alkyl-; in another specific embodiment, R 1b is -C(O)NH-C 1-6 alkyl-, preferably -C(O)NH-C 1-4 alkyl-.

[0228] R 1c

[0229] In one specific embodiment, R 1c is H; in another specific embodiment, R 1c is C 1-6 alkyl, preferably C 1-4 alkyl; in another specific embodiment, R 1c is C 1-6 haloalkyl, preferably C 1-4 haloalkyl.

[0230] m

[0231] In one specific embodiment, m is an integer ranging from 1 to 30000; in another specific embodiment, m is independently an integer ranging from 1 to 5000; in another specific embodiment, m is independently an integer ranging from 1 to 3000; in another specific embodiment, m is independently an integer ranging from 1 to 1000; in another specific embodiment, m is independently an integer ranging from 100 to 500; in another specific embodiment, m is independently an integer ranging from 400 to 500.

[0232] R*

[0233] In one specific embodiment, R* is H; in another specific embodiment, R* is a halogen; in another specific embodiment, R* is C 1-6 alkyl, preferably C 1-4 alkyl; in another specific embodiment, R* is C 1-6 haloalkyl, preferably C 1-4 haloalkyl; in another specific embodiment, R* is C 1-6 alkoxy, preferably C 1-4 alkoxy; in another specific embodiment, R* is C 2-6 alkenyl; in another specific embodiment, R* is C 2-6 alkynyl.

[0234] R2

[0235] In one specific embodiment, R2 is a linker cleavable in a tumor environment; in another specific embodiment, R2 is a cleavable linker, and the cleavable linker is selected from matrix metalloproteinase (MMP)-cleavable linkers, metalloproteinase-cleavable linkers containing an a disintegrin and metalloproteinase (ADAM) domain, prostate-specific antigen (PSA) protease-cleavable linkers, urokinase-type plasminogen activator (uPA) protease-cleavable linkers, membrane-type serine protease 1 (MT-SP1) protease-cleavable linkers, matriptase (ST14) protease-cleavable linkers, and legumain protease-cleavable linkers.

[0236] In a specific embodiment, R2 is a matrix metalloproteinase (MMP)-cleavable linker, and the matrix metalloproteinase (MMP)-cleavable linker comprises the amino acid sequence of AHGL (SEQ ID NO:54) or PRQV (SEQ ID NO:61).

[0237] In one specific embodiment, R2 is a prostate-specific antigen (PSA) protease-cleavable linker, and the prostate-specific antigen (PSA) protease-cleavable linker comprises the amino acid sequence of HSSKLQ (SEQ ID NO:15).

[0238] In one specific embodiment, R2 is a urokinase-type plasminogen activator (uPA) protease-cleavable linker, and the urokinase-type plasminogen activator (uPA) protease-cleavable linker comprises the amino acid sequence of SGRSA (SEQ ID NO:11).

[0239] In one specific embodiment, R2 is a legumain protease-cleavable linker, and the legumain protease-cleavable linker comprises the amino acid sequence of AAN.

[0240] R3

[0241] In one specific embodiment, R3 is In another specific embodiment, R3 is

[0242] X

[0243] In one specific embodiment, X is -NH-; in another specific embodiment, X is -O-.

[0244] Y

[0245] In one specific embodiment, Y is -NH-; in another specific embodiment, Y is -O-; in another specific embodiment, Y is -C(O)-; in another specific embodiment, Y is -OC(O)-; in another specific embodiment, Y is -C(O)-C(O)-; in another specific embodiment, Y is -NHC(O)-; provided that it is chemically feasible.

[0246] R x

[0247] In one specific embodiment, R x is H; in another specific embodiment, R x is halogen; in another specific embodiment, R x is C 1-6 alkyl, preferably C 1-4 alkyl; in another specific embodiment, R x is C 1-6 alkoxy, preferably C 1-4 alkoxy; in another specific embodiment, R x is C 1-6 haloalkyl, preferably C 1-4Halogenated alkyl; in another specific embodiment, R x is C 2-6 alkenyl; in another specific embodiment, R x is C 2-6 alkynyl.

[0248] R y

[0249] In one specific embodiment, R y is H; in another specific embodiment, R y is halogen; in another specific embodiment, R y is C 1-6 alkyl, preferably C 1-4 alkyl; in another specific embodiment, R y is C 1-6 alkoxy, preferably C 1-4 alkoxy; in another specific embodiment, R y is C 1-6 halogenated alkyl, preferably C 1-4 halogenated alkyl; in another specific embodiment, R y is C 2-6 alkenyl; in another specific embodiment, R y is C 2-6 alkynyl.

[0250] n

[0251] In one specific embodiment, n is 0; in another specific embodiment, n is 1; in another specific embodiment, n is 2; in another specific embodiment, n is 3; in another specific embodiment, n is 4; in another specific embodiment, n is 5.

[0252] R4

[0253] In one specific embodiment, R4 is

[0254] R a

[0255] In one specific embodiment, R a is H; in another specific embodiment, R a is C 1-6 alkyl, preferably C 1-4 alkyl; in another specific embodiment, R a is C 1-6 halogenated alkyl, preferably C 1-4 halogenated alkyl; in another specific embodiment, R a is C 1-6Alkoxy, preferably C 1-4 alkoxy.

[0256] R b

[0257] In one specific embodiment, R b is H; in another specific embodiment, R b is C 1-6 alkyl, preferably C 1-4 alkyl; in another specific embodiment, R b is C 1-6 haloalkyl, preferably C 1-4 haloalkyl; in another specific embodiment, R b is C 1-6 alkoxy, preferably C 1-4 alkoxy.

[0258] R 4a

[0259] In one specific embodiment, R 4a is absent; in another specific embodiment, R 4a is -(CH2) 1-12 -; in another specific embodiment, R 4a is -(CH2) 1-12 -O-; in another specific embodiment, R 4a is -O-(CH2) 1-12 -; in another specific embodiment, R 4a is -O-(CH2) 1-12 -O-; in another specific embodiment, R 4a is -(CH2) 1-12 -O-(CH2) 1-12 -; in another specific embodiment, R 4a is (C 1-6 alkylene-O) p -(CH2) 1-12 ; in another specific embodiment, R 4a is -(CH2) 1-12 -C 3-8 cycloalkylene-; in another specific embodiment, R 4a is -(CH2) 1-12 -C 3-8 cycloalkylene-(CH2) 1-12 ; in another specific embodiment, R 4a is -(CH2) 1-12 -C 6-10 arylene-; in another specific embodiment, R 4ais -(CH2) 1-12 -C 6-10 arylene-(CH2) 1-12 .

[0260] In a specific embodiment, R 4a is not further substituted by an R# group; in a specific embodiment, R 4a is further substituted by 1 R# group; in a specific embodiment, R 4a is further substituted by 2 R# groups; in a specific embodiment, R 4a is further substituted by 3 R# groups; in a specific embodiment, R 4a is further substituted by 4 R# groups; in a specific embodiment, R 4a is further substituted by 5 R# groups.

[0261] R 4b

[0262] In one specific embodiment, R 4a is -NH-; in another specific embodiment, R 4a is -O-; in another specific embodiment, R 4a is -C(O)-; in another specific embodiment, R 4a is -C(O)O; in another specific embodiment, R 4a is -OC(O)-; in another specific embodiment, R 4a is -C(O)-C(O)-; in another specific embodiment, R 4a is -C(O)NH-; in another specific embodiment, R 4a is -NHC(O)-, provided that it is chemically feasible.

[0263] R#

[0264] In one specific embodiment, R# is H; in another specific embodiment, R# is a halogen; in another specific embodiment, R# is C 1-6 alkyl, preferably C 1-4 alkyl; in another specific embodiment, R# is C 1-6 haloalkyl, preferably C 1-4 haloalkyl; in another specific embodiment, R# is C 1-6 alkoxy, preferably C 1-4 alkoxy; in another specific embodiment, R# is C 2-6 alkenyl; in another specific embodiment, R# is C 2-6 alkynyl.

[0265] p

[0266] In one specific embodiment, p is 1; in another specific embodiment, p is 2; in another specific embodiment, p is 3; in another specific embodiment, p is 4; in another specific embodiment, p is 5.

[0267] The definitions of specific functional groups and chemical terms are described in more detail below.

[0268] When a range of values is listed, it is intended to cover every value and sub-range within the range. For example, "C 1-6 alkyl" is intended to include C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C 3-6 、C 3-5 、C 3-4 、C 4-6 、C 4-5 and C 5-6 alkyl.

[0269] “C 1-6 alkyl” refers to a group of a straight-chain or branched-chain saturated hydrocarbon group having 1-6 carbon atoms. In some embodiments, C 1-4 alkyl is substitutable. Examples of C 1-6 alkyl include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5) and n-hexyl (C6). The term “C 1-6 alkyl” also includes heteroalkyls in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl can be optionally substituted by one or more substituents, e.g., substituted by 1-5 substituents, 1-3 substituents or 1 substituent. Conventional abbreviations of alkyl include Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3) or i-Bu (-CH2CH(CH3)2).

[0270] “C 2-6"Alkenyl" refers to a group of straight-chain or branched-chain hydrocarbon groups having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-4 alkenyl is optional. C 2-6 Examples of alkenyl include vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. The term "C 2-6 alkenyl" also includes heteroalkenyl, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl may optionally be substituted with one or more substituents, e.g., substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0271] "C 2-6 Alkynyl" refers to a group of straight-chain or branched-chain hydrocarbon groups having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds present. In some embodiments, C 2-4 alkynyl is optional. C 2-6 Examples of alkynyl include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), etc. The term "C 2-6 alkynyl" also includes heteroalkynyl, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkynyl may be substituted with one or more substituents, e.g., substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0272] "C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 Alkynylene" refers to a divalent group of "C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl" as defined above.

[0273] "C 1-6 Alkylene" refers to a divalent group formed by removing one hydrogen from C 1-6 alkyl, and may be substituted or unsubstituted. In some embodiments, C 1-4The alkylene group is also substitutable. Unsubstituted alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Examples of substituted alkylene groups, such as those substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3)2-), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like.

[0274] “C 2-6 “Vinylene” means removing one hydrogen from a 2-6 vinyl group to provide a divalent vinylene group, and it can be substituted or unsubstituted. In some embodiments, 2-4 the vinylene group is also substitutable. Exemplary unsubstituted vinylene groups include, but are not limited to, vinylene (-CH=CH-) and propenylene (e.g., -CH=CHCH2-, -CH2-CH=CH-). Exemplary substituted vinylene groups, such as those substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted vinylene (-C(CH3)=CH-, -CH=C(CH3)-), substituted propenylene (e.g., -C(CH3)=CHCH2-, -CH=C(CH3)CH2-, -CH=CHCH(CH3)-, -CH=CHC(CH3)2-, -CH(CH3)-CH=CH-, -C(CH3)2-CH=CH-, -CH2-C(CH3)=CH-, -CH2-CH=C(CH3)-), and the like.

[0275] “C 2-6 “Vinylene” means removing one hydrogen from a 2-6 vinyl group to provide a divalent vinylene group, and it can be substituted or unsubstituted. In some embodiments, 2-4 the vinylene group is also substitutable. Exemplary vinylene groups include, but are not limited to, acetylene (-C≡C-), substituted or unsubstituted propyne (-C≡CCH2-), and the like.

[0276] “Halogenated” or “halogen” means fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). Thus, “C1-6 "Halogenated alkyl" refers to the above-mentioned "C 1-6 alkyl" substituted by one or more halogen atoms. In some embodiments, C 1-4 halogenated alkyl is also substitutable, and C 1-2 halogenated alkyl is also substitutable. Exemplary halogenated alkyls include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethylethyl, etc. The halogenated alkyl can be substituted at any available attachment point, for example, by 1-5 substituents, 1-3 substituents, or 1 substituent.

[0277] "C 3-8 "Cycloalkyl" refers to a group of non-aromatic cyclic hydrocarbon groups having 3-8 ring carbon atoms and zero heteroatoms. In some embodiments, C 3-6 cycloalkyl is also substitutable, and C 5-6 cycloalkyl is also substitutable. Cycloalkyl also includes ring systems in which the cycloalkyl described herein is fused to one or more aryl or heteroaryl groups, where the attachment point is on the cycloalkyl ring, and in such cases, the number of carbon atoms still represents the number of carbon atoms in the cycloalkyl system. Exemplary cycloalkyls include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), etc. The cycloalkyl can be substituted by one or more substituents, for example, by 1-5 substituents, 1-3 substituents, or 1 substituent.

[0278] "C 3-8 "Cycloalkylidene" refers to removing one hydrogen atom from C 3-8 cycloalkyl to provide a divalent cycloalkylidene group, and it can be substituted or unsubstituted. In some embodiments, C 3-7 cycloalkylidene is also substitutable.

[0279] "C 6-10 "Aryl" refers to a group of monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring systems having 6-10 ring carbon atoms and zero heteroatoms (e.g., having 6 or 10 shared π electrons in a cyclic arrangement). In some embodiments, the aryl has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl has ten ring carbon atoms ("C 10"Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above aromatic ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment is on the aromatic ring, in which case the number of carbon atoms still refers to the number of carbon atoms in the aromatic ring system. Aryl can be substituted with one or more substituents, for example, 1-5 substituents, 1-3 substituents or 1 substituent.

[0280] “C 6-10 "Arylene" means a C 6-10 The aryl group further removes one hydrogen to provide a divalent arylene group, and it can be substituted or unsubstituted. In some embodiments, phenylene is also substituted.

[0281] "Oxo" means =O.

[0282] Alkyl, alkenyl, alkynyl, cycloalkyl and aryl as defined herein are optionally substituted groups. Exemplary substituents on carbon atoms include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa 、-ON(R bb )2、-N(R bb )2、-N(R bb )3 + X - 、-N(OR cc )R bb , -SH, -SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc 2. -CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2, -OC(=O)N(R bb )2.-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2, -C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb)N(R bb )2, -OC(=NR bb )N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa , -NR bb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R aa , -S(=O)R aa , -OS(=O)R aa , -Si(R aa )3, -OSi(R aa )3, -C(=S)N(R bb )2, -C(=O)SR aa , -C(=S)SR aa , -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R dd groups; or two geminal hydrogens on a carbon atom are replaced by =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NR bb or =NOR cc groups; Each of R aa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, or two of the R aa groups are combined to form a heterocyclic group or a heteroaryl ring, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R dd groups; Each of R bb is independently selected from hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, or two R bbThe groups are combined to form a heterocyclic or heteroaryl ring, wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups; each R cc is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl, or two R cc groups are combined to form a heterocyclic or heteroaryl ring, wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups; each R dd is independently selected from halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3 + X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff )OR ee , -OC(=NR ff )R ee , -OC(=NR ff )OR ee , -C(=NR ff )N(R ff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee, -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee , -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg groups, or two geminal R dd substituents may combine to form =O or =S; each of R ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic group and heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg groups.

[0283] Each of R ff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, or two R ff groups are combined to form a heterocyclic group or heteroaryl ring, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg groups; each of R gg is independently selected from halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 alkyl, -ON(C 1-6 alkyl)2, -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6(alkyl) + X - 、 -NH3 + X - 、 -N(OC 1-6 (alkyl)(C 1-6 alkyl)、 -N(OH)(C 1-6 alkyl)、 -NH(OH)、 -SH、 -SC 1-6 alkyl、 -SS(C 1-6 alkyl)、 C(=O)(C 1-6 alkyl)、 -CO2H、 -CO2(C 1-6 alkyl)、 -OC(=O)(C 1-6 alkyl)、 -OCO2(C 1-6 alkyl)、 -C(=O)NH2、 C(=O)N(C 1-6 alkyl)2、 -OC(=O)NH(C 1-6 alkyl)、 -NHC(=O)(C 1-6 alkyl)、 -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl)、 -NHCO2(C 1-6 alkyl)、 -NHC(=O)N(C 1-6 alkyl)2、 -NHC(=O)NH(C 1-6 alkyl)、 -NHC(=O)NH2、 -C(=NH)O(C 1-6 alkyl)、 -OC(=NH)(C 1-6 alkyl)、 -OC(=NH)OC 1-6 alkyl、 -C(=NH)N(C 1-6 alkyl)2、 -C(=NH)NH(C 1-6 alkyl)、 -C(=NH)NH2、 -OC(=NH)N(C 1-6 alkyl)2、 -OC(NH)NH(C 1-6 alkyl)、 -OC(NH)NH2、 -NHC(NH)N(C 1-6 alkyl)2、 -NHC(=NH)NH2、 -NHSO2(C 1-6 alkyl)、 -SO2N(C 1-6 alkyl)2、 -SO2NH(C 1-6 alkyl)、 -SO2NH2、 -SO2C 1-6 alkyl、 -SO2OC 1-6 alkyl、 -OSO2C 1-6 alkyl、 -SOC 1-6 alkyl、 -Si(C 1-6 alkyl)3、 -OSi(C 1-6 alkyl)3、 C(=S)N(C1-6 (alkyl)2, C(=S)NH(C 1-6 (alkyl), C(=S)NH2, -C(=O)S(C 1-6 (alkyl), -C(=S)SC 1-6 alkyl, -SC(=S)SC 1-6 (alkyl), -P(=O)2(C 1-6 (alkyl), -P(=O)(C 1-6 (alkyl)2, -OP(=O)(C 1-6 (alkyl)2, -OP(=O)(OC 1-6 (alkyl)2, C 1-6 alkyl, C 1-6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 aryl, C3-C7 heterocyclic group, C5-C 10 heteroaryl, or two geminal R gg substituents may combine to form =O or =S; wherein X - is a counterion.

[0284] Exemplary substituents on the nitrogen atom include, but are not limited to, hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, or two R attached to the nitrogen atom ccThe groups combine to form a heterocyclic or heteroaryl ring, wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups, and wherein R aa , R bb , R cc , and R dd are as described herein.

[0285] In some embodiments of the polypeptides of the present application, the chemical moiety comprises a structure of formula (I): R1-R2-R3-R4 (I), wherein R1 is optionally further substituted with 1, 2, or 3 R* groups; L1 is selected from CH or N; R 1a is absent, or is selected from -C(O)-, -OC(O)-, and -NHC(O)-; each R 1b is independently absent, or is independently selected from -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)NH-, -NH-C 1-4 alkyl-, -O-C 1-4 alkyl-, -C(O)-C 1-4 alkyl-, -OC(O)-C 1-4 alkyl-, -C(O)O-C 1-4 alkyl-, and -C(O)NH-C 1-4 alkyl-; each R 1c is independently selected from H, C 1-4 alkyl, and C 1-4 haloalkyl; each m is independently an integer in the range of 1 - 5000; R* is selected from H, halogen, C 1-6 alkyl, or C 1-6 haloalkyl; or, R1 is optionally further substituted with 1, 2, or 3 R* groups; R 1a is absent, or is -C(O)-; R 1c is selected from H, C 1-4 alkyl, and C 1-4 haloalkyl; m is an integer in the range of 1 - 3000; R* is selected from H, halogen, or C 1-4 alkyl; or alternatively, R1 is R 1c is selected from H, C 1-4 alkyl, and C 1-4 haloalkyl, m is an integer in the range of 1 - 1000, 100 - 500, or 400 - 500; or still alternatively, R1 is a linear polyethylene glycol having a molecular weight of about 20000.

[0286] In some embodiments of the polypeptides of the present application, the chemical moiety comprises a structure of formula (I): R1-R2-R3-R4 (I), wherein R2 is selected from matrix metalloproteinase (MMP) cleavable linkers, a disintegrin and metalloproteinase (ADAM) domain-containing metalloproteinase cleavable linkers, prostate specific antigen (PSA) protease cleavable linkers, urokinase-type plasminogen activator (uPA) protease cleavable linkers, membrane-type serine protease 1 (MT-SP1) protease cleavable linkers, matriptase (ST14) protease cleavable linkers, and legumain protease cleavable linkers. In some embodiments, R2 is a matrix metalloproteinase (MMP) cleavable linker. In some embodiments, the MMP cleavable linker comprises an amino acid sequence selected from any one of SEQ ID NO: 21-35. In some embodiments, R2 is a prostate specific antigen (PSA) protease cleavable linker. In some embodiments, the PSA protease cleavable linker comprises an amino acid sequence selected from any one of SEQ ID NO: 14-16. In some embodiments, R2 is a urokinase-type plasminogen activator (uPA) protease cleavable linker. In some embodiments, the uPA protease cleavable linker comprises an amino acid sequence selected from any one of SEQ ID NO: 11-13. In some embodiments, R2 is a legumain protease cleavable linker. In some embodiments, the legumain protease cleavable linker comprises an amino acid sequence selected from any one of SEQ ID NO: 18-19 or AAN or Cbz-AAN-AMC.

[0287] In some embodiments, the chemical moiety comprises a structure of formula (II): wherein R1, R3, and R4 are as defined above.

[0288] In some embodiments of the polypeptides of the present application, the chemical moiety comprises a structure of formula (I): R1-R2-R3-R4 (I), wherein R3 is wherein X is selected from -NH- or -O-; Y is selected from -NH-, -O-, -C(O)-, -OC(O)-, or -NHC(O)-; each R x and R y is independently selected from H, halogen, C 1-4 alkyl or C 1-4 haloalkyl; n is 0, 1, 2, or 3; or, R3 is X is selected from NH or O; Y is selected from -NH-, -O-, -C(O)-, -OC(O)-, or -NHC(O)-. In some embodiments, the chemical moiety comprises a structure of formula (III):

[0289]

[0290] wherein R1, R2, and R4 are as defined above. In some embodiments, the chemical moiety comprises a structure of formula (IV) wherein R1 and R4 are as defined above.

[0291] In some embodiments of the polypeptides of the present application, the chemical moiety comprises a structure of formula (I): R1-R2-R3-R4 (I), wherein R4 is R a and R b are each independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, and C 1-6 alkoxy; R 4a is absent or is selected from -(CH2) 1-12 -, -(CH2) 1-12 -O-, -O-(CH2) 1-12 -, -O-(CH2) 1-12 -O-, -(CH2) 1-12 -O-(CH2) 1-12 -, (C 1-6 alkylene-O) p -(CH2) 1-12 -, -(CH2) 1-12 -C 3-8 cycloalkylene and -(CH2) 1-12 -C 3-8 cycloalkylene-(CH2) 1-12 ; R 4a is optionally further substituted with 1, 2, 3, or 4 R# groups; R 4b is -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)NH-, or -NHC(O)-; R# is selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, or C 2-6 alkynyl; p is 1, 2, 3, or 4; or, R a and R b are each independently selected from H and C 1-4 alkyl; R 4a is selected from -(CH2) 1-12 -, -(CH2) 1-12 -O-, -O-(CH2) 1-12 -, -O-(CH2) 1-12 -O-, -(CH2) 1-12 -O-(CH2) 1-12 - and (C1-4 (alkylene-O) p -(CH2) 1-12 ; R 4a optionally further substituted with 1, 2 or 3 R# groups; R 4b is -NH-, -O- or -C(O)-; R# is selected from H, halogen, C 1-6 alkyl and C 1-6 haloalkyl; p is 1, 2 or 3; alternatively, R a and R b are H; R 4a is -(CH2) 2-12 -; R 4b is -NH-; R 4a optionally further substituted with 1 or 2 R# groups; R# is selected from H, halogen and C 1-4 alkyl. In some embodiments, the chemical moiety comprises a structure of formula (V):

[0292]

[0293] wherein R1, R2 and R3 are as defined above.

[0294] In some embodiments, the chemical moiety comprises a structure of formula (VI): wherein PEG m is R 1c is selected from H, C 1-4 alkyl and C 1-4 haloalkyl, and m is an integer in the range of 1 - 1000, 100 - 500 or 400 - 500. In some embodiments, PEG m is linear polyethylene glycol with a molecular weight of about 20000.

[0295] In some embodiments of the polypeptides of the present application, IFNα2b or a functional fragment is chemically modified at one or more residues corresponding to those selected from R12, L15, M16, R22, L26, F27, L30, R33, R33, H34, D35, A145, M148, R149, S152, L153 and N156 of SEQ ID NO:1. In some embodiments, IFNα2b or a functional fragment is chemically modified at the residue corresponding to S152 of SEQ ID NO:1.

[0296] In some embodiments, IFNα2b or a functional fragment thereof comprises an amino acid substitution at a position corresponding to a position selected from R12, L15, M16, R22, L26, F27, L30, R33, R33, H34, D35, A145, M148, R149, S152, L153, and N156 of SEQ ID NO:1, and the chemical moiety is attached to IFNα2b or the functional fragment at the substituted amino acid. In some embodiments, IFNα2b or a functional fragment thereof comprises an amino acid substitution with cysteine at a position corresponding to a position selected from R12, L15, M16, R22, L26, F27, L30, R33, R33, H34, D35, A145, M148, R149, S152, L153, and N156 of SEQ ID NO:1, and the chemical moiety is attached to IFNα2b or the functional fragment via cysteine. In some embodiments, IFNα2b or the functional fragment comprises an amino acid substitution with cysteine at a position corresponding to the position of S152 of SEQ ID NO:1, and the chemical moiety is attached to IFNα2b or the functional fragment via cysteine.

[0297] In some embodiments, R4 of the chemical moiety is attached to the S atom of the cysteine residue via maleimide, acetylene, vinyl, monosubstituted maleic acid, or disubstituted maleimide. In some embodiments, R4 of the chemical moiety is attached to the S atom of the cysteine residue via maleimide.

[0298] In some embodiments, IFNα2b or the functional fragment comprises an amino acid substitution with cysteine at the position corresponding to the position of S152 of SEQ ID NO:1, and the chemical moiety is attached to cysteine via R4 of the chemical moiety via maleimide, acetylene, vinyl, monosubstituted maleic acid, or disubstituted maleimide. In some embodiments, IFNα2b or the functional fragment comprises an amino acid substitution with cysteine at the position corresponding to the position of S152 of SEQ ID NO:1, and the chemical moiety is attached to cysteine via R4 of the chemical moiety via maleimide. In some embodiments, IFNα2b or the functional fragment comprises an amino acid substitution with cysteine at the position corresponding to the position of S152 of SEQ ID NO:1, and the chemical moiety comprises a structure of formula (VI) which is attached to cysteine via maleimide: where PEG m is R 1c is selected from H, C 1-4 alkyl and C 1-4A haloalkyl group, where m is an integer in the range of 1 - 1000, 100 - 500, or 400 - 500; or, PEG m is a linear polyethylene glycol with a molecular weight of approximately 20,000.

[0299] Bifunctional immunoconjugate

[0300] In another aspect, the present application provides an immunoconjugate comprising IFNα2b or a functional fragment thereof of the present application and a biomolecule. In some embodiments, the biomolecule specifically binds to a target. In some embodiments, the biomolecule comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from full-length antibodies, Fab, Fab’, F(ab’)2, Fd, Fd’, Fv, scFv, ds-scFv, sdAb, and nanobodies. In some embodiments, the antibody specifically targets PD-L1 or PD-1. In some embodiments, the biomolecule is a PD-L1 / PD-1 antibody as described herein.

[0301] In some embodiments of the immunoconjugate of the present application, the biomolecule is a PD-L1 antibody. In some embodiments, the PD-L1 antibody is an sdAb. In some embodiments, the PD-L1 antibody is an sdAb comprising CDR1, CDR2, and CDR3 of SEQ ID NO:6. In some embodiments, the PD-L1 antibody is an sdAb comprising CDR1, CDR2, and CDR3 of SEQ ID NO:6, and CDR1, CDR2, and CDR3 are according to the Kabat numbering scheme. In some embodiments, the biomolecule is a PD-L1 antibody, and the PD-L1 antibody comprises: (1) CDR1, which comprises the amino acid sequence of FRHYVMG (SEQ ID NO:3) or an amino acid sequence having one or more substitutions compared to SEQ ID NO:3; (2) CDR2, which comprises the amino acid sequence of AISWSGSGSYYADSVKG (SEQ ID NO:4) or an amino acid sequence having one or more substitutions compared to SEQ ID NO:4; and (3) CDR3, which comprises the amino acid sequence of DMTTRMSQASREYDY (SEQ ID NO:5) or an amino acid sequence having one or more substitutions compared to SEQ ID NO:5.

[0302] In some embodiments, the biomolecule is a PD-L1 antibody, and the PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:6.

[0303] In some embodiments, the immunoconjugate comprises any suitable number of sdAbs as described above. In some embodiments, the immunoconjugate comprises 1-4 of the sdAbs as described above. In some embodiments, the immunoconjugate comprises one of the sdAbs as described above. In some embodiments, the immunoconjugate comprises two of the sdAbs as described above. In some embodiments, the immunoconjugate comprises four of the sdAbs as described above.

[0304] In some embodiments, the immunoconjugate comprises IFNα2b or a functional fragment thereof as described herein. In some embodiments, IFNα2b or a functional fragment thereof is modified to attenuate the binding affinity of IFNα2b or a functional fragment thereof for the interferon α / β receptor. In some embodiments, the binding affinity of IFNα2b or a functional fragment thereof for the interferon α / β receptor is attenuated to reduce adverse reactions of the immunoconjugate upon administration to a subject in need thereof. In some embodiments, IFNα2b or a functional fragment thereof is modified to attenuate the activation of type I interferon signaling induced by IFNα2b or a functional fragment thereof. In some embodiments, the activation of type I interferon signal transduction is attenuated to reduce adverse reactions of the immunoconjugate upon administration to a subject in need thereof.

[0305] In some embodiments, the adverse reactions include acute toxicity, subacute side effects, and chronic side effects. In some embodiments, the acute toxicity includes flu-like symptoms such as fever, chills, myalgia, headache, and nausea. In some embodiments, the subacute side effects include, but are not limited to, hematological side effects such as anemia, reduction in white blood cell and platelet counts, and reduced platelet aggregation; liver side effects such as elevated transaminases and inhibition of cytochrome P450 enzymes; gastrointestinal side effects such as anorexia, nausea, vomiting, and diarrhea; mental side effects such as depression, cognitive impairment, and delirium; neurological side effects such as seizures, neuropathy, multiple sclerosis-like diseases, and myasthenia gravis; renal side effects such as proteinuria; cardiovascular side effects such as arrhythmia, ischemic heart disease, cardiomyopathy, and retinal abnormalities; pulmonary side effects such as pneumonia; endocrine side effects such as thyroid diseases, diabetes, decreased sex hormone levels, and hypopituitarism; skin side effects such as hair loss, erythema, induration at the injection site, rash, pruritus, vitiligo, lichen planus, and psoriasis.

[0306] In some embodiments, side effects include autoimmune diseases such as thyroid diseases, liver dysfunction, connective tissue diseases, skin diseases, blood system diseases, nervous system diseases, lung diseases, and metabolic diseases. In some embodiments, the side effects result in conditions including but not limited to subacute lymphocytic thyroiditis, Graves' disease, permanent hypothyroidism, autoimmune hepatitis, primary biliary cirrhosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, leukocytoclastic vasculitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, inflammatory demyelinating polyneuropathy, multiple sclerosis-like diseases, axonal neuropathy, and hearing loss, Bell palsy, multiple mononeuropathy, myasthenia gravis, abducent nerve paralysis, interstitial pneumonia, and diabetes.

[0307] In some embodiments of the immunoconjugates of the present application, IFNα2b or a functional fragment thereof is modified to improve the performance of the immunoconjugate after administration to a subject in need thereof. In some embodiments, the performance includes but is not limited to pharmacokinetics (PK), pharmacodynamics (PD), potency, and safety. In some embodiments, the potency includes anti-tumor potency. In some embodiments, the anti-tumor potency includes but is not limited to inhibiting tumor growth, reducing tumor volume, increasing the survival period of the subject, and inducing protection against tumor recurrence.

[0308] In some embodiments, the immunoconjugates of the present application comprise IFNα2b or a functional fragment thereof modified by amino acid substitution at one or more residues corresponding to one or more of R12, L15, M16, R22, L26, F27, L30, R33, R33, H34, D35, A145, M148, R149, S152, L153, and N156 selected from SEQ ID NO:1. In some embodiments, the immunoconjugates of the present application comprise IFNα2b or a functional fragment thereof modified by amino acid substitution at R12, L15, M16, R22, L26, F27, L30, R33, R33, H34, D35, A145, M148, R149, S152, L153, and N156 of SEQ ID NO:1. In some embodiments, the immunoconjugates of the present application comprise IFNα2b or a functional fragment thereof modified by amino acid substitution at S152 of SEQ ID NO:1. In some embodiments, the immunoconjugates of the present application comprise IFNα2b or a functional fragment thereof having an amino acid substitution with cysteine at a residue corresponding to S152 of SEQ ID NO:1.

[0309] In some embodiments, the immunoconjugates of the present application comprise IFNα2b or a functional fragment, and IFNα2b comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:1. In some embodiments, the immunoconjugates of the present application comprise IFNα2b or a functional fragment, and IFNα2b comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the immunoconjugate comprises IFNα2b, and IFNα2b comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:2. In some embodiments, the immunoconjugates of the present application comprise IFNα2b or a functional fragment, and IFNα2b comprises the amino acid sequence of SEQ ID NO:2.

[0310] In some embodiments, the immunoconjugates of the present application comprise IFNα2b or a functional fragment, wherein IFNα2b comprises the amino acid sequence of SEQ ID NO:2 and is further chemically modified at residue C152 of SEQ ID NO:2, and the chemical moiety comprises the structure of formula (VI): (VI), and PEG m is R 1c selected from H, C 1-4 alkyl and C 1-4 haloalkyl, m is an integer ranging from 1 - 1000, 100 - 500 or 400 - 500; or, PEG m is a linear polyethylene glycol having a molecular weight of about 20000.

[0311] In some embodiments, the immunoconjugate comprises any suitable number of IFNα2b or functional fragments as described above. In some embodiments, the immunoconjugate comprises 1 - 4 of the IFNα2b or functional fragments as described above. In some embodiments, the immunoconjugate comprises two of the IFNα2b or functional fragments as described above. In some embodiments, the immunoconjugate comprises four of the IFNα2b or functional fragments as described above.

[0312] In some embodiments of the immunoconjugates of the present application, the immunoconjugates further comprise an Fc region. As used herein, the term "Fc region" refers to the C-terminal region of a human immunoglobulin heavy chain, which contains at least a portion of the hinge region, CH2 domain, and CH3 domain. Any suitable Fc region known in the art can be used in the immunoconjugates of the present application. In some embodiments, the Fc region can be derived from any suitable IgG isotype, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is derived from IgG1. In some embodiments, the Fc region further comprises one or more mutations to enhance ADCC function.

[0313] In some embodiments, the Fc region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the sequence of SEQ ID NO:7. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:7.

[0314] In some embodiments of the immunoconjugates of the present application, IFNα2b or a functional fragment thereof is conjugated to the N-terminus of a biomolecule. In some embodiments, IFNα2b or a functional fragment thereof is conjugated to the C-terminus of a biomolecule. In some embodiments, IFNα2b or a functional fragment thereof is conjugated to the C-terminus of the Fc region. In some embodiments of the immunoconjugate, IFNα2b or a functional fragment thereof is conjugated to the N-terminus of the Fc region. In some embodiments, IFNα2b or a functional fragment thereof is conjugated to a biomolecule through a linker. In some embodiments, IFNα2b or a functional fragment thereof is conjugated to the Fc region through a linker. Any suitable linker known in the art can be used for conjugation. In some embodiments, the linker is selected from (GGGGS)n (SEQ ID NO:53), (GGGS)n (SEQ ID NO:54), (GGS)n, (GmS)n, and (XmS)n, and n is an integer from 1 to 8.

[0315] Exemplary forms of the immunoconjugates of the present application are as Figure 1 shown.

[0316] In some embodiments, the immunoconjugate comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the sequence of SEQ ID NO:9. In some embodiments, the immunoconjugate comprises the amino acid sequence of SEQ ID NO:9.

[0317] In some embodiments, the immunoconjugate comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:10. In some embodiments, the immunoconjugate comprises the amino acid sequence of SEQ ID NO:10.

[0318] In some embodiments, the immunoconjugate comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:36 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:37. In some embodiments, the immunoconjugate comprises the amino acid sequences of SEQ ID NO:36 and 37.

[0319] In some embodiments, the immunoconjugate comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:38 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:39. In some embodiments, the immunoconjugate comprises the amino acid sequences of SEQ ID NO:38 and 39.

[0320] In some embodiments, the immunoconjugate comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:40 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:41. In some embodiments, the immunoconjugate comprises the amino acid sequences of SEQ ID NO:40 and 41.

[0321] In some embodiments, the immunoconjugate comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:42 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:43. In some embodiments, the immunoconjugate comprises the amino acid sequences of SEQ ID NO:42 and 43.

[0322] In some embodiments, the immunoconjugate comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:44 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:45. In some embodiments, the immunoconjugate comprises the amino acid sequences of SEQ ID NO:44 and 45.

[0323] In some embodiments, the immunoconjugate comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:46 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:47. In some embodiments, the immunoconjugate comprises the amino acid sequences of SEQ ID NO:46 and 47.

[0324] In another aspect, provided herein is a polynucleotide encoding a polypeptide or immunoconjugate of the present application. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to SEQ ID NO:8. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:8.

[0325] In another aspect, provided herein is a vector comprising a polynucleotide as described in the present application. In some embodiments, the term "vector" refers to a DNA molecule for introducing and directing the expression of a particular gene operably linked thereto in a target cell. In some embodiments, the vector comprises an expression cassette comprising a polynucleotide sequence encoding a polypeptide or immunoconjugate of the present application.

[0326] On the other hand, the present disclosure provides a host cell comprising a vector as described in the present application. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce the polypeptides or immunoconjugates of the present application. Host cells suitable for replicating and supporting the expression of polypeptides or immunoconjugates are well known in the art. Such cells can be appropriately transfected or transduced with a specific expression vector, and large amounts of vector-containing cells can be grown to inoculate large-scale fermenters to obtain sufficient amounts of polypeptides or immunoconjugates for clinical use. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like. For example, particularly when glycosylation is not required, polypeptides can be produced in bacteria. After expression, the polypeptides can be separated from the bacterial cell paste as a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable as cloning or expression hosts for vectors encoding polypeptides, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of polypeptides with a partially or fully human glycosylation pattern. Suitable host cells for expressing (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension growth may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney cell lines (293 or 293T cells), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells, monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells), MRC5 cells, and FS4 cells.

[0327] Treatment

[0328] On the other hand, the present disclosure provides a method for treating a disorder in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide, immunoconjugate, or nucleic acid of the present application. In some embodiments, the disorder is cancer. In some embodiments, the disorder is cancer resistant to PD-1 / PD-L1 antibody therapy.

[0329] All publications and patent applications cited in this specification are hereby incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0330] Example

[0331] The following examples are intended to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure, nor are they intended to represent that the experiments below are all or the only experiments conducted. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric.

[0332] Example 1. Preparation of Bifunctional Polypeptides and Immunoconjugates

[0333] Design bifunctional molecules that include IFNα2b or functional fragments and are also capable of targeting PD-L1 / PD-1, and express them in a mammalian expression system based on HEK293. The structures of these polypeptides are shown in Figure 1 and their sequences are shown in Table 2 below.

[0334] Table 2. Amino Acid Sequences of Immunoconjugates

[0335]

[0336]

[0337]

[0338]

[0339] As Figure 1 shown, various forms of bifunctional polypeptides are employed. Two of these forms are symmetric, each containing two anti-PD-L1 nanobodies and two IFNα2b molecules. In the first symmetric form (L1IF) on the far left, the two nanobodies are placed on the N-terminal side of the IgG1 Fc fragment, while the two IFNα2b molecules are on the C-terminal side of the Fc fragment. In contrast, in L1-G4S-IF-Fc (Form 1), the two IFNα2b molecules are between the nanobody and the Fc fragment.

[0340] Five different asymmetric bifunctional polypeptides were prepared. In L1-Fc(H)+L1-Fc-IF(K) (Form 2), only a single IFNα2b molecule was included and fused to the C-terminus of one of the CH3 domains of the Fc fragment. In all asymmetric formats, a staphylococcal nuclease substitution was performed to reduce mismatches. In Form 2, the IFNα2b molecule was fused to the CH3 including the staphylococcal nuclease. In Form 3 (L1-Fc(K)+L1-Fc-IF(H)), the IFNα2b molecule was fused to the CH3 including the nuclease-resistant domain.

[0341] In Form 4 (L1*2-Fc(H)+IF-Fc(K)), two anti-PD-L1 nanobodies were tandemly linked to one of the chains (nuclease-resistant domain) of the Fc fragment, while a single IFNα2b was fused to the N-terminus of the other chain (staphylococcal nuclease). In Form 5 (L1-Fc(H)+IF-G4S-L1-Fc(K)), the two anti-PD-L1 nanobodies were separately fused to the two Fc chains, and one of the chains also contained an IFNα2b molecule at the most N-terminus. Finally, in Form 6 (L1-Fc(H)+L1-G4S-IF-Fc(K)), a single IFNα2b molecule was between the N-terminal anti-PD-L1 nanobody and the C-terminal Fc.

[0342] In some versions of the bifunctional polypeptide, the IFNα2b molecule was wild-type (WT). In some versions of the bifunctional polypeptide, the IFNα2b molecule included an S152C substitution (position according to SEQ ID NO:1). The bifunctional polypeptide with the S152C substitution was designated L1IF M205.

[0343] Immunoconjugates (L1IF M205 DAR1) based on these bifunctional polypeptides and further including the chemical moiety of the present application were also designed and prepared, as Figure 2A and 2B shown. The chemical moiety was mainly prepared as previously described (see WO2019091384A1).

[0344] As Figure 2A shown, L1IF M205 adopted a symmetric form with a bivalent L1-Fc-IF arm, in which the residue S152 of the IFNα2b component was replaced with a cysteine (C) (position according to SEQ ID NO:1). Then, the chemical moiety containing a maleimide chemical linker, PABC, a legumain-cleavable linker, and 20K PEG was linked to the cysteine introduced at residue 152 of IFNα2b in one arm (L1IFM205 DAR1) or both arms (L1IF M205 DAR2) of L1IF M205. The PEG structure could be cleaved by legumain accumulated in the tumor environment, thereby releasing the active form of the molecule.Figure 2B SDS-PAGE of L1IF M205 DAR1 and L1IF M205 DAR2 is shown under conditions with DTT (left) or without DTT (right).

[0345] Example 2. Binding of Immunoconjugate to PD-L1

[0346] Biacore assays were performed to evaluate the binding affinity of the immunoconjugate to human PD-L1.

[0347] Briefly, PD-L1 mAb (SEQ ID NO:6), L1IF WT, L1IF M205, and L1IF M205 DAR1 were immobilized on a Protein A chip respectively, and the flow rate was 10 μL / min. Recombinant human PD-L1 protein was used as the analyte. The concentrations of the analyte were 400, 200, 100, 50, 25, 0 nM, the injection rate of the analyte was 30 μL / min, and the binding time was 180 seconds. The results were analyzed by using a 1:1 binding fitting model. Then the K of the L1IF WT, L1IF M205 group and L1IF M205 DAR1 group D Normalized to PD-L1 mAb respectively, as shown in Table 3 below.

[0348] Table 3. Binding Affinity of the Immunoconjugate of the Present Application to PD-L1

[0349]

[0350] As shown in Table 3, the binding affinities of L1IF WT, L1IF M205, and L1IF M205 DAR1 to PD-L1 are comparable to those of the PD-L1 mAb used in the immunoconjugate, indicating that conjugation of IFNα2b or IFNα2b chemically modified in the present application does not affect the binding affinity of the PD-L1 antibody to PD-L1.

[0351] Then the binding of the PD-L1 arm of the immunoconjugate was further evaluated by ELISA. Briefly, recombinant human PD-L1 protein was coated in a 96-well plate. PD-L1 mAb (SEQ ID NO:6), L1IF WT, L1IF M205, and L1IF M205 DAR1 were serially diluted and added to the plate. Goat anti-human IgG Fc HRP was further added as the secondary antibody for analysis. As Figure 3A shown, the EC50 of the L1IF M205 DAR1 group was 1.292 nM, a 3.6-fold change compared to the PD-L1 mAb, while the EC50 values of the L1IF WT and L1IF M205 groups were comparable to those of the PD-L1 mAb.

[0352] To more accurately simulate PD-L1 antigen exposure, the PD-L1 antigen protein was expressed on CHO-K1 cells. Then, the binding activity of the immunoconjugates of the present application to PD-L1 on CHO-K1 cells was detected by FACS. As Figure 3B shown, L1IFM205 and L1IF WT exhibited comparable binding ability to PD-L1 mAb, while L1IF M205 DAR1 exhibited slightly reduced binding ability.

[0353] Example 3. Activation of PD-L1 / PD-1 Signaling by Immunoconjugates

[0354] To evaluate the activation of PD-L1 / PD-1 signaling by the immunoconjugates of the present application, an artificial functional assay system was established, which included CHO-K1 APC stably expressing human PD-L1 and OKT3 and Jurkat effector cells stably expressing PD-1, CD3, and NFAT reporter gene. In this system, when an antagonist of PD1 / PD-L1 signaling was introduced into the system, the inhibition of the CD3 pathway by PD1 could be eliminated, and the CD3-induced NFAT reporter gene could be detected.

[0355] As Figure 4 shown, after incubation, the expression of the NFAT reporter gene induced by L1IF WT and L1IF M205 was comparable to that of PD-L1mAb, while L1IF M205 DAR1 caused slightly reduced expression of the NFAT reporter gene compared to PD-L1mAb.

[0356] Example 4. Binding of Immunoconjugates to IFN Receptors

[0357] Biacore assays were performed to test the binding affinity of the immunoconjugates of the present application to IFN receptors IFNAR1 and IFNAR2. The results are summarized in Table 4 below.

[0358] Table 4. Binding Affinity of Immunoconjugates to IFNAR1 and IFNAR2

[0359]

[0360] As shown in Table 4, compared with the control (commercially available PEG-IFNα2b without the modification of the present application), L1IF WT exhibited comparable binding affinity to IFNAR1 and IFNAR2; L1IF M205 with S152C substitution in the IFNα2b component of the immunoconjugate exhibited comparable binding affinity to IFNAR1 but weakened binding to IFNAR2, where K D increased by 10 4-fold; L1IF M205 DAR1, which has an S152C substitution in the IFNα2b component of the immunoconjugate and an additional chemical moiety linked to the substituted residue, showed further diminished binding to both IFNAR1 and IFNAR2 compared to L1IF M205.

[0361] The binding ability of the immunoconjugate to IFNAR1 and IFNAR2 was then evaluated by ELISA. Briefly, recombinant human IFNAR1 or IFNAR2 protein (1 μg / ml) was coated on a 96-well plate overnight at 4 °C. Then 100 μl of the immunoconjugate diluted in 1% BSA / PBST was added and incubated with IFNAR1 or IFNAR2 for 1 hour. 100 μl of goat anti-human IgG Fc HRP was added and analyzed by spectrophotometer at 450 nm.

[0362] As Figure 5A and Figure 5B shown, consistent with the Biacore results, L1IF M205 with an S152C substitution in the IFNα2b component of the immunoconjugate showed comparable binding affinity to IFNAR1 but diminished binding to IFNAR2 compared to L1IF WT, and L1IF M205 DAR1, which has an S152C substitution in the IFNα2b component of the immunoconjugate and an additional chemical moiety linked to the substituted residue, showed further diminished binding to both IFNAR1 and IFNAR2 compared to L1IF M205.

[0363] Example 5. Activation of IFNα Signaling by Immunoconjugates

[0364] An IFNα2b signaling reporter gene assay and a Daudi proliferation inhibition assay were performed to evaluate the activation of IFNα signaling by the immunoconjugates of the present application. Briefly, HEK-Blue IFN-α / β cells (at a density of 5.0 x 10 4 cells per well) were plated in white 96-well plates. Then immunoconjugates containing L1IF WT, L1IF M205, L1IF M205 DAR1, and L1IF M205 DAR2 were added separately and incubated with the cells at 37 °C for 6 hours, followed by measurement of luminescence.

[0365] As Figure 6AAs shown, compared to L1IF WT, L1IF M205 with an S152C substitution in the IFNα2b component of the immunoconjugate exhibits reduced activation of type I interferon signaling, and L1IF M205 DAR1 and L1IF M205 DAR2, which have an S152C substitution in the IFNα2b component of the immunoconjugate and an additional chemical moiety linked to the substituted residue, exhibit further reduced activation of type I interferon signaling compared to L1IF M205.

[0366] Daudi cells (at a density of 2.0 x 10 4 cells per well) were plated in 96-well plates. Then, immunoconjugates comprising L1IF WT, L1IF M205, L1IF M205 DAR1, and L1IF M205 DAR2 were added separately and incubated with the cells at 37 °C for 3 days. Then, titer-glo detection buffer (Promega) was added and incubated for 3 minutes, followed by measurement of luminescence by Envision.

[0367] As Figure 6B shown, L1IF M205 with an S152C substitution in the IFNα2b component of the immunoconjugate exhibits significant inhibition of Daudi proliferation, and L1IF M205 DAR1 and L1IF M205 DAR2, which have an S152C substitution in the IFNα2b component of the immunoconjugate and an additional chemical moiety linked to the substituted residue, exhibit further inhibition of Daudi proliferation compared to L1IF M205.

[0368] Example 7. In vitro potency of immunoconjugates

[0369] The in vitro tumor killing potency of the immunoconjugates of the present application was evaluated by using the human colorectal cancer cell line RKO cells as target cells and primary CD8 T cells as effector cells, as Figure 7A shown.

[0370] Briefly, primary CD8 T cells were isolated from human PBMC using a Milteny kit and then co-cultured with 1.0 x 10 4 CellTrace-labeled RKO cells at a density of 5.0 x 10 4 cells / well in 96-well plates. The immunoconjugates of the present application comprising L1IF WT, L1IF M205, L1IF M205 DAR1, and L1IF M205 DAR2 were serially diluted and added to the plates and incubated with the cells for 3 days, followed by analysis of the number of live RKO cells by FACS.

[0371] AsFigure 7B As shown, similar to L1IF WT, L1IF M205 exhibits dose-dependent tumor-killing activity. In contrast, L1IF M205 DAR1 and L1IF M205 DAR2 do not exhibit tumor-killing activity, indicating that the activation of type I interferon signaling is attenuated due to the modification of the chemical part of this application. After cleavage by Legumain, L1IF M205DAR1 restored dose-dependent tumor-killing activity, as shown in the L1IF M205 active group.

[0372] Example 8. In Vivo Efficacy of Immunoconjugates

[0373] The in vivo anti-tumor activity of the immunoconjugates of this application was evaluated in a CB-17 SCID RKO mouse model.

[0374] Briefly, RKO cells (a colorectal cancer cell line that is PD-L1 positive but resistant to anti-PD-L1 treatment) were subcutaneously inoculated into human PBMC-reconstituted CB-17 mice (purchased from Vital River Laboratory). When the tumor volume reached approximately 40 mm 3 , the mice were randomly divided into 6 groups and administered immunoconjugates including L1IF WT, L1IF M205, and L1IFM205 DAR1 according to the Figure 8 shown protocol.

[0375] As Figure 8 shown, in the model, L1IF WT, L1IF M205, and L1IF M205 DAR1 all showed stronger TGI anti-tumor efficacy compared to the Tecentriq group, and the anti-tumor efficacy of L1IF WT was superior to the Tecentriq plus pegylated IFNα2b group (TGI 47.81% vs. 18.10%). At a dose of 3 mg / kg, L1IF M205DAR1 showed more excellent anti-tumor efficacy compared to L1IF WT (TGI 66.73% vs. 47.81%), indicating that the restoration of L1IF M205 DAR1 improved the anti-tumor activity.

[0376] Example 9. Toxicity of Immunoconjugates

[0377] The in vivo toxicity of the immunoconjugates of this application was further evaluated. As Figure 9 shown, after administration, L1IFM205DAR1 showed more stable lymphocyte and platelet counts, indicating a reduction in hematological toxicity induced by L1IF M205 DAR1.

[0378] Example 10. Bifunctional Polypeptides with a New Anti-PD-L1 Antibody

[0379] In this example, a novel anti-PD-L1 nanobody (112_08) was generated and used to prepare immunoconjugates similar to those described above. The 112_08 nanobody has the amino acid sequence of SEQ ID NO:49.

[0380] Two forms of bifunctional polypeptides were prepared, as Figure 10 shown, and their sequences are shown in Table 6 below. One of them (112_08-IFM) is symmetric and contains two copies of the IFNα2b S152C variant at the C-terminus of the Fc fragment. The other (112_08-IFM-knob) contains a single IFNα2b S152C variant and a knob-into-hole substitution to reduce mismatching. Immunoconjugates based on these two bifunctional polypeptides were prepared, each containing a single chemical moiety of 112_08-IFM DAR1 and 112_08-IFM-knob DAR1.

[0381] Table 6. Amino acid sequences of immunoconjugates

[0382]

[0383]

[0384] As described above, the activities of these bifunctional polypeptides and the corresponding immunoconjugates were tested using ELISA, IFNα reporter gene assay, and Daudi cell binding assay. As Figure 11 shown, the bifunctional polypeptides exhibited high activity, while the immunoconjugates showed significantly reduced affinity for IFNAR2.

[0385] 112_08-IFM-knob was also tested for binding and inhibition of PD-L1. Encouragingly, it showed stronger binding and higher inhibitory activity than atezolizumab ( Figure 12 ).

[0386] In addition, the in vivo antitumor activities of these bifunctional polypeptides and immunoconjugates were tested in the CB-17 SCID RKO model (see the method in Example 8). As Figure 13 and Table 5 shown, at least the asymmetric 112_08-IFM-knob and the two immunoconjugates were superior to the combination of atezolizumab and PEG-IFNα.

[0387] Table 5. In vivo antitumor efficacy

[0388] Treatment TGI(%) Day 17 PBS / Tecentriq + PEG-IFNα 45 112_08-IFM 27 112_08-IFM-DAR1 48 112_08-IFM-knob 54 112_08-IFM-knob-DAR1 62

[0389] Therefore, this example demonstrated the excellent performance of the new anti-PD-L1 nanobody in bifunctional polypeptides and immunoconjugates.

[0390] ***

[0391] The present disclosure has been described in terms of specific embodiments found or proposed by the inventors to include preferred ways of practicing the present disclosure. Those skilled in the art will understand that, in accordance with the present disclosure, many modifications and changes can be made in the specific embodiments of the examples without departing from the intended scope of the present disclosure. For example, due to codon redundancy, changes can be made to the underlying DNA sequence without affecting the protein sequence. In addition, based on considerations of biological functional equivalence, changes can be made to the protein structure without affecting the nature or degree of biological action. All such modifications are intended to be included within the scope of the appended claims.

[0392] Sequence Listing

[0393]

[0394]

[0395]

[0396]

[0397]

Claims

1. A polypeptide comprising IFNα2b or a functional fragment thereof, wherein the IFNα2b or the functional fragment thereof is modified compared to the wild-type IFNα2b protein to attenuate the binding affinity to the interferon α / β receptor, wherein (a) the polypeptide is a multifunctional polypeptide, or (b) the polypeptide is conjugated to a chemical moiety.

2. The polypeptide according to claim 1, wherein the interferon α / β receptor is selected from IFNAR1, IFNAR2, and combinations thereof.

3. The polypeptide according to claim 1 or 2, wherein the binding affinity of the IFNα2b or its functional fragment to the interferon α / β receptor is reduced by at least 10-fold, 10 2 -fold, 10 3 -fold, 10 4 -fold or 10 5 -fold.

4. A polypeptide comprising IFNα2b or a functional fragment thereof, wherein the IFNα2b or the functional fragment thereof is modified compared to the wild-type IFNα2b protein to attenuate the activity of inducing type I interferon signaling.

5. The polypeptide according to claim 4, wherein the activity of inducing type I interferon signal transduction is reduced by at least 10-fold, 10 2 -fold, 10 3 -fold, 10 4 -fold or 10 5 -fold.

6. The polypeptide according to any one of claims 1-5, wherein the IFNα2b or the functional fragment thereof is modified by amino acid substitution at one or more residues corresponding to the residues selected from R12, L15, M16, R22, L26, F27, L30, R33, H34, D35, A145, M148, R149, S152, L153, and N156 of SEQ ID NO:

1.

7. The polypeptide according to any one of claims 1-6, wherein the modification comprises an amino acid substitution at the residue corresponding to S152 of SEQ ID NO:

1.

8. The polypeptide according to any one of claims 1-7, wherein the modification comprises an amino acid substitution with cysteine at the residue corresponding to S152 of SEQ ID NO:

1.

9. The polypeptide according to any one of claims 1-8, wherein the IFNα2b comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO:1 or SEQ ID NO:

2.

10. The polypeptide according to any one of claims 1-9, wherein the IFNα2b or the functional fragment thereof is conjugated to a chemical moiety.

11. The polypeptide according to claim 10, wherein the chemical moiety comprises a structure of formula (I): R1-R2-R3-R4 (I), wherein, R1 is optionally further substituted by 1, 2 or 3 R* groups; L1 is selected from CH or N; R 1a is absent or selected from -(CH2) 0-3 -C(O)-, -(CH2) 0-3 -OC(O)- and -(CH2) 0-3 -NHC(O)-; Each R 1b independently does not exist, or is independently selected from -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)NH-, -NH-C 1-6 alkyl-, -O-C 1-6 alkyl-, -C(O)-C 1-6 alkyl-, -OC(O)-C 1-6 alkyl-, -C(O)O-C 1-6 alkyl- and -C(O)NH-C 1-6 alkyl-; Each R 1c is independently selected from H, C 1-6 alkyl, and C 1-6 haloalkyl; m is an integer ranging from 1 to 30000; R* is selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 2-6 alkenyl or C 2-6 alkynyl; R2 is a linker cleavable in the tumor environment; R3 is X is selected from -NH- or -O-; Y is selected from -NH-, -O-, -C(O)-, -OC(O)-, -C(O)-C(O)-, or -NHC(O)-; Each R x and R y are independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl; n is 0, 1, 2, 3, 4, or 5; R4 is R a and R b each independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, and C 1-6 alkoxy; R 4a is absent or selected from -(CH2) 1-12 -, -(CH2) 1-12 -O-, -O-(CH2) 1-12 -, -O-(CH2) 1-12 -O-, -(CH2) 1-12 -O-(CH2) 1-12 -, (C 1-6 alkylene - O) p -(CH2) 1-12 , -(CH2) 1-12 -C 3-8 cycloalkylene-, -(CH2) 1-12 -C 3-8 cycloalkylene-(CH2) 1-12 , -(CH2) 1-12 -C 6-10 arylene- and -(CH2) 1-12 -C 6-10 arylene-(CH2) 1-12 ; R 4a optionally further substituted by 1, 2, 3, 4 or 5 R# groups; R 4b selected from -NH-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-C(O)-, -C(O)NH- or -NHC(O)-; R is selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 2-6 alkenyl or C 2-6 alkynyl; p is 1, 2, 3, 4, or 5; wherein R3 is linked to R2 through X of R3 and linked to R4 through Y of R3.

12. The polypeptide according to claim 11, wherein, R1 is optionally further substituted by 1, 2 or 3 R* groups; L1 is selected from CH or N; R 1a is absent or selected from -C(O)-, -OC(O)- and -NHC(O)-; Each R 1b independently does not exist or is independently selected from -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)NH-, -NH-C 1-4 alkyl-, -O-C 1-4 alkyl-, -C(O)-C 1-4 alkyl-, -OC(O)-C 1-4 alkyl-, -C(O)O-C 1-4 alkyl- and -C(O)NH-C 1-4 alkyl-; Each R 1c is independently selected from H, C 1-4 alkyl, and C 1-4 haloalkyl; each m is independently an integer ranging from 1 to 5000; R* is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; or, R1 is optionally further substituted by 1, 2 or 3 R* groups; R 1a is absent or is -C(O)-; R 1c selected from H, C 1-4 alkyl and C 1-4 haloalkyl; m is an integer ranging from 1 to 3000; R* is selected from H, halogen or C 1-4 alkyl; or alternatively, R1 is R 1c selected from H, C 1-4 alkyl and C 1-4 haloalkyl, and m is an integer in the range of 1 - 1000, 100 - 500, or 400 - 500; or still alternatively, R1 is a linear polyethylene glycol with a molecular weight of about 20000.

13. The polypeptide according to claim 11 or 12, wherein R2 is selected from a matrix metalloproteinase (MMP) cleavable linker, a metalloprotease cleavable linker containing a disintegrin and metalloprotease (ADAM) domain, a prostate specific antigen (PSA) protease cleavable linker, a urokinase type plasminogen activator (uPA) protease cleavable linker, a membrane type serine protease 1 (MT-SP1) protease cleavable linker, a matriptase (ST14) protease cleavable linker, and a legumain protease cleavable linker.

14. The polypeptide according to claim 13, wherein R2 is a matrix metalloproteinase (MMP) cleavable linker and comprises an amino acid sequence selected from any one of SEQ ID NO: 21-35.

15. The polypeptide according to claim 13, wherein R2 is a prostate specific antigen (PSA) protease cleavable linker and comprises an amino acid sequence selected from any one of SEQ ID NO: 14-16.

16. The polypeptide according to claim 13, wherein R2 is a urokinase type plasminogen activator (uPA) protease cleavable linker and comprises an amino acid sequence selected from any one of SEQ ID NO: 11-13.

17. The polypeptide according to claim 13, wherein R2 is a legumain protease cleavable linker and comprises an amino acid sequence selected from any one of SEQ ID NO: 18-19 or AAN or Cbz-AAN-AMC.

18. The method according to any one of claims 11-13 and 17, wherein the chemical moiety comprises a structure of formula (II): wherein R1, R3, and R4 are as defined in any one of claims 11-13 and 17.

19. The polypeptide according to any one of claims 11-18, wherein R3 is wherein X is selected from -NH- or -O-; Y is selected from -NH-, -O-, -C(O)-, -OC(O)-, or -NHC(O)-; Each R x and R y are independently selected from H, halogen, C 1-4 alkyl or C 1-4 haloalkyl; n is 0, 1, 2, or 3; alternatively, R3 is X is selected from NH or O; Y is selected from -NH-, -O-, -C(O)-, -OC(O)-, or -NHC(O)-.

20. The method according to any one of claims 11-19, wherein the chemical moiety comprises a structure of formula (III): wherein, X is selected from NH or O; Y is selected from -NH-, -O-, -C(O)-, -OC(O)-, or -NHC(O)-; wherein R1, R2, and R4 are as defined in any one of claims 11-19.

21. The polypeptide according to any one of claims 11-20, wherein the chemical moiety comprises a structure of formula (IV): wherein R1 and R4 are as defined in any one of claims 11-20.

22. The polypeptide according to any one of claims 11-21, wherein: R4 is R a and R b each independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, and C 1-6 alkoxy; R 4a is absent or selected from -(CH2) 1-12 -, -(CH2) 1-12 -O-, -O-(CH2) 1-12 -, -O-(CH2) 1-12 -O-, -(CH2) 1-12 -O-(CH2) 1-12 -, (C 1-6 alkylene - O) p -(CH2) 1-12 , -(CH2) 1-12 -C 3-8 cycloalkylene and -(CH2) 1-12 -C 3-8 cycloalkylene-(CH2) 1-12 ; R 4a optionally further substituted by 1, 2, 3 or 4 R# groups; R 4b is -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)NH- or -NHC(O)-; R is selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl; p is 1, 2, 3, or 4; alternatively, R a and R b each independently selected from H and C 1-4 alkyl; R 4a Selected from -(CH2) 1-12 -,-(CH2) 1-12 -O-,-O-(CH2) 1-12 -,-O-(CH2) 1-12 -O-,-(CH2) 1-12 -O-(CH2) 1-12 - and (C 1-4 alkylene - O) p -(CH2) 1-12 ; R 4a optionally further substituted by 1, 2 or 3 R# groups; R 4b is -NH-, -O- or -C(O)-; R is selected from H, halogen, C 1-6 alkyl and C 1-6 haloalkyl; p is 1, 2, or 3; alternatively, R a and R b is H; R 4a is -(CH2) 2-12 -; R 4b is -NH-; R 4a Optionally further substituted by one or two R# groups; R is selected from H, halogen and C 1-4 alkyl group.

23. The polypeptide according to any one of claims 11-22, wherein the chemical moiety comprises a structure of formula (V): wherein R1, R2 and R3 are as defined in any one of claims 11 - 22.

24. The polypeptide according to any one of claims 11 - 23, wherein the chemical moiety comprises a structure of formula (VI): Among them, PEG m is R 1c selected from H, C 1-4 alkyl and C 1-4 haloalkyl, and m is an integer in the range of 1 - 1000, 100 - 500, or 400 - 500.

25. The polypeptide according to claim 24, wherein the PEG m is a linear polyethylene glycol having a molecular weight of about 20,000.

26. The polypeptide according to any one of claims 11 - 25, wherein the IFNα2b or its functional fragment is modified with the chemical moiety at residues corresponding to the residues selected from R12, L15, M16, R22, L26, F27, L30, R33, H34, D35, A145, M148, R149, S152, L153 and N156 residues of SEQ ID NO:

1.

27. The polypeptide according to claim 26, wherein the IFNα2b or its functional fragment comprises an amino acid substitution with cysteine at positions corresponding to the positions selected from R12, L15, M16, R22, L26, F27, L30, R33, H34, D35, A145, M148, R149, S152, L153 and N156 positions of SEQ ID NO:1, and the chemical moiety is linked to the IFNα2b or functional fragment through cysteine.

28. The polypeptide according to claim 27, wherein R4 of the chemical moiety is linked to the S atom of the cysteine residue through maleimide, acetylene, vinyl, monosubstituted maleic acid or disubstituted maleimide.

29. The polypeptide according to any one of claims 1 - 28, wherein the IFNα2b or its functional fragment is conjugated to a biomolecule that specifically binds to a target.

30. The polypeptide according to claim 29, wherein the IFNα2b or its functional fragment is directly conjugated to the biomolecule.

31. The polypeptide according to claim 29, wherein the IFNα2b or its functional fragment is conjugated to the biomolecule through a linker.

32. The polypeptide according to any one of claims 29 - 31, wherein the biomolecule comprises an antibody or an antigen - binding fragment thereof.

33. The polypeptide according to claim 32, wherein the antibody or an antigen - binding fragment thereof is selected from full - length antibody, Fab, Fab’, F(ab’)2, Fd, Fd’, Fv, scFv, ds - scFv, sdAb and nanobody.

34. The polypeptide according to any one of claims 28 - 33, wherein the target is PD - L1 or PD - 1.

35. The polypeptide according to claim 34, wherein the biomolecule comprises an sdAb, the sdAb comprises CDR1, CDR2 and CDR3 of SEQ ID NO:49, and the CDR1, CDR2 and CDR3 are according to the Kabat numbering scheme.

36. The polypeptide according to claim 34, wherein the biomolecule comprises an sdAb, the sdAb comprises: (i) CDR1, which comprises the amino acid sequence of SEQ ID NO:50, (ii) CDR2, which comprises the amino acid sequence of SEQ ID NO:51, and (iii) CDR3, which comprises the amino acid sequence of SEQ ID NO:

52.

37. The polypeptide according to claim 35 or 36, wherein the biomolecule comprises an sdAb, and the sdAb comprises a sequence having at least 80% identity to SEQ ID NO:

49.

38. The polypeptide according to any one of claims 35-37, wherein the biomolecule further comprises an Fc region.

39. A multifunctional polypeptide comprising IFNα2b or a functional fragment thereof according to any one of claims 1-28, and an antibody or an antigen-binding fragment thereof.

40. The multifunctional polypeptide according to claim 39, which comprises a single copy of IFNα2b or a functional fragment thereof.

41. The multifunctional polypeptide according to claim 39 or 40, which comprises an Fc fragment.

42. The multifunctional polypeptide according to claim 41, wherein the IFNα2b or a functional fragment thereof is located on the C-terminal side of the Fc fragment.

43. The multifunctional polypeptide according to any one of claims 39-42, wherein the antibody or an antigen-binding fragment thereof is located on the N-terminal side of the Fc fragment.

44. The multifunctional polypeptide according to any one of claims 41-43, wherein the Fc fragment comprises a knobs-into-holes substitution compared to the corresponding wild-type Fc fragment.

45. The multifunctional polypeptide according to any one of claims 41-44, wherein the antibody or an antigen-binding fragment thereof is specific for a tumor-associated antigen or an immune checkpoint protein.

46. The multifunctional polypeptide according to claim 45, wherein the tumor-associated antigen is selected from EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, and tight junction protein 18.

2.

47. The multifunctional polypeptide according to claim 45, wherein the immune checkpoint protein is selected from PD-1, PD-L1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOS-L, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR, and CD47.

48. The multifunctional polypeptide according to claim 47, wherein the antibody or an antigen-binding fragment thereof is an anti-PD-L1 or anti-PD-1 single-domain antibody (sdAb).

49. The multifunctional polypeptide according to claim 48, wherein the anti-PD-L1 sdAb comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 49, according to the Kabat numbering scheme.

50. The multifunctional polypeptide according to claim 48, wherein the anti-PD-L1 sdAb comprises: (i) CDR1, which comprises the amino acid sequence of SEQ ID NO:50, (ii) CDR2, which comprises the amino acid sequence of SEQ ID NO:51, and (iii) CDR3, which comprises the amino acid sequence of SEQ ID NO:

52.

51. The multifunctional polypeptide according to claim 49 or 50, wherein the anti-PD-L1 sdAb comprises a sequence having at least 80% identity to SEQ ID NO:

49.

52. The multifunctional polypeptide according to any one of claims 39-51, wherein the IFNα2b comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1 or SEQ ID NO:

2.

53. An immunoconjugate comprising the multifunctional polypeptide according to any one of claims 39-52, wherein the multifunctional polypeptide is conjugated to a chemical moiety.

54. The immunoconjugate according to claim 53, wherein the chemical moiety comprises the structure of formula (VI): Among them, PEG m is R 1c selected from H, C 1-4 alkyl and C 1-4 haloalkyl, and m is an integer in the range of 1 - 1000, 100 - 500 or 400 - 500; Or, PEG m is a linear polyethylene glycol with a molecular weight of about 20,000.

55. A polynucleotide encoding the polypeptide according to any one of claims 1-38 or the multifunctional polypeptide according to any one of claims 39-52.

56. A vector comprising the polynucleotide according to claim 55.

57. A host cell comprising the vector according to claim 56.

58. A pharmaceutical composition comprising the polypeptide according to any one of claims 1-38, the multifunctional polypeptide according to any one of claims 39-52, the immunoconjugate according to claim 53 or 54, or the polynucleotide according to claim 55.

59. A method for treating a disorder in a subject in need thereof, comprising administering to the subject an effective amount of the polypeptide according to any one of claims 1-38, the multifunctional polypeptide according to any one of claims 39-52, the immunoconjugate according to claim 53 or 54, or the polynucleotide according to claim 55.

60. The method according to claim 59, wherein the disorder is cancer.

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