Interferon α2b mutant, fusion polypeptide comprising same, and use

Abstract

An interferon α2b mutant, and a fusion polypeptide and polypeptide construct comprising same. The fusion polypeptide or polypeptide construct comprising the interferon α2b mutant has weakened affinity to an interferon receptor, reduced off-target toxicity, and a tumor killing effect.

Description

Interferon α2b mutants, fusion peptides containing them, and their uses Technical Field

[0001] This disclosure relates to the field of protein drugs, and more specifically, to an interferon α2b (IFNα2b) mutant and a fusion polypeptide comprising the same, and its use in the treatment of tumors. Background Technology

[0002] Anti-PD-1 antibodies are immunotherapies approved for cancer treatment in recent years, demonstrating certain clinical benefits in the treatment of different types of cancer. However, due to CD8... + The immunosuppressive receptor FcγRIIB on the surface of T cells binds to the Fc fragment of anti-PD-1 antibodies. Up to 40% of patients do not respond to immune checkpoint inhibitors (Bennion KB et al., Science Translational Medicine, 2023, 15(710)). Overall, only about 20% of patients are able to obtain long-term benefits after administration of anti-PD-1 antibodies.

[0003] IFNα2b is a highly active cytokine protein belonging to the IFNα family. It has been approved for use as a drug to treat renal cell carcinoma. It can act directly or indirectly on tumor cells, inhibiting their proliferation and inducing apoptosis. However, its clinical application is limited because IFNα2b may also bind to other cells (leading to off-target toxicity) and trigger non-specific inflammatory responses.

[0004] US10544199B2 describes a fusion protein designed to combine a deglycosylated and inactivated IFNα2b mutant with a cell surface-associated antigen-binding peptide, aiming to reduce the off-target toxicity of IFNα-based drugs while retaining their on-target therapeutic effects. However, the dosage of the fusion protein TAK-573, involving the inactivated IFNα2b mutant and an anti-CD38 antibody, is currently limited in clinical applications, and the therapeutic window needs to be improved. Summary of the Invention

[0005] This disclosure describes the fusion of an affinity-weakened IFNα2b mutant with an anti-PD-1 antibody, rather than a tumor-associated antigen, and demonstrates its high activity on PD-1-positive cells and the difference in selectivity between PD-1-positive and negative cells, potentially targeting more tumor types. Currently, there are no PD-1 and IFNα2b fusion proteins in clinical trials. Since PD-1 expression is very low in normal tissues, it is beneficial to reduce off-target toxicity and increase tolerable dose. This disclosure improves the druggability and activity of the fusion protein by mutating near, rather than directly, the glycosylation site of IFNα2b or by removing O-glycosylation through CD loop sequence substitution.

[0006] This disclosure, through screening different mutation sites and combinations of IFNα2b and different molecular forms of fusion peptides, can obtain fusion peptide molecules with good drug-like properties, high activity, and a larger therapeutic window.

[0007] In one aspect, this disclosure relates to an IFNα2b mutant, wherein, compared to human wild-type IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant comprises:

[0008] (a) A mutation at position 105, and mutations at any one or more of the following positions: position 15, position 19, position 26, position 30, position 33, position 57, position 58, position 61, position 80, position 120, position 142, position 145, position 146, position 148, position 149;

[0009] (b) A mutation at position 145, and a mutation at any one or more of the following positions: position 105, position 107, position 108, position 109, the CD ring sequence or a portion thereof;

[0010] (c) A mutation selected from any one or more of the following positions: position 105, position 106, position 108, position 109, a mutation in the CD ring sequence or a portion thereof; or

[0011] (d) Mutations selected from any one or more of the following positions: position 15, position 26, position 30, position 142, position 148, position 149.

[0012] In one aspect, this disclosure relates to the use of the IFNα2b mutant in the preparation of fusion peptides, for example, said fusion peptides comprising an anti-PD-1 antibody or a portion thereof.

[0013] In one aspect, this disclosure relates to a fusion polypeptide comprising: (1) an IFNα2b mutant, and (2) an anti-PD-1 antibody or a portion thereof, wherein, compared to human wild-type IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant comprises:

[0014] (a) A mutation at position 105, and mutations at any one or more of the following positions: position 15, position 19, position 26, position 30, position 33, position 57, position 58, position 61, position 80, position 120, position 142, position 146, position 148, position 149;

[0015] (b) A mutation at position 145, and a mutation at any one or more of the following positions: position 105, position 107, position 108, position 109, the CD ring sequence or a portion thereof;

[0016] (c) A mutation selected from any one or more of the following positions: position 105, position 106, position 108, position 109, a mutation in the CD ring sequence or a portion thereof; or

[0017] (d) Mutations selected from any one or more of the following positions: position 15, position 26, position 30, position 142, position 148, position 149.

[0018] In one aspect, this disclosure relates to a polypeptide construct comprising:

[0019] (1) Peptide chain 1, from N-terminus to C-terminus, includes: the heavy chain variable region (VH), CH1 and Fc regions of the anti-PD-1 antibody, and the IFNα2b mutant; and

[0020] Peptide chain 2, from N-terminus to C-terminus, sequentially includes the light chain variable region (VL) and CL of the anti-PD-1 antibody;

[0021] (2) Peptide chain 1, from N-terminus to C-terminus, contains: the heavy chain variable region (VH), CH1 and Fc region of the anti-PD-1 antibody, and the IFNα2b mutant;

[0022] Peptide chain 2, from N-terminus to C-terminus, sequentially includes the light chain variable region (VL) and CL of the anti-PD-1 antibody; and

[0023] Peptide chain 3, from N-terminus to C-terminus, sequentially includes the VH, CH1 and Fc regions of the anti-PD-1 antibody;

[0024] (3) The peptide chain 1' contains, from the N-terminus to the C-terminus, the Fc region of the IFNα2b mutant and the anti-PD-1 antibody;

[0025] Peptide chain 2, from N-terminus to C-terminus, sequentially comprises the VL and CL of the anti-PD-1 antibody; and

[0026] Peptide chain 3, from N-terminus to C-terminus, sequentially includes the VH, CH1, and Fc regions of the anti-PD-1 antibody; or

[0027] (4) The peptide chain 1' contains, from the N-terminus to the C-terminus, the Fc region of the IFNα2b mutant and the anti-PD-1 antibody;

[0028] Peptide chain 2, from N-terminus to C-terminus, sequentially comprises the VL and CL of the anti-PD-1 antibody; and

[0029] The peptide chain 3' contains, from the N-terminus to the C-terminus, the VH, CH1, VH, CH1 and Fc regions of the anti-PD-1 antibody;

[0030] Among them, the IFNα2b mutant, compared with human wild-type IFNα2b (e.g., wild-type IFNα2b containing the amino acid sequence shown in SEQ ID NO:4), comprises:

[0031] (a) A mutation at position 105, and mutations at any one or more of the following positions: position 15, position 19, position 26, position 30, position 33, position 57, position 58, position 61, position 80, position 120, position 142, position 146, position 148, position 149;

[0032] (b) A mutation at position 145, and a mutation at any one or more of the following positions: position 105, position 107, position 108, position 109, the CD ring sequence or a portion thereof;

[0033] (c) A mutation selected from any one or more of the following positions: position 105, position 106, position 108, position 109, a mutation in the CD ring sequence or a portion thereof; or

[0034] (d) Mutations selected from any one or more of the following positions: position 15, position 26, position 30, position 142, position 148, position 149.

[0035] In one aspect, this disclosure relates to a polynucleotide, wherein the polynucleotide encodes the IFNα2b mutant, the fusion polypeptide, or the polypeptide construct.

[0036] In one aspect, this disclosure relates to a recombinant vector comprising the polynucleotide, or encoding the IFNα2b mutant, the fusion polypeptide, or the polypeptide construct.

[0037] In one aspect, this disclosure relates to a recombinant cell comprising the polynucleotide or recombinant vector, or expressing the IFNα2b mutant, the fusion polypeptide, or the polypeptide construct.

[0038] In one aspect, this disclosure relates to a pharmaceutical composition comprising: the IFNα2b mutant, the fusion polypeptide or the polypeptide construct, and optionally pharmaceutically acceptable excipients.

[0039] In one aspect, this disclosure relates to the use of the aforementioned IFNα2b mutant, fusion peptide, peptide construct, or pharmaceutical composition in the preparation of a medicament for treating tumors. Alternatively, this disclosure relates to a method of treating a tumor in a subject of need, comprising administering to the subject a therapeutically effective amount of the IFNα2b mutant, the fusion peptide, the peptide construct, or the pharmaceutical composition. Alternatively, this disclosure relates to an IFNα2b mutant, fusion peptide, peptide construct, or pharmaceutical composition for treating tumors. Alternatively, this disclosure relates to the use of the aforementioned IFNα2b mutant, fusion peptide, peptide construct, or pharmaceutical composition for treating tumors. Beneficial effects

[0040] Compared with existing technologies, the fusion peptide in this disclosure has weaker receptor affinity, lower off-target toxicity in in vitro experiments, comparable on-target activity, greater selectivity for both PD-1 positive and PD-1 negative cells, and better tolerability in vivo. This study confirms that the mouse alternative molecule of the fusion peptide described herein exhibits tumor-killing effects in different tumor models in vivo, showing reduced off-target toxicity, higher tolerable doses in animals, and a prolonged in vivo half-life, indicating that the fusion peptide described herein has the potential to effectively treat tumors. Attached Figure Description

[0041] Figure 1 shows the molecular form of the IFNα2b fusion peptide.

[0042] Figure 2 shows the crystal structure of the IFNα2b complex with the IFNα / β receptor (composed of IFNAR1 and IFNAR2 subunits).

[0043] Figures 3A-3D illustrate the different αPD1-IFNα2b-m fusion peptides for HEK-Blue. TM IFN-α / β reporter cell line signaling activation status.

[0044] Figures 4A-4F show the effects of different αPD1-IFNα2b-m fusion peptides on pSTAT1 (also known as "P-STAT1") signaling in activated T lymphocytes.

[0045] Figures 5A and 5B show the results of the PD1 blocking assay for the αPD1-IFNα2b-m fusion peptide.

[0046] Figure 6 shows the detection results of CXCL10 release from PBMC cells stimulated by the αPD1-IFNα2b-m fusion peptide.

[0047] Figure 7 illustrates the effect of the αPD1-IFNα2b mouse alternative molecule (αmPD1-IFNα2b-m) on B16-Blue. TM IFN-α / β reporter cell line signaling activation status.

[0048] Figure 8 illustrates the antitumor effect of the αPD1-IFNα2b mouse substitute molecule on the B16F10 tumor-bearing mouse model.

[0049] Figure 9 shows the effect of the αPD1-IFNα2b mouse substitute molecule on the change in body weight in a B16F10 tumor-bearing mouse model.

[0050] Figure 10 shows the effect of the αPD1-IFNα2b mouse substitute molecule on the survival of the B16F10 tumor-bearing mouse model.

[0051] Figure 11 illustrates the antitumor effect of the αPD1-IFNα2b mouse substitute molecule on the EMT6 tumor-bearing mouse model.

[0052] Figure 12 shows the effect of the αPD1-IFNα2b mouse substitute molecule on the change in body weight in the EMT6 tumor-bearing mouse model.

[0053] Figure 13 illustrates the antitumor effect of the αPD1-IFNα2b mouse substitute molecule on the Pan02 tumor-bearing mouse model.

[0054] Figure 14 shows the effect of the αPD1-IFNα2b mouse substitute molecule on the change in body weight in the Pan02 tumor-bearing mouse model.

[0055] Figure 15 illustrates the antitumor effect of the αPD1-IFNα2b mouse substitute molecule on the Pan02 tumor-bearing mouse model.

[0056] Figure 16 shows the effect of the αPD1-IFNα2b mouse substitute molecule on the change in body weight in the Pan02 tumor-bearing mouse model.

[0057] Figure 17 shows the effect of the αPD1-IFNα2b mouse substitute molecule on the weight change of C57 mice.

[0058] Figure 18 shows the effect of the αPD1-IFNα2b mouse substitute molecule on the survival of C57 mice.

[0059] Figure 19 illustrates the antitumor effect of the αPD1-IFNα2b mouse substitute molecule on the Hepa1.6 tumor-bearing mouse model.

[0060] Figure 20 shows the effect of the αPD1-IFNα2b mouse substitute molecule on the change in body weight in the Hepa1.6 tumor-bearing mouse model.

[0061] Figure 21 illustrates the antitumor effects of combined administration of the αPD1-IFNα2b mouse substitute molecule and Bevacizumab, as well as the αPD1-IFNα2b mouse substitute molecule alone, on the Pan02 tumor-bearing mouse model.

[0062] Figure 22 shows the effects of combined administration of αPD1-IFNα2b mouse substitute molecule and Bevacizumab, as well as administration of αPD1-IFNα2b mouse substitute molecule alone, on the changes in body weight in the Pan02 tumor-bearing mouse model.

[0063] Figure 23 shows the effect of combined administration of αPD1-IFNα2b mouse substitute molecule and Bevacizumab, as well as administration of αPD1-IFNα2b mouse substitute molecule or Bevacizumab alone, on the survival of the Pan02 tumor-bearing mouse model.

[0064] Figure 24 shows the pharmacokinetic results of the αPD1-IFNα2b-m fusion peptide in a cynomolgus monkey model. Detailed Implementation

[0065] The present application has been described in detail below with reference to embodiments, but this does not imply any adverse limitations on the present application. The present application has been described in detail herein, and specific embodiments thereof have been disclosed. For those skilled in the art, various changes and modifications to the specific implementation methods of the present application without departing from the spirit and scope of the present application are also included within the scope of protection of the present application.

[0066] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Perbal, A Practical Guide to Molecular Cloning (1984).

[0067] In this document, unless the context clearly indicates otherwise, the term "or" is intended to include "and" and vice versa. In this document, unless otherwise stated, singular terms cover plural referents and vice versa.

[0068] In this document, unless otherwise stated, the terms “comprise”, “comprises”, and “comprising” or their equivalents (e.g., contain, containing, include, including) are open-ended expressions and should be understood as “including but not limited to”, meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be covered.

[0069] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0070] As used herein, the terms “fusion polypeptide” and “fusion protein” are used interchangeably and refer to a polypeptide construct formed by the optional fusion of at least two heterologous polypeptide sequences through a linker (i.e., with or without a linker).

[0071] Unless otherwise stated, the term "subject" as used herein encompasses any vertebrate, such as mammals, including humans, non-human primates, sheep, dogs, cats, horses, cattle, chickens, pigs, rats, etc. Preferably, the subject in this application is a human.

[0072] Unless otherwise stated, the term "treatment" as used herein means that, after administration, it can inhibit, suppress, reduce, improve, alleviate, relieve, or eliminate a disease or its related symptoms; it can delay, slow, stop, or terminate the progression of a disease or its related symptoms; or it can prevent, control, or reduce the recurrence of a disease or its related symptoms. For the prevention and treatment of disease, the effective therapeutic dose can be determined by a clinician based on the subject's individual condition, disease severity, sex, age, weight, route of administration, etc., using routine methods or experience.

[0073] The "percentage of amino acid sequence identity" refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the reference sequence, relative to the total number of amino acid residues in the reference sequence, after aligning a candidate sequence with a reference sequence and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. The percentage of amino acid sequence identity can be determined by aligning two or more sequences using tools known in the art, such as BLASTp, ClustalW2 (see Higgins DG et al., Methods Enzymol 1996, 266:383-402; Larkin MA et al., Bioinformatics 2007, 23:2947-2948), ALIGN, or Megalign (DNASTAR) software.

[0074] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene that is capable of binding to an antigen. The term "complementarity-determining region" can refer to a portion of the variable region of an antibody that confers antigen-binding specificity, and can refer to an amino acid sequence found in the highly variable region of the heavy or light chain of an immunoglobulin. The heavy chain may include three complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3); and the light chain may include three complementarity-determining regions (CDRs) (LCDR1, LCDR2, and LCDR3). These CDRs can provide contact residues that play an important role in the binding of the antibody to its antigen or antigenic epitope.

[0075] The term "half antibody" refers to a molecule assembled from one heavy chain and one light chain of a monoclonal antibody.

[0076] The Fab fragment refers to a fragment containing the complete light chain and the VH and CH1 domains of the heavy chain, possessing antigen-binding activity. The Fc fragment refers to a fragment containing the CH2 and CH3 domains that can form crystals at low temperatures or low ionic strength. The F(ab')2 fragment refers to a fragment containing the complete light chain, the VH and CH1 domains of the heavy chain, and the hinge region, produced by the cleavage of IgG at the carboxyl side of the disulfide bond in the hinge region during pepsin digestion, possessing antigen-binding activity.

[0077] Unless otherwise stated, the terms "optional" or "optionally" as used herein mean that the object or event it modifies exists or does not exist, or occurs or does not occur. For example, "optionally contains A" means that A is included or not included.

[0078] "Pharmaceutical acceptable" means that compounds, materials, compositions, and / or dosage forms are suitable for use in contact with human and animal tissues, within the limits of reliable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0079] The terms "peptide chain 1," "peptide chain 2," "peptide chain 3," and peptide chain n (n is any value) or similar terms like "first chain" and "second chain" are used only to number the peptide chains for easier and clearer description, and are not intended to indicate the order of the peptide chains. When multiple "peptide chains 1" appear in the same paragraph or section, for example, to facilitate differentiation, they can be named as peptide chain 1, peptide chain 1', etc. Similarly, "peptide chain 2," "peptide chain 3," etc., can also be named for the same purpose. When multiple peptide chains exist, they can be named peptide chain 2, peptide chain 2', etc., and peptide chain 3, peptide chain 3', etc.

[0080] The terms "peptide construct" and "fusion peptide" in this article have essentially the same meaning, referring to a peptide product formed by the combination of multiple peptide chains.

[0081] The term “CDR” (complementarity-determining region), also known as “hypervariant region (HVR)”, as used in this article, refers to each region of an antibody variable domain that is highly variable in sequence and / or forms a structurally defined loop. Natural antibodies typically contain three CDRs (i.e., HCDR1 to HCDR3) located in the heavy chain variable region and three CDRs (LCDR1 to LCDR3) located in the light chain variable region. Several well-known definitions in the art can be used to identify heavy and light chain CDRs, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop; Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987); AbM (University of Bath); Contact (University College London); the International ImMunoGeneTics (IMGT) database (the international ImMunoGeneTics information system, http: / / imgt.cines.fr); and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)). CDRs determined using different numbering schemes are shown in the table below: *Based on Kabat numbering**Based on Chothia / Martin numbering

[0082] Next, the technical solutions of this disclosure will be described with the aid of different implementation methods. However, those skilled in the art will understand that the scope of protection of this disclosure is not limited thereto.

[0083] IFNα2b mutant

[0084] Since the affinity and O-glycosylation of wild-type IFNα2b can lead to adverse consequences such as off-target toxicity and non-specific inflammatory responses, reasonable mutation design of wild-type IFNα2b is expected to enhance its clinical application.

[0085] In some embodiments, this disclosure relates to an IFNα2b mutant, wherein, compared to human wild-type IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant comprises:

[0086] (a) A mutation at position 105, and mutations at any one or more of the following positions: position 15, position 19, position 26, position 30, position 33, position 57, position 58, position 61, position 80, position 120, position 142, position 145, position 146, position 148, position 149;

[0087] (b) A mutation at position 145, and a mutation at any one or more of the following positions: position 105, position 107, position 108, position 109, the CD ring sequence or a portion thereof;

[0088] (c) A mutation selected from any one or more of the following positions: position 105, position 106, position 108, position 109, a mutation in the CD ring sequence or a portion thereof; or

[0089] (d) Mutations selected from any one or more of the following positions: position 15, position 26, position 30, position 142, position 148, position 149.

[0090] The mutations described herein include substitutions, deletions, etc., of amino acids at certain positions in the amino acid sequence relative to wild-type IFNα2b. These mutations can result in reduced affinity for IFNARs (including IFNAR1 and / or IFNAR2) and / or partial or complete removal of O-glycosylation.

[0091] In some embodiments, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant, in addition to the amino acid substitution mutation at V105, also comprises amino acid substitution mutations at at least one of the following positions: L15, A19, L26, L30, R33, H57, E58, Q61, L80, R120, V142, A145, E146, M148, R149.

[0092] In some embodiments, the IFNα2b mutant comprises, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b containing the amino acid sequence shown in SEQ ID NO:4), the following mutation: V105E, and at least one mutation selected from:

[0093] L15A, L15E, L15Q, L15S, L15V, L15G, A19G, L26A, L30V, L30G, L30I, L30S, L30Q, R33A, R33K, R33H, R33N, R33S, H57Y, E58N, Q61S, L80A, R120G, R120A, V142A, V142D, V142E, V142G, V142H, V142I, V1 42K, V142L, V142M, V142N, V142Q, V142R, V142S, V142T, V142Y, A145D, E146A, E146G, E146H, E146I, E146K, E146L, E146R, E146S, E146T, M148A, M148D, M148G, M148K, M148S, R149E, R149G, or R149S.

[0094] In some embodiments, the IFNα2b mutant comprises a combination of V105E and at least one of the following mutations, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4):

[0095] (1) L15A, L15E, L15Q, L15S, L15V or L15G;

[0096] (2) L30V, L30G, L30I, L30S or L30Q;

[0097] (3) R33A, R33K, R33H, R33N or R33S;

[0098] (4) R120G;

[0099] (5) V142A, V142D, V142E, V142G, V142H, V142I, V142K, V142L, V142M, V142N, V142Q, V142R, V142S, V142T or V142Y;

[0100] (6)A145D;

[0101] (7) E146A, E146G, E146H, E146I, E146K, E146L, E146R, E146S or E146T;

[0102] (8) M148A, M148D, M148G, M148K or M148S;

[0103] (9) R149E, R149G, or R149S; or

[0104] (10) L15G+A19G, L15A+V142A, L15A+V142N, L15A+L26A+V142A, L15G+M148G, L26A+V142A, R33A+H57Y+E58N+Q61S, V142A+E146A, L26A+V142A+L80A or L26A+V142A+R120A.

[0105] In some embodiments, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant, in addition to the amino acid substitution mutation at A145, also comprises amino acid substitution mutations at at least one of the following positions: V105, E107, T108, P109, CD loop sequence or a portion thereof.

[0106] In some embodiments, the IFNα2b mutant comprises, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b containing the amino acid sequence shown in SEQ ID NO:4), the following mutation: A145D, and at least one mutation selected from:

[0107] V105E, E107D, E107K, P109A, P109R, P109N, T108A, T108I, T108G, and the CD ring sequence “IQGVGVTETPLMKED” (SEQ ID NO:174; which may be referred to as “the fragment shown in SEQ ID NO:174”) are replaced with “EEKLEKEDFTRGKLM” (SEQ ID NO:175); “IQGVGVTETPLMKED” (SEQ ID NO:174) in the CD ring sequence is replaced with “EEKLEKTDFTRGKLM” (SEQ ID NO:176); “GVTETPLM” (SEQ ID NO:177; which may be referred to as “the fragment shown in SEQ ID NO:177”) in the CD ring sequence is replaced with “EKEDFTRG” (SEQ ID NO:178); or “GVTETPLM” (SEQ ID NO:177) in the CD ring sequence is replaced with “EKTDFTRG” (SEQ ID NO:178). ID NO:179).

[0108] In some embodiments, the IFNα2b mutant comprises, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), a combination of A145D and at least one of the following mutations:

[0109] (1) V105E, V105E+P109R or V105E+T108I+P109A;

[0110] (2) E107D, E107K or E107D+T108A;

[0111] (3) T108G, T108A or T108I+P109A;

[0112] (4) P109R or P109N; or

[0113] (5) Replace “IQGVGVTETPLMKED” (SEQ ID NO:174) in the CD ring region sequence with “EEKLEKEDFTRGKLM” (SEQ ID NO:175).

[0114] Replace “IQGVGVTETPLMKED” (SEQ ID NO:174) with “EEKLEKTDFTRGKLM” (SEQ ID NO:176) in the CD ring sequence.

[0115] Replace “GVTETPLM” (SEQ ID NO:177) in the CD ring region sequence with “EKEDFTRG” (SEQ ID NO:178), or

[0116] In the CD ring region sequence, “GVTETPLM” (SEQ ID NO:177) is replaced with “EKTDFTRG” (SEQ ID NO:179).

[0117] In some embodiments, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant contains an amino acid substitution or deletion mutation at at least one of the following positions:

[0118] V105, T106, T108, P109, CD ring sequence or part thereof.

[0119] In some embodiments, the IFNα2b mutant contains at least one mutation compared to wild-type human IFNα2b (e.g., wild-type IFNα2b containing the amino acid sequence shown in SEQ ID NO:4):

[0120] Replace “GVTETPLM” (SEQ ID NO:177) in V105E, T106A, ΔT106, T108I, P109A, P109R, and CD ring sequences with “EKTDFTRG” (SEQ ID NO:179), or replace “IQGVGVTETPLMKED” (SEQ ID NO:174) in CD ring sequences with “EEKLEKTDFTRGKLM” (SEQ ID NO:176).

[0121] In some embodiments, the IFNα2b mutant contains at least one mutation compared to wild-type human IFNα2b (e.g., wild-type IFNα2b containing the amino acid sequence shown in SEQ ID NO:4):

[0122] (1) V105E, V105E+P109R or V105E+T108I+P109A;

[0123] (2) T106A or ΔT106;

[0124] (3) P109R or T108I+P109A; or

[0125] (4) Replace “GVTETPLM” (SEQ ID NO:177) in the CD ring region sequence with “EKTDFTRG” (SEQ ID NO:179), or

[0126] In the CD ring region sequence, “IQGVGVTETPLMKED” (SEQ ID NO:174) is replaced with “EEKLEKTDFTRGKLM” (SEQ ID NO:176).

[0127] In some embodiments, the IFNα2b mutant, compared to human wild-type IFNα2b, contains at least one of the following mutations:

[0128] (1)L26A+V142A;

[0129] (2) M148K;

[0130] (3)R149G;

[0131] (4) L15G+M148G;

[0132] (5) V142L or V142M; or

[0133] (6)L30S.

[0134] Examples of IFNα2b mutations in this article: "L15" means leucine L at position 15; "L15A" means leucine L at position 15 is mutated to alanine A; "L15G+A19G" means a combination of leucine L at position 15 and glycine A at position 19 being mutated to alanine G; "ΔT106" means threonine deletion at position 106; "CD loop region" refers to the loop region connecting the C-helix and D-helix in the human IFNa2b structure.

[0135] In some embodiments, the IFNα2b mutant comprises an amino acid sequence shown in any of SEQ ID NOs:98-173 and 226-227 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0136] In some embodiments, the IFNα2b mutant comprises an amino acid sequence shown in SEQ ID NO:147 or 161 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0137] Compared to wild-type IFNα2b, the IFNα2b mutant of this disclosure exhibits a reduction in the O-glycosylation ratio of more than 10%, more than 20%, more than 50%, or complete de-O-glycosylation; and / or, the IFNα2b mutant of this disclosure has a weakened affinity for the receptor IFNAR, for example, K... D Value > 1×10 -9 M, >1×10 -8 M.

[0138] In some embodiments, this disclosure relates to the use of the IFNα2b mutant in the preparation of fusion peptides (e.g., fusion peptides comprising an anti-PD-1 antibody or a portion thereof). In some embodiments, the fusion peptide is used for the treatment of tumors, such as PD-1-resistant tumors.

[0139] The anti-PD-1 antibody described herein may be any anti-PD-1 antibody known in the art or any anti-PD-1 antibody developed in the future. In some embodiments, preferred anti-PD-1 antibodies will be described below.

[0140] In the fusion peptide, the anti-PD-1 antibody and the IFNα2b mutant may or may not have a linker, such as a flexible linker (including but not limited to (G5)). n (G4S) nRigid joints (including but not limited to (PA)) n P-head, (AP) n ), cuttable joints (including disulfide bonds, etc.), where n is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5 or 6.

[0141] In some embodiments, constructing fusion peptides using the IFNα2b mutant can reduce off-target toxicity, decrease charge heterogeneity, and exhibit greater selectivity for PD-1 positive and PD-1 negative cells, thus demonstrating good tolerability in vivo.

[0142] Fusion peptides and peptide constructs

[0143] In some embodiments, this disclosure relates to a fusion polypeptide comprising: (1) an IFNα2b mutant, and (2) an anti-PD-1 antibody or a portion thereof, wherein, compared to human wild-type IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant comprises:

[0144] (a) A mutation at position 105, and mutations at any one or more of the following positions: position 15, position 19, position 26, position 30, position 33, position 57, position 58, position 61, position 80, position 120, position 142, position 146, position 148, position 149;

[0145] (b) A mutation at position 145, and a mutation at any one or more of the following positions: position 105, position 107, position 108, position 109, the CD ring sequence or a portion thereof;

[0146] (c) A mutation selected from any one or more of the following positions: position 105, position 106, position 108, position 109, a mutation in the CD ring sequence or a portion thereof; or

[0147] (d) Mutations selected from any one or more of the following positions: position 15, position 26, position 30, position 142, position 148, position 149.

[0148] This disclosure provides a peptide construct that, by linking an antibody to a ligand with weakened affinity, can significantly reduce selectivity for antigen-negative cells and thereby exhibit stronger activity against antigen-positive cells (i.e., reduced off-target toxicity and increased on-target activity). In some embodiments, this disclosure relates to a peptide construct comprising an IFNα2b mutant operatively linked to an antibody or a portion thereof (e.g., an anti-PD-1 antibody or a portion thereof), said IFNα2b mutant comprising mutations (including, but not limited to, amino acid substitutions and deletions) that cause it to have weakened affinity for IFNAR and / or at least partially remove O glycosylation. The resulting peptide constructs exhibit stronger selectivity for PD-1 positive cells. Compared to PD-1 negative cells, the peptide constructs described herein show selectivity for PD-1 positive cells that is thousands or even tens of thousands of times higher (e.g., more than 3,000, 6,000, 8,000, 10,000, or 20,000 times higher). They also exhibit comparable or stronger on-target activity and significantly weaker off-target toxicity, and can achieve higher in vivo tolerable doses.

[0149] In some embodiments, this disclosure relates to a polypeptide construct comprising:

[0150] (1) Peptide chain 1 (e.g., two peptide chains 1), from N-terminus to C-terminus, comprises: the heavy chain variable region (VH), CH1 and Fc regions of the anti-PD-1 antibody, and the IFNα2b mutant; and

[0151] Peptide chain 2 (e.g., two peptide chains 2) contains, from the N-terminus to the C-terminus, the light chain variable region (VL) and CL of the anti-PD-1 antibody;

[0152] (2) Peptide chain 1 (e.g., 1 peptide chain 1) contains, from N-terminus to C-terminus, the heavy chain variable region (VH), CH1 and Fc regions of the anti-PD-1 antibody, and the IFNα2b mutant.

[0153] Peptide chain 2 (e.g., two peptide chains 2) sequentially includes, from the N-terminus to the C-terminus, the light chain variable region (VL) and CL of the anti-PD-1 antibody; and

[0154] The peptide chain 3 (e.g., one peptide chain 3) contains, from the N-terminus to the C-terminus, the VH, CH1 and Fc regions of the anti-PD-1 antibody;

[0155] (3) Peptide chain 1' (e.g., 1 peptide chain 1') contains, from N-terminus to C-terminus, the IFNα2b mutant and the Fc region of anti-PD-1 antibody;

[0156] Peptide chain 2 (e.g., one peptide chain 2) comprises, from the N-terminus to the C-terminus, the VL and CL of the anti-PD-1 antibody; and

[0157] Peptide chain 3 (e.g., one peptide chain 3) contains, from the N-terminus to the C-terminus, the VH, CH1, and Fc regions of the anti-PD-1 antibody; or

[0158] (4) The peptide chain 1' contains, from the N-terminus to the C-terminus, the Fc region of the IFNα2b mutant and the anti-PD-1 antibody;

[0159] Peptide chain 2 (e.g., two peptide chains 2), comprising, from the N-terminus to the C-terminus, the VL and CL of the anti-PD-1 antibody; and

[0160] The peptide chain 3' (e.g., one peptide chain 3') contains, from the N-terminus to the C-terminus, the VH, CH1, VH, CH1 and Fc regions of the anti-PD-1 antibody;

[0161] Among them, the IFNα2b mutant, compared with human wild-type IFNα2b (e.g., wild-type IFNα2b containing the amino acid sequence shown in SEQ ID NO:4), comprises:

[0162] (a) A mutation at position 105, and mutations at any one or more of the following positions: position 15, position 19, position 26, position 30, position 33, position 57, position 58, position 61, position 80, position 120, position 142, position 146, position 148, position 149;

[0163] (b) A mutation at position 145, and a mutation at any one or more of the following positions: position 105, position 107, position 108, position 109, the CD ring sequence or a portion thereof;

[0164] (c) A mutation selected from any one or more of the following positions: position 105, position 106, position 108, position 109, a mutation in the CD ring sequence or a portion thereof; or

[0165] (d) Mutations selected from any one or more of the following positions: position 15, position 26, position 30, position 142, position 148, position 149.

[0166] In this document, the anti-PD-1 antibody may be any anti-PD-1 antibody known in the art or any anti-PD-1 antibody developed in the future.

[0167] In some embodiments, the anti-PD-1 antibody may comprise a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, the HCDR1 comprising the amino acid sequence shown in SEQ ID NO:187 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, the HCDR2 comprising the amino acid sequence shown in SEQ ID NO:188 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the HCDR3 comprising the amino acid sequence shown in SEQ ID NO:188. The amino acid sequence shown in NO:189 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0168] The VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:190 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:191 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:190. The amino acid sequence shown in NO:192 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0169] In some embodiments, the anti-PD-1 antibody may comprise:

[0170] (1) HCDR1 having the amino acid sequence shown in SEQ ID NO:187,

[0171] (2) HCDR2 having the amino acid sequence shown in SEQ ID NO:188,

[0172] (3) HCDR3 having the amino acid sequence shown in SEQ ID NO:189,

[0173] (4) LCDR1 having the amino acid sequence shown in SEQ ID NO:190,

[0174] (5) LCDR2 having the amino acid sequence shown in SEQ ID NO:191, and

[0175] (6) LCDR3 having the amino acid sequence shown in SEQ ID NO:192.

[0176] In some embodiments, the anti-PD-1 antibody may comprise:

[0177] (1) The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and / or

[0178] (2) Light chain variable region, comprising an amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0179] In some embodiments, amino acids that differ from the amino acid sequence shown as SEQ ID NO:3 or 6 by at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of sequence identity are located in the backbone region (FR).

[0180] In some embodiments, the anti-PD-1 antibody further comprises a heavy chain constant region (CH) comprising a constant region of human IgG or a variant thereof, such as a constant region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof. In some embodiments, the anti-PD-1 antibody further comprises a constant region of human IgG1 or IgG4 or a variant thereof (e.g., having an S228P mutation, LALA mutation, YTE mutation, LS mutation, and / or one heavy chain constant region is designed to have a knock and the other heavy chain constant region is designed to have a hole). In some embodiments, the anti-PD-1 antibody further comprises a heavy chain constant region having an amino acid sequence shown as SEQ ID NO: 205, 206, 207, 208, 210, 242, 243, 244, 245, 246, or 247.

[0181] In some implementations, the knock-in-hole (KIH) technique can be used, which involves engineered CH3 domains to create a “knob” or “hole” in each heavy chain, thereby promoting heterodimerization. For example, the KIH technique is described in Xu, Yiren, et al., “Production of bispecific antibodies in ‘knobs-into-holes’ using a cell-free expression system,” MAbs. Vol. 7. No. 1, Taylor & Francis, 2015, which is incorporated herein by reference in its entirety. In some implementations, one heavy chain has the S354C and T366W (knob) mutations (EU number), and the other heavy chain has the Y349C, T366S, L368A, and Y407V (hole) mutations (EU number).

[0182] In some embodiments, the anti-PD-1 antibody further comprises a light chain constant region (CL), which comprises a human κ chain or λ chain constant region, preferably a human κ chain constant region. In some embodiments, the anti-PD-1 antibody further comprises a light chain constant region having the amino acid sequence shown in SEQ ID NO:209.

[0183] The anti-PD-1 antibody described herein may contain two identical heavy chains and two identical light chains, or it may contain two different heavy chains and two identical light chains.

[0184] In some embodiments, the anti-PD-1 antibody may comprise:

[0185] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:2, 93, 94, 204, 236, 237, 238, 239, 240 or 241, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; and / or

[0186] (2) A light chain comprising an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0187] In some embodiments, amino acids that differ from the amino acid sequences shown as SEQ ID NO:2, 5, 93, 94, 204, 236, 237, 238, 239, 240, or 241 in sequence identity at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) are located in the backbone region (FR) or constant region.

[0188] In some embodiments, the anti-PD-1 antibody may comprise:

[0189] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and / or

[0190] (2) A light chain comprising an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0191] In some embodiments, the anti-PD-1 antibody may comprise:

[0192] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:240 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and / or

[0193] (2) A light chain comprising an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0194] In some embodiments, the anti-PD-1 antibody may comprise:

[0195] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:241 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and / or

[0196] (2) A light chain comprising an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0197] In some embodiments, the anti-PD-1 antibody may comprise:

[0198] (1) A first heavy chain comprising the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and a second heavy chain comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and / or

[0199] (2) A light chain comprising an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0200] In some embodiments, the anti-PD-1 antibody may comprise:

[0201] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:204 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and / or

[0202] (2) A light chain comprising an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0203] In some embodiments, the anti-PD-1 antibody may comprise:

[0204] (1) A first heavy chain comprising the amino acid sequence shown in SEQ ID NO:238 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and a second heavy chain comprising the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and / or

[0205] (2) A light chain comprising an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0206] In some embodiments, the anti-PD-1 antibody may comprise:

[0207] (1) A first heavy chain comprising the amino acid sequence shown in SEQ ID NO:239 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and a second heavy chain comprising the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and / or

[0208] (2) A light chain comprising an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0209] In this document, CDRs are defined according to the Kabat numbering rules, but CDRs defined according to other numbering rules such as Chothia, AbM, and Contact are also within the scope of protection of this application.

[0210] In some embodiments, a portion of the anti-PD-1 antibody may comprise an antigen-binding fragment or a non-antigen-binding fragment of the antibody. In some embodiments, a portion of the anti-PD-1 antibody may comprise an anti-PD-1 antibody portion lacking one Fab fragment, a half-antibody (i.e., a molecule composed of one heavy chain and one light chain of an anti-PD-1 antibody), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd fragment, an Fv fragment, a dAb fragment, isolated CDR, CH1, CH2, CH3, Fc region, scFv, nanobodies, and any combination thereof.

[0211] In some embodiments, the anti-PD-1 antibody portion may be an anti-PD-1 antibody portion lacking one Fab fragment, a hapten, an Fc region, fused CH2 and CH3 regions, a Fab fragment, an F(ab')2 fragment, or any combination thereof. In some embodiments, the anti-PD-1 antibody portion may be an anti-PD-1 antibody portion lacking one Fab fragment, or an anti-PD-1 antibody portion with one arm lacking one Fab fragment and the other arm additionally fused with one Fab fragment (i.e., wherein one arm of the molecule is the Fc region of the anti-PD-1 antibody, or the fused CH2 and CH3 regions, and the other arm is a hapten fused with the Fab fragment of the anti-PD-1 antibody).

[0212] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0213] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0214] (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0215] (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:195, 196, 197, 252 or 253 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0216] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0217] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0218] (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0219] (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:195, 198, 252 or 253 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0220] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0221] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0222] (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0223] (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0224] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0225] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0226] (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0227] (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0228] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0229] (1) A heavy chain comprising an amino acid sequence shown in SEQ ID NO:238 or 239 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0230] (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0231] (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO: 195, 198, 250, 251, 252 or 253 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0232] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0233] (1) A heavy chain comprising an amino acid sequence shown in SEQ ID NO:236 or 237 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0234] (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0235] (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO: 195, 196, 197, 248, 249, 252 or 253 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0236] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0237] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:238 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0238] (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0239] (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:250 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0240] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0241] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:239 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0242] (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0243] (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:251 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0244] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0245] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0246] (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0247] (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:248 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0248] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0249] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0250] (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0251] (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:249 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0252] In some embodiments, a portion of the anti-PD-1 antibody may comprise: a polypeptide chain containing CH2 and CH3 regions (or Fc regions), two light chains of the anti-PD-1 antibody, and a polypeptide chain comprising a heavy chain of the anti-PD-1 antibody fused to the Fd segment (containing VH and CH1 domains) of the anti-PD-1 antibody.

[0253] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0254] (1) A first polypeptide chain comprising an amino acid sequence shown in SEQ ID NO:195, 196 or 197 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0255] (2) Two second polypeptide chains, each comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0256] (3) A third polypeptide chain comprising the following amino acid sequences linked together in the direction from the N-terminus to the C-terminus: an amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; an amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and an amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0257] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0258] (1) A first polypeptide chain comprising an amino acid sequence shown in SEQ ID NO:195 or 198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0259] (2) Two second polypeptide chains, each comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0260] (3) A third polypeptide chain comprising the following amino acid sequences linked together in the direction from the N-terminus to the C-terminus: an amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity therewith; an amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity therewith; and an amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity therewith.

[0261] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0262] (1) A first polypeptide chain comprising an amino acid sequence shown in SEQ ID NO:195, 196 or 197 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0263] (2) Two second polypeptide chains, each comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0264] (3) A third polypeptide chain comprising the following amino acid sequences linked together in the direction from the N-terminus to the C-terminus: an amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; an amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; a linker; and an amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0265] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0266] (1) A first polypeptide chain comprising an amino acid sequence shown in SEQ ID NO:195 or 198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0267] (2) Two second polypeptide chains, each comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0268] (3) A third polypeptide chain comprising the following amino acid sequences linked together in the direction from the N-terminus to the C-terminus: an amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; an amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; a linker; and an amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0269] In some embodiments, the linker in the third polypeptide chain may be selected from (G5). n (G4S) n Or D(G4S) n Where n is an integer selected from 1 to 5, such as 1, 2, 3, 4, or 5. Preferably, the connector is D(G4S). n n is an integer selected from 1 to 3.

[0270] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0271] (1) A first polypeptide chain comprising an amino acid sequence shown in SEQ ID NO:195, 196 or 197 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0272] (2) Two second polypeptide chains, each comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0273] (3) A third polypeptide chain comprising the following amino acid sequences linked together in the direction from the N-terminus to the C-terminus: the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; the amino acid sequence shown in SEQ ID NO:216 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and the amino acid sequence shown in SEQ ID NO:3. The amino acid sequence shown in NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0274] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0275] (1) A first polypeptide chain comprising an amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0276] (2) Two second polypeptide chains, each comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0277] (3) A third polypeptide chain comprising the following amino acid sequences linked together in the direction from the N-terminus to the C-terminus: the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; the amino acid sequence shown in SEQ ID NO:216 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and the amino acid sequence shown in SEQ ID NO:3. The amino acid sequence shown in NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0278] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0279] (1) A first polypeptide chain comprising an amino acid sequence shown in SEQ ID NO:195 or 198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0280] (2) Two second polypeptide chains, each comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0281] (3) A third polypeptide chain comprising the following amino acid sequences linked together in the direction from the N-terminus to the C-terminus: the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; the amino acid sequence shown in SEQ ID NO:216 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and the amino acid sequence shown in SEQ ID NO:3. The amino acid sequence shown in NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0282] In some embodiments, a portion of the anti-PD-1 antibody comprises:

[0283] (1) A first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0284] (2) Two second polypeptide chains, each comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0285] (3) A third polypeptide chain comprising the following amino acid sequences linked together in the direction from the N-terminus to the C-terminus: the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; the amino acid sequence shown in SEQ ID NO:216 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and the amino acid sequence shown in SEQ ID NO:3. The amino acid sequence shown in NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0286] In some embodiments, the fusion polypeptide may further include a linker for connecting the anti-PD-1 antibody or a portion thereof to the IFNα2b mutant. In some embodiments, the linker is located at the N-terminus or C-terminus of the anti-PD-1 antibody or a portion thereof. In some embodiments, the linker is located at the C-terminus of one or both heavy chains of the anti-PD-1 antibody (i.e., there may be one or two linkers).

[0287] In some embodiments, the fusion polypeptide may comprise: an IFNα2b mutant, a linker, and the portion of the anti-PD-1 antibody described above.

[0288] In some embodiments, the fusion polypeptide comprises an IFNα2b mutant, a linker, and the Fc fragment of the aforementioned anti-PD-1 antibody portion, linked sequentially from the N-terminus to the C-terminus. Alternatively, in some embodiments, the fusion polypeptide comprises the first polypeptide chain of an IFNα2b mutant, a linker, and the aforementioned anti-PD-1 antibody portion, linked sequentially from the N-terminus to the C-terminus.

[0289] In some embodiments, the connector may be selected from a flexible connector (including but not limited to (G5)). n (G4S) n Rigid joints (including but not limited to (PA)) n P, (AP) nThe connector can be cut (including disulfide bonds, etc.), where n is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, or 6. Preferably, the connector can be selected from (G5). n (G4S) n 、or (PA) n P, where n is an integer selected from 1 to 5, such as 1, 2, 3, 4, or 5. In some embodiments, the adapter comprises an amino acid sequence shown in any of SEQ ID NOs:182-186 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; preferably, the adapter comprises an amino acid sequence shown in any of SEQ ID NOs:182-186.

[0290] In some embodiments, the polypeptide construct comprises:

[0291] (1) Peptide chain 1, comprising, from N-terminus to C-terminus, the following: a heavy chain variable region (VH) of the anti-PD-1 antibody, CH1, a partial hinge region and Fc region comprising the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant; and

[0292] Peptide chain 2, from N-terminus to C-terminus, sequentially includes the light chain variable region (VL) and CL of the anti-PD-1 antibody;

[0293] (2) Peptide chain 1, comprising, from N-terminus to C-terminus, the following: heavy chain variable region (VH) of the anti-PD-1 antibody, CH1, a partial hinge region and Fc region comprising the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and an IFNα2b mutant;

[0294] Peptide chain 2, from N-terminus to C-terminus, sequentially includes the light chain variable region (VL) and CL of the anti-PD-1 antibody; and

[0295] Peptide chain 3, from N-terminus to C-terminus, sequentially includes VH, CH1, a portion of the anti-PD-1 antibody, a hinge region and an Fc region containing the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0296] (3) The peptide chain 1' contains, from the N-terminus to the C-terminus, the Fc region of the IFNα2b mutant and the anti-PD-1 antibody;

[0297] Peptide chain 2, from N-terminus to C-terminus, sequentially comprises the VL and CL of the anti-PD-1 antibody; and

[0298] Peptide chain 3, from N-terminus to C-terminus, sequentially comprises VH, CH1, a partial hinge region and Fc region containing the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; or

[0299] (4) The peptide chain 1' contains, from the N-terminus to the C-terminus, the Fc region of the IFNα2b mutant and the anti-PD-1 antibody;

[0300] Peptide chain 2, from N-terminus to C-terminus, sequentially comprises the VL and CL of the anti-PD-1 antibody; and

[0301] The peptide chain 3', from the N-terminus to the C-terminus, sequentially comprises VH, CH1, VH, CH1 of the anti-PD-1 antibody, a partial hinge region and an Fc region comprising the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[0302] Among them, the IFNα2b mutant, compared with human wild-type IFNα2b (e.g., wild-type IFNα2b containing the amino acid sequence shown in SEQ ID NO:4), comprises:

[0303] (a) A mutation at position 105, and mutations at any one or more of the following positions: position 15, position 19, position 26, position 30, position 33, position 57, position 58, position 61, position 80, position 120, position 142, position 146, position 148, position 149;

[0304] (b) A mutation at position 145, and mutations at one or more of the following positions: position 105, position 107, position 108, position 109, the CD ring sequence or a portion thereof; or

[0305] (c) Mutations selected from any one or more of the following positions: position 105, position 106, position 108, position 109, CD ring sequence or a portion thereof.

[0306] In some embodiments, the polypeptide construct comprises:

[0307] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0308] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0309] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0310] The Fc region comprises the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and

[0311] IFNα2b mutant; and

[0312] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0313] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0314] CL comprises an amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0315] In some embodiments, the polypeptide construct comprises:

[0316] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0317] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0318] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0319] The Fc region comprises the amino acid sequence shown in SEQ ID NO: 248, 249, 250, 251, 252 or 253, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and

[0320] IFNα2b mutant; and

[0321] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0322] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0323] CL comprises an amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0324] In some embodiments, the polypeptide construct comprises:

[0325] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0326] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0327] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0328] The Fc region comprises the amino acid sequence shown in SEQ ID NO:252 or 253, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0329] IFNα2b mutant; and

[0330] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0331] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0332] CL comprises an amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0333] In some embodiments, the polypeptide construct comprises:

[0334] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0335] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0336] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0337] A portion of the hinge region contains the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0338] The Fc region comprises the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and

[0339] IFNα2b mutant; and

[0340] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0341] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0342] CL comprises an amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0343] In some embodiments, the polypeptide construct comprises:

[0344] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0345] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0346] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0347] A portion of the hinge region contains the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0348] The Fc region comprises the amino acid sequence shown in SEQ ID NO: 248, 249, 250, 251, 252 or 253, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and

[0349] IFNα2b mutant; and

[0350] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0351] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0352] CL comprises an amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0353] In some embodiments, the polypeptide construct comprises:

[0354] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0355] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0356] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0357] A portion of the hinge region contains the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0358] The Fc region comprises the amino acid sequence shown in SEQ ID NO:252 or 253, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0359] IFNα2b mutant; and

[0360] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0361] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0362] CL comprises an amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0363] In some embodiments, the polypeptide construct comprises:

[0364] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0365] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0366] CH, comprising the amino acid sequence shown in SEQ ID NO: 205, 206, 207, 208 or 210, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and

[0367] IFNα2b mutant; and

[0368] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0369] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0370] CL comprises an amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0371] In some embodiments, the polypeptide construct comprises:

[0372] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0373] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0374] CH, comprising the amino acid sequence shown in SEQ ID NO:242, 243, 244, 245, 246 or 247, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and

[0375] IFNα2b mutant; and

[0376] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0377] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0378] CL comprises an amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0379] In some embodiments, the polypeptide construct comprises:

[0380] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0381] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0382] CH, comprising the amino acid sequence shown in SEQ ID NO:244 or 245, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0383] IFNα2b mutant; and

[0384] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0385] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0386] CL comprises an amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0387] In some embodiments, the polypeptide construct comprises:

[0388] Peptide chain 1, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:2, 93, 94, 204, 236, 237, 238, 239, 240, or 241, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant; and

[0389] Peptide chain 2 comprises an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0390] In some embodiments, the polypeptide construct comprises:

[0391] Peptide chain 1, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant; and

[0392] Peptide chain 2 comprises an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0393] In some embodiments, the polypeptide construct comprises:

[0394] Peptide chain 1, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant; and

[0395] Peptide chain 2 comprises an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0396] In some embodiments, the polypeptide construct comprises:

[0397] Peptide chain 1, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant; and

[0398] Peptide chain 2 comprises an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0399] In some embodiments, the polypeptide construct comprises:

[0400] Peptide chain 1, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:240 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant; and

[0401] Peptide chain 2 comprises an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0402] In some embodiments, the polypeptide construct comprises:

[0403] Peptide chain 1, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:241 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant; and

[0404] Peptide chain 2 comprises an amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0405] In some embodiments, the polypeptide construct comprises:

[0406] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0407] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0408] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0409] The Fc region comprises the amino acid sequence shown in SEQ ID NO: 195, 196, 197, 198, 248, 249, 250, 251, 252, or 253, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0410] IFNα2b mutant;

[0411] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0412] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0413] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0414] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0415] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0416] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0417] The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197, 198, 248, 249, 250, 251, 252 or 253, or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0418] In some embodiments, the polypeptide construct comprises:

[0419] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0420] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0421] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0422] A portion of the hinge region contains the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0423] The Fc region comprises the amino acid sequence shown in SEQ ID NO: 195, 196, 197, 198, 248, 249, 250, 251, 252, or 253, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0424] IFNα2b mutant;

[0425] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0426] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0427] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0428] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0429] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0430] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0431] A partial hinge region comprising the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0432] The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197, 198, 248, 249, 250, 251, 252 or 253, or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0433] In some embodiments, the polypeptide construct comprises:

[0434] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0435] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0436] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0437] The Fc region contains the amino acid sequence shown in SEQ ID NO:195 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0438] IFNα2b mutant;

[0439] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0440] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0441] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0442] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0443] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0444] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0445] The Fc region contains the amino acid sequence shown in SEQ ID NO:195 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0446] In some embodiments, the polypeptide construct comprises:

[0447] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0448] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0449] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0450] The Fc region contains the amino acid sequence shown in SEQ ID NO: 196 or 197, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0451] IFNα2b mutant;

[0452] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0453] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0454] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0455] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0456] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0457] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0458] The Fc region contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0459] In some embodiments, the polypeptide construct comprises:

[0460] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0461] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0462] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0463] The Fc region contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0464] IFNα2b mutant;

[0465] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0466] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0467] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0468] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0469] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0470] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0471] The Fc region contains an amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0472] In some embodiments, the polypeptide construct comprises:

[0473] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0474] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0475] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0476] The Fc region comprises the amino acid sequence shown in SEQ ID NO:248 or 249, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0477] IFNα2b mutant;

[0478] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0479] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0480] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0481] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0482] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0483] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0484] The Fc region contains an amino acid sequence shown in SEQ ID NO:250 or 251 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0485] In some embodiments, the polypeptide construct comprises:

[0486] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0487] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0488] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0489] The Fc region comprises the amino acid sequence shown in SEQ ID NO: 250 or 251, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0490] IFNα2b mutant;

[0491] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0492] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0493] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0494] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0495] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0496] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0497] The Fc region contains an amino acid sequence shown in SEQ ID NO:248 or 249 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0498] In some embodiments, the polypeptide construct comprises:

[0499] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0500] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0501] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0502] The Fc region contains the amino acid sequence shown in SEQ ID NO:248 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0503] IFNα2b mutant;

[0504] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0505] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0506] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0507] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0508] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0509] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0510] The Fc region contains the amino acid sequence shown in SEQ ID NO:250 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0511] In some embodiments, the polypeptide construct comprises:

[0512] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0513] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0514] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0515] The Fc region comprises the amino acid sequence shown in SEQ ID NO:250 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0516] IFNα2b mutant;

[0517] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0518] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0519] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0520] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0521] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0522] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0523] The Fc region contains the amino acid sequence shown in SEQ ID NO:248 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0524] In some embodiments, the polypeptide construct comprises:

[0525] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0526] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0527] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0528] The Fc region contains the amino acid sequence shown in SEQ ID NO:249 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0529] IFNα2b mutant;

[0530] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0531] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0532] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0533] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0534] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0535] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0536] The Fc region contains the amino acid sequence shown in SEQ ID NO:251 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0537] In some embodiments, the polypeptide construct comprises:

[0538] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0539] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0540] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0541] The Fc region contains the amino acid sequence shown in SEQ ID NO:251 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0542] IFNα2b mutant;

[0543] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0544] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0545] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0546] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0547] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0548] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0549] The Fc region contains the amino acid sequence shown in SEQ ID NO:249 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0550] In some embodiments, the polypeptide construct comprises:

[0551] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0552] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0553] CH, comprising the amino acid sequence shown in SEQ ID NO: 205, 206, 207, 208, 210, 242, 243, 244, 245, 246 or 247, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and

[0554] IFNα2b mutant; and

[0555] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0556] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0557] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0558] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0559] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0560] CH, comprising an amino acid sequence shown in SEQ ID NO: 205, 206, 207, 208, 210, 242, 243, 244, 245, 246 or 247, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0561] In some embodiments, the polypeptide construct comprises:

[0562] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0563] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0564] CH, comprising the amino acid sequence shown in SEQ ID NO:205 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0565] IFNα2b mutant; and

[0566] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0567] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0568] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0569] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0570] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0571] CH, comprising the amino acid sequence shown in SEQ ID NO:205 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0572] In some embodiments, the polypeptide construct comprises:

[0573] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0574] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0575] CH, comprising the amino acid sequence shown in SEQ ID NO:206 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0576] IFNα2b mutant; and

[0577] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0578] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0579] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0580] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0581] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0582] CH, comprising the amino acid sequence shown in SEQ ID NO:207 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0583] In some embodiments, the polypeptide construct comprises:

[0584] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0585] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0586] CH, comprising the amino acid sequence shown in SEQ ID NO:207 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0587] IFNα2b mutant; and

[0588] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0589] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0590] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0591] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0592] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0593] CH, comprising the amino acid sequence shown in SEQ ID NO:206 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0594] In some embodiments, the polypeptide construct comprises:

[0595] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0596] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0597] CH, comprising the amino acid sequence shown in SEQ ID NO:210 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0598] IFNα2b mutant; and

[0599] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0600] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0601] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0602] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0603] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0604] CH, comprising the amino acid sequence shown in SEQ ID NO:207 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0605] In some embodiments, the polypeptide construct comprises:

[0606] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0607] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0608] CH, comprising the amino acid sequence shown in SEQ ID NO:207 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0609] IFNα2b mutant; and

[0610] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0611] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0612] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0613] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0614] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0615] CH, comprising the amino acid sequence shown in SEQ ID NO:210 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0616] In some embodiments, the polypeptide construct comprises:

[0617] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0618] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0619] CH, comprising the amino acid sequence shown in SEQ ID NO:242 or 243, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0620] IFNα2b mutant; and

[0621] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0622] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0623] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0624] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0625] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0626] CH, comprising an amino acid sequence shown in SEQ ID NO:246 or 247 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0627] In some embodiments, the polypeptide construct comprises:

[0628] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0629] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0630] CH, comprising the amino acid sequence shown in SEQ ID NO:246 or 247, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0631] IFNα2b mutant; and

[0632] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0633] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0634] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0635] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0636] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0637] CH, comprising an amino acid sequence shown in SEQ ID NO:242 or 243 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0638] In some embodiments, the polypeptide construct comprises:

[0639] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0640] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0641] CH, comprising the amino acid sequence shown in SEQ ID NO:242 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0642] IFNα2b mutant; and

[0643] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0644] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0645] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0646] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0647] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0648] CH, comprising the amino acid sequence shown in SEQ ID NO:246 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0649] In some embodiments, the polypeptide construct comprises:

[0650] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0651] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0652] CH, comprising the amino acid sequence shown in SEQ ID NO:243 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0653] IFNα2b mutant; and

[0654] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0655] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0656] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0657] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0658] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0659] CH, comprising the amino acid sequence shown in SEQ ID NO:247 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0660] In some embodiments, the polypeptide construct comprises:

[0661] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0662] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0663] CH, comprising the amino acid sequence shown in SEQ ID NO:246 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0664] IFNα2b mutant; and

[0665] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0666] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0667] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0668] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0669] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0670] CH, comprising the amino acid sequence shown in SEQ ID NO:242 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0671] In some embodiments, the polypeptide construct comprises:

[0672] (1) Peptide chain 1, from the N-terminus to the C-terminus, contains:

[0673] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0674] CH, comprising the amino acid sequence shown in SEQ ID NO:247 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0675] IFNα2b mutant; and

[0676] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0677] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0678] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0679] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0680] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0681] CH, comprising the amino acid sequence shown in SEQ ID NO:243 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0682] In some embodiments, the polypeptide construct comprises:

[0683] The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:2, 93, 94, 204, 236, 237, 238, 239, 240 or 241 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and an IFNα2b mutant;

[0684] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0685] The peptide chain 3, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:2, 93, 94, 204, 236, 237, 238, 239, 240 or 241, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0686] In some embodiments, the polypeptide construct comprises:

[0687] The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant;

[0688] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0689] Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0690] In some embodiments, the polypeptide construct comprises:

[0691] The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant;

[0692] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0693] Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0694] In some embodiments, the polypeptide construct comprises:

[0695] The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant;

[0696] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0697] Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0698] In some embodiments, the polypeptide construct comprises:

[0699] The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant;

[0700] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0701] Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:238 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0702] In some embodiments, the polypeptide construct comprises:

[0703] The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:238 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant;

[0704] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0705] Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0706] In some embodiments, the polypeptide construct comprises:

[0707] The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant;

[0708] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0709] Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:239 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0710] In some embodiments, the polypeptide construct comprises:

[0711] The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:239 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant;

[0712] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0713] Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0714] In some embodiments, the polypeptide construct comprises:

[0715] The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:240 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant;

[0716] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0717] Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:240 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0718] In some embodiments, the polypeptide construct comprises:

[0719] The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:241 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and an IFNα2b mutant;

[0720] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0721] Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:241 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0722] In some embodiments, the polypeptide construct comprises:

[0723] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0724] IFNα2b mutant,

[0725] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0726] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0727] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0728] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0729] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0730] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0731] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0732] The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0733] In some embodiments, the polypeptide construct comprises:

[0734] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0735] IFNα2b mutant,

[0736] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0737] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0738] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0739] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0740] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0741] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0742] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0743] A partial hinge region comprising the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0744] The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0745] In some embodiments, the polypeptide construct comprises:

[0746] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0747] IFNα2b mutant,

[0748] The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:195 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0749] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0750] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0751] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0752] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0753] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0754] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0755] The Fc region contains the amino acid sequence shown in SEQ ID NO:195 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0756] In some embodiments, the polypeptide construct comprises:

[0757] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0758] IFNα2b mutant,

[0759] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0760] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0761] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0762] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0763] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0764] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0765] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0766] The Fc region contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0767] In some embodiments, the polypeptide construct comprises:

[0768] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0769] IFNα2b mutant,

[0770] The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0771] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0772] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0773] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0774] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0775] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0776] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0777] The Fc region contains an amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0778] In some embodiments, the polypeptide construct comprises:

[0779] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0780] IFNα2b mutant,

[0781] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0782] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0783] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0784] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0785] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0786] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0787] CH, comprising an amino acid sequence shown in SEQ ID NO:205, 206, 207, 208 or 210 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0788] In some embodiments, the polypeptide construct comprises:

[0789] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0790] IFNα2b mutant,

[0791] The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:195 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0792] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0793] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0794] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0795] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0796] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0797] CH, comprising the amino acid sequence shown in SEQ ID NO:205 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0798] In some embodiments, the polypeptide construct comprises:

[0799] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0800] IFNα2b mutant,

[0801] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0802] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0803] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0804] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0805] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0806] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0807] CH, comprising the amino acid sequence shown in SEQ ID NO:207 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0808] In some embodiments, the polypeptide construct comprises:

[0809] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0810] IFNα2b mutant,

[0811] The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0812] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0813] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0814] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0815] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0816] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0817] CH, comprising the amino acid sequence shown in SEQ ID NO:206 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0818] In some embodiments, the polypeptide construct comprises:

[0819] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0820] IFNα2b mutant,

[0821] The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0822] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0823] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0824] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0825] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[0826] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0827] CH, comprising the amino acid sequence shown in SEQ ID NO:210 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0828] In some embodiments, the polypeptide construct comprises:

[0829] The peptide chain 1' comprises, from the N-terminus to the C-terminus, an Fc region of an anti-PD-1 antibody containing an IFNα2b mutant, an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0830] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0831] Peptide chain 3 comprises an amino acid sequence shown in SEQ ID NO:2, 93, 94 or 204 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0832] In some embodiments, the polypeptide construct comprises:

[0833] The peptide chain 1' comprises, from the N-terminus to the C-terminus, an IFNα2b mutant, an Fc region of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:195 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[0834] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0835] Peptide chain 3 comprises an amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0836] In some embodiments, the polypeptide construct comprises:

[0837] The peptide chain 1' comprises, from the N-terminus to the C-terminus, an IFNα2b mutant, an Fc region of an anti-PD-1 antibody comprising the amino acid sequence shown in SEQ ID NO: 196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[0838] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0839] Peptide chain 3 comprises an amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0840] In some embodiments, the polypeptide construct comprises:

[0841] The peptide chain 1' comprises, from the N-terminus to the C-terminus, an IFNα2b mutant, an Fc region of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[0842] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0843] Peptide chain 3 comprises an amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0844] In some embodiments, this disclosure relates to a polypeptide construct comprising:

[0845] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0846] IFNα2b mutant,

[0847] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0848] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0849] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0850] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0851] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0852] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0853] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0854] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0855] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0856] The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0857] In some embodiments, this disclosure relates to a polypeptide construct comprising:

[0858] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0859] IFNα2b mutant,

[0860] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0861] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0862] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0863] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0864] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0865] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0866] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0867] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0868] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0869] A partial hinge region comprising the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0870] The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0871] In some embodiments, this disclosure relates to a polypeptide construct comprising:

[0872] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0873] IFNα2b mutant,

[0874] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0875] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0876] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0877] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0878] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0879] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0880] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0881] The connector is selected from (G5). n (G4S) n Or D(G4S) n n is an integer selected from 1 to 5.

[0882] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0883] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0884] The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0885] In some embodiments, this disclosure relates to a polypeptide construct comprising:

[0886] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0887] IFNα2b mutant,

[0888] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0889] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0890] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0891] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0892] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0893] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0894] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0895] The connector is selected from (G5). n (G4S) n Or D(G4S) n n is an integer selected from 1 to 5.

[0896] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0897] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0898] A partial hinge region comprising the amino acid sequence shown in SEQ ID NO:217 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0899] The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0900] In some embodiments, this disclosure relates to a polypeptide construct comprising:

[0901] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0902] IFNα2b mutant,

[0903] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0904] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0905] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0906] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0907] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0908] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0909] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0910] The connector is a D(G4S) connector. n n is an integer selected from 1 to 3.

[0911] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0912] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0913] The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0914] In some embodiments, this disclosure relates to a polypeptide construct comprising:

[0915] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0916] IFNα2b mutant,

[0917] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0918] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0919] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0920] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0921] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0922] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0923] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0924] A connector comprising the amino acid sequence shown in SEQ ID NO:216.

[0925] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0926] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0927] The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0928] In some embodiments, this disclosure relates to a polypeptide construct comprising:

[0929] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0930] IFNα2b mutant,

[0931] The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:195 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0932] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0933] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0934] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0935] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0936] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0937] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0938] A connector comprising the amino acid sequence shown in SEQ ID NO:216.

[0939] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0940] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0941] The Fc region contains the amino acid sequence shown in SEQ ID NO:195 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0942] In some embodiments, this disclosure relates to a polypeptide construct comprising:

[0943] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0944] IFNα2b mutant,

[0945] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0946] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0947] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0948] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0949] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0950] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0951] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0952] A connector comprising the amino acid sequence shown in SEQ ID NO:216.

[0953] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0954] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0955] The Fc region contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0956] In some embodiments, this disclosure relates to a polypeptide construct comprising:

[0957] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0958] IFNα2b mutant,

[0959] The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0960] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0961] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0962] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0963] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0964] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0965] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0966] A connector comprising the amino acid sequence shown in SEQ ID NO:216.

[0967] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0968] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0969] The Fc region contains an amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0970] In some embodiments, the polypeptide construct comprises:

[0971] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0972] IFNα2b mutant,

[0973] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0974] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0975] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0976] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0977] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0978] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0979] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0980] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0981] CH, comprising an amino acid sequence shown in SEQ ID NO:205, 206, 207, 208 or 210 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0982] In some embodiments, the polypeptide construct comprises:

[0983] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0984] IFNα2b mutant,

[0985] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0986] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[0987] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0988] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0989] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0990] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0991] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0992] The connector is selected from (G5). n (G4S) n Or D(G4S) n n is an integer selected from 1 to 5.

[0993] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[0994] CH, comprising an amino acid sequence shown in SEQ ID NO:205, 206, 207, 208 or 210 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0995] In some embodiments, the polypeptide construct comprises:

[0996] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[0997] IFNα2b mutant,

[0998] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[0999] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[1000] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1001] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1002] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1003] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1004] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1005] The connector is a D(G4S) connector. n n is an integer selected from 1 to 3.

[1006] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1007] CH, comprising an amino acid sequence shown in SEQ ID NO:205, 206, 207, 208 or 210 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[1008] In some embodiments, the polypeptide construct comprises:

[1009] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1010] IFNα2b mutant,

[1011] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1012] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[1013] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1014] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1015] (3) Peptide chain 3, from the N-terminus to the C-terminus, contains:

[1016] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1017] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1018] A connector comprising the amino acid sequence shown in SEQ ID NO:216.

[1019] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1020] CH, comprising an amino acid sequence shown in SEQ ID NO:205, 206, 207, 208 or 210 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[1021] In some embodiments, the polypeptide construct comprises:

[1022] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1023] IFNα2b mutant,

[1024] The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:195 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1025] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[1026] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1027] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1028] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1029] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1030] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1031] A connector comprising the amino acid sequence shown in SEQ ID NO:216.

[1032] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1033] CH, comprising the amino acid sequence shown in SEQ ID NO:205 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1034] In some embodiments, the polypeptide construct comprises:

[1035] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1036] IFNα2b mutant,

[1037] The Fc region of the anti-PD-1 antibody contains an amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1038] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[1039] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1040] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1041] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1042] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1043] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1044] A connector comprising the amino acid sequence shown in SEQ ID NO:216.

[1045] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1046] CH, comprising the amino acid sequence shown in SEQ ID NO:207 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1047] In some embodiments, the polypeptide construct comprises:

[1048] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1049] IFNα2b mutant,

[1050] The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1051] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[1052] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1053] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1054] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1055] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1056] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1057] A connector comprising the amino acid sequence shown in SEQ ID NO:216.

[1058] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1059] CH, comprising the amino acid sequence shown in SEQ ID NO:206 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1060] In some embodiments, the polypeptide construct comprises:

[1061] (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1062] IFNα2b mutant,

[1063] The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1064] (2) Peptide chain 2, from the N-terminus to the C-terminus, contains:

[1065] VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1066] CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1067] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1068] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1069] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1070] A connector comprising the amino acid sequence shown in SEQ ID NO:216.

[1071] VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1072] CH, comprising the amino acid sequence shown in SEQ ID NO:210 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1073] In some embodiments, the polypeptide construct comprises:

[1074] (1) Peptide chain 1', comprising, from N-terminus to C-terminus, an Fc region of an anti-PD-1 antibody containing an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1075] (2) Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1076] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1077] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1078] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and

[1079] The amino acid sequence shown in SEQ ID NO:2, 93, 94 or 204 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[1080] In some embodiments, the polypeptide construct comprises:

[1081] (1) Peptide chain 1', comprising, from N-terminus to C-terminus, an Fc region of an anti-PD-1 antibody containing an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1082] (2) Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1083] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1084] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1085] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1086] The connector is selected from (G5). n (G4S) n Or D(G4S) n n is an integer selected from 1 to 5, and

[1087] The amino acid sequence shown in SEQ ID NO:2, 93, 94 or 204 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[1088] In some embodiments, the polypeptide construct comprises:

[1089] (1) Peptide chain 1', comprising, from N-terminus to C-terminus, an Fc region of an anti-PD-1 antibody containing an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1090] (2) Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1091] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1092] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1093] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1094] The connector, which is (G4S), is a connector. n n is an integer selected from 1 to 3, and

[1095] The amino acid sequence shown in SEQ ID NO:2, 93, 94 or 204 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[1096] In some embodiments, the polypeptide construct comprises:

[1097] (1) Peptide chain 1', comprising, from N-terminus to C-terminus, an Fc region of an anti-PD-1 antibody containing an amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1098] (2) Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1099] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1100] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1101] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1102] A connector comprising the amino acid sequence shown in SEQ ID NO:216, and

[1103] The amino acid sequence shown in SEQ ID NO:2, 93, 94 or 204 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[1104] In some embodiments, the polypeptide construct comprises:

[1105] (1) Peptide chain 1', comprising, from N-terminus to C-terminus, an IFNα2b mutant, an Fc region of an anti-PD-1 antibody comprising the amino acid sequence shown in SEQ ID NO:195 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1106] (2) Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1107] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1108] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1109] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1110] A connector comprising the amino acid sequence shown in SEQ ID NO:216, and

[1111] The amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1112] In some embodiments, the polypeptide construct comprises:

[1113] (1) Peptide chain 1', comprising, from N-terminus to C-terminus, an IFNα2b mutant, an Fc region of an anti-PD-1 antibody comprising the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1114] (2) Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1115] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1116] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1117] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1118] A connector comprising the amino acid sequence shown in SEQ ID NO:216, and

[1119] The amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1120] In some embodiments, the polypeptide construct comprises:

[1121] (1) Peptide chain 1', comprising, from N-terminus to C-terminus, an IFNα2b mutant, an Fc region of an anti-PD-1 antibody comprising the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1122] (2) Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1123] (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains:

[1124] VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1125] CH1 comprises the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1126] A connector comprising the amino acid sequence shown in SEQ ID NO:216, and

[1127] The amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1128] In some embodiments, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant, in addition to the amino acid substitution mutation at V105, also comprises amino acid substitution mutations at at least one of the following positions: L15, A19, L26, L30, R33, H57, E58, Q61, L80, R120, V142, A145, E146, M148, R149.

[1129] In some embodiments, the IFNα2b mutant, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), comprises the mutation V105E, and a mutation selected from at least one of the following positions:

[1130] L15A, L15E, L15Q, L15S, L15V, L15G, A19G, L26A, L30V, L30G, L30I, L30S, L30Q, R33A, R33K, R33H, R33N, R33S, H57Y, E58N, Q61S, L80A, R120G, R120A, V142A, V142D, V142E, V142G, V142H, V142I, V1 42K, V142L, V142M, V142N, V142Q, V142R, V142S, V142T, V142Y, A145D, E146A, E146G, E146H, E146I, E146K, E146L, E146R, E146S, E146T, M148A, M148D, M148G, M148K, M148S, R149E, R149G, or R149S.

[1131] In some embodiments, the IFNα2b mutant comprises a combination of V105E and at least one of the following mutations, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4):

[1132] (1) L15A, L15E, L15Q, L15S, L15V or L15G;

[1133] (2) L30V, L30G, L30I, L30S or L30Q;

[1134] (3) R33A, R33K, R33H, R33N or R33S;

[1135] (4) R120G;

[1136] (5) V142A, V142D, V142E, V142G, V142H, V142I, V142K, V142L, V142M, V142N, V142Q, V142R, V142S, V142T or V142Y;

[1137] (6)A145D;

[1138] (7) E146A, E146G, E146H, E146I, E146K, E146L, E146R, E146S or E146T;

[1139] (8) M148A, M148D, M148G, M148K or M148S;

[1140] (9) R149E, R149G, or R149S; or

[1141] (10) L15G+A19G, L15A+V142A, L15A+V142N, L15A+L26A+V142A, L15G+M148G, L26A+V142A, R33A+H57Y+E58N+Q61S, V142A+E146A, L26A+V142A+L80A or L26A+V142A+R120A.

[1142] In some embodiments, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant, in addition to the amino acid substitution mutation at A145, also comprises amino acid substitution mutations at at least one of the following positions: V105, E107, T108, P109, CD loop sequence or a portion thereof.

[1143] In some embodiments, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant comprises the mutation A145D, and a mutation selected from at least one of the following positions:

[1144] The following sequences in the CD ring region are replaced with “IQGVGVTETPLMKED” (SEQ ID NO:174) and “EEKLEKEDFTRGKLM” (SEQ ID NO:175), “GVTETPLM” (SEQ ID NO:177) and “EKEDFTRG” (SEQ ID NO:178), or “GVTETPLM” (SEQ ID NO:177) and “EKTDFTRG” (SEQ ID NO:179).

[1145] In some embodiments, the IFNα2b mutant comprises, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), a combination of A145D and at least one of the following mutations:

[1146] (1) V105E, V105E+P109R or V105E+T108I+P109A;

[1147] (2) E107D, E107K or E107D+T108A;

[1148] (3) T108G, T108A or T108I+P109A;

[1149] (4) P109R or P109N; or

[1150] (5) Replace “IQGVGVTETPLMKED” (SEQ ID NO:174) in the CD ring region sequence with “EEKLEKEDFTRGKLM” (SEQ ID NO:175).

[1151] Replace “IQGVGVTETPLMKED” (SEQ ID NO:174) with “EEKLEKTDFTRGKLM” (SEQ ID NO:176) in the CD ring sequence.

[1152] Replace “GVTETPLM” (SEQ ID NO:177) in the CD ring region sequence with “EKEDFTRG” (SEQ ID NO:178), or

[1153] In the CD ring region sequence, “GVTETPLM” (SEQ ID NO:177) is replaced with “EKTDFTRG” (SEQ ID NO:179).

[1154] In some embodiments, compared to wild-type human IFNα2b (e.g., wild-type IFNα2b comprising the amino acid sequence shown in SEQ ID NO:4), the IFNα2b mutant contains an amino acid substitution or deletion mutation at at least one of the following positions:

[1155] V105, T106, T108, P109, CD ring sequence or part thereof.

[1156] In some embodiments, the IFNα2b mutant contains at least one mutation compared to wild-type human IFNα2b (e.g., wild-type IFNα2b containing the amino acid sequence shown in SEQ ID NO:4):

[1157] Replace “GVTETPLM” (SEQ ID NO:177) in V105E, T106A, ΔT106, T108I, P109A, P109R, and CD ring sequences with “EKTDFTRG” (SEQ ID NO:179), or replace “IQGVGVTETPLMKED” (SEQ ID NO:174) in CD ring sequences with “EEKLEKTDFTRGKLM” (SEQ ID NO:176).

[1158] In some embodiments, the IFNα2b mutant contains at least one mutation compared to wild-type human IFNα2b (e.g., wild-type IFNα2b containing the amino acid sequence shown in SEQ ID NO:4):

[1159] (1) V105E, V105E+P109R or V105E+T108I+P109A;

[1160] (2) T106A or ΔT106;

[1161] (3) P109R or T108I+P109A; or

[1162] (4) Replace “GVTETPLM” (SEQ ID NO:177) in the CD ring region sequence with “EKTDFTRG” (SEQ ID NO:179), or

[1163] In the CD ring region sequence, “IQGVGVTETPLMKED” (SEQ ID NO:174) is replaced with “EEKLEKTDFTRGKLM” (SEQ ID NO:176).

[1164] In some embodiments, the IFNα2b mutant, compared to human wild-type IFNα2b, contains at least one of the following mutations:

[1165] (1)L26A+V142A;

[1166] (2) M148K;

[1167] (3)R149G;

[1168] (4) L15G+M148G;

[1169] (5) V142L or V142M; or

[1170] (6)L30S.

[1171] In some embodiments, the IFNα2b mutant comprises an amino acid sequence shown in any of SEQ ID NOs:98-173 and 226-227 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1172] In some embodiments, the IFNα2b mutant comprises an amino acid sequence shown in SEQ ID NO:147 or 161 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1173] In the peptide construct, the anti-PD-1 antibody (preferably the Fc region of the anti-PD-1 antibody) and the IFNα2b mutant may or may not have a linker, such as a flexible linker (including but not limited to (G5)). n (G4S) n Rigid joints (including but not limited to (PA)) n P, (AP) n The connector can be cut (including disulfide bonds, etc.), where n is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, or 6. Preferably, the connector can be selected from (G5). n (G4S) n 、or (PA) n P, where n is an integer selected from 1 to 5, such as 1, 2, 3, 4 or 5.

[1174] In some embodiments, a linker may be present between the anti-PD-1 antibody (preferably the Fc region of the anti-PD-1 antibody) and the IFNα2b mutant, the linker being selectable from PAPAP (SEQ ID NO:182) or PAPAPAPAPAP (SEQ ID NO:183), GGGGG (SEQ ID NO:184), GGGGGGGGGG (SEQ ID NO:185) or GGGGSGGGGS (SEQ ID NO:186).

[1175] In some embodiments, the N-terminus of the IFNα2b mutant is connected to the C-terminus of the CH3 region of the anti-PD-1 antibody via the adapter. In some embodiments, in peptide chain 1, the N-terminus of the IFNα2b mutant is connected to the C-terminus of the CH3 region of the anti-PD-1 antibody via the adapter.

[1176] In some embodiments, the fusion peptide or peptide construct exists in the structural form of a 2+2 antibody-IFNα2b mutant (Format 1 or Format 1') or a 2+1 antibody-IFNα2b mutant (Format 2).

[1177] In some embodiments, the polypeptide construct comprises:

[1178] (1) Peptide chain 1, comprising an amino acid sequence shown in any of SEQ ID NOs: 9-84, 211, 214, 234 and 235, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; and

[1179] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1180] (2) Peptide chain 1, comprising an amino acid sequence shown in any of SEQ ID NOs:85-90 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1181] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1182] (3) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:91 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1183] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1184] Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1185] (4) Peptide chain 1, comprising an amino acid sequence shown in SEQ ID NO: 219, 220, 221, 222, 223, 224 or 225 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1186] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1187] Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1188] (5) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:232 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1189] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1190] Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1191] (6) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:233 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1192] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1193] Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1194] (7) Peptide chain 1', comprising the amino acid sequence shown in SEQ ID NO:92 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1195] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1196] Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; or

[1197] (8) Peptide chain 1', comprising the amino acid sequence shown in SEQ ID NO:92 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1198] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1199] The peptide chain 3' comprises an amino acid sequence shown in SEQ ID NO:95 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1200] In some embodiments, the polypeptide construct comprises:

[1201] (1) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:56 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1202] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1203] (2) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:67 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1204] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1205] (3) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:71 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1206] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1207] (4) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:72 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1208] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1209] (5) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:77 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1210] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1211] (6) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:79 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1212] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1213] (7) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:234 ​​or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1214] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1215] (8) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:235 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1216] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1217] (9) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:91 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1218] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1219] Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1220] (10) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:220 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1221] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1222] Peptide chain 3, comprising the heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1223] (11) Peptide chain 1, comprising an amino acid sequence shown in SEQ ID NO: 219, 221, 222, 223, 224 or 225 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1224] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1225] Peptide chain 3, comprising the heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1226] (12) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:232 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1227] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1228] Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1229] (13) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:233 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1230] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1231] Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[1232] (14) Peptide chain 1', comprising the amino acid sequence shown in SEQ ID NO:92 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1233] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1234] Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; or

[1235] (15) Peptide chain 1', comprising the amino acid sequence shown in SEQ ID NO:92 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it;

[1236] Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[1237] The peptide chain 3' comprises an amino acid sequence shown in SEQ ID NO:95 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[1238] Polynucleotides, carriers and cells

[1239] In some embodiments, this disclosure relates to a polynucleotide, wherein the polynucleotide encodes the IFNα2b mutant, fusion polypeptide, or polypeptide construct.

[1240] In some embodiments, this disclosure relates to a recombinant vector comprising the polynucleotide, or encoding the IFNα2b mutant, fusion polypeptide, or polypeptide construct.

[1241] Recombinant vectors can be constructed using any expression vector known in the art capable of expressing the IFNα2b mutant, fusion peptide, or peptide construct described herein. The vectors described herein include, but are not limited to, naked plasmids, phage particles, yeast plasmids, adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), retroviruses (such as lentiviruses), poxviruses, papillomaviruses, papillomaviruses (such as SV40), rod-shaped viruses, or baculoviruses. For ease of production and purification, the expression vectors may also contain secretory signal peptides, expression tags, etc.

[1242] It is known to those skilled in the art that, due to codon degeneracy, each amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequences encoding the IFNα2b mutant, fusion peptide, or peptide construct of this disclosure can be synthesized using methods known in the art (e.g., de novo solid-phase DNA synthesis, PCR amplification). Given that specific amino acid sequences have been described in this disclosure, those skilled in the art can easily modify one or more codons of their respective coding sequences without altering the amino acid sequences of the IFNα2b mutant, fusion peptide, or peptide construct of this disclosure to prepare a variety of different nucleic acids capable of expressing proteins having the same amino acid sequence.

[1243] In some embodiments, this disclosure relates to a recombinant cell comprising the polynucleotide or recombinant vector, or expressing the IFNα2b mutant, fusion polypeptide, or polypeptide construct.

[1244] The cells used in this document may be prokaryotic or eukaryotic cells. In some embodiments, the cells are selected from yeast cells, mammalian cells, or any other cells suitable for expressing the relevant proteins. In some examples, the mammalian cells are, for example, Chinese hamster ovary (CHO) cells (e.g., ExpiCHO cells, GS-CHO-K1 cells, or CHO-K1 cells), CHO-S cells, 293 cells (e.g., Expi293F cells), and monkey kidney cells.

[1245] The vector described above can be introduced into suitable cells to express the desired target protein product using any conventional means known in the art, such as protoplast fusion, calcium phosphate precipitation, electroporation, viral transfection, gene gun, liposome transfection or other conventional techniques, but not limited thereto.

[1246] Under conditions suitable for the expression of the target protein, the cells described above were cultured under conventional conditions, and then the target protein of this paper was recovered from the cells or the cell culture medium by conventional protein separation and purification methods (such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (such as Protein A column affinity chromatography), size exclusion chromatography, etc.).

[1247] Pharmaceutical Composition

[1248] In some embodiments, this disclosure relates to a pharmaceutical composition comprising: the IFNα2b mutant, a fusion peptide or a peptide construct, and optionally pharmaceutically acceptable excipients.

[1249] The pharmaceutical compositions described herein can be formulated in any manner known in the art and can be formulated to suit any route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The pharmaceutical compositions described herein comprise therapeutically effective amounts of the aforementioned components.

[1250] The excipients described herein may be any pharmaceutically acceptable excipient known in the art, such as, but not limited to, solvents. Those skilled in the art can select appropriate conventional excipients to formulate the pharmaceutical composition into the desired formulation, such as an injection, suspension, etc., according to the specific formulation requirements.

[1251] In some embodiments, the pharmaceutical composition may be in the form of a sterile aqueous solution, microemulsion, liposome, or powder. In some embodiments, the pharmaceutical composition may be in the form of a unit dose to facilitate administration to the patient at the desired dosage.

[1252] The dosage range of the IFNα2b mutant, fusion peptide or peptide construct, and pharmaceutical composition described in this disclosure can be determined by clinicians based on experience, taking into account factors such as the method of administration (including administration time, administration interval, and route of administration), the patient's age, weight, sex or pathological condition, diet, excretion rate, and sensitivity to the drug.

[1253] The pharmaceutical compositions described herein may be included in containers, packages or dispensers together with the instructions for use.

[1254] Medical Use

[1255] In some embodiments, this disclosure relates to the use of the aforementioned IFNα2b mutant, fusion peptide, peptide construct, or pharmaceutical composition in the preparation of a medicament for treating tumors. Alternatively, this disclosure relates to a method of treating a tumor in a subject of need, comprising administering to the subject a therapeutically effective amount of the IFNα2b mutant, fusion peptide, peptide construct, or pharmaceutical composition. Alternatively, this disclosure relates to an IFNα2b mutant, fusion peptide, peptide construct, or pharmaceutical composition for treating tumors. Alternatively, this disclosure relates to the use of the aforementioned IFNα2b mutant, fusion peptide, peptide construct, or pharmaceutical composition for treating tumors.

[1256] The IFNα2b mutants, fusion peptides, peptide constructs, or pharmaceutical compositions described herein can be formulated into any dosage form known in the art, such as injections, suspensions, solutions, powders, emulsions, sprays, tablets, pills, capsules, granules, ointments, suppositories, gels, etc.

[1257] The IFNα2b mutants, fusion peptides, peptide constructs, or pharmaceutical compositions described herein are suitable for intravenous, intramuscular, intra-articular, intra-articular, subcapsular, subarachnoid, intraorbital, intracardiac, subcutaneous, extracorporeal, intraperitoneal, intraspinal, intranasal, or epidermal administration (e.g., by injection or infusion). The IFNα2b mutants, fusion peptides, peptide constructs, or pharmaceutical compositions described herein may be in the form of sterile aqueous solutions, microemulsions, liposomes, or powders.

[1258] Therapeutic doses can be administered via single or multiple doses. For example, therapeutic doses of the IFNα2b mutant, fusion peptide, peptide construct, or pharmaceutical composition described herein can be formulated into unit formulations for convenient administration to subjects in need.

[1259] Subjects described herein include subjects at risk of developing tumors. Administration of the IFNα2b mutant, fusion peptide, peptide construct, or pharmaceutical composition described herein may reduce the rate of tumor volume increase over time in subjects, reduce the risk of metastasis, or reduce the risk of recurrence in subjects, or may prevent, slow, delay, or inhibit cancer progression, or may cause a reduction in the severity and / or duration of one or more symptoms of tumor in subjects.

[1260] In some embodiments, the tumors described in this disclosure are preferably PD-1 resistant tumors.

[1261] In some embodiments, the tumor is melanoma, breast cancer, or pancreatic cancer.

[1262] Methods for preparing fusion peptides or peptide constructs

[1263] Using any conventional method known in the art, the polynucleotide or expression vector encoding the fusion polypeptide or polypeptide construct described herein is transferred into a suitable expression system (e.g., CHO cells or 293 cells), and then the cells are cultured under conventional conditions suitable for the expression of the target protein. After an appropriate period of culture, the target protein is recovered from the cells or the cell culture medium using conventional protein separation and purification methods (e.g., high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A column affinity chromatography), size exclusion chromatography, etc.).

[1264] Combined drugs

[1265] In some embodiments, this disclosure relates to combination drugs, including: (1) the fusion peptide, peptide construct or pharmaceutical composition described in this disclosure; and (2) VEGF inhibitors.

[1266] In some embodiments, in the combination drug, the fusion peptide, peptide construct, or pharmaceutical composition is packaged separately from the VEGF inhibitor. In some embodiments, in the combination drug, the fusion peptide, peptide construct, or pharmaceutical composition is packaged together with the VEGF inhibitor.

[1267] In this article, the term "separate packaging" means that two or more active ingredients are packaged separately in different packaging units within the same medicine box, or packaged separately in different medicine boxes and sold as individual products.

[1268] In this article, the term "packaged together" generally means that two or more active ingredients are packaged in the same unit, such as in the same box, the same bag, the same vial, etc.

[1269] In some embodiments, the VEGF inhibitor is in the form of a pharmaceutical composition.

[1270] In some embodiments, when the fusion peptide, peptide construct, or pharmaceutical composition is administered to a patient in need along with the VEGF inhibitor, the fusion peptide, peptide construct, or pharmaceutical composition and the VEGF inhibitor may be administered simultaneously, sequentially, or at regular or irregular intervals.

[1271] In some embodiments, the VEGF inhibitor may include an anti-VEGF antibody, a VEGF receptor (VEGFR) antagonist, and a tyrosine kinase inhibitor targeting VEGFR.

[1272] In some embodiments, the dosing cycle of the fusion peptide, peptide construct, or pharmaceutical composition may be the same as or different from that of the VEGF inhibitor.

[1273] In some embodiments, the fusion peptide, peptide construct, or pharmaceutical composition may be administered at the same or different frequencies as the VEGF inhibitor.

[1274] In some embodiments, the effective therapeutic dose of the fusion peptide, peptide construct, or pharmaceutical composition with the VEGF inhibitor can be determined by a clinician based on the patient's sex, age, weight, general health condition, family history, disease severity, etc. In this document, an effective therapeutic dose refers to a dose that can reduce the rate of tumor volume increase over time in a subject, reduce the risk of metastasis, or reduce the risk of recurrence in a subject, or can prevent, slow, delay, or inhibit cancer progression, or can cause a reduction in the severity and / or duration of one or more symptoms of the tumor in a subject.

[1275] In some embodiments, the VEGF inhibitor is Bevacizumab.

[1276] Example

[1277] Unless otherwise specified, the experimental methods in the following examples were performed under standard conditions. Unless otherwise stated, reagents, materials, or instruments used in the following examples that do not specify the manufacturer are all commercially available products.

[1278] Example 1. Construction of IFNα2b mutant and design of anti-PD-1 antibody-IFNα2b fusion peptide

[1279] Interferons are glycoproteins with antiviral, antitumor, and cellular regulatory activities. They can be classified into type I, type II, and type III interferons, which conduct signal transduction by binding to their receptors. For example, they can promote cross-presentation by dendritic cells and T cell activation. Human type I interferons include IFNα and IFNβ, among which IFNα includes several subtypes such as IFNα1a, IFNα2a, IFNα1b, and IFNα2b.

[1280] Wild-type (WT) IFNα2b is a single-chain polypeptide composed of 165 amino acids (SEQ ID NO:4). It can play an anti-proliferative role directly or indirectly in the treatment of certain malignant tumors (Fei Xiong et al., Biomarker Research, vol.10, (2022)). However, due to its short half-life and other defects, its clinical application is greatly limited.

[1281] Construction of IFNα2b mutant

[1282] Wild-type IFNα2b contains an O-glycosylation site, which may cause off-target toxicity of the fusion protein, generate more heterogeneous IFNα2b products in mammalian expression systems, and reduce affinity for FcRn. In this experiment, we attempted to reduce or remove the proportion of O-glycosylation at the O-glycosylation site (T106) of IFNα2b by mutating the amino acids near this site; alternatively, we could replace the CD loop region sequence of the O-glycosylated IFNα2b (i.e., IQGVGVTETPLMKED, SEQ ID NO:174) or a portion thereof with the CD loop region sequence of human IFNβ1, which is less prone to O-glycosylation, to achieve de-O-glycosylation. Based on the crystal structure 3se3 of the IFNα2b-receptor complex (Figure 2), the inventors selected specific mutation sites at the receptor binding interface to construct multiple IFNα2b mutants (see Table 1 below) to reduce the binding of IFNα2b to its receptor.

[1283] Design of anti-PD-1 antibody-IFNα2b fusion peptide

[1284] Based on the IFNα2b mutant obtained above, this disclosure specifically designs a fusion polypeptide that can specifically bind to human PD-1 and block the binding of PD-1 to PD-L1, while also binding to interferon-α / β receptor (IFNAR) to activate T cells or NK cells. This fusion polypeptide can enhance the effect of PD-1 antibody immunotherapy. The PD-1 binding antibody sequence is an anti-PD-1 antibody derived from WO2017024465A1 (its heavy and light chains are shown in SEQ ID NO:2 and SEQ ID NO:5; αPD-1), or an anti-PD-1 antibody that further includes a knob / hole mutation in the constant region of the heavy chain of the aforementioned antibody (its heavy chain containing a knob mutation, its heavy chain containing a hole mutation, and its light chain are shown in SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:5, respectively), or an anti-PD-1 antibody that further includes a YTE mutation in the constant region of the heavy chain of the aforementioned antibody (its heavy and light chains are shown in SEQ ID NO:240 and SEQ ID NO:5), or an anti-PD-1 antibody that further includes an LS mutation in the constant region of the heavy chain of the aforementioned antibody (its heavy and light chains are shown in SEQ ID NO:241 and SEQ ID NO:5). (Shown NO:5), or anti-PD-1 antibodies that further contain knob / hole mutations and YTE mutations in the heavy chain constant region of the aforementioned antibody (the heavy chain containing knob mutations, the heavy chain containing hole mutations, and the light chain are shown in SEQ ID NO:238, SEQ ID NO:236, and SEQ ID NO:5, respectively), or anti-PD-1 antibodies that further contain knob / hole mutations and LS mutations in the heavy chain constant region of the aforementioned antibody (the heavy chain containing knob mutations, the heavy chain containing hole mutations, and the light chain are shown in SEQ ID NO:239, SEQ ID NO:237, and SEQ ID NO:5, respectively).

[1285] The fusion peptide designed above may include a first domain and a second domain, wherein the first domain contains a polypeptide ligand that binds to cell surface-associated antigens, and the second domain contains an IFNα2b mutant (deglycosylated interferon α2b). The deglycosylated IFNα2b may further include one or more amino acid substitutions or deletions to weaken the activity of IFNα2b. The molecular form of the fusion peptide provided in this disclosure is shown in Figure 1.

[1286] The mutation status of the constructed IFNα2b mutant in this embodiment, as well as the molecular forms of the constructed anti-PD-1 antibody-IFNα2b mutant fusion polypeptide (αPD1-IFNα2b-m) and its control molecule (anti-PD-1 antibody fusion polypeptide constructed based on wild-type IFNα2b or the IFNα2b mutant disclosed in US10544199B2) are shown in Table 1.

[1287] Table 1. αPD1-IFNα2b-m fusion peptide and control molecules

[1288] Example 2. Preparation of anti-PD-1 antibody-IFNα2b fusion peptide and its mouse surrogate for in vivo experiments; expression and purification of the fusion peptide.

[1289] Based on the amino acid sequences of the fusion peptides constructed in Example 1, heavy chain genes and light chain genes of the fusion peptides were designed respectively. After synthesis, the heavy chain genes and light chain genes of the fusion peptides were respectively constructed into the pcDNA3.1 vector by Suzhou Genewiz Biotechnology Co., Ltd.

[1290] Following the procedure described below, recombinant vectors of heavy and light chains were co-transfected into mammalian cell expression systems using transient transfection. Different expression systems were used for different fusion peptides (see Table 2 below).

[1291] Expression in Expi293F cells: The recombinant vector and transfection reagent PEI MAX (Polysciences, 24765) were prepared in a clean bench. 10 mL of Opti-MEM medium (Gibco, 31985-070) was added to a 50 mL centrifuge tube, followed by 100 μg of the corresponding recombinant vector (light chain + heavy chain recombinant vector). The mixture was then mixed, followed by 300 μg of PEI MAX (1 g / L). The mixture was allowed to stand for 10 min. The recombinant vector / PEI MAX mixture was slowly added to 100 mL of Expi293F cells and mixed well. The resulting cell suspension was cultured at 37°C, 8% CO2, and 120 rpm for 18-22 h. After that, 1% (v / v) of a 200 g / L feed solution (100 g / L Phytone Peptone (BD, 211906) + 100 g / L Difco Select Phytone (BD, 210931), a glucose solution (Merck, G7021-1KG) to a final concentration of 5 g / L, and a valproic acid sodium salt solution (Merk, Cat#P4543-100G) to a final concentration of 2 mM were added to the cell suspension. The cells were then cultured at 37°C, 8% CO2, and 120 rpm for 5 days. The cell supernatant was then obtained for purification.

[1292] Expression in ExpiCHO cells: Take 2 mL of OptiPRO SFM Serum Free medium (Gibco, 12309-019), add 20 μg of the corresponding recombinant vector (light chain + heavy chain recombinant vector), mix well, then add 60 μL of FectoPRO Transfection Reagent (Genesee Scientific, 101000019), and let stand for 10 min; slowly add the recombinant vector / FectoPRO mixture to 25 mL of ExpiCHO cells and mix well. Incubate the resulting cell suspension at 37℃, 8% CO2, and 120 rpm for 18-22 h. After that, add Enhancer (Gibco, 100033019) at a volume of 6 μL / mL of cell suspension, and add Feed (Gibco, A29101-01) at a volume of 240 μL / mL of cell suspension. Incubate for 6 days at 32℃, 8% CO2, and 120 rpm.

[1293] Expression in GS-CHO-K1 cells or wild-type CHO-K1 cells: except for the following adjustments, follow the method of the ExpiCHO system described above: recombinant vector 1.5 mg / L, transfection reagent PEI MAX (Polysciences, 24765), 10 mg / L.

[1294] Collect the cell cultures obtained above and purify them according to the molecular form of the fusion peptides as follows.

[1295] Format 1 and Format 1' molecules were purified using Mabselect affinity chromatography: Cells were centrifuged at 2500g for 20 min, and the supernatant was collected and purified using Mabselect Sure LX (Cytiva, 17-5474-02) protein A affinity chromatography packing material. The packing material column was equilibrated with 20 column volumes of equilibration buffer (PBS) before purification. The collected supernatant was passed through the column, and the column was washed with another 20 column volumes of equilibration buffer to remove non-specific binding proteins. The packing material was washed with 3 column volumes of elution buffer (0.1M sodium citrate, pH 3.2), and the eluent was collected. 2M Tris was added to the eluent to adjust the pH to approximately 6.0.

[1296] Molecules of Format 2-4 were obtained through Mabselect affinity purification (method as described above) and ion exchange purification. Ion exchange purification method: Heterodimolecular molecules in the bispecific molecules were separated by ion exchange chromatography to remove homodimeric impurities. The Mabselect affinity-purified sample was first desalted using a desalting column (HiPrep 26 / 10 Desaulting, Cytiva, 17-5087-01) or diluted multiple times to a buffer of 20 mM NaH2PO4 / Na2HPO4 (pH = 6.0). Then, it was purified by gradient elution using an ion exchange column (Mono S 5 / 50GL, Cytiva, 17516801) with low-salt and high-salt buffers. The low-salt and high-salt buffers were 20 mM NaH2PO4 / Na2HPO4 pH = 6.0 and 20 mM NaH2PO4 / Na2HPO4, 1 M NaCl, pH = 6.0, respectively.

[1297] Preparation of mouse surrogate for in vivo experiments

[1298] Since the natural human IFNα2b molecule has no significant activity in mouse cells, previous studies have reported that the hybrid molecules IFNα-AD (the amino acid sequence of which is shown in SEQ ID NO:199) and IFNα-AD' (the amino acid sequence of which is shown in SEQ ID NO:213), obtained by splicing a certain length of the N-terminal sequence of human IFNα2 (IFNα-A) and the C-terminal sequence of IFNα1 (IFNα-D), have significantly enhanced antiviral and antiproliferative activities in mouse cells (Rehberg et al., (1982) J. Biol. Chem., 257, 11497-11502); or that mutating some amino acid residues in human IFNα2 to amino acid residues of IFNα1 can also improve the activity of the molecule in mouse cells (Weber H et al., (1987) EMBO J., 6, 591-598.).

[1299] Following the molecular format Format 1 described in Example 1, an anti-mouse PD-1 antibody-IFNα-AD fusion polypeptide was constructed using an anti-mouse PD-1 antibody (αmPD-1, the heavy and light chain sequences of which are shown in SEQ ID NO:200 and SEQ ID NO:201, respectively) and the aforementioned IFNα-AD or IFNα-AD'. The heavy chain and light chain genes of the anti-mouse PD-1 antibody-IFNα-AD fusion polypeptide (αPD1-IFNα2b mouse substitute molecules: molecule 116, the heavy and light chain sequences of which are shown in SEQ ID NO:202 and SEQ ID NO:201, respectively; molecule 118, the heavy and light chain sequences of which are shown in SEQ ID NO:203 and SEQ ID NO:201, respectively) were synthesized and then constructed into the pcDNA3.1 vector. The vectors containing the heavy chain gene and the vector containing the light chain gene were co-transfected into Expi293F cells for expression using the transient transfection method described above. The supernatant of the cell culture was purified by Mabselect affinity chromatography (Cytiva, 17-5474-02; method as described above) and ion exchange chromatography (Mono S 5 / 50GL, Cytiva, 17516801).

[1300] Following the molecular form Format 1 described in Example 1, a mouse alternative molecule 183 of the fusion peptide was constructed using the anti-mouse PD-1 antibody and the IFNα2 mutant described by Weber H et al. (the sequences of its heavy chain and light chain are shown in SEQ ID NO:228 and SEQ ID NO:201, respectively). Additionally, following the molecular form Format 2 described in Example 1, a mouse alternative molecule 209 of the fusion peptide was constructed using the anti-mouse PD-1 antibody and the IFNα2 mutant described by Weber H et al. (the sequences of peptide chain 1, peptide chain 2, and light chain are shown in SEQ ID NO:229, SEQ ID NO:230, and SEQ ID NO:201, respectively). After synthesis, the heavy chain and light chain genes were respectively constructed into the pcDNA3.1 vector, and the vector containing the heavy chain gene and the vector containing the light chain gene were co-transfected into Expi293F cells for expression using the transient transfection method described above. The supernatant of the cell culture was purified by Mabselect affinity chromatography (Cytiva, 17-5474-02; method as described above) and ion exchange chromatography (Mono S 5 / 50GL, Cytiva, 17516801).

[1301] Example 3. Detection of the O-glycosylation ratio of the fusion peptide by liquid chromatography-mass spectrometry (LC-MS)

[1302] The method for analyzing the proportion of O-glycosylation modification at specific sites of the fusion peptide is as follows:

[1303] 1) After digesting the proteome sample or a single protein sample with peptide N-glycosidase F, the interchain disulfide bonds are reduced by the mild reducing agent DTT to obtain N-glucose-free single-chain protein sample groups or single groups of single-chain protein samples.

[1304] 2) The obtained single-chain protein sample groups or single groups of single-chain protein samples were separated using a Polyphenyl reversed-phase chromatography column, and then detected by a high-resolution mass spectrometer in positive ion mode to obtain high-resolution mass spectra.

[1305] 3) The obtained mass spectrometry data is deconvolved, and the O-glycosylation modification components of specific single chains are determined by molecular weight. Then, all components are integrated to obtain the O-glycosylation modification ratio of specific sites.

[1306] Table 2 summarizes the proportion of O-glycosylation in fusion peptides or control molecules of IFNα2b mutants (removed glycosylation through mutation or CD loop sequence substitution) as detected by LC-MS.

[1307] As shown in Table 2, the O-glycosylation rate in wild-type molecules (peptide numbers: 011, 214) constructed from wild-type IFNα2b or the control molecule (peptide number: 012) constructed from an IFNα2b mutant containing only the affinity-weakened A145D mutation was very high, exceeding 50%. In contrast, by mutating valine (V105) at position 105 near threonine 106 of the glycosylation site of IFNα2b to glutamate (V105E, peptide numbers: 042, 177), a mutation at just one site could reduce the O-glycosylation rate of the resulting fusion peptides from greater than 50% to 0% or very low levels. Furthermore, mutating proline at position 109 to arginine (V105E+P109R) or mutating threonine at position 108 and proline at position 109 to isoleucine and alanine respectively (V105E+T108I+P109A) based on V105E can achieve complete de-glycosylation. Replacing the IFNα2b CD loop region sequence (SEQ ID NO:174) containing O-glycosylation or a portion thereof with the human IFNβ1CD loop region sequence, which is less prone to O-glycosylation, can also completely remove O-glycosylation.

[1308] Table 2: Proportion of O-glycosylation in IFNα2b mutant fusion peptides or control molecules as detected by LC-MS Note: NA indicates not detected.

[1309] Example 4. Detection of molecular charge heterogeneity by whole-column imaging capillary isoelectric focusing electrophoresis (iCIEF).

[1310] iCIEF establishes a stable pH gradient in a capillary, causing proteins to migrate to their isoelectric point under the influence of an electric field and focus into a sharp band. At the isoelectric point, the protein's net charge is zero, and its migration rate slows down, thus achieving the separation of different charge variants. iCIEF has become the gold standard for characterizing charge heterogeneity in all stages of biopharmaceutical development, including formulation and stability studies, process optimization, and quality control.

[1311] The sample to be tested was thoroughly mixed with a mixture of methylcellulose (Protein Simple, 102505) and stabilizing additive urea (Sigma, 15604), carrier amphoteric electrolyte Pharmalyte 3-10 (GE, 17-0456-01), isoelectric point standard proteins pI marker-5.41 and pI marker-9.91 (Shanghai Shuangmo Biotechnology, C19100023), and cathode stabilizer arginine (Sigma, A5006), and then loaded into a 96-well plate. iCIEF detection was performed using a panoramic imaging protein isoelectric focusing electrophoresis system (Protein Simple, Maurice C). The iCIEF separation conditions were: pre-focusing voltage 1.5 kV, focusing time 1 min; focusing voltage 3 kV, focusing time 8 min; detection wavelength: 280 nm. Pre-focusing refers to the application of voltage for a predetermined period of time, causing the carrier amphoteric electrolyte to form a pH gradient within the capillary. The longer focusing time in the second phase causes protein charge variants to migrate within the capillary until they reach a point where the total charge of the variants is neutral. The sample-loading capillary is connected to a digital camera, enabling direct detection and quantification of protein charge variants.

[1312] Table 3 summarizes the proportion of charge variants detected by iCIEF for fusion peptides or control molecules of IFNα2b mutants (removed glycosylation through mutation or CD loop region sequence substitution).

[1313] The fusion peptides of IFNα2b mutants with reduced or eliminated O-glycosylation were obtained by mutations in V105E (peptide IDs: 042, 177), T108I+P109A (peptide IDs: 049, 173), V105E+P109R (peptide IDs: 052, 200), and V105E+T108I+P109A (peptide IDs: 053, 202) or CD loop sequence substitutions (peptide IDs: 048, 051, 201, 176). These peptides showed reduced charge heterogeneity and increased main peak proportion on iCIEF (compared to control molecule 012 and wild-type molecule 011, respectively), and this trend was consistent across different expression systems. Furthermore, peptides from this study (numbers: 097, 204, 216) that underwent further affinity-weakening mutations after O-glycosylation removal showed significantly improved charge heterogeneity, comparable to the control molecule (number: 018).

[1314] Table 3: Charge variants of IFNα2b mutant fusion peptides or control molecules detected by iCIEF Note: NA indicates not detected.

[1315] Example 5. Affinity assay (BLI) of anti-PD-1 antibody-IFNα2b mutant fusion peptide with IFNα / β receptor and PD-1.

[1316] Biomembrane layer interferometry (BLI, ForteBio) was used to determine the equilibrium dissociation constant (K0) of the fusion peptides binding to human IFN receptors IFNAR2 (Acro) or IFNAR1 (kactusbio) and human PD-1 in this study. D BLI affinity determination was performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8).

[1317] After equilibrating the HIS1K (Sartorius, 18-5120) sensor in analysis buffer (PBS 1x, BSA 0.1%, Tween-20 0.05%) for 30 minutes, a baseline was established after 120 seconds of detection. Then, human IFNAR2 or IFNAR1 antigen was loaded onto the HIS1K sensor, and the sensor was reequilibrated to a new baseline using analysis buffer. The antigen-loaded sensor was then exposed to an immunoconjugate or control molecule for binding (100 seconds), followed by transfer to analysis buffer for dissociation rate measurement (120 seconds). K was analyzed using ForteBio analysis software. D value.

[1318] After equilibrating the AHC (ForteBio, 18-5060) sensor in analysis buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%) for 30 minutes, a baseline was established by detection for 60 seconds. Then, the target antibody was loaded onto the AHC sensor, and the sensor was reequilibrated to a new baseline using analysis buffer. The antibody-loaded sensor was then exposed to the antigen-human PD-1 for association. Finally, the sensor was transferred to analysis buffer for dissociation rate measurement. The run time for each step depends on the sample binding and dissociation rates. K was analyzed using ForteBio analysis software. D value.

[1319] Table 4 shows the binding constants of the fusion peptide of the IFNα2b mutant or the control molecule to IFNAR2 and IFNAR1.

[1320] Engineered molecules with O-glycosylation removed from the A145D mutation (peptide numbers: 037, 042, 049, 045, 046, 047, 048, 050, 051) showed no significant change in affinity for human receptors (including IFNAR2 and IFNAR1) compared to the control molecule without glycosylation removal (peptide number: 012). Fusion peptides of IFNα2b mutants engineered with only O-glycosylation on wild-type IFNα2b (peptide numbers: 177, 172, 173, 200, 202, 201, 176) showed comparable affinity for human receptors to the wild-type molecule (peptide number: 011). Therefore, the removal of O-glycosylation in this disclosure has a relatively small impact on the affinity of IFNα2b molecules for IFNAR2 and IFNAR1.

[1321] In this study, the fusion peptides of the affinity-weakening mutant combined with the O-glycosylation removal mutant showed a weaker affinity for IFNAR2 than the wild-type molecule (number: 011) or were comparable to the wild-type molecule. Based on the affinity assay results, fusion peptides with a weaker affinity than the control molecule (peptide number: 018) or comparable to it were selected for further in vitro functional experiments.

[1322] Table 5 shows the affinity assay results of the anti-PD-1 antibody-IFNα2b mutant fusion peptide or control molecule to human PD-1. The fusion peptides in this study all showed strong affinity for human PD-1, comparable to or better than the PD-1 monoclonal antibody sintilimab.

[1323] Table 4: Binding constants of IFNα2b mutant fusion peptides or control molecules with human IFNAR2 and IFNAR1 Note: NB indicates not bound, NA indicates not detected.

[1324] Table 5. Affinity of anti-PD-1 antibody-IFNα2b mutant fusion peptide or control molecule to human PD-1

[1325] Example 6. Detection of molecular thermal stability using differential scanning calorimetry (DSC), differential scanning fluorescence (DSF), and dynamic light scattering (DLS).

[1326] Differential scanning calorimetry (DSC) is a thermal analysis method. It measures the energy difference between a sample and a reference material as a function of temperature under programmed temperature conditions. As temperature increases, protein structure changes, and the accompanying thermal changes are recorded as a DSC curve by a differential scanning calorimeter. The Tm value of the protein is obtained by analyzing the DSC curve. Samples were diluted with PBS to 0.5-1 mg / mL. After degassing the diluted samples and PBS buffer, the samples were added to the left sample plate, and PBS buffer was added to the corresponding position in the right reference plate. The initial temperature was 30℃, the equilibration time was 10 min, and the heating rate was 1℃ / min, ending at 90℃.

[1327] Table 6 shows the results of the melting temperature (Tm) of the anti-PD-1 antibody-IFNα2b mutant fusion peptide or control molecule in PBS buffer, as detected by differential scanning calorimetry (DSC).

[1328] Table 6. Midpoint temperature (Tm) of thermal denaturation of fusion peptides or control molecules in solution as detected by DSC.

[1329] Differential scanning fluorescence (DSF) was used to detect the Tm of the samples: The fusion peptide sample was diluted with ultrapure water to 1 mg / mL. 90 μL of the sample was taken, and 10 μL of 200x Sypro Orange (Gibco, S6640) fluorescent reagent was added and mixed well. Then, 30 μL of the resulting solution was added to a 96-well PCR plate for measurement. An ABI 7500 qPCR instrument was used for detection. The reaction program was 25℃ for 5 minutes, with a ramp-up to 99℃ at a rate of 0.5%. The lowest point temperature of the first derivative curve (Melt Curve) after the temperature ramp-up was taken as the Tm value of the sample. The detection results are shown in Table 7.

[1330] Table 7. Midpoint temperature (Tm) of thermal denaturation of fusion peptides in solution as determined by DSF.

[1331] Dynamic light scattering (DLS) was used to detect the thermal stability of fusion peptides. DLS utilizes laser irradiation of small particles undergoing Brownian motion in solution and detects changes in the intensity of scattered light. DLS can be used to detect protein particle size in solution and the stability under different temperatures or concentrations. The fusion peptide sample was prepared using ultrapure water and 200x Sypro Orange fluorescent reagent as described above. After centrifugation at 13000 G / min for 5 min, the sample was added to a 96-well PCR plate and centrifuged for 1 min to remove air bubbles. Using Tagg, a continuous temperature ramp experiment was selected, the readout wells were chosen, and the DLS acquisition time was set to 5 s, the number of acquisitions to 5, and the experimental temperature to 25-85℃. The change in sample particle size with temperature was analyzed after the experiment. The results are shown in Table 8.

[1332] Table 8. DLS detection of the initial aggregation temperature of fusion peptides in solution (Tagg)

[1333] Therefore, the fusion peptides of Format 1 and Format 2 in the embodiments of this disclosure can both exhibit good thermal stability. Specifically, according to the results of DSF and DLS detection, the thermal denaturation temperature and the initiation aggregation temperature of the fusion peptides 204, 214, 216, 217, 218, 219 and 223 of Format 2 are significantly higher than those of fusion peptide 097 of Format 1.

[1334] Example 7. Using HEK-Blue TM Detection of biological activity of αPD1-IFNα2b-m fusion peptide in IFN-α / β reporter cell lines

[1335] Utilizing HEK-Blue TM Functional assays were performed on the αPD1-IFNα2b-m fusion peptide using an IFN-α / β reporter cell line. It is expected that the αPD1-IFNα2b-m fusion peptide will interact with HEK-Blue cells. TM IFN-α / β reporter cell lines bind to the IFNα receptor, activating the JAK-STAT pathway and producing secreted embryonic alkaline phosphatase (SEAP). This pathway activation can be detected using the QUANTI-Blue-Solution (a SEAP detection reagent). This study used the HEK-Blue reporter cell line provided by InvivoGen. TM The IFN-α / β reporter cell line (HKB-IFNABV2) was administered via HEK-Blue according to the instructions. TM IFN-α / β reporter cell lines were used to evaluate the fusion peptides and control molecules disclosed herein.

[1336] Cell thawing: 1) Prepare DMEM complete medium in advance: DMEM (ATCC, 30-2002), 10% FBS (Gibco, 10091-148), 1% penicillin-streptomycin (Gibco, 15140-122); 2) Remove the frozen cells from the liquid nitrogen tank and gently shake them in a 37°C water bath until thawed, keeping the caps of the cryovials above the water surface to prevent contamination; 3) After thawing, immediately sterilize with 75% alcohol and transfer to a clean bench, strictly following aseptic techniques thereafter; 4) Transfer the cell suspension from the cryovials to a 15mL centrifuge tube, add 10mL of DMEM complete medium, mix gently, centrifuge at 1000rpm for 5 minutes to precipitate the cells, and discard the supernatant; 5) Use 5mL of... Resuspend the cell pellet in DMEM complete medium, take 20 μL of cell suspension, and count the viable cells using trypan blue (Gibco, 15250-061); 6) Adjust the viable cell density to 5 × 10⁶ cells / mL. 5 7) Inoculate the cell suspension into T75 cell culture flasks (NUNC, 156499) at 1000 cells / mL; 8) Incubate statically in a 37°C, 5% CO2 incubator.

[1337] Cell culture: HEK-Blue TM When passaged, IFN-α / β reporter cells should be selected with a pressure of 0.5 μg / mL puromycin (Gibco, A11138-02), and passaged every 2-3 days, maintaining a passage density of 5 × 10⁻⁶ cells / year. 5 Cells / mL.

[1338] Viability assay: 1) Cell plating: Cells were digested with 0.25% Trypsin-EDTA trypsin (Gibco, 25200-072), then DMEM complete medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 minutes, resuspended in DMEM complete medium, and the cell density was adjusted to 2.8 × 10⁻⁶ cells / cm². 51) Cells / mL were collected to obtain a cell suspension. 180 μL of this cell suspension was added to each well of a 96-well flat-bottom cell culture plate (NEST, 701001). 2) Preparation of αPD1-IFNα2b-m fusion peptide: The fusion peptide was diluted with DMEM complete medium in a sterile 96-well V-bottom plate (Beyotime, FPT019). The initial concentration of the fusion peptide was 3000 nM, diluted 5-fold, for a total of 16 gradients. 36 μL of the diluted fusion peptide was added to each well of the 96-well flat-bottom cell culture plate prepared in step 1). 3) Microplate reader detection: Prepare QUANTI-Blue-Solution reagent (Invitrogen, rep-qbs3) in advance according to the instructions, equilibrate at room temperature for 15 minutes, add 180μL of QUANTI-Blue-Solution to a new 96-well flat-bottom cell culture plate per well, then add 20μL of the supernatant of the incubated fusion peptide to this 96-well flat-bottom cell culture plate, gently shake, and incubate at 37℃ with 5% CO2 for 2-4 hours. Measure the absorbance at 630nm using a multi-functional microplate reader (SPARK, SZ-A2-TM-LAB09).

[1339] The experimental results are shown in Figures 3A-3D. Figure 3A shows the fusion peptides of αPD-1 with different IFNα2b mutants in Format 1 molecular form (peptide molecules 090, 079, 095, 104, 163, 165, 088, 097, 164, 166, 167, 080, 081, 086, 088, and 111) in HEK-Blue. TM Results of assays in IFN-α / β reporter cell lines. Compared with the wild-type molecule (αPD-1-IFNα2b-WT molecule, code: 011) used as a control, the αPD-1-IFNα2b-m fusion peptide in this study showed reduced activity in PD-1 negative cells, suggesting that the affinity-weakened IFNα2b mutant in this study reduces the off-target toxicity of the fusion peptide and increases its potential for in vivo tolerance.

[1340] Figure 3B shows the αPD-1-IFNα2b-m fusion peptides 097, 174, 177, 204, 205, and 206 in HEK-Blue with the same IFNα2b mutation combination but different molecular forms (Format 1 to Format 4, see Figure 1). TMResults of IFN-α / β assays in reporter cell lines. Compared with control molecule 018 and fusion peptide 097, fusion peptides 174, 177, 204, 205, and 206 showed enhanced activity in both PD-1 negative and PD-1 positive cells, and enhanced selectivity between these two cell types. The IFNα2b mutants in fusion peptides 174 (T106A) and 177 (V105E) are molecules modified by removing only O-glycosylation from wild-type IFNα2b. No significant difference was observed in protein affinity compared to the wild-type molecule (peptide number: 011) (Table 4). In this example, HEK-Blue... TM IFN-α / β reporter cell lines showed better selectivity than wild-type molecules.

[1341] Figure 3C shows the wild-type molecule 214 and mutant molecules 204, 215, 216, 217, 218, 219, 220 and 221 of the αPD-1-IFNα2b fusion peptide in Format 2 in HEK-Blue. TM Results of assays in IFN-α / β reporter cell lines; and the results of assays in HEK-Blue cells with the αPD-1-IFNα2b fusion polypeptide wild-type molecule 011 and mutant molecule 097 in the same experiment. TM Results of assays in IFN-α / β reporter cell lines. Compared with wild-type molecules 214 and 011, the αPD-1-IFNα2b-m fusion peptide of this disclosure exhibits reduced activity in PD-1 negative cells and high selectivity between PD-1 positive and PD-1 negative cells.

[1342] Figure 3D shows the wild-type molecule 214, mutant molecules 216 and 223 of IFNα2b and αPD-1-IFNα2b fusion peptides in Format 2 in HEK-Blue. TM The assay results in IFN-α / β reporter cell lines showed that, compared with IFNα2b and wild-type molecule 214, the activity of the disclosed αPD-1-IFNα2b-m fusion peptide was reduced in PD-1 negative cells, and it exhibited high selectivity between PD-1 positive and PD-1 negative cells.

[1343] Example 8. Detection of pSTAT1 signal in activated PBMC cells using the αPD1-IFNα2b-m fusion peptide.

[1344] Isolation of CD8+ T cells: 1) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10091-148), 1% penicillin-streptomycin (Gibco, 15140-122); 2) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS, catalog number PB100C-W) from the liquid nitrogen tank, and quickly place them in a 37℃ water bath, gently shaking until thawed, keeping the cap of the cryovial above the water surface to prevent contamination; 3) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench, strictly following aseptic techniques thereafter; 4) Transfer the cell suspension from the cryovial to a 15mL centrifuge tube, and add 10mL of DNase 1 (SIG) 5) In 1640 complete medium (MA, D5025-375KU), gently mix and centrifuge at 1000 rpm for 5 minutes to precipitate the cells. Discard the supernatant and then wash once with PBS (Shanghai Sangon Biotech, E607008-0500); 6) Isolate CD8+ T cells from PBMC according to the instructions of the CD8+ T cell isolation kit (stemcell, 19053); 7) Resuspend the CD8+ T cells in 15 mL of 1640 complete medium, take 20 μL of the cell suspension, and count the viable cells using trypan blue (Gibco, 15250-061).

[1345] CD8+ T cell activation and pSTAT1 signal detection (P-STAT1 assay): 1) Coat T75 culture flasks (NUNC, 156499) with 1 μg / mL anti-human CD3 antibody (Acro, CDE-H5223) and incubate at 37℃ for 2-3 hours; 2) Take the isolated CD8+ T cells, resuspend the cells in 1640 complete medium containing 2 μg / mL anti-CD28 antibody (Invitrogen, 16-0289-85), and dilute the cells to 2.0 × 10⁻⁶. 6 The cell suspension obtained was added to a coated T75 culture flask and incubated at 37°C with 5% CO2 for 72 hours to activate CD8+ T cells; 3) After 3 days, the activated CD8+ T cells were collected, washed once with 1640 complete medium, and the cell density was adjusted to 2.0 × 10⁶ cells / mL. 6 4) Take the incubated cell suspension, wash once with PBS, resuspend in PBS, and stain the cells with AF488-αPD1 (obtained by labeling αPD-1 with AF488, AF488 purchased from Thermo Fisher, A10235) in an incubator for 20 minutes. Wash once with PBS and dilute the labeled cells to 3.0 × 10⁶ cells / mL. 65) Dilute the αPD1-IFNα2b fusion peptide with 1640 complete medium in a sterile 96-well V plate. The initial concentration was 3000 nM, and it was diluted 6-fold in 10 steps. Add 88 μL of the diluted fusion peptide solution to each well of the cell-containing 96-well V plate and incubate at 37°C for 20 minutes with 5% CO2. 6) Add 25 μL of 16% Formaldehyde Solution (ThermoScientific, 28908) to each well. 7) Incubate at 37°C with CO2 for 10 minutes, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant; 8) Resuspend the cells in 50 μL of 0.05% Triton-X100 (Beyotime, ST797-500 mL) per well, incubate at 4°C for 10 minutes, and then wash once with 200 μL of Perm / wash buffer (BD Pharmingen, 554723) per well; 9) Prepare pSTAT1 staining antibody (BD Pharmingen, 612597) in Perm / wash buffer at a volume ratio of 1:50 (antibody:buffer). Add 2 μL of antibody + 100 μL of buffer to each well, incubate at room temperature for 1.5 hours, wash twice with Perm / wash buffer, and then add 120 μL of Perm / wash buffer to each well for FACS detection.

[1346] The experimental results are shown in Figures 4A-4F. Figure 4A shows the effect of fusion peptides (097, 164, 166, 167) with different affinity-weakened IFNα2b mutants in Format 1 molecular form on pSTAT1 signaling in activated T lymphocytes. Fusion peptides 011 and 018 are wild-type and control molecules, respectively. In PD-1-negative (PD-1-) T cells, the activity of fusion peptide 097 in this study was weaker than that of control molecule 018, while in PD-1-positive (PD-1-) T cells, the activity of fusion peptide 097 was weaker than that of control molecule 018. + On T cells, the activity of fusion peptide 097 was comparable to that of control molecule 018, indicating that fusion peptide 097 has greater selectivity for PD-1 than control molecule 018, less off-target toxicity than control molecule 018, and a higher tolerable dose in vivo than control molecule 018.

[1347] Figure 4B illustrates the effects of fusion peptides 097, 174, 177, 204, 205, and 206, representing different molecular forms (Format 1 to Format 4) of the affinity-weakened IFNα2b mutant, on pSTAT1 signaling in activated T lymphocytes. Formats 2-4 all showed stronger phosphorylation signals and enhanced pSTAT1 signaling in PD-1 positive cells. + The fusion peptides 174 and 177, with only the O-glycosylation removed, showed better selectivity on T cells and PD-1 negative T cells. The pSTAT1 signaling on activated T lymphocytes was comparable to that of wild-type molecule 011 with only the O-glycosylation removed. However, further O-glycosylation removal (control molecule 018, with the A145D+T106A mutation) significantly enhanced pSTAT1 signaling on activated T lymphocytes compared to control molecule 012 (with only attenuated affinity) which showed a weakened affinity.

[1348] Figure 4C shows the effects of fusion peptides 097, 204, 205, and 206, IFNα2b mutants with different molecular forms (Format 1 to Format 4), on pSTAT1 signaling in activated T lymphocytes from another PBMC donor. Among them, fusion peptides 204, 205, and 206, with molecular forms of Format 2 to Format 4, showed stronger on-target activity, suggesting potentially stronger tumor therapeutic effects; fusion peptide 097, with molecular form of Format 1, showed weaker off-target toxicity, potentially leading to better tolerability.

[1349] Figures 4D and 4E show the effects of different molecular forms of αPD-1-IFNα2b fusion peptides 097, 204, 214, 215, 216, 217, 218, 219, 220, and 221 on pSTAT1 signaling in activated T lymphocytes from two different PBMC donors (fusion peptides 011 and 097 are wild-type and mutant molecules of Format 1, respectively; fusion peptide 214 is wild-type molecule of Format 2; and the remaining fusion peptides are mutant molecules of Format 2).

[1350] Figure 4F shows the results of detecting the effects of IFNα2b and Format 2 molecular forms of αPD-1-IFNα2b fusion peptides 214, 216 and 223 on pSTAT1 signaling in activated T lymphocytes from PBMC donors. Compared with IFNα2b and wild-type molecule 214, the αPD-1-IFNα2b-m fusion peptides disclosed herein showed reduced activity on PD-1 negative cells and high selectivity between PD-1 positive and PD-1 negative cells.

[1351] Example 9. Verification of the blocking function of the αPD1-IFNα2b-m fusion peptide using CHO-K1-PD-L1 and Jurkat hPD1-NFAT-Luc reporter cell lines.

[1352] The function of the disclosed αPD1-IFNα2b-m fusion peptide was assessed using CHO-K1-PD-L1 and Jurkat hPD1-NFAT-Luc reporter cell lines. It is anticipated that the disclosed αPD1-IFNα2b fusion peptide will bind to PD-1, thereby blocking the binding of CHO-K1-PD-L1 and Jurkat hPD-1-NFAT-Luc reporter cell lines. The blocking effect of the αPD1-IFNα2b-m fusion peptide on these two reporter cell lines can be detected using Bio-Glo luciferase. This study used PD-L1αAPC / CHO-K1 cells (Promega, J1250; CHO-K1-PD-L1 cell line) and PD-1 effector Jurkat cells (Promega, J1250; Jurkat hPD1-NFAT-Luc cell line) to verify the PD-1 blocking function of the αPD1-IFNα2b fusion peptide.

[1353] Cell resuscitation: 1) Prepare F12K complete medium and 1640 complete medium in advance. F12K complete medium: F12K (ATCC, 30-2004), 10% FBS (Gibco, 10091-148), 1% penicillin-streptomycin (Gibco, 15140-122), 200 μg / mL hygromycin B (Basal Media, S160J7), 250 μg / mL GENECTICIN (Gibco, 10131-027); and 1640 complete medium: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% penicillin-streptomycin (Gibco, 15140-122), 1% NEAA (Hyclone, SH30238.01), 1%... 1) Dissolve NaP (Gibco, 11360-070), 200 μg / mL hygromycin B (Basal Media, S160J7), and 500 μg / mL GENECTICIN (Gibco, 10131-027); 2) Remove the frozen cells from the liquid nitrogen tank and gently shake them in a 37°C water bath until thawed, keeping the cap of the cryovial above the water surface to prevent contamination; 3) After thawing, immediately sterilize with 75% alcohol and transfer to a clean bench, strictly following aseptic techniques thereafter; 4) Transfer the cell suspension from the cryovial to a 15 mL centrifuge tube, add 10 mL of F12K complete medium (CHO-K1-PDL1 reporter cell line) and 10 mL of 1640 complete medium (Jurkat hPD1-NFAT-Luc reporter cell line), gently mix, centrifuge at 1000 rpm for 5 minutes to precipitate the cells, and discard the supernatant; 5) Use 5 mL Resuspend the cell pellet in F12K complete medium and 5 mL of 1640 complete medium. Take 20 μL of the cell suspension and count the viable cells using trypan blue (Gibco, 15250-061); 6) Adjust the viable cell density to 5 × 10⁶ cells / mL using the above-mentioned medium. 5 7) Incubate the cells at 37°C and 5% CO2 in a constant temperature incubator.

[1354] Cell culture: For the CHO-K1-PD-L1 reporter cell line, maintain a selection pressure of 200 μg / mL hygromycin B and 250 μg / mL GENECTICIN during passage; for the Jurkat hPD1-NFAT-Luc reporter cell line, maintain a selection pressure of 200 μg / mL hygromycin B and 500 μg / mL GENECTICIN during passage. Passage should be performed every 2-3 days, with a passage density controlled at 5 × 10⁻⁶ cells / year. 5 Cells / mL.

[1355] Activity assay: 1) The night before the assay, adjust the density of CHO-K1-PD-L1 reporter cells to 4 × 10⁻⁶ cells using F12K complete medium. 5 1) Spread the obtained cell suspension at 100 μL per well into 60 wells of a 96-well flat-bottom cell culture plate (NEST, 701001). Spread 100 μL / well of PBS into each well at the outer edge of the plate. Incubate overnight at 37°C with 5% CO2. 2) Prepare assay buffer (99% RPMI 1640 medium (Gibco, 22400-071) + 1% FBS). Dilute the test samples (anti-PD-1 monoclonal antibody sintilimab, fusion peptide) and control Isotype (hIgG) with assay buffer. The initial concentration of the test samples and control is 300 nM, 4-fold dilution, for a total of 10 gradients, with three replicates for each concentration. After overnight cell culture, remove the supernatant from the overnight culture and use assay buffer. Wash once with buffer, then add 50 μL / well of the diluted test sample to the culture plate containing cells; 3) Adjust the Jurkat hPD-1-NFAT-Luc reporter cell line density to 5 × 10⁻⁶ cells / well with 1640 complete medium. 5 100 μL of the culture plate was added to each well at a concentration of 100 cells / mL, mixed thoroughly, and incubated at 37°C and 5% CO2 for 6 hours. 4) After incubation for 6 hours, 100 μL of thawed Bio-Glo luciferase was added to each well and immediately detected using a microplate reader.

[1356] The experimental results are shown in Figures 5A and 5B. Figure 5A shows that the anti-PD-1 monoclonal antibody sintilimab (its heavy chain and light chain are shown as SEQ ID NO:204 and SEQ ID NO:5, respectively) and the fusion peptides 097, 204, 205, and 206 of Format 1 to Format 4 all have PD-1 blocking functions. The PD-1 blocking functions of fusion peptides 097 and 204 are comparable to those of sintilimab. The PD-1 blocking function of fusion peptide 206 is slightly weaker than that of sintilimab, and the PD-1 blocking function of fusion peptide 205 is relatively weaker.

[1357] Figure 5B shows that the anti-PD-1 monoclonal antibody sintilimab, as well as the fusion peptides 216 and 223 of Format 2, all have PD-1 blocking functions, and their PD-1 blocking functions are comparable to those of sintilimab.

[1358] Example 10. Study on the release of CXCL10 signaling from primary PBMC cells stimulated by αPD1-IFNα2b-m fusion peptide.

[1359] Resuscitating PBMC cells: 1) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10091-148), 1% penicillin-streptomycin (Gibco, 15140-122); 2) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS, catalog number PB100C-W) from the liquid nitrogen tank and quickly place them in a 37°C water bath, gently shaking until thawed. Keep the cap of the cryovial above the water surface to prevent contamination; 3) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench, strictly following aseptic techniques thereafter; 4) Transfer the cell suspension from the cryovial to... Add 10 mL of 1640 complete medium containing DNase1 (SIGMA, D5025-375KU) to a 15 mL centrifuge tube, mix gently, centrifuge at 1000 rpm for 5 minutes to precipitate the cells, discard the supernatant, and then wash once with PBS (Shanghai Sangon Biotech, E607008-0500); 5) Resuspend PBMC cells in 15 mL of 1640 complete medium, take 20 μL of cell suspension, and count the viable cells using trypan blue (Gibco, 15250-061).

[1360] CXCL10 signal detection: 1) Dilute cells to 3.0 × 10⁻⁶ using 1640 complete medium. 6 1) Cells / mL, diluted cell suspension was added to 88 μL per well of a 96-well V plate (Beyotime, FPT019); 2) The αPD1-IFNα2b fusion peptide was diluted with 1640 complete medium in a sterile 96-well V plate at an initial concentration of 3000 nM, 6-fold dilution, for a total of 10 gradients. The diluted fusion peptide solution was added to 88 μL per well of the 96-well V plate containing cells. Wells containing the same volume of P1AI429 (US2024376205A1) and isotype were set as controls. The plate was incubated for three days in a 5% CO2, 37°C incubator; 3) After three days of incubation in a 5% CO2, 37°C incubator, the plate was centrifuged at 1500 rpm for 5 minutes and the supernatant was collected; 4) The supernatant was diluted twice with PBS and detected using a CXCL10 assay kit (R&D, DY266).

[1361] The experimental results are shown in Figure 6. The fusion peptides 216 and 223 of this disclosure stimulated PBMC to release CXCL10 less than the control molecules 010 and P1AI4295, indicating that the off-target toxicity of the fusion peptides 216 and 223 of this disclosure is less than that of the control molecules, and the tolerable dose in vivo is also higher than that of the control molecules.

[1362] Example 11. Using B16-Blue TMScreening of IFN-α / β reporter cell lines for αPD1-IFNα2b mouse alternative molecules

[1363] Using B16-Blue TM The IFN-α / β reporter cell line was used to functionally test the αPD1-IFNα2b mouse substitute molecules (molecules 116 and 118) prepared in Example 2. It is expected that the αPD1-IFNα2b mouse substitute molecules will interact with B16-Blue. TM The binding of the mIFNα receptor to the IFN-α / β reporter cell line activates the JAK / STAT / ISGF3 pathway, leading to the production of secretory embryonic alkaline phosphatase (SEAP). This pathway activation can be detected using the QUANTI-Blue-Solution (a SEAP detection reagent). This study used the B16-Blue reporter cell line provided by InvivoGen. TM The IFN-α / β reporter cell line (bb-ifnt1) was administered via B16-Blue according to the instructions. TM IFN-α / β reporter cell lines were used to evaluate the function of the αPD1-IFNα2b mouse substitute molecule.

[1364] Cell resuscitation: 1) Prepare DMEM complete medium in advance: DMEM (ATCC, 30-2002), 10% 1) Use FBS (Gibco, 10091-148) and 1% penicillin-streptomycin (Gibco, 15140-122); 2) Remove the frozen cells from the liquid nitrogen tank and gently shake them in a 37°C water bath until thawed, keeping the cap of the cryovial above the water surface to prevent contamination; 3) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench, then strictly follow aseptic techniques; 4) Transfer the cell suspension from the cryovial to a 15mL centrifuge tube, add 10mL of DMEM complete medium, mix gently, centrifuge at 1000rpm for 5 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 5mL of DMEM complete medium, take 20μL of the cell suspension, and count the viable cells using trypan blue (Gibco, 15250-061); 6) Adjust the viable cell density to 5×10⁶ cells / mL. 5 7) Incubate the cells at 37°C and 5% CO2 in a constant temperature incubator.

[1365] Cell culture: Maintain a selection pressure of 1.0 μg / mL puromycin (Gibco, A11138-02) during passage, and perform passage every 2-3 days, controlling the passage density at 5 × 10⁻⁶ cells / year. 5 Cells / mL.

[1366] Viability assay: 1) Cell plating: Cells were digested with 0.25% Trypsin-EDTA trypsin (Gibco, 25200-072), then DMEM complete medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 minutes, resuspended in DMEM complete medium, and the cell density was adjusted to 4.2 × 10⁻⁶ cells / cm². 5 Cells / mL, to obtain cell suspension, add 180 μL of the cell suspension to each well of a 96-well flat-bottom cell culture plate (NEST, 701001); 2) Prepare αPD1-IFNα2b mouse substitute molecule: Dilute the fusion peptide with DMEM complete medium in a sterile 96-well V-bottom plate (Beyotime, FPT019). The initial concentration of the fusion peptide is 3000 nM, 5-fold dilution, for a total of 16 gradients. Add 36 μL of the diluted fusion peptide to each well of the 96-well flat-bottom cell culture plate from step 1), 5% 3) Microplate reader detection: Prepare QUANTI-Blue-Solution reagent (Invitrogen, rep-qbs3) in advance according to the instructions, equilibrate at room temperature for 15 minutes, add 180μL of QUANTI-Blue-Solution to a new 96-well flat-bottom cell culture plate per well, then add 20μL of the supernatant of the incubated αPD1-IFNα2b mouse substitute molecule to this 96-well flat-bottom cell culture plate, gently shake well, incubate at 37℃ with 5% CO2 for 4-6 hours, and measure the absorbance at 630nm using a multi-functional microplate reader (SPARK, SZ-A2-TM-LAB09).

[1367] The experimental results are shown in Figure 7. The αPD1-IFNα2b mouse substitute molecules (116, 118, 183, and 209) can effectively activate the JAK / STAT / ISGF3 pathway, and mPD1 overexpression of B16-Blue... TM Signaling in IFN-α / β reporter cell lines and wild-type B16-Blue TM There is a certain window between the signaling of IFN-α / β reporter cell lines. Compared with αPD1-IFNα2b mouse substitute molecules 183 and 209, which have the same IFNα2b mutation but different molecular forms (Format1 and Format2, see Figure 1), mouse substitute molecule 209 has stronger in vitro activity.

[1368] Example 12. Antitumor efficacy of αPD1-IFNα2b mouse alternative molecule in B16F10 mouse melanoma model

[1369] Subcutaneous inoculation in mice: On day 0, C57 mice were shaved on the right dorsoventral side and inoculated with B16F10 cells at a dose of 5 × 10⁶ cells. 5Cells per mouse, inoculated at a volume of 200 μL per mouse (cells suspended in PBS). On day 7 post-inoculation, the maximum width and length axes of mouse tumors were measured using calipers to calculate tumor volume. Based on tumor volume, mice were grouped in a serpentine pattern for drug administration: Isotype (h-IgG, isotype control antibody, purchased from Equitech-Bio, batch number 161206-0656), 32 mg / kg; anti-mouse PD-1 antibody (αmPD-1, same as in Example 2), 32 mg / kg; αPD1-IFNα2b mouse substitute molecule 118, 20 mg / kg; αPD1-IFNα2b mouse substitute molecule 116, 40 mg / kg, for a total of 4 groups, 6 mice per group. Drugs were administered intraperitoneally on days 7, 10, 14, and 17, and monitoring continued until day 28 (following animal ethics, mice with tumors exceeding 2000 mm² were monitored). 3 The patient will be euthanized at that time; B16F10 is a malignant tumor that is prone to ulceration / excessive tumor size can lead to death.

[1370] The relative tumor inhibition rate (TGI%) was calculated on day 17 post-vaccination using the following formula: TGI% = 100% × (Tumor volume in the control group - Tumor volume in the treatment group) / (Tumor volume in the control group - Tumor volume in the control group before drug administration)

[1371] Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using vernier calipers. The tumor volume was calculated using the following formula: V = (L * W) 2 ) / 2.

[1372] The results are shown in Table 9 and Figures 8-10. At the same dose, the αPD1-IFNα2b mouse substitute molecule has better anti-tumor efficacy than αmPD-1, indicating that the fusion peptide of this disclosure, which has comparable in vitro activity to the substitute molecule, has the potential to treat melanoma.

[1373] Table 9. Tumor suppression rate for the B16F10 model

[1374] Example 13. Antitumor efficacy of αPD1-IFNα2b mouse alternative molecule in EMT6 mouse breast cancer model

[1375] To demonstrate the in vivo efficacy of the αPD1-IFNα2b mouse substitute molecule prepared in Example 2, we constructed a breast cancer model in Balb / C mice by inoculating EMT6 cells to determine the antitumor efficacy of the disclosed mouse substitute molecule, thereby showcasing the antitumor potential of the fusion peptide of this disclosure. SPF-grade female mice (16-19g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the experiment. Certificate No.: 110011241105390638. After arrival, the mice were acclimatized and fed for 3 days before the experiment began. Body weight was measured using an electronic balance.

[1376] Subcutaneous inoculation in mice: On day 0, Balb / C mice were shaved on the right dorsoventral side and inoculated with EMT6 cells at a dose of 1×10⁻⁶. 6 Cells per mouse, inoculated at a volume of 200 μL per mouse (cells su...

Claims

1. An IFNα2b mutant, wherein, Compared to human wild-type IFNα2b, the IFNα2b mutant comprises: (a) A mutation at position 105, and mutations at any one or more of the following positions: position 15, position 19, position 26, position 30, position 33, position 57, position 58, position 61, position 80, position 120, position 142, position 145, position 146, position 148, position 149; (b) A mutation at position 145, and a mutation at any one or more of the following positions: position 105, position 107, position 108, position 109, the CD ring sequence or a portion thereof; (c) A mutation selected from any one or more of the following positions: position 105, position 106, position 108, position 109, a mutation in the CD ring sequence or a portion thereof; or (d) Mutations selected from any one or more of the following positions: position 15, position 26, position 30, position 142, position 148, position 149.

2. The IFNα2b mutant as described in claim 1, wherein, Compared to the wild-type human IFNα2b, in addition to the amino acid substitution mutation at V105, the IFNα2b mutant also contains amino acid substitution mutations at at least one of the following positions: L15, A19, L26, L30, R33, H57, E58, Q61, L80, R120, V142, A145, E146, M148, or R149.

3. The IFNα2b mutant as described in claim 1 or 2, wherein, Compared to human wild-type IFNα2b, the IFNα2b mutant contains the following mutation: V105E, and at least one mutation selected from the following: L15A, L15E, L15Q, L15S, L15V, L15G, A19G, L26A, L30V, L30G, L30I, L30S, L30Q, R33A, R33K, R33H, R33N, R33S, H57Y, E58N, Q61S, L80A, R120G, R120A, V142A, V142D, V142E, V142G, V142H, V142I, V1 42K, V142L, V142M, V142N, V142Q, V142R, V142S, V142T, V142Y, A145D, E146A, E146G, E146H, E146I, E146K, E146L, E146R, E146S, E146T, M148A, M148D, M148G, M148K, M148S, R149E, R149G, or R149S.

4. The IFNα2b mutant as described in any one of claims 1-3, wherein, Compared to wild-type human IFNα2b, the IFNα2b mutant comprises a combination of V105E and at least one of the following mutations: (1) L15A, L15E, L15Q, L15S, L15V or L15G; (2) L30V, L30G, L30I, L30S or L30Q; (3) R33A, R33K, R33H, R33N or R33S; (4) R120G; (5) V142A, V142D, V142E, V142G, V142H, V142I, V142K, V142L, V142M, V142N, V142Q, V142R, V142S, V142T or V142Y; (6)A145D; (7) E146A, E146G, E146H, E146I, E146K, E146L, E146R, E146S or E146T; (8) M148A, M148D, M148G, M148K or M148S; (9) R149E, R149G, or R149S; or (10) L15G+A19G, L15A+V142A, L15A+V142N, L15A+L26A+V142A, L15G+M148G, L26A+V142A, R33A+H57Y+E58N+Q61S, V142A+E146A, L26A+V142A+L80A or L26A+V142A+R120A.

5. The IFNα2b mutant as described in claim 1, wherein, Compared to wild-type human IFNα2b, in addition to the amino acid substitution mutation at A145, the IFNα2b mutant also contains amino acid substitution mutations at at least one of the following positions: V105, E107, T108, P109, CD ring sequence or part thereof.

6. The IFNα2b mutant as described in claim 1 or 5, wherein, Compared to wild-type human IFNα2b, the IFNα2b mutant contains the following mutation: A145D, and at least one mutation selected from the following: The following sequences in the CD ring region are replaced with "EEKLEKEDFTRGKLM" (SEQ ID NO:175), "IQGVGVTETPLMKED" (SEQ ID NO:174), "EEKLEKTDFTRGKLM" (SEQ ID NO:176), "GVTETPLM" (SEQ ID NO:177) and "EKEDFTRG" (SEQ ID NO:178), or "GVTETPLM" (SEQ ID NO:177) and "EKTDFTRG" (SEQ ID NO:179).

7. The IFNα2b mutant as described in any one of claims 1, 5, and 6, wherein, Compared to wild-type human IFNα2b, the IFNα2b mutant contains a combination of A145D and at least one of the following mutations: (1) V105E, V105E+P109R or V105E+T108I+P109A; (2) E107D, E107K or E107D+T108A; (3) T108G, T108A or T108I+P109A; (4) P109R or P109N; or (5) Replace "IQGVGVTETPLMKED" (SEQ ID NO:174) in the CD ring region sequence with "EEKLEKEDFTRGKLM" (SEQ ID NO:175). Replace "IQGVGVTETPLMKED" (SEQ ID NO:174) in the CD ring sequence with "EEKLEKTDFTRGKLM" (SEQ ID NO:176). Replace "GVTETPLM" (SEQ ID NO:177) in the CD ring region sequence with "EKEDFTRG" (SEQ ID NO:178), or In the CD ring region sequence, "GVTETPLM" (SEQ ID NO:177) is replaced with "EKTDFTRG" (SEQ ID NO:179).

8. The IFNα2b mutant as described in claim 1, wherein, Compared to the human wild-type IFNα2b, the IFNα2b mutant contains an amino acid substitution or deletion mutation at at least one of the following positions: V105, T106, T108, P109, CD loop sequence or a portion thereof.

9. The IFNα2b mutant as described in claim 1 or 8, wherein, Compared to human wild-type IFNα2b, the IFNα2b mutant contains at least one of the following mutations: Replace "GVTETPLM" (SEQ ID NO:177) in V105E, T106A, ΔT106, T108I, P109A, P109R, and CD ring sequences with "EKTDFTRG" (SEQ ID NO:179), or replace "IQGVGVTETPLMKED" (SEQ ID NO:174) in CD ring sequences with "EEKLEKTDFTRGKLM" (SEQ ID NO:176).

10. The IFNα2b mutant as described in any one of claims 1, 8, and 9, wherein, Compared to human wild-type IFNα2b, the IFNα2b mutant contains at least one of the following mutations: (1) V105E, V105E+P109R or V105E+T108I+P109A; (2) T106A or ΔT106; (3) P109R or T108I+P109A; or (4) Replace "GVTETPLM" (SEQ ID NO:177) in the CD ring region sequence with "EKTDFTRG" (SEQ ID NO:179), or Replace "IQGVGVTETPLMKED" (SEQ ID NO:174) in the CD ring sequence with "EEKLEKTDFTRGKLM" (SEQ ID NO:176).

11. The IFNα2b mutant as described in claim 1, wherein, Compared to human wild-type IFNα2b, the IFNα2b mutant contains at least one of the following mutations: (1)L26A+V142A; (2) M148K; (3)R149G; (4) L15G+M148G; (5) V142L or V142M; or (6)L30S.

12. The IFNα2b mutant according to any one of claims 1-11, wherein, The human wild-type IFNα2b contains the amino acid sequence shown in SEQ ID NO:

4.

13. The IFNα2b mutant according to any one of claims 1-12, wherein, The IFNα2b mutant comprises an amino acid sequence shown in either SEQ ID NOs:98-173 or 226-227, or an amino acid sequence having at least 80% sequence identity with it.

14. Use of the IFNα2b mutant of any one of claims 1-13 in the preparation of a fusion polypeptide, for example, said fusion polypeptide comprising an anti-PD-1 antibody or a portion thereof.

15. A fusion polypeptide comprising: (1) an IFNα2b mutant, and (2) an anti-PD-1 antibody or a portion thereof, wherein, Compared to human wild-type IFNα2b, the IFNα2b mutant comprises: (a) A mutation at position 105, and mutations at any one or more of the following positions: position 15, position 19, position 26, position 30, position 33, position 57, position 58, position 61, position 80, position 120, position 142, position 146, position 148, position 149; (b) A mutation at position 145, and a mutation at any one or more of the following positions: position 105, position 107, position 108, position 109, the CD ring sequence or a portion thereof; (c) A mutation selected from any one or more of the following positions: position 105, position 106, position 108, position 109, a mutation in the CD ring sequence or a portion thereof; or (d) Mutations selected from any one or more of the following positions: position 15, position 26, position 30, position 142, position 148, position 149.

16. A polypeptide construct comprising: (1) Peptide chain 1, from N-terminus to C-terminus, includes: the heavy chain variable region (VH), CH1 and Fc regions of the anti-PD-1 antibody, and the IFNα2b mutant; and Peptide chain 2, from N-terminus to C-terminus, sequentially includes the light chain variable region (VL) and CL of the anti-PD-1 antibody; (2) Peptide chain 1, from N-terminus to C-terminus, comprises: the heavy chain variable region (VH), CH1 and Fc region of the anti-PD-1 antibody, and the IFNα2b mutant; Peptide chain 2, from N-terminus to C-terminus, sequentially includes the light chain variable region (VL) and CL of the anti-PD-1 antibody; and Peptide chain 3, from N-terminus to C-terminus, sequentially includes the VH, CH1 and Fc regions of the anti-PD-1 antibody; (3) Peptide chain 1', from N-terminus to C-terminus, contains the IFNα2b mutant and the Fc region of the anti-PD-1 antibody in sequence; Peptide chain 2, from N-terminus to C-terminus, sequentially comprises the VL and CL of the anti-PD-1 antibody; and Peptide chain 3, from N-terminus to C-terminus, sequentially includes the VH, CH1, and Fc regions of the anti-PD-1 antibody; or (4) Peptide chain 1', from N-terminus to C-terminus, contains the IFNα2b mutant and the Fc region of the anti-PD-1 antibody in sequence; Peptide chain 2, from N-terminus to C-terminus, sequentially comprises the VL and CL of the anti-PD-1 antibody; and The peptide chain 3' contains, from the N-terminus to the C-terminus, the VH, CH1, VH, CH1 and Fc regions of the anti-PD-1 antibody; in, Compared to human wild-type IFNα2b, the IFNα2b mutant comprises: (a) A mutation at position 105, and mutations at any one or more of the following positions: position 15, position 19, position 26, position 30, position 33, position 57, position 58, position 61, position 80, position 120, position 142, position 146, position 148, position 149; (b) A mutation at position 145, and mutations at one or more of the following positions: position 105, position 107, position 108, position 109, the CD ring sequence or a portion thereof; or (c) A mutation selected from any one or more of the following positions: position 105, position 106, position 108, position 109, a mutation in the CD ring sequence or a portion thereof; or (d) Mutations selected from any one or more of the following positions: position 15, position 26, position 30, position 142, position 148, position 149.

17. The fusion polypeptide of claim 15 or the polypeptide construct of claim 16, wherein, The anti-PD-1 antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein, The VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:187 or an amino acid sequence having at least 80% sequence identity with it, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:188 or an amino acid sequence having at least 80% sequence identity with it, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:189 or an amino acid sequence having at least 80% sequence identity with it. The VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:190 or an amino acid sequence having at least 80% sequence identity with it, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:191 or an amino acid sequence having at least 80% sequence identity with it, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:192 or an amino acid sequence having at least 80% sequence identity with it.

18. The fusion polypeptide of claim 15 or the polypeptide construct of claim 16, wherein, The anti-PD-1 antibody may contain: (1) HCDR1 having the amino acid sequence shown in SEQ ID NO:187, (2) HCDR2 having the amino acid sequence shown in SEQ ID NO:188, (3) HCDR3 having the amino acid sequence shown in SEQ ID NO:189, (4) LCDR1 having the amino acid sequence shown in SEQ ID NO:190, (5) LCDR2 having the amino acid sequence shown in SEQ ID NO:191, and (6) LCDR3 having the amino acid sequence shown in SEQ ID NO:

192.

19. The fusion polypeptide of claim 15 or the polypeptide construct of claim 16, wherein, The anti-PD-1 antibody may contain: (1) Heavy chain variable region, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it; and / or (2) Light chain variable region, containing the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it.

20. The fusion polypeptide of claim 15 or the polypeptide construct of claim 16, wherein, The anti-PD-1 antibody further comprises a heavy chain constant region (CH), which comprises the constant region of human IgG or a variant thereof.

21. The fusion polypeptide of claim 15 or the polypeptide construct of claim 16, wherein, The anti-PD-1 antibody further comprises a constant region of human IgG1 or IgG4 or a variant thereof.

22. The fusion polypeptide of claim 15 or the polypeptide construct of claim 16, wherein, The anti-PD-1 antibody further comprises a heavy chain constant region having an amino acid sequence shown in SEQ ID NO: 205, 206, 207, 208, 210, 242, 243, 244, 245, 246 or 247.

23. The fusion polypeptide of claim 15 or the polypeptide construct of claim 16, wherein, The anti-PD-1 antibody further comprises a light chain constant region (CL), which includes a human κ chain or λ chain constant region.

24. The fusion polypeptide of claim 15 or the polypeptide construct of claim 16, wherein, The anti-PD-1 antibody further comprises a light chain constant region having the amino acid sequence shown in SEQ ID NO:

209.

25. The fusion polypeptide of claim 15 or the polypeptide construct of claim 16, wherein, The anti-PD-1 antibody comprises: (1) A heavy chain comprising an amino acid sequence shown in SEQ ID NO:2, 93, 94, 204, 236, 237, 238, 239, 240 or 241, or an amino acid sequence having at least 80% sequence identity with it; and / or (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the anti-PD-1 antibody may comprise: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% sequence identity with it; and / or (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the anti-PD-1 antibody may comprise: (1) A first heavy chain comprising the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% sequence identity with it, and a second heavy chain comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it; and / or (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the anti-PD-1 antibody may comprise: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:204 or an amino acid sequence having at least 80% sequence identity with it; and / or (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the anti-PD-1 antibody may comprise: 1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:240 or an amino acid sequence having at least 80% sequence identity with it; and / or (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the anti-PD-1 antibody may comprise: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:241 or an amino acid sequence having at least 80% sequence identity with it; and / or (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the anti-PD-1 antibody may comprise: (1) A first heavy chain comprising the amino acid sequence shown in SEQ ID NO:238 or an amino acid sequence having at least 80% sequence identity with it, and a second heavy chain comprising the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% sequence identity with it; and / or (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the anti-PD-1 antibody may comprise: (1) A first heavy chain comprising the amino acid sequence shown in SEQ ID NO:239 or an amino acid sequence having at least 80% sequence identity with it, and a second heavy chain comprising the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% sequence identity with it; and / or (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it.

26. The fusion polypeptide of claim 15, wherein, The anti-PD-1 antibody portion includes an anti-PD-1 antibody portion lacking one Fab fragment, a half antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd fragment, an Fv fragment, a dAb fragment, isolated CDR, CH1, CH2, CH3, Fc region, scFv, nanobodies, and any combination thereof.

27. The fusion polypeptide of claim 15 or 26, wherein, The anti-PD-1 antibody comprises: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it; (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:195, 196, 197, 252 or 253 or an amino acid sequence having at least 80% sequence identity with it.

28. The fusion polypeptide of claim 15 or 26, wherein, The anti-PD-1 antibody comprises: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% sequence identity with it; (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:195, 198, 252 or 253 or an amino acid sequence having at least 80% sequence identity with it.

29. The fusion polypeptide according to any one of claims 15 and 26-28, wherein, The anti-PD-1 antibody comprises: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it; (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) One Fc fragment containing the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, a portion of the anti-PD-1 antibody may include: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% sequence identity with it; (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) One Fc fragment containing the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, a portion of the anti-PD-1 antibody may include: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:238 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and (3) One Fc fragment comprising the amino acid sequence shown in SEQ ID NO:250 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; Alternatively, a portion of the anti-PD-1 antibody may include: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:239 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and (3) One Fc fragment comprising the amino acid sequence shown in SEQ ID NO:251 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; Alternatively, a portion of the anti-PD-1 antibody may include: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and (3) One Fc fragment comprising the amino acid sequence shown in SEQ ID NO:248 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; Alternatively, a portion of the anti-PD-1 antibody may include: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; (2) A light chain comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and (3) One Fc fragment containing an amino acid sequence shown in SEQ ID NO:249 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

30. The fusion polypeptide of claim 15 or 26, wherein, The anti-PD-1 antibody may include: a polypeptide chain containing a CH2 region and a CH3 region, two light chains of the anti-PD-1 antibody, and a polypeptide chain containing a heavy chain of the anti-PD-1 antibody fused to the Fd segment of the anti-PD-1 antibody.

31. The fusion polypeptide according to any one of claims 15, 26, and 30, wherein, The anti-PD-1 antibody comprises: (1) A first polypeptide chain comprising an amino acid sequence shown in SEQ ID NO:195, 196 or 197 or an amino acid sequence having at least 80% sequence identity with it; (2) Two second polypeptide chains, each containing the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) A third polypeptide chain comprising the following amino acid sequence linked together from the N-terminus to the C-terminus: The amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it.

32. The fusion polypeptide according to any one of claims 15, 26, and 30, wherein, The anti-PD-1 antibody comprises: (1) A first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:195 or 198 or an amino acid sequence having at least 80% sequence identity with it; (2) Two second polypeptide chains, each containing the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) A third polypeptide chain comprising the following amino acid sequence linked together from the N-terminus to the C-terminus: The amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% sequence identity with it.

33. The fusion polypeptide according to any one of claims 15, 26, 30, and 31, wherein, The anti-PD-1 antibody comprises: (1) A first polypeptide chain comprising an amino acid sequence shown in SEQ ID NO:195, 196 or 197 or an amino acid sequence having at least 80% sequence identity with it; (2) Two second polypeptide chains, each containing the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) A third polypeptide chain comprising the following amino acid sequence linked together from the N-terminus to the C-terminus: The amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, Connector, and The amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it.

34. The fusion polypeptide according to any one of claims 15, 26, 30, and 32, wherein, The anti-PD-1 antibody comprises: (1) A first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:195 or 198 or an amino acid sequence having at least 80% sequence identity with it; (2) Two second polypeptide chains, each containing the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) A third polypeptide chain comprising the following amino acid sequence linked together from the N-terminus to the C-terminus: The amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, Connector, and The amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% sequence identity with it.

35. The fusion polypeptide of claim 33 or 34, wherein, The connector is selected from (G5). n (G4S) n Or D(G4S) n n is an integer selected from 1 to 5.

36. The fusion polypeptide of claim 35, wherein, The connector is D(G4S). n n is an integer selected from 1 to 3.

37. The fusion polypeptide according to any one of claims 15, 26, 30, 31, and 33, wherein, The anti-PD-1 antibody comprises: (1) A first polypeptide chain comprising an amino acid sequence shown in SEQ ID NO:195, 196 or 197 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; (2) Two second polypeptide chains, each comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and (3) A third polypeptide chain comprising the following amino acid sequence linked together from the N-terminus to the C-terminus: The amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:216 or an amino acid sequence having at least 80% sequence identity with it, and The amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it.

38. The fusion polypeptide according to any one of claims 15, 26, 30, 31, 33, and 37, wherein, The anti-PD-1 antibody comprises: (1) A first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% sequence identity with it; (2) Two second polypeptide chains, each containing the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) A third polypeptide chain comprising the following amino acid sequence linked together in the direction from the N-terminus to the C-terminus: the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:216 or an amino acid sequence having at least 80% sequence identity with it, and The amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it.

39. The fusion polypeptide according to any one of claims 15, 26, 30, 32 and 34, wherein, The anti-PD-1 antibody comprises: (1) A first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:195 or 198 or an amino acid sequence having at least 80% sequence identity with it; (2) Two second polypeptide chains, each containing the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) A third polypeptide chain comprising the following amino acid sequence linked together in the direction from the N-terminus to the C-terminus: the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:216 or an amino acid sequence having at least 80% sequence identity with it, and The amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% sequence identity with it.

40. The fusion polypeptide according to any one of claims 15, 26, 30, 32, 34, and 39, wherein, The anti-PD-1 antibody comprises: (1) A first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it; (2) Two second polypeptide chains, each containing the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) A third polypeptide chain comprising the following amino acid sequence linked together in the direction from the N-terminus to the C-terminus: the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, The amino acid sequence shown in SEQ ID NO:216 or an amino acid sequence having at least 80% sequence identity with it, and The amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% sequence identity with it.

41. The fusion polypeptide according to any one of claims 15-40, wherein, The fusion polypeptide also includes a linker for connecting the anti-PD-1 antibody or a portion thereof to the IFNα2b mutant.

42. The fusion polypeptide of claim 41, wherein, The linker is located at the N-terminus or C-terminus of the anti-PD-1 antibody or a portion thereof.

43. The fusion polypeptide of claim 41 or 42, wherein, The fusion polypeptide comprises the IFNα2b mutant, a linker, and the Fc fragment of the anti-PD-1 antibody, sequentially linked from the N-terminus to the C-terminus; or The fusion polypeptide comprises the first polypeptide chain of the IFNα2b mutant, the linker, and a portion of the anti-PD-1 antibody, connected sequentially from the N-terminus to the C-terminus.

44. The fusion polypeptide according to any one of claims 41-43, wherein, The joint is selected from flexible joints, rigid joints, and cuttable joints.

45. The fusion polypeptide of claim 44, wherein, The connector is selected from (G5). n (G4S) n (PA) n P, (AP) n And disulfide bonds, where n is an integer selected from 1 to 10, preferably an integer selected from 1 to 5.

46. ​​The fusion polypeptide of claim 44 or 45, wherein, The connector comprises an amino acid sequence shown in any of SEQ ID NOs:182-186.

47. The polypeptide construct according to any one of claims 16-25, wherein, The polypeptide construct comprises: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. The Fc region contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198, or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. The Fc region contains the amino acid sequence shown in SEQ ID NO: 248, 249, 250, 251, 252 or 253, or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it.

48. The polypeptide construct according to any one of claims 16-25 and 47, wherein, The polypeptide construct comprises: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH, comprising the amino acid sequence shown in SEQ ID NO: 205, 206, 207, 208 or 210, or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it. CH, comprising the amino acid sequence shown in SEQ ID NO:242, 243, 244, 245, 246 or 247, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and The IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and CL comprises an amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

49. The polypeptide construct according to any one of claims 16-25 and 47-48, wherein, The polypeptide construct comprises: Peptide chain 1, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:2, 93, 94, 204, 236, 237, 238, 239, 240, or 241, or an amino acid sequence having at least 80% sequence identity with it; and the IFNα2b mutant; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it.

50. The polypeptide construct according to any one of claims 16-25 and 47-49, wherein, The polypeptide construct comprises: Peptide chain 1, from N-terminus to C-terminus, comprises: a heavy chain containing an anti-PD-1 antibody having an amino acid sequence shown in SEQ ID NO:2 or having at least 80% sequence identity with it, and the IFNα2b mutant; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: Peptide chain 1, from N-terminus to C-terminus, comprises, in sequence: a heavy chain containing an anti-PD-1 antibody of the amino acid sequence shown in SEQ ID NO:240 or having at least 80% sequence identity with it, and the IFNα2b mutant; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: Peptide chain 1, from N-terminus to C-terminus, comprises: a heavy chain containing an anti-PD-1 antibody having an amino acid sequence shown in SEQ ID NO:241 or having at least 80% sequence identity with it, and the IFNα2b mutant; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it.

51. The polypeptide construct according to any one of claims 16-25, wherein, The polypeptide construct comprises: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. The Fc region contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197, 198, 248, 249, 250, 251, 252 or 253, or an amino acid sequence having at least 80% sequence identity with it. The IFNα2b mutant; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains an amino acid sequence shown in SEQ ID NO: 195, 196, 197, 198, 248, 249, 250, 251, 252 or 253, or an amino acid sequence having at least 80% sequence identity with it.

52. The polypeptide construct according to any one of claims 16-25 and 51, wherein, The polypeptide construct comprises: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. The Fc region contains the amino acid sequence shown in SEQ ID NO: 196 or 197, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it. The IFNα2b mutant; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. The Fc region contains the amino acid sequence shown in SEQ ID NO:248 or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO: 250 or 251, or an amino acid sequence having at least 80% sequence identity with it. Alternatively, the polypeptide construct may contain: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. The Fc region contains the amino acid sequence shown in SEQ ID NO:249 or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO:250 or 251 or an amino acid sequence having at least 80% sequence identity with it.

53. The polypeptide construct according to any one of claims 16-25 and 51, wherein, The polypeptide construct comprises: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. The Fc region contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. The Fc region contains the amino acid sequence shown in SEQ ID NO:250 or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO:248 or 249 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. The Fc region contains the amino acid sequence shown in SEQ ID NO:251 or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO:248 or 249 or an amino acid sequence having at least 80% sequence identity with it.

54. The polypeptide construct according to any one of claims 16-25 and 51, wherein, The polypeptide construct comprises: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH, comprising the amino acid sequence shown in SEQ ID NO: 205, 206, 207, 208, 210, 242, 243, 244, 245, 246 or 247, or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising an amino acid sequence shown in SEQ ID NO:205, 206, 207, 208, 210, 242, 243, 244, 245, 246 or 247, or an amino acid sequence having at least 80% sequence identity with it.

55. The polypeptide construct of claim 54, wherein, The polypeptide construct comprises: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH, comprising the amino acid sequence shown in SEQ ID NO:206 or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising the amino acid sequence shown in SEQ ID NO:207 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH, comprising the amino acid sequence shown in SEQ ID NO:242 or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising the amino acid sequence shown in SEQ ID NO:246 or 247 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH, comprising the amino acid sequence shown in SEQ ID NO:243 or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising the amino acid sequence shown in SEQ ID NO:246 or 247 or an amino acid sequence having at least 80% sequence identity with it.

56. The polypeptide construct of claim 54, wherein, The polypeptide construct comprises: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH, comprising the amino acid sequence shown in SEQ ID NO:207 or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising the amino acid sequence shown in SEQ ID NO:206 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH, comprising the amino acid sequence shown in SEQ ID NO:246 or an amino acid sequence having at least 80% sequence identity with it, and The IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising the amino acid sequence shown in SEQ ID NO:242 or 243 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: (1) Peptide chain 1, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH, comprising the amino acid sequence shown in SEQ ID NO:247 or an amino acid sequence having at least 80% sequence identity with it, and IFNα2b mutant; and (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising the amino acid sequence shown in SEQ ID NO:242 or 243 or an amino acid sequence having at least 80% sequence identity with it.

57. The polypeptide construct according to any one of claims 16-25 and 51, wherein, The polypeptide construct comprises: The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:2, 93, 94, 204, 236, 237, 238, 239, 240 or 241 or an amino acid sequence having at least 80% sequence identity with it, and the IFNα2b mutant; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and The peptide chain 3, from the N-terminus to the C-terminus, comprises, in sequence, a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:2, 93, 94, 204, 236, 237, 238, 239, 240 or 241, or an amino acid sequence having at least 80% sequence identity with it.

58. The polypeptide construct of claim 57, wherein, The polypeptide construct comprises: The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain containing an anti-PD-1 antibody having an amino acid sequence shown in SEQ ID NO:93 or having at least 80% sequence identity with it, and the IFNα2b mutant; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and The peptide chain 3, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain containing an anti-PD-1 antibody having the amino acid sequence shown in SEQ ID NO:236 or having at least 80% sequence identity with it, and the IFNα2b mutant; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:238 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain containing an anti-PD-1 antibody having the amino acid sequence shown in SEQ ID NO:237 or having at least 80% sequence identity with it, and the IFNα2b mutant; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:239 or an amino acid sequence having at least 80% sequence identity with it.

59. The polypeptide construct of claim 57, wherein, The polypeptide construct comprises: The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain containing an anti-PD-1 antibody having the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it, and the IFNα2b mutant; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain containing an anti-PD-1 antibody having the amino acid sequence shown in SEQ ID NO:238 or an amino acid sequence having at least 80% sequence identity with it, and the IFNα2b mutant; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and The peptide chain 3, from the N-terminus to the C-terminus, comprises: a heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% sequence identity with it; Alternatively, the polypeptide construct may contain: The peptide chain 1, from the N-terminus to the C-terminus, comprises: a heavy chain containing an anti-PD-1 antibody having the amino acid sequence shown in SEQ ID NO:239 or an amino acid sequence having at least 80% sequence identity with it, and the IFNα2b mutant; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, from N-terminus to C-terminus, comprises: a heavy chain containing an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% sequence identity with it.

60. The polypeptide construct according to any one of claims 16-25, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it. (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it.

61. The polypeptide construct of claim 60, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it.

62. The polypeptide construct of claim 60, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% sequence identity with it.

63. The polypeptide construct according to any one of claims 16-25 and 60, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it. (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising an amino acid sequence shown in SEQ ID NO:205, 206, 207, 208 or 210 or an amino acid sequence having at least 80% sequence identity with it.

64. The polypeptide construct of claim 63, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising the amino acid sequence shown in SEQ ID NO:207 or an amino acid sequence having at least 80% sequence identity with it.

65. The polypeptide construct of claim 63, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) Peptide chain 3, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising the amino acid sequence shown in SEQ ID NO:206 or an amino acid sequence having at least 80% sequence identity with it.

66. The polypeptide construct according to any one of claims 16-25, 60, and 63, wherein, The polypeptide construct comprises: The peptide chain 1' contains, from the N-terminus to the C-terminus, an Fc region of an IFNα2b mutant, an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198, or an amino acid sequence having at least 80% sequence identity with it. Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, comprising an amino acid sequence shown in SEQ ID NO:2, 93, 94 or 204 or an amino acid sequence having at least 80% sequence identity with it.

67. The polypeptide construct of claim 66, wherein, The polypeptide construct comprises: The peptide chain 1' contains, from the N-terminus to the C-terminus, an IFNα2b mutant, an Fc region of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO: 196 or 197 or an amino acid sequence having at least 80% sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it.

68. The polypeptide construct of claim 66, wherein, The polypeptide construct comprises: The peptide chain 1' contains, from the N-terminus to the C-terminus, an IFNα2b mutant and an Fc region of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it. Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% sequence identity with it.

69. The polypeptide construct according to any one of claims 16-25, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it. (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it.

70. The polypeptide construct of claim 69, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it. (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. The connector is selected from (G5). n (G4S) n Or D(G4S) n n is an integer selected from 1 to 5. VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it.

71. The polypeptide construct of claim 69 or 70, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it. (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. A connector comprising the amino acid sequence shown in SEQ ID NO:

216. VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it.

72. The polypeptide construct according to any one of claims 69-71, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. A connector comprising the amino acid sequence shown in SEQ ID NO:

216. VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it.

73. The polypeptide construct according to any one of claims 69-71, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. A connector comprising the amino acid sequence shown in SEQ ID NO:

216. VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1, comprising the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it, and The Fc region contains the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% sequence identity with it.

74. The polypeptide construct according to any one of claims 69-71, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO: 195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it. (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. A connector comprising the amino acid sequence shown in SEQ ID NO:

216. VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising an amino acid sequence shown in SEQ ID NO:205, 206, 207, 208 or 210 or an amino acid sequence having at least 80% sequence identity with it.

75. The polypeptide construct of claim 74, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. A connector comprising the amino acid sequence shown in SEQ ID NO:

216. VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising the amino acid sequence shown in SEQ ID NO:207 or an amino acid sequence having at least 80% sequence identity with it.

76. The polypeptide construct of claim 75, wherein, The polypeptide construct comprises: (1) The 1' of the peptide chain, from the N-terminus to the C-terminus, contains: The IFNα2b mutant The Fc region of the anti-PD-1 antibody contains the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, from the N-terminus to the C-terminus, contains: VL, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80% sequence identity with it, and CL, comprising the amino acid sequence shown in SEQ ID NO:209 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. A connector comprising the amino acid sequence shown in SEQ ID NO:

216. VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, and CH, comprising the amino acid sequence shown in SEQ ID NO:206 or an amino acid sequence having at least 80% sequence identity with it.

77. The polypeptide construct according to any one of claims 69-71, wherein, The polypeptide construct comprises: (1) Peptide chain 1', from N-terminus to C-terminus, contains the Fc region of an IFNα2b mutant, an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:195, 196, 197 or 198 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. A connector comprising the amino acid sequence shown in SEQ ID NO:216, and The amino acid sequence shown in SEQ ID NO:2, 93, 94 or 204 or an amino acid sequence having at least 80% sequence identity with it.

78. The polypeptide construct of claim 77, wherein, The polypeptide construct comprises: (1) Peptide chain 1', from N-terminus to C-terminus, contains the Fc region of an IFNα2b mutant, an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:196 or 197 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. A connector comprising the amino acid sequence shown in SEQ ID NO:216, and The amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it.

79. The polypeptide construct of claim 77, wherein, The polypeptide construct comprises: (1) Peptide chain 1', from N-terminus to C-terminus, contains the IFNα2b mutant, the Fc region of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:198 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and (3) The 3' of the peptide chain, from the N-terminus to the C-terminus, contains: VH, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with it, CH1 contains the amino acid sequence shown in SEQ ID NO:193 or an amino acid sequence having at least 80% sequence identity with it. A connector comprising the amino acid sequence shown in SEQ ID NO:216, and The amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80% sequence identity with it.

80. The polypeptide construct according to any one of claims 16-25 and 47-79, wherein, Compared with the human wild-type IFNα2b, in addition to the amino acid substitution mutation at V105, the IFNα2b mutant also contains amino acid substitution mutations at at least one of the following positions: L15, A19, L26, L30, R33, H57, E58, Q61, R120, V142, A145, E146, M148, R149.

81. The polypeptide construct according to any one of claims 16-25 and 47-80, wherein, Compared to wild-type human IFNα2b, the IFNα2b mutant contains the following mutation: V105E, and a mutation selected from at least one of the following positions: L15A, L15E, L15Q, L15S, L15V, L15G, A19G, L26A, L30V, L30G, L30I, L30S, L30Q, R33A, R33K, R3 3H, R33N, R33S, H57Y, E58N, Q61S, R120G, V142A, V142D, V142E, V142G, V142H, V142I, V142K, V 142L, V142M, V142N, V142Q, V142R, V142S, V142T, V142Y, A145D, E146A, E146G, E146H, E146I, E146K, E146L, E146R, E146S, E146T, M148A, M148D, M148G, M148K, M148S, R149E, R149G, or R149S.

82. The polypeptide construct according to any one of claims 16-25 and 47-81, wherein, Compared to wild-type human IFNα2b, the IFNα2b mutant comprises a combination of V105E and at least one of the following mutations: (1) L15A, L15E, L15Q, L15S, L15V or L15G; (2) L30V, L30G, L30I, L30S or L30Q; (3) R33A, R33K, R33H, R33N or R33S; (4) R120G; (5) V142A, V142D, V142E, V142G, V142H, V142I, V142K, V142L, V142M, V142N, V142Q, V142R, V142S, V142T or V142Y; (6)A145D; (7) E146A, E146G, E146H, E146I, E146K, E146L, E146R, E146S or E146T; (8) M148A, M148D, M148G, M148K or M148S; (9) R149E, R149G, or R149S; or (10) L15G+A19G, L15A+V142A, L15A+V142N, L15A+L26A+V142A, L15G+M148G, L26A+V142A, R33A+H57Y+E58N+Q61S or V142A+E146A.

83. The polypeptide construct according to any one of claims 16-25 and 47-79, wherein, Compared to wild-type human IFNα2b, in addition to the amino acid substitution mutation at A145, the IFNα2b mutant also contains amino acid substitution mutations at at least one of the following positions: V105, E107, T108, P109, CD ring sequence or part thereof.

84. The polypeptide construct according to any one of claims 16-25, 47-79, and 83, wherein, Compared to wild-type human IFNα2b, the IFNα2b mutant contains the following mutation: A145D, and a mutation selected from at least one of the following positions: The following sequences in the CD ring region are replaced with "EEKLEKEDFTRGKLM" (SEQ ID NO:175), "IQGVGVTETPLMKED" (SEQ ID NO:174), "EEKLEKTDFTRGKLM" (SEQ ID NO:176), "GVTETPLM" (SEQ ID NO:177), "EKEDFTRG" (SEQ ID NO:178), or "GVTETPLM" (SEQ ID NO:177) or "EKTDFTRG" (SEQ ID NO:179).

85. The polypeptide construct according to any one of claims 16-25, 47-79, and 83-84, wherein, Compared to wild-type human IFNα2b, the IFNα2b mutant contains a combination of A145D and at least one of the following mutations: (1) V105E, V105E+P109R or V105E+T108I+P109A; (2) E107D, E107K or E107D+T108A; (3) T108G, T108A or T108I+P109A; (4) P109R or P109N; or (5) Replace "IQGVGVTETPLMKED" (SEQ ID NO:174) in the CD ring region sequence with "EEKLEKEDFTRGKLM" (SEQ ID NO:175). Replace "IQGVGVTETPLMKED" (SEQ ID NO:174) in the CD ring sequence with "EEKLEKTDFTRGKLM" (SEQ ID NO:176). Replace "GVTETPLM" (SEQ ID NO:177) in the CD ring region sequence with "EKEDFTRG" (SEQ ID NO:178), or In the CD ring region sequence, "GVTETPLM" (SEQ ID NO:177) is replaced with "EKTDFTRG" (SEQ ID NO:179).

86. The polypeptide construct according to any one of claims 16-25 and 47-79, wherein, Compared to the human wild-type IFNα2b, the IFNα2b mutant contains an amino acid substitution or deletion mutation at at least one of the following positions: V105, T106, T108, P109, CD loop sequence or a portion thereof.

87. The polypeptide construct according to any one of claims 16-25, 47-79, and 86, wherein, Compared to human wild-type IFNα2b, the IFNα2b mutant contains at least one of the following mutations: Replace "GVTETPLM" (SEQ ID NO:177) in V105E, T106A, ΔT106, T108I, P109A, P109R, and CD ring sequences with "EKTDFTRG" (SEQ ID NO:179), or replace "IQGVGVTETPLMKED" (SEQ ID NO:174) in CD ring sequences with "EEKLEKTDFTRGKLM" (SEQ ID NO:176).

88. The polypeptide construct according to any one of claims 16-25, 47-79, and 86-87, wherein, Compared to human wild-type IFNα2b, the IFNα2b mutant contains at least one of the following mutations: (1) V105E, V105E+P109R or V105E+T108I+P109A; (2) T106A or ΔT106; (3) P109R or T108I+P109A; or (4) Replace "GVTETPLM" (SEQ ID NO:177) in the CD ring region sequence with "EKTDFTRG" (SEQ ID NO:179), or Replace "IQGVGVTETPLMKED" (SEQ ID NO:174) in the CD ring sequence with "EEKLEKTDFTRGKLM" (SEQ ID NO:176).

89. The polypeptide construct according to any one of claims 16-25 and 47-79, wherein, Compared to human wild-type IFNα2b, the IFNα2b mutant contains at least one of the following mutations: (1)L26A+V142A; (2) M148K; (3)R149G; (4) L15G+M148G; (5) V142L or V142M; or (6)L30S.

90. The polypeptide construct according to any one of claims 16-25 and 47-89, wherein, The IFNα2b mutant comprises an amino acid sequence shown in either SEQ ID NOs:98-173 or 226-227, or an amino acid sequence having at least 80% sequence identity with it.

91. The polypeptide construct according to any one of claims 16-25 and 47-90, wherein, The polypeptide construct also includes a linker between the anti-PD-1 antibody and the IFNα2b mutant.

92. The polypeptide construct of claim 91, wherein, The joint is selected from flexible joints, rigid joints, and cuttable joints.

93. The polypeptide construct of claim 91 or 92, wherein, The connector is selected from (G5). n (G4S) n (PA) n P, (AP) n And disulfide bonds, where n is an integer selected from 1 to 10, preferably an integer selected from 1 to 5.

94. The polypeptide construct according to any one of claims 91-93, wherein, The connector comprises an amino acid sequence shown in any of SEQ ID NOs:182-186.

95. The polypeptide construct according to any one of claims 91-94, wherein, The N-terminus of the IFNα2b mutant is connected to the C-terminus of the CH3 region of the anti-PD-1 antibody via the adapter.

96. The fusion polypeptide as described in any one of claims 15-46, or the polypeptide construct as described in any one of claims 16-25 and 47-90, wherein, The fusion polypeptide or the polypeptide construct exists in the structural form of a 2+2 antibody-IFNα2b mutant or a 2+1 antibody-IFNα2b mutant.

97. The polypeptide construct according to any one of claims 16-25 and 47-96, wherein, The polypeptide construct comprises: (1) Peptide chain 1, comprising an amino acid sequence shown in any one of SEQ ID NOs: 9-84, 211, 214, 234 and 235, or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 1, comprising an amino acid sequence shown in any of SEQ ID NOs:85-90 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; (3) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:91 or an amino acid sequence having at least 80% sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it; (4) Peptide chain 1, comprising an amino acid sequence shown in SEQ ID NO: 219, 220, 221, 222, 223, 224 or 225 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; as well as Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; (5) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:232 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; as well as Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; (6) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:233 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; as well as Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; (7) Peptide chain 1', comprising the amino acid sequence shown in SEQ ID NO:92 or an amino acid sequence having at least 80% sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it; or (8) Peptide chain 1', comprising the amino acid sequence shown in SEQ ID NO:92 or an amino acid sequence having at least 80% sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and The peptide chain 3' contains the amino acid sequence shown in SEQ ID NO:95 or an amino acid sequence having at least 80% sequence identity with it.

98. The polypeptide construct according to any one of claims 16-25 and 47-97, wherein, The polypeptide construct comprises: (1) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:56 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; (2) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:67 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; (3) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:71 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; (4) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:72 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; (5) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:77 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; (6) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:79 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; (7) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:234 ​​or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; as well as Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; (8) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:235 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; as well as Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; (9) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:91 or an amino acid sequence having at least 80% sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it; (10) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:220 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; as well as Peptide chain 3, comprising the heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; (11) Peptide chain 1, comprising an amino acid sequence shown in SEQ ID NO: 219, 221, 222, 223, 224 or 225 or an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; as well as Peptide chain 3, comprising the heavy chain of an anti-PD-1 antibody containing the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; (12) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:232 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; as well as Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; (13) Peptide chain 1, comprising the amino acid sequence shown in SEQ ID NO:233 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; as well as Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:237 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; (14) Peptide chain 1', comprising the amino acid sequence shown in SEQ ID NO:92 or an amino acid sequence having at least 80% sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and Peptide chain 3, comprising the amino acid sequence shown in SEQ ID NO:94 or an amino acid sequence having at least 80% sequence identity with it; or (15) Peptide chain 1', comprising the amino acid sequence shown in SEQ ID NO:92 or an amino acid sequence having at least 80% sequence identity with it; Peptide chain 2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with it; and The peptide chain 3' contains the amino acid sequence shown in SEQ ID NO:95 or an amino acid sequence having at least 80% sequence identity with it.

99. A polynucleotide, wherein, The polynucleotide encodes the IFNα2b mutant as described in any one of claims 1-13, the fusion polypeptide as described in any one of claims 15-46 and 96, or the polypeptide construct as described in any one of claims 16-25 and 47-98.

100. A recombinant vector comprising the polynucleotide of claim 99, or encoding an IFNα2b mutant of any one of claims 1-13, a fusion polypeptide of any one of claims 15-46 and 96, or a polypeptide construct of any one of claims 16-25 and 47-98.

101. A recombinant cell comprising the polynucleotide of claim 99 or the recombinant vector of claim 100, or expressing the IFNα2b mutant of any one of claims 1-13, the fusion polypeptide of any one of claims 15-46 and 96, or the polypeptide construct of any one of claims 16-25 and 47-98.

102. A pharmaceutical composition comprising: an IFNα2b mutant as claimed in any one of claims 1-13, a fusion polypeptide as claimed in any one of claims 15-46 and 96, or a polypeptide construct as claimed in any one of claims 16-25 and 47-98, and optionally pharmaceutically acceptable excipients.

103. Use of the IFNα2b mutant as described in any one of claims 1-13, the fusion polypeptide as described in any one of claims 15-46 and 96, or the polypeptide construct as described in any one of claims 16-25 and 47-98 in the preparation of a medicament for treating tumors.

104. A combination drug comprising: (1) the fusion polypeptide as described in any one of claims 15-46 and 96, the polypeptide construct as described in any one of claims 16-25 and 47-98, or the pharmaceutical composition as described in claim 102; and (2) a VEGF inhibitor.

105. The combination drug as described in claim 104, wherein, The fusion peptide, peptide construct, or pharmaceutical composition is packaged separately from the VEGF inhibitor.

106. The combination drug as described in claim 104 or 105, wherein, The VEGF inhibitors include anti-VEGF antibodies, VEGF receptor antagonists, and tyrosine kinase inhibitors that target VEGFR.

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