Fusion proteins containing IL-21 variants for the treatment of cancer and viral diseases

Abstract

The present invention relates to a fusion protein comprising an anti-PD-1 antibody moiety (having a C-terminus) and an IL-21 variant bound to the C-terminus of the anti-PD-1 antibody moiety. The IL-21 variant contains at least one of the following mutations relative to wild-type IL-21: (1) any of K72Y substitution, K72M substitution, or K72Q substitution, and (2) a 75-80 amino acid deletion. For example, the anti-PD-1 antibody may employ an ETYY format, with the first heavy chain containing the VH sequence shown in SEQ ID NO: 11, and the second heavy chain having a VH containing any of the sequences in SEQ ID NOs: 8-10. Furthermore, the present invention also provides a pharmaceutical composition containing the fusion protein, a nucleic acid encoding the fusion protein, a vector containing the nucleic acid, and a method for treating a disease using the fusion protein.

Description

[Technical Field]

[0001] Interleukin-21 (hereinafter referred to as "IL-21" or "IL21") is a multifunctional cytokine belonging to the common gamma chain (γc) family and plays an important role in the regulation of both innate and adaptive immune responses. IL-21 was discovered in 2000 and is primarily associated with activated CD4 + IL-21 is produced by T cells, particularly follicular helper T cells (Tfh cells) and natural killer T cells (NKT cells). IL-21 acts on various immune cells, including T cells, B cells, natural killer (NK) cells, and dendritic cells, and is known to regulate diverse immunological processes, such as T cell differentiation, B cell proliferation, antibody production, and enhancement of NK cell cytotoxicity. In recent years, IL-21 has attracted attention for its potential therapeutic applications based on its ability to modulate immune responses in chronic viral infections and cancer.

[0002] Chronic hepatitis B virus (HBV) infection is a major public health challenge affecting hundreds of millions of people worldwide, leading to significant morbidity and mortality rates due to the development of cirrhosis and hepatocellular carcinoma. Currently used antiviral therapies, such as nucleoside analogs and interferon-alpha, show limited efficacy in achieving functional cure in many patients. IL-21 is attracting attention as a promising candidate factor for the treatment of chronic HBV infection due to its ability to enhance the antiviral immune response and promote viral clearance. Research reports have shown that IL-21 enhances the function of HBV-specific T cells, increasing their proliferation, cytotoxic activity, and cytokine production. Furthermore, IL-21 has been confirmed to suppress HBV replication in human hepatocytes and animal models, suggesting its potential as a novel therapeutic agent for achieving functional cure in chronic HBV patients.

[0003] IL-21 is considered promising in the field of cancer immunotherapy due to its ability to induce a potent anti-tumor immune response. In preclinical studies, IL-21 has been shown to stimulate NK cells and CD8 + IL-21 has been shown to enhance the cytotoxic activity of T cells and induce the elimination of tumor cells in various tumor models. Furthermore, IL-21 is known to induce differentiation of T cells into the Th1 phenotype, contributing to improved anti-tumor immunity. Clinical trials involving the administration of IL-21 as a monotherapy or in combination with other immunotherapies have reported promising results in patients with metastatic melanoma and renal cell carcinoma. Further research is needed to fully elucidate the therapeutic potential of IL-21 in cancer treatment and to optimize its combination with other novel immunotherapies.

[0004] Furthermore, IL-21 is attracting attention as a potential therapeutic factor for human immunodeficiency virus (HIV) infection due to its immunomodulatory effects and ability to enhance virus-specific immune responses. HIV infection is a progressive CD4 infection. + Characterized by T cell reduction, immunodeficiency, and the formation of a viral reservoir, this is a major obstacle to achieving functional healing. Studies have shown that IL-21 is associated with CD4 in HIV-infected individuals. + and CD8 + IL-21 has been shown to promote T cell survival, proliferation, and function, and to enhance antiviral immunity by increasing the cytotoxic activity of NK cells. Furthermore, IL-21 has shown a direct antiviral effect against HIV replication in vitro, and administration to primates infected with simian immunodeficiency virus (SIV) has been reported to reduce viral load and improve immune reconstitution. These findings suggest that IL-21 holds promise as a novel immunotherapy strategy to be used in combination with antiretroviral therapy (ART) and other immunotherapeutic interventions. [Overview of the project]

[0005] In one aspect of this disclosure, a fusion protein is provided comprising the C-terminus of an anti-PD1 antibody moiety and an IL-21 variant (mutei) conjugated to the anti-PD1 antibody moiety at its C-terminus. The IL-21 variant comprises one or both of the following mutations compared to wild-type IL-21: (1) any of the K72Y substitution, K72M substitution, or K72Q substitution, and / or (2) deletion of residues 75-80. As an example, the IL-21 variant may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-6.

[0006] In some embodiments of the present invention, the IL-21 mutant includes both (1) a K72Y substitution, a K72M substitution, or a K72Q substitution, and (2) a deletion of residues 75-80. In other embodiments, the IL-21 mutant includes only one of the above two types of mutations.

[0007] In some embodiments, the anti-PD1 antibody has an IgG1 format and comprises a primary heavy chain and a secondary heavy chain, the primary heavy chain containing the T366Y mutation and the secondary heavy chain containing the Y407T mutation.

[0008] In other embodiments, the anti-PD1 antibody has an IgG1 format and comprises a primary heavy chain and a secondary heavy chain, the primary heavy chain containing K360, S354Y and T366Y mutations, and the secondary heavy chain containing G347E, Y349 and Y407T mutations.

[0009] In some embodiments, the anti-PD1 antibody comprises a first heavy chain containing a VH having the sequence of SEQ ID NO: 7. Furthermore, in some of these embodiments, the anti-PD1 antibody further comprises a second heavy chain containing a VH having a sequence selected from any of SEQ ID NOs: 8 to 10.

[0010] In other embodiments, the anti-PD1 antibody comprises a first heavy chain containing a VH having the sequence of SEQ ID NO: 11. Furthermore, in some of these embodiments, the anti-PD1 antibody further comprises a second heavy chain containing a VH having a sequence selected from any of SEQ ID NOs: 8 to 10.

[0011] In some embodiments, the fusion protein comprises a primary heavy chain containing a mutant Fc having the sequence of SEQ ID NO: 13 and a secondary heavy chain containing a mutant Fc having the sequence of SEQ ID NO: 12.

[0012] Furthermore, in some embodiments, the Fc of the first heavy chain in the KIH structure (Knobs-into-holes structure) includes the sequence of SEQ ID NO: 15, and the Fc of the second heavy chain in the same structure includes the sequence of SEQ ID NO: 14.

[0013] In another embodiment, the Fc of the first heavy chain in the ETYY structure includes the sequence of SEQ ID NO: 17, and the Fc of the second heavy chain in the structure includes the sequence of SEQ ID NO: 16.

[0014] In some embodiments, only the primary heavy chain is bound to the IL-21 variant, while the secondary heavy chain is not.

[0015] In one embodiment of the present invention, the fusion protein (or antibody-cytokine fusion) may be linked to the anti-PD-1 antibody moiety and the IL-21 mutant via a linker. The linker is ligated to the C-terminus of the heavy chain of the anti-PD-1 antibody, to which the IL-21 mutant can be bound (see Figures 3e and 3f). In some embodiments, the linker is composed of (GnS)x, where n is an integer from 2 to 4 and x is an integer from 1 to 4. In other embodiments, the anti-PD-1 antibody moiety (e.g., the heavy chain) may be directly bound to the IL-21 mutant without a linker (see Figures 1a-1f, 2a-2f, and 3a-3d), thereby simplifying the fusion structure.

[0016] In some embodiments of the fusion protein of the present invention, the primary heavy chain comprises an amino acid sequence selected from the group of SEQ ID NOs: 17-25, for example, SEQ ID NOs: 20, 22, 23, 24, or 25. The IL-21 variant bound thereto may contain an amino acid sequence selected from the group of SEQ ID NOs: 2-6, for example, SEQ ID NO: 6.

[0017] In some embodiments, the primary heavy chain includes an Fc region selected from the group of SEQ ID NOs: 17-22. In such embodiments, the primary heavy chain includes the VH sequence of SEQ ID NO: 11, and the dual chain may include the VH sequence of any of SEQ ID NOs: 8-10, for example, SEQ ID NO: 10.

[0018] Furthermore, the present invention provides nucleic acids that encode the aforementioned fusion protein.

[0019] Furthermore, the present invention provides a vector containing the nucleic acid.

[0020] Furthermore, the present invention provides a pharmaceutical composition comprising the fusion protein and a pharmaceutically acceptable carrier.

[0021] Furthermore, the present invention provides a method for treating cancer in a subject, the method comprising administering an effective amount of the fusion protein of the present invention or a pharmaceutically acceptable composition thereof to the subject. The cancer may be a solid tumor, a hematological malignancy, or a lymphoid malignancy. Examples include lung cancer, head and neck cancer, kidney cancer, breast cancer, brain tumor, melanoma and other skin cancers, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, colorectal cancer, prostate cancer, gastrointestinal cancer, bladder cancer, hematological cancer, lymphoma, testicular cancer, gynecological cancer, and sarcoma.

[0022] Furthermore, in one aspect of the present invention, a method for treating a chronic viral infection in a subject is provided. The method includes administering to the subject an effective amount of the fusion protein or pharmaceutical composition described herein. In some embodiments, the chronic viral infection may be hepatitis B virus (HBV) infection or human immunodeficiency virus (HIV) infection. In other embodiments, the chronic viral infection may include viral infections selected from herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Kaposi's sarcoma-associated herpesvirus (KSHV), human herpesvirus 7 (HHV-7), human herpesvirus 6 (HHV-6), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), hepatitis D virus (HDV), and human papillomavirus (HPV).

Brief Description of the Drawings

[0023] Figures 1a to 1f are diagrams showing some exemplary schematic structures of a fusion protein of an anti-PD-1 antibody and an IL-21 variant according to an embodiment of the present invention.

[0024] Figures 2a to 2f are diagrams showing yet another exemplary schematic structure of a fusion protein of an anti-PD-1 antibody and an IL-21 variant according to another embodiment of the present invention.

[0025] Figures 3a to 3f are diagrams showing a series of exemplary schematic structures of a fusion protein of an anti-PD-1 antibody and an IL-21 variant, including changes based on the Fc region structure of the anti-PD-1 antibody (shown in Figure 2e), according to another embodiment of the present invention.

[0026] Figure 4a is a graph showing a comparison of the tumor control dynamics between an isotype control and the anti-PD-1 / IL-21 variant of the present invention in a humanized mouse colon cancer model.

[0027] Figure 4b is a graph showing the comparison of the tumor growth inhibition rate (TGI) on the 11th and 20th days after administration of the isotype control and the anti-PD-1 / IL-21 variant of the present invention in a humanized mouse colorectal cancer model.

Mode for Carrying Out the Invention

[0028] In one aspect of the present invention, a fusion protein (or protein fusion) is provided that includes an anti-PD-1 antibody portion having a C-terminus and an IL-21 variant bound to the anti-PD-1 antibody portion at its C-terminus. These are collectively referred to herein as "anti-PD-1×IL-21 fusion protein" (or "anti-PD-1 / IL-21 fusion protein", "anti-PD-1-IL-21 fusion protein"). The amino acid sequence of wild-type human IL-21 is shown as SEQ ID NO: 1 (SEQ ID NO: 1) in the sequence listing.

[0029] The IL-21 variant described herein is designed to have a reduced affinity for the IL-21 receptor (IL-21R) compared to wild-type human IL-21. The fusion protein of the present invention does not respond to binding to IL-21R unless co-expression of the PD-1 receptor is present. Since PD-1 is expressed in response to the activation of immune cells, it is present only in immune cells within diseased tissues or sites of inflammatory activity. Therefore, the anti-PD-1 / IL-21 variant fusion protein according to the present invention can achieve selective IL-21 activation and improve safety and therapeutic index (therapeutic index) in patients with chronic viral infections or cancer. This selective activity increases the exposure level in plasma and enables strengthening of PD-1 blockade and IL-21R activation in immune subsets present at the diseased site. Therefore, the anti-PD-1 / IL-21 variant fusion protein of the present invention can selectively induce immune activation at the diseased site while suppressing binding to PD-1-negative immune cells in peripheral blood, immune organs, or non-diseased tissues.

[0030] Various exemplary structures of the double-stranded fusion protein of the present invention are shown in Figures 1 to 3. The characteristics of the fusion protein can be described based on the structure or properties of its constituent elements, namely the antibody moiety (and its subcomponents, such as the heavy chain, light chain, VH region, Fc region, CH3 region, etc.), the linker, and IL-21 variants. Structural variations of each element constituting the fusion protein can be used in combination with each other.

[0031] As shown in Figures 1 to 3, in an example of a fusion protein according to some embodiments of the present invention, the anti-PD-1 antibody portion of the fusion protein (also simply referred to as "anti-PD-1 antibody") has an IgG1 format and includes a first heavy chain and a second heavy chain (and corresponding light chains such as κ-type light chains). In the fusion proteins shown in Figures 1 to 3, the IL-21 variant is bound only to the first of the two heavy chains, but it is conceivable that the fusion protein according to the present invention may have the IL-21 variant bound to both heavy chains.

[0032] The IL-21 mutant included in the fusion protein of the present invention differs from wild-type IL-21 in that it has at least one of the following mutations: (1) a K72Y substitution, a K72M substitution, or a K72Q substitution, and / or (2) a deletion of amino acid residues 75-80. For example, an IL-21 mutant may have only one of the K72Y, K72M, or K72Q substitutions and no deletion between 75 and 80. Another example is an IL-21 mutant which may have only the deletion between 75 and 80 and no K72 substitution. Yet another example is an IL-21 mutant which may have both a K72 substitution (one of K72Y, K72M, or K72Q) and a deletion between 75 and 80.

[0033] The amino acid sequence of an IL-21 mutant (mutant) with the K72Y mutation is shown as Sequence ID No. 2 (SEQ ID NO: 2) in the sequence listing. The amino acid sequence of an IL-21 mutant with deletions of amino acid residues 75-80 is shown as Sequence ID No. 3 (SEQ ID NO: 3) in the sequence listing. The amino acid sequence of an IL-21 mutant with a K72Y substitution and deletions of amino acid residues 75-80 is shown as Sequence ID No. 4 (SEQ ID NO: 4) in the sequence listing. The amino acid sequence of an IL-21 mutant with a K72M substitution and deletions of amino acid residues 75-80 is shown as Sequence ID No. 5 (SEQ ID NO: 5) in the sequence listing. The amino acid sequence of an IL-21 mutant with a K72Q substitution and deletions of amino acid residues 75-80 is shown as Sequence ID No. 6 (SEQ ID NO: 6) in the sequence listing.

[0034] In one embodiment, the two heavy chains of the anti-PD-1 moiety of the fusion protein of the present invention may have a "knob-into-hole" (KIH) structure. For example, the first heavy chain of the anti-PD-1 moiety may contain the T366Y mutation, and the second heavy chain may contain the Y407T mutation, as shown in Figure 1 as KIH-K72Y, KIH-del, and KIH-K72Y&del.

[0035] In other embodiments, the first heavy chain may contain K360, S354Y, and T366Y mutations, and the second heavy chain may contain G347E, Y349, and Y407T mutations. These combinations are also referred to as "ETYY structures" and are illustrated in Figure 1 as ETYY-K72Y, ETYY-del, and ETYY-K72Y&del, as well as in Figures 2 and 3.

[0036] In one embodiment, the Fc(CH3) region of the first heavy chain in the KIH structure includes the sequence shown as sequence number 15 in the sequence listing, and the Fc(CH3) region of the second heavy chain in the KIH structure includes the sequence shown as sequence number 14 in the sequence listing.

[0037] In one embodiment, the Fc(CH3) region of the first heavy chain in the ETYY structure includes the sequence shown as sequence number 17 in the sequence listing, and the Fc(CH3) region of the second heavy chain in the ETYY structure includes the sequence shown as sequence number 16 in the sequence listing.

[0038] In one embodiment, the anti-PD-1 portion of the fusion protein includes a hinge region having an LALA substitution, as shown in Figures 1-3.

[0039] In one embodiment, the anti-PD-1 antibody portion of the fusion protein contains the VH sequence of pembrolizumab (Keytruda) (wild-type parent strain, SEQ ID NO: 7) in the first heavy chain on the "knob" side of the dual chain. In some of these embodiments, the anti-PD-1 antibody portion further contains a mutant pembrolizumab VH sequence in the second heavy chain on the "hole" side that expands the pI difference between the two half-antibodies of asymmetric bispecific IgG, enabling better heterodimerization and CMC production. For example, mutant pembrolizumab VH having the Q1E, V11L, K12V, K84S mutations (referred to as ELVS mutations) has the sequence shown in SEQ ID NO: 8 (SEQ ID NO: 8) in the sequence listing. As another example, having the E mutation (Q1E, V11L, K12V, K84S, Q87E) in addition to the ELVS mutation has the sequence shown in SEQ ID NO: 9 (SEQ ID NO: 9) in the sequence listing. As yet another example, mutant pembrolizumab VH with ELVS+EE mutations (Q1E, V11L, K12V, K84S, K13E, Q87E) has the sequence shown in sequence number 10 (sequence number 10) of the sequence listing.

[0040] In one embodiment, the first heavy chain may include, for example, a mutant pembrolizumab VH having the Q87R mutation (sequence number 11 in the sequence listing), and the second heavy chain may have the ELVS+E or ELVS+EE mutation (see Figures 2a-2f). In some embodiments, both the first and second heavy chains include non-mutant (wild-type) pembrolizumab VH.

[0041] In one embodiment, the second heavy chain contains a mutant Fc(hIgG1-Fc) having an S400C substitution, and its CH3 sequence is shown as sequence number 12 in the sequence listing. The first heavy chain also contains a mutant Fc(hIgG1-Fc) having a Q386C substitution, and its CH3 sequence is shown as sequence number 13 in the sequence listing.

[0042] In one embodiment, the anti-PD-1 antibody portion of the fusion protein and the IL-21 variant may be linked via a linker. In one embodiment, the linker is (G n S) x This consists of n being an integer between 2 and 4, and x being an integer between 1 and 4. In other embodiments, the anti-PD-1 antibody moiety and the IL-21 mutant may be directly bound without a linker.

[0043] In one embodiment, the first heavy chain of the fusion protein includes a sequence selected from sequence numbers 17-25 (e.g., sequence numbers 20, 22, 23, 24, and 25) in the sequence listing. The IL-21 variant bound to the first heavy chain may include a sequence selected from sequence numbers 2-6 (e.g., sequence number 6) in the sequence listing.

[0044] In one embodiment, the first heavy chain may have an Fc region (CH3) containing a sequence selected from sequence numbers 17 to 22 in the sequence listing. In some of these embodiments, the first heavy chain may contain the VH sequence of sequence number 7 or 11 (sequence number 7 or 11) in the sequence listing, and the second heavy chain may contain the VH sequence of any of sequence numbers 8 to 10 (e.g., sequence number 10) in the sequence listing. The IL-21 variant bound to the first heavy chain may contain a sequence selected from sequence numbers 2 to 6 (e.g., sequence number 6) in the sequence listing.

[0045] In another embodiment, the present invention provides a nucleic acid molecule encoding any of the fusion proteins described herein. The fusion proteins of this disclosure, preferably derived from a single clone, can be produced using a host cell (e.g., CHO cells, human embryonic kidney cells, lymphoid cells, or microorganisms such as Escherichia coli, or fungi such as yeast) that holds an expression vector containing the nucleic acid molecule.

[0046] In one embodiment, DNA encoding a partial or full-length fusion protein of the present invention (e.g., HC1, HC2, LC, and an IL-21 variant bound to HC1) is obtained by standard molecular biological techniques and inserted into one or more expression vectors so as to be operatively linked to transcriptional and translational regulatory sequences. "Operatally linked" as used herein means that the DNA sequence encoding the fusion protein is linked within the vector, and that the transcriptional and translational regulatory sequences within the vector are positioned to perform their intended functions in regulating the transcription and translation of the DNA sequence. "Regulatory sequence" refers to a sequence, including promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals), that controls the transcription or translation of the DNA sequence encoding the fusion protein. These regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990). Suitable regulatory sequences for high expression in mammalian host cells include viral elements such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus main late promoter: AdMLP), or polyomavirus-derived promoters / enhancers. Non-viral regulatory sequences (e.g., ubiquitin promoters or β-globin promoters) may also be used. Furthermore, regulatory elements composed of sequences from different origins (e.g., the SRα promoter system containing the SV40 early promoter and the long-terminal repeat sequence (LTR) of human T-cell leukemia virus type 1, Takebe et al., Mol. Cell. Biol., 8:466-472, 1988) can also be used. Expression vectors and regulatory sequences should be selected to be compatible with the host cells being used.

[0047] The DNA encoding the fusion protein can be inserted into an expression vector. The recombinant expression vector may encode a signal peptide that promotes the secretion of the fusion protein from host cells. The DNA encoding the fusion protein can be cloned into the vector such that the signal peptide is in-frame linked to the amino terminus of the fusion protein. The signal peptide may be an immunoglobulin-derived signal peptide or a heterologous signal peptide derived from a protein other than immunoglobulin.

[0048] In yet another embodiment, the present invention provides a pharmaceutical composition comprising one or more fusion proteins of the present invention and a pharmaceutically acceptable carrier. In this specification, “pharmaceutically acceptable carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers. These include, but are not limited to, physiologically compatible substances such as solvents, dispersion media, coatings, isotonic agents, absorption retarders, surfactants, thickeners or emulsifiers, solid binders, dispersion / suspension aids, solubilizers, disintegrants, lubricants, and isotonic agents. The selection of a suitable carrier is readily available to those skilled in the art. The composition may further contain other pharmacologically active ingredients (e.g., another antibody, fusion protein, or cytotoxic or antitumor agent). The pharmaceutical composition of the present invention may be administered, for example, in combination therapy with other anticancer or anti-inflammatory agents.

[0049] The pharmaceutical compositions of the present invention may be suitable for administration via intravenous, intramuscular, subcutaneous, parenteral, transdermal, or other routes. Depending on the route of administration, the active ingredient may be coated with a covering material or encapsulated in a protective structure to protect it from inactivating factors such as acids and the internal environment. As used herein, "parenteral administration" refers to a form of administration other than oral and topical administration, and is usually performed by injection, and includes, but is not limited to, injection or infusion into intravenous, intramuscular, intra-arterial, subarachnoid, intra-articular, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, intra-articular, intracapsular, subarachnoid, intraspinal, epidural, and intrasternal regions. The compositions of the present invention can also be administered via routes other than parenteral administration, i.e., topical, transdermal, or mucosal routes. Examples include nasal administration, oral administration, vaginal administration, transrectal administration, sublingual administration, or topical administration.

[0050] Furthermore, the present invention provides a method of treatment. In a representative embodiment, the method comprises administering a therapeutically effective amount of the fusion protein or pharmaceutical composition of the present disclosure to a subject in need of treatment. In one embodiment, the subject has a tumor (e.g., a solid tumor, a hematopoietic malignancy, or a lymphoid malignancy), and the pharmaceutical composition is administered in an amount effective to treat the tumor of the subject. The tumor may include lung cancer, head and neck cancer, kidney cancer, breast cancer, brain tumor, melanoma and other skin cancers, ovarian cancer, liver cancer, pancreatic cancer, colorectal cancer, prostate cancer, gastrointestinal cancer, bladder cancer, hematological cancer, lymphoma, testicular cancer, gynecological cancer, and sarcoma. In other embodiments, the subject may have a chronic viral infection, which may be a hepatitis B virus (HBV) infection, a human immunodeficiency virus (HIV) infection, or an infection with herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Kaposi's sarcoma-associated herpesvirus (KSHV), human herpesvirus type 7 (HHV-7), human herpesvirus type 6 (HHV-6), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), hepatitis D virus (HDV), and / or human papillomavirus (HPV).

[0051] Example 1 Unless otherwise noted, all VHs in the anti-PD-1 moieties (wild-type and mutant) of the fusion proteins in this embodiment are either Keytruda VH or mutants based on mutations in Keytruda VH, and IL-21 or IL-21 mutants are directly bound only to the C-terminus of the primary heavy chain of the anti-PD-1 antibody. Examples of structures are shown in Figures 1 and 2.

[0052] Human and cynomolgus monkey PD-1 binding affinity PD-1 binding affinity was measured by biolayer interferometry (BLI). Binding experiments were performed using Gator Bio Prime (30°C) (see Gatorbio.com / products / instruments). Anti-PD-1 / IL-21 or anti-PD-1 / IL-21 mutants were loaded onto the tip of an anti-human IgG Fc (HFC) biosensor and incubated with serial dilutions of human or cynomolgus monkey PD-1 antigen. Binding kinetics constants were calculated using a unit-price (1:1) model.

[0053] Human and cynomolgus monkey IL-21R binding affinity IL-21R binding affinity was also measured by BLI. Binding experiments were performed in Gator Bio Prime (30°C). Human or cynomolgus monkey IL-21R protein was loaded onto the tip of an anti-His biosensor, and an anti-PD-1 antibody / IL-21 (or IL-21 mutant) fusion protein was incubated in a 3-fold dilution series (6 steps from 300 nM). Binding kinetics constants were calculated based on a unit price (1:1) model.

[0054] PD-1 reporter assay PD-1 / PD-L1 checkpoint activity was evaluated using the PD-1 / PD-L1 Blockade Bioassay (Promega, J1250). For evaluation, GloResponse Jurkat NFAT-luc2 / PD-1 stable effector cells (Promega, #CS187102) and CHO PD-L1 stable cell line (Promega, #CS178103) were co-cultured in a 1.25:1 ratio. Antibodies or fusion proteins were added in triple dilutions and incubated at 37°C and 5% CO2 for 6 hours. Luminescence was measured using the Bio-Glo luciferase assay system (Promega, #G7940), and PD-1 / PD-L1 interaction inhibition ability was evaluated as an increase in Jurkat NFAT activation. EC50 was calculated from the administration curve.

[0055] HEK-Blue IL-21R Signaling Assay HEK-Blue IL-21 reporter cells are specifically designed to overexpress IL-21R, activate the JAK / STAT signaling pathway (STAT3 phosphorylation), and induce transcriptional activation of the SEAP reporter gene. SEAP secretion can be measured using Quanti-Blue reagent. To evaluate the function of IL-21R signaling, free WT human IL-21, anti-PD-1 / IL-21, or anti-PD-1 / IL-21 mutants were co-cultured with HEK-Blue IL-21 reporter cells, and EC50 was calculated from the administration curve. For PD-1-dependent selectivity evaluation, HEK-Blue IL-21R cells were transiently co-expressed with human PD-1, and anti-PD-1 / IL-21 mutants were co-cultured with PD-1 overexpressing cells, and EC50 was calculated from the administration curve.

[0056] Hut78 T-cell lineage IL-21R / pSTAT3 signaling assay HuT78 (ATCC, TIB-161) is a T cell line that expresses human IL-21R. To evaluate the efficacy and selectivity of the anti-PD-1 / IL-21 mutant fusion antibody, a Hut78-PD-1 cell line was generated by stably expressing human PD-1 in the Hut78 parental strain. For the parental strain and the PD-1-expressing strain, control and antibody fusion proteins were administered at dilutions, and the phosphorylation level of STAT3 Tyr705 was measured using the AlphaLISA Surefire Ultra pSTAT3 (Tyr705) Assay Kit (Perkin Elmer, #ALSU-PST3-A10K). The EC50 was calculated from the dose-response curve.

[0057] in vivo MC38 colorectal tumor mouse model The in vivo activity of the anti-PD-1 / IL-21 mutant fusion antibody was evaluated in a syngeneic MC38 mouse colorectal tumor model. Mice (B-hPD-1 plus / hIL21R; C57BL / 6 background) in which the extracellular domains of human PD-1 and IL-21R were genetically fused with the transmembrane and signaling domains of mouse PD-1 and IL-21R were used to reproduce human binding specificity and physiological signaling.

[0058] Human PD-1-expressing MC38 colon tumors were subcutaneously inoculated into B-hPD-1 plus / hIL-21R mice, and after the tumors reached approximately 100 mm 3 the anti-PD-1 / IL-21 mutant fusion antibody or a control isotype antibody was administered. The antibody was administered at 2.5 mg / kg each on days 0, 3, and 6 when the tumors reached 100 mm 3 After administration, weight loss, morbidity signs, mortality, and tumor growth inhibition rate (TGI (%) = [1-(Tt-T0) / (Ct-C0)] × 100%) were monitored. The test was terminated when the average tumor volume of the control group reached approximately 2000 mm 3 or on the final observation day.

[0059] PD-1 binding The binding affinity of anti-PD-1 / IL-21 and anti-PD-1 / IL-21 mutant fusion antibodies to human PD-1 was measured by BLI (see Table 1). Keytruda, the parental control strain of the anti-PD-1 monoclonal antibody, bound to human PD-1 with high affinity in the single order of nM (Table 1; 4 nM). As expected, mutant fusion proteins containing K72Y, 75-80 deletion, or K72Y / 75-80 deletion while retaining the parental antibody PD-1 binding domain also bound to human PD-1 (Table 1; range 1.5–3 nM), showing affinity comparable to the Keytruda control. Furthermore, mutants containing K72M / 75-80 deletion, K72Q / 75-80 deletion, or additional R+E, R+EE, or cysteine ​​mutations similarly maintained single-order nM binding to human PD-1 (Table 1; 2.1–2.7 nM). In this embodiment, the R+E mutation refers to a combination of the Q87R mutation in the first-chain VH and the ELVS+E mutation in the second-chain (see Figure 2d), and the R+EE mutation refers to a combination of the Q87R mutation in the first-chain VH and the ELVS+EE mutation in the second-chain (see Figure 2e). The cysteine ​​mutation refers to the mutation combination shown in Figure 2f.

[0060] IL-21R binding The binding affinity of anti-PD-1 / IL-21 and anti-PD-1 / IL-21 mutant fusion antibodies to human IL-21R was also measured by BLI (see Table 2). Wild-type (wt) IL-21 bound to IL-21R with sub-nM affinity (Table 2; 0.1 nM). This was nearly equivalent for PD-1 / IL-21 fusion antibodies retaining the wt IL-21 sequence (within 5 times that of wt IL-21, Table 2; 0.5 nM). IL-21 K72Y, 75-80 deletion, and K72Y / 75-80 deletion mutants were designed to reduce IL-21R affinity, and anti-PD-1 / IL-21 mutant fusion antibodies containing these mutations showed a decrease in affinity to IL-21R in the range of 6-152 nM (Table 2; 60-1520-fold decrease compared to wt IL-21). Furthermore, the combinations of K72M and K72Q with 75-80 deletions showed the optimal affinity range (Table 2; 12-34 nM, 120-340-fold decrease compared to wt IL-21). R+E, R+EE, and cysteine ​​mutations did not affect the affinity range.

[0061] PD-1 checkpoint inhibitor reporter assay Antibodies exhibiting optimal IL-21R binding characteristics while maintaining PD-1 binding were evaluated for PD-1 / PD-L1 checkpoint inhibitory activity using the Jurkat-NFAT reporter assay. Checkpoint inhibitory activity was indicated by increased NFAT signaling and luciferase measurement. Table 3 shows the semi-maximal effective concentrations (EC50) of antibodies and antibody fusion proteins measured in this assay. As expected, K72Y or K72Q alone, or fusion molecules combined with the 75-80 deletion, retained PD-1 / PD-L1 inhibitory activity at EC50 values ​​within 2x of those of the Keytruda parent strain (Table 3; ETYY 1.1-2 nM, KIH 2.4-4.7 nM). K72Y / 75-80 deletion on a KIH background showed the largest shift (Table 3; 11 nM). Combinations of IL-21 mutant mutations and pI-reducing mutations (R+E, R+EE, cysteine ​​mutations) also did not affect PD-1 / PD-L1 inhibitory activity (Table 3; 1.1-< 2*). Therefore, anti-PD-1 / IL-21 mutant fusion molecules maintain the checkpoint inhibitory properties of the parental Keytruda antibody.

[0062] HEK293 pSTAT3 IL-21R activation assay To evaluate whether an anti-PD-1 / IL-21 fusion antibody can selectively activate the IL-21R signaling pathway in the presence of PD-1, a pSTAT3 reporter assay was performed on HEK293 cells expressing only human IL-21R (HEK parent strain) and HEK-PD-1 cells co-expressing human PD-1. Free WT-IL-21 was used as a positive control, and comparable EC50 values ​​were expected in PD-1-negative and PD-1-positive cells. As shown in Tables 4 and 5, WT-IL-21 showed nearly equivalent activity in HEK parent strain (Table 4; EC50 0.06 nM) and HEK-PD-1 (Table 5; EC50 0.05 nM). For comparison, PD-1 / IL-21 fusions (containing WT-IL-21) also showed similar activity to the HEK parent strain (Table 4; EC50 0.01 nM) and HEK-PD-1 (Table 5; EC50 0.03 nM). PD-1 / IL-21 mutants, including K72Y, 75-80 deletion, and K72Y / 75-80 deletion, showed EC50 fluctuations compared to WT-IL-21 and PD-1 / IL-21WT, exhibiting a range consistent with IL-21R affinity (Table 2) (0.2 nM -> 100 nM).

[0063] Hut78 pSTAT3 IL-21R Activation Assay To evaluate whether PD-1 / IL-21 mutants can be activated in cells expressing endogenous IL-21R, we used Hut78 parental cell lines expressing low levels of IL-21R and Hut78-PD-1 cells overexpressing PD-1. WT-IL-21 showed nearly equivalent activity in Hut78 parental cell lines (Table 6; EC50 0.09 nM) and Hut78-PD-1 cells (Table 7; EC50 0.11 nM). PD-1 fusion antibodies containing the WT-IL-21 sequence also showed similar activity in Hut78 parental cell lines (Table 6; EC50 0.7 nM) and Hut78-PD-1 cells (Table 7; EC50 0.12 nM). PD-1 / IL-21 mutants, including those with K72Y / 75-80, K72M / 75-80, and K72Q / 75-80 deletions, showed little activity in the Hut78 parental strain, regardless of the presence or absence of pI-reducing mutations (R+E, R+EE) or cysteine ​​mutations (Table 6; EC50 > 100 nM). In contrast, all PD-1 / IL-21 mutants showed activity within 10 times that of WT-IL-21 in Hut78-PD-1 cells (Table 7; EC50 0.1–0.9 nM). These data support the idea that PD-1 / IL-21 mutants express equivalent signaling to WT-IL-21 in IL-21R-positive and PD-1-positive cells, and do not selectively act on PD-1-negative cells.

[0064] in vivo MC38 colorectal tumor mouse model As expected, MC38 tumors in B-hPD-1 plus / hIL-21R mice proliferated. In the IgG1 isotype control antibody group, tumors grew steadily, reaching a tumor volume (TV; mean ± SEM) of 2090.25 ± 629.62 mm³ by day 20 (Figure 4a, 4b; Table 8). The isotype control group was euthanized by day 20 based on tumor burden and morbidity criteria. In contrast, in the PD-1 / IL-21 mutant fusion antibody group, the tumor volume (TV; mean ± SEM) at day 20 was 893.48 ± 237.81 mm³, and tumor growth was significantly suppressed (Figure 4a, 4b; Table 8). This corresponds to 58.66% tumor control compared to the isotype control (Figure 4a, 4b; Table 8). All animals treated with the PD-1 / IL-21 mutant fusion antibody showed a therapeutic response, and complete response (TV=0 mm^3) was observed in one case (Table 8).

[0065] The data is summarized in the table below. Table 1 summarizes data on IL-21, IL-21 variants, and fusion proteins (anti-PD-1 / IL-21 variants) that bind to human PD-1. [Table 1]

[0066] Table 2 summarizes data on IL-21, IL-21 variants, and fusion proteins (anti-PD-1 / IL-21 variants) that bind to human IL-21R. [Table 2]

[0067] Table 3 summarizes the data on IL-21, IL-21 variants, and fusion proteins (anti-PD-1 / IL-21 variants) that bind to human PD-1. [Table 3]

[0068] Table 4 summarizes the in vitro activity data of the fusion protein (anti-PD-1 / IL-21 mutant) in the HEK293 PD-1 negative cell line. [Table 4]

[0069] Table 5 summarizes the in vitro activity data of the fusion protein (anti-PD-1 / IL-21 mutant) in the HEK293 PD-1 positive cell line. [Table 5]

[0070] Table 6 summarizes the in vitro activity data of the fusion protein (anti-PD-1 / IL-21 mutant) in the Hut78 PD-1 negative cell line. [Table 6]

[0071] Table 7 summarizes the in vitro activity data of the fusion protein (anti-PD-1 / IL-21 mutant) in the Hut78 PD-1 positive cell line. [Table 7]

[0072] Table 8 summarizes the data on tumor suppression by anti-PD-1 / IL-21 mutants in a humanized mouse model of colorectal cancer. [Table 8]

[0073] All patent applications, patents, and publications cited herein are incorporated herein in their entirety, except for definitions, disclaimers, or denials relating to the subject matter of the invention. In the event of any conflict between the content of incorporated documents and the express disclosures herein, the content of this specification shall prevail.

[0074] Sequence List Sequence ID 1 Human wild-type IL-21 QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0075] Sequence ID 2 Human IL-21 mutant 1:K72Y QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIYKLKRKPPSTNAGRRQKHRLTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0076] Sequence ID 3 Human IL-21 mutant 2: Deletion of amino acids 75-80 QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLTNAGRRQKHRLTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0077] Sequence ID 4 Human IL-21 mutant 3: K72Y + deletions at positions 75-80 QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIYKLTNAGRRQKHRLTTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0078] Sequence ID 5 Human IL-21 mutant 4:K72M+ with deletions at positions 75-80 QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIMKLTNAGRRQKHRLTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0079] Sequence ID 6 Human IL-21 mutant 5:K72Q+ with deletions at positions 75-80 QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIQKLTNAGRRQKHRLTTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0080] Sequence ID 7 Keytruda VH (wild type parent chain) QVQLVQSVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS

[0081] Sequence ID 8 Keytruda VH (pI lower mutant ELVS) EVQLVQSGVELVKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELSSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS

[0082] Sequence ID 9 Keytruda VH (pI-reduced mutant ELVS+E) EVQLVQSGVELVKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELSSLEFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS

[0083] Sequence ID 10 Keytruda VH (pI-reduced mutant ELVS+EE) EVQLVQSGVELVEPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELSSLEFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS

[0084] Sequence ID 11 Keytruda VH (a pI-reduced mutant, HC1 Q87R, part of R+EE) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLRFDDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS

[0085] Sequence ID 12 hIgG1-Fc (CH3 region, HC2 S400C mutant) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPEVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0086] Sequence ID 13 hIgG1-Fc (CH3 region, HC1 Q386C mutant) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPEVKTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0087] Sequence ID 14 KIH HC2 (double hemisphere Fc in a knob-into-hole structure) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0088] Sequence ID 15 KIH HC1 (single-strand Fc with a knob-into-hole structure) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0089] Sequence ID 16 ETYY HC2 (double-strand Fc in the ETYY variant structure) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPEVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0090] Sequence ID 17 ETYY HC1 (single heavy chain Fc in the ETYY mutant structure) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0091] Sequence ID 18 ETYY HC1 mutA (C-terminal K deletion) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0092] sequence number 19 ETYY HC1 mutB(C terminalK→R) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGR

[0093] sequence no. 20 ETYY HC1 mutC(CterminalK→A) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA

[0094] Sequence number 21 ETYY HC1 mutD (C-terminal K→Q) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGQ

[0095] Sequence number 22 ETYY HC1 mutG (C-terminal K deletion - S terminal) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGS

[0096] Sequence ID 23 ETYY HC1 mutE (C-terminal K → A+G4S addition) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGS

[0097] Sequence ID 24 ETYY HC1 mutF (C-terminal K deletion + G4S addition) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS

[0098] Sequence number 24 ETYY HC1 mutH (C-terminal K deletion + SG4S addition) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGS

Claims

1. A fusion protein comprising an anti-PD-1 antibody having a C-terminus and an IL-21 variant bound to the anti-PD-1 antibody site at its C-terminus, wherein the IL-21 variant contains at least one of the following mutations relative to wild-type human IL-21: (1) one of K72Y substitution, K72M substitution, or K72Q substitution, and (2) deletion of positions 75-80.

2. The fusion protein according to claim 1, wherein the IL-21 mutant comprises both (1) one of the K72Y substitution, the K72M substitution, and the K72Q substitution, and (2) a deletion between positions 75 and 80.

3. The fusion protein according to claim 1, wherein the anti-PD-1 antibody is in IgG1 format and comprises a primary heavy chain and a secondary heavy chain, the primary heavy chain comprising a T366Y mutation and the secondary heavy chain comprising a Y407T mutation.

4. The fusion protein according to claim 1, wherein the anti-PD-1 antibody is in IgG1 format and comprises a primary heavy chain and a secondary heavy chain, the primary heavy chain comprising mutations K360, S354Y, and T366Y, and the secondary heavy chain comprising mutations G347E, Y349, and Y407T.

5. The fusion protein according to claim 1, wherein the anti-PD-1 antibody comprises a primary heavy chain containing a VH having the sequence of SEQ ID NO:

7.

6. The fusion protein according to claim 5, wherein the anti-PD-1 antibody further comprises a double chain containing a VH having a sequence selected from the group consisting of SEQ ID NOs: 8 to 10.

7. The fusion protein according to claim 1, wherein the anti-PD-1 antibody comprises a primary heavy chain containing a VH having the sequence of SEQ ID NO:

11.

8. The fusion protein according to claim 7, wherein the anti-PD-1 antibody further comprises a double chain containing a VH having a sequence selected from the group consisting of SEQ ID NOs: 8 to 10.

9. The fusion protein according to any one of claims 3 to 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 13 and the second heavy chain comprises the sequence of SEQ ID NO:

12.

10. The fusion protein according to claim 9, wherein only the first heavy chain is bound to the IL-21 variant, and the second heavy chain is not bound to any IL-21 variant.

11. The fusion protein according to claim 1, wherein the anti-PD-1 antibody site and the IL-21 variant are linked via a linker.

12. The fusion protein according to claim 11, wherein the linker comprises (GnS)x, where n is an integer from 2 to 4 and x is an integer from 1 to 4.

13. The fusion protein according to claim 1, wherein the anti-PD-1 antibody site is directly linked to the IL-21 mutant.

14. The fusion protein according to claim 9, wherein the first heavy chain comprises a sequence selected from the group consisting of SEQ ID NOs: 17 to 22.

15. The fusion protein according to claim 14, wherein the double chain comprises the sequence shown in SEQ ID NO:

16.

16. A nucleic acid encoding a fusion protein according to any one of claims 1 to 15.

17. A vector comprising the nucleic acid described in claim 16.

18. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

19. A method for treating a target cancer, comprising administering an effective amount of the fusion protein described in any one of claims 1 to 15, or the pharmaceutical composition described in claim 18, to the target.

20. The method according to claim 19, wherein the cancer is a solid tumor, a hematological malignancy, or a lymphoid malignancy.

21. The method according to claim 19, wherein the cancer includes lung cancer, head and neck cancer, kidney cancer, breast cancer, brain tumor, melanoma and other skin cancers, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, prostate cancer, gastrointestinal cancer, bladder cancer, hematological cancer, lymphoma, testicular cancer, gynecological cancer, and sarcoma.

22. A method for treating a target chronic viral infection, comprising administering an effective amount of the fusion protein described in any one of claims 1 to 15, or the pharmaceutical composition described in claim 18, to the target.

23. The method according to claim 22, wherein the chronic viral infection is caused by one or more of the following: HBV, HIV, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Kaposi's sarcoma-associated herpesvirus (KSHV), human herpesvirus 7 (HHV-7), human herpesvirus 6 (HHV-6), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), hepatitis delta virus (HDV), and human papillomavirus (HPV).

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