Antibody-cytokine fusion protein and uses thereof

Abstract

The present invention provides an antibody-cytokine fusion protein comprising an anti-PD-L1 antibody and Interleukin-21, methods for its preparation, and use of the fusion protein for therapeutic purposes.

Description

[Technical field]

[0001] The present disclosure relates to the field of biopharmaceuticals, in particular to an antibody-cytokine fusion protein comprising an anti-PD-L1 antibody and interleukin-21, its preparation method and its use. [Background technology]

[0002] Interleukin 21 (IL21) is a cytokine that mainly stimulates CD4 + IL21 is a four-alpha helix bundle cytokine secreted by T cells, B cells, myeloid cells, and natural killer (NK) T cells. By signaling through the heterodimer of the IL21 receptor (IL21R) and the common cytokine receptor gamma chain, IL21 exerts pleiotropic effects on a wide range of immune cell types and regulates both innate and adaptive immune responses. It has been reported that intratumoral injection of recombinant IL21 can promote the conversion of tumor-associated macrophages in the tumor microenvironment from an immunosuppressive M2 phenotype to a tumor-inhibitory M1 phenotype. The antitumor activity of IL21 has been demonstrated in phase I or II clinical trials in renal cell carcinoma, non-Hodgkin's lymphoma, and melanoma, either as monotherapy or in combination with other therapeutic agents. However, IL21 has a very short half-life and causes significant side effects when administered systemically. Although intratumoral administration of recombinant IL21 may offer advantages such as higher efficacy and lower systemic toxicity, it remains quite challenging for most patients.

[0003] Programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are immunoinhibitory receptors / ligands that regulate T-cell proliferation and activity. The PD-1 / PD-L1 pathway is exploited by cancer cells to suppress antitumor immunity and is generally associated with poor clinical outcomes. PD-1 / PD-L1 blockade using antibodies against PD-1 or PD-L1 has shown promising therapeutic effects in the treatment of various cancer types. Although anti-PD-1 / PD-L1 immunotherapy shows promising clinical efficacy, only a small number of patients benefit from it. Summary of the Invention

[0004] In one embodiment, a fusion protein having the structure of formula (I) or (I'): [B-Sp1-Lk-Sp2] m -A (I) A-[Sp1-Lk-Sp2-B] m (I') During the ceremony, A is an anti-PD-L1 antibody, B is interleukin 21 (IL21), Sp1 and Sp2 are each independently a spacer or absent; Lk is a linker formed by conjugation of the acceptor substrate recognition sequence and the donor substrate recognition sequence of the sortase; The present invention provides the fusion protein as described above, wherein m is an integer of 1 to 4.

[0005] In some embodiments, the acceptor substrate recognition sequence comprises 1 to 100 tandemly connected structural units selected from the group consisting of glycine (G) and alanine (A). In certain embodiments, the acceptor substrate recognition sequence comprises (G) n where n is an integer of 2 to 20, particularly 3.

[0006] In some embodiments, the donor substrate recognition sequence comprises a sequence of X1X2X3TX4X5, where X1 is leucine (L) or asparagine (N), X2 is proline (P) or alanine (A), X3 is any natural or unnatural amino acid, X4 is glycine (G), serine (S) or asparagine (N) or absent, and X5 is an amino acid sequence comprising 1 to 10 amino acids or absent.

[0007] In certain embodiments, X1X2X3TX4X5 is LPX3TG, LPX3TGG, NPQTN, NPKTG, LAX3TG, or LPQTSEQ, where X3 is any natural or unnatural amino acid. In preferred embodiments, X1X2X3TX4X5 is LPETG or LPETGG.

[0008] In some embodiments, the spacer comprises 1 to 100 tandemly connected structural units selected from the group consisting of glycine (G), alanine (A), and serine (S).

[0009] In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, 3F2, or an engineered antibody thereof. In a preferred embodiment, the anti-PD-L1 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 1 and a heavy chain having the amino acid sequence of SEQ ID NO: 2, or a light chain having the amino acid sequence of SEQ ID NO: 3 and a heavy chain having the amino acid sequence of SEQ ID NO: 4.

[0010] In some embodiments, the IL21 is wild-type IL21 or a variant thereof. In preferred embodiments, the IL21 comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0011] In some embodiments, the IL21 is conjugated to the N-terminus of the light chain and / or heavy chain of the anti-PD-L1 antibody. In a preferred embodiment, the IL21 is conjugated to the N-terminus of the heavy chain of the anti-PD-L1 antibody.

[0012] In some embodiments, Lk is X1X2X3T(G). n and the fusion protein has a structure of (II) or (II'): [B-Sp1-X1X2X3T(G) n -Sp2] m -A (II) A-[Sp1- X1X2X3T(G) n -Sp2-B] m (II') wherein X1 is leucine (L) or asparagine (N), X2 is proline (P) or alanine (A), X3 is any natural or unnatural amino acid, and n is an integer from 2 to 20; m is 1 or 2; A, B, Sp1 and Sp2 are as defined above.

[0013] In certain embodiments, the fusion protein has the structure (III) or (III'): [B-GALPETGGGGSGGGGS] m -A (III) A-[GALPETGGGGSGGGGS-B] m (III') wherein A and B are as defined above.

[0014] In certain embodiments, the fusion protein has the structure of formula (III): [B-GALPETGGGGSGGGGS] m -A (III) During the ceremony, A is an anti-PD-L1 antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 1 and a heavy chain having the amino acid sequence of SEQ ID NO: 2, or a light chain having the amino acid sequence of SEQ ID NO: 3 and a heavy chain having the amino acid sequence of SEQ ID NO: 4; B is IL21 comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; m is 1 or 2.

[0015] In certain embodiments, the fusion protein comprises two identical heavy chain portions and two identical light chain portions, of which: (1) each of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20 and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:1; or (2) each of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:21, and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:3.

[0016] In another specific embodiment, the fusion protein comprises two different heavy chain portions and two identical light chain portions, of which: (1) one of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20, the other of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:14, and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:1; or (2) one of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:21, the other of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:15, and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:3.

[0017] In another aspect, there is provided a use of a fusion protein according to the present disclosure in the manufacture of a medicament for treating a disease in a subject in need thereof. Preferably, said disease is cancer. More preferably, the cancer is selected from melanoma, ovarian cancer, breast cancer, Merkel cell carcinoma, lung cancer, renal cell carcinoma, bladder cancer, colon cancer, colon adenocarcinoma, colorectal cancer, squamous cell carcinoma of the head and neck, mesothelioma, virus-induced cancer, cervical cancer, nasopharyngeal carcinoma, soft tissue sarcoma, hematological malignancies, gastric cancer, gastric or gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, squamous cell carcinoma of the esophagus, endometrial cancer, primary mediastinal B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors, hepatocellular carcinoma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, renal cell carcinoma (RCC), squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), non-small cell lung cancer and bladder cancer.

[0018] In a further aspect, there is provided a method for producing a fusion protein according to the present disclosure, comprising: (a-1) providing an antibody moiety having a structure of formula (I-1) and a cytokine moiety having a structure of formula (I-2); [Lk a -Sp2] m -A (I-1) B-Sp1-Lk d (I-2) (b-1) conjugating the antibody portion with the cytokine portion in the presence of a sortase to obtain a fusion protein having the structure of formula (I), [B-Sp1-Lk-Sp2] m -A (I) or (a-2) preparing an antibody moiety having a structure of formula (I'-1) and a cytokine moiety having a structure of formula (I'-2); A-[Sp1-Lk d ] m (I'-1) Lk a -Sp2-B (I'-2) (b-2) conjugating the antibody portion with the cytokine portion in the presence of a sortase to obtain a fusion protein having the structure of formula (I'), A-[Sp1-Lk-Sp2-B] m (I') During the ceremony, Lk a is the acceptor substrate recognition sequence of the sortase defined above, and Lk d is the donor substrate recognition sequence of the sortase defined above, wherein A, B, Sp1, Sp2, Lk and m are as defined above. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows a schematic diagram of a fusion protein according to some embodiments of the present disclosure. [Figure 2A] Figure 2 shows the binding activity of Ab0030 and Ab0033 to hPD-L1 as analyzed by ELISA, respectively. [Figure 2B] Figure 2 shows the binding activity of Ab0030 and Ab0033 to hPD-L1 as analyzed by ELISA, respectively. [Diagram 3] 1 shows SDS-PAGE analysis of antibody-cytokine conjugation mixtures containing fusion proteins prepared from Ab0030 and Ab0033. [Figure 4] Shown is an SDS-PAGE analysis of purified fusion proteins prepared from Ab0030 and Ab0033. [Diagram 5] 4 shows the HIC-HPLC profiles of fusion proteins prepared from Ab0030 and Ab0033. [Figure 6] 4 shows the HIC-HPLC profiles of fusion proteins prepared from Ab0030 and Ab0033. [Figure 7] Figure 2 shows the binding activity of antibodies alone or fusion proteins to hPD-L1 as analyzed by ELISA. [Figure 8] Binding activity of antibodies alone or fusion proteins to IL21R analyzed by ELISA. [Figure 9] Flow cytometry analysis of STAT3 phosphorylation (pSTAT3) signals in PBMCs stimulated with control (open histograms) or hIL21, Ab0033 and LC0033-1 (filled histograms). [Figure 10] Body weights of experimental animals from the first efficacy study at the indicated time points are shown. [Figure 11] Tumor volumes at the indicated time points for experimental animals from the first efficacy study are shown. [Figure 12] 4 shows the body weights of experimental animals at the indicated time points from the second efficacy study. [Figure 13] Tumor volumes at the indicated time points for experimental animals from the second efficacy study are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.In addition, terms and experimental procedures related to biochemistry, molecular biology, cell and tissue culture, microbiology and immunology are widely used in the art.In addition, in order to better understand this disclosure, the following provides definitions and explanations of relevant terms.

[0021] As used herein, the phrases "at least one" or "one or more" mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Unless expressly indicated to the contrary, the terms "a" and "an" as used herein should be understood to mean "at least one."

[0022] When a particular amount, concentration, value, or parameter is described in the form of a range, a preferred range, or a preferred upper limit or a preferred lower limit, it should be understood that any range formed by combining any upper limit or preferred value with any lower limit or preferred value is equivalent to being specifically stated, regardless of whether the range is explicitly stated or not. Unless otherwise specified, numerical ranges recited herein are intended to include both ends of the range and all integers and fractions (decimals) within the range. For example, the expression "i is an integer from 1 to 20" means that i is any integer from 1 to 20, for example, i may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar expressions, such as n and m, should be understood in the same manner.

[0023] As used in this specification, the term "optional" or "optionally" means that the event subsequently described may occur, but does not necessarily occur, and the description includes cases where the event or circumstance does or does not occur.

[0024] The terms "comprising," "including," "containing," and "having" are open-ended and do not exclude additional, unrecited elements, steps, or ingredients. The term "consisting of" excludes any unspecified elements, steps, or ingredients. "Consisting essentially of" means that the scope is limited to the specified elements, steps, or ingredients, or to a group of optionally present elements, steps, or ingredients that do not materially affect the essential and novel characteristics of the claimed subject matter. The term "comprising" should be understood to encompass terms such as "consisting essentially of" and "consisting of."

[0025] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule or fragment thereof that specifically binds to an epitope of an antigen through at least one antigen-binding site (i.e., paratope). As used herein, the definition of "antibody (Ab)" encompasses conventional antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, diabodies, anti-idiotypic antibodies, and antigen-binding fragments. The antigen-binding fragments described herein can be produced by enzymatic processing of full-length antibodies and / or synthetic methods, e.g., recombinant antibodies produced by grafting the CDRs of one antibody onto the framework of another antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, scFv, dsFv, scdsFv, diabodies, Fab, scFab, Fab', F(ab')2, and other fragments. The antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass (e.g., IgG2a and IgG2b). In some embodiments, the antibody is an anti-PD-L1 antibody that recognizes at least a portion of PD-L1. In some embodiments, the anti-PD-L1 antibody is a conventional antibody comprising two identical light chains and two identical heavy chains.

[0026] A "conventional" or "full-length" antibody typically consists of four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain contains, from amino to carboxyl terminus, a light chain variable region (VL) and a light chain constant region (CL). Each heavy chain contains, from amino to carboxyl terminus, a heavy chain variable region (VH) and one or more heavy chain constant regions (CH).

[0027] Each VL and VH typically has three hypervariable "complementarity determining regions (CDRs)" and four relatively conserved "framework regions (FRs)", arranged from amino-terminus to carboxyl-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Depending on their position in the VL or VH, the CDRs are designated as heavy chain variable region CDRs (CDRHs), such as CDRH1, CDRH2 and CDRH3, or light chain variable region CDRs (CDRLs), such as CDRL1, CDRL2 and CDRL3.

[0028] Those of skill in the art will be familiar with CDRs and can sequence CDRs using methodologies well known in the art, such as those described in Kabat (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), Chothia (see, e.g., Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917), AbM (see, e.g., Martin, ACR and Allen, J. (2007). Bioinformatics Tools for Antibody Engineering. In Handbook of Therapeutic Antibodies, S. Dubel (Ed.)) and IMGT (see, e.g., Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev. Comp. A variety of numbering schemes can be used to define the CDRs, including, for example, the CDR numbering scheme (see, for example, Immunol., 27, 55-77 (2003)). One of skill in the art will appreciate that the CDR sequences of a given antibody may have different boundaries depending on the numbering scheme used. The CDR sequences of the anti-PD-L1 antibodies described herein are defined using the Kabat numbering scheme, although CDR sequences defined by any other numbering scheme are intended to be within the scope of protection of this disclosure.

[0029] As used herein, the term "binding affinity" is a measure of the strength between two binding partners, such as an antibody and its cognate antigen (e.g., an anti-PD-L1 antibody and PD-L1) or a ligand and its receptor (e.g., IL21 and IL21R). Typically, affinity between proteins is usually measured using the equilibrium dissociation constant, K DIt is measured and reported by K D can be calculated from kinetic analysis using the following equation: K D =k off / k on In the formula, k off and k on are the dissociation rate constant and the association rate constant between the antigen and the antibody, respectively. K D Typically, the K between an antibody and its cognate antigen is D is about 10 -6 M ~ about 10 -9 M or less, e.g. 10 -8 Below, 10 -9 Less than or equal to 10 -10 The following is true: k off and k on The methods include immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, e.g., Paul, WE, Fundamental Immunology, 2 nd ed., Raven Press, New York, pages 332-336 (1989). See also U.S. Pat. No. 7,229,619. Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (e.g., BiaCore2000, BiacoreAB, Upsala, Sweden).

[0030] As used herein, the term "polypeptide" refers to two or more amino acids covalently linked by a peptide bond. The terms "polypeptide" and "protein" are used interchangeably herein. A polypeptide can be modified, for example, to include one or more spacers or functional moieties, such as polyethylene glycol (PEGylation) and glycans (glycosylation). Methods for modifying polypeptides are well known to those of skill in the art.

[0031] As used herein, "natural amino acid" refers to a naturally occurring amino acid in the L-configuration that is used in protein synthesis in living organisms. Natural amino acids are represented herein by the standard one-letter or three-letter codes known to those skilled in the art, such as alanine (A, Ala), arginine (R, Arg), asparagine (N, Asn), arginic acid (D, Asp), cysteine ​​(C, Cys), glutamine (Q, Gln), glutamic acid (E, Glu), glycine (G, Gly), histidine (H, His), isoleucine (I, Ile), leucine (L, Leu), lysine (K, Lys), methionine (M, Met), phenylalanine (F, Phe), proline (P, Pro), serine (S, Ser), threonine (T, Thr), tryptophan (W, Trp), tyrosine (Y, Tyr), valine (V, Val), pyrrolysine (O, Pyl), and selenocysteine ​​(U, Sec). Examples of unnatural amino acids include D-isomers and derivatives of the natural amino acids listed above, as well as unusual amino acids. Unnatural amino acids include, but are not limited to, homoamino acids, β-homoamino acids, N-methyl amino acids, α-methyl amino acids, citrulline (Cit), hydroxyproline (Hyp), norleucine (Nle), 3-nitrotyrosine, nitroarginine, ornithine (Orn), naphthylalanine (Nal), aminobutyric acid (Abu), diaminobutyric acid (Dab), methionine sulfoxide, and methionine sulfone.

[0032] As used herein, the numbering of amino acid positions in a polypeptide is defined such that the first amino acid position from the N-terminus is designated as position 1.

[0033] As used herein, "spacer" refers to a structure located between two moieties in a fusion protein to spatially separate the two moieties. Examples of spacers include, but are not limited to, amino acids, amino acid derivatives or analogs, and amino acid sequences. Preferred spacers include GA, GGG, GAG, and (G) o S((G) s S) t where o is an integer from 1 to 5, s is an integer from 1 to 10, particularly 4, and t is 0 or an integer from 1 to 10. Spacers may be incorporated into a polypeptide (such as an antibody moiety or cytokine moiety described herein) by, but are not limited to, chemical synthesis, chemical or enzymatic reaction, or recombinant DNA technology.

[0034] As used herein, the terms "recognition sequence" and "recognition motif" are used interchangeably to refer to an amino acid sequence that is recognized by an enzyme (e.g., a sortase) and through which the enzyme exerts its enzymatic action. For example, two polypeptides that contain donor and acceptor substrate recognition sequences, respectively, of a sortase can be conjugated to form a fusion polypeptide by the enzymatic action of a sortase.

[0035] As used herein, "sequence identity" has its art-recognized meaning, and the percent sequence identity between two nucleic acids or polypeptides can be calculated by aligning the two sequences using published algorithms, such as the Basic Local Alignment Search Tool (BLAST) and the Fast Adaptive Shrinkage / Thresholding Algorithm (FASTA) (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994). Although there are many methods for measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0036] As used herein, a "variant" of a reference polypeptide (such as wild-type IL21) comprises one or more amino acid additions, deletions, insertions and / or substitutions compared to the reference polypeptide.

[0037] As used herein, "treating" a subject with a disease or condition means administering or administering a composition, procedure or regimen to a subject in an attempt to cure the disease or condition or to prevent, alleviate, ameliorate or eliminate the symptoms of the disease or condition. Thus, the term "treatment" encompasses prophylaxis, therapy and / or cure. The term "prevention" refers to the prevention of underlying disease and / or the prevention of worsening symptoms or progression of disease. As used herein, treatment also encompasses any pharmaceutical use of the fusion protein or pharmaceutical composition provided herein.

[0038] As used herein, the definition of "subject" includes human and non-human subjects, such as laboratory animals (eg, mice, rabbits, and rats and non-human primates), and preferably refers to humans.

[0039] As used herein, "therapeutic effect" means an effect resulting from treating a subject that alters, typically ameliorates or eliminates, the symptoms of a disease or condition.

[0040] As used herein, a "therapeutically effective amount" refers to an amount of a drug, compound, or composition containing one or more active agents that is at least sufficient to produce a therapeutic effect after administration to a subject, and thus is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or disorder.

[0041] Fusion proteins In one embodiment, a fusion protein having the structure of formula (I) or (I') is provided: [B-Sp1-Lk-Sp2] m -A (I) A-[Sp1-Lk-Sp2-B] m (I') During the ceremony, A is an anti-PD-L1 antibody, B is interleukin 21 (IL21), Sp1 and Sp2 are each independently a spacer or absent; Lk is a linker formed by conjugation of the acceptor substrate recognition sequence and the donor substrate recognition sequence of the sortase; m is an integer from 1 to 4.

[0042] The fusion protein is an antibody-cytokine fusion protein (also referred to as an immunocytokine) comprising an anti-PD-L1 antibody and interleukin 21 (IL21) conjugated via a linker, where the linker comprises an acceptor substrate recognition sequence conjugated to a donor substrate recognition sequence of a sortase. In some embodiments, the linker is formed by sortase-mediated site-specific conjugation of the acceptor substrate recognition sequence recognized by the sortase and the donor substrate recognition sequence, where one of the recognition sequences is linked to the anti-PD-L1 antibody to form the antibody portion and the other of the recognition sequence is linked to IL21 to form the cytokine portion.

[0043] In formula (I), IL21 is linked to a donor substrate recognition sequence, and an anti-PD-L1 antibody is linked to an acceptor substrate recognition sequence. In formula (I'), an anti-PD-L1 antibody is linked to a donor substrate recognition sequence, and IL21 is linked to an acceptor substrate recognition sequence. Through the enzymatic action of sortase, the acceptor and donor substrate recognition sequences form a linker between the anti-PD-L1 antibody and IL21. Optionally, a spacer is inserted between the recognition sequence and the anti-PD-L1 antibody and / or IL21 to provide spatial separation.

[0044] Formulas (I) and (I') define the linkage between IL21 or the anti-PD-L1 antibody and the linker, respectively, but are not intended to limit the position at which IL21 is conjugated to the anti-PD-L1 antibody. In either formula (I) or (I'), IL21 can be conjugated to any position on the anti-PD-L1 antibody.

[0045] In some embodiments, the anti-PD-L1 antibody comprises a light chain and a heavy chain. In one embodiment, IL21 is conjugated at the N-terminus of the light chain and / or heavy chain of the anti-PD-L1 antibody. In one embodiment, IL21 is conjugated at the C-terminus of the light chain and / or heavy chain of the anti-PD-L1 antibody. In some embodiments, IL21 is conjugated at a non-terminal position of the anti-PD-L1 antibody.

[0046] In some preferred embodiments, in Formula (I), IL21 is conjugated to the N-terminus of the light chain and / or the heavy chain of the anti-PD-L1 antibody. In certain embodiments, IL21 is conjugated to the N-terminus of the heavy chain of the anti-PD-L1 antibody.

[0047] In some preferred embodiments, in Formula (I'), IL21 is conjugated to the C-terminus of the light chain and / or the heavy chain of the anti-PD-L1 antibody. In certain embodiments, IL21 is conjugated to the C-terminus of the heavy chain of the anti-PD-L1 antibody.

[0048] m represents the number of IL21 conjugated to the anti-PD-L1 antibody. One or more IL21 can be conjugated to the anti-PD-L1 antibody by controlling the number of donor or acceptor substrate recognition sequences linked to the anti-PD-L1 antibody. In some preferred embodiments, m is an integer selected from 1, 2, 3, and 4. In a particular embodiment, m is 1. In another particular embodiment, m is 2.

[0049] Anti-PD-L1 antibody "Programmed cell death ligand 1 (PD-L1)", also known as "B7H1" or "CD274", is a ligand for the immunosuppressive receptor Programmed cell death 1 (PD-1). PD-L1 can be from any species, such as human, monkey, chimpanzee, cow, pig, goat, sheep, horse, rat, mouse, guinea pig, rabbit, etc. Preferably, the PD-L1 is human PD-L1. The full amino acid sequence of human PD-L1 (hPD-L1) is available from public databases, such as Uniprot and GenBank. The anti-PD-L1 antibody can be any antibody against PD-L1 (e.g., PD-L1 comprising the amino acid sequence shown in Uniprot identifier Q9NZQ7-1, Q9NZQ7-2, or Q9NZQ7-3) or a fragment thereof. Preferably, the anti-PD-L1 antibody blocks the binding between PD-L1 and PD-1 and inhibits PD-L1 signaling. Examples of anti-PD-L1 antibodies include, but are not limited to, atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi) or 3F2 (see WO2020169062A1, the entire contents of which are incorporated herein by reference). The anti-PD-L1 antibody may include further modifications. The modifications may include one or more amino acid additions, deletions, insertions and / or substitutions compared to an unmodified antibody. The anti-PD-L1 antibody may be an engineered antibody prepared from atezolizumab, avelumab, durvalumab or 3F2.

[0050] In some embodiments, the anti-PD-L1 antibody is a polyclonal antibody. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. Methods for producing polyclonal and monoclonal antibodies are known in the art. For example, a PD-L1 fragment as an antigen can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, hamster, goat, or sheep, and polyclonal anti-PD-L1 antibodies can be purified from the serum of the immunized mammal. Anti-PD-L1 antibody-secreting B cells can be isolated from the immunized mammal and fused with myeloma cells to produce monoclonal anti-PD-L1 antibodies. Alternatively, monoclonal anti-PD-L1 antibodies can be generated using phage display and recombinant DNA technology.

[0051] In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, 3F2, or an engineered antibody thereof. In a particular embodiment, the anti-PD-L1 antibody is atezolizumab or an engineered antibody thereof. In another particular embodiment, the anti-PD-L1 antibody is the anti-PD-L1 antibody 3F2 or an engineered antibody thereof.

[0052] In some embodiments, the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH). In one embodiment, the VL comprises the CDRL1 sequence set forth in SEQ ID NO:22, the CDRL2 sequence set forth in SEQ ID NO:23, and the CDRL3 sequence set forth in SEQ ID NO:24, and the VH comprises the CDRH1 sequence set forth in SEQ ID NO:25, the CDRH2 sequence set forth in SEQ ID NO:26, and the CDRH3 sequence set forth in SEQ ID NO:27. In another embodiment, the VL comprises the CDRL1 sequence set forth in SEQ ID NO:28, the CDRL2 sequence set forth in SEQ ID NO:29, and the CDRL3 sequence set forth in SEQ ID NO:30, and the VH comprises the CDRH1 sequence set forth in SEQ ID NO:31, the CDRH2 sequence set forth in SEQ ID NO:32, and the CDRH3 sequence set forth in SEQ ID NO:33.

[0053] In another embodiment, the VL comprises the amino acid sequence of SEQ ID NO: 34 and the VH comprises the amino acid sequence of SEQ ID NO: 35. In yet another embodiment, the VL comprises the amino acid sequence of SEQ ID NO: 36 and the VH comprises the amino acid sequence of SEQ ID NO: 37.

[0054] In some embodiments, the antibody comprises a light chain and a heavy chain. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 1 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 3 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 4.

[0055] Anti-PD-L1 antibodies may be obtained by methods known in the art, including but not limited to phage display technology, hybridoma technology, recombinant DNA technology, and chemical synthesis. Anti-PD-L1 antibodies may be isolated from a variety of sources, such as yeast display libraries or phage display libraries (see, e.g., Winter et al., (1994) Annu. Rev. Immunol. 12:433-455 and McCafferty et al., (1990) Nature 348:552-553) or from human peripheral blood. For example, antibodies may be expressed in host cells that are generally used for antibody production, such as yeast cells, bacteria, insect cells, or mammalian cells. Suitable mammalian host cells for antibody expression include, but are not limited to, myeloma cells, Chinese hamster ovary (CHO) cells, HEK293 cells, and other mammalian cells suitable for antibody expression. In a specific embodiment, mammalian host cells are transformed with expression vectors encoding the light and heavy chains of an anti-PD-L1 antibody, and the expressed antibody is purified using a Protein A affinity column. Signal peptides can be used to improve antibody quality, yield and purification by aiding in the expression, correct folding and extracellular secretion of the antibody. Exemplary sequences of signal peptides that can be added to the light and heavy chains are set forth in SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0056] Interleukin 21 (IL21) Interleukin 21 (IL21) is a four alpha helix bundle cytokine of approximately 14 kDa. IL21 signals by binding to a complex receptor consisting of the IL21 receptor (IL21R) and the common cytokine gamma chain (gamma c). Without wishing to be bound by a particular theory, IL21 signaling is thought to mediate activation of macrophages, B cells, CD4 + T cells, CD8 + They enhance both humoral and cell-mediated immune responses by inducing the proliferation, differentiation, or activity of a variety of target immune cells, including T cells, cytotoxic T cells, and natural killer cells.

[0057] IL21 can be from any species, e.g. monkey, chimpanzee, cow, pig, goat, sheep, horse, rat, mouse, guinea pig, rabbit, etc. Preferably, IL21 is human IL21 (hIL21). Two hIL21 precursors have been identified, UniProt identifier Q9HBE4-1 (SEQ ID NO: 7, isoform 1 precursor) and UniProt identifier Q9HBE4-2 (SEQ ID NO: 8, isoform 2 precursor), with the first 24 aa being the signal peptide. Wild-type IL21 is often secreted as a mature protein lacking the signal peptide. Exemplary amino acid sequences of mature hIL21 are shown in SEQ ID NO: 9 (isoform 1) and SEQ ID NO: 10 (isoform 2).

[0058] IL21 can be wild-type IL21 or a variant thereof. Wild-type IL21 can have the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. IL21 variants can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, or deletions compared to SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, IL21 variants include an amino acid sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, IL21 variants bind to IL21R with lower affinity compared to wild-type IL21. Examples of such IL21 variants may include IL21 muteins described in Shen et. al., Front. Immunol., (2020) 11:832 and WO2019028316A1, the entire relevant contents of which are incorporated by reference herein.

[0059] IL21 can be obtained by methods known in the art. For example, the coding sequence of IL21 can be produced by recombinant DNA technology and introduced into a suitable host cell for expression of IL21. Exemplary host cells can include, but are not limited to, yeast cells, bacteria, fungi, insect cells and mammalian cells. In one embodiment, IL21 is expressed in E. coli BL21(DE3).

[0060] Acceptor and donor substrate recognition sequences The acceptor and donor substrate recognition sequences correspond to the type of sortase used. The sortase may be a naturally occurring sortase, a non-naturally occurring sortase, or a combination thereof. Naturally occurring sortases may include sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), sortase F (SrtF), etc. (see, e.g., US20110321183A1 and EP3647419A1).

[0061] A typical acceptor substrate recognition sequence includes an N-terminal amino group as a nucleophile, such as the N-terminal amino group of glycine and alanine. In some embodiments, the acceptor substrate recognition sequence includes 1 to 100 tandemly connected structural units selected from the group consisting of glycine (G) and alanine (A). In a preferred embodiment, the acceptor substrate recognition sequence includes (G) n where n is an integer from 2 to 20, particularly 3.

[0062] In some embodiments, the donor substrate recognition sequence is X1X2X3TX4X5, where X1 is leucine (L) or asparagine (N), X2 is proline (P) or alanine (A), X3 is any natural or unnatural amino acid, X4 is glycine (G), serine (S) or asparagine (N) or absent, and X5 is an amino acid sequence comprising 1-10 amino acids or absent. In a preferred embodiment, X3 is glutamic acid (E). In one embodiment, X5 is absent. In another embodiment, X5 is an amino acid sequence comprising 1-10 amino acids, where each amino acid is independently any natural or unnatural amino acid.

[0063] In some embodiments, the sortase is SrtA, such as SrtA from Staphylococcus aureus, and X1X2X3TX4X5 can be LPX3TGG (SEQ ID NO: 39), where X3 is any natural or unnatural amino acid. In some embodiments, the sortase is SrtB, and X1X2X3TX4X5 can be NPX3TGG (SEQ ID NO: 46), where X3 is any natural or unnatural amino acid. In some embodiments, the sortase is SrtC, and X1X2X3TX4X5 can be LPX3TGG, where X3 is any natural or unnatural amino acid. In some other embodiments, the sortase is SrtD, and X1X2X3TX4X5 can be LPX3TA (SEQ ID NO: 47), where X3 is any natural or unnatural amino acid. In still other embodiments, the sortase is SrtE and X1X2X3TX4X5 may be LAX3TGG (SEQ ID NO: 48), where X3 is any natural or unnatural amino acid. In some other embodiments, the sortase is SrtF and X1X2X3TX4X5 may be LPX3TG (SEQ ID NO: 49), where X3 is selected from the group consisting of A, R, E, N, D, Q, I, L, and K. In some other embodiments, the sortase is SrtA from Streptococcus pyogenes and X1X2X3TX4X5 may be LPX3TGX5 (SEQ ID NO: 50), where X5 is as defined above. In some embodiments, the sortase is SrtE from Streptomyces coelicolor and X1X2X3TX4X5 may be LAX3TG (SEQ ID NO: 42). In yet another embodiment, the sortase is SrtA from Lactobacillus plantarum and X1X2X3TX4X5 may be LPQTSEQ (SEQ ID NO: 43). In one embodiment, the sortase is SrtB from Staphylococcus aureus and X1X2X3TX4X5 may be NPQTN (SEQ ID NO: 40). In another embodiment, the sortase is SrtB from Bacillus anthracis and X1X2X3TX4X5 may be NPKTG (SEQ ID NO: 41).

[0064] In some particular embodiments, the sortase is SrtA from Staphylococcus aureus. Thus, X1X2X3TX4X5 may be the typical donor substrate recognition sequence of the enzyme LPX3TGG, where X3 may be any single amino acid, natural or unnatural. In some embodiments, X1X2X3TX4X5 is LPX3TGX5, where X3 is any single amino acid, natural or unnatural, and X5 is absent or an amino acid sequence comprising 1-10 amino acids. In one embodiment, X5 is absent. In another embodiment, X5 is an amino acid sequence comprising 1-10 amino acids, where each amino acid is independently any natural or unnatural amino acid. In particular embodiments, X5 is (G) i wherein i is an integer from 1 to 10. In another specific embodiment, X1X2X3TX4X5 is LPETG (SEQ ID NO: 44). In yet another specific embodiment, X1X2X3TX4X5 is LPETGG (SEQ ID NO: 45).

[0065] In a typical sortase-catalyzed conjugation reaction, sortase cleaves the donor substrate recognition sequence X1X2X3TX4X5 at a threonine residue. Upon cleavage, the downstream fragment (-X4X5) of the threonine residue is released, and the upstream fragment (X1X2X3T-) forms a threonine-thioester with the active site cysteine ​​of the sortase to generate an acyl-enzyme intermediate. The intermediate is subsequently deacylated by ligating the sortase-bound thioester to the N-terminal amino group of the acceptor substrate recognition sequence. (G) n is used as the acceptor substrate recognition sequence, the resulting linker Lk is X1X2X3T(G) n where X1 is leucine (L) or asparagine (N), X2 is proline (P) or alanine (A), X3 is any natural or unnatural amino acid, and n is an integer from 2 to 20, particularly 3.

[0066] In some embodiments, X1X2X3T(G) n is LPX3T(G) n(SEQ ID NO:51), NPX3T(G) n (SEQ ID NO: 52) or LAX3T(G) n (SEQ ID NO:53), where L is leucine, P is proline, N is asparagine, A is alanine, X3 is any natural or unnatural amino acid, T is threonine, G is glycine, and n is an integer from 2 to 20, in particular 3. In one embodiment, n is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In a preferred embodiment, n is 3.

[0067] In some embodiments, X1X2X3T(G) n is LPX3T(G) n wherein X3 is any natural or unnatural amino acid and n is as defined above. In one embodiment, X3 is glutamic acid (E). In a particular embodiment, X1X2X3T(G) n is LPETGGG (SEQ ID NO: 54). In another embodiment, X3 is glutamine (Q). In a particular embodiment, X1X2X3T(G) n is LPQTGGG (SEQ ID NO:55).

[0068] In yet another embodiment, X1X2X3T(G) n is LAX3T(G) n wherein X3 is any natural or unnatural amino acid and n is as defined above. In a preferred embodiment, X3 is E.

[0069] In another embodiment, X1X2X3T(G) n is NPQT(G) n (SEQ ID NO: 56) or NPKT(G) n (SEQ ID NO:57), where n is as defined above.

[0070] Spacer In some embodiments, at least one of Sp1 and Sp2 is a spacer. In some embodiments, Sp1 and Sp2 are each independently a spacer.

[0071] In some embodiments, the spacer comprises 1 to 100 tandemly connected structural units selected from the group consisting of glycine (G), alanine (A) and serine (S). In some embodiments, the spacer comprises GA, GGG, GAG and (G) o S((G) s S) t (SEQ ID NO: 59), where o is an integer from 1 to 5, s is an integer from 1 to 10, in particular 4, and t is 0 or an integer from 1 to 10. In a particular embodiment, Sp1 is a spacer having the sequence GA and Sp2 is a spacer having the sequence GSGGGGS (SEQ ID NO: 58).

[0072] Specific embodiments of the fusion protein Exemplary embodiments of fusion proteins according to the present disclosure are provided below.

[0073] In some embodiments, Lk is X1X2X3T(G). n and the fusion protein has the structure (II) or (II'): [B-Sp1-X1X2X3T(G) n -Sp2] m -A (II) A-[Sp1-X1X2X3T(G) n -Sp2-B] m (II') wherein X1 is leucine (L) or asparagine (N), X2 is proline (P) or alanine (A), X3 is any natural or unnatural amino acid, and n is an integer from 2 to 20; m is 1 or 2; A, B, Sp1 and Sp2 are as defined above.

[0074] In some embodiments, X1X2X3T(G) nis LPX3T(G) n In one embodiment, X3 is E. In another embodiment, X3 is Q.

[0075] In some embodiments, X1X2X3T(G) n is LAX3T(G) n In one embodiment, X3 is E or Q.

[0076] In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, 3F2, or engineered antibodies thereof. In a particular embodiment, the anti-PD-L1 antibody is Ab0001 (atezolizumab) and comprises a light chain having the amino acid sequence of SEQ ID NO: 1 and a heavy chain having the amino acid sequence of SEQ ID NO: 2. In another embodiment, the anti-PD-L1 antibody is Ab0002 (3F2) and comprises a light chain having the amino acid sequence of SEQ ID NO: 3 and a heavy chain having the amino acid sequence of SEQ ID NO: 4.

[0077] In some embodiments, IL21 is wild-type IL21 or a variant thereof. In one embodiment, IL21 comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0078] In one embodiment, in formula (II) or (II'), n is 3.

[0079] In some embodiments, Sp1 and Sp2 are absent. In some embodiments, at least one of Sp1 and Sp2 is a spacer described herein. In some embodiments, Sp1 and Sp2 are each independently a spacer. In one embodiment, Sp1 is GA and Sp2 is GSGGGGS.

[0080] In one embodiment, Sp1 is GA, the donor substrate recognition sequence is LPETGG, the acceptor substrate recognition sequence is GGG, Sp2 is GSGGGGS, and the fusion protein has the structure of (III) or (III'). [B-GALPETGGGGSGGGGS] m -A (III) A-[GALPETGGGGSGGGGS-B] m (III') GALPETGGGGSGGGGS is represented as sequence number 60.

[0081] In one embodiment, in any of Formulas (I), (I'), (II), (II'), (III) and (III'), the anti-PD-L1 antibody comprises a light chain having the amino acid sequence of SEQ ID NO:1 and a heavy chain having the amino acid sequence of SEQ ID NO:2, or a light chain having the amino acid sequence of SEQ ID NO:3 and a heavy chain having the amino acid sequence of SEQ ID NO:4; IL21 comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; and m is 1 or 2.

[0082] In some embodiments, the anti-PD-L1 antibody in any of Formulas (I), (II) and (III) comprises a light chain and a heavy chain. In one embodiment, IL21 is conjugated to the N-terminus of the light chain and / or heavy chain of the anti-PD-L1 antibody. In a specific embodiment, IL21 is conjugated to the N-terminus of the heavy chain of the anti-PD-L1 antibody.

[0083] In some other embodiments, the anti-PD-L1 antibody in any of Formula (I'), (II'), and (III') comprises a light chain and a heavy chain. In one embodiment, IL21 is conjugated to the C-terminus of the light chain and / or heavy chain of the anti-PD-L1 antibody. In a specific embodiment, IL21 is conjugated to the C-terminus of the heavy chain of the anti-PD-L1 antibody.

[0084] In some embodiments, the anti-PD-L1 antibody comprises two identical light chains and two identical heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO:1 and each heavy chain comprises the amino acid sequence of SEQ ID NO:2, or each light chain comprises the amino acid sequence of SEQ ID NO:3 and each heavy chain comprises the amino acid sequence of SEQ ID NO:4. In one embodiment, IL21 is conjugated to the N-terminus of at least one of the heavy chains of the anti-PD-L1 antibody. In one embodiment, a fusion protein according to the disclosure comprises two identical light chain portions and two identical heavy chain portions, wherein each heavy chain portion comprises IL21 conjugated to the N-terminus of each of the heavy chains of the anti-PD-L1 antibody. In another embodiment, a fusion protein according to the disclosure comprises two identical light chain portions and two different heavy chain portions, wherein only one of the heavy chain portions comprises IL21 conjugated to one of the heavy chains of the anti-PD-L1 antibody.

[0085] In certain embodiments, the fusion protein comprises two identical heavy chain portions and two identical light chain portions, of which: (1) each of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20 and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:1; or (2) each of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:21, and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:3.

[0086] In another specific embodiment, the fusion protein comprises two different heavy chain portions and two identical light chain portions, wherein (1) one of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20, the other of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:14, and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:1; or (2) one of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:21, the other heavy chain portion comprises the amino acid sequence of SEQ ID NO:15, and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:3.

[0087] Methods for preparing fusion proteins In some embodiments, the fusion protein is produced by conjugating an antibody moiety comprising an anti-PD-L1 antibody to a cytokine moiety comprising IL21 under sortase catalysis, where one of the antibody moiety and the cytokine moiety further comprises a donor substrate recognition sequence for a sortase, and the other further comprises an acceptor substrate recognition sequence for a sortase, whereby the donor substrate recognition sequence and the acceptor substrate recognition sequence are conjugated by the enzymatic action of the sortase. Suitable sortases, acceptor and donor substrate recognition sequences are described above.

[0088] Thus, in another aspect, a method for producing a fusion protein according to the present disclosure is provided.

[0089] In one embodiment, the method comprises: (a-1) providing an antibody moiety having a structure of formula (I-1) and a cytokine moiety having a structure of formula (I-2); [Lk a -Sp2] m -A (I-1) B-Sp1-Lk d (I-2) (b-1) conjugating the antibody moiety to the cytokine moiety in the presence of a sortase to obtain a fusion protein having the structure of formula (I), [B-Sp1-Lk-Sp2] m -A (I) During the ceremony, Lk a is the acceptor substrate recognition sequence of sortase, and Lk d is the donor substrate recognition sequence for sortase, A, B, Sp1, Sp2, Lk and m are as defined above.

[0090] In another embodiment, the method comprises: (a-2) preparing an antibody moiety having a structure of formula (I'-1) and a cytokine moiety having a structure of formula (I'-2); A-[Sp1-Lk d ] m(I'-1) Lk a -Sp2-B (I'-2) (b-2) conjugating the antibody moiety to the cytokine moiety in the presence of a sortase to obtain a fusion protein having the structure of formula (I'), A-[Sp1-Lk-Sp2-B] m (I') During the ceremony, Lk a is the acceptor substrate recognition sequence of sortase, and Lk d is the donor substrate recognition sequence for sortase, A, B, Sp1, Sp2, Lk and m are as defined above.

[0091] In one particular embodiment, step (a-1) comprises: (a-1-1)Lk a - adding Sp2 to the N-terminus of the heavy chain of an anti-PD-L1 antibody to obtain an antibody moiety having the structure of formula (I-1); [Lk a -Sp2] m -A (I-1) (a-1-2) Sp1-Lk d to the C-terminus of IL21 to obtain a cytokine moiety having the structure of formula (I-2). B-Sp1-Lk d (I-2)

[0092] In another particular embodiment, step (a-2) comprises: (a-2-1) Sp1-Lk d to the C-terminus of the heavy chain of an anti-PD-L1 antibody to obtain an antibody moiety having the structure of Formula (I'-1): A-[Sp1-Lk d ] m (I'-1) (a-2-2)Lk a - adding Sp2 to the N-terminus of IL21 to obtain a cytokine moiety having the structure of formula (I'-2). Lk a -Sp2-B (I'-2)

[0093] In one embodiment, Lk a comprises 1 to 100 tandemly connected structural units selected from the group consisting of glycine (G) and alanine (A). a (G) n where n is an integer from 2 to 20, particularly 3.

[0094] In one embodiment, Lk d comprises the sequence of X1X2X3TX4X5, where X1 is L or N, X2 is P or A, X3 is any natural or unnatural amino acid, X4 is G, S, N or absent, and X5 is an amino acid sequence comprising 1-10 amino acids or absent. In certain embodiments, X1X2X3TX4X5 is LPX3TG (SEQ ID NO: 38), LPX3TGG, NPQTN, NPKTG, LAX3TG, or LPQTSEQ, where X3 is any natural or unnatural amino acid. In certain embodiments, X1X2X3TX4X5 is LPETG or LPETGG.

[0095] In one embodiment, Sp1 and Sp2 are absent. In one embodiment, at least one of Sp1 and Sp2 is a spacer as described herein. In another embodiment, Sp1 and Sp2 are each independently a spacer. In an embodiment, the spacer comprises 1 to 100 tandemly connected structural units selected from the group consisting of G, A, and S. In a preferred embodiment, the spacer is selected from GA, GGG, GAG, and (G) o S((G) s S) t where o is an integer from 1 to 5, s is an integer from 1 to 10, in particular 4, and t is 0 or an integer from 1 to 10. In a particular embodiment, Sp1 is a spacer having the sequence GA and Sp2 is a spacer having the sequence GSGGGGS.

[0096] In one embodiment, the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, 3F2, or engineered antibodies thereof. In one embodiment, the anti-PD-L1 antibody comprises a VL and a VH, wherein the VL and VH comprise the CDR sequences described herein. In another embodiment, the anti-PD-L1 antibody comprises a VL and a VH having sequences described herein. In a specific embodiment, the anti-PD-L1 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 1 and a heavy chain having the amino acid sequence of SEQ ID NO: 2, or a light chain having the amino acid sequence of SEQ ID NO: 3 and a heavy chain having the amino acid sequence of SEQ ID NO: 4.

[0097] In one embodiment, IL21 is wild-type IL21 or a variant thereof. In a specific embodiment, IL21 comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0098] antibody part The antibody moiety may be generated by modifying an anti-PD-L1 antibody described above with an acceptor substrate recognition sequence or a donor substrate recognition sequence as described herein. The modification may occur at any position of the anti-PD-L1 antibody. In some embodiments, the modification occurs at the N-terminus and / or C-terminus of the anti-PD-L1 antibody, e.g., the N-terminus of the antibody light chain and / or the N-terminus of the antibody heavy chain. In some embodiments, the modification occurs at a non-terminal position of the anti-PD-L1 antibody, e.g., the side chains of the antibody light chain and / or heavy chain.

[0099] In some embodiments, m is an integer selected from 1, 2, 3, and 4. In particular embodiments, m is 1. In another particular embodiment, m is 2.

[0100] In one embodiment, (G) n is introduced at the N-terminus of the light chain of the anti-PD-L1 antibody. n In yet another embodiment, (G) n is introduced into the N-terminus of the light and heavy chains of an anti-PD-L1 antibody. (G) nand the anti-PD-L1 antibody is as defined above.

[0101] In one embodiment, X1X2X3TX4X5 is introduced at the C-terminus of the light chain of the anti-PD-L1 antibody. In another embodiment, X1X2X3TX4X5 is introduced at the C-terminus of the heavy chain of the anti-PD-L1 antibody. In yet another embodiment, X1X2X3TX4X5 is introduced at the C-terminus of both the light chain and the heavy chain of the anti-PD-L1 antibody. X1X2X3TX4X5 and the anti-PD-L1 antibody are as defined above.

[0102] In some embodiments, the antibody moiety comprises a light chain, where the light chain is: 1) a light chain of an anti-PD-L1 antibody (designated LC); 2) a light chain of a (G) n or 3) an N-terminally modified light chain (referred to as LCNT), comprising an anti-PD-L1 antibody light chain having at its N-terminus n -N-terminally modified light chains (LCNTs) containing anti-PD-L1 antibody light chains with Sp2 introduced at the N-terminus L In some other embodiments, the antibody moiety comprises a heavy chain, where the heavy chain can be: 1) a heavy chain of an anti-PD-L1 antibody (denoted as HC); 2) (G) n or 3) an N-terminally modified heavy chain (referred to as HCNT), comprising an anti-PD-L1 antibody heavy chain having at its N-terminus: n -N-terminally modified heavy chains (HCNTs) containing anti-PD-L1 antibody heavy chains with Sp2 introduced at the N-terminus L By combining the light chain and the heavy chain of the antibody moiety, eight types of antibody moieties can be obtained, and m in formulas (I) and (I′) can be 1, 2, 3, and 4.

[0103] In a specific embodiment, the antibody portion is Ab0030 and is derived from the antibody Ab0001. Ab0030 comprises a light chain of SEQ ID NO:1 and a heavy chain of SEQ ID NO:14, where the heavy chain comprises, from N-terminus to C-terminus, the acceptor substrate recognition sequence GGG, the spacer GSGGGGS, and the heavy chain of Ab0001 (SEQ ID NO:2).

[0104] In another specific embodiment, the antibody portion is Ab0033 and is derived from antibody Ab0002. Ab0033 comprises a light chain of SEQ ID NO:3 and a heavy chain of SEQ ID NO:15, where the heavy chain comprises, from N-terminus to C-terminus, the acceptor substrate recognition sequence GGG, the spacer GSGGGGS, and the heavy chain of Ab0002 (SEQ ID NO:4).

[0105] Cytokine part The cytokine portion may be generated by modifying IL21, as described above, with a donor substrate recognition sequence or an acceptor substrate recognition sequence, respectively.

[0106] In some embodiments, the antibody portion comprises an anti-PD-L1 antibody and an acceptor substrate recognition sequence, such as (G): n and the cytokine portion comprises IL21 and a donor substrate recognition sequence, e.g., X1X2X3TX4X5. In some embodiments, the donor substrate recognition sequence is located at the C-terminus of the cytokine portion. In some embodiments, the donor substrate recognition sequence is located at the N-terminus of the cytokine portion. In some embodiments, the donor substrate recognition sequence is located at a non-terminal position of the cytokine portion.

[0107] In some embodiments, the antibody portion comprises an anti-PD-L1 antibody and a donor substrate recognition sequence, such as X1X2X3TX4X5, and the cytokine portion comprises IL21 and an acceptor substrate recognition sequence, such as (G) n In some embodiments, the acceptor substrate recognition sequence is located at the N-terminus of the cytokine moiety. In some embodiments, the acceptor substrate recognition sequence is located at the C-terminus of the cytokine moiety. In some embodiments, the acceptor substrate recognition sequence is located at a non-terminal position of the cytokine moiety.

[0108] The antibody and cytokine moieties can be obtained by various methods known in the art, including but not limited to chemical synthesis, recombinant DNA technology, and combinations thereof. In a preferred embodiment, polynucleotides encoding the light and heavy chains of the antibody moiety are produced by recombinant DNA technology and introduced into a suitable host cell, after which the antibody moiety can be expressed in the host cell and purified from the host cell or culture medium. The host cell may be stably or transiently transformed. Isolation and purification of the antibody and cytokine moieties can be performed using techniques well known in the art. For example, the antibody moiety can be purified on a protein A affinity column.

[0109] Exemplary Fusion Proteins In one embodiment, the cytokine portion is obtained by modifying IL21 (SEQ ID NO:9 or SEQ ID NO:10) with the spacer GA and the donor substrate recognition sequence LPETGG (SEQ ID NO:6) at the C-terminus, and the cytokine portion comprises the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12.

[0110] In one embodiment, the antibody portion is Ab0030 and comprises a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 14. Ab0030 is derived from the anti-PD-L1 antibody Ab0001 (atezolizumab) by modification of the heavy chain of Ab0001 (SEQ ID NO: 2) at the N-terminus with the acceptor substrate recognition sequence GGG and the spacer GSGGGGS (SEQ ID NO: 5). In another specific embodiment, the antibody portion is Ab0033 and comprises a light chain of SEQ ID NO: 3 and a heavy chain of SEQ ID NO: 15. Ab0033 is derived from the antibody Ab0002 (3F2) by modification of the heavy chain of Ab0002 (SEQ ID NO: 4) at the N-terminus with the acceptor substrate recognition sequence GGG and the spacer GSGGGGS (SEQ ID NO: 5).

[0111] According to some embodiments of the disclosure, the sequences of the cytokine IL21 and anti-PD-L1 antibodies, and the sequences of the cytokine and antibody moieties derived therefrom, are set forth in the table below.

[0112] [Table 1]

[0113] Exemplary fusion proteins according to the present disclosure are set forth in Table 2.

[0114] Pharmaceutical Compositions Also provided is a pharmaceutical composition comprising a fusion protein according to the present disclosure and a pharma- ceutically acceptable carrier.

[0115] Examples of physiologically acceptable carriers include diluents (e.g., lactose, microcrystalline cellulose, and dextrose), binders and adhesives (e.g., acacia, gelatin, starch paste, carboxymethylcellulose), lubricants (e.g., polyethylene glycol, calcium stearate, and stearic acid), disintegrants (e.g., starch, cellulose, and crosslinking polymers), preservatives, vehicles, glidants (e.g., corn starch), sweeteners (e.g., mannitol and saccharin), coating materials (e.g., povidone, ethylcellulose, and synthetic polymers), and plasticizers (e.g., castor oil, diacetylated monoglycerides, and polyethylene glycol), structure-forming excipients (e.g., cetostearyl alcohol and mineral oil), preservatives (e.g., benzyl alcohol, propylparaben, and sodium benzoate), antioxidants (e.g., butyl hydroxypropyl cellulose, butyl hydroxypropyl cellulose, and butyl hydroxypropyl cellulose). Examples of suitable preservatives include, but are not limited to, antioxidants (e.g., hydroxyltoluene and butyl hydroxyl anisole), solubilizing agents (e.g., lanolin and cholesterol), gelling agents (e.g., carboxymethylcellulose, hydroxypropylcellulose and xanthan gum), and emollients (e.g., glycerin, mineral oil, petrolatum and isopropyl palmitate), solvents (e.g., water, alcohol, acetic acid and syrup), buffers (e.g., phosphate buffers and acetate buffers), antimicrobial preservatives (e.g., benzyl alcohol, butylparaben, phenol and thiomersal), antioxidants (e.g., ascorbic acid, sodium hydrogen sulfate, thiourea and butyl hydroxytoluene), chelating agents (e.g., disodium EDTA, dihydroxyethylglycine and citric acid), and emulsifiers (e.g., sodium lauryl sulfate, cetrimide and clogol esters).

[0116] The pharmaceutical compositions provided herein may be in a variety of dosage forms, such as solid, semi-solid, liquid, powder, aqueous, or lyophilized form.

[0117] The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, for example, by systemic or local administration. Routes of administration include, but are not limited to, parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intracavitary), topical, epidural, or transmucosal (e.g., intranasal or oral) routes. The exact dose administered will vary depending on a variety of factors, such as the therapeutic objectives, the route of administration, and the subject's condition, such as the patient's health condition, weight, sex, diet, etc. Thus, in order to obtain the optimal therapeutic effect, the therapist must determine the dose of the pharmaceutical composition and modify the route of administration as necessary. In general, the dosage range of the pharmaceutical compositions provided herein for administration is an amount large enough to produce the desired effect of eliminating PD-L1 positive disease cells.

[0118] In a preferred embodiment, the pharmaceutical composition of the present disclosure can be administered to a human subject in a therapeutically effective amount. The therapeutic dose of the antibody is, for example, preferably 0.1-25 mg / kg body weight per therapeutic dose, and most preferably 0.1-10 mg / kg body weight per therapeutic dose. In certain embodiments, any fusion protein of the present disclosure can be formulated according to conventional practices for administration by any suitable route, and generally may be in liquid form (e.g., a solution of the fusion protein in a physiologically acceptable sterile buffer) for administration by, for example, intravenous, intraperitoneal, subcutaneous, or intramuscular routes.

[0119] treatment Further provided is a method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a fusion protein or pharmaceutical composition according to the present disclosure.

[0120] The present disclosure also relates to a fusion protein or a pharmaceutical composition according to the present disclosure for use in treating a disease in a subject in need thereof.

[0121] The present disclosure also relates to the use of a fusion protein or a pharmaceutical composition according to the present disclosure for the manufacture of a medicament for treating a disease in a subject in need thereof.

[0122] In some embodiments, the subject is a mammal, such as a mouse, rat, rabbit, goat, sheep, pig, chimpanzee, ape, cow, and human, hi some preferred embodiments, the subject is a human.

[0123] In some embodiments, the disease is cancer. Preferably, the cancer is a PD-L1 positive cancer. In some embodiments, the cancer is selected from melanoma, ovarian cancer, breast cancer, Merkel cell carcinoma, lung cancer (including small cell lung cancer and non-small cell lung cancer, such as squamous cell carcinoma and non-squamous non-small cell lung cancer), renal cell carcinoma, bladder cancer, colon cancer, colon adenocarcinoma, colorectal cancer, squamous cell carcinoma of the head and neck, mesothelioma, virus-induced cancer, cervical cancer, nasopharyngeal carcinoma, soft tissue sarcoma, hematological malignancies, gastric cancer, gastric or gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, squamous cell carcinoma of the esophagus, endometrial cancer, primary mediastinal B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors, hepatocellular carcinoma, lymphoma, Hodgkin's disease (Hodgkin's lymphoma), non-Hodgkin's disease (non-Hodgkin's lymphoma), and diffuse large B-cell lymphoma. In some embodiments, the cancer is a malignant cancer selected from colorectal cancer, melanoma, renal cell carcinoma (RCC), squamous cell carcinoma (cSCC), triple negative breast cancer (TNBC), non-small cell lung cancer, and bladder cancer. In some preferred embodiments, the cancer is selected from gastric cancer, breast cancer, colon cancer, colon adenocarcinoma, urothelial carcinoma, lung cancer, liver cancer, endometrial cancer, head and neck cancer, and ovarian cancer.

[0124] Although the present disclosure has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the application and principles of the present disclosure. Modifications are possible without departing from the spirit and scope of the present disclosure.

[0125] Beneficial effects The fusion protein according to the present disclosure comprises: (1) Specific inhibition of PD-L1 signaling and activation of IL21 signaling, (2) CD4 + Effective activation of T cells and (3) Achieving at least one of the following technical effects: higher antitumor activity and lower toxicity.

[0126] Furthermore, conventional recombinant DNA technology is often used to generate antibody-cytokine fusion proteins (immunocytokines). However, the yield of recombinant immunocytokines prepared using conventional methods can be very low compared to naked antibodies (not conjugated to cytokines), hindering the clinical application of many immunocytokines. Using the enzyme-mediated conjugation method of the present disclosure, fusion proteins can be prepared with very high yields and purity.

[0127] Working Example Unless otherwise stated, the preparation of modified or unmodified anti-PD-L1 antibodies and IL21 utilizes commonly used techniques in molecular biology, biochemistry, and cell biology. Unless otherwise stated, the instruments and reagents used in the examples are commercially available. The reagents can be used directly without further purification.

[0128] Flow Cytometry: CytoFLEX S HIC-HPLC: Butyl HIC. Mobile phase A: 25 mM PB, 2 M (NH4)2SO4, pH 7.0. Mobile phase B: 25 mM PB, pH 7.0. Flow rate: 0.8 mL / min. Acquisition time: 25 min. Injection volume: 20 μg. Column temperature: 25 °C. Detection wavelength: 280 nm. Sample injector temperature: 8 °C. SEC-HPLC: Column: TSK-gel G3000 SWXL, TOSOH 7.8 mm ID x 300 mm, 5 μm. Mobile phase: 0.2 M KH2PO4, 0.25 M KCl, pH 6.2. Flow rate: 0.5 mL / min. Acquisition time: 30 min. Injection volume: 50 μL. Column temperature: 25 °C. Detection wavelength: 280 nm. Sample tray temperature: 8 °C. CHO was obtained from Thermo Fisher Scientific, pcDNA3.3 was obtained from Life Technology, and MabSelect Sure ProA was obtained from GE. The optimized recombinant enzyme sortase A from Staphylococcus aureus was prepared in Escherichia coli.

[0129] Example 1 Construction and expression of antibody moieties 1.1 Construction of expression plasmids for Ab0030 and Ab0033 Two antibody moieties, Ab0030 and Ab0033, were prepared based on the anti-PD-L1 antibodies Ab0001 (atezolizumab) and Ab0002 (3F2), respectively. The amino acid sequences (SEQ ID NOs) of Ab0001 and Ab0002 are shown in Table 1A.

[0130] [Table 1A]

[0131] Specifically, the N-terminus of the heavy chain of Ab0001 (SEQ ID NO:2) and the heavy chain of Ab0002 (SEQ ID NO:4) were modified by the addition of the peptide sequence GGGGSGGGGS (SEQ ID NO:5, i.e., the acceptor substrate recognition sequence with a spacer).

[0132] To promote expression and extracellular secretion of the antibody moiety, coding sequences for a light chain signal peptide (SEQ ID NO: 16) and a heavy chain signal peptide (SEQ ID NO: 17) were added to the N-terminus of the coding sequences for the light and heavy chains, respectively.

[0133] The coding sequences of Ab0030 and Ab0033 were codon-optimized for efficient expression in CHO cells and cloned into pcDNA3.3 vector to obtain plasmid pairs for each antibody moiety.

[0134] 1.2 Expression of Ab0030 and Ab0033 The expression plasmids of Ab0030 and Ab0033 obtained in Example 1.1 were transiently transfected into CHO cells at a mass ratio of 2:1. After fermentation, the cell culture supernatant containing the antibodies was collected. The expression levels of Ab0030 and Ab0033 measured by ELISA assay were 757.60mg / L and 622.96mg / L, respectively.

[0135] 1.3 Purification of Ab0030 and Ab0033 A Protein A column (MabSelect Sure ProA, 20 cm x 5.0 cm) was washed with equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). The cell culture supernatant from Example 1.2 was filtered through a 0.22 μm filter and loaded onto the column. Once sample loading was complete, the column was washed with 5-10 column volumes of equilibration buffer. The antibody was eluted in 50 mM citrate buffer, pH 3.5, and collected. The pH of the antibody solution was adjusted to 5.5-6.5 by adding 6% (v / v) 1 M Tris, pH 10.5. Finally, the antibody solution buffer was exchanged into equilibration buffer by ultrafiltration. The concentrations of Ab0030 and Ab0033 were measured using a UV spectrometer. Ab0030 and Ab0033 were aliquoted and stored at -80°C. The purified antibody portion Ab0030 comprises a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 14. The purified antibody portion Ab0033 comprises a light chain of SEQ ID NO: 3 and a heavy chain of SEQ ID NO: 15 (see Table 1B).

[0136] [Table 1B]

[0137] 1.4 Characterization of Ab0030 and Ab0033 Ab0030 and Ab0033 were analyzed by SDS-PAGE electrophoresis. As expected, the molecular weights of the heavy and light chains measured by SDS-PAGE are about 50 kDa and about 25 kDa, respectively. SEC-HPLC analysis demonstrates that the percentages of Ab0030 and Ab0033 monomers are about 97.5% and 96.2%, respectively, meeting the preparation requirements of fusion proteins.

[0138] The PD-L1 binding activity of Ab0030 and Ab0033 was measured by enzyme-linked immunosorbent assay (ELISA). Recombinant hPD-L1 was diluted to 0.5μg / mL in coating buffer (1x PBS) and coated onto ELISA plates overnight at 4℃. The plates were blocked with 5% nonfat dry milk in coating buffer at 37℃ for 1h. Serially diluted Ab0030 and Ab0033 were added to the plates, and then the plates were incubated at 37℃ for 1h. Finally, anti-human-IgG(H+L)-HRP diluted 1:5000 was added. After incubation at 37℃ for 1h, TMB substrate was added for color development. Data were collected using a microplate reader.

[0139] The results are shown in Figure 2. Ab0030 (Figure 2A) and Ab0033 (Figure 2B) can specifically bind to the antigen hPD-L1.

[0140] Example 2 Construction and expression of the cytokine moiety Using overlap PCR, a polynucleotide encoding the peptide sequence GALPETGG (SEQ ID NO:6, including the sortase donor substrate recognition sequence A (LPETGG) and the spacer GA) was added 3' to a polynucleotide encoding hIL21 (whose amino acid sequence is shown in SEQ ID NO:9 or SEQ ID NO:10) to generate a polynucleotide encoding the cytokine portion (whose amino acid sequence is shown in SEQ ID NO:11 or SEQ ID NO:12, respectively). To promote expression and extracellular secretion of the cytokine portion, a coding sequence for a signal peptide (SEQ ID NO:13) was added 5' to the polynucleotide encoding SEQ ID NO:11 or SEQ ID NO:12. The purified cytokine portion has the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12.

[0141] hIL21 was expressed in E. coli BL21(DE3). After expression, recombinant hIL21 was purified on a Ni-NTA column. Purified hIL21 was stored in 1x PBS buffer.

[0142] Example 3 Preparation of fusion proteins The antibody moieties Ab0030 and Ab0033 prepared in Example 1 were conjugated to the cytokine moiety prepared in Example 2 by sortase-mediated site-specific conjugation to obtain a fusion protein.

[0143] Step 1) Pretreatment Ab0030 and Ab0033 and the cytokine moiety were pretreated using ultrafiltration membranes with suitable pore sizes, then concentrated and buffer exchanged into the conjugation buffer.

[0144] Step 2) Enzyme-mediated conjugation Ab0030 and Ab0033 (1-100 mg / mL) and the cytokine moiety (SEQ ID NO:11, 1-100 mg / mL) were prepared separately in reaction buffer. A conjugation column containing immobilized recombinant SrtA from Staphylococcus aureus was pretreated with NaOH and high salt and prewarmed to 10-40°C in an air or water bath for 30 min or more. The reaction solution containing the antibody moiety and the cytokine moiety was mixed in a certain ratio and slowly passed through the conjugation column to carry out the conjugation reaction. Alternatively, the conjugation reaction may be carried out by mixing the reaction solution with immobilized recombinant SrtA or purified recombinant SrtA for 5 min-24 h.

[0145] The antibody-cytokine conjugation mixture containing the fusion protein was collected for further purification and DAR (DAR=average number of hIL21 conjugated to anti-PD-L1 antibody) measurement.

[0146] Step 3) Purification of the fusion protein The fusion protein was purified by cation exchange chromatography. Briefly, a chromatography column was filled with Capto S ImpAct medium and equilibrated with citric acid-sodium citrate buffer at pH 5.0-6.0. The conjugation mixture from step 2) was loaded onto the column. The column was washed with citric acid-sodium citrate buffer at pH 5.0-6.0 until the desired offset (baseline) was reached. The unconjugated antibody and cytokine moieties were eluted with citric acid-sodium citrate buffer at pH 5.0-6.0, 100-500 mM NaCl. The fusion protein was eluted by increasing the NaCl concentration in the citric acid-sodium citrate buffer (pH 5.0-6.0) from 0.5 M to 1 M. Unexpectedly, at this step, two fractions representing two different fusion protein populations were eluted. The two fractions were then subjected to SDS-PAGE analysis. It was confirmed that the first fraction represents a major population with a DAR value of 1 (called the DAR1 population, each fusion protein contains one hIL21), and the second fraction represents a minor population with a DAR value of 2 (called the DAR2 population, each fusion protein contains two hIL21). The fusion proteins containing only one hIL21 (isoform 1) are designated LC0030-1 (prepared from Ab0030) and LC0033-1 (prepared from Ab0033), respectively. The fusion proteins containing two hIL21 (isoform 1) are designated LC0030-2, prepared from Ab0030, and LC0033-2, prepared from Ab0033, respectively. Figure 3 shows the SDS-PAGE analysis of the two fractions containing LC0030-1 and LC0030-2, respectively. Figure 4 shows the SDS-PAGE analysis of the fraction containing LC0033-1.

[0147] The column was regenerated using citric acid-sodium citrate buffer, pH 5.0-6.0, 1 M NaCl, and clean-in-place (CIP) was performed using 1 M NaOH.

[0148] Step 4) Ultrafiltration of the fusion protein The purified fusion protein was concentrated using an ultrafiltration membrane with a suitable pore size, buffer exchanged into a storage buffer, and then stored at 2-8°C or -80°C.

[0149] Fusion proteins containing hIL21 (isoform 2) (the cytokine portion has the amino acid sequence of SEQ ID NO: 12) were prepared in the same manner as above. The fusion proteins containing only one hIL21 (isoform 2) are named LC0030'-1 (prepared from Ab0030) and LC0033'-1 (prepared from Ab0033), respectively. The fusion proteins containing two hIL21 (isoform 2) are named LC0030'-2 (prepared from Ab0030) and LC0033'-2 (prepared from Ab0033), respectively.

[0150] The fusion proteins prepared are listed in Table 2.

[0151] [Table 2]

[0152] Example 4 HIC-HPLC analysis of fusion proteins The fusion protein prepared in Example 3 was subjected to butyl HIC HPLC analysis to measure the DAR distribution.

[0153] The results are shown in Figures 5 and 6. The peak position of LC0030-1 is shifted forward compared to Ab0030, and the peak position of LC0030-2 is shifted forward compared to LC0030-1, indicating that the hydrophobicity of the fusion proteins LC0030-1 and LC0030-2 is lower than that of Ab0030 (Figure 5). Interestingly, the peak position of LC0033-1 is shifted backward compared to Ab0033, and the peak position of LC0033-2 is shifted backward compared to LC0033-1, indicating that the hydrophobicity of LC0033-1 and LC0033-2 is stronger than that of Ab0033 (Figure 6). These differences may be due to the amino acid and structural differences between Ab0030 and Ab0033.

[0154] Example 5 Measurement of the binding activity of fusion proteins to hPD-L1 The binding activity of the fusion proteins to human PD-L1 (hPD-L1) was measured by ELISA. Recombinant hPD-L1 protein (Acrobiosystems, PD1-H5253) was diluted to 0.5μg / mL in coating buffer (1x PBS) and coated onto ELISA plates overnight at 4℃. The plates were blocked with 5% nonfat dry milk in coating buffer at 37℃ for 1h. Serial dilutions of Ab0030, Ab0033, LC0030-1 and LC0033-1 were added, and then the plates were incubated at 37℃ for 1h. Finally, anti-human-IgG(H+L)-HRP diluted 1:5000 was added. After incubation at 37℃ for 1h, TMB substrate was added for color development. Data were collected using a microplate reader.

[0155] The results are shown in Figure 7. Both LC0030-1 and LC0033-1 could specifically bind to the antigen PD-L1 with similar affinity as Ab0030 and Ab0033, indicating that the PD-L1-binding activity of the anti-PD-L1 antibodies in the fusion proteins is retained.

[0156] Example 6 Measurement of binding activity of fusion protein to IL21 receptor (IL21R) The binding activity of the fusion proteins to human IL21 receptor (hIL21R or Hu-IL21R) was measured by ELISA. Recombinant hIL21R protein was diluted to 0.5 μg / mL in coating buffer (1x PBS) and coated on ELISA plates overnight at 4°C. The plates were blocked with 5% nonfat dry milk in coating buffer at 37°C for 1 h. Serially diluted Ab0030, Ab0033, LC0030-1 and LC0033-1 were added to the plates, and then the plates were incubated at 37°C for 1 h. Finally, anti-human-IgG(H+L)-HRP diluted 1:5000 was added. After incubation at 37°C for 1 h, TMB substrate was added for color development. Data were collected using a microplate reader.

[0157] The results are shown in Figure 8. LC0030-1 and LC0033-1 can specifically bind to hIL21R, while Ab0030 and Ab0033 do not bind to hIL21R. Because anti-human-IgG(H+L)-HRP binds to the human Fc portion of the antibody, the results suggest that the conjugation of anti-PD-L1 antibody to hIL21 was successful and the fusion protein specifically binds to hIL21R.

[0158] Example 7 Measurement of the binding affinity of fusion proteins to hPD-L1 and hIL21R Surface plasmon resonance (SPR) was performed using Biacore (Biacore, INC, Piscataway NJ) to measure the binding affinity of LC0033-1 and Ab0033 to hPD-L1 or hIL21R. A Biacore T200 biosensor was equipped with a sensor chip Protein A. The protein to be tested (diluted to 1 μg / mL) was injected over the Biacore T200 biosensor at 10 μL / min for 50 sec to reach the desired protein density (approximately 300 RU). Subsequently, His-tagged human IL21R (hIL21R-His, 200 nM) or human PD-L1 (hPD-L1-His, 100 nM) was injected into the system at 30 μL / min for 180 sec. Dissociation was monitored for 600 sec. After each cycle, the chip was regenerated using glycine buffer pH 1.7 (30 μL / min, 30 sec).

[0159] Data were processed with Biacore T200 Evaluation Software 3.0. Binding kinetics were analyzed by globally fitting the data to a 1:1 Langmuir binding model to determine association rates (k on ) and dissociation rate (k off ) is obtained. D ) to the formula: K D =k off / k on The calculation was made as follows.

[0160] The results are shown in Table 3. LC0033-1 and Ab0033 can bind to hPD-L1-His with similar affinity. LC0033-1 can bind to IL21R with high affinity.

[0161] [Table 3]

[0162] Example 8 Fusion Protein CD4 + Measurement of the ability to activate T cells The in vitro PBMC cell stimulatory activity of LC0033-1 was measured using a STAT3 phosphorylation (pSTAT3) assay. STAT3 phosphorylation was measured by CD4 + It is an indicator of T cell stimulation. Commercially available PBMC cells (AllCells, Cat. No. FPB003F-C) were thawed and allowed to recover at 37°C, then suspended in pre-warmed culture drum (RPMI1640) and centrifuged at 300g for 3 min. The suspension and centrifugation process was repeated three times. The cells were then plated at 10 5Cells / well were added. Serially diluted Ab0033, LC0033-1 or hIL21 were then added to each well at 100 μL (an equal volume of PBS was added to control wells as a blank control) and incubated at 37°C for 30 min. An equal volume of fixative was added to the cells to a working concentration of 4% formaldehyde in PBS and the cells were fixed at room temperature for 15 min. Cells were centrifuged at 300g for 3 min, the supernatant was removed and washed with 200 μL PBS. For intracellular staining, cells were resuspended in 100% ice-cold methanol and incubated on ice for 30 min. After centrifugation at 300g for 3 min, cells were washed three times with PBS+0.5% BSA. For staining, 5 μL of directly labeled antibodies (Phospho-STAT3 (Tyr705) monoclonal antibody (LUVNKLA), APC, eBioscience™, Cat. No. 17-9033-42 and CD4 monoclonal antibody (RPA-T4), FITC, eBioscience™, Cat. No. 11-0049-42) were added to each well. After 30 min incubation at 4° C., cells were analyzed using a Sartorius-IntelliCyt® iQue flow cytometer.

[0163] The results are shown in Figure 9. Compared to the blank control (open histograms), the peaks of LC0033-1 and hIL21 (filled histograms) were clearly shifted to the right, which indicates that the two molecules inhibit CD4 + It is effective in inducing phosphorylation in T cells, and therefore CD4 + Shown to be effective in stimulating T cells.

[0164] Example 9. Evaluation of in vivo efficacy of fusion proteins To evaluate the in vivo efficacy of the fusion proteins of the present disclosure and predict therapeutic efficacy in humans, a mouse model of colon adenocarcinoma was established using humanized PD-1 / PD-L1 mice (PD-1 / PD-L1 gene humanized mice, see WO2018041118A1 and WO2018001241A1, the relevant contents of which are incorporated herein by reference in their entirety).

[0165] Two in vivo efficacy studies were performed using the homozygous humanized PD-1 / PD-L1 mice (6-7 weeks old) (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Cat. No. 120522) described above. Briefly, mice were inoculated with mouse colon adenocarcinoma cells MC38-hPD-L1 (5 × 10 5 The tumors were subcutaneously inoculated with 100-150 mm2 of Biocytogen Pharmaceuticals (Beijing) Co., Ltd. (100 μL PBS). 3 At tumor volume 1, mice were randomly assigned to treatment or control groups based on their tumor volume. Treatment mice were randomized to receive Ab0033, hIL21, LC0033-1, LC0033-2, or a combination of hIL21 and Ab0033 (equimolar). Control mice were injected with an equal volume of saline. Mice were dosed twice weekly (BIW) for a total of six doses (intravenous (iv), first efficacy study; or intraperitoneal (ip), second efficacy study). Tumor volumes and body weights of mice were measured twice weekly. Tumor volumes of one mouse were 3000 mm 3 When the dose reached 0.01 mg / kg, the mice were euthanized and the experiment was terminated. The dosing regimens for the two studies are summarized in Tables 4 and 6, respectively.

[0166] 9.1 First Efficacy Study The dosing regimen for the first efficacy study is shown in Table 4.

[0167] [Table 4]

[0168] The tumor volumes on the day of grouping (day 0) and 14 days (day 14) or 26 days (day 26) after grouping are summarized in Table 5. Tumor growth inhibition values ​​(TGI TV %) to the formula: TGI TV (%)=[1-(T i -T0) / (V i -V0)] × 100. T i is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume of the treatment group on day 0. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day 0.

[0169] [Table 5]

[0170] Overall, the mice in each group were in good health during the experiment. The treatment and control groups showed no significant differences in body weight and body weight change (Figure 10). At the end of the experiment, the mice in each group gained weight and there was no significant difference in body weight between the treatment and control groups, indicating that the drugs were well tolerated. The results indicate that LC0033-1, LC0033-2 and Ab0033 are safe and have no significant side effects in the animals.

[0171] As can be seen from Figure 11, tumors in control mice continued to grow during the experiment, while tumor growth in mice from treatment groups was inhibited to different degrees. In particular, tumor growth in LC0033-1 and LC0033-2 groups (G6, G8 and G10) was significantly inhibited compared to other treatment groups (G2, G3, G4, G5, G7 and G9), indicating that LC0033-1 and LC0033-2 showed superior in vivo tumor inhibitory activity compared to Ab0033 or hIL21 monotherapy or their combination therapy.

[0172] As can be seen from Table 5 and Figure 11, at the end of the experiment, the tumor volumes of all mice treated with LC0033-1 (G6 and G8) or LC0033-2 (G10) were significantly smaller than those of the control mice (G1), indicating that LC0033-1 and LC0033-2 have tumor-inhibiting effects at low doses. Furthermore, LC0033-1 and LC0033-2 showed similar efficacy at the same dose (TGI TV %: 60.5% vs. 56.2%). When the dose of LC0033-1 was increased from 1 mg / kg to 3 mg / kg, the TGI TVThe TGI from groups treated with Ab0033 alone, hIL21 alone, or the combination of Ab0033 and hIL21 increased from 60.5% to 69.9%, indicating a slight increase in the tumor inhibition effect. TV % was less than 25% (except for G7), indicating that Ab0033, hIL21 and their combination did not exhibit significant antitumor activity.

[0173] 9.2 Secondary Efficacy Study The dosing regimen for the second efficacy study is shown in Table 6.

[0174] [Table 6]

[0175] The tumor volumes on the day of grouping (day 0) and 14 days (day 14) or 24 days (day 24) after grouping are summarized in Table 7, and the tumor growth inhibition values ​​(TGI TV %) was calculated.

[0176] [Table 7]

[0177] Overall, the mice in each group were in good health during the experiment. The treatment and control groups showed no significant differences in body weight and body weight change (Figure 12). At the end of the experiment, the mice in each group gained weight and there was no significant difference in body weight between the treatment and control groups, indicating that the drugs were well tolerated. The results indicate that LC0033-1 and Ab0033 are safe and have no significant side effects in the animals.

[0178] As can be seen from Table 7 and Figure 13, tumors in the control mice continued to grow during the experiment, while tumor growth in mice from the treatment groups was inhibited to different degrees. At the end of the experiment (day 24), the tumor volumes of mice from G2 to G7 were smaller than those of the control mice (G1), indicating that LC0033-1 and Ab0033 have different tumor inhibitory effects at different doses. LC0033-1 treatment also showed a dose-dependent tumor inhibition, with significant tumor inhibitory effects at low doses (G5, TGI TV %=80.3%). High doses of Ab0033 (G4, TGI TV %=84.1%) showed antitumor activity equivalent to that of medium-dose LC0033-1 (G6, TGI TV %=85.3%), but at low doses (G2, TGI TV %=40.0%) and medium dose (G3, TGI TV %=22.9%) failed to inhibit tumor growth.

[0179] At the same dose and dosing frequency, LC0033-1 (G5-G7) showed higher efficacy than Ab0033 (G2-G4), indicating that the in vivo tumor inhibitory effect of anti-PD-L1 antibody was effectively improved by fusing with IL21.

[0180] As can be seen from FIG. 13, LC0033-1 (G5) at 3 mg / kg effectively inhibited tumor growth compared to equimolar Ab0033 (G2).

[0181] Data from one mouse within each treatment group was also studied. The 3 mg / kg LC0033-1 group (G5) slowed tumor growth and inhibited tumor growth in all mice. Only one mouse in the 2.73 mg / kg Ab0033 group (G2) had significant tumor inhibition.

[0182] For LC0033-1, the antitumor activity in the medium dose group (G6) was higher than that in the low dose group (G5), and Ab0033 at the corresponding dose (G3 vs. G2) did not show significant antitumor activity. High dose Ab0033 (G4) effectively inhibited tumor growth, and the antitumor activity was comparable to that of low dose LC0033-1 (G5). The antitumor activity of LC0033-1 was further increased at high dose (G7), but the tumor inhibition was not significantly increased compared to the low dose group (G5), i.e., no significant dose-dependent effect was observed, which may be because LC0033-1 at 3 mg / kg already showed significant antitumor activity, so for the MC38 tumor model, further increase in LC0033-1 dose did not completely eliminate the tumor.

[0183] Sequence Listing [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14]

Claims

1. A fusion protein having a structure of formula (I) or (I'): [B-Sp １ -Lk-Sp ２ ] ｍ -A (I) A-[Sp １ -Lk-Sp ２ -B] ｍ (I’) During the ceremony, A is an anti-PD-L1 antibody, B is interleukin 21 (IL21), Sp １ and Sp ２ are each independently a spacer or absent, Lk is a linker formed by conjugation of the acceptor substrate recognition sequence and the donor substrate recognition sequence of the sortase; The fusion protein, wherein m is an integer of 1 to 4.

2. The fusion protein of claim 1, wherein the acceptor substrate recognition sequence comprises 1 to 100 tandemly connected structural units selected from the group consisting of glycine (G) and alanine (A).

3. The acceptor substrate recognition sequence is (G): ｎ 3. The fusion protein according to claim 1 or 2, comprising the sequence:

4. The donor substrate recognition sequence is X １ X ２ X ３ T.X. ４ X ５ where X １ is leucine (L) or asparagine (N), and X ２ is proline (P) or alanine (A), and X ３ is any natural or unnatural amino acid, and X ４ is glycine (G), serine (S), asparagine (N), or absent; X ５ The fusion protein according to any one of claims 1 to 3, wherein is an amino acid sequence comprising 1 to 10 amino acids or is absent.

5. X １ X ２ X ３ T.X. ４ X ５ is LPX ３ T.G., L.P.X. ３ TGG, NPQTN, NPKTG, LAX ３ TG or LPQTSEQ, where X ３ is any natural or unnatural amino acid.

6. X １ X ２ X ３ T.X. ４ X ５ The fusion protein of claim 4, wherein is LPETG or LPETGG.

7. the spacer comprises 1 to 100 tandemly connected structural units selected from the group consisting of glycine (G), alanine (A) and serine (S); Preferably, the spacer is selected from the group consisting of GA, GGG, GAG and (G) ｏ S ((G) ｓ S) ｔ 6. The fusion protein according to any one of claims 1 to 5, selected from the group consisting of:

8. Lk is X １ X ２ X ３ T(G) ｎ and the fusion protein has the structure (II) or (II'): [B-Sp １ -X １ X ２ X ３ T(G) ｎ -Sp ２ ] ｍ -A (II) A-[Sp １ -X １ X ２ X ３ T(G) ｎ -Sp ２ -B] ｍ (II’) In the formula, X １ is leucine (L) or asparagine (N), and X ２ is proline (P) or alanine (A), and X ３ is any natural or unnatural amino acid, and n is an integer from 2 to 20; m is 1 or 2; A, B, Sp １ and Sp ２ The fusion protein of claim 1, wherein:

9. X １ X ２ X ３ T(G) ｎ is LPX ３ T(G) ｎ , N.P.X. ３ T(G) ｎ or LAX ３ T(G) ｎ And X ３ The fusion protein of claim 8, wherein n is any natural or unnatural amino acid and n is an integer from 2 to 20.

10. The fusion protein has the structure (III) or (III'): [B-GALPETGGGGSGGGGS] ｍ -A (III) A-[GALPETGGGGSGGGGS-B] ｍ (III’) 10. The fusion protein of claim 9, wherein A and B are as defined in claim 1.

11. the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, 3F2, or an engineered antibody thereof; Preferably, the anti-PD-L1 antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), among which: (1) The VL comprises a CDRL1 sequence shown in SEQ ID NO: 22, a CDRL2 sequence shown in SEQ ID NO: 23, and a CDRL3 sequence shown in SEQ ID NO: 24, and the VH comprises a CDRH1 sequence shown in SEQ ID NO: 25, a CDRH2 sequence shown in SEQ ID NO: 26, and a CDRH3 sequence shown in SEQ ID NO: 27, or (2) The VL comprises a CDRL1 sequence shown in SEQ ID NO: 28, a CDRL2 sequence shown in SEQ ID NO: 29, and a CDRL3 sequence shown in SEQ ID NO: 30, and the VH comprises a CDRH1 sequence shown in SEQ ID NO: 31, a CDRH2 sequence shown in SEQ ID NO: 32, and a CDRH3 sequence shown in SEQ ID NO: 33; More preferably, the VL comprises the amino acid sequence of SEQ ID NO: 34 and the VH comprises the amino acid sequence of SEQ ID NO: 35, or the VL comprises the amino acid sequence of SEQ ID NO: 36 and the VH comprises the amino acid sequence of SEQ ID NO: 37; Most preferably, the anti-PD-L1 antibody comprises a light chain and a heavy chain, among which: (1) the light chain comprises the amino acid sequence of SEQ ID NO:1 and the heavy chain comprises the amino acid sequence of SEQ ID NO:2; or (2) The fusion protein according to any one of claims 1 to 10, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 3 and the heavy chain comprises the amino acid sequence of SEQ ID NO:

4.

12. The IL21 is a wild-type IL21 or a mutant thereof, The fusion protein according to any one of claims 1 to 11, wherein the IL21 comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

10.

13. The fusion protein has the structure of formula (III): [B-GALPETGGGGSGGGGS] ｍ -A (III) During the ceremony, A is an anti-PD-L1 antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 1 and a heavy chain having the amino acid sequence of SEQ ID NO: 2, or a light chain having the amino acid sequence of SEQ ID NO: 3 and a heavy chain having the amino acid sequence of SEQ ID NO: 4; B is IL21 comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; The fusion protein of claim 1 , wherein m is 1 or 2.

14. the IL21 is conjugated at the N-terminus of the light chain and / or the heavy chain of the anti-PD-L1 antibody; The fusion protein of any one of claims 11 to 13, wherein preferably said IL21 is conjugated at the N-terminus of the heavy chain of said anti-PD-L1 antibody.

15. The fusion protein comprises: (a) two identical heavy chain portions and two identical light chain portions, in which: (1) each of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20 and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:1; or (2) each of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:21, and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:3; Alternatively, the fusion protein comprises: (b) two different heavy chain portions and two identical light chain portions, of which: (1) one of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20, the other of the heavy chain portions comprises the amino acid sequence of SEQ ID NO:14, and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:1; or (2) The fusion protein described in claim 1, wherein one of the heavy chain portions comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 21, the other of the heavy chain portions comprises the amino acid sequence of SEQ ID NO: 15, and each of the light chain portions comprises the amino acid sequence of SEQ ID NO:

3.

16. 20. Use of a fusion protein according to any one of claims 1 to 15 in the manufacture of a medicament for treating a disease in a subject in need thereof, comprising Preferably, the disease is cancer, More preferably, the cancer is melanoma, ovarian cancer, breast cancer, Merkel cell carcinoma, lung cancer, renal cell carcinoma, bladder cancer, colon cancer, colon adenocarcinoma, colorectal cancer, head and neck squamous cell carcinoma, mesothelioma, virus-induced cancer, cervical cancer, nasopharyngeal carcinoma, soft tissue sarcoma, hematological malignancies, gastric cancer, gastric or gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, squamous cell carcinoma of the esophagus, endometrial cancer, primary mediastinal B-cell lymphoma (PMB) CL), urothelial carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumor, hepatocellular carcinoma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, renal cell carcinoma (RCC), squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), non-small cell lung cancer, and bladder cancer.

17. A method for producing a fusion protein according to any one of claims 1 to 15, comprising the steps of: (a-1) providing an antibody moiety having a structure of formula (I-1) and a cytokine moiety having a structure of formula (I-2); [Lk ａ -Sp ２ ] ｍ -A (I-1) B-Sp １ -Lk ｄ (I-2) (b-1) conjugating the antibody portion to the cytokine portion in the presence of a sortase to obtain a fusion protein having the structure of formula (I), [B-Sp １ -Lk-Sp ２ ] ｍ -A (I) or (a-2) providing an antibody moiety having the structure of formula (I'-1) and a cytokine moiety having the structure of formula (I'-2); A-[Sp １ -Lk ｄ ] ｍ (I’-1) Lk ａ -Sp ２ -B (I’-2) (b-2) conjugating the antibody portion with the cytokine portion in the presence of a sortase to obtain a fusion protein having the structure of formula (I'), A-[Sp １ -Lk-Sp ２ -B] ｍ (I’) During the ceremony, Lk ａ is the acceptor substrate recognition sequence of the sortase, and Lk ｄ is a donor substrate recognition sequence for the sortase, A, B, Sp １ , Sp ２ 16. The method of claim 1 , wherein Lk and m are as defined in any one of claims 1 to 15.

18. Step (a-1) is (a-1-1) Lk ａ -Sp ２ to the N-terminus of the heavy chain of the anti-PD-L1 antibody to obtain the antibody portion having the structure of formula (I-1); [Lk ａ -Sp ２ ] ｍ -A (I-1) (a-1-2) Sp １ -Lk ｄ to the C-terminus of IL21 to obtain the cytokine moiety having the structure of formula (I-2). B-Sp １ -Lk ｄ (I-2)

19. Step (a-2) is (a-2-1) Sp １ -Lk ｄ to the C-terminus of the heavy chain of the anti-PD-L1 antibody to obtain the antibody moiety having the structure of formula (I'-1); A-[Sp １ -Lk ｄ ] ｍ (I’-1) (a-2-2) Lk ａ -Sp ２ to the N-terminus of IL21 to obtain the cytokine moiety having the structure of formula (I'-2). Lk ａ -Sp ２ -B (I’-2)

20. 20. The method of any one of claims 17 to 19, wherein the acceptor substrate recognition sequence is as defined in claim 2 or 3.

21. 21. The method of any one of claims 17 to 20, wherein the donor substrate recognition sequence is as defined in claims 4 to 6.

22. 22. The method of any one of claims 17 to 21, wherein the spacer is as defined in claim 7.

23. The method according to any one of claims 17 to 22, wherein the anti-PD-L1 antibody is as defined in claim 11.

24. 24. The method of any one of claims 17 to 23, wherein said IL21 is as defined in claim 12.

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