New Immunocytokines For The Treatment Of Cancer

Abstract

The present invention relates to new immunocytokines which are useful for the treatment of cancer. These fusion proteins comprise (i) an antibody or antigen-binding fragment thereof fused to (ii) a cleavable peptide linker, and (iii) cytokine, or functional fragments thereof. Methods of treatment using these immunocytokines are also disclosed.

Description

INTRODUCTION

[0001] The invention relates to a new immunocytokines and its use for treating cancer.

[0002] While therapeutic success has been achieved for various types of haematological malignancies and some solid tumours (e.g., metastatic testicular cancer), the majority of disseminated forms of solid cancer remain incurable. The therapeutic efficacy of conventional cancer therapeutics is often limited by the inability of small organic molecules to accumulate in sufficient amounts at the site of disease (see e.g., van der Veldt A A, et al. Clin Cancer Res. 19: 4163-4173, 2013).

[0003] New strategies are now developed that preferentially activate relevant immune subsets, such as T effectors, monocytes and NK cells, while limiting the activation of regulatory T cells. However, substantial side effects and unfavourable pharmacokinetic properties have been a major drawback hampering the administration of therapeutically relevant doses. Notably, cytokine immunotherapy often results in the development of severe dose-limiting side effects (Pachella et al., Pract Oncol 6:212-221, 2015). Two properties shared by most cytokines are thought to play a crucial role in the development of treatment-associated adverse effects. Firstly, cytokines are pleiotropic, meaning they are able to influence more than a single cell type. Furthermore, cytokines have a short serum half-life and, thus, need to be administered at high doses to achieve their therapeutic effects. While effectively enhancing therapeutic efficacy, high doses exacerbate pleiotropic activities that manifest as adverse effects in patients.

[0004] One approach aimed at increasing efficacy attempts to deliver cytokines to tumour sites by genetically fusing cytokines to antibodies, or antibody components such as a single chain variable fragment (scFv). Such fusion proteins, designated immunocytokines, combine the binding specificity of an antibody with the potency of cytokines such as, for example, IL-2 (Sondel .di-elect cons.t Gillies, Antibodies 1: 149-171, 2012; Skrombolas .di-elect cons.t Frelinger, Expert Rev Clin Immunol. 10(2): 207-217, 2014; Kiefer .di-elect cons.t Neri, Immunol Rev. 270(1): 178-192, 2016). Delivery of the cytokine to the tumour site is improved by the use of immunocytokines, notably for cancers with easily accessible tumours. In another instance, the immunocytokine comprises a cytokine (IL-12) joined to a specific inhibitory anti-IL-12 scFv by a MMP9-cleavage site (Skrombolas et al., J Interferon Cytokine Res. 39(4): 233-245, 2019). However, the treatment of disseminated, systemic diseases might benefit from immunocytokines that have been optimised for tumour targeting and activation at the tumour site (Sondel .di-elect cons.t Gillies, Antibodies 1: 149-171, 2012). In particular, binding of the antibody outside of the tumour may result in unwanted cytokine activity and potential side effects. This problem is all the more crucial as certain payloads have been reported to completely abrogate the tumour-targeting potential of the parental antibody in mouse models of cancer (see e.g., Hess, Doctoral Thesis, ETH Zurich, 2015).

[0005] Thus, there is still a need for an immunocytokine which can deliver and activate the cytokine safely and efficiently to the tumour site.

FIGURE LEGENDS

[0006] FIG. 1. Fusion sites for generating immunocytokines (ICC).

[0007] FIG. 2: Deconvoluted MS spectrum of c9G4PVGLIG-IL15 obtained after deglycosylation RP-LC separation.

[0008] FIG. 3: Deconvoluted MS spectrum of Fc/2 cG4PVGLIG-IL15 obtained after deglycosylation, IdEs digestion and RP-LC separation.

[0009] FIG. 4. Evaluation of MMP-9/2 linkers cleavability when fused to the C-terminus of a mAb heavy chain. The GIVGPL linker reported as non-cleavable by MMP-9/2 was used as negative control for cleavage specificity.

[0010] FIG. 5. Evaluation of MMP-9/2 linkers cleavability when fused to the N-terminus of a mAb heavy chain. The GIVGPL linker reported as non-cleavable by MMP-9/2 was used as negative control for cleavage specificity. HC: Heavy Chain, LC: Light Chain, Ck: Cytokine.

[0011] FIG. 6. Evaluation of cleavability of c9G4 based immunocytokines as well as H161L16-IL15 and HHS76-IL15 immunocytokines by human and murine MMP-9 and MMP-2 (HC C-term fusion, linker PVGLIG). (A) c9G4-1115, H161L16-IL15, and HHS76-IL15; (B) c9G4-CCL4 and c9G4-IFN.alpha.. HC: Heavy Chain, LC: Light Chain, Ck: Cytokine. The NanoLuc.RTM. fusion was used as positive control for cleavage efficiency. Note 1: IL15 and IFN.alpha. visualisation post-cleavage in impaired by the high level of glycosylation of the proteins. Sample deglycosylation prior to cleavage allows visualisation of the released cytokines, indicating the proteins are not proteolysed by MMP-912 (data not shown). Note 3: The partial cleavage observed for the IL15 fusion is likely due to the heterogeneity of the tested sample (.apprxeq.50% monomer by Size-Exclusion Chromatography, data not shown).

[0012] FIG. 7: Summary of the MMP-912 linkers cleavability evaluation.

[0013] FIG. 8: PVGLIG and GIVGPL linker stability in presence of MMP-9 activity in 50 mM Tris pH7.5, 150 mM NaCl, 20 mM CaCl.sub.2) buffer: LC/MS fragment profile of anti-PDL1-PVGLIG-NanoLuc.RTM. (A) and anti-PDL1-GIVGPL-NanoLuc.RTM. (B) antibodies obtained after immunoprecipitation and reduction and reverse phase separation

[0014] FIG. 9: Analysis of ICC cleavage in mouse serum: LC/MS profile of anti-PDL1-PVGLIG-NanoLuc.RTM. fragments obtained after immunoprecipitation, reduction and reverse phase separation at T0 (A) and T24 (B) without MMP-9 spiking, at T0 (C) and T24 (D) with MMP-9 spiking.

[0015] FIG. 10. IL15 induced dimerisation of the IL2R.beta. and IL2R.gamma. receptor subunits. Representative data from three independent experiments.

[0016] FIG. 11: Western blot analysis of plasma samples (RENCA engrafted mice).

[0017] FIG. 12: Densitometric analysis of plasma samples western blots. X indicates that sample is missing.

[0018] FIG. 13: Statistical analysis on circulating ICC (plasma samples) (RENCA engrafted mice)

[0019] FIG. 14: Western blot analysis of tumour samples (RENCA engrafted mice).

[0020] FIG. 15: Densitometric analysis of tumour samples western blots. X indicates that sample is missing.

[0021] FIG. 16: Statistical analysis of ICC addressed to the RENCA tumours.

[0022] FIG. 17: Statistical analysis of ICC-PVGLIG behaviour in plasma versus tumour of RENCA engrafted mice.

[0023] FIG. 18: Deconvoluted MS spectrum of NHS67-PVGLIG-IL15 obtained after deglycosylation RP-LC separation.

[0024] FIG. 19: Deconvoluted MS spectrum of Fc/2 NHS67-PVGLIG-IL15 obtained after deglycosylation, IdEs digestion and RP-LC separation.

[0025] FIG. 20: Deconvoluted MS spectrum of H16L16-PVGLIG-IL15 obtained after deglycosylation RP-LC separation.

[0026] FIG. 21: Deconvoluted MS spectrum of Fc/2 H16L16-PVGLIG-IL15 obtained after deglycosylation, IdEs digestion and RP-LC separation.

[0027] FIG. 22: SDS-PAGE analysis of purified c9G4-PVGLIG-hIL15, NHS76-PVGLIG-hIL15 and H16L16-PVGLIG-hIL15 ICC in non-reduced/heated (NRH) and reduced/heated (RH) conditions.

[0028] FIG. 23: Murine T cell activation with ICC compared to controls. Activation measured by T cells expression of CD69 (A) or CD25 (B) in presence of cleaved and uncleaved NHS76-PVGLIG-IL15 or controls and by T cell expression of CD69 (C) or CD25 (D) in presence of cleaved and uncleaved H16L16-PVGLIG-IL15 or controls.

[0029] FIG. 24: Human T cell activation with ICC compared to controls. Activation measured by T cells expression of CD69 (A) or CD25 (B) in presence of cleaved and uncleaved NHS76-PVGLIG-IL15 or controls and by T cell expression of CD69 (C) or CD25 (D) in presence of cleaved and uncleaved H16L16-PVGLIG-IL15 or controls.

[0030] FIG. 25: Human T cell activation with ICC compared to controls. Activation measured by T cells secretion of INF.gamma. in presence of cleaved and uncleaved NHS76-PVGLIG-IL15 or controls for two different donors (Donor 1 (A) and Donor 2 (B)). Upper panel: activation measured by T cell secretion of INF.gamma. in presence of cleaved and uncleaved H16L16-PVGLIG-IL15 or controls for two different donors (Donor 1 (C) and Donor 2 (D)).

[0031] FIG. 26: Analysis of IL-8 production levels in A431 conditioned culture media after a 24 h incubation with the different samples. IL8 relative content is determined using DUOSET ELISA and is expressed in optical unit at 450 nm.

[0032] FIG. 27: Induction of ISRE-dependent luciferase dependent production by hIFN.alpha.2a. hIFN.alpha.2a activity was assayed in c9G4-PVGLIG-hIFN.alpha.2a (A), NHS76-PVGLIG-hIFN.alpha.2a (B), and H16/L16-PVGLIG-hIFN.alpha.2a, with (C) or without (D) preincubation of the cells with 10 .mu.g/ml H16/L16 antibody, by monitoring luminescence produced in the GloResponse.TM. ISRE-luc2P/HEK293 (Promega).

[0033] FIG. 28: IL15 activity after a 6 h incubation with/without urokinase. IL15 relative content is determined using IL15 Bioassay and is expressed in luminescence.

[0034] FIG. 29A: Evaluation of hIFN.alpha. activity after uPA-mediated cleavage of H16/L16-SGRSA hIFN.alpha.2a (A) and H16/L16-PSSRRRVN hIFN.alpha.2a (B). hIFN.alpha. activity was assayed after a 24 h-incubation of H16/L16-SGRSA hIFN.alpha.2a (A) and H16/L16-PSSRRRVN hIFN.alpha.2a (B) with/without urokinase and after IGF1R receptor saturation in ISRE-luc2/HEK293. Relative hIFN.alpha. activity is determined using GloResponse ISRE-luc2P Bioassay and is expressed in luminescence.

[0035] FIG. 30: Evaluation of hCXCL10 activity after uPA-mediated cleavage of c9G4-GSRS-CXCL10 (A), NHS76-GSRS-CXCL10 (B), and H16/L16-SGRS-CXCL10 (C). Relative hCXCL10 activity is determined using PathHunter eXpress CXCR3 CHOK1 .beta.-arrestin GPCR assay and is expressed in luminescence.

DESCRIPTION

[0036] It was surprisingly found that the specific combination of an antibody fused to a cytokine moiety which can be selectively released upon cleavage of a cleavable peptide linker, provides for a new and therapeutically effective fusion protein.

[0037] The present invention relates to an "immunocytokine", i.e., a fusion between an antibody or a fragment or a derivative thereof and a cytokine. The antibody moiety in the present immunocytokine targets the tumour where the cytokine is released to exert its action. This confers greater specificity to the fusion protein, i.e. it generates fewer side effects than immunocytokines of the prior art which merely rely on localised proteolysis for targeting cytokine activity to the tumour (Skrombolas et al., 2019).

[0038] Whereas other immunocytokines of the prior art either did not contain any linker or contained a merely structural linker (i.e., a linker without any specific biological activity) between the antibody and the cytokine, the present fusion protein comprises a peptide linker which can be cleaved between the two moieties, allowing better control of the therapeutic activity of the molecule. Indeed, the inventors have found that the fusion protein is surprisingly inactive in the blood but is activated upon reaching the tumour site. The cleavable peptide linker is preferentially cleaved in the tumour microenvironment, thus releasing the cytokine. Targeted delivery of the cytokine thus potentiates its anti-tumour activity, whilst reducing the risks of cytokine-associated toxicity.

[0039] In a first aspect, the invention relates to a fusion protein comprising an antibody, or antigen-binding protein thereof, a cleavable peptide linker, and a cytokine or a functional fragment thereof.

[0040] A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety. This term is meant to encompass all conjugates, wherein said antibody, or antigen-binding protein thereof is somehow bound to the cleavable peptide linker and the cytokine or functional fragment thereof, by, e.g. covalent and/or non-covalent, e.g. ionic bonds.

[0041] The term encompasses all binding arrangements. Preferred arrangements include antibody-linker-cytokine and cytokine-linker-antibody.

[0042] Antibodies

[0043] An "antibody" as used herein refers to an immunoglobulin (Ig) molecule capable of specific binding to a target, the "antigen", such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. The antibody or antigen-binding protein thereof of the present fusion protein mediates the targeted delivery of immunocytokines into disease environments and/or to specific cell subsets. Preferred target antigens are those that are overexpressed in diseased tissues, while remaining at low levels elsewhere. Such antigens are well-known to the skilled person, as-they have been the subject of numerous studies over the years. For example, the antibody moiety of the present immunocytokine may target antigens overexpressed on the surface of malignant cells (e.g., epithelial cell adhesion molecule, EGFR, IGF-1R, GD2 disialoganglioside, HER2/neu, CD20 and CD30), as well as targeting of neoangiogenic antigens found in tumours and chronic inflammation sites (e.g., fibronectin, splice variants EDA/EDB and A1 domain of tenascin C).

[0044] As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also any antigen binding fragment (i.e., "antigen-binding fragment") or single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site including, for example without limitation, scFv, single domain antibodies {e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv. As used herein, the term "antibody" encompasses both full-length antibodies and their antigen-binding fragments, as well as any derivative thereof. Preferably, the antibody according to the invention, or its derived compounds or antigen-binding fragments, is a monoclonal antibody.

[0045] A "monoclonal antibody", as used herein, means an antibody arising from a nearly homogeneous antibody population. More particularly, the individual antibodies of a population are identical except for a few possible naturally-occurring mutations which can be found in minimal proportions. In other words, a monoclonal antibody consists of a homogeneous antibody arising from the growth of a single cell clone (for example a hybridoma, a eukaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody, a prokaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody, etc.) and is generally characterized by heavy chains of one and only one class and subclass, and light chains of only one type. Monoclonal antibodies are highly specific and are directed against a single antigen. In addition, in contrast with preparations of polyclonal antibodies which typically include various antibodies directed against various determinants, or epitopes, each monoclonal antibody is directed against a single epitope of the antigen. Since these antibodies are directed against a single epitope, they are highly specific.

[0046] An "epitope" is the site on the antigen to which binds the antibody. It can be formed by contiguous residues or by non-contiguous residues brought into close proximity by the folding of an antigenic protein. Epitopes formed by contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by non-contiguous amino acids are typically lost under said exposure.

[0047] The generation of the antibody reactive with a specific antigen can be realised by any method known by the man skilled in the art, such as for example, fusion of a myeloma cell with spleen cells from immunized mice or other species compatible with the selected myeloma cells (Kohler .di-elect cons.t Milstein, Nature, 256:495-497, 1975). The immunized animals could include transgenic mice with human immunoglobulin loci which then directly produce human antibodies. Alternatively, an antibody can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors. In general, for the preparation of monoclonal antibodies or their functional fragments, especially of murine origin, it is possible to refer to techniques which are described in particular in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 726, 1988) or to the technique of preparation from hybridomas described by Kohler and Milstein (Nature, 256:495-497, 1975).

[0048] An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or subclass thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes.

[0049] A typical IgG antibody is composed of two identical heavy chains and two identical light chains that are joined by disulphide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called "complementarity-determining regions" ("CDRs") or "hypervariable regions", which are primarily responsible for binding an epitope of an antigen. They are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The more highly conserved portions of the variable regions are called the "framework regions".

[0050] There are three heavy-chain CDRs and 3 light-chain CDRs. The term "CDR" or "CDRs" is used here in order to indicate, according to the case, one of these regions or several, or even the whole, of these regions which contain the majority of the amino acid residues responsible for the binding by affinity of the antibody for the antigen or the epitope which it recognises.

[0051] As used herein, "VH" or "V.sub.H" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv, Fab, Fab', or F(ab')2 fragment. Reference to "VL" or "V.sub.L" refers to the variable region of the immunoglobulin light chain of an antibody, including the light chain of an Fv, scFv, dsFv, Fab, Fab', or F(ab')2 fragment.

[0052] Antibody constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called .alpha., .delta., .epsilon., .gamma., and .mu., respectively. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes, i.e., IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2 (see, W. E. Paul, ed., 1993, Fundamental Immunology, Raven Press, New York, N.Y.).

[0053] Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual "Fc" fragment. The crystal structure of the human IgG Fc domain has been determined (Deisenhofer, Biochemistry, 20, 2361-2370, 1981). As used in the specification and claims, "immunoglobulin Fc domain or Fc" means the carboxyl-terminal portion of the immunoglobulin heavy chain constant region. A "native sequence Fc domain", as used herein, comprises an amino acid sequence identical to the amino acid sequence of a Fc domain found in nature. Native sequence human Fc domains include a native sequence human IgG1 Fc domain (non-A and A allotypes); native sequence human IgG2 Fc domain; native sequence human IgG3 Fc domain; and native sequence human IgG4 Fc domain as well as naturally occurring variants thereof.

[0054] Although the boundaries of the Fc domain of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc domain is usually defined to stretch from an amino acid residue at position Cys226 or Pro230 in the hinge region, to the carboxyl-terminus thereof containing the CH2 and CH3 domain of the heavy chain. Throughout the present specification and claims, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0055] The term "hinge region" is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton, Mol Immunol, 22: 161-206, 1985). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S--S bonds in the same positions. The "CH2 domain" of a human IgG Fc portion (also referred to as "Cy2" domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain (Burton, Mol Immunol, 22: 161-206, 1985). The "CH3 domain" comprises the stretch of residues C-terminal to a CH2 domain in an Fc portion (i.e., from about amino acid residue 341 to about amino acid residue 447 of an IgG).

[0056] The Fc domains are central in determining the biological functions of the immunoglobulin and these biological functions are termed "effector functions". These Fc domain-mediated activities are mediated via immunological effector cells, such as killer cells, natural killer cells, and activated macrophages, or various complement components. These effector functions involve activation of receptors on the surface of said effector cells, through the binding of the Fc domain of an antibody to the said receptor or to complement component(s). The antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities involve the binding of the Fc domain to Fc-receptors such as Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RIII of the effector cells or complement components such as C1q. Of the various human immunoglobulin classes, human IgG1l and IgG3 mediate ADCC more effectively than IgG2 and IgG4.

[0057] The antibodies of the invention also comprise chimeric or humanised antibodies.

[0058] A chimeric antibody is one containing a natural variable region (light chain and heavy chain) derived from an antibody of a given species in combination with constant regions of the light chain and the heavy chain of an antibody of a species heterologous to said given species.

[0059] The antibodies, or chimeric fragments of same, can be prepared by using the techniques of recombinant genetics. For example, the chimeric antibody could be produced by cloning recombinant DNA containing a promoter and a sequence coding for the variable region of a nonhuman monoclonal antibody of the invention, notably murine, and a sequence coding for the human antibody constant region. A chimeric antibody according to the invention coded by one such recombinant gene could be, for example, a mouse-human chimera, the specificity of this antibody being determined by the variable region derived from the murine DNA and its isotype determined by the constant region derived from human DNA. It will be appreciated that in this case, the Fc domain of the chimeric antibody is of human origin. Refer to Verhoeyn et al. (BioEssays, 8:74, 1988) for methods for preparing chimeric antibodies.

[0060] In addition, the invention also relates to humanised antibodies arising from the murine antibodies described above. "Humanised antibody" refers herein to an antibody that contains CDR regions derived from an antibody of nonhuman origin, the other parts of the antibody molecule being derived from one (or several) human antibodies. In addition, some of the skeleton segment residues (called FR) can be modified to preserve binding affinity (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al., Nature, 332:323-327, 1988). The Fc domain of a humanised antibody will be of human origin, as in chimeric antibodies.

[0061] The humanised antibodies of the invention or fragments of same can be prepared by techniques known to a person skilled in the art (such as, for example, those described in the documents Singer et al., J. Immun., 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10:1-142, 1992; and Bebbington et al., Bio/Technology, 10: 169-175, 1992). Such humanised antibodies are preferred for their use in methods involving in vitro diagnoses or preventive and/or therapeutic treatment in vivo. Other humanisation techniques, also known to a person skilled in the art, such as, for example, the "CDR grafting" technique described by PDL in patents EP 0 451 261, EP 0 682 040, EP 0 939 127, EP 0 566 647 or U.S. Pat. Nos. 5,530,101, 6,180,370, 5,585,089 and 5,693,761. U.S. Pat. No. 5,639,641 or 6,054,297, 5,886,152 and 5,877,293 can also be cited.

[0062] Although it is possible to use antibody fragments in the present immunocytokines, it is preferred to use full-length, bivalent antibodies. A monovalent antibody such as a Fab or a scFv has only a single binding site for an antigen (as distinct from natural `bivalent` antibodies), i.e., is composed of a single antigen-binding arm. As known to the skilled person, the greater an immunoglobulin's valency (number of antigen binding sites), the greater the amount of antigen it can bind. There is a significant affinity change between monovalent and bivalent bindings with a 1,500-fold change in Kd values. Bivalent antibodies can thus be used at a lower dose to attain similar therapeutic efficiency as monovalent Fab or scFv fragments, thus limiting the risks of secondary effects.

[0063] Preferably, the antibody which can be used in the immunocytokines described herein is an antibody which does not bind specifically the cytokine moiety of said immunocytokine. For example, if the cytokine is IL-12, the antibody according to this embodiment is not an antibody which binds IL-12.

[0064] In another embodiment the antibody used in the present immunocytokine is an antibody which binds specifically a tumour-associated antigens (TAA) or a tumour-specific antigens (TSA). As used herein, a "tumour-specific antigen" is a protein or other molecule that is found on cancer cells whilst a "tumour-specific antigen" is a protein or other molecule that is found on cancer cells and not on normal cells.

[0065] Tumour-specific antigens are known in the art Tumour antigens can be classified in a variety of ways. Tumour antigens include antigens encoded by genes that have undergone chromosomal alteration. Many of these antigens are found in lymphoma and leukaemia. Even within this classification, antigens can be characterized as those that involve activation of quiescent genes. These include BCL-1 and IgH (Mantel cell lymphoma), BCL-2 and IgH (Follicular lymphoma), BCL-6 (Diffuse large B-cell lymphoma), TAL-1 and TCR delta or SIL (T-cell acute lymphoblastic leukaemia), c-MYC and IgH or IgL (Burkitt lymphoma), MUN/IRF4 and IgH (Myeloma), PAX-5 (BSAP) (Immunocytoma).

[0066] Other tumour antigens that involve chromosomal alteration and thereby create a novel fusion gene and/or protein include RARoa, PML, PLZF, NPMor NuM4 (Acute promyelocytic leukaemia), BCR and ABL (Chronic myeloid/acute lymphoblastic leukaemia), MLL (HRX) (Acute leukaemia), E2A and PBX or HLF (B-cell acute lymphoblastic leukaemia), NPM, ALK (Anaplastic large cell leukaemia), and NPM, MLF-1 (Myelodysplastic syndrome/acute myeloid leukaemia).

[0067] Other tumour antigens are specific to a tissue or cell lineage. These include cell surface proteins such as CD20, CD22 (Non-Hodgkin's lymphoma, B-cell lymphoma, Chronic lymphocytic leukaemia (CLL)), CD52 (B-cell CLL), CD33 (Acute myelogenous leukaemia (AML)), CD 10 (gp100) (Common (pre-B) acute lymphocytic leukaemia and malignant melanoma), CD3/T-cell receptor (TCR) (T-cell lymphoma and leukaemia), CD79/B-cell receptor (BCR) (B-cell lymphoma and leukaemia), CD26 (Epithelial and lymphoid malignancies), Human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ (Lymphoid malignancies), RCAS1 (Gynaecological carcinomas, biliary adenocarcinomas and ductal adenocarcinomas of the pancreas), and Prostate specific membrane antigen (Prostate cancer).

[0068] Tissue- or lineage-specific tumour antigens also include epidermal growth factor receptors (high expression) such as EGFR (HER1 or erbB1) and EGFRvIII (Brain, lung, breast, prostate and stomach cancer), erbB2 (HER2 or HER2/neu) (Breast cancer and gastric cancer), erbB3 (HER3) (Adenocarcinoma), and erbB4 (HER4) (Breast cancer).

[0069] Tissue- or lineage-specific tumour antigens also include cell-associated proteins such as Tyrosinase, Melan-A/MART-1, tyrosinase related protein (TRP)-1/gp75 (Malignant melanoma), Polymorphic epithelial mucin (PEM) (Breast tumours), and Human epithelial mucin (MUC1) (Breast, ovarian, colon and lung cancers).

[0070] Tissue- or lineage-specific tumour antigens also include secreted proteins such as Monoclonal immunoglobulin (Multiple myeloma and plasmacytoma), Immunoglobulin light chains (Multiple Myeloma), alpha-fetoprotein (Liver carcinoma), Kallikreins 6 and 10 (Ovarian cancer), Gastrin-releasing peptide/bombesin (Lung carcinoma), and Prostate specific antigen (Prostate cancer).

[0071] Still other tumour antigens are cancer testis (CT) antigens that are expressed in some normal tissues such as testis and in some cases placenta. Their expression is common in tumours of diverse lineages and as a group the antigens form targets for immunotherapy. Examples of tumour expression of CT antigens include MAGE-A1, -A3, -A6, -A12, BAGE, GAGE, HAGE, LAGE-1, NY-ESO-1, RAGE, SSX-1, -2, -3, -4, -5, -6, -7, -8, -9, HOM-TES-14/SCP-1, HOM-TES-85 and PRAME. Still other examples of CT antigens and the cancers in which they are expressed include SSX-2, and -4 (Neuroblastoma), SSX-2 (HOM-MEL-40), MAGE, GAGE, BAGE and PRAME (Malignant melanoma), HOM-TES-14/SCP-1 (Meningioma), SSX-4 (Oligodendroglioma), HOM-TES-14/SCP-1, MAGE-3 and SSX-4 (Astrocytoma), SSX member (Head and neck cancer, ovarian cancer, lymphoid tumours, colorectal cancer and breast cancer), RAGE-1, -2, -4, GAGE-1-2, -3, -4, -5, -6, -7 and -8 (Head and neck squamous cell carcinoma (HNSCC)), HOM-TES14/SCP-1, PRAME, SSX-1 and CT-7 (Non-Hodgkin's lymphoma), and PRAME (Acute lymphoblastic leukaemia (ALL), acute myelogenous leukaemia (AML) and chronic lymphocytic leukaemia (CLL)).

[0072] Other tumour antigens are not specific to a particular tissue or cell lineage. These include members of the carcinoembryonic antigen (CEA) family: CD66a, CD66b, CD66c, CD66d and CD66e. These antigens can be expressed in many different malignant tumours and can be targeted by immunotherapy.

[0073] Still other tumour antigens are viral proteins and these include Human papilloma virus protein (cervical cancer), and EBV-encoded nuclear antigen (EBNA)-1 (lymphomas of the neck and oral cancer).

[0074] Still other tumour antigens are mutated or aberrantly expressed molecules such as but not limited to CDK4 and beta-catenin (melanoma).

[0075] In some embodiments, the antigen is a tumour antigen. The tumour antigen may be selected from the group consisting of MART-1/Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal associated antigen (CRC)-C017-1A/GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, and CD20. The tumour antigen may also be selected from the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5). In still another embodiment, the tumour antigen is selected from the group consisting of GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9. And in yet a further embodiment, the tumour antigen is selected from the group consisting of BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21 ras, RCAS 1, .alpha.-fetoprotein, E-cadherin, .alpha.-catenin, .beta.-catenin, .gamma.-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papilloma virus proteins, Smad family of tumour antigens, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.

[0076] Cancer or tumour antigens can also be classified according to the cancer or tumour they are associated with (i.e., expressed by). Cancers or tumours associated with tumour antigens include acute lymphoblastic leukaemia (etv6; am11; cyclophilin b), B cell lymphoma (Ig-idiotype); Burkitt's (Non-Hodgkin's) lymphoma (CD20); glioma (E-cadherin; .alpha.-catenin; .beta.-catenin; .gamma.-catenin; pi20ctn), bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUC family; HER2/neu; c-erbB-2), cervical carcinoma (p53; p21ras), colon carcinoma (p21ras; HER2/neu; c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRC)-0017-1A/GA733; APC), choriocarcinoma (CEA), epithelial cell-cancer (cyclophilin b), gastric cancer (HER2/neu; c-erbB-2; ga733 glycoprotein), hepatocellular cancer (.alpha.-fetoprotein), Hodgkin's lymphoma (lmp-1; EBNA-1), lung cancer (CEA; MAGE-3; NY-ESO-1), lymphoid cell-derived leukaemia (cyclophilin b), melanoma (p15 protein, gp75, oncofoetal antigen, GM2 and GD2 gangliosides), myeloma (MUC family; p21 ras), non-small cell lung carcinoma (HER2/neu; c-erbB-2), nasopharyngeal cancer (lmp-1; EBNA-1), ovarian cancer (MUC family; HER2/neu; c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3; PSMA; HER2/neu; c-erbB-2), pancreatic cancer (p21ras; MUC family; HER2/neu; c-erbB-2; ga733 glycoprotein), renal (HER2/neu; c-erbB-2), squamous cell cancers of cervix and oesophagus (viral products such as human papilloma virus proteins and non-infectious particles), testicular cancer (NY-ESO-1), T cell leukaemia (HTLV-1 epitopes), and melanoma (Melan-A/MART-1; cdc27; MAGE-3; p21ras; gp100.sup.Pme117). More preferably, the antibody of the present immunocytokine is an antibody which does not bind specifically the cytokine moiety of said immunocytokine, but binds specifically a tumour-associated antigens (TAA) or a tumour-specific antigens (TSA).

[0077] Examples of antibodies which can be used in the present invention include: Alemtuzumab (CD52), Alirocumab (PCSK9), Arcitumomab (Human CEA (carcinoembryonic antigen)), Atezolizumab (PD-L1), Avelumab (PD-L1), Basiliximab (CD25 (a chain of IL2 receptor)), Belimumab (BLyS), Bevacizumab (CD19), Bevacizumab (VEGF), Brodalumab (IL-17RA), Capromab (Tumour surface antigen PSMA), Catumaxomab (EpCAM and CD3), Catumaxomab (EpCAM), Certolizumab pegol (TNFa), Cetuximab (EGFR), Daratumumab (CD38), Dinutuximab (GD2), Dupilumab (IL-4Ra), Durvalumab (PD-L1), Efalizumab (CD11a), Elotuzumab (SLAMF7), Evolocumab (LDL-C/PCSK9), Fanolesomab (CD15), Golimumab (TNFa), Ibritumomab tiuxetan (CD20), Infliximab (TNF.alpha.), Ipilimumab (CTLA-4), Necitumumab (EGFR), Necitumumab (CD25 (a chain of IL2 receptor)), Nivolumab (PD-1), Nofetumomab (Carcinoma-associated antigen), Obinutuzumab (CD20), Ocrelizumab (CD20), Ofatumumab (CD20), Olaratumab (PDGFR-.alpha.), Panitumumab (EGFR), Pembrolizumab (PD-1), Pertuzumab (HER2), Ramucirumab (VEGF), Ranibizumab (VEGF-A), Rituximab (CD20), Siltuximab (cCLB8), Tocilizumab (IL-6 receptor), Trastuzumab (HER-2), Vedolizumab (Integrin-.alpha.4B7), Votumumab (Cytokeratin tumour-associated antigen).

[0078] Cleavable Peptide Linkers

[0079] A "cleavable peptide linker" as used herein refers to a polyvalent linker covalently bonded to an antibody, or an antigen-binding fragment thereof, and covalently bonded to a cytokine, or fragment thereof, which is enzymatically cleavable (e.g. at a cleavage site). According to the invention, upon hydrolysis (proteolytic cleavage) of the cleavable peptide linker, the cytokine moiety, preferably IL-15, is released, enabling it to exert its therapeutic activity. In preferred embodiments the cleavable peptide linker is recombinantly expressed as part of the immunocytokine. In other embodiments, the cleavable peptide linker is a linker formed by reacting a functional (reactive) group attached to the linker with an antibody, or an antigen-binding fragment thereof using, for example, conjugate chemistry. In yet other embodiments, the cleavable peptide linker is a linker formed by reacting a functional (reactive) group attached to the linker with a cytokine, or fragment thereof, using, for example, conjugate chemistry. In a preferred embodiment, the cleavable peptide linker connects the cytokine, or fragment thereof, to the heavy chain of the antibody, or an antigen-binding fragment thereof. In another embodiment, the cleavable peptide linker connects the cytokine, or fragment thereof, to the light chain of the antibody, or an antigen-binding fragment thereof. The cleavable peptide linker may connect the cytokine, or fragment thereof, to the N-terminus of one of the heavy and light chains of the antibody, or an antigen-binding fragment thereof. It also possible that the cleavable peptide linker connects the cytokine, or fragment thereof, to the C-terminus of the heavy and light chains of the antibody, or an antigen-binding fragment thereof. Most preferably, the cleavable peptide linker connects the cytokine, or fragment thereof, to the N-terminus or C-terminus of the heavy chain of the antibody, or an antigen-binding fragment thereof.

[0080] The cleavable peptide linker provided herein may include a protease cleavage site.

[0081] A "cleavage site" as used herein, refers to a recognisable site for cleavage of a portion of a linker (e.g. cleavable peptide linker as described hereinabove) present in an immunocytokine described herein. Thus, a cleavage site may be found in the sequence of a cleavable peptide linker as described herein, including embodiments thereof. In embodiments, the cleavage site is an amino acid sequence that is recognised and cleaved by a cleaving agent (e.g. a peptidyl sequence). Exemplary cleaving agents include proteins, enzymes, DNAzymes, RNAzymes, metals, acids, and bases. Exemplary cleavage sites are defined herein (see FIG. 7).

[0082] A "protease cleavage site" as used herein is a cleavage site which is recognised and specifically cleaved by a protease. According to a preferred embodiment, the protease cleavage site is a tumour-associated protease cleavage site. A "tumour-associated protease cleavage site" as used herein refers to an amino acid sequence recognised by a protease, whose expression is specific for a tumour cell or tumour cell environment thereof or mainly expressed in the tumour cell or tumour environment compared to healthy tissues or is only/mainly active in the tumour cell or tumour environment. In embodiments, the protease cleavage site is a matrix metalloprotease (MMP) cleavage site, a prostate specific antigen (PSA) protease cleavage site, a membrane type serine protease 1 (MT-SP1) protease cleavage site, a uPA urokinase plasminogen activator cleavage site, or a legumain protease cleavage site. In some embodiments, the matrix metalloprotease (MMP) cleavage site is a MMP 9 cleavage site, a MMP 13 cleavage site, or a MMP 2 cleavage site. Protease cleavage sites may be designated by a specific amino acid sequence but may also encompass any variation of this canonical amino acid sequence which is still recognised and cleaved by the protease of interest.

[0083] Preferably, the cleavable peptide linker is a matrix metalloprotease (MMP) cleavage site. More preferably, the cleavable peptide linker comprises a MMP 9 cleavage site or a MMP 2 cleavage site. Examples of MMP cleavage sites include GPLGIAGQ, GPLGLWAQ, GPLGMLSQ, PLGLAG, and PVGLIG.

[0084] In another preferred embodiment, the cleavable peptide linker is a urokinase plasminogen activator (uPA) cleavage site. Examples of uPA cleavage sites include SGRS, SGRSA, and PSSSRRRVN.

[0085] The term "MMP 2" or "MMP 2 protease" as used herein refers to the matrix metalloproteinase 2 (MMP 2). MMP-2 is the protein identified by the NCBI sequence reference GI: 189217853. The term "MMP-9" or "MMP9 protease" as used herein refers to the matrix metalloproteinase 9 (MMP-9). MMP9 is the protein identified by the NCBI sequence reference GI: 74272287. The term "MMP 13" or "MMP 13 protease" as used herein refers the matrix metalloproteinase 13 (MMP 13). MMP 13 is the protein identified by the NCBI sequence reference GL4505209. The term "PSA" or "PSA protease" as used herein refers to the prostate-specific antigen (PSA), also known as gamma seminoprotein or kallikrein-3. PSA is the protein identified by the NCBI sequence reference GL71834853. The term "PSMA" or "PSMA protease" as used herein refers to the prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) or NAAG peptidase. PSMA is the protein identified by the NCBI sequence reference GL62548858. The term "fibroblast associated protein" as used herein refers to the fibroblast associated protein. Fibroblast associated protein is the protein as identified by the NCBI sequence reference GL 1888316. The term "MT-SPL" or "MT-SPL protease" as used herein refers to the membrane type serine protease 1 (MT-SPL). MT-SPL is the protein identified by the NCBI sequence reference GI: 1 1415040. The term "legumain" or "legumain protease" as used herein refers to the legumain protein. Legumain is the protein identified by the NCBI sequence reference GL2842759. The term uPA as used herein refers to the urokinase-type plasminogen activator identified by the NCBI sequence reference Gene ID: 5328.

[0086] In some embodiment, the cleavable peptide linker comprises at Least 2, at Least 3, at Least 4, at Least 5, at Least 6, at Least 7, at Least 8, at Least 9, at Least 10, at Last 11, at Least 12, or at Least 13 amino acids. In other embodiments, cleavable peptide Linkers are 5-mers (i.e. peptides 5 amino acids in Length), 6-mers (i.e. peptides 6 amino acids in Length), 7-mers (i.e. peptides 7 amino acids in Length), 8-mers (i.e. peptides 8 amino acids in Length), 9-mers (i.e. peptides 9 amino acids in Length), 10-mers (i.e. peptides 10 amino acids in Length), 11-mers (i.e. peptides 11 amino acids in Length), 12-mers (i.e. peptides 12 amino acids in Length), or 13-mers (i.e. peptides 13 amino acids in Length).

[0087] Most preferably, said the sequence of said cleavage peptide linker is selected from the group consisting of: GPLGIAGQ, GPLGLWAQ, GPLGMLSQ, PLGLAG, PVGLIG, SGRS, SGRSA, and PSSSRRRVN.

[0088] Cytokines

[0089] The term "cytokine" as used herein refers to a member of a family of small secreted regulatory proteins which have an effect on the immune system. Cytokines are involved in cell-to-cell communication and regulate many cellular functions, such as cell survival and growth, as well as induction of the expression of many genes. Secretion of cytokines thus enables the rapid propagation of immune signalling in a multifaceted and efficient manner. Cytokines regulate the nature, intensity and duration of the immune response by exerting a variety of effects on Lymphocytes and/or other cells. Indeed, cytokines are usually classified into pro- and anti-inflammatory cytokines. Some cytokines are also capable of mobilising the immune system to fight cancer (see e.g., Floros .di-elect cons.t Tarhini, Semin Oncol. 42(4): 539-548, 2015). Cytokines can be produced by many cell types, including immune and non-immune cells. Examples of cytokines include interleukins, lymphokines, monokines, interferons, colony stimulating factors, and chemokines, inter alia. A "cytokine" as used herein may be any one of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26, IL-28, IL-29, IL-33, IL-36, IL37, IL-38, IFN-.alpha. (including IFN-.alpha.1/13, IFN-.alpha.2, IFN-.alpha.4, IFN-.alpha.5, IFN-.alpha.6, IFN-.alpha.7, IFN-.alpha.8, IFN-.alpha.10, IFN-.alpha.14, IFN-.alpha.16, IFN-.alpha.17, and IFN-.alpha.21), IFN-.beta., IFN-.gamma., IFN-.lamda., TNF-.alpha., TNF-.beta., TGF-.beta.1, M-CSF, G-CSF, GM-CSF, and CXL10. According to the invention, the cytokine is preferably inactivated or attenuated when linked to the ICC and became active only after cleavage of ICC by the protease.

[0090] The term "functional fragment" with regard to said cytokines is to be interpreted essentially in analogy to the same term for antibodies (see below). Functional fragments and derivatives of cytokines are those that essentially have the same physiological function/activity as the naturally occurring cytokines.

[0091] In a preferred embodiment, the cytokine is IL-15. By "IL-15" or "interleukin-15", it is herein referred to a cytokine that regulates T and natural killer cell activation and proliferation. Interleukin-15 (IL-15) is a 14 to 15 kDa member of the 4.alpha.-helix bundle family of cytokines composed of 114 amino acids whose sequence is available under the accession number NP_000576.1. There is 97% sequence identity between human and simian IL-15 and 73% between human and mouse. This appears to be sufficient for hulL-15 to render it biologically active on simian and murine cells.

[0092] IL-15 displays high structural similarity to Interleukin-2 (IL-2). Like IL-2, IL-15 binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). Specificity of the signalling is ensured by IL15 being recognised by the alpha unit of its receptor (IL15RA), whilst IL-2 binds IL2RA. IL-15 stimulates the production of proinflammatory cytokines (e.g. TNF.alpha., IL-1, IFN.gamma.), the proliferation and lg synthesis of activated B cells, the activation of T.sub.H1, monocytes and lymphokine activated killer cells, the proliferation of mast cells and T cells and inhibits the apoptosis of T and B cells. In addition to the mentioned functional activities IL-15 plays a pivotal role in the development, survival and function of NK cells [Joost J. Oppenheim et al., Cytokine Reference; 213-221, (2002)]. IL-15 is a cytokine that primarily stimulates the proliferation and cytotoxic functions of CD8T cells and NK cells leading to enhanced anti-tumour responses (Waldmann, J Investig Dermatol Symp Proc. 16(1): S28-30, 2013). While initially showing promise as a cancer therapeutic, the efficacy of IL-15 was limited by its short in vivo half-life. However, new IL-15-based therapies have been developed and are currently in clinical trials (Robinson .di-elect cons.t Schluns, Immunol Lett. 190: 159-168, 2017). The inventors have now shown that IL-15 is not active when fused to an antibody moiety and becomes activated only when released by the cleavage of the linker. Immunocytokines comprising IL-15 localise in vivo to the tumour where they are cleaved. This allows for circumventing the short half-life problem. In addition, the active cytokine is delivered to the site where it is needed, reducing the risks of side effects.

[0093] In another preferred embodiment, the cytokine is CXCL10. By "CXCL10" or "C-X-C motif chemokine 10" or "interferon gamma-induced protein 10" or "IP10", it is herein referred to an 8.7-kDa CXC chemokine which functions to recruit activated and memory lymphocytes to sites of inflammation. The secreted bioactive form (after cleavage of the signal peptide) is a polypeptide of 77 residues (corresponding to positions 22-98 of NP_001556), herein designated "long CXCL10", which binds the CXCR3 receptor. CXCL10 signalling through the chemokine receptor CXCR3 has an important role in lymphocyte migration and function. Notably, CXCL10 appears to enhance T cell-dependent anti-cancer immunity (Karin et Razon, Cytokine, 109:24-28, 2018).

[0094] In another preferred embodiment, the cytokine is IL-36. As used herein, the expressions "IL-36" or "hIL-36" or "Interleukin-36" refers to a subgroup of the IL-1 family with pro-inflammatory properties (see e.g. Murrieta-Coxca, Int J Mol Sci., 20(7). pii: E1649, 2019). By "IL-36", it is notably referred to IL-36.alpha. (IL-1F6), IL-36.beta. (IL-1F8), and IL-36.gamma. (IL-1F9). As used herein, "IL-36.alpha." refers to a 158-amino acid protein whose sequence is available under the accession number NP_055255, "IL-36.beta." a 157-amino acid protein with 2 isoforms (accession numbers: NP_055253 and NP_775270), and "IL-36.gamma." a 169-amino acid protein with 2 isoforms (accession numbers: NP_001265497 and NP_062564). IL-36.alpha., IL-36.beta., and IL-36.gamma. are agonist ligands with pro-inflammatory activity. They promote the induction of various inflammatory mediators including cytokines, chemokines, growth factors, and antimicrobial peptides. All of them use the same receptor, IL-36R, which dimerises with IL-1 RAcP to activate intracellular signalling cascades. This pathway culminates with the expression of inflammatory cytokines driven by AP-1 (activator protein 1) and NF-kB transcription factors.

[0095] In yet another preferred embodiment, the cytokine is IFN.alpha.. As used herein, the expressions "IFN.alpha." or "IFN-.alpha." or "Interferon .alpha." refer to a subtype of human type-I interferons (IFN-I), a large subgroup of interferon proteins that help regulate the activity of the immune system. All IFN-I, including IFN-.alpha., bind to a specific cell surface receptor complex known as the IFN-.alpha. receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains (see e.g., Lopez de Padilla .di-elect cons.t Niewold, Gene, 576(1 Pt 1): 14-21, 2016). There are 12 functional human IFN-.alpha. proteins (IFN-.alpha.1/13, IFN-.alpha.2, IFN-.alpha.4, IFN-.alpha.5, IFN-.alpha.6, IFN-.alpha.7, IFN-.alpha.8, IFN-.alpha.10, IFN-.alpha.14, IFN-.alpha.16, IFN-.alpha.17, and IFN-.alpha.21), all of which exhibit high homology in their primary, secondary, and tertiary structures. Each of the IFNA genes (IFNA1, IFNA2, IFNA3, IFN4, IFN5, IFN6, IFN7, IFN8, IFNA10, IFNA14, IFNA16, IFNA17, and IFNA21) encode a pre-protein comprising a 23 amino acid signal peptide which is cleaved upon secretion, resulting in a mature 166-residue protein (Uniprot accession number: Q6QNB6) except for IFN-2 which is composed of 165 amino acids only (Uniprot accession number: P01563). Indeed, the aspartic acid residue present at position 44 in other subtypes of IFN-.alpha. is missing in IFN-.alpha.2.

[0096] According to this embodiment, the invention relates to a fusion protein comprising:

[0097] (i) an antibody or antigen-binding fragment thereof fused to

[0098] (ii) a cleavable peptide linker, and

[0099] (iii) a cytokine, preferably IL-15, CXCL10, IL-36, or IFN-.alpha., or functional fragments thereof.

[0100] Methods of Identification of Peptide Cleavage Linkers and Cytokines

[0101] The inventors have shown that it is possible to identify linkers suitable for use in the present fusion proteins. The experimental data show that several linkers can be discriminated according to whether they are cleaved or not in vitro. The relevance of these in vitro results is emphasised by the fact that all the linkers thus identified are cleaved in vivo, thus liberating an active cytokine at the site of the tumour.

[0102] Thus, in another aspect, a method of selecting a peptide cleavable linker is herein provided. This method comprises the steps of: [0103] (i) providing a fusion protein as described herein, said fusion protein comprising the peptide cleavable linker to be tested; [0104] (ii) contacting said fusion protein with the relevant protease; and [0105] (iii) detecting the cleavage of said fusion protein.

[0106] In an embodiment, the method is carried out in vitro. In another embodiment, the method is carried out in vivo. According to this specific embodiment, step (ii) of the method comprises administering the fusion protein of step (i) to a mammal, preferably a rodent, most preferably a mouse.

[0107] Detection of the cleavage of the linker can be performed by any means available to the person of skills in the art. It may be notably performed using specific antibodies, in particular using well known technologies such as immunoprecipitation with specific antibodies, western blot, ELISA or ELISPOT, antibodies microarrays, or tissue microarrays coupled to immunohistochemistry. Other suitable techniques include FRET or BRET, single cell microscopic or histochemistry methods using single or multiple excitation wavelength and applying any of the adapted optical methods, such as electrochemical methods (voltammetry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g. multipolar resonance spectroscopy, confocal and non-confocal, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry), cell ELISA, flow cytometry, radioisotopic, magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE); HPLC-Mass Spectroscopy; Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS)).

[0108] Alternatively, the cleavage of the linker can also be detected with a functional test. Notably, the hydrolysis of the linker releases an active cytokine moiety, whereas said cytokine moiety was attenuated when part of the fusion protein. In this embodiment, step (iii) of the method comprises measuring the activity of the cytokine moiety. Preferably, step (iii) further comprises comparing the activity of the cytokine moiety of said fusion protein which has been contacted with the relevant protease in step (ii), with the activity of the cytokine moiety of said fusion protein which has not been contacted with the protease. According to a preferred embodiment, the linker is cleaved if the activity of the cytokine moiety of said fusion protein which has been contacted with the relevant protease in step (ii), is increased at least 2-fold, preferably at least 3-fold, preferably at least 4-fold, preferably at least 5-fold, preferably at least 10-fold, preferably at least 20-fold, preferably at least 50-fold, preferably at least 100-fold, relative to the activity of the cytokine moiety of the untreated fusion protein. More preferably, the linker is cleaved if the activity of the cytokine moiety is increased at least 10-fold.

[0109] In addition, it is immediately apparent that cytokines and variants thereof can be easily tested for their suitability in the present immunocytokines. A "cytokine variant" as used herein refers to a cytokine which differs from a main species cytokine. For example, a cytokine variant may have an amino acid sequence which differs from a main species cytokine. Ordinarily, variants will possess at least about 70% homology with the main species cytokine, and preferably, they will be at least about 80%, and more preferably at least about 90% homologous with the main species cytokine. The cytokine variants possess substitutions, deletions, and/or additions at certain positions within or adjacent to the amino acid sequence of the main species cytokines. Alternatively, the cytokine variant may differ from a main species cytokine in at least one post-translational modification. For example, the cytokine variant may carry one or more carbohydrate moieties attached thereto which differ from one or more carbohydrate moieties attached to a main species antibody.

[0110] Thus, in another aspect, a method of selecting a cytokine or a variant thereof is herein provided. This method comprises the steps of: [0111] (i) providing a fusion protein as described herein, said fusion protein comprising the cytokine or variant thereof to be tested; [0112] (ii) contacting said fusion protein with the relevant protease; and [0113] (iii) detecting the activity of said cytokine.

[0114] In an embodiment, the method is carried out in vitro. In another embodiment, the method is carried out in vivo. According to this specific embodiment, step (ii) of the method comprises administering the fusion protein of step (i) to a mammal, preferably a rodent, most preferably a mouse.

[0115] If the cytokine or the variant is suitable for use in an immunocytokine as described herein, the hydrolysis of the linker will release an active cytokine moiety, whereas said cytokine moiety is attenuated when part of the fusion protein. In this embodiment, step (iii) of the method comprises measuring the activity of the cytokine moiety. Preferably, step (iii) further comprises comparing the activity of the cytokine moiety of said fusion protein which has been contacted with the relevant protease in step (ii), with the activity of the cytokine moiety of said fusion protein which has not been contacted with the protease. According to a preferred embodiment, the cytokine or cytokine variant is active if the activity of the cytokine moiety of said fusion protein which has been contacted with the relevant protease in step (ii), is increased at least 2-fold, preferably at least 3-fold, preferably at least 4-fold, preferably at least 5-fold, preferably at least 10-fold, preferably at least 20-fold, preferably at least 50-fold, preferably at least 100-fold, relative to the activity of the cytokine moiety of the untreated fusion protein. More preferably, the cytokine or cytokine variant is active if said activity is increased at least 10-fold.

[0116] The skilled person will know how to measure the activity of the cytokine moiety depending upon the nature of said cytokine. Cytokine activity can be determined by a variety of methods including but not limited to the techniques of enzyme fragment complementation (Eglen J Biomol Screen. 2004 August; 9(5):398-408), proximity ligation assay (Andersen et al. Cytokine. 2013 October; 64(1):54-7) NF-.kappa.B translocation (Trask 2012, Assay Guidance Manual [Internet]. Bethesda (Md.): Eli Lilly .di-elect cons.t Company and the National Center for Advancing Translational Sciences; 2004-2012 Oct. 1.) Beta-arrestin recruitment (Wang Assay Guidance Manual [Internet]. Bethesda (Md.): Eli Lilly .di-elect cons.t Company and the National Center for Advancing Translational Sciences; 2004-2017 Nov. 20.) Bioluminescence Resonance Energy Transfer (Compan Methods Mol Biol. 2016; 1417:89-95). These methods have been used for a wide range of cytokines and can be easily adapted to the needs of the particular cytokine of interest as required.

[0117] The skilled person will in particular refer to the experimental section of the present application, wherein instances of such assays are described.

[0118] Polynucleotides Encoding an Immunocytokine

[0119] Also provided herein are polynucleotides comprising a nucleotide sequence encoding a fusion protein as described above. Also provided herein are polynucleotides that hybridise under high stringency, intermediate or lower stringency hybridisation conditions, e.g., as defined supra, to polynucleotides that encode a fusion protein or modified fusion protein provided herein.

[0120] In certain embodiments, nucleic acid molecules provided herein comprise or consist of a nucleic acid sequence encoding a V.sub.H or a V.sub.L amino acid sequence fused to a cleavable peptide linker and a cytokine, as provided herein. In other embodiments, nucleic acid molecules provided herein comprise or consist of a nucleic acid sequence encoding a V.sub.H or a V.sub.L amino acid sequence which is not fused to additional sequences. In yet other embodiments, the nucleic acid molecules provided herein comprise or consist of combinations of a nucleic acid sequence encoding a V.sub.H or a V.sub.L amino acid sequence fused to a cleavable peptide linker and a cytokine and of a nucleic acid sequence encoding a V.sub.H or a V.sub.L amino acid sequence which is not fused to additional sequences. Preferably, a nucleic acid sequence encoding a V.sub.H amino acid sequence fused to a cleavable peptide linker and a cytokine is combined with a nucleic acid sequence encoding a V.sub.L amino acid sequence which is not fused to additional sequences. Alternatively, said combination comprises a nucleic acid sequence encoding a V.sub.L amino acid sequence fused to a cleavable peptide linker and a cytokine and a nucleic acid sequence encoding a V.sub.H amino acid sequence which is not fused to additional sequences.

[0121] Recombinant Expression of an Antibody

[0122] A variety of expression systems may be used to express the present immunocytokines as described herein. In one aspect, such expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transiently transfected with the appropriate nucleotide coding sequences, express an antibody of the invention in situ.

[0123] The invention provides vectors comprising the polynucleotides described herein. In one embodiment, the vector contains a polynucleotide encoding a heavy chain of the immunocytokine of the invention, wherein said heavy chain is fused or not to a cleavable peptide linker and a cytokine. In another embodiment, said polynucleotide encodes the light chain of an immunocytokine of the invention, wherein said light chain is fused or not to a cleavable peptide linker and a cytokine. The invention also provides vectors comprising polynucleotide molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof.

[0124] In order to express the heavy and/or light chain of an immunocytokine disclosed herein, the polynucleotides encoding said heavy and/or light chains are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational sequences.

[0125] "Operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0126] The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0127] Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such forms of expression vectors, such as bacterial plasmids, YACs, cosmids, retrovirus, EBV-derived episomes, and all the other vectors that the one skilled in the art will know to be convenient for ensuring the expression of the heavy and/or light chains of the antibodies of the invention. The skilled man will realise that the polynucleotides encoding the heavy and the light chains can be cloned into different vectors or in the same vector. In a preferred embodiment, said polynucleotides are cloned into two vectors.

[0128] Polynucleotides of the invention and vectors comprising these molecules can be used for the transformation of a suitable host cell. The term "host cell", as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced in order to express the present immunocytokine. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0129] Transformation can be performed by any known method for introducing polynucleotides into a cell host. Such methods are well known of the man skilled in the art and include dextran-mediated transformation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, biolistic injection and direct microinjection of DNA into nuclei.

[0130] The host cell may be co-transfected with two or more expression vectors, including the vector expressing the protein of the invention. In particular, the other expression vectors may encode enzymes involved in post-translational modifications, such as glycosylation. For example, a host cell can be transfected with a first vector encoding an immunocytokine as described above, and a second vector encoding a glycosyltransferase polypeptide. Alternatively, the host cell can be transformed with a first vector encoding an immunocytokine, a second vector encoding a glycosyltransferase, as described above, and a third vector encoding another glycosyltransferase. Mammalian cells are commonly used for the expression of a recombinant therapeutic immunoglobulins, especially for the expression of whole recombinant antibodies. For example, mammalian cells such as HEK293 or CHO cells, in conjunction with a vector, containing the expression signal such as one carrying the major intermediate early gene promoter element from human cytomegalovirus, are an effective system for expressing the present immunocytokine (Foecking et al., 1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).

[0131] It is also possible to select a host cell which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing of protein products may be important for the function of the protein. Different host cells have features and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems are chosen to ensure the correct modification and processing of the expressed antibody of interest. Hence, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, COS, HEK293, NS/0, BHK, Y2/0, 3T3 or myeloma cells (all these cell lines are available from public depositories such as the Collection Nationale des Cultures de Microorganismes, Paris, France, or at the American Type Culture Collection, Manassas, Va., U.S.A.).

[0132] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In one embodiment of the invention, cell lines which stably express the immunocytokine may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells are transformed with DNA under the control of the appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other appropriate sequences known to the person skilled in art, and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for one to two days in an enriched media, and then are moved to a selective media. The selectable marker on the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into a chromosome and be expanded into a cell line. Other methods for constructing stable cell lines are known in the art. In particular, methods for site-specific integration have been developed. According to these methods, the transformed DNA under the control of the appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other appropriate sequences is integrated in the host cell genome at a specific target site which has previously been cleaved (Moele et al., Proc. Natl. Acad. Sci. U.S.A., 104(9): 3055-3060; U.S. Pat. Nos. 5,792,632; 5,830,729; 6,238,924; WO 2009/054985; WO 03/025183; WO 2004/067753, all of which are incorporated herein by reference).

[0133] A number of selection systems may be used, including but not limited to the Herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., Proc Natl Acad Sci USA 48:202, 1992), glutamate synthase selection in the presence of methionine sulfoximide (Adv Drug Del Rev, 58:671, 2006, and website or literature of Lonza Group Ltd.) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genes in tk, hgprt or aprt cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Natl Acad Sci USA 77: 357, 1980); gpt, which confers resistance to mycophenolic acid (Mulligan et al., Proc Natl Acad Sci USA 78: 2072, 1981); neo, which confers resistance to the aminoglycoside, G-418 (Wu et al., Biotherapy 3: 87, 1991); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147, 1984). Methods known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley Ft Sons (1993). The expression levels of an immunocytokine can be increased by vector amplification. When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in the culture will increase the number of copies of the marker gene. Since the amplified region is associated with the gene encoding the immunocytokine of interest, production of said immunocytokine will also increase (Crouse et al., Mol Cell Biol 3: 257, 1983). Alternative methods of expressing the gene of the invention exist and are known to the person of skills in the art. For example, a modified zinc finger protein can be engineered that is capable of binding the expression regulatory elements upstream of the gene of the invention; expression of the said engineered zinc finger protein (ZFN) in the host cell of the invention leads to increases in protein production (see e.g., Reik et al., Biotechnol. Bioeng., 97(5), 1180-1189, 2006). Moreover, ZFN can stimulate the integration of a DNA into a predetermined genomic location, resulting in high-efficiency site-specific gene addition (Moehle et al, Proc Natl Acad Sci USA 104:3055, 2007).

[0134] The immunocytokine of the invention may be prepared by growing a culture of the transformed host cells under culture conditions necessary to express the desired antibody. The resulting expressed immunocytokine may then be purified from the culture medium or cell extracts. Soluble forms of the immunocytokine can be recovered from the culture supernatant. It may then be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by Protein A affinity for Fc, and so on), centrifugation, differential solubility or by any other standard technique for the purification of proteins. Suitable methods of purification will be apparent to a person of ordinary skills in the art.

[0135] When addressed to the tumour site, the immunocytokines provided herein are cleaved, thus releasing a cytokine such as IL-15, which has anti-tumour activity. In addition, some of the antibody moieties are capable of inducing ADCC and/or CDC responses and/or have an intrinsic anti-tumour activity. It will thus be appreciated by the skilled person that the immunocytokines provided herein are useful in the treatment of metastatic tumours and diseases such as cancer.

[0136] The terms "treating" or "treatment" refer to administering or the administration of a composition described herein in an amount, manner, and/or mode effective to improve a condition, symptom, or parameter associated with a disorder or to prevent progression or exacerbation of the disorder (including secondary damage caused by the disorder) to either a statistically significant degree or to a degree detectable to one skilled in the art.

[0137] Another aspect of the invention relates to pharmaceutical compositions of the immunocytokines described herein.

[0138] The pharmaceutical composition of the invention may contain, in addition to the immunocytokine of the invention, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.

[0139] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, salt solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The type of carrier can be selected based upon the intended route of administration. In various embodiments, the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of media and agents for pharmaceutically active substances is well known in the art. As detailed below, additional active compounds can also be incorporated into the compositions, such as anti-cancer and/or anti-angiogenesis agents; in particular, the additional active compound can be an anti-angiogenic agent, a chemotherapeutic agent, or a low-molecular weight agent. A typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of the combination. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example, Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), and the 18.sup.th and 19.sup.th editions thereof, which are incorporated herein by reference.

[0140] The immunocytokine present in the composition preferably is formulated in an effective amount. An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result, such as induction of apoptosis in tumour cells. A "therapeutically effective amount" means an amount sufficient to influence the therapeutic course of a particular disease state. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.

[0141] For therapeutic applications, the immunocytokine is administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The immunocytokine is also suitably administered by intratumoural, peritumoural, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. The intraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumours. Dosage regimens may be adjusted to provide the optimum response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased. One skilled in the art in the field of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the product selected, the disease or condition to be treated, the stage of the disease or condition and other relevant circumstances

[0142] The compositions of the invention can be administered to a subject to effect cell growth activity in a subject. As used herein, the term "subject" is intended to include living organisms in which apoptosis can be induced, and specifically includes mammals, such as rabbits, dogs, cats, mice, rats, monkey transgenic species thereof, and preferably humans.

[0143] The effectiveness of the immunocytokine in preventing or treating cancer may be improved by administering said immunocytokine serially or in combination with another agent that is effective for those purposes, such as tumour necrosis factor (TNF), an antagonist capable of inhibiting or neutralising the angiogenic activity of acidic or basic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), or hepatocyte growth factor (HGF), an antagonist capable of inhibiting or neutralising the coagulant activities of tissue factor, protein C, or protein S (see WO 91/01753), an antagonist such as an antibody capable of binding to HER2 receptor (see U.S. Pat. No. 5,772,997), or one or more conventional therapeutic agents such as, for example, alkylating agents, folic acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil, cisplatin, purine nucleosides, amines, amino acids, triazol nucleosides, or corticosteroids.

[0144] In addition, the pharmaceutical composition of the invention may also comprise another agent which is capable of modulating immune cell, notably T cell or monocyte, activation and/or function. In particular, the pharmaceutical composition of the invention may further comprise a therapeutically effective amount of an antagonist to a co-inhibitory molecule. In some embodiments, the co-inhibitory molecule is selected from the group consisting of CD86, CD80, PDL-1, PDL-2, CTLA-4, PD1, LAG3, BTNL2, B7-H3, B7-H4, a butyrophilin, CD48, CD244, TIM-3, CD200R, CD200, CD160, BTLA, HVEM, LAIR1, TIM1, Galectin 9, TIM3, CD48, 2B4, CD155, CD112, CD113 and TIGIT. The antagonist to the co-inhibitory molecule includes an antibody against the co-inhibitory molecule. It is recognised that antagonist to other co-inhibitory molecules are well known in the art, such as those described in Mercier et al., Frontiers in Immunology, 6:418 (2015), Kyi et al., FEBS Letters, 588:368-376 (2014) and Pardoll, Nature Reviews, 12:252-264 (2012). In some other embodiments, the pharmaceutical composition described herein further comprises a therapeutically effective amount of an agonist to a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule is selected from the group consisting of CD154, TNFRSF25, GITR, 4-1BB, OX40, CD27, TMIGD2, ICOS, CD28, CD40, TL1A, GITRL, 41BBL, OX40L, CD70, HHLA2, ICOSL, a cytokine, LIGHT, HVEM, CD30, CD30L, B7-H2, CD80, CD86, CD40L, TIM4, TIM1, SLAM, CD48, CD58, CD155, CD112, DR3, GITR, CD2, and CD226. The agonist to the co-stimulatory molecule includes an agonistic antibody against the co-stimulatory molecule. It is recognised that agonists to co-stimulatory molecules are well known in the art, such as those described in Mercier et al., Frontiers in Immunology, 6:418 (2015), Kyi et al., FEBS Letters, 588:368-376 (2014) and Capece et al., J. Biomed. Biotechnol. 2012:926321, 17 pages (2012).

[0145] In another aspect of the invention, the administration is combined with an administration of therapeutically effective amount of chemotherapeutic agent, such as for example, taxol (paclitaxel) or taxotere (docetaxel).

[0146] Chemotherapeutic agents include without any limitations, anti-microtubule agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents such as nitrogen mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents such as anthracyclins, actinomycins and bleomycins; topoisomerase II inhibitors such as epipodophyllotoxins; antimetabolites such as purine and pyrimidine analogues and antifolate compounds; topoisomerase I inhibitors such as camptothecins; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; and cell cycle signalling inhibitors. In addition, the methods of the invention can be combined with another anti-cancer treatment, anti-angiogenic agent, or chemotherapeutic agent or radiation therapy. A preferred example is docetaxel or taxotere. Other examples include, gemcitabine, cisplatin diterpenoids and vinca alkaloids, paclitaxel, vinblastine, vincristine, and vinorelbine, carboplatin, cyclophosphamide, melphalan, and chlorambucil, busulfan, carmustine, dacarbazine, cyclophosphamide, melphalan, chlorambucil, busulfan, carmustine, dacarbazine, anti-neoplastic agents including, but not limited to, actinomycins such as dactinomycin, anthrocyclins such as daunorubicin and doxorubicin, bleomycins, epipodophyllotoxins, etoposide and teniposide; antimetabolite neoplastic agents, 5-fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, camptothecins, irinotecan HCl, and topotecan HCl.

[0147] A variety of different chemotherapeutic agents or anti-cancer polypeptides can also be selected. Information sources such as www.clinicaltrials.gov, www.ncbi.nlm.nih and www.drugs.com, include references to polypeptides and agents that can be selected.

[0148] The immunocytokine and the pharmaceutical compositions of the invention are especially useful in the treatment or prevention of several types of cancers.

[0149] Another aspect of the invention thus relates to the immunocytokine described herein for use in the treatment of cancer.

[0150] The invention also relates to a pharmaceutical composition comprising the immunocytokine described herein for use in the treatment of cancer.

[0151] The cancers which may be treated by the present immunocytokine are cancers in which the antigen recognised by the antibody moiety of said immunocytokine is expressed. These cancers include (but not limited to) the following: carcinomas and adenocarcinomas, including that of the bladder, breast, colon, head-and-neck, prostate, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin, and including squamous cell carcinoma; hematopoietic tumours of lymphoid lineage, including multiple myeloma, leukaemia, acute and chronic lymphocytic (or lymphoid) leukaemia, acute and chronic lymphoblastic leukaemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin lymphoma (e.g. Burkitt's lymphoma); hematopoietic tumours of myeloid lineage, including acute and chronic myelogenous (myeloid or myelocytic) leukaemias, and promyelocytic leukaemia; tumours of mesenchymal origin, including fibrosarcoma, osteosarcoma and rhabdomyosarcoma; tumours of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; and other tumours, including melanoma, teratocarcinoma, xeroderma pigmentosum, keratoacanthoma, and seminoma, and other cancers yet to be determined in which said antigen is expressed. By cancers in which the antigen recognised by the antibody moiety of said immunocytokine is expressed, it is herein referred to cancers displaying high expression of said antigen, relative to the expression level of said antigen on a normal adult cell.

[0152] Other agents described above, e.g. anti-angiogenic agents or chemotherapeutic agents may be present in the composition being administered or may be administered separately. In one aspect of the invention, the administration is performed with the other active principle, either simultaneously, separately or sequentially over time. When the administration is performed simultaneously, the two active principles may be combined in a single pharmaceutical composition, comprising the two compositions, such as a tablet or a gel capsule. On the other hand, the two active principles may, whether or not they are administered simultaneously, be present in separate pharmaceutical compositions. To this end, the combination may be in the form of a kit comprising, on the one hand, the immunocytokine described herein and, on the other hand, the second active principle, the immunocytokine described herein and the second active principle being in separate compartments and being intended to be administered simultaneously, separately, or sequentially over time.

[0153] The present combination can be administered especially for treating cancer in combination with chemotherapy, protein therapy (i.e., using a therapeutic agent such as an antibody or recombinant protein), gene therapy, radiotherapy, immunotherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above.

[0154] Cytokines such as IL-15 are capable of stimulating tumour-specific T cell responses that are highly-specific. For example, administration of IL-15 induces the selective activation and proliferation in CD8 T cells and NK cells, the very cell types most amenable to mediating anti-tumour responses (Waldmann, J Investig Dermatol Symp Proc. 16(1): S28-30, 2013).

[0155] In some embodiments, the immunocytokine can be used in a method of modulating Immune cell function, mediated by binding of the cytokine moiety, preferably IL-15, of said immunocytokine. Preferably, said immune cell is a T cell or a monocyte. Such methods can include contacting the immune cell, preferably a T cell or a monocyte, with the immunocytokine described herein. In some embodiments, the method for modulating the immune cell (notably T cell or monocyte) function includes administering an effective amount of a composition comprising an immunocytokine provided herein to a subject. In some aspects, the T cell function that is modulated includes increasing T cell activation. Such T cell activation can further include increasing T cell proliferation. In some aspects, the monocyte function that is modulated includes increasing secretion of anti-cancer cytokines. Methods for assaying the modulation of an immune response are well known to one of skill in the art, and it is understood that a skilled artisan would be able to readily conduct such assays.

[0156] In some embodiments, an immunocytokine or a composition comprising an immunocytokine, including as described herein, can be used either alone or in combination with another compound or treatment. For example, in some embodiments, the other compound is an antagonist to a co-inhibitory molecule or an agonist to a co-stimulatory molecule. In such embodiments, the combined therapy leads to reinvigoration or de novo activation of the immune system through activated T cells that is greater than the administration of either compound or treatment individually. This activation of the immune system will result in a highly beneficial physiological response in the treatment of cancer.

[0157] In some embodiments, the methods described herein can include administering a therapeutically effective amount of an immunocytokine in combination with a therapeutically effective amount of an antagonist to a co-inhibitory molecule. In some embodiments, the co-inhibitory molecule is selected from the group consisting of CD86, CD80, PDL-1, PDL-2, CTLA-4, PD1, LAG3, BTNL2, B7-H3, B7-H4, a butyrophilin, CD48, CD244, TIM-3, CD200R, CD200, CD160, BTLA, HVEM, LAIR1, TIM1, Galectin 9, TIM3, CD48, 2B4, CD155, CD112, CD113 and TIGIT. The antagonist to the co-inhibitory molecule includes an antibody against the co-inhibitory molecule. It is recognised that antagonist to other co-inhibitory molecules are well known in the art, such as those described in Mercier et al., Frontiers in Immunology, 6:418 (2015), Kyi et al., FEBS Letters, 588:368-376 (2014) and Pardoll, Nature Reviews, 12:252-264 (2012). According to this embodiment, the invention relates to an immunocytokine for use in treatment of cancer as described above, said use further comprising the administration of an antagonist to a co-inhibitory molecule, wherein said co-inhibitory molecule is selected from the group consisting of CD86, CD80, PDL-1, PDL-2, CTLA-4, PD1, LAG3, BTNL2, B7-H3, B7-H4, a butyrophilin, CD48, CD244, TIM-3, CD200R, CD200, CD160, BTLA, HVEM, LAIR1, TIM1, Galectin 9, TIM3, CD48, 2B4, CD155, CD112, CD113 and TIGIT.

[0158] In some embodiments, the methods described herein can include administering a therapeutically effective amount of an immunocytokine in combination with a therapeutically effective amount of an agonist to a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule is selected from the group consisting of CD154, TNFRSF25, GITR, 4-1BB, OX40, CD27, TMIGD2, ICOS, CD28, CD40, TL1A, GITRL, 41BBL, OX40L, CD70, HHLA2, ICOSL, a cytokine, LIGHT, HVEM, CD30, CD30L, B7-H2, CD80, CD86, CD40L, TIM4, TIM1, SLAM, CD48, CD58, CD155, CD112, DR3, GITR, CD2, and CD226. The agonist to the co-stimulatory molecule includes an agonistic antibody against the co-stimulatory molecule. It is recognised that agonists to co-stimulatory molecules are well known in the art, such as those described in Mercier et al., Frontiers in Immunology, 6:418 (2015), Kyi et al., FEBS Letters, 588:368-376 (2014) and Capece et al., J. Biomed. Biotechnol. 2012:926321, 17 pages (2012). According to this embodiment, the invention relates to an immunocytokine for use in treatment of cancer as described above, said use further comprising the administration of an agonist to a co-stimulatory molecule, wherein said co-stimulatory molecule is selected from the group consisting of CD154, TNFRSF25, GITR, 4-1BB, OX40, CD27, TMIGD2, ICOS, CD28, CD40, TL1A, GITRL, 41BBL, OX40L, CD70, HHLA2, ICOSL, a cytokine, LIGHT, HVEM, CD30, CD30L, B7-H2, CD80, CD86, CD40L, TIM4, TIM1, SLAM, CD48, CD58, CD155, CD112, DR3, GITR, CD2, and CD226.

[0159] The examples that follow are merely exemplary of the scope of this invention and content of this disclosure. One skilled in the art can devise and construct numerous modifications to the examples listed below without departing from the scope of this invention.

EXAMPLES

Example 1: Sequences of the Constructions

[0160] 1. Sequences

TABLE-US-00001 TABLE 1 Antibodies variable regions. HC (variable HC (variable HC (variable LC (variable region): SEQ ID NO: region): DNA region): protein region): DNA protein Anti-PDL1 1 2 3 4 H16/L16 5 6 7 8 NHS76 9 10 11 12 C9G4 13 14 15 16

TABLE-US-00002 TABLE 2 Constant regions. SEQ ID NO: DNA Protein Human IgG1.DELTA..kappa. 17 18 Human IgG4.DELTA..kappa. 19 20 Human Kappa constant 21 22 Human Lambda constant 23 24

[0161] Similar results were obtained with a human IgG1 instead of a human IgG1.DELTA.K

TABLE-US-00003 TABLE 3 Cytokines. SEQ ID NO: DNA Protein Human IL15 25 26 Human IL2 27 28 Human CCL4 29 30 Human INF.alpha.2a 31 32 NanoLuc .RTM. 33 34 Human IL36.gamma. 171 172

TABLE-US-00004 TABLE 4 Linkers. SEQ ID NO: DNA Protein L1 35 36 L2 37 38 L3 39 40 L4 41 42 L5 43 44 L6 45 46 L7 165 166 L8 167 168 L9 169 170

TABLE-US-00005 TABLE 5 Cloning sites (for some NanoLuc .RTM. constructions, two amino acids were added to create a new and necessary restriction site). SEQ ID NO: DNA Protein Cloning site 47 48

TABLE-US-00006 TABLE 6 ICC. SEQ ID NO: DNA Protein HC: H16/L16-PVGLIG-IL15 49 50 HC: NHS76-PVGLIG-hIL15 51 52 H16/L16-PVGLIG-hIL15 HC: 49 HC: 50 LC: 53 LC: 54 NHS76-PVGLIG-hIL15 HC: 51 HC: 52 LC: 55 LC: 56

TABLE-US-00007 TABLE 7 ICC-full constructs. SEQ ID NO: DNA Protein PDL1-GPLGMLSQ- C-term HC 57 58 NanoLuc C-term LC 59 60 N-term HC 61 62 N-term LC 63 64 PD-L1-GPLGMLSQ-I C-term HC 65 66 C-term LC 67 68 N-term HC 69 70 N-term LC 71 72 PD-L1-GPLGIAGQ- C-term HC 73 74 NanoLuc C-term LC 75 76 N-term HC 77 78 N-term LC 79 80 PD-L1-GPLGIAGQ- C-term HC 81 82 IL2 C-term LC 83 84 N-term HC 85 86 N-term LC 87 88 PD-L1-GPLGLWAQ- C-term HC 89 90 NanoLuc C-term LC 91 92 N-term HC 93 94 N-term LC 95 96 PD-L1-GPLGLWAQ- C-term HC 97 98 IL2 C-term LC 99 100 N-term HC 101 102 N-term LC 103 104 PD-L1-PVGLIG- C-term HC 105 106 NanoLuc C-term LC 107 108 N-term HC 109 110 N-term LC 111 112 PD-L1-PVGLIG-IL2 C-term HC 113 114 C-term LC 115 116 N-term HC 117 118 N-term LC 119 120 PD-L1-PLGLAG- C-term HC 121 122 NanoLuc C-term LC 123 124 N-term HC 125 126 N-term LC 127 128 PD-L1-PLGLAG -IL2 C-term HC 129 130 C-term LC 131 132 N-term HC 133 134 N-term LC 135 136 PD-L1-GIVGPL- C-term HC 137 138 NanoLuc C-term LC 139 140 N-term HC 141 142 N-term LC 143 144 PD-L1-GIVGPL-IL2 C-term HC 145 146 C-term LC 147 148 N-term HC 149 150 N-term LC 151 152 9G4-PVGLIG- C-term HC 153 154 NanoLuc 9G4-PVGLIG-CCL4 C-term HC 155 156 9G4-PVGLIG-IL15 C-term HC 157 158 9G4-PVGLIG-IFN.alpha. C-term HC 159 160 NHS76-PVGLIG- C-term HC 161 162 IL15 H16/L16-PVGLIG- C-term HC 163 164 IL15 H16/L16-PVGLIG- 173 174 IL36G heavy chain 9G4-SGRS-hCXCL10 175 176 heavy chain NHS76-SGRS- 177 178 hCXCL10 heavy chain H16L16-SGRS- 179 180 hCXCL10 heavy chain H16L16-SGRS-hIL15 181 182 heavy chain H16L16-SGRSA- 183 184 hIFNa heavy chain H16L16-PSSRRRVN- 185 186 hIFNa heavy chain

[0162] 1.2. Obtention of the Immunocytokines (ICC)

[0163] Sequences coding for the whole ICC (see point 1. Sequences) were cloned into pCDNA3.4 vectors by using the HindIII/BamHI restriction sites, including a signal peptide (Thermo Fisher Scientific). The NanoLuc.RTM. DNA sequence was obtained from Promega. Linker modification was achieved by Q5 mutagenesis (New England Biolabs). Fusion proteins were obtained by transient protein expression in Expi HEK293 cells (Thermo Fisher Scientific) grown to a density of 2.5 10.sup.6/ml in Expi293 Expression Medium (Thermo Fisher Scientific) and co-transfected with 1.25 .mu.g/ml DNA (HC/LC: 1/1 w/w) using polyethyleneimine (PEI, Polyscience, DNA/PEI ratio: 1/4). 2 mM valproic acid (VPA, Sigma-Aldrich) was then added 3 hours post transfection. Supernatants containing the produced fusion proteins were harvested 6 days post-transfection. Proteins were purified by affinity chromatography on Protein A-Sepharose and formulated by overnight dialysis against 25 mM sodium citrate, 150 mM NaCl, 6% Saccharose pH 5.5. Some of the constructions realised are resumed in FIG. 1.

[0164] The positions (i.e., the fusion sites) of the linker-cytokine on the Ab are explained on FIG. 2.

Example 2: Proof of Immunocytokines Formation and Integrity by LC-MS Analysis

[0165] 2.1. Material and Methods:

[0166] All the purified ICC were characterised by SDS-PAGE in non-reducing, heated conditions and in reducing and heated condition. The SDS-PAGE migration of c9G4-PVGLIG-hIL15, NHS76-PVGLIG-hIL15 and H16/L16-PVGLIG-hIL15 are shown in FIG. 22. The apparent ICC molecular weights deduced from the SDS PAGE were in accordance of what was expected with theoretical calculations. At least of 80% for each ICC were complete ICC (H2L2). The monomeric content of ICC was determined by Size Exclusion Chromatography.

[0167] ICC integrity was also verified by LC-MS on glycosylated ICC and on deglycosylated ICC after IdeS digestion. Reverse phase separation was performed on an ultra-high-performance liquid chromatography (UHPLC) system (Acquity UPLC H-Class Bio system, Waters) coupled to a Synapt G2si mass spectrometer, instrument control was performed using MassLynx.RTM.software (Waters).

[0168] For c9G4PVGLIG-IL15, c9G4PVGLIG-hINFa, c9G4PVGLIG-hCCL4, deglycosylation of the Fc region was performed by incubating ICC solution 30 min at 37.degree. C. with IgGZERO.RTM. Enzyme (Genovis) at the concentration of 1 unit of enzyme per .mu.g of ICC according with the manufacturer's instructions. For NHS76-PVGLIG-hIL15 and H16L16-PVGLIG-hIL15 which carry out N-glycosylation on the IL15 part, deglycosylation was performed by adding 2 .mu.L of PNGaseF (New England Biolabs, 500000 U/mL) and 2 .mu.L of neuraminidase (New England Biolabs, 50000 U/mL) to 25 .mu.g of sample solution followed by incubation at 37.degree. C. overnight. The deglycosylated ICC were injected on a zorbax diphenyl column (Agilent) heated at 80.degree. C. Elution was performed with water as eluent A and acetonitrile as eluent B, both containing 0.1% FA and 0.02 TFA. The gradient condition was maintained at 30% B for 0.5 min, ramped to 46.9% in 6.5 min and increased to 95% in 0.1 min.

[0169] Subunit Fc/2 fragments were obtained by incubating ICC solution during 30 minutes at 37.degree. C. with IdeS enzyme (FabRICATOR.RTM., Genovis) at the concentration of 1 Unit of enzyme per .mu.g of ICC according to the manufacturer's instructions. Deglycosylated and digested ICC were injected on a PLRP-S column (Agilent) heated at 80.degree. C. Elution was performed with water as eluent A and acetonitrile as eluent B, both containing 0.05% TFA. The gradient condition was maintained at 5% B for 5 min, ramped to 50% in 45 min and increased to 95% in 2 min

[0170] 2.2. Results

[0171] On MS chromatogram peaks, m/z spectrum was extracted and mass determined after m/z spectrum deconvolution and calculated masses of deglycosylated ICC and deglycosylated fc/2 were compared with experimental masses.

[0172] The LS-MS analysis of deglycosylated c9G4-PVGLIG-hIL15, NHS76-PVGLIG-hIL15 and H16/L16-PVGLIG-hIL15 immunocytokines is represented in (FIG. 2 and FIG. 3). The pattern is consistent with what was expected for a fully formed ICC, as shown in Table 8:

TABLE-US-00008 TABLE 8 C-term HC ICC: Comparison of experimental vs. theoretical masses of ICC and Fc/2. ICC Subunit: c9G4-Fc/2 + Cytokine Calculated MW Experimental Calculated MW Experimental (Da) MW (Da) (Da) MW (Da) c9G4-PVGLIG- 171746 171760 37076 37081 hIL15 c9G4-PVGLIG- 158378 158379 43892 43892 hINFa c9G4-PVGLIG- 164725 164727 32470 32469 hCCL4 Subunits: NHS76 or H16/L16-Fc/2 + ICC Cytokine Calculated MW Experimental Calculated MW Experimental (Da) MW (Da) (Da) MW (Da) NHS76-PVGLIG- 169559 169566 37084 37085 hIL15 H16/L16- 172120 172138 37084 37083 PVGLIG-hIL15

[0173] Whole experimental masses confirmed the ICC structures (2 cytokines I antibody) and Fc/2+Cytokine masses confirmed the cytokine position.

[0174] Similar experiments were performed and similar conclusions were obtained for other ICCs.

Example 3: Linker Sequences and Cleavability by MMP-9/2

[0175] Five linkers from the literature were evaluated as substrates for MMP-9 when fused to four different sites on the PDL1 Ab sequence: N-term HC, N-term LC, C-term HC and C-term LC. The `GIVGPL` linker described as non-cleavable (Chau et al. Bioconjug Chem (2004) 15(4):931-41) was used as negative control. The summary of the constructions and results are displayed in FIG. 7.

[0176] 3.1. Materials and Methods

[0177] 2 .mu.g of purified ICC were incubated in presence of 40 ng recombinant MMP-9 or MMP-2 (molar ratio 25:1) in assay buffer containing 20 mM Tris pH 7.5, 10 mM CaCl.sub.2 and 100 mM NaCl in 20 .mu.l total volume. Samples were incubated at 30.degree. C. for 2.5 hrs under gentle agitation (300 rpm). Cleavage reaction was stopped by addition of loading buffer+reducing agent (4.times.XT sample Buffer and 20.times. Reducing Agent, BioRad) and sample heating at 90.degree. C. for 5 min.

[0178] Cleavage efficiency was then evaluated by SDS-PAGE. 20 .mu.l of sample were run onto Criterion TGX 4-15% Stain Free gels (BioRad) in Tris-Glycine-SDS buffer (BioRad) at 300 V for 20 min with protein standards (Precision Plus Protein Standards, Unstained, BioRad). Protein bands were then revealed using a ChemiDoc Touch Imager (BioRad).

[0179] 3.2. Results

3.2.1. Cleavability with different linkers in HC or LC C-terminal fusions of anti-PDL1 antibodies and two different molecules: IL2 and NanoLuc.RTM.:

[0180] The results are shown in FIG. 4

3.2.2. Cleavability with different linkers in HC or LC N-terminal fusions of anti-PDL1 antibodies and two different molecules: IL2 and NanoLuc.RTM.:

[0181] The results are shown in FIG. 5.

3.2.3. Cleavability of different c9G4-based ICC and H16/L16-IL15 and HHS76-IL15 ICC by human and murine MMP-9/2 (HC C-term fusions and PVGLIG linker). The results are shown in FIG. 6.

[0182] Note 1: IL15 and IFN.alpha. visualisation post-cleavage in impaired by the high level of glycosylation of the proteins. Sample deglycosylation prior cleavage allows the visualisation of the released cytokines, indicating the proteins are not proteolysed by MMP-912 (data not shown).

[0183] 3.3. Conclusion

[0184] Results of FIGS. 4, 5 and 6 are summarized in FIG. 7. Two fusions sites were identified, which allowed correct cleavage by the protease: N-term and C-term of the Ab heavy chain.

[0185] On the other hand, MMP-9 linkers were only weakly cleaved by either MMP-2 or MMP-9 when fused on N-term or C-term of the Ab light chains.

[0186] All five c9G4-fusion proteins are cleaved by murine and human MMP-9 and MMP-2 (FIG. 6).

Example 4: Analysis of the MMP-9 In Vitro Stability by LC-MS

[0187] 4.1. Materials and Methods

[0188] ICC were spiked in buffer (50 mM Tris pH7.5, 150 mM NaCl, 20 mM CaCl2)), sera, plasma, and fresh blood at a concentration of 100 .mu.g/ml with and without 12 nM MMP-9. Aliquots of 100 .mu.l were incubated at +37.degree. C. in Protein LoBind Tube (Eppendorf). After 24 hrs samples were removed from the oven and stored at -80.degree. C. until sample processing and analysis.

[0189] The ICC was immunoprecipitated from biological fluid by immunoprecipitation using M280 streptavidin magnetics beads coated with 4 .mu.g of CaptureSelect.TM. human IgG-Fc Biotin. After washing, ICC was eluted with 0.4% TFA in water and freeze drying. The samples were reconstituted in denaturing buffer (6 M Guanidine, 0.1 M Tris, 2 mM EDTA pH 8.0) and reduced in presence of DTT for 45 min at 56.degree. C. Acetic acid was then added to quench the reaction and samples were analysed by LC-MS.

[0190] Reverse phase separation was performed on an ultra-high-performance liquid chromatography (UHPLC) system (Acquity UPLC H-Class Bio system, Waters) coupled to a Synapt G2si mass spectrometer, instrument control was performed using MassLynx.RTM.software (Waters).

[0191] The reduced samples were diluted volume to volume with eluent A and injected on a PLRP-S column heated at 80.degree. C. with a flow rate of 0.5 mL/min. Elution was performed with water as eluent A and acetonitrile as eluent B, both containing 0.05% TFA. The gradient condition was maintained at 5% B for 5 min, ramped to 70% in 45 min and increased to 95% in 2 min.

[0192] For each peak of MS chromatogram, m/z spectrum was extracted and mass determined after m/z spectrum deconvolution.

[0193] 4.2. Results

[0194] 4.2.1 Validation of MMP-9 Cleavage Site in ICC.

[0195] In MMP-9 absence, no cleaved fragments were observed for both anti-PDL1-PVGLIG-NanoLuc.RTM. and anti-PDL1-GIVGPL-NanoLuc.RTM., thus both linkers were stable in buffer at the concentration of 12 nM.

[0196] Cleavage in presence of MMP-9 was checked by LC/MS as described in Example 2. The results are shown in FIGS. 8 A and B.

[0197] In the presence of MMP-9, 100% of antibody anti-PDL1-PVGLIG-NanoLuc.RTM. were cleaved in buffer after 24 hrs at 37.degree. C. (FIG. 8A). In fact, in FIG. 8A, the peak observed at 29.47 min which corresponds to the heavy chain of the anti-PDL1 antibody linked to a fragment of the PVGLIG linker (PVG). In comparison, no such a fragment is observed in FIG. 8B where the major signal corresponds to the full heavy chain of the anti-PDL1 antibody linked to the GIVGPL linker and NanoLuc.RTM.. MMP-9 is thus unable to cleave the GIVGPL peptide as opposed to PVGLIG peptide.

[0198] 4.2.2. MMP-9 Cleavage in Sera, Plasma and Fresh Blood

[0199] The results are shown in FIG. 9. With or without MMP-9 spiking, similar profiles were obtained at T0 and T24 in mouse heparin plasma, in mouse, cynomolgus, human sera and in whole mouse heparin blood. MMP-9 was inhibited in all the biological fluids tested, LC-MS profiles example presented in FIG. 9.

[0200] In conclusion, monitoring of In vitro antibody anti-PDL1 hkappa/hIgG4-PVGLIG-NanoLuc.RTM. cleavage was not possible in plasma heparin, sera and whole heparin blood (data not shown), probably because MMP-9 was inhibited. In fact, MMP-9 is known to be naturally attenuated in serum, plasma and blood, notably because of the presence of protease inhibitors like A2M.

Example 5: Attenuation of hIL15 when Fused to an Antibody

[0201] In order to evaluate and to compare the biological activity of hIL15, when linked to the ICC or after cleavage by MMP9, a test of IL-15 receptor dimerisation was performed. IL15 in its active form signals through the dimerisation of its two receptor subunits IL2R.beta./IL2R.gamma..

[0202] 5.1. Materials and Methods

[0203] 5.1.1. Materials and Reagents Recombinant human recombinant pro-MMP-9 was purchased from R&D Systems and activated by 1 mM 4-Aminophenylmercuric acetate (APMA) in buffer containing 50 mM Tris, 150 mM NaCl, 10 mM CaCl2), 0.05% Brij-35 (w/v), pH7.5. APMA was then removed using Zeba.TM. Spin Desalting Columns (ThermoFisher Scientific) and APMA-free MMP-9 was immediately stored at -80.degree. C. until needed. Recombinant human IL15 was purchased from PeproTech. IL15 activity was monitored using the PathHunter.RTM. U2OS IL2R.beta./IL2R.gamma./(IL2R.alpha.) Dimerisation Bioassay (DiscoverX, Eurofins). The assay allows the detection of the IL15-induced dimerisation of the two receptor subunits IL2R.beta. and IL2R.gamma..

[0204] 5.1.2. Methods

[0205] The activity of three IL15-based immunocytokines was assessed: NHS76-PVGLIG-hIL15, H16/L16-PVGLIG-hIL15 and c9G4-PVGLIG-hIL15. The effect of recombinant hIL15 and hMMP-9 was also evaluated using the same procedure. All samples were incubated for two hours in presence (+MMP-9) or absence (-MMP-9) of recombinant hMMP-9 in an assay buffer containing 50 mM Tris, 150 mM NaCl, 10 mM CaCl2) pH 7.5. Cleavage efficiency was controlled by SDS-PAGE analysis and samples were immediately stored at -20.degree. C. until processing. U2OS IL2R.beta./IL2R.gamma./IL2R.alpha. cells were treated by either cleaved or uncleaved ICC and controls for 6 hours. Detection reagent was then added and chemiluminescence intensity was recorded with a microplate reader (Infinite M1000Pro, Tecan). Data analysis was performed with the Prism 7.01 software (GraphPad).

[0206] 5.2. Results

[0207] The results of the hIL15 induced receptor dimerisation for the three tested ICC are presented in FIG. 10 as function of the hIL15 concentration.

[0208] The EC.sub.50 of each compound or ICC are shown in Table 9.

[0209] As seen in FIG. 10 and Table 9, hIL15 and hIL15+MMP-9 highly induce receptor dimerisation and thus validate the experiment. On the contrary MMP-9 alone has no effect on IL2R.beta./IL2R.gamma. dimerisation as expected.

[0210] In the absence of MMP-9, the three evaluated ICC had no significant activity on receptor dimerisation. On the other hand, pre-treatment of the same molecules with MMP-9 results in activity recovery through IL15-induced receptor dimerisation in a dose dependent manner.

[0211] It appears thus clearly from these experiments that IL-15 shows a highly attenuated activity when linked to the three antibodies. After cleavage of the ICC by MMP-9, IL15 is liberated in its active form and is again able to fulfil its biological activity.

TABLE-US-00009 TABLE 9 EC.sub.50 of Ab-bound hIL15 vs free hIL15 (recombinant or released after linker cleavage). IL15 NHS76 IL15 H16/L16 IL15 c9G4 IL15 -MMP-9 +MMP-9 -MMP-9 +MMP-9 -MMP-9 +MMP-9 -MMP-9 +MMP-9 IC.sub.50 0.72 .+-. 0.57 0.71 .+-. 0.42 ND.dagger. 8.84 .+-. 1.08 ND.dagger. 2.84 .+-. 1.06 ND.dagger. 2.07 .+-. 1.79 (nM) .dagger.EC.sub.50 values could not be determined due to impaired IL15 ability to trigger receptor dimerisation. function.

Example 6: In Vivo Stability of ICC

[0212] 6.1. Materials and Methods:

[0213] 6.1.1. Engraftment of Mice and Injection of ICC

[0214] Six-week-old immunocompetent BALB/c mice were used for all in vivo assessments. They were housed in sterilized filter-topped cages, maintained in sterile conditions and manipulated according to French and European guidelines.

[0215] RENCA (ATCC: CRL-2947), a murine renal carcinoma cell line expressing PDL1 was selected for in vivo evaluations. Mice were injected subcutaneously at DO with 0.5.times.10.sup.6 cells. When tumours reached 100 mm.sup.3 (11-12 days post tumour cell injection), animals were divided into 9 groups of 6 mice with comparable tumour size and administered intraperitoneally with PDL1-PVGLIG-NanoLuc.RTM. or the control isotype c9G4-PVGLIG-NanoLuc.RTM., 200 .mu.g/mouse Q1d1.

[0216] 6.1.2. Serum Samples

[0217] Animals were sacrificed at 0, 3, 6, 24 and 48 hours post administration and blood samples were collected in Na-heparinised tubes by cardiac puncture. Blood samples were centrifuged for 15 min at 1500 g, 4.degree. C. and plasma was collected in 96 well U bottomed plates. Plasma samples were frozen (-80.degree. C.) prior to analysis.

[0218] 6.1.3. Tumour Samples

[0219] Tumours were sampled and snap frozen in liquid nitrogen and were stored at -80.degree. C.

[0220] Prior to evaluation, the tumours are "resuspended" in 50 mM Tris-HCL buffer, 150 mM NaCl, 0.5% DOC, 1% Igepal, 1% Triton X100 containing an inhibitor cocktail (lysis buffer) at 200 mg of tumour per ml of buffer.

[0221] The tumours are disrupted with Minilys by 3 cycles of agitation at 5000 rpm in the presence of steel balls for 15 seconds. Between 2 cycles the tubes are kept on ice for 2 min. The solution is placed in a 2 ml conical Eppendorf tube and centrifuged at 11500 g at 4.degree. C. for 10 min. The supernatant, i.e., the cell lysate, is recovered. Its protein content is measured by Bicinconinic Acid assay according to the manufacturer's instructions.

[0222] 6.1.4. Western Blot Analysis

[0223] Serum samples as well as tumour samples were analysed by Western Blot. For each in vivo experiment the Western blots were done in triplicates.

[0224] Briefly, samples to be analysed are loaded on precast 4-15% polyacrylamide gel, at a rate of 0.1 .mu.l for the plasma samples and 2 .mu.l for the tumour lysates. Samples are migrated under heated, non-reduced conditions. Proteins are then transferred on nitrocellulose membranes using Trans-Blot.RTM. Turbo.TM. Midi Nitrocellulose Transfer Packs (2.5 A, 25 V, 7 min). After a 1 h, room-temperature saturation step in Tris buffered saline, 0.05% Tween 20, 1% milk (TBS-T 1% milk), membranes are incubated 1 h at room temperature with goat anti-hlgG-HRP, Fc.gamma. Fragment specific (1/50000). After three washes, membranes labelling is analysed on ChemiDoc.TM. Touch Gel Imaging System.

[0225] 6.1.5. Softwares

[0226] Densitometric analysis of western blot are carried out with Image Lab (Bio-Rad).

[0227] Statistical analyses are carried out with GraphPad Prism6.

[0228] 6.2. Results:

[0229] 6.2.1 In Vivo Stability of ICC in Mice Sera.

[0230] The stability of the ICC was evaluated in mice sera as a function of time. FIG. 11 shows the results obtained by western blot at 3 h, 6 h and 24 h post ICC injection. The amount of circulating ICC dramatically decreases as a function of time for each ICC construct tested. Without being bound by theory, this result is most easily explained by the sequestration of the ICC in the tumour and/or by the natural in vivo Ab degradation.

[0231] The western blots were submitted to densitometric analysis. The graphic representations (FIG. 12) shows the percentage of uncleaved ICC calculated as follow: [Intensity of the uncleaved ICC/(cleaved+uncleaved)ICC].times.100. Both model ICC behaved similarly in blood circulation, i.e. they looked stable over a 6 hours period, before losing progressively their NanoLuc.RTM. counterpart.

[0232] Overall, there was no significant difference between circulating c9G4 ICC and PDL1 ICC.

[0233] 6.2.2. In Vivo Stability of ICC in Mice Tumour Samples.

[0234] The stability of the ICC was evaluated in mice tumour samples as a function of time. FIG. 14 shows the results obtained by western blot at 3 h, 6 h and 24 h post ICC injection.

[0235] The Western Blots were submitted to densitometric analysis, the graphic representations (FIG. 15) show the percentage of uncleaved ICC calculated as previously described.

[0236] Both model ICC behaved similarly, i.e., the NanoLuc.RTM. counterpart was cleaved in RENCA tumours over a 48-hour period. PDL1 ICC cleavage was detected very quickly (less than 60% of uncleaved form in the tumour as early as 3 hours post-injection). At 24 hour-post injection, more than 80% of the total ICC present in the tumour was cleaved.

[0237] The cleavage of the c9G4 ICC was slower, with about 20% of the ICC cleaved in the tumour 6 hours post-injection. However, at 24 hours post injection, more than 80% of the total ICC present in the tumour is cleaved. Statistical analysis was performed on tumour-sequestered ICC. The amount of total tumour sequestered ICC was determined by adding of the signals obtained for the uncleaved ICC and the cleaved ICC at each time point.

[0238] FIG. 16 shows a quantitative analysis of total ICC (cleaved and uncleaved) present in the tumours as a function of time.

[0239] Overall the PDL1-ICC was more sequestered in the RENCA tumours than the control c9G4 ICC.

[0240] These results demonstrate that a tumour-specific ICC is able to accumulate quickly in the tumour environment. After accumulation, the ICC is cleaved by the tumour-specific protease releasing specifically the cytokine in the tumour environment.

[0241] 6.2.3. In Vivo Stability of ICC in Mice Sera Compared to Tumour Samples.

[0242] The behaviour of the two ICC was compared in plasma versus tumour samples in RENCA engrafted mice as a function of time.

[0243] FIG. 17 shows that regardless of the nature of the antibody moiety, ICC are significantly more cleaved in the RENCA tumours than in the mouse plasma.

Example 7: In Vitro T Cell Activation with ICC

[0244] 7.1. Materials and Methods:

[0245] 7.1.1 Materials and Methods.

[0246] Recombinant human IL15 was purchased from PeproTech.

[0247] The following ICC constructions were tested: [0248] NHS76-PVGLIG-IL15; [0249] H16/L16-PVGLIG-IL15; and [0250] c9G4-PVGLIG-IL15

[0251] The following controls were added: hIL15, MMP9, hIL15+MMP9 and individual antibodies.

[0252] 7.1.2. Methods

[0253] The activity of three IL15-based immunocytokines, i.e. NHS76-IL15, H16/L16-IL15, and c9G4-IL15, was assessed on their ability to activate murine and human CD3' T cells. The effect of hIL15+/-MMP9, MMP9 and individual antibodies was also evaluated using the same procedure.

[0254] Murine CD3.sup.+ T cells were isolated by negative selection from the spleen of balb/c mice (Charles River) using the murine pan T cells isolation kit II (Miltenyi, 130-095-130) according to the manufacturer's instructions. Two spleens per experiment were used for the T cells isolation.

[0255] Human peripheral blood mononuclear cells (PBMC) were isolated from fresh blood from healthy donors' cytapheresis rings by density gradient centrifugation. CD3 T cells were then purified by negative selection from PBMC using the human pan T cells isolation kit II (Miltenyi, 130-096-535).

[0256] Murine and human CD3.sup.+ T cells were seeded at 200 000 cells/well in culture medium, RPMI1640+10% FCS+2 mM L-glutamine+10 mM Hepes+1% Penicillin/streptomycin+0.1 mM beta-2-mercaptoethanol or RPMI 1640+10% FCS+1% L-glutamine+1% Sodium Pyruvate+1% Penicillin/streptomycin, respectively.

[0257] They were then treated with human IL-15 as a positive control at 100 and 200 ng/ml or with the different ICC constructions at 6 .mu.g/ml with an equivalent of 200 ng/ml for IL-15 in the presence or not of MMP9. Cells were incubated at 37.degree. C. 5% C02 and culture medium was refreshed after 3 days.

[0258] Activation of CD3.sup.+ T cells was monitored after 6 days of treatment through the expression of CD25 and CD69 surface markers. The expression of these markers was assessed by flow cytometry (Novocyte, ACEA). Cell culture supernatants were transferred into 96-wells plates for interferon-.gamma. secretion analysis by flow cytometry (BD, CBA IFN Flex Set).

[0259] For each murine and human T cell activation experiment, the analysis was performed in triplicate; three human healthy donors were tested.

[0260] Data analysis was performed with the Prism 7.01 software (GraphPad).

[0261] 7.2. Results

[0262] The ability of ICC to activate murine T cells was compared to controls. T cell activation was measured by T cell expression of CD69 or CD25. FIGS. 23A and B, show that NHS76-PVGLIG-IL15 and c9G4-PVLGIG-IL15 induce the expression of CD69 and CD25 in presence of MMP-9 but not in the absence of MMP-9. Activation of murine T cells is thus not observed when the ICC are uncleaved. Negative control (antibody alone, MMP-9 alone, buffer alone) or positive control (IL15 and IL15+MMP-9 alone) were also tested to confirm the validity of the experiment.

[0263] As shown in FIGS. 23 B and C, cleaved H16/L16-PVGLIG-IL15 (but not the ICC uncleaved form) is also able to activate murine T cell activation in a similar way.

[0264] The same experiments were performed with human T cells from healthy donors. T cell activation was measured by T cells expression of CD69 or CD25. NHS76-PVGLIG-IL15, c9G4-PVLGIG-IL15 and H16L16-PVGLIG-IL15 induce expression of CD69 and CD25 in presence of MMP-9 but not in absence of MMP-9 (FIGS. 24 A, B, C and D). Activation of the human T cells was thus not observed when the ICC are uncleaved. Negative control (antibody alone, MMP-9 alone, buffer alone) or positive control (IL15 and IL15+MMP-9 alone) were also tested to confirm the validity of the experiment.

[0265] This activation of T cells in presence of cleaved (but not of uncleaved ICC) was also measured by the secretion of IFN-.gamma. by human T cells from two different donors.

Example 8: Attenuation of hIL36.gamma. when Fused to an Antibody

[0266] To evaluate and compare the biological activity of hIL36.gamma. when part of an ICC or after cleavage by MMP9, IL36.gamma. activity was assayed by measuring CXCL8 production in human epidermoid cancer A431 cell culture. This assay is based on the finding that IL36.gamma. signaling pathway can trigger CXCL8 secretion.

[0267] 8.1. Materials and Methods

[0268] 8.1.1. Materials and Reagents

[0269] Recombinant human recombinant pro-MMP-9 was purchased from R&D Systems and activated by 1 mM 4-Aminophenylmercuric acetate (APMA) in buffer containing 50 mM Tris, 150 mM NaCl, 10 mM CaCl.sub.2, 0.05% Brij-35 (w/v), pH7.5. APMA was then removed using Zeba.TM. Spin Desalting Columns (ThermoFisher Scientific) and APMA-free MMP-9 was immediately stored at -80.degree. C. until needed. Recombinant human IL36.gamma. was purchased from R&D Systems. IL8 was dosed using the Human IL8 DuoSet ELISA (R&D Systems).

[0270] 8.1.2. Methods

[0271] To assess the activity of an IL36 .gamma.-based immunocytokine (H16/L16-PVGLIG-IL-36), squamous carcinoma A431 cells were incubated for 24 hours with the ICC or the different control molecules. All the samples were tested in the presence (+MMP-9) or absence (-MMP-9) of recombinant hMMP-9 in an assay buffer containing 50 mM Tris, 150 mM NaCl, 10 mM CaCl2) pH 7.5. The cell culture medium was then collected and hIL8 dosed by ELISA. The effect of recombinant hIL36.gamma. and H16/L16 antibody (+/-hMMP-9) was also evaluated using the same procedure. Data analysis was performed with the Prism 7.01 software (GraphPad).

[0272] 8.2. Results

[0273] The results of the hIL8 production in A431 cultures for the ICC and controls are presented in FIG. 26. Data are expressed in O.D (450 nm) corresponding to hIL8 expression level as a function of the hIL36.gamma. concentration.

[0274] As seen in FIG. 26, rhIL36.gamma. and rhIL36.gamma.+MMP-9 highly induced hIL8 production by A431 cells and thus validated the experimental format. On the other hand, neither MMP-9 alone nor the antibody H16/L16 (+/-MMP9) had any effect on hIL8 production as expected.

[0275] In the absence of MMP-9, the H16/L16-PVGLIG-hIL36.gamma. ICC had no significant activity on hIL8 production. On the other hand, pre-treatment of the same molecules with MMP-9 resulted in activity recovery as observed through IL-8 production in a dose dependent manner.

[0276] In summary, the C-terminal linkage of hIL36.gamma. using PVGLIG to H16/L16 antibody totally attenuates the cytokine activity. A MMP9 cleavage within the PVGLIG peptide restores it at a level comparable those of the free hIL36.gamma. cytokine at an equivalent dose.

Example 9: Attenuation of hIFN.alpha.2a when Fused to an Antibody

[0277] In order to evaluate and compare hIFN.alpha.2a biological activity when linked to the ICC or after cleavage by MMP9, IFN.alpha.2a activity was assayed with a cell-based luciferase reporter bioassay.

[0278] 9.1. Materials and Methods

[0279] 9.1.1. Materials and Reagents

[0280] Recombinant human recombinant pro-MMP-9 was purchased from R&D Systems and activated by 1 mM 4-Aminophenylmercuric acetate (APMA) in buffer containing 50 mM Tris, 150 mM NaCl, 10 mM CaCl2), 0.05% Brij-35 (w/v), pH7.5. APMA was then removed using Zeba.TM. Spin Desalting Columns (ThermoFisher Scientific) and APMA-free MMP-9 was immediately stored at -80.degree. C. until needed. Recombinant human IFN.alpha.2a was purchased from Pbl Assay Science (rhIFN.alpha.2a).

[0281] 9.1.2. Methods

[0282] IFN.alpha.2a activity was monitored using the GloResponse.TM. ISRE-luc2P/HEK293 (Promega). The ISRE-luc2P/HEK293 cell line contains the firefly luciferase gene under the control of ISRE stably integrated into HEK293 cells. Binding of IFN.alpha.2a to its receptor leads to the activation of the JAK pathway, thereby promoting ISRE-dependent transcription of the luciferase gene. Luminescence can then be detected upon addition of a substrate and quantified with a luminometer.

[0283] The activity of three hIFN.alpha.2a-based immunocytokines was assessed: NHS76-PVGLIG-h IFN.alpha.2a, H16/L16-PVGLIG-hIFN.alpha.2a and c9G4-PVGLIG-hIFN.alpha.2a. The effect of recombinant hIFN.alpha.2a was also evaluated using the same procedure. All samples were incubated for one hour in presence (+MMP-9) or absence (-MMP-9) of recombinant hMMP-9 in an assay buffer containing 50 mM Tris, 150 mM NaCl, 10 mM CaCl2) pH 7.5.

[0284] Cleavage efficiency was controlled by SDS-PAGE analysis and samples were immediately stored at -20.degree. C. until processing. ISRE-luc2P/HEK293 cells were treated with either cleaved or uncleaved ICC or controls overnight. Detection reagent (Bio-Glo.TM. luciferase Assay reagent) was then added and chemiluminescence intensity was recorded with a microplate reader (Infinite M1000Pro, Tecan). Data analysis was performed with the Prism 7.01 software (GraphPad).

[0285] For H16/L16-PVGLIG-hIFN.alpha.2a, cells were pre-treated or not with 10 .mu.g of H16/L16 antibody lacking a cytokine fusion in order to saturate IGF1-R binding sites on the ISRE-luc2P/HEK293 cell surface.

[0286] 9.2. Results

[0287] The results of the hIFN.alpha.2a activity assay for the three tested ICC are shown in FIG. 27 as a function of the hIFN.alpha.2a concentration. The EC50 of each compound or ICC are shown in Table 10.

[0288] Both hIFN.alpha.2a and hIFN.alpha.2a+MMP-9 strongly induce signalling response, as shown by the luciferase expression, thus validating the experiment (FIG. 27 and Table 10).

[0289] In the absence of MMP-9, each of the three assessed ICC: c9G4-PVGLIG-hIFN.alpha.2a (FIG. 27A), NHS76-PVGLIG-hIFN.alpha.2a (FIG. 27B), and H16/L16-PVGLIG-hIFN.alpha.2a, with (FIG. 27C) or without (FIG. 27D) preincubation of the cells with 10 .mu.g/ml H16/L16 antibody, showed a highly attenuated activity compared to equimolar concentrations of hIFN.alpha.2a activity. On the other hand, pre-treatment of the same molecules with MMP-9 results in activity recovery in a dose dependent manner.

[0290] It appears thus clearly from these experiments that hIFN.alpha.2a shows an attenuated activity when linked to any of the three antibodies. After cleavage of the ICC by MMP-9, hIFN.alpha.2a is liberated in its active form and is again able to fulfil its biological activity.

TABLE-US-00010 TABLE 10 EC50 (nM) of Ab-bound hIFN.alpha.2a vs free hIFNa H16L16-hIFN.alpha.2a Pre incubation c9G4- NHS76- No pre with hIFN.alpha.2a hIFN.alpha.2a hIFN.alpha.2a incubation H16/L16 -MMP9 0.08 .+-. 4.00 .+-. 5.33 .+-. 2.6 0.03 .+-. 0.009 0.95 .+-. 0.05 0.01 1.40 +MMP9 0.05 .+-. 0.02 .+-. 0.03 .+-. 0.008 0.02 .+-. 0 0.02 .+-. 0 0.01 0.005

Example 10: Attenuation of hIL15 when Fused to an Antibody

[0291] To evaluate and to compare the biological activity of hIL15, when linked to the ICC or after cleavage by uPA, a biological test of IL15 activity was used (Promega IL15 Bioassay)

[0292] 10.1. Materials and Methods

[0293] 10.1.1. Materials and Reagents

[0294] Recombinant urokinase (=uPA) was purchased from Abcam: ab92767 for the human. Recombinant human IL15 was purchased from PeproTech (200-15).

[0295] IL15 activity was monitored using the IL-15 Bioassay from Promega (Cat. #JA2015). This is a bioluminescent cell-based assay designed to measure IL-15 stimulation or inhibition using the STAT-5 response element as a readout. When IL-15 binds to its receptor, receptor-mediated pathway signalling induces luminescence that can be detected upon addition of a substrate and quantified with a luminometer.

[0296] 10.1.2. Cleavage

[0297] Constructions to evaluate are incubated in PBS with uPA during 24 h at 37.degree. C.

[0298] Cleavage efficiency was controlled by SDS-PAGE analysis and samples were immediately stored at -20.degree. C. until processing.

[0299] 10.1.2. Methods

[0300] IL15 cells were treated for 6 h with either cleaved or uncleaved ICC or controls. Detection reagent (Bio-Glo.TM. luciferase Assay reagent) was then added and chemiluminescence intensity was recorded with a microplate reader (Mithras, Berthold). Data analysis was performed with the Prism 7.01 software (GraphPad).

[0301] 10.2. Results

[0302] The results of the hIL15 activity for the ICC and controls are presented in FIG. 28. Data are expressed in relative luminescence (RLU) corresponding to hIL15 activity level as a function of the STAT-5 activation.

[0303] Both rhIL15 and rhIL15+ uPA highly activated luminescence, thereby validating the experiment format. In the absence of uPA, the H16L16-SGRSA-hIL15 ICC had no significant IL15 activity. Pre-treatment of the same molecule with uPA resulted in a significant recovery of activity as observed through the STAT-5 activation (FIG. 28).

[0304] In summary, the C-terminal linkage of hIL15 using SGRSA to H16/L16 antibody totally attenuates its activity. Cleavage by uPA within the SGRSA peptide significantly restores IL15 activity.

Example 11: Attenuation of hIFN.alpha. when Fused to an Antibody

[0305] To evaluate and to compare the biological activity of hIFN.alpha., when linked to the ICC or after cleavage by uPA, a biological test of IFN.alpha. activity was used (GloResponse ISRE-luc2P).

[0306] 11.1. Materials and Methods

[0307] 11.1.1. Materials and Reagents

[0308] Recombinant human urokinase (=uPA) was purchased from Abcam (ab92767). Recombinant human IFN.alpha. was purchased from PBL (11101-02). IFN.alpha. activity was monitored using the GloResponse ISRE-luc2P (Promega CS190701). This is a bioluminescent HEK293 cell-based assay designed to measure IFN.alpha. stimulation or inhibition using interferon-stimulated response element (ISRE) that drives transcription of the luciferase reporter gene luc2P (Photinus pyralis). When IFN.alpha. binds to its receptor, receptor-mediated pathway signalling induces luminescence that can be detected upon addition of a substrate and quantified with a luminometer.

[0309] 11.1.2. Cleavage

[0310] Constructions to evaluate are incubated in PBS with uPA during 24 h at 37.degree. C. Cleavage efficiency was controlled by SDS-PAGE analysis and samples were immediately stored at -20.degree. C. until processing.

[0311] 11.1.2. Methods

[0312] ISRE-luc2P/HEK293 cells were pre-treated for 1 h with 10 .mu.g of anti IGF1R antibody in order to saturate IGF1R binding sites on the cell surface.

[0313] ISRE-luc2P/HEK293 cells were treated with either cleaved or uncleaved ICC or controls overnight. Detection reagent (Bio-Glo.TM. luciferase Assay reagent) was then added and chemiluminescence intensity was recorded with a microplate reader (Mithras, Berthold). Data analysis was performed with the Prism 7.01 software (GraphPad).

[0314] 11.2. Results

[0315] The results of the hIFN.alpha. activity for the ICC and controls are presented in FIGS. 29A and 29B. Data are expressed in relative luminescence (RLU) corresponding to hIFN.alpha. activity level as a function of the ISRE activation.

[0316] 11.2.1 H16L16-SGRSA-hIFN.alpha.

[0317] As seen in FIG. 29A, rhIFN.alpha. and rhIFN.alpha.+uPA highly activated luminescence and thus validated the experiment format.

[0318] In the absence of uPA, the IFN.alpha. EC50 of H16/L16-SGRSA-hIFN.alpha. ICC showed a 2-log reduction with only 75% maximal activity. Pre-treatment of the same molecule with uPA resulted in a similar activity to rhIFN.alpha. as determined by ISRE activation.

[0319] In summary, the C-terminal linkage of hIFN.alpha. using SGRSA to H16/L16 antibody attenuates its activity. Cleavage by uPA within the SGRSA peptide restores it at a level similar to that of the free hIFN.alpha. cytokine.

[0320] 11.2.2 H16/L16-PSSRRRVN-hIFN.alpha.

[0321] As seen in FIG. 29B, r hIFN.alpha. and r hIFN.alpha.+uPA highly activated luminescence and thus validated the experiment format.

[0322] In the absence of uPA, the IFN.alpha. EC50 H16/L16-PSSRRRVN-hIFN.alpha. ICC displayed a 2-log reduction with a 75% maximal activity. Pre-treatment of the same molecule with uPA resulted in an activity recovery as observed through the ISRE activation.

[0323] In summary, the C-terminal linkage of hIFN.alpha. using PSSRRRVN to H16/L16 antibody attenuates its activity. Cleavage by uPA within the PSSRRRVN peptide restores activity to a level comparable to the free hIFN.alpha. cytokine.

Example 12: Attenuation of hCXCL10 when Fused to an Antibody

[0324] To evaluate and compare the biological activity of hCXCL10 when linked to the ICC or after cleavage by uPA, a biological test of .beta.-arrestin recruitment induced by CXCL10 binding to the CXCR3 receptor was performed (PathHunter eXpress CXCR3 CHOK1 .beta.-arrestin GPCR assay).

[0325] 12.1. Materials and Methods

[0326] 12.1.1. Materials and Reagents

[0327] Recombinant human urokinase (=uPA) was purchased from Abcam (ab92767). Recombinant human CXCL10 was purchased from Peprotech (300-12). CXCL10 activity was monitored using the PathHunter eXpress CXCR3 CHOK1 .beta.-arrestin GPCR assay (DiscoverX 93-0271E2). This is a bioluminescent CHO cell-based assay designed to measure .beta.-arrestin recruitment induced by CXCL10 binding to the CXCR3 receptor.

[0328] Activation of the GPCR-PK induces .beta.-arrestin-EA recruitment, forcing complementation of the two .beta.-galactosidase enzyme fragments (EA and PK). The resulting functional enzyme hydrolyzes substrate to generate a chemiluminescent signal that can be detected upon addition of a substrate and quantified with a luminometer.

[0329] 12.1.2. Cleavage

[0330] Constructions to evaluate are incubated in PBS with uPA during 1 h at 37.degree. C. Cleavage efficiency was controlled by SDS-PAGE analysis and samples were immediately stored at -20.degree. C. until processing.

[0331] 12.1.2. Methods

[0332] CHO-K1 CXCR3 cells were treated with either cleaved or uncleaved ICC or controls for 1 h30. Detection reagent was then added and chemiluminescence intensity was recorded 1 h later with a microplate reader (Mithras, Berthold). Data analysis was performed with the Prism 7.01 software (GraphPad).

[0333] 12.2. Results

[0334] The results of the hCXCL10 activity for the ICC and controls are presented in FIGS. 30A, 30B and 30C. Data are expressed relative luminescence (RLU) corresponding to hCXCL10 activity level as function of the CXCR3 .beta.-arrestin recruitment.

[0335] 12.2.1 9G4-SGRS-CXCL10

[0336] Both recombinant hCXCL10 and recombinant hCXCL10+uPA led to a strong activation of the luminescence signal, thereby validating the experiment format. In the absence of uPA, the 9G4-SGRS-CXCL10 ICC had no significant activity. Pre-treatment of the same molecules with uPA resulted in an activity recovery as observed through the CXCR3 .beta.-arrestin recruitment (FIG. 30A).

[0337] In summary, the C-terminal linkage of hCXCL10 using the SGRS linker to the 9G4 antibody attenuates its activity. Cleavage by uPA within the SGRS peptide restores CXCL10 activity almost to that of the free hCXCL10 cytokine.

[0338] 12.2.2 NHS76-SGRS-CXCL10

[0339] Both recombinant hCXCL10 and recombinant hCXCL10+uPA led to a strong activation of the luminescence signal, thereby validating the experiment format. In the absence of uPA, the NHS76-SGRS-CXCL10 ICC had no significant activity. Pre-treatment of the same molecule with uPA resulted in a partial recovery of activity as observed through the CXCR3 .beta.-arrestin recruitment (FIG. 30B). Full activity was not observed probably due to a partial proteolysis of the parent NHS76-SGRS-CXCL10.

[0340] In summary, the C-term linkage of hCXCL10 using SGRS to the NHS76 antibody attenuates its activity. Cleavage by uPA within the SGRS peptide restores CXCL10 activity.

[0341] 12.2.3 H16L16-SGRS-CXCL10

[0342] Both recombinant hCXCL10 and recombinant hCXCL10+uPA led to a strong activation of the luminescence signal, thereby validating the experiment format. In the absence of uPA, the H16/L16-SGRS-CXCL10 ICC had no significant activity. Pre-treatment of the same molecule with uPA resulted in a partially recovery activity as observed through the CXCR3 .beta.-arrestin recruitment (FIG. 30C). Full activity was not observed probably due to a partial proteolysis of the parent H16/L16-SGRS-CXCL10.

[0343] In summary, the C-term linkage of hCXCL10 using SGRS to H16/L16 antibody attenuates its activity. Cleavage by uPA within the SGRS peptide restores CXCL10 activity.

Sequence CWU 1

1

1861354DNAArtificialAtezolizumab Heavy Chain Variable Region 1gaggtccagc ttgtcgagtc aggtggaggt cttgtacagc caggaggaag tttgcgtttg 60agctgcgctg cttctgggtt cacttttagc gactcctgga tccactgggt cagacaggca 120cctggaaagg ggctggaatg ggttgcatgg atttccccct acggcggctc tacttattac 180gctgacagtg ttaaaggcag gtttaccata agcgccgaca cctccaaaaa caccgcctac 240ctgcagatga atagtctgcg agccgaagat actgccgtgt actattgcgc ccggcgccat 300tggcccggcg gctttgatta ttgggggcaa gggacactgg tgacagtgtc tgct 3542118PRTArtificialAtezolizumab Heavy Chain Variable Region 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala 1153320DNAArtificialAtezolizumab Light Chain Variable Region 3gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa 3204107PRTArtificialAtezolizumab Light Chain Variable Region 4Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 1055357DNAArtificialGanitumab Heavy Chain Variable Region 5caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggggac cctgtccctc 60acctgcgctg tctctggtgg ctccatcagc agtagtaact ggtggagttg ggtccgccag 120cccccaggga aggggctgga gtggattggg gaaatctatc atagtgggag caccaactac 180aacccgtccc tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc 240ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagatggacc 300gggcgtactg atgcttttga tatctggggc caagggacaa tggtcaccgt ctcaagc 3576119PRTArtificialGanitumab Heavy Chain Variable Region 6Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Thr Gly Arg Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser 1157336DNAArtificialGanitumab Light Chain Variable Region 7gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagggtgg aggctgagga tgttggggtt tattactgca tgcaaggtac acactggcct 300ctgacgttcg gccaagggac caaggtggag atcaaa 3368112PRTArtificialGanitumab Light Chain Variable Region 8Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 1109351DNAArtificialNHS76 Heavy Chain Variable Region 9caggtgcagc tgcaggagtc cggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcgctg tctctggtta ctccatcagc agtggttact actggggctg gattcggcag 120cccccaggga aggggctgga gtggattggg agtatctatc atagtgggag cacctactac 180aacccgtccc tcaagagtcg agtcaccata tcagtagaca cgtccaagaa ccagttctcc 240ctgaagctga gctctgtgac cgccgcagac acggccgtgt attactgtgc aagagggaag 300tggtcgaagt ttgactattg gggccaaggc accctggtca ccgtctcttc a 35110117PRTArtificialNHS76 Heavy Chain Variable Region 10Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Lys Trp Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser 11511324DNAArtificialNHS76 Light Chain Variable Region 11tcctctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60acatgccaag gagacagcct cagaagctat tatgcaagct ggtaccagca gaagccagga 120caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat tccagaccga 180ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattactg taactcccgg gacagcagtg gtaaccatgt ggtattcggc 300ggagggacca agctgaccgt ccta 32412108PRTArtificialNHS76 Light Chain Variable Region 12Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His 85 90 95Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 10513339DNAArtificial9G4 Heavy Chain Variable Region 13gaagtgatgt tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60tcctgtacag cctctggatt cactttcagt acctatgaca tgtcttgggt tcgccagact 120ccggagaaga ggctggagtg ggtctcaacc attagtagta gtcgtactta cacctactat 180tcagccagtg tgaaggggcg attcaccatc tccagagaca atgccaggaa catcctgtac 240ctgcaaatga gcagtctgag gtctgaggac acggccatgt attactgtgc aagacaattt 300tctcactggg gccaagggac tctggtcact gtctctgca 33914113PRTArtificial9G4 Heavy Chain Variable Region 14Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ser Thr Ile Ser Ser Ser Arg Thr Tyr Thr Tyr Tyr Ser Ala Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gln Phe Ser His Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110Ala15336DNAArtificial9G4 Light Chain Variable Region 15gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctcc 60atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120ttgttacaga ggccaggcca gtctccaacg cgcctaatct atctggtgtc taaactggac 180tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaagatc 240agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttcct 300cacacgttcg gaggggggac caagctggaa ataaaa 33616112PRTArtificial9G4 Light Chain Variable Region 16Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45Pro Thr Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro His Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 11017987DNAHuman 17gcgtccacaa agggcccaag cgtgttcccg ctagccccca gcagcaagag caccagcggc 60ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 120tggaacagcg gagccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagagcagc 180ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 240tacatctgta acgtgaacca caagcccagc aacaccaagg tggacaagag agtggagccc 300aagagctgtg acaagaccca cacctgtccc ccctgcccag cccccgagct gctgggcgga 360cccagcgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatcag cagaaccccc 420gaggtgacct gtgtggtggt ggacgtgtcc cacgaggacc cagaggtgaa gttcaactgg 480tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagagagga gcagtacaac 540agcacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 600gagtacaagt gtaaggtgtc caacaaggcc ctgccagccc caatcgaaaa gaccatcagc 660aaggccaagg gccagccaag agagccccag gtgtacaccc tgccacccag cagggaggag 720atgaccaaga accaggtgtc cctgacctgt ctggtgaagg gcttctaccc aagcgacatc 780gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac ccccccagtg 840ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gagcagatgg 900cagcagggca acgtgttcag ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 960cagaagagcc tgagcctgtc cccaggc 98718329PRTHuman 18Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32519978DNAHuman 19gcgtccacaa agggccccag cgtgttccct ctggccccct gcagcagaag caccagcgag 60tctacagccg ccctgggatg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 120tggaactctg gcgccctgac cagcggcgtg cacacctttc cagccgtgct gcagagcagc 180ggcctgtaca gcctgagcag cgtcgtgacc gtgcctagca gcagcctggg caccaagacc 240tacacctgta acgtggacca caagcccagc aacaccaagg tggacaagcg ggtggaatct 300aagtacggcc ctccctgccc cccctgccca gcccctgaat ttctgggcgg accctccgtg 360ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 420tgcgtggtgg tggacgtgtc ccaggaagat cccgaggtcc agttcaactg gtacgtggac 480ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagttcaa cagcacctac 540cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 600tgcaaggtct ccaacaaggg cctgcccagc tccatcgaga aaaccatcag caaggccaag 660ggccagcccc gcgagcccca ggtgtacaca ctgcccccca gccaggaaga gatgaccaag 720aaccaggtgt ccctgacctg cctcgtgaag ggcttctacc ccagcgatat cgccgtggaa 780tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 840gacggcagct tcttcctgta ctcccggctg accgtcgaca agtctagatg gcaggaaggc 900aacgtcttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 960ctgagcctga gcctggga 97820326PRTHuman 20Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295

300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly 32521321DNAHuman 21cgtacggtgg ccgctcccag cgtgttcatc ttcccaccca gcgacgagca gctgaagtct 60ggcaccgcca gcgtcgtgtg cctgctgaac aacttctacc cccgcgaggc caaggtgcag 120tggaaggtgg acaacgccct gcagtccggc aacagccagg aaagcgtcac cgagcaggac 180agcaaggact ccacctacag cctgagcagc accctgaccc tgagcaaggc cgactacgag 240aagcacaagg tgtacgcctg cgaagtgacc caccagggcc tgtccagccc cgtgaccaag 300agcttcaacc ggggcgagtg c 32122107PRTHuman 22Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10523318DNAHuman 23ggtcaaccca aggccgctcc cagcgtgacc ctgttccccc cctcgagcga ggagctgcag 60gccaacaagg ccaccctggt gtgtctgatc agcgacttct acccaggcgc cgtgaccgtg 120gcctggaagg ccgacagcag ccccgtgaag gccggcgtgg agaccaccac ccccagcaag 180cagagcaaca acaagtacgc cgccagcagc tacctgagcc tgaccccaga gcagtggaag 240agccacagga gctacagctg ccaggtcacc cacgagggca gcaccgtgga aaagaccgtg 300gccccaaccg agtgctcc 31824106PRTHuman 24Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys65 70 75 80Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 10525342DNAHuman 25aactgggtga atgtaataag tgatttgaaa aaaattgaag atcttattca atctatgcat 60attgatgcta ctttatatac ggaaagtgat gttcacccca gttgcaaagt aacagcaatg 120aagtgctttc tcttggagtt acaagttatt tcacttgagt ccggagatgc aagtattcat 180gatacagtag aaaatctgat catcctagca aacaacagtt tgtcttctaa tgggaatgta 240acagaatctg gatgcaaaga atgtgaggaa ctggaggaaa aaaatattaa agaatttttg 300cagagttttg tacatattgt ccaaatgttc atcaacactt ct 34226114PRTHuman 26Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser27399DNAHuman 27gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgact 39928133PRTHuman 28Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr 13029207DNAHuman 29gcaccaatgg gctcagaccc tcccaccgcc tgctgctttt cttacaccgc gaggaaactt 60cctcgcaact ttgtggtaga ttactatgag accagcagcc tctgctccca gccagctgtg 120gtattccaaa ccaaaagaag caagcaagtc tgtgctgatc ccagtgaatc ctgggtccag 180gagtacgtgt atgacctgga actgaac 2073069PRTHuman 30Ala Pro Met Gly Ser Asp Pro Pro Thr Ala Cys Cys Phe Ser Tyr Thr1 5 10 15Ala Arg Lys Leu Pro Arg Asn Phe Val Val Asp Tyr Tyr Glu Thr Ser 20 25 30Ser Leu Cys Ser Gln Pro Ala Val Val Phe Gln Thr Lys Arg Ser Lys 35 40 45Gln Val Cys Ala Asp Pro Ser Glu Ser Trp Val Gln Glu Tyr Val Tyr 50 55 60Asp Leu Glu Leu Asn6531495DNAHuman 31tgtgatctgc ctcaaaccca cagcctgggt agcaggagga ccttgatgct cctggcacag 60atgaggaaaa tctctctttt ctcctgcttg aaggacagac atgactttgg atttccccag 120gaggagtttg gcaaccagtt ccaaaaggct gaaaccatcc ctgtcctcca tgagatgatc 180cagcagatct tcaatctctt cagcacaaag gactcatctg ctgcttggga tgagaccctc 240ctagacaaat tctacactga actctaccag cagctgaatg acctggaagc ctgtgtgata 300cagggggtgg gggtgacaga gactcccctg atgaaggagg actccattct ggctgtgagg 360aaatacttcc aaagaatcac tctctatctg aaagagaaga aatacagccc ttgtgcctgg 420gaggttgtca gagcagaaat catgagatct ttttctttgt caacaaactt gcaagaaagt 480ttaagaagta aggaa 49532165PRTHuman 32Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu 16533513DNAOplophorus gracilirostris 33atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcg 51334173PRTOplophorus gracilirostris 34Leu Glu Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln1 5 10 15Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser 20 25 30Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile 35 40 45Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile 50 55 60Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile65 70 75 80Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu 85 90 95His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp 100 105 110Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys 115 120 125Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu 130 135 140Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn145 150 155 160Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala 165 1703518DNAArtificiallinker 35ccgctgggcc tggcgggc 18366PRTArtificiallinker 36Pro Leu Gly Leu Ala Gly1 53724DNAArtificiallinker 37ggcccgctgg gcattgcggg ccag 24388PRTArtificiallinker 38Gly Pro Leu Gly Ile Ala Gly Gln1 53924DNAArtificiallinker 39ggcccgctgg gcctgtgggc gcag 24408PRTArtificiallinker 40Gly Pro Leu Gly Leu Trp Ala Gln1 54124DNAArtificiallinker 41ggcccgctgg gcatgctgag ccag 24428PRTArtificiallinker 42Gly Pro Leu Gly Met Leu Ser Gln1 54318DNAArtificiallinker 43ccggtgggcc tgattggc 18446PRTArtificiallinker 44Pro Val Gly Leu Ile Gly1 54518DNAArtificiallinker 45ggcattgtgg gaccgctg 18466PRTArtificiallinker 46Gly Ile Val Gly Pro Leu1 5476DNAArtificiallinker 47ctcgag 6482PRTArtificiallinker 48Leu Glu1491704DNAArtificialHeavy Chain of Ganitumab-PVGLIG-IL15 49caggtgcagc tgcaggagtc gggaccagga ctggtgaagc cttcggggac cctgtccctc 60acctgcgctg tctctggtgg ctccatcagc agtagtaact ggtggagttg ggtccgccag 120cccccaggga aggggctgga gtggattggg gaaatctatc atagtgggag caccaactac 180aacccgtccc tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc 240ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagatggacc 300gggcgtactg atgcttttga tatctggggc caagggacaa tggtcaccgt ctcaagcgcg 360tccacaaagg gcccaagcgt gttcccgcta gcccccagca gcaagagcac cagcggcggc 420acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 480aacagcggag ccctgacctc cggcgtgcac accttccccg ccgtgctgca gagcagcggc 540ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 600atctgtaacg tgaaccacaa gcccagcaac accaaggtgg acaagagagt ggagcccaag 660agctgtgaca agacccacac ctgtcccccc tgcccagccc ccgagctgct gggcggaccc 720agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 780gtgacctgtg tggtggtgga cgtgtcccac gaggacccag aggtgaagtt caactggtac 840gtggacggcg tggaggtgca caacgccaag accaagccca gagaggagca gtacaacagc 900acctacaggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960tacaagtgta aggtgtccaa caaggccctg ccagccccaa tcgaaaagac catcagcaag 1020gccaagggcc agccaagaga gccccaggtg tacaccctgc cacccagcag ggaggagatg 1080accaagaacc aggtgtccct gacctgtctg gtgaagggct tctacccaag cgacatcgcc 1140gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc cccagtgctg 1200gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag cagatggcag 1260cagggcaacg tgttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320aagagcctga gcctgtcccc aggcccggtg ggcctgattg gcaactgggt gaatgtaata 1380agtgatttga aaaaaattga agatcttatt caatctatgc atattgatgc tactttatat 1440acggaaagtg atgttcaccc cagttgcaaa gtaacagcaa tgaagtgctt tctcttggag 1500ttacaagtta tttcacttga gtccggagat gcaagtattc atgatacagt agaaaatctg 1560atcatcctag caaacaacag tttgtcttct aatgggaatg taacagaatc tggatgcaaa 1620gaatgtgagg aactggagga aaaaaatatt aaagaatttt tgcagagttt tgtacatatt 1680gtccaaatgt tcatcaacac ttct 170450568PRTArtificialHeavy Chain of Ganitumab-PVGLIG-IL15 50Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Thr Gly Arg Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Pro Val Gly Leu Ile Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys 450 455 460Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr465 470 475 480Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys 485 490 495Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser 500 505 510Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu 515 520 525Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu 530 535 540Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile545 550 555 560Val Gln Met Phe Ile Asn Thr Ser 565511698DNAArtificialHeavy Chain of NHS76-PVGLIG-IL15 51caggtgcagc tgcaggagtc cggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcgctg tctctggtta ctccatcagc agtggttact actggggctg gattcggcag 120cccccaggga aggggctgga gtggattggg agtatctatc atagtgggag cacctactac 180aacccgtccc tcaagagtcg agtcaccata tcagtagaca cgtccaagaa ccagttctcc 240ctgaagctga gctctgtgac cgccgcagac acggccgtgt attactgtgc aagagggaag 300tggtcgaagt ttgactattg gggccaaggc accctggtca ccgtctcttc agcgtccaca 360aagggcccaa

gcgtgttccc gctagccccc agcagcaaga gcaccagcgg cggcacagcc 420gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgtc ctggaacagc 480ggagccctga cctccggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac 540agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcacccagac ctacatctgt 600aacgtgaacc acaagcccag caacaccaag gtggacaaga gagtggagcc caagagctgt 660gacaagaccc acacctgtcc cccctgccca gcccccgagc tgctgggcgg acccagcgtg 720ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcagaacccc cgaggtgacc 780tgtgtggtgg tggacgtgtc ccacgaggac ccagaggtga agttcaactg gtacgtggac 840ggcgtggagg tgcacaacgc caagaccaag cccagagagg agcagtacaa cagcacctac 900agggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 960tgtaaggtgt ccaacaaggc cctgccagcc ccaatcgaaa agaccatcag caaggccaag 1020ggccagccaa gagagcccca ggtgtacacc ctgccaccca gcagggagga gatgaccaag 1080aaccaggtgt ccctgacctg tctggtgaag ggcttctacc caagcgacat cgccgtggag 1140tgggagagca acggccagcc cgagaacaac tacaagacca cccccccagt gctggacagc 1200gacggcagct tcttcctgta cagcaagctg accgtggaca agagcagatg gcagcagggc 1260aacgtgttca gctgctccgt gatgcacgag gccctgcaca accactacac ccagaagagc 1320ctgagcctgt ccccaggccc ggtgggcctg attggcaact gggtgaatgt aataagtgat 1380ttgaaaaaaa ttgaagatct tattcaatct atgcatattg atgctacttt atatacggaa 1440agtgatgttc accccagttg caaagtaaca gcaatgaagt gctttctctt ggagttacaa 1500gttatttcac ttgagtccgg agatgcaagt attcatgata cagtagaaaa tctgatcatc 1560ctagcaaaca acagtttgtc ttctaatggg aatgtaacag aatctggatg caaagaatgt 1620gaggaactgg aggaaaaaaa tattaaagaa tttttgcaga gttttgtaca tattgtccaa 1680atgttcatca acacttct 169852566PRTArtificialHeavy Chain of NHS76-PVGLIG-IL15 52Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Lys Trp Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Pro Val 435 440 445Gly Leu Ile Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile 450 455 460Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu465 470 475 480Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu 485 490 495Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His 500 505 510Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser 515 520 525Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu 530 535 540Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln545 550 555 560Met Phe Ile Asn Thr Ser 56553657DNAArtificialGanitumab Light Chain 53gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagggtgg aggctgagga tgttggggtt tattactgca tgcaaggtac acactggcct 300ctgacgttcg gccaagggac caaggtggag atcaaacgta cggtggccgc tcccagcgtg 360ttcatcttcc cacccagcga cgagcagctg aagtctggca ccgccagcgt cgtgtgcctg 420ctgaacaact tctacccccg cgaggccaag gtgcagtgga aggtggacaa cgccctgcag 480tccggcaaca gccaggaaag cgtcaccgag caggacagca aggactccac ctacagcctg 540agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 600gtgacccacc agggcctgtc cagccccgtg accaagagct tcaaccgggg cgagtgc 65754219PRTArtificialGanitumab Light Chain 54Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 21555645DNAArtificialNHS76 Light Chain 55tcctctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60acatgccaag gagacagcct cagaagctat tatgcaagct ggtaccagca gaagccagga 120caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat tccagaccga 180ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattactg taactcccgg gacagcagtg gtaaccatgt ggtattcggc 300ggagggacca agctgaccgt cctaggtcaa cccaaggccg ctcccagcgt gaccctgttc 360cccccctcga gcgaggagct gcaggccaac aaggccaccc tggtgtgtct gatcagcgac 420ttctacccag gcgccgtgac cgtggcctgg aaggccgaca gcagccccgt gaaggccggc 480gtggagacca ccacccccag caagcagagc aacaacaagt acgccgccag cagctacctg 540agcctgaccc cagagcagtg gaagagccac aggagctaca gctgccaggt cacccacgag 600ggcagcaccg tggaaaagac cgtggcccca accgagtgct cctga 64556214PRTArtificialNHS76 Light Chain 56Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His 85 90 95Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly145 150 155 160Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205Ala Pro Thr Glu Cys Ser 210571875DNAArtificialheavy chain of PD-L1-GPLGMLSQ-NanoLuc (C-terminal fusion) 57gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaggcccgct gggcatgctg agccagctcg agatggtctt cacactcgaa 1380gatttcgttg gggactggcg acagacagcc ggctacaacc tggaccaagt ccttgaacag 1440ggaggtgtgt ccagtttgtt tcagaatctc ggggtgtccg taactccgat ccaaaggatt 1500gtcctgagcg gtgaaaatgg gctgaagatc gacatccatg tcatcatccc gtatgaaggt 1560ctgagcggcg accaaatggg ccagatcgaa aaaattttta aggtggtgta ccctgtggat 1620gatcatcact ttaaggtgat cctgcactat ggcacactgg taatcgacgg ggttacgccg 1680aacatgatcg actatttcgg acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag 1740atcactgtaa cagggaccct gtggaacggc aacaaaatta tcgacgagcg cctgatcaac 1800cccgacggct ccctgctgtt ccgagtaacc atcaacggag tgaccggctg gcggctgtgc 1860gaacgcattc tggcg 187558625PRTArtificialheavy chain of PD-L1-GPLGMLSQ-NanoLuc (C-terminal fusion) 58Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Pro Leu Gly 435 440 445Met Leu Ser Gln Leu Glu Met Val Phe Thr Leu Glu Asp Phe Val Gly 450 455 460Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln465 470 475 480Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro 485 490 495Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile 500 505 510His Val Ile Ile Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln 515 520 525Ile Glu Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe 530 535 540Lys Val Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro545 550 555 560Asn Met Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe 565 570 575Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys 580 585 590Ile Ile Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg 595 600 605Val Thr Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu 610 615 620Ala625591185DNAArtificiallight chain of PD-L1-GPLGMLSQ-NanoLuc (C-terminal fusion) 59gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc

tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcggcccgct gggcatgctg 660agccagctcg agatggtctt cacactcgaa gatttcgttg gggactggcg acagacagcc 720ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt ccagtttgtt tcagaatctc 780ggggtgtccg taactccgat ccaaaggatt gtcctgagcg gtgaaaatgg gctgaagatc 840gacatccatg tcatcatccc gtatgaaggt ctgagcggcg accaaatggg ccagatcgaa 900aaaattttta aggtggtgta ccctgtggat gatcatcact ttaaggtgat cctgcactat 960ggcacactgg taatcgacgg ggttacgccg aacatgatcg actatttcgg acggccgtat 1020gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa cagggaccct gtggaacggc 1080aacaaaatta tcgacgagcg cctgatcaac cccgacggct ccctgctgtt ccgagtaacc 1140atcaacggag tgaccggctg gcggctgtgc gaacgcattc tggcg 118560395PRTArtificiallight chain of PD-L1-GPLGMLSQ-NanoLuc (C-terminal fusion) 60Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Gly Pro Leu Gly Met Leu Ser Gln Leu Glu 210 215 220Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala225 230 235 240Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 245 250 255Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 260 265 270Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 275 280 285Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys 290 295 300Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr305 310 315 320Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 325 330 335Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 340 345 350Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 355 360 365Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val 370 375 380Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala385 390 395611869DNAArtificialheavy chain of PD-L1-GPLGMLSQ-NanoLuc (N-terminal fusion) 61atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgggcccgc tgggcatgct gagccaggaa 540gtccaactgg tcgaaagcgg tggaggtctg gtacagccag gaggaagttt gagactgtca 600tgcgctgctt ccgggtttac tttcagcgac tcctggattc actgggttcg tcaggcacct 660ggaaaggggc ttgagtgggt tgcatggatt tccccatacg gcggctctac atactacgct 720gacagcgtca agggcaggtt tacaataagc gccgatacct ctaaaaacac cgcctacctg 780cagatgaata gtttgcgagc cgaggatacc gccgtgtatt attgtgcccg gcgccattgg 840cccggcggct tcgattattg ggggcagggg acacttgtga cagtgtctgc agcctctacc 900aagggcccca gcgtgttccc tctggccccc tgcagcagaa gcaccagcga gtctacagcc 960gccctgggat gcctggtcaa ggactacttc cccgagcccg tgaccgtgtc ctggaactct 1020ggcgccctga ccagcggcgt gcacaccttt ccagccgtgc tgcagagcag cggcctgtac 1080agcctgagca gcgtcgtgac cgtgcctagc agcagcctgg gcaccaagac ctacacctgt 1140aacgtggacc acaagcccag caacaccaag gtggacaagc gggtggaatc taagtacggc 1200cctccctgcc ccccctgccc agcccctgaa tttctgggcg gaccctccgt gttcctgttc 1260cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 1320gtggacgtgt cccaggaaga tcccgaggtc cagttcaact ggtacgtgga cggcgtggaa 1380gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcaccta ccgggtggtg 1440tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtc 1500tccaacaagg gcctgcccag ctccatcgag aaaaccatca gcaaggccaa gggccagccc 1560cgcgagcccc aggtgtacac actgcccccc agccaggaag agatgaccaa gaaccaggtg 1620tccctgacct gcctcgtgaa gggcttctac cccagcgata tcgccgtgga atgggagagc 1680aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggcagc 1740ttcttcctgt actcccggct gaccgtcgac aagagccggt ggcaggaagg caacgtcttc 1800agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1860agcctggga 186962623PRTArtificialheavy chain of PD-L1-GPLGMLSQ-NanoLuc (N-terminal fusion) 62Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly Pro Leu Gly Met 165 170 175Leu Ser Gln Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 180 185 190Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 195 200 205Ser Asp Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 210 215 220Glu Trp Val Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala225 230 235 240Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 245 250 255Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 260 265 270Tyr Tyr Cys Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly 275 280 285Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser 290 295 300Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala305 310 315 320Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 325 330 335Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 340 345 350Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 355 360 365Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 370 375 380Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly385 390 395 400Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 405 410 415Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 420 425 430Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 435 440 445Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 450 455 460Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val465 470 475 480Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 485 490 495Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 500 505 510Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 515 520 525Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 530 535 540Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser545 550 555 560Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 565 570 575Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 580 585 590Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 595 600 605Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 610 615 620631179DNAArtificiallight chain of PD-L1-GPLGMLSQ-NanoLuc (N-terminal fusion) 63atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgggcccgc tgggcatgct gagccaggac 540atccagatga cccaatcccc atcctccttg agcgcatctg taggagacag agtcaccatc 600acctgcaggg cttcacagga tgttagcaca gctgtggctt ggtaccagca gaaacctgga 660aaggccccaa aactgctgat atacagcgcc tcattcctgt acagcggagt gccctctcgc 720tttagtggct ctgggagtgg cactgatttc acactgacta tttctagtct gcagcccgaa 780gatttcgcca cttactattg ccagcagtat ctctatcacc ccgccacctt tgggcagggc 840acaaaggtcg agattaagcg tacggtggcc gctcccagcg tgttcatctt cccacccagc 900gacgagcagc tgaagtctgg caccgccagc gtcgtgtgcc tgctgaacaa cttctacccc 960cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa cagccaggaa 1020agcgtcaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctgaccctg 1080agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca ccagggcctg 1140tccagccccg tgaccaagag cttcaaccgg ggcgagtgc 117964393PRTArtificiallight chain of PD-L1-GPLGMLSQ-NanoLuc (N-terminal fusion) 64Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly Pro Leu Gly Met 165 170 175Leu Ser Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 180 185 190Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 195 200 205Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 210 215 220Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg225 230 235 240Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 245 250 255Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr 260 265 270His Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 275 280 285Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 290 295 300Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro305 310 315 320Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 325 330 335Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 340 345 350Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 355 360 365Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 370 375 380Thr Lys Ser Phe Asn Arg Gly Glu Cys385 390651755DNAArtificialheavy chain of PD-L1-GPLGMLSQ-IL2 (C-terminal fusion) 65gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaggcccgct gggcatgctg agccaggcac ctacctcgag ttctacaaag 1380aaaacacagc tacaactgga gcatttactg ctggatttac agatgatttt gaatggaatt 1440aataattaca agaatcccaa actcaccagg atgctcacat ttaagtttta catgcccaag 1500aaggccacag aactgaaaca tcttcagtgt ctagaagaag aactcaaacc tctggaggaa 1560gtgctaaatt tagctcaaag caaaaacttt cacttaagac ccagggactt aatcagcaat 1620atcaacgtaa tagttctgga actaaaggga tctgaaacaa cattcatgtg tgaatatgct 1680gatgagacag caaccattgt agaatttctg aacagatgga ttaccttttg tcaaagcatc 1740atctcaacac tgact 175566585PRTArtificialheavy chain of PD-L1-GPLGMLSQ-IL2 (C-terminal fusion) 66Glu Val Gln Leu Val Glu Ser

Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Pro Leu Gly 435 440 445Met Leu Ser Gln Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 450 455 460Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile465 470 475 480Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 485 490 495Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 500 505 510Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 515 520 525Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 530 535 540Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala545 550 555 560Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 565 570 575Cys Gln Ser Ile Ile Ser Thr Leu Thr 580 585671065DNAArtificiallight chain of PD-L1-GPLGMLSQ-IL2 (C-terminal fusion) 67gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcggcccgct gggcatgctg 660agccaggcac ctacctcgag ttctacaaag aaaacacagc tacaactgga gcatttactg 720ctggatttac agatgatttt gaatggaatt aataattaca agaatcccaa actcaccagg 780atgctcacat ttaagtttta catgcccaag aaggccacag aactgaaaca tcttcagtgt 840ctagaagaag aactcaaacc tctggaggaa gtgctaaatt tagctcaaag caaaaacttt 900cacttaagac ccagggactt aatcagcaat atcaacgtaa tagttctgga actaaaggga 960tctgaaacaa cattcatgtg tgaatatgct gatgagacag caaccattgt agaatttctg 1020aacagatgga ttaccttttg tcaaagcatc atctcaacac tgact 106568355PRTArtificiallight chain of PD-L1-GPLGMLSQ-IL2 (C-terminal fusion) 68Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Gly Pro Leu Gly Met Leu Ser Gln Ala Pro 210 215 220Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu225 230 235 240Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro 245 250 255Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala 260 265 270Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu 275 280 285Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro 290 295 300Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly305 310 315 320Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile 325 330 335Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser 340 345 350Thr Leu Thr 355691755DNAArtificialheavy chain of PD-L1-GPLGMLSQ-IL2 (N-terminal fusion) 69gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactg gcccgctggg catgctgagc 420caggaagtcc aactggtcga aagcggtgga ggtctggtac agccaggagg aagtttgaga 480ctgtcatgcg ctgcttccgg gtttactttc agcgactcct ggattcactg ggttcgtcag 540gcacctggaa aggggcttga gtgggttgca tggatttccc catacggcgg ctctacatac 600tacgctgaca gcgtcaaggg caggtttaca ataagcgccg atacctctaa aaacaccgcc 660tacctgcaga tgaatagttt gcgagccgag gataccgccg tgtattattg tgcccggcgc 720cattggcccg gcggcttcga ttattggggg caggggacac ttgtgacagt gtctgcagcc 780tctaccaagg gccccagcgt gttccctctg gccccctgca gcagaagcac cagcgagtct 840acagccgccc tgggatgcct ggtcaaggac tacttccccg agcccgtgac cgtgtcctgg 900aactctggcg ccctgaccag cggcgtgcac acctttccag ccgtgctgca gagcagcggc 960ctgtacagcc tgagcagcgt cgtgaccgtg cctagcagca gcctgggcac caagacctac 1020acctgtaacg tggaccacaa gcccagcaac accaaggtgg acaagcgggt ggaatctaag 1080tacggccctc cctgcccccc ctgcccagcc cctgaatttc tgggcggacc ctccgtgttc 1140ctgttccccc caaagcccaa ggacaccctg atgatcagcc ggacccccga agtgacctgc 1200gtggtggtgg acgtgtccca ggaagatccc gaggtccagt tcaactggta cgtggacggc 1260gtggaagtgc acaacgccaa gaccaagccc agagaggaac agttcaacag cacctaccgg 1320gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaaaga gtacaagtgc 1380aaggtctcca acaagggcct gcccagctcc atcgagaaaa ccatcagcaa ggccaagggc 1440cagccccgcg agccccaggt gtacacactg ccccccagcc aggaagagat gaccaagaac 1500caggtgtccc tgacctgcct cgtgaagggc ttctacccca gcgatatcgc cgtggaatgg 1560gagagcaacg gccagcccga gaacaactac aagaccaccc cccctgtgct ggacagcgac 1620ggcagcttct tcctgtactc ccggctgacc gtcgacaaga gccggtggca ggaaggcaac 1680gtcttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1740agcctgagcc tggga 175570585PRTArtificialheavy chain of PD-L1-GPLGMLSQ-IL2 (N-terminal fusion) 70Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Gly Pro Leu Gly Met Leu Ser Gln Glu Val Gln 130 135 140Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg145 150 155 160Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser Trp Ile His 165 170 175Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile 180 185 190Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 195 200 205Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 210 215 220Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg225 230 235 240His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 245 250 255Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 260 265 270Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 275 280 285Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 290 295 300Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly305 310 315 320Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 325 330 335Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 340 345 350Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 355 360 365Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 370 375 380Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys385 390 395 400Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 405 410 415Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 420 425 430Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 435 440 445His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 450 455 460Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly465 470 475 480Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 485 490 495Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 500 505 510Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 515 520 525Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 530 535 540Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn545 550 555 560Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 565 570 575Gln Lys Ser Leu Ser Leu Ser Leu Gly 580 585711065DNAArtificiallight chain of PD-L1-GPLGMLSQ-IL2 (N-terminal fusion) 71gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactg gcccgctggg catgctgagc 420caggacatcc agatgaccca atccccatcc tccttgagcg catctgtagg agacagagtc 480accatcacct gcagggcttc acaggatgtt agcacagctg tggcttggta ccagcagaaa 540cctggaaagg ccccaaaact gctgatatac agcgcctcat tcctgtacag cggagtgccc 600tctcgcttta gtggctctgg gagtggcact gatttcacac tgactatttc tagtctgcag 660cccgaagatt tcgccactta ctattgccag cagtatctct atcaccccgc cacctttggg 720cagggcacaa aggtcgagat taagcgtacg gtggccgctc ccagcgtgtt catcttccca 780cccagcgacg agcagctgaa gtctggcacc gccagcgtcg tgtgcctgct gaacaacttc 840tacccccgcg aggccaaggt gcagtggaag gtggacaacg ccctgcagtc cggcaacagc 900caggaaagcg tcaccgagca ggacagcaag gactccacct acagcctgag cagcaccctg 960accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaagt gacccaccag 1020ggcctgtcca gccccgtgac caagagcttc aaccggggcg agtgc 106572355PRTArtificiallight chain of PD-L1-GPLGMLSQ-IL2 (N-terminal fusion) 72Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Gly Pro Leu Gly Met Leu Ser Gln Asp Ile Gln 130 135 140Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val145 150 155 160Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp 165 170 175Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 180 185 190Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 195 200 205Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 210 215 220Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala Thr Phe Gly225

230 235 240Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 245 250 255Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 260 265 270Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 275 280 285Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 290 295 300Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu305 310 315 320Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 325 330 335Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 340 345 350Gly Glu Cys 355731875DNAArtificialheavy chain of PD-L1-GPLGIAGQ -Nanoluc (C-terminal fusion) 73gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaggcccgct gggcattgcg ggccagctcg agatggtctt cacactcgaa 1380gatttcgttg gggactggcg acagacagcc ggctacaacc tggaccaagt ccttgaacag 1440ggaggtgtgt ccagtttgtt tcagaatctc ggggtgtccg taactccgat ccaaaggatt 1500gtcctgagcg gtgaaaatgg gctgaagatc gacatccatg tcatcatccc gtatgaaggt 1560ctgagcggcg accaaatggg ccagatcgaa aaaattttta aggtggtgta ccctgtggat 1620gatcatcact ttaaggtgat cctgcactat ggcacactgg taatcgacgg ggttacgccg 1680aacatgatcg actatttcgg acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag 1740atcactgtaa cagggaccct gtggaacggc aacaaaatta tcgacgagcg cctgatcaac 1800cccgacggct ccctgctgtt ccgagtaacc atcaacggag tgaccggctg gcggctgtgc 1860gaacgcattc tggcg 187574625PRTArtificialheavy chain of PD-L1-GPLGIAGQ -Nanoluc (C-terminal fusion) 74Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Pro Leu Gly 435 440 445Ile Ala Gly Gln Leu Glu Met Val Phe Thr Leu Glu Asp Phe Val Gly 450 455 460Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln465 470 475 480Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro 485 490 495Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile 500 505 510His Val Ile Ile Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln 515 520 525Ile Glu Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe 530 535 540Lys Val Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro545 550 555 560Asn Met Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe 565 570 575Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys 580 585 590Ile Ile Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg 595 600 605Val Thr Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu 610 615 620Ala625751185DNAArtificiallight chain of PD-L1-GPLGIAGQ -Nanoluc (C-terminal fusion) 75gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcggcccgct gggcattgcg 660ggccagctcg agatggtctt cacactcgaa gatttcgttg gggactggcg acagacagcc 720ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt ccagtttgtt tcagaatctc 780ggggtgtccg taactccgat ccaaaggatt gtcctgagcg gtgaaaatgg gctgaagatc 840gacatccatg tcatcatccc gtatgaaggt ctgagcggcg accaaatggg ccagatcgaa 900aaaattttta aggtggtgta ccctgtggat gatcatcact ttaaggtgat cctgcactat 960ggcacactgg taatcgacgg ggttacgccg aacatgatcg actatttcgg acggccgtat 1020gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa cagggaccct gtggaacggc 1080aacaaaatta tcgacgagcg cctgatcaac cccgacggct ccctgctgtt ccgagtaacc 1140atcaacggag tgaccggctg gcggctgtgc gaacgcattc tggcg 118576395PRTArtificiallight chain of PD-L1-GPLGIAGQ -Nanoluc (C-terminal fusion) 76Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Gly Pro Leu Gly Ile Ala Gly Gln Leu Glu 210 215 220Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala225 230 235 240Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 245 250 255Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 260 265 270Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 275 280 285Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys 290 295 300Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr305 310 315 320Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 325 330 335Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 340 345 350Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 355 360 365Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val 370 375 380Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala385 390 395771869DNAArtificialheavy chain of PD-L1-GPLGIAGQ -Nanoluc (N-terminal fusion) 77atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgggcccgc tgggcattgc gggccaggaa 540gtccaactgg tcgaaagcgg tggaggtctg gtacagccag gaggaagttt gagactgtca 600tgcgctgctt ccgggtttac tttcagcgac tcctggattc actgggttcg tcaggcacct 660ggaaaggggc ttgagtgggt tgcatggatt tccccatacg gcggctctac atactacgct 720gacagcgtca agggcaggtt tacaataagc gccgatacct ctaaaaacac cgcctacctg 780cagatgaata gtttgcgagc cgaggatacc gccgtgtatt attgtgcccg gcgccattgg 840cccggcggct tcgattattg ggggcagggg acacttgtga cagtgtctgc agcctctacc 900aagggcccca gcgtgttccc tctggccccc tgcagcagaa gcaccagcga gtctacagcc 960gccctgggat gcctggtcaa ggactacttc cccgagcccg tgaccgtgtc ctggaactct 1020ggcgccctga ccagcggcgt gcacaccttt ccagccgtgc tgcagagcag cggcctgtac 1080agcctgagca gcgtcgtgac cgtgcctagc agcagcctgg gcaccaagac ctacacctgt 1140aacgtggacc acaagcccag caacaccaag gtggacaagc gggtggaatc taagtacggc 1200cctccctgcc ccccctgccc agcccctgaa tttctgggcg gaccctccgt gttcctgttc 1260cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 1320gtggacgtgt cccaggaaga tcccgaggtc cagttcaact ggtacgtgga cggcgtggaa 1380gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcaccta ccgggtggtg 1440tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtc 1500tccaacaagg gcctgcccag ctccatcgag aaaaccatca gcaaggccaa gggccagccc 1560cgcgagcccc aggtgtacac actgcccccc agccaggaag agatgaccaa gaaccaggtg 1620tccctgacct gcctcgtgaa gggcttctac cccagcgata tcgccgtgga atgggagagc 1680aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggcagc 1740ttcttcctgt actcccggct gaccgtcgac aagagccggt ggcaggaagg caacgtcttc 1800agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1860agcctggga 186978623PRTArtificialheavy chain of PD-L1-GPLGIAGQ -Nanoluc (N-terminal fusion) 78Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly Pro Leu Gly Ile 165 170 175Ala Gly Gln Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 180 185 190Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 195 200 205Ser Asp Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 210 215 220Glu Trp Val Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala225 230 235 240Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 245 250 255Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 260 265 270Tyr Tyr Cys Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly 275 280 285Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser 290 295 300Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala305 310 315 320Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 325 330 335Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 340 345 350Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 355 360 365Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 370 375 380Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly385 390 395 400Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 405 410 415Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 420 425

430Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 435 440 445Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 450 455 460Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val465 470 475 480Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 485 490 495Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 500 505 510Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 515 520 525Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 530 535 540Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser545 550 555 560Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 565 570 575Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 580 585 590Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 595 600 605Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 610 615 620791179DNAArtificiallight chain of PD-L1-GPLGIAGQ -Nanoluc (N-terminal fusion) 79atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgggcccgc tgggcattgc gggccaggac 540atccagatga cccaatcccc atcctccttg agcgcatctg taggagacag agtcaccatc 600acctgcaggg cttcacagga tgttagcaca gctgtggctt ggtaccagca gaaacctgga 660aaggccccaa aactgctgat atacagcgcc tcattcctgt acagcggagt gccctctcgc 720tttagtggct ctgggagtgg cactgatttc acactgacta tttctagtct gcagcccgaa 780gatttcgcca cttactattg ccagcagtat ctctatcacc ccgccacctt tgggcagggc 840acaaaggtcg agattaagcg tacggtggcc gctcccagcg tgttcatctt cccacccagc 900gacgagcagc tgaagtctgg caccgccagc gtcgtgtgcc tgctgaacaa cttctacccc 960cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa cagccaggaa 1020agcgtcaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctgaccctg 1080agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca ccagggcctg 1140tccagccccg tgaccaagag cttcaaccgg ggcgagtgc 117980393PRTArtificiallight chain of PD-L1-GPLGIAGQ -Nanoluc (N-terminal fusion) 80Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly Pro Leu Gly Ile 165 170 175Ala Gly Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 180 185 190Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 195 200 205Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 210 215 220Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg225 230 235 240Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 245 250 255Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr 260 265 270His Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 275 280 285Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 290 295 300Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro305 310 315 320Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 325 330 335Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 340 345 350Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 355 360 365Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 370 375 380Thr Lys Ser Phe Asn Arg Gly Glu Cys385 390811755DNAArtificialheavy chain of PD-L1-GPLGIAGQ -IL2 (C-terminal fusion) 81gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaggcccgct gggcattgcg ggccaggcac ctacctcgag ttctacaaag 1380aaaacacagc tacaactgga gcatttactg ctggatttac agatgatttt gaatggaatt 1440aataattaca agaatcccaa actcaccagg atgctcacat ttaagtttta catgcccaag 1500aaggccacag aactgaaaca tcttcagtgt ctagaagaag aactcaaacc tctggaggaa 1560gtgctaaatt tagctcaaag caaaaacttt cacttaagac ccagggactt aatcagcaat 1620atcaacgtaa tagttctgga actaaaggga tctgaaacaa cattcatgtg tgaatatgct 1680gatgagacag caaccattgt agaatttctg aacagatgga ttaccttttg tcaaagcatc 1740atctcaacac tgact 175582585PRTArtificialheavy chain of PD-L1-GPLGIAGQ -IL2 (C-terminal fusion) 82Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Pro Leu Gly 435 440 445Ile Ala Gly Gln Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 450 455 460Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile465 470 475 480Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 485 490 495Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 500 505 510Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 515 520 525Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 530 535 540Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala545 550 555 560Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 565 570 575Cys Gln Ser Ile Ile Ser Thr Leu Thr 580 585831065DNAArtificiallight chain of PD-L1-GPLGIAGQ -IL2 (C-terminal fusion) 83gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcggcccgct gggcattgcg 660ggccaggcac ctacctcgag ttctacaaag aaaacacagc tacaactgga gcatttactg 720ctggatttac agatgatttt gaatggaatt aataattaca agaatcccaa actcaccagg 780atgctcacat ttaagtttta catgcccaag aaggccacag aactgaaaca tcttcagtgt 840ctagaagaag aactcaaacc tctggaggaa gtgctaaatt tagctcaaag caaaaacttt 900cacttaagac ccagggactt aatcagcaat atcaacgtaa tagttctgga actaaaggga 960tctgaaacaa cattcatgtg tgaatatgct gatgagacag caaccattgt agaatttctg 1020aacagatgga ttaccttttg tcaaagcatc atctcaacac tgact 106584355PRTArtificiallight chain of PD-L1-GPLGIAGQ -IL2 (C-terminal fusion) 84Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Gly Pro Leu Gly Ile Ala Gly Gln Ala Pro 210 215 220Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu225 230 235 240Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro 245 250 255Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala 260 265 270Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu 275 280 285Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro 290 295 300Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly305 310 315 320Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile 325 330 335Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser 340 345 350Thr Leu Thr 355851755DNAArtificialheavy chain of PD-L1-GPLGIAGQ -IL2 (N-terminal fusion) 85gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactg gcccgctggg cattgcgggc 420caggaagtcc aactggtcga aagcggtgga ggtctggtac agccaggagg aagtttgaga 480ctgtcatgcg ctgcttccgg gtttactttc agcgactcct ggattcactg ggttcgtcag 540gcacctggaa aggggcttga gtgggttgca tggatttccc catacggcgg ctctacatac 600tacgctgaca gcgtcaaggg caggtttaca ataagcgccg atacctctaa aaacaccgcc 660tacctgcaga tgaatagttt gcgagccgag gataccgccg tgtattattg tgcccggcgc 720cattggcccg gcggcttcga ttattggggg caggggacac ttgtgacagt gtctgcagcc 780tctaccaagg gccccagcgt gttccctctg gccccctgca gcagaagcac cagcgagtct 840acagccgccc tgggatgcct ggtcaaggac tacttccccg agcccgtgac cgtgtcctgg 900aactctggcg ccctgaccag cggcgtgcac acctttccag ccgtgctgca gagcagcggc 960ctgtacagcc tgagcagcgt cgtgaccgtg cctagcagca gcctgggcac caagacctac 1020acctgtaacg tggaccacaa gcccagcaac accaaggtgg acaagcgggt ggaatctaag 1080tacggccctc cctgcccccc ctgcccagcc cctgaatttc tgggcggacc ctccgtgttc 1140ctgttccccc caaagcccaa ggacaccctg atgatcagcc ggacccccga agtgacctgc 1200gtggtggtgg acgtgtccca ggaagatccc gaggtccagt

tcaactggta cgtggacggc 1260gtggaagtgc acaacgccaa gaccaagccc agagaggaac agttcaacag cacctaccgg 1320gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaaaga gtacaagtgc 1380aaggtctcca acaagggcct gcccagctcc atcgagaaaa ccatcagcaa ggccaagggc 1440cagccccgcg agccccaggt gtacacactg ccccccagcc aggaagagat gaccaagaac 1500caggtgtccc tgacctgcct cgtgaagggc ttctacccca gcgatatcgc cgtggaatgg 1560gagagcaacg gccagcccga gaacaactac aagaccaccc cccctgtgct ggacagcgac 1620ggcagcttct tcctgtactc ccggctgacc gtcgacaaga gccggtggca ggaaggcaac 1680gtcttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1740agcctgagcc tggga 175586585PRTArtificialheavy chain of PD-L1-GPLGIAGQ -IL2 (N-terminal fusion) 86Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Gly Pro Leu Gly Ile Ala Gly Gln Glu Val Gln 130 135 140Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg145 150 155 160Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser Trp Ile His 165 170 175Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile 180 185 190Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 195 200 205Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 210 215 220Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg225 230 235 240His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 245 250 255Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 260 265 270Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 275 280 285Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 290 295 300Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly305 310 315 320Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 325 330 335Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 340 345 350Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 355 360 365Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 370 375 380Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys385 390 395 400Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 405 410 415Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 420 425 430Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 435 440 445His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 450 455 460Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly465 470 475 480Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 485 490 495Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 500 505 510Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 515 520 525Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 530 535 540Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn545 550 555 560Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 565 570 575Gln Lys Ser Leu Ser Leu Ser Leu Gly 580 585871065DNAArtificiallight chain of PD-L1-GPLGIAGQ -IL2 (N-terminal fusion) 87gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactg gcccgctggg cattgcgggc 420caggacatcc agatgaccca atccccatcc tccttgagcg catctgtagg agacagagtc 480accatcacct gcagggcttc acaggatgtt agcacagctg tggcttggta ccagcagaaa 540cctggaaagg ccccaaaact gctgatatac agcgcctcat tcctgtacag cggagtgccc 600tctcgcttta gtggctctgg gagtggcact gatttcacac tgactatttc tagtctgcag 660cccgaagatt tcgccactta ctattgccag cagtatctct atcaccccgc cacctttggg 720cagggcacaa aggtcgagat taagcgtacg gtggccgctc ccagcgtgtt catcttccca 780cccagcgacg agcagctgaa gtctggcacc gccagcgtcg tgtgcctgct gaacaacttc 840tacccccgcg aggccaaggt gcagtggaag gtggacaacg ccctgcagtc cggcaacagc 900caggaaagcg tcaccgagca ggacagcaag gactccacct acagcctgag cagcaccctg 960accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaagt gacccaccag 1020ggcctgtcca gccccgtgac caagagcttc aaccggggcg agtgc 106588355PRTArtificiallight chain of PD-L1-GPLGIAGQ -IL2 (N-terminal fusion) 88Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Gly Pro Leu Gly Ile Ala Gly Gln Asp Ile Gln 130 135 140Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val145 150 155 160Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp 165 170 175Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 180 185 190Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 195 200 205Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 210 215 220Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala Thr Phe Gly225 230 235 240Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 245 250 255Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 260 265 270Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 275 280 285Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 290 295 300Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu305 310 315 320Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 325 330 335Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 340 345 350Gly Glu Cys 355891875DNAArtificialheavy chain of PD-L1-GPLGLWAQ -NanoLuc (C-terminal fusion) 89gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaggcccgct gggcctgtgg gcgcagctcg agatggtctt cacactcgaa 1380gatttcgttg gggactggcg acagacagcc ggctacaacc tggaccaagt ccttgaacag 1440ggaggtgtgt ccagtttgtt tcagaatctc ggggtgtccg taactccgat ccaaaggatt 1500gtcctgagcg gtgaaaatgg gctgaagatc gacatccatg tcatcatccc gtatgaaggt 1560ctgagcggcg accaaatggg ccagatcgaa aaaattttta aggtggtgta ccctgtggat 1620gatcatcact ttaaggtgat cctgcactat ggcacactgg taatcgacgg ggttacgccg 1680aacatgatcg actatttcgg acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag 1740atcactgtaa cagggaccct gtggaacggc aacaaaatta tcgacgagcg cctgatcaac 1800cccgacggct ccctgctgtt ccgagtaacc atcaacggag tgaccggctg gcggctgtgc 1860gaacgcattc tggcg 187590625PRTArtificialheavy chain of PD-L1-GPLGLWAQ -NanoLuc (C-terminal fusion) 90Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Pro Leu Gly 435 440 445Leu Trp Ala Gln Leu Glu Met Val Phe Thr Leu Glu Asp Phe Val Gly 450 455 460Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln465 470 475 480Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro 485 490 495Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile 500 505 510His Val Ile Ile Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln 515 520 525Ile Glu Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe 530 535 540Lys Val Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro545 550 555 560Asn Met Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe 565 570 575Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys 580 585 590Ile Ile Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg 595 600 605Val Thr Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu 610 615 620Ala625911185DNAArtificiallight chain of PD-L1-GPLGLWAQ -NanoLuc (C-terminal fusion) 91gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcggcccgct gggcctgtgg 660gcgcagctcg agatggtctt cacactcgaa gatttcgttg gggactggcg acagacagcc 720ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt ccagtttgtt tcagaatctc 780ggggtgtccg taactccgat ccaaaggatt gtcctgagcg gtgaaaatgg gctgaagatc 840gacatccatg tcatcatccc gtatgaaggt ctgagcggcg accaaatggg ccagatcgaa 900aaaattttta aggtggtgta ccctgtggat gatcatcact ttaaggtgat cctgcactat 960ggcacactgg taatcgacgg ggttacgccg aacatgatcg actatttcgg acggccgtat 1020gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa cagggaccct gtggaacggc 1080aacaaaatta tcgacgagcg cctgatcaac cccgacggct ccctgctgtt ccgagtaacc 1140atcaacggag tgaccggctg gcggctgtgc gaacgcattc tggcg 118592395PRTArtificiallight chain of PD-L1-GPLGLWAQ -NanoLuc (C-terminal fusion) 92Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Gly Pro Leu Gly Leu Trp Ala Gln Leu Glu 210 215 220Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala225 230 235 240Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 245 250 255Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 260 265 270Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 275 280 285Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys 290 295 300Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr305 310 315 320Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 325 330 335Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 340 345 350Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 355 360 365Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val 370 375 380Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala385 390 395931869DNAArtificialheavy chain of PD-L1-GPLGLWAQ -NanoLuc (N-terminal fusion) 93atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgggcccgc tgggcctgtg ggcgcaggaa 540gtccaactgg tcgaaagcgg tggaggtctg gtacagccag gaggaagttt gagactgtca 600tgcgctgctt ccgggtttac tttcagcgac tcctggattc actgggttcg tcaggcacct 660ggaaaggggc ttgagtgggt tgcatggatt tccccatacg gcggctctac atactacgct 720gacagcgtca agggcaggtt tacaataagc gccgatacct ctaaaaacac cgcctacctg 780cagatgaata gtttgcgagc cgaggatacc gccgtgtatt attgtgcccg gcgccattgg 840cccggcggct tcgattattg ggggcagggg acacttgtga cagtgtctgc agcctctacc 900aagggcccca gcgtgttccc tctggccccc tgcagcagaa gcaccagcga gtctacagcc 960gccctgggat gcctggtcaa ggactacttc cccgagcccg tgaccgtgtc ctggaactct 1020ggcgccctga ccagcggcgt gcacaccttt ccagccgtgc tgcagagcag cggcctgtac 1080agcctgagca gcgtcgtgac cgtgcctagc agcagcctgg gcaccaagac ctacacctgt 1140aacgtggacc acaagcccag caacaccaag gtggacaagc gggtggaatc taagtacggc 1200cctccctgcc ccccctgccc agcccctgaa tttctgggcg gaccctccgt gttcctgttc 1260cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 1320gtggacgtgt cccaggaaga tcccgaggtc cagttcaact ggtacgtgga cggcgtggaa 1380gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcaccta ccgggtggtg 1440tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtc 1500tccaacaagg gcctgcccag ctccatcgag aaaaccatca gcaaggccaa gggccagccc 1560cgcgagcccc aggtgtacac actgcccccc agccaggaag agatgaccaa gaaccaggtg 1620tccctgacct gcctcgtgaa gggcttctac cccagcgata tcgccgtgga atgggagagc 1680aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggcagc 1740ttcttcctgt actcccggct gaccgtcgac aagagccggt ggcaggaagg caacgtcttc 1800agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1860agcctggga 186994623PRTArtificialheavy chain of PD-L1-GPLGLWAQ -NanoLuc (N-terminal fusion) 94Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly Pro Leu Gly Leu 165 170 175Trp Ala Gln Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 180 185 190Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 195 200 205Ser Asp Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 210 215 220Glu Trp Val Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala225 230 235 240Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 245 250 255Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 260 265 270Tyr Tyr Cys Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly 275 280 285Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser 290 295 300Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala305 310 315 320Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 325 330 335Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 340 345 350Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 355 360 365Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 370 375 380Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly385 390 395 400Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 405 410 415Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 420 425 430Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 435 440 445Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 450 455 460Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val465 470 475 480Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 485 490 495Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 500 505 510Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 515 520 525Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 530 535 540Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser545 550 555 560Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 565 570 575Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 580 585 590Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 595 600 605Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 610 615 620951179DNAArtificiallight chain of PD-L1-GPLGLWAQ -NanoLuc (N-terminal fusion) 95atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgggcccgc tgggcctgtg ggcgcaggac 540atccagatga cccaatcccc atcctccttg agcgcatctg taggagacag agtcaccatc 600acctgcaggg cttcacagga tgttagcaca gctgtggctt ggtaccagca gaaacctgga 660aaggccccaa aactgctgat atacagcgcc tcattcctgt acagcggagt gccctctcgc 720tttagtggct ctgggagtgg cactgatttc acactgacta tttctagtct gcagcccgaa 780gatttcgcca cttactattg ccagcagtat ctctatcacc ccgccacctt tgggcagggc 840acaaaggtcg agattaagcg tacggtggcc gctcccagcg tgttcatctt cccacccagc 900gacgagcagc tgaagtctgg caccgccagc gtcgtgtgcc tgctgaacaa cttctacccc 960cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa cagccaggaa 1020agcgtcaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctgaccctg 1080agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca ccagggcctg 1140tccagccccg tgaccaagag cttcaaccgg ggcgagtgc 117996393PRTArtificiallight chain of PD-L1-GPLGLWAQ -NanoLuc (N-terminal fusion) 96Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly Pro Leu Gly Leu 165 170 175Trp Ala Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 180 185 190Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 195 200 205Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 210 215 220Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg225 230 235 240Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 245 250 255Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr 260 265 270His Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 275 280 285Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 290 295 300Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro305 310 315 320Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 325 330 335Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 340 345 350Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 355 360 365Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 370 375 380Thr Lys Ser Phe Asn Arg Gly Glu Cys385 390971755DNAArtificialheavy chain of PD-L1-GPLGLWAQ -IL2 (C-terminal fusion) 97gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaggcccgct gggcctgtgg gcgcaggcac ctacctcgag ttctacaaag 1380aaaacacagc tacaactgga gcatttactg ctggatttac agatgatttt gaatggaatt 1440aataattaca agaatcccaa actcaccagg atgctcacat ttaagtttta catgcccaag 1500aaggccacag aactgaaaca tcttcagtgt ctagaagaag aactcaaacc tctggaggaa 1560gtgctaaatt tagctcaaag caaaaacttt cacttaagac ccagggactt aatcagcaat 1620atcaacgtaa tagttctgga actaaaggga tctgaaacaa cattcatgtg tgaatatgct 1680gatgagacag caaccattgt agaatttctg aacagatgga ttaccttttg tcaaagcatc 1740atctcaacac tgact 175598585PRTArtificialheavy chain of PD-L1-GPLGLWAQ -IL2 (C-terminal fusion) 98Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225

230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Pro Leu Gly 435 440 445Leu Trp Ala Gln Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 450 455 460Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile465 470 475 480Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 485 490 495Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 500 505 510Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 515 520 525Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 530 535 540Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala545 550 555 560Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 565 570 575Cys Gln Ser Ile Ile Ser Thr Leu Thr 580 585991065DNAArtificiallight chain of PD-L1-GPLGLWAQ -IL2 (C-terminal fusion) 99gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcggcccgct gggcctgtgg 660gcgcaggcac ctacctcgag ttctacaaag aaaacacagc tacaactgga gcatttactg 720ctggatttac agatgatttt gaatggaatt aataattaca agaatcccaa actcaccagg 780atgctcacat ttaagtttta catgcccaag aaggccacag aactgaaaca tcttcagtgt 840ctagaagaag aactcaaacc tctggaggaa gtgctaaatt tagctcaaag caaaaacttt 900cacttaagac ccagggactt aatcagcaat atcaacgtaa tagttctgga actaaaggga 960tctgaaacaa cattcatgtg tgaatatgct gatgagacag caaccattgt agaatttctg 1020aacagatgga ttaccttttg tcaaagcatc atctcaacac tgact 1065100355PRTArtificiallight chain of PD-L1-GPLGLWAQ -IL2 (C-terminal fusion) 100Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Gly Pro Leu Gly Leu Trp Ala Gln Ala Pro 210 215 220Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu225 230 235 240Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro 245 250 255Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala 260 265 270Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu 275 280 285Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro 290 295 300Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly305 310 315 320Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile 325 330 335Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser 340 345 350Thr Leu Thr 3551011755DNAArtificialheavy chain of PD-L1-GPLGLWAQ -IL2 (N-terminal fusion) 101gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactg gcccgctggg cctgtgggcg 420caggaagtcc aactggtcga aagcggtgga ggtctggtac agccaggagg aagtttgaga 480ctgtcatgcg ctgcttccgg gtttactttc agcgactcct ggattcactg ggttcgtcag 540gcacctggaa aggggcttga gtgggttgca tggatttccc catacggcgg ctctacatac 600tacgctgaca gcgtcaaggg caggtttaca ataagcgccg atacctctaa aaacaccgcc 660tacctgcaga tgaatagttt gcgagccgag gataccgccg tgtattattg tgcccggcgc 720cattggcccg gcggcttcga ttattggggg caggggacac ttgtgacagt gtctgcagcc 780tctaccaagg gccccagcgt gttccctctg gccccctgca gcagaagcac cagcgagtct 840acagccgccc tgggatgcct ggtcaaggac tacttccccg agcccgtgac cgtgtcctgg 900aactctggcg ccctgaccag cggcgtgcac acctttccag ccgtgctgca gagcagcggc 960ctgtacagcc tgagcagcgt cgtgaccgtg cctagcagca gcctgggcac caagacctac 1020acctgtaacg tggaccacaa gcccagcaac accaaggtgg acaagcgggt ggaatctaag 1080tacggccctc cctgcccccc ctgcccagcc cctgaatttc tgggcggacc ctccgtgttc 1140ctgttccccc caaagcccaa ggacaccctg atgatcagcc ggacccccga agtgacctgc 1200gtggtggtgg acgtgtccca ggaagatccc gaggtccagt tcaactggta cgtggacggc 1260gtggaagtgc acaacgccaa gaccaagccc agagaggaac agttcaacag cacctaccgg 1320gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaaaga gtacaagtgc 1380aaggtctcca acaagggcct gcccagctcc atcgagaaaa ccatcagcaa ggccaagggc 1440cagccccgcg agccccaggt gtacacactg ccccccagcc aggaagagat gaccaagaac 1500caggtgtccc tgacctgcct cgtgaagggc ttctacccca gcgatatcgc cgtggaatgg 1560gagagcaacg gccagcccga gaacaactac aagaccaccc cccctgtgct ggacagcgac 1620ggcagcttct tcctgtactc ccggctgacc gtcgacaaga gccggtggca ggaaggcaac 1680gtcttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1740agcctgagcc tggga 1755102585PRTArtificialheavy chain of PD-L1-GPLGLWAQ -IL2 (N-terminal fusion) 102Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Gly Pro Leu Gly Leu Trp Ala Gln Glu Val Gln 130 135 140Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg145 150 155 160Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser Trp Ile His 165 170 175Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile 180 185 190Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 195 200 205Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 210 215 220Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg225 230 235 240His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 245 250 255Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 260 265 270Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 275 280 285Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 290 295 300Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly305 310 315 320Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 325 330 335Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 340 345 350Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 355 360 365Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 370 375 380Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys385 390 395 400Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 405 410 415Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 420 425 430Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 435 440 445His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 450 455 460Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly465 470 475 480Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 485 490 495Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 500 505 510Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 515 520 525Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 530 535 540Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn545 550 555 560Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 565 570 575Gln Lys Ser Leu Ser Leu Ser Leu Gly 580 5851031065DNAArtificiallight chain of PD-L1-GPLGLWAQ -IL2 (N-terminal fusion) 103gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactg gcccgctggg cctgtgggcg 420caggacatcc agatgaccca atccccatcc tccttgagcg catctgtagg agacagagtc 480accatcacct gcagggcttc acaggatgtt agcacagctg tggcttggta ccagcagaaa 540cctggaaagg ccccaaaact gctgatatac agcgcctcat tcctgtacag cggagtgccc 600tctcgcttta gtggctctgg gagtggcact gatttcacac tgactatttc tagtctgcag 660cccgaagatt tcgccactta ctattgccag cagtatctct atcaccccgc cacctttggg 720cagggcacaa aggtcgagat taagcgtacg gtggccgctc ccagcgtgtt catcttccca 780cccagcgacg agcagctgaa gtctggcacc gccagcgtcg tgtgcctgct gaacaacttc 840tacccccgcg aggccaaggt gcagtggaag gtggacaacg ccctgcagtc cggcaacagc 900caggaaagcg tcaccgagca ggacagcaag gactccacct acagcctgag cagcaccctg 960accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaagt gacccaccag 1020ggcctgtcca gccccgtgac caagagcttc aaccggggcg agtgc 1065104355PRTArtificiallight chain of PD-L1-GPLGLWAQ -IL2 (N-terminal fusion) 104Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Gly Pro Leu Gly Leu Trp Ala Gln Asp Ile Gln 130 135 140Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val145 150 155 160Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp 165 170 175Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 180 185 190Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 195 200 205Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 210 215 220Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala Thr Phe Gly225 230 235 240Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 245 250 255Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 260 265 270Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 275 280 285Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 290 295 300Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu305 310 315 320Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 325 330 335Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 340 345 350Gly Glu Cys 3551051869DNAArtificialheavy chain of PD-L1-PVGLIG -NanoLuc (C-terminal fusion) 105gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg

tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaccggtggg cctgattggc ctcgagatgg tcttcacact cgaagatttc 1380gttggggact ggcgacagac agccggctac aacctggacc aagtccttga acagggaggt 1440gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc cgatccaaag gattgtcctg 1500agcggtgaaa atgggctgaa gatcgacatc catgtcatca tcccgtatga aggtctgagc 1560ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat 1620cactttaagg tgatcctgca ctatggcaca ctggtaatcg acggggttac gccgaacatg 1680atcgactatt tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact 1740gtaacaggga ccctgtggaa cggcaacaaa attatcgacg agcgcctgat caaccccgac 1800ggctccctgc tgttccgagt aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc 1860attctggcg 1869106623PRTArtificialheavy chain of PD-L1-PVGLIG -NanoLuc (C-terminal fusion) 106Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Pro Val Gly Leu 435 440 445Ile Gly Leu Glu Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp 450 455 460Arg Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly465 470 475 480Val Ser Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln 485 490 495Arg Ile Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val 500 505 510Ile Ile Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu 515 520 525Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val 530 535 540Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met545 550 555 560Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly 565 570 575Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile 580 585 590Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr 595 600 605Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala 610 615 6201071179DNAArtificiallight chain of PD-L1-PVGLIG -NanoLuc (C-terminal fusion) 107gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcccggtggg cctgattggc 660ctcgagatgg tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac 720aacctggacc aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg 780tccgtaactc cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc 840catgtcatca tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt 900tttaaggtgg tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca 960ctggtaatcg acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc 1020atcgccgtgt tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa 1080attatcgacg agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac 1140ggagtgaccg gctggcggct gtgcgaacgc attctggcg 1179108393PRTArtificiallight chain of PD-L1-PVGLIG -NanoLuc (C-terminal fusion) 108Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Pro Val Gly Leu Ile Gly Leu Glu Met Val 210 215 220Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr225 230 235 240Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln 245 250 255Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly 260 265 270Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu Gly 275 280 285Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val 290 295 300Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly Thr305 310 315 320Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly Arg 325 330 335Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr 340 345 350Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn 355 360 365Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly 370 375 380Trp Arg Leu Cys Glu Arg Ile Leu Ala385 3901091863DNAArtificialheavy chain of PD-L1-PVGLIG -NanoLuc (N-terminal fusion) 109atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgccggtgg gcctgattgg cgaagtccaa 540ctggtcgaaa gcggtggagg tctggtacag ccaggaggaa gtttgagact gtcatgcgct 600gcttccgggt ttactttcag cgactcctgg attcactggg ttcgtcaggc acctggaaag 660gggcttgagt gggttgcatg gatttcccca tacggcggct ctacatacta cgctgacagc 720gtcaagggca ggtttacaat aagcgccgat acctctaaaa acaccgccta cctgcagatg 780aatagtttgc gagccgagga taccgccgtg tattattgtg cccggcgcca ttggcccggc 840ggcttcgatt attgggggca ggggacactt gtgacagtgt ctgcagcctc taccaagggc 900cccagcgtgt tccctctggc cccctgcagc agaagcacca gcgagtctac agccgccctg 960ggatgcctgg tcaaggacta cttccccgag cccgtgaccg tgtcctggaa ctctggcgcc 1020ctgaccagcg gcgtgcacac ctttccagcc gtgctgcaga gcagcggcct gtacagcctg 1080agcagcgtcg tgaccgtgcc tagcagcagc ctgggcacca agacctacac ctgtaacgtg 1140gaccacaagc ccagcaacac caaggtggac aagcgggtgg aatctaagta cggccctccc 1200tgccccccct gcccagcccc tgaatttctg ggcggaccct ccgtgttcct gttcccccca 1260aagcccaagg acaccctgat gatcagccgg acccccgaag tgacctgcgt ggtggtggac 1320gtgtcccagg aagatcccga ggtccagttc aactggtacg tggacggcgt ggaagtgcac 1380aacgccaaga ccaagcccag agaggaacag ttcaacagca cctaccgggt ggtgtccgtg 1440ctgaccgtgc tgcaccagga ctggctgaac ggcaaagagt acaagtgcaa ggtctccaac 1500aagggcctgc ccagctccat cgagaaaacc atcagcaagg ccaagggcca gccccgcgag 1560ccccaggtgt acacactgcc ccccagccag gaagagatga ccaagaacca ggtgtccctg 1620acctgcctcg tgaagggctt ctaccccagc gatatcgccg tggaatggga gagcaacggc 1680cagcccgaga acaactacaa gaccaccccc cctgtgctgg acagcgacgg cagcttcttc 1740ctgtactccc ggctgaccgt cgacaagagc cggtggcagg aaggcaacgt cttcagctgc 1800agcgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag cctgagcctg 1860gga 1863110621PRTArtificialheavy chain of PD-L1-PVGLIG -NanoLuc (N-terminal fusion) 110Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Pro Val Gly Leu Ile 165 170 175Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 180 185 190Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp 195 200 205Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 210 215 220Val Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser225 230 235 240Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 245 250 255Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 260 265 270Cys Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly 275 280 285Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 290 295 300Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu305 310 315 320Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 325 330 335Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 340 345 350Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 355 360 365Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 370 375 380Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro385 390 395 400Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 405 410 415Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 420 425 430Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 435 440 445Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 450 455 460Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val465 470 475 480Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 485 490 495Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 500 505 510Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 515 520 525Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 530 535 540Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly545 550 555 560Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 565 570 575Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 580 585 590Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 595 600 605Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 610 615 6201111173DNAArtificiallight chain of PD-L1-PVGLIG -NanoLuc (N-terminal fusion) 111atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt

cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgccggtgg gcctgattgg cgacatccag 540atgacccaat ccccatcctc cttgagcgca tctgtaggag acagagtcac catcacctgc 600agggcttcac aggatgttag cacagctgtg gcttggtacc agcagaaacc tggaaaggcc 660ccaaaactgc tgatatacag cgcctcattc ctgtacagcg gagtgccctc tcgctttagt 720ggctctggga gtggcactga tttcacactg actatttcta gtctgcagcc cgaagatttc 780gccacttact attgccagca gtatctctat caccccgcca cctttgggca gggcacaaag 840gtcgagatta agcgtacggt ggccgctccc agcgtgttca tcttcccacc cagcgacgag 900cagctgaagt ctggcaccgc cagcgtcgtg tgcctgctga acaacttcta cccccgcgag 960gccaaggtgc agtggaaggt ggacaacgcc ctgcagtccg gcaacagcca ggaaagcgtc 1020accgagcagg acagcaagga ctccacctac agcctgagca gcaccctgac cctgagcaag 1080gccgactacg agaagcacaa ggtgtacgcc tgcgaagtga cccaccaggg cctgtccagc 1140cccgtgacca agagcttcaa ccggggcgag tgc 1173112391PRTArtificiallight chain of PD-L1-PVGLIG -NanoLuc (N-terminal fusion) 112Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Pro Val Gly Leu Ile 165 170 175Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 180 185 190Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr 195 200 205Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 210 215 220Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser225 230 235 240Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 245 250 255Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro 260 265 270Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 275 280 285Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 290 295 300Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu305 310 315 320Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 325 330 335Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 340 345 350Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 355 360 365Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 370 375 380Ser Phe Asn Arg Gly Glu Cys385 3901131749DNAArtificialheavy chain of PD-L1-PVGLIG-IL2 (C-terminal fusion) 113gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaccggtggg cctgattggc gcacctacct cgagttctac aaagaaaaca 1380cagctacaac tggagcattt actgctggat ttacagatga ttttgaatgg aattaataat 1440tacaagaatc ccaaactcac caggatgctc acatttaagt tttacatgcc caagaaggcc 1500acagaactga aacatcttca gtgtctagaa gaagaactca aacctctgga ggaagtgcta 1560aatttagctc aaagcaaaaa ctttcactta agacccaggg acttaatcag caatatcaac 1620gtaatagttc tggaactaaa gggatctgaa acaacattca tgtgtgaata tgctgatgag 1680acagcaacca ttgtagaatt tctgaacaga tggattacct tttgtcaaag catcatctca 1740acactgact 1749114583PRTArtificialheavy chain of PD-L1-PVGLIG-IL2 (C-terminal fusion) 114Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Pro Val Gly Leu 435 440 445Ile Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 450 455 460Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn465 470 475 480Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 485 490 495Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 500 505 510Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 515 520 525His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 530 535 540Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu545 550 555 560Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln 565 570 575Ser Ile Ile Ser Thr Leu Thr 5801151059DNAArtificiallight chain of PD-L1-PVGLIG-IL2 (C-terminal fusion) 115gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcccggtggg cctgattggc 660gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 720ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 780acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 840gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 900agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 960acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 1020tggattacct tttgtcaaag catcatctca acactgact 1059116353PRTArtificiallight chain of PD-L1-PVGLIG-IL2 (C-terminal fusion) 116Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Pro Val Gly Leu Ile Gly Ala Pro Thr Ser 210 215 220Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp225 230 235 240Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu 245 250 255Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu 260 265 270Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu 275 280 285Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp 290 295 300Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu305 310 315 320Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 325 330 335Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu 340 345 350Thr1171749DNAArtificialheavy chain of PD-L1-PVGLIG-IL2 (N-terminal fusion) 117gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactc cggtgggcct gattggcgaa 420gtccaactgg tcgaaagcgg tggaggtctg gtacagccag gaggaagttt gagactgtca 480tgcgctgctt ccgggtttac tttcagcgac tcctggattc actgggttcg tcaggcacct 540ggaaaggggc ttgagtgggt tgcatggatt tccccatacg gcggctctac atactacgct 600gacagcgtca agggcaggtt tacaataagc gccgatacct ctaaaaacac cgcctacctg 660cagatgaata gtttgcgagc cgaggatacc gccgtgtatt attgtgcccg gcgccattgg 720cccggcggct tcgattattg ggggcagggg acacttgtga cagtgtctgc agcctctacc 780aagggcccca gcgtgttccc tctggccccc tgcagcagaa gcaccagcga gtctacagcc 840gccctgggat gcctggtcaa ggactacttc cccgagcccg tgaccgtgtc ctggaactct 900ggcgccctga ccagcggcgt gcacaccttt ccagccgtgc tgcagagcag cggcctgtac 960agcctgagca gcgtcgtgac cgtgcctagc agcagcctgg gcaccaagac ctacacctgt 1020aacgtggacc acaagcccag caacaccaag gtggacaagc gggtggaatc taagtacggc 1080cctccctgcc ccccctgccc agcccctgaa tttctgggcg gaccctccgt gttcctgttc 1140cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 1200gtggacgtgt cccaggaaga tcccgaggtc cagttcaact ggtacgtgga cggcgtggaa 1260gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcaccta ccgggtggtg 1320tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtc 1380tccaacaagg gcctgcccag ctccatcgag aaaaccatca gcaaggccaa gggccagccc 1440cgcgagcccc aggtgtacac actgcccccc agccaggaag agatgaccaa gaaccaggtg 1500tccctgacct gcctcgtgaa gggcttctac cccagcgata tcgccgtgga atgggagagc 1560aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggcagc 1620ttcttcctgt actcccggct gaccgtcgac aagagccggt ggcaggaagg caacgtcttc 1680agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1740agcctggga 1749118583PRTArtificialheavy chain of PD-L1-PVGLIG-IL2 (N-terminal fusion) 118Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Pro Val Gly Leu Ile Gly Glu Val Gln Leu Val 130 135 140Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser145 150 155 160Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser Trp Ile His Trp Val 165 170 175Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile Ser Pro 180 185 190Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 195 200 205Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 210 215 220Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg His Trp225 230 235 240Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 245 250 255Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 260 265 270Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 275 280 285Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 290 295 300Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr305 310 315 320Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 325 330 335Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 340 345 350Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 355 360 365Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 370 375 380Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val385 390 395 400Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 405 410 415Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 420 425 430Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 435 440 445Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 450 455 460Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro465 470 475 480Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 485 490 495Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 500 505 510Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 515 520 525Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 530 535 540Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe545 550 555 560Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 565 570 575Ser Leu Ser Leu Ser Leu Gly 5801191059DNAArtificiallight chain of PD-L1-PVGLIG-IL2 (N-terminal fusion) 119gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactc cggtgggcct gattggcgac 420atccagatga cccaatcccc atcctccttg agcgcatctg taggagacag agtcaccatc 480acctgcaggg cttcacagga tgttagcaca gctgtggctt ggtaccagca gaaacctgga 540aaggccccaa aactgctgat atacagcgcc tcattcctgt acagcggagt gccctctcgc 600tttagtggct ctgggagtgg cactgatttc acactgacta tttctagtct gcagcccgaa 660gatttcgcca cttactattg ccagcagtat ctctatcacc ccgccacctt tgggcagggc 720acaaaggtcg agattaagcg tacggtggcc gctcccagcg tgttcatctt cccacccagc 780gacgagcagc tgaagtctgg caccgccagc gtcgtgtgcc tgctgaacaa cttctacccc 840cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa cagccaggaa 900agcgtcaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctgaccctg 960agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca ccagggcctg 1020tccagccccg tgaccaagag cttcaaccgg ggcgagtgc 1059120353PRTArtificiallight chain of PD-L1-PVGLIG-IL2 (N-terminal fusion) 120Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Pro Val Gly Leu Ile Gly Asp Ile Gln Met Thr 130 135 140Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile145 150 155 160Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln 165 170 175Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 180 185 190Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 210 215 220Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala Thr Phe Gly Gln Gly225 230 235 240Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 245 250 255Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 260 265 270Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 275 280 285Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 290 295 300Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu305 310 315 320Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 325 330 335His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 340 345 350Cys1211869DNAArtificialheavy chain of PD-L1-PLGLAG-NanoLuc (C-terminal fusion) 121gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaccgctggg cctggcgggc ctcgagatgg tcttcacact cgaagatttc 1380gttggggact ggcgacagac agccggctac aacctggacc aagtccttga acagggaggt 1440gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc cgatccaaag gattgtcctg 1500agcggtgaaa atgggctgaa gatcgacatc catgtcatca tcccgtatga aggtctgagc 1560ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat 1620cactttaagg tgatcctgca ctatggcaca ctggtaatcg acggggttac gccgaacatg 1680atcgactatt tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact 1740gtaacaggga ccctgtggaa cggcaacaaa attatcgacg agcgcctgat caaccccgac 1800ggctccctgc tgttccgagt aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc 1860attctggcg 1869122623PRTArtificialheavy chain of PD-L1-PLGLAG-NanoLuc (C-terminal fusion) 122Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Pro Leu Gly Leu 435 440 445Ala Gly Leu Glu Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp 450 455 460Arg Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly465 470 475 480Val Ser Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln 485 490 495Arg Ile Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val 500 505 510Ile Ile Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu 515 520 525Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val 530 535 540Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met545 550 555 560Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly 565 570 575Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile 580 585 590Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr 595 600 605Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala 610 615 6201231179DNAArtificiallight chain of PD-L1-PLGLAG-NanoLuc (C-terminal fusion) 123gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcccgctggg cctggcgggc 660ctcgagatgg tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac 720aacctggacc aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg 780tccgtaactc cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc 840catgtcatca tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt 900tttaaggtgg tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca 960ctggtaatcg acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc 1020atcgccgtgt tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa 1080attatcgacg agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac 1140ggagtgaccg gctggcggct gtgcgaacgc attctggcg 1179124393PRTArtificiallight chain of PD-L1-PLGLAG-NanoLuc (C-terminal fusion) 124Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Pro Leu Gly Leu Ala Gly Leu Glu Met Val 210 215 220Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr225 230 235 240Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln 245 250 255Asn Leu Gly Val Ser

Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly 260 265 270Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu Gly 275 280 285Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val 290 295 300Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly Thr305 310 315 320Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly Arg 325 330 335Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr 340 345 350Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn 355 360 365Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly 370 375 380Trp Arg Leu Cys Glu Arg Ile Leu Ala385 3901251863DNAArtificialheavy chain of PD-L1-PLGLAG-NanoLuc (N-terminal fusion) 125atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgccgctgg gcctggcggg cgaagtccaa 540ctggtcgaaa gcggtggagg tctggtacag ccaggaggaa gtttgagact gtcatgcgct 600gcttccgggt ttactttcag cgactcctgg attcactggg ttcgtcaggc acctggaaag 660gggcttgagt gggttgcatg gatttcccca tacggcggct ctacatacta cgctgacagc 720gtcaagggca ggtttacaat aagcgccgat acctctaaaa acaccgccta cctgcagatg 780aatagtttgc gagccgagga taccgccgtg tattattgtg cccggcgcca ttggcccggc 840ggcttcgatt attgggggca ggggacactt gtgacagtgt ctgcagcctc taccaagggc 900cccagcgtgt tccctctggc cccctgcagc agaagcacca gcgagtctac agccgccctg 960ggatgcctgg tcaaggacta cttccccgag cccgtgaccg tgtcctggaa ctctggcgcc 1020ctgaccagcg gcgtgcacac ctttccagcc gtgctgcaga gcagcggcct gtacagcctg 1080agcagcgtcg tgaccgtgcc tagcagcagc ctgggcacca agacctacac ctgtaacgtg 1140gaccacaagc ccagcaacac caaggtggac aagcgggtgg aatctaagta cggccctccc 1200tgccccccct gcccagcccc tgaatttctg ggcggaccct ccgtgttcct gttcccccca 1260aagcccaagg acaccctgat gatcagccgg acccccgaag tgacctgcgt ggtggtggac 1320gtgtcccagg aagatcccga ggtccagttc aactggtacg tggacggcgt ggaagtgcac 1380aacgccaaga ccaagcccag agaggaacag ttcaacagca cctaccgggt ggtgtccgtg 1440ctgaccgtgc tgcaccagga ctggctgaac ggcaaagagt acaagtgcaa ggtctccaac 1500aagggcctgc ccagctccat cgagaaaacc atcagcaagg ccaagggcca gccccgcgag 1560ccccaggtgt acacactgcc ccccagccag gaagagatga ccaagaacca ggtgtccctg 1620acctgcctcg tgaagggctt ctaccccagc gatatcgccg tggaatggga gagcaacggc 1680cagcccgaga acaactacaa gaccaccccc cctgtgctgg acagcgacgg cagcttcttc 1740ctgtactccc ggctgaccgt cgacaagagc cggtggcagg aaggcaacgt cttcagctgc 1800agcgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag cctgagcctg 1860gga 1863126621PRTArtificialheavy chain of PD-L1-PLGLAG-NanoLuc (N-terminal fusion) 126Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Pro Leu Gly Leu Ala 165 170 175Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 180 185 190Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp 195 200 205Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 210 215 220Val Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser225 230 235 240Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 245 250 255Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 260 265 270Cys Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly 275 280 285Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 290 295 300Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu305 310 315 320Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 325 330 335Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 340 345 350Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 355 360 365Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 370 375 380Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro385 390 395 400Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 405 410 415Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 420 425 430Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 435 440 445Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 450 455 460Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val465 470 475 480Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 485 490 495Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 500 505 510Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 515 520 525Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 530 535 540Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly545 550 555 560Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 565 570 575Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 580 585 590Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 595 600 605Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 610 615 6201271173DNAArtificiallight chain of PD-L1-PLGLAG-NanoLuc (N-terminal fusion) 127atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgccgctgg gcctggcggg cgacatccag 540atgacccaat ccccatcctc cttgagcgca tctgtaggag acagagtcac catcacctgc 600agggcttcac aggatgttag cacagctgtg gcttggtacc agcagaaacc tggaaaggcc 660ccaaaactgc tgatatacag cgcctcattc ctgtacagcg gagtgccctc tcgctttagt 720ggctctggga gtggcactga tttcacactg actatttcta gtctgcagcc cgaagatttc 780gccacttact attgccagca gtatctctat caccccgcca cctttgggca gggcacaaag 840gtcgagatta agcgtacggt ggccgctccc agcgtgttca tcttcccacc cagcgacgag 900cagctgaagt ctggcaccgc cagcgtcgtg tgcctgctga acaacttcta cccccgcgag 960gccaaggtgc agtggaaggt ggacaacgcc ctgcagtccg gcaacagcca ggaaagcgtc 1020accgagcagg acagcaagga ctccacctac agcctgagca gcaccctgac cctgagcaag 1080gccgactacg agaagcacaa ggtgtacgcc tgcgaagtga cccaccaggg cctgtccagc 1140cccgtgacca agagcttcaa ccggggcgag tgc 1173128391PRTArtificiallight chain of PD-L1-PLGLAG-NanoLuc (N-terminal fusion) 128Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Pro Leu Gly Leu Ala 165 170 175Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 180 185 190Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr 195 200 205Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 210 215 220Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser225 230 235 240Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 245 250 255Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro 260 265 270Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 275 280 285Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 290 295 300Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu305 310 315 320Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 325 330 335Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 340 345 350Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 355 360 365Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 370 375 380Ser Phe Asn Arg Gly Glu Cys385 3901291749DNAArtificialheavy chain of PD-L1-PLGLAG-IL2 (C-terminal fusion) 129gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaccgctggg cctggcgggc gcacctacct cgagttctac aaagaaaaca 1380cagctacaac tggagcattt actgctggat ttacagatga ttttgaatgg aattaataat 1440tacaagaatc ccaaactcac caggatgctc acatttaagt tttacatgcc caagaaggcc 1500acagaactga aacatcttca gtgtctagaa gaagaactca aacctctgga ggaagtgcta 1560aatttagctc aaagcaaaaa ctttcactta agacccaggg acttaatcag caatatcaac 1620gtaatagttc tggaactaaa gggatctgaa acaacattca tgtgtgaata tgctgatgag 1680acagcaacca ttgtagaatt tctgaacaga tggattacct tttgtcaaag catcatctca 1740acactgact 1749130583PRTArtificialheavy chain of PD-L1-PLGLAG-IL2 (C-terminal fusion) 130Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Pro Leu Gly Leu 435 440 445Ala

Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 450 455 460Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn465 470 475 480Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 485 490 495Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 500 505 510Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 515 520 525His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 530 535 540Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu545 550 555 560Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln 565 570 575Ser Ile Ile Ser Thr Leu Thr 5801311059DNAArtificiallight chain of PD-L1-PLGLAG-IL2 (C-terminal fusion) 131gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcccgctggg cctggcgggc 660gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 720ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 780acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 840gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 900agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 960acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 1020tggattacct tttgtcaaag catcatctca acactgact 1059132353PRTArtificiallight chain of PD-L1-PLGLAG-IL2 (C-terminal fusion) 132Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Pro Leu Gly Leu Ala Gly Ala Pro Thr Ser 210 215 220Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp225 230 235 240Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu 245 250 255Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu 260 265 270Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu 275 280 285Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp 290 295 300Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu305 310 315 320Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 325 330 335Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu 340 345 350Thr1331749DNAArtificialheavy chain of PD-L1-PLGLAG-IL2 (N-terminal fusion) 133gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactc cgctgggcct ggcgggcgaa 420gtccaactgg tcgaaagcgg tggaggtctg gtacagccag gaggaagttt gagactgtca 480tgcgctgctt ccgggtttac tttcagcgac tcctggattc actgggttcg tcaggcacct 540ggaaaggggc ttgagtgggt tgcatggatt tccccatacg gcggctctac atactacgct 600gacagcgtca agggcaggtt tacaataagc gccgatacct ctaaaaacac cgcctacctg 660cagatgaata gtttgcgagc cgaggatacc gccgtgtatt attgtgcccg gcgccattgg 720cccggcggct tcgattattg ggggcagggg acacttgtga cagtgtctgc agcctctacc 780aagggcccca gcgtgttccc tctggccccc tgcagcagaa gcaccagcga gtctacagcc 840gccctgggat gcctggtcaa ggactacttc cccgagcccg tgaccgtgtc ctggaactct 900ggcgccctga ccagcggcgt gcacaccttt ccagccgtgc tgcagagcag cggcctgtac 960agcctgagca gcgtcgtgac cgtgcctagc agcagcctgg gcaccaagac ctacacctgt 1020aacgtggacc acaagcccag caacaccaag gtggacaagc gggtggaatc taagtacggc 1080cctccctgcc ccccctgccc agcccctgaa tttctgggcg gaccctccgt gttcctgttc 1140cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 1200gtggacgtgt cccaggaaga tcccgaggtc cagttcaact ggtacgtgga cggcgtggaa 1260gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcaccta ccgggtggtg 1320tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtc 1380tccaacaagg gcctgcccag ctccatcgag aaaaccatca gcaaggccaa gggccagccc 1440cgcgagcccc aggtgtacac actgcccccc agccaggaag agatgaccaa gaaccaggtg 1500tccctgacct gcctcgtgaa gggcttctac cccagcgata tcgccgtgga atgggagagc 1560aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggcagc 1620ttcttcctgt actcccggct gaccgtcgac aagagccggt ggcaggaagg caacgtcttc 1680agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1740agcctggga 1749134583PRTArtificialheavy chain of PD-L1-PLGLAG-IL2 (N-terminal fusion) 134Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Pro Leu Gly Leu Ala Gly Glu Val Gln Leu Val 130 135 140Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser145 150 155 160Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser Trp Ile His Trp Val 165 170 175Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile Ser Pro 180 185 190Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 195 200 205Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 210 215 220Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg His Trp225 230 235 240Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 245 250 255Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 260 265 270Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 275 280 285Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 290 295 300Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr305 310 315 320Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 325 330 335Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 340 345 350Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 355 360 365Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 370 375 380Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val385 390 395 400Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 405 410 415Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 420 425 430Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 435 440 445Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 450 455 460Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro465 470 475 480Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 485 490 495Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 500 505 510Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 515 520 525Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 530 535 540Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe545 550 555 560Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 565 570 575Ser Leu Ser Leu Ser Leu Gly 5801351059DNAArtificiallight chain of PD-L1-PLGLAG-IL2 (N-terminal fusion) 135gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactc cgctgggcct ggcgggcgac 420atccagatga cccaatcccc atcctccttg agcgcatctg taggagacag agtcaccatc 480acctgcaggg cttcacagga tgttagcaca gctgtggctt ggtaccagca gaaacctgga 540aaggccccaa aactgctgat atacagcgcc tcattcctgt acagcggagt gccctctcgc 600tttagtggct ctgggagtgg cactgatttc acactgacta tttctagtct gcagcccgaa 660gatttcgcca cttactattg ccagcagtat ctctatcacc ccgccacctt tgggcagggc 720acaaaggtcg agattaagcg tacggtggcc gctcccagcg tgttcatctt cccacccagc 780gacgagcagc tgaagtctgg caccgccagc gtcgtgtgcc tgctgaacaa cttctacccc 840cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa cagccaggaa 900agcgtcaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctgaccctg 960agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca ccagggcctg 1020tccagccccg tgaccaagag cttcaaccgg ggcgagtgc 1059136353PRTArtificiallight chain of PD-L1-PLGLAG-IL2 (N-terminal fusion) 136Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Pro Leu Gly Leu Ala Gly Asp Ile Gln Met Thr 130 135 140Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile145 150 155 160Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln 165 170 175Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 180 185 190Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 210 215 220Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala Thr Phe Gly Gln Gly225 230 235 240Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 245 250 255Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 260 265 270Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 275 280 285Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 290 295 300Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu305 310 315 320Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 325 330 335His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 340 345 350Cys1371869DNAArtificialheavy chain of PD-L1-GIVGPL-NanoLuc (C-terminal fusion) 137gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaggcattgt gggaccgctg ctcgagatgg tcttcacact cgaagatttc 1380gttggggact ggcgacagac agccggctac aacctggacc aagtccttga acagggaggt 1440gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc cgatccaaag gattgtcctg 1500agcggtgaaa atgggctgaa gatcgacatc catgtcatca tcccgtatga aggtctgagc 1560ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat 1620cactttaagg tgatcctgca ctatggcaca ctggtaatcg acggggttac gccgaacatg 1680atcgactatt tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact 1740gtaacaggga ccctgtggaa cggcaacaaa attatcgacg agcgcctgat caaccccgac 1800ggctccctgc tgttccgagt aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc 1860attctggcg

1869138623PRTArtificialheavy chain of PD-L1-GIVGPL-NanoLuc (C-terminal fusion) 138Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ile Val Gly 435 440 445Pro Leu Leu Glu Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp 450 455 460Arg Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly465 470 475 480Val Ser Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln 485 490 495Arg Ile Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val 500 505 510Ile Ile Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu 515 520 525Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val 530 535 540Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met545 550 555 560Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly 565 570 575Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile 580 585 590Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr 595 600 605Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala 610 615 6201391179DNAArtificiallight chain of PD-L1-GIVGPL-NanoLuc (C-terminal fusion) 139gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcggcattgt gggaccgctg 660ctcgagatgg tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac 720aacctggacc aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg 780tccgtaactc cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc 840catgtcatca tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt 900tttaaggtgg tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca 960ctggtaatcg acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc 1020atcgccgtgt tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa 1080attatcgacg agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac 1140ggagtgaccg gctggcggct gtgcgaacgc attctggcg 1179140393PRTArtificiallight chain of PD-L1-GIVGPL-NanoLuc (C-terminal fusion) 140Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Gly Ile Val Gly Pro Leu Leu Glu Met Val 210 215 220Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr225 230 235 240Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln 245 250 255Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly 260 265 270Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu Gly 275 280 285Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val 290 295 300Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly Thr305 310 315 320Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly Arg 325 330 335Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr 340 345 350Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn 355 360 365Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly 370 375 380Trp Arg Leu Cys Glu Arg Ile Leu Ala385 3901411863DNAArtificialheavy chain of PD-L1-GIVGPL-NanoLuc (N-terminal fusion) 141atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgggcattg tgggaccgct ggaagtccaa 540ctggtcgaaa gcggtggagg tctggtacag ccaggaggaa gtttgagact gtcatgcgct 600gcttccgggt ttactttcag cgactcctgg attcactggg ttcgtcaggc acctggaaag 660gggcttgagt gggttgcatg gatttcccca tacggcggct ctacatacta cgctgacagc 720gtcaagggca ggtttacaat aagcgccgat acctctaaaa acaccgccta cctgcagatg 780aatagtttgc gagccgagga taccgccgtg tattattgtg cccggcgcca ttggcccggc 840ggcttcgatt attgggggca ggggacactt gtgacagtgt ctgcagcctc taccaagggc 900cccagcgtgt tccctctggc cccctgcagc agaagcacca gcgagtctac agccgccctg 960ggatgcctgg tcaaggacta cttccccgag cccgtgaccg tgtcctggaa ctctggcgcc 1020ctgaccagcg gcgtgcacac ctttccagcc gtgctgcaga gcagcggcct gtacagcctg 1080agcagcgtcg tgaccgtgcc tagcagcagc ctgggcacca agacctacac ctgtaacgtg 1140gaccacaagc ccagcaacac caaggtggac aagcgggtgg aatctaagta cggccctccc 1200tgccccccct gcccagcccc tgaatttctg ggcggaccct ccgtgttcct gttcccccca 1260aagcccaagg acaccctgat gatcagccgg acccccgaag tgacctgcgt ggtggtggac 1320gtgtcccagg aagatcccga ggtccagttc aactggtacg tggacggcgt ggaagtgcac 1380aacgccaaga ccaagcccag agaggaacag ttcaacagca cctaccgggt ggtgtccgtg 1440ctgaccgtgc tgcaccagga ctggctgaac ggcaaagagt acaagtgcaa ggtctccaac 1500aagggcctgc ccagctccat cgagaaaacc atcagcaagg ccaagggcca gccccgcgag 1560ccccaggtgt acacactgcc ccccagccag gaagagatga ccaagaacca ggtgtccctg 1620acctgcctcg tgaagggctt ctaccccagc gatatcgccg tggaatggga gagcaacggc 1680cagcccgaga acaactacaa gaccaccccc cctgtgctgg acagcgacgg cagcttcttc 1740ctgtactccc ggctgaccgt cgacaagagc cggtggcagg aaggcaacgt cttcagctgc 1800agcgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag cctgagcctg 1860gga 1863142621PRTArtificialheavy chain of PD-L1-GIVGPL-NanoLuc (N-terminal fusion) 142Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly Ile Val Gly Pro 165 170 175Leu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 180 185 190Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp 195 200 205Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 210 215 220Val Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser225 230 235 240Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 245 250 255Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 260 265 270Cys Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly 275 280 285Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 290 295 300Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu305 310 315 320Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 325 330 335Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 340 345 350Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 355 360 365Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 370 375 380Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro385 390 395 400Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 405 410 415Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 420 425 430Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 435 440 445Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 450 455 460Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val465 470 475 480Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 485 490 495Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 500 505 510Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 515 520 525Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 530 535 540Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly545 550 555 560Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 565 570 575Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 580 585 590Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 595 600 605Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 610 615 6201431173DNAArtificiallight chain of PD-L1-GIVGPL-NanoLuc (N-terminal fusion) 143atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgggcattg tgggaccgct ggacatccag 540atgacccaat ccccatcctc cttgagcgca tctgtaggag acagagtcac catcacctgc 600agggcttcac aggatgttag cacagctgtg gcttggtacc agcagaaacc tggaaaggcc 660ccaaaactgc tgatatacag cgcctcattc ctgtacagcg gagtgccctc tcgctttagt 720ggctctggga gtggcactga tttcacactg actatttcta gtctgcagcc cgaagatttc 780gccacttact attgccagca gtatctctat caccccgcca cctttgggca gggcacaaag 840gtcgagatta agcgtacggt ggccgctccc agcgtgttca tcttcccacc cagcgacgag 900cagctgaagt ctggcaccgc cagcgtcgtg tgcctgctga acaacttcta cccccgcgag 960gccaaggtgc agtggaaggt ggacaacgcc ctgcagtccg gcaacagcca ggaaagcgtc 1020accgagcagg acagcaagga ctccacctac agcctgagca gcaccctgac cctgagcaag 1080gccgactacg agaagcacaa ggtgtacgcc tgcgaagtga cccaccaggg cctgtccagc 1140cccgtgacca agagcttcaa ccggggcgag tgc 1173144391PRTArtificiallight chain of PD-L1-GIVGPL-NanoLuc (N-terminal fusion) 144Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe

Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly Ile Val Gly Pro 165 170 175Leu Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 180 185 190Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr 195 200 205Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 210 215 220Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser225 230 235 240Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 245 250 255Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro 260 265 270Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 275 280 285Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 290 295 300Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu305 310 315 320Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 325 330 335Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 340 345 350Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 355 360 365Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 370 375 380Ser Phe Asn Arg Gly Glu Cys385 3901451749DNAArtificialheavy chain of PD-L1-GIVGPL-IL2 (C-terminal fusion) 145gaagtccaac tggtcgaaag cggtggaggt ctggtacagc caggaggaag tttgagactg 60tcatgcgctg cttccgggtt tactttcagc gactcctgga ttcactgggt tcgtcaggca 120cctggaaagg ggcttgagtg ggttgcatgg atttccccat acggcggctc tacatactac 180gctgacagcg tcaagggcag gtttacaata agcgccgata cctctaaaaa caccgcctac 240ctgcagatga atagtttgcg agccgaggat accgccgtgt attattgtgc ccggcgccat 300tggcccggcg gcttcgatta ttgggggcag gggacacttg tgacagtgtc tgcagcctct 360accaagggcc ccagcgtgtt ccctctggcc ccctgcagca gaagcaccag cgagtctaca 420gccgccctgg gatgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480tctggcgccc tgaccagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tacagcctga gcagcgtcgt gaccgtgcct agcagcagcc tgggcaccaa gacctacacc 600tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agcgggtgga atctaagtac 660ggccctccct gccccccctg cccagcccct gaatttctgg gcggaccctc cgtgttcctg 720ttccccccaa agcccaagga caccctgatg atcagccgga cccccgaagt gacctgcgtg 780gtggtggacg tgtcccagga agatcccgag gtccagttca actggtacgt ggacggcgtg 840gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaacagcac ctaccgggtg 900gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960gtctccaaca agggcctgcc cagctccatc gagaaaacca tcagcaaggc caagggccag 1020ccccgcgagc cccaggtgta cacactgccc cccagccagg aagagatgac caagaaccag 1080gtgtccctga cctgcctcgt gaagggcttc taccccagcg atatcgccgt ggaatgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1200agcttcttcc tgtactcccg gctgaccgtc gacaagagcc ggtggcagga aggcaacgtc 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320ctgagcctgg gaggcattgt gggaccgctg gcacctacct cgagttctac aaagaaaaca 1380cagctacaac tggagcattt actgctggat ttacagatga ttttgaatgg aattaataat 1440tacaagaatc ccaaactcac caggatgctc acatttaagt tttacatgcc caagaaggcc 1500acagaactga aacatcttca gtgtctagaa gaagaactca aacctctgga ggaagtgcta 1560aatttagctc aaagcaaaaa ctttcactta agacccaggg acttaatcag caatatcaac 1620gtaatagttc tggaactaaa gggatctgaa acaacattca tgtgtgaata tgctgatgag 1680acagcaacca ttgtagaatt tctgaacaga tggattacct tttgtcaaag catcatctca 1740acactgact 1749146583PRTArtificialheavy chain of PD-L1-GIVGPL-IL2 (C-terminal fusion) 146Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ile Val Gly 435 440 445Pro Leu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 450 455 460Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn465 470 475 480Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 485 490 495Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 500 505 510Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 515 520 525His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 530 535 540Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu545 550 555 560Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln 565 570 575Ser Ile Ile Ser Thr Leu Thr 5801471059DNAArtificiallight chain of PD-L1-GIVGPL-IL2 (C-terminal fusion) 147gacatccaga tgacccaatc cccatcctcc ttgagcgcat ctgtaggaga cagagtcacc 60atcacctgca gggcttcaca ggatgttagc acagctgtgg cttggtacca gcagaaacct 120ggaaaggccc caaaactgct gatatacagc gcctcattcc tgtacagcgg agtgccctct 180cgctttagtg gctctgggag tggcactgat ttcacactga ctatttctag tctgcagccc 240gaagatttcg ccacttacta ttgccagcag tatctctatc accccgccac ctttgggcag 300ggcacaaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttcccaccc 360agcgacgagc agctgaagtc tggcaccgcc agcgtcgtgt gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gcggcattgt gggaccgctg 660gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 720ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 780acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 840gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 900agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 960acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 1020tggattacct tttgtcaaag catcatctca acactgact 1059148353PRTArtificiallight chain of PD-L1-GIVGPL-IL2 (C-terminal fusion) 148Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Gly Ile Val Gly Pro Leu Ala Pro Thr Ser 210 215 220Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp225 230 235 240Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu 245 250 255Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu 260 265 270Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu 275 280 285Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp 290 295 300Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu305 310 315 320Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 325 330 335Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu 340 345 350Thr1491749DNAArtificialheavy chain of PD-L1-GIVGPL-IL2 (N-terminal fusion) 149gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactg gcattgtggg accgctggaa 420gtccaactgg tcgaaagcgg tggaggtctg gtacagccag gaggaagttt gagactgtca 480tgcgctgctt ccgggtttac tttcagcgac tcctggattc actgggttcg tcaggcacct 540ggaaaggggc ttgagtgggt tgcatggatt tccccatacg gcggctctac atactacgct 600gacagcgtca agggcaggtt tacaataagc gccgatacct ctaaaaacac cgcctacctg 660cagatgaata gtttgcgagc cgaggatacc gccgtgtatt attgtgcccg gcgccattgg 720cccggcggct tcgattattg ggggcagggg acacttgtga cagtgtctgc agcctctacc 780aagggcccca gcgtgttccc tctggccccc tgcagcagaa gcaccagcga gtctacagcc 840gccctgggat gcctggtcaa ggactacttc cccgagcccg tgaccgtgtc ctggaactct 900ggcgccctga ccagcggcgt gcacaccttt ccagccgtgc tgcagagcag cggcctgtac 960agcctgagca gcgtcgtgac cgtgcctagc agcagcctgg gcaccaagac ctacacctgt 1020aacgtggacc acaagcccag caacaccaag gtggacaagc gggtggaatc taagtacggc 1080cctccctgcc ccccctgccc agcccctgaa tttctgggcg gaccctccgt gttcctgttc 1140cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 1200gtggacgtgt cccaggaaga tcccgaggtc cagttcaact ggtacgtgga cggcgtggaa 1260gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcaccta ccgggtggtg 1320tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtc 1380tccaacaagg gcctgcccag ctccatcgag aaaaccatca gcaaggccaa gggccagccc 1440cgcgagcccc aggtgtacac actgcccccc agccaggaag agatgaccaa gaaccaggtg 1500tccctgacct gcctcgtgaa gggcttctac cccagcgata tcgccgtgga atgggagagc 1560aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggcagc 1620ttcttcctgt actcccggct gaccgtcgac aagagccggt ggcaggaagg caacgtcttc 1680agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1740agcctggga 1749150583PRTArtificialheavy chain of PD-L1-GIVGPL-IL2 (N-terminal fusion) 150Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Gly Ile Val Gly Pro Leu Glu Val Gln Leu Val 130 135 140Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser145 150 155 160Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser Trp Ile His Trp Val 165 170 175Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile Ser Pro 180 185 190Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 195 200 205Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 210 215 220Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg His Trp225 230 235 240Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 245 250 255Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 260 265 270Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 275 280 285Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 290 295 300Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr305 310 315 320Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 325 330 335Thr Tyr Thr Cys Asn

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 340 345 350Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 355 360 365Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 370 375 380Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val385 390 395 400Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 405 410 415Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 420 425 430Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 435 440 445Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 450 455 460Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro465 470 475 480Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 485 490 495Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 500 505 510Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 515 520 525Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 530 535 540Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe545 550 555 560Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 565 570 575Ser Leu Ser Leu Ser Leu Gly 5801511059DNAArtificiallight chain of PD-L1-GIVGPL-IL2 (N-terminal fusion) 151gcacctacct cgagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360tggattacct tttgtcaaag catcatctca acactgactg gcattgtggg accgctggac 420atccagatga cccaatcccc atcctccttg agcgcatctg taggagacag agtcaccatc 480acctgcaggg cttcacagga tgttagcaca gctgtggctt ggtaccagca gaaacctgga 540aaggccccaa aactgctgat atacagcgcc tcattcctgt acagcggagt gccctctcgc 600tttagtggct ctgggagtgg cactgatttc acactgacta tttctagtct gcagcccgaa 660gatttcgcca cttactattg ccagcagtat ctctatcacc ccgccacctt tgggcagggc 720acaaaggtcg agattaagcg tacggtggcc gctcccagcg tgttcatctt cccacccagc 780gacgagcagc tgaagtctgg caccgccagc gtcgtgtgcc tgctgaacaa cttctacccc 840cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa cagccaggaa 900agcgtcaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctgaccctg 960agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca ccagggcctg 1020tccagccccg tgaccaagag cttcaaccgg ggcgagtgc 1059152353PRTArtificiallight chain of PD-L1-GIVGPL-IL2 (N-terminal fusion) 152Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr Gly Ile Val Gly Pro Leu Asp Ile Gln Met Thr 130 135 140Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile145 150 155 160Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln 165 170 175Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 180 185 190Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 210 215 220Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala Thr Phe Gly Gln Gly225 230 235 240Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 245 250 255Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 260 265 270Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 275 280 285Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 290 295 300Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu305 310 315 320Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 325 330 335His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 340 345 350Cys1531863DNAArtificialheavy chain of 9G4-PVGLIG-NanoLuc (C-terminal fusion) 153gaagtgatgt tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60tcctgtacag cctctggatt cactttcagt acctatgaca tgtcttgggt tcgccagact 120ccggagaaga ggctggagtg ggtctcaacc attagtagta gtcgtactta cacctactat 180tcagccagtg tgaaggggcg attcaccatc tccagagaca atgccaggaa catcctgtac 240ctgcaaatga gcagtctgag gtctgaggac acggccatgt attactgtgc aagacaattt 300tctcactggg gccaagggac tctggtcact gtctctgcag cgtccacaaa gggcccaagc 360gtgttcccgc tagcccccag cagcaagagc accagcggcg gcacagccgc cctgggctgc 420ctggtgaagg actacttccc cgagcccgtg accgtgtcct ggaacagcgg agccctgacc 480tccggcgtgc acaccttccc cgccgtgctg cagagcagcg gcctgtacag cctgagcagc 540gtggtgaccg tgcccagcag cagcctgggc acccagacct acatctgtaa cgtgaaccac 600aagcccagca acaccaaggt ggacaagaga gtggagccca agagctgtga caagacccac 660acctgtcccc cctgcccagc ccccgagctg ctgggcggac ccagcgtgtt cctgttcccc 720cccaagccca aggacaccct gatgatcagc agaacccccg aggtgacctg tgtggtggtg 780gacgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggacgg cgtggaggtg 840cacaacgcca agaccaagcc cagagaggag cagtacaaca gcacctacag ggtggtgtcc 900gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg taaggtgtcc 960aacaaggccc tgccagcccc aatcgaaaag accatcagca aggccaaggg ccagccaaga 1020gagccccagg tgtacaccct gccacccagc agggaggaga tgaccaagaa ccaggtgtcc 1080ctgacctgtc tggtgaaggg cttctaccca agcgacatcg ccgtggagtg ggagagcaac 1140ggccagcccg agaacaacta caagaccacc cccccagtgc tggacagcga cggcagcttc 1200ttcctgtaca gcaagctgac cgtggacaag agcagatggc agcagggcaa cgtgttcagc 1260tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgtcc 1320ccaggcccgg tgggcctgat tggcctcgag atggtcttca cactcgaaga tttcgttggg 1380gactggcgac agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc 1440agtttgtttc agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt 1500gaaaatgggc tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac 1560caaatgggcc agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt 1620aaggtgatcc tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac 1680tatttcggac ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca 1740gggaccctgt ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc 1800ctgctgttcc gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg 1860gcg 1863154621PRTArtificialheavy chain of 9G4-PVGLIG-NanoLuc (C-terminal fusion) 154Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ser Thr Ile Ser Ser Ser Arg Thr Tyr Thr Tyr Tyr Ser Ala Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gln Phe Ser His Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr145 150 155 160Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro225 230 235 240Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser305 310 315 320Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe385 390 395 400Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Pro Val Gly Leu Ile Gly 435 440 445Leu Glu Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln 450 455 460Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser465 470 475 480Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile 485 490 495Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile 500 505 510Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile 515 520 525Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu 530 535 540His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp545 550 555 560Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys 565 570 575Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu 580 585 590Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn 595 600 605Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala 610 615 6201551551DNAArtificialheavy chain of 9G4-PVGLIG-CCL4 (C-terminal fusion) 155gaagtgatgt tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60tcctgtacag cctctggatt cactttcagt acctatgaca tgtcttgggt tcgccagact 120ccggagaaga ggctggagtg ggtctcaacc attagtagta gtcgtactta cacctactat 180tcagccagtg tgaaggggcg attcaccatc tccagagaca atgccaggaa catcctgtac 240ctgcaaatga gcagtctgag gtctgaggac acggccatgt attactgtgc aagacaattt 300tctcactggg gccaagggac tctggtcact gtctctgcag cgtccacaaa gggcccaagc 360gtgttcccgc tagcccccag cagcaagagc accagcggcg gcacagccgc cctgggctgc 420ctggtgaagg actacttccc cgagcccgtg accgtgtcct ggaacagcgg agccctgacc 480tccggcgtgc acaccttccc cgccgtgctg cagagcagcg gcctgtacag cctgagcagc 540gtggtgaccg tgcccagcag cagcctgggc acccagacct acatctgtaa cgtgaaccac 600aagcccagca acaccaaggt ggacaagaga gtggagccca agagctgtga caagacccac 660acctgtcccc cctgcccagc ccccgagctg ctgggcggac ccagcgtgtt cctgttcccc 720cccaagccca aggacaccct gatgatcagc agaacccccg aggtgacctg tgtggtggtg 780gacgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggacgg cgtggaggtg 840cacaacgcca agaccaagcc cagagaggag cagtacaaca gcacctacag ggtggtgtcc 900gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg taaggtgtcc 960aacaaggccc tgccagcccc aatcgaaaag accatcagca aggccaaggg ccagccaaga 1020gagccccagg tgtacaccct gccacccagc agggaggaga tgaccaagaa ccaggtgtcc 1080ctgacctgtc tggtgaaggg cttctaccca agcgacatcg ccgtggagtg ggagagcaac 1140ggccagcccg agaacaacta caagaccacc cccccagtgc tggacagcga cggcagcttc 1200ttcctgtaca gcaagctgac cgtggacaag agcagatggc agcagggcaa cgtgttcagc 1260tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgtcc 1320ccaggcccgg tgggcctgat tggcgcacca atgggctcag accctcccac cgcctgctgc 1380ttttcttaca ccgcgaggaa acttcctcgc aactttgtgg tagattacta tgagaccagc 1440agcctctgct cccagccagc tgtggtattc caaaccaaaa gaagcaagca agtctgtgct 1500gatcccagtg aatcctgggt ccaggagtac gtgtatgacc tggaactgaa c 1551156517PRTArtificialheavy chain of 9G4-PVGLIG-CCL4 (C-terminal fusion) 156Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ser Thr Ile Ser Ser Ser Arg Thr Tyr Thr Tyr Tyr Ser Ala Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gln Phe Ser His Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr145 150 155 160Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro225 230 235 240Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser305 310 315 320Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe385 390 395 400Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425

430Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Pro Val Gly Leu Ile Gly 435 440 445Ala Pro Met Gly Ser Asp Pro Pro Thr Ala Cys Cys Phe Ser Tyr Thr 450 455 460Ala Arg Lys Leu Pro Arg Asn Phe Val Val Asp Tyr Tyr Glu Thr Ser465 470 475 480Ser Leu Cys Ser Gln Pro Ala Val Val Phe Gln Thr Lys Arg Ser Lys 485 490 495Gln Val Cys Ala Asp Pro Ser Glu Ser Trp Val Gln Glu Tyr Val Tyr 500 505 510Asp Leu Glu Leu Asn 5151571686DNAArtificialheavy chain of 9G4-PVGLIG-IL15 (C-terminal fusion) 157gaagtgatgt tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60tcctgtacag cctctggatt cactttcagt acctatgaca tgtcttgggt tcgccagact 120ccggagaaga ggctggagtg ggtctcaacc attagtagta gtcgtactta cacctactat 180tcagccagtg tgaaggggcg attcaccatc tccagagaca atgccaggaa catcctgtac 240ctgcaaatga gcagtctgag gtctgaggac acggccatgt attactgtgc aagacaattt 300tctcactggg gccaagggac tctggtcact gtctctgcag cgtccacaaa gggcccaagc 360gtgttcccgc tagcccccag cagcaagagc accagcggcg gcacagccgc cctgggctgc 420ctggtgaagg actacttccc cgagcccgtg accgtgtcct ggaacagcgg agccctgacc 480tccggcgtgc acaccttccc cgccgtgctg cagagcagcg gcctgtacag cctgagcagc 540gtggtgaccg tgcccagcag cagcctgggc acccagacct acatctgtaa cgtgaaccac 600aagcccagca acaccaaggt ggacaagaga gtggagccca agagctgtga caagacccac 660acctgtcccc cctgcccagc ccccgagctg ctgggcggac ccagcgtgtt cctgttcccc 720cccaagccca aggacaccct gatgatcagc agaacccccg aggtgacctg tgtggtggtg 780gacgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggacgg cgtggaggtg 840cacaacgcca agaccaagcc cagagaggag cagtacaaca gcacctacag ggtggtgtcc 900gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg taaggtgtcc 960aacaaggccc tgccagcccc aatcgaaaag accatcagca aggccaaggg ccagccaaga 1020gagccccagg tgtacaccct gccacccagc agggaggaga tgaccaagaa ccaggtgtcc 1080ctgacctgtc tggtgaaggg cttctaccca agcgacatcg ccgtggagtg ggagagcaac 1140ggccagcccg agaacaacta caagaccacc cccccagtgc tggacagcga cggcagcttc 1200ttcctgtaca gcaagctgac cgtggacaag agcagatggc agcagggcaa cgtgttcagc 1260tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgtcc 1320ccaggcccgg tgggcctgat tggcaactgg gtgaatgtaa taagtgattt gaaaaaaatt 1380gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag tgatgttcac 1440cccagttgca aagtaacagc aatgaagtgc tttctcttgg agttacaagt tatttcactt 1500gagtccggag atgcaagtat tcatgataca gtagaaaatc tgatcatcct agcaaacaac 1560agtttgtctt ctaatgggaa tgtaacagaa tctggatgca aagaatgtga ggaactggag 1620gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgtccaaat gttcatcaac 1680acttct 1686158562PRTArtificialheavy chain of 9G4-PVGLIG-IL15 (C-terminal fusion) 158Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ser Thr Ile Ser Ser Ser Arg Thr Tyr Thr Tyr Tyr Ser Ala Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gln Phe Ser His Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr145 150 155 160Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro225 230 235 240Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser305 310 315 320Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe385 390 395 400Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Pro Val Gly Leu Ile Gly 435 440 445Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 450 455 460Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His465 470 475 480Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 485 490 495Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 500 505 510Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 515 520 525Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 530 535 540Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn545 550 555 560Thr Ser1591839DNAArtificialheavy chain of 9G4-PVGLIG-IFNalpha (C-terminal fusion) 159gaagtgatgt tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60tcctgtacag cctctggatt cactttcagt acctatgaca tgtcttgggt tcgccagact 120ccggagaaga ggctggagtg ggtctcaacc attagtagta gtcgtactta cacctactat 180tcagccagtg tgaaggggcg attcaccatc tccagagaca atgccaggaa catcctgtac 240ctgcaaatga gcagtctgag gtctgaggac acggccatgt attactgtgc aagacaattt 300tctcactggg gccaagggac tctggtcact gtctctgcag cgtccacaaa gggcccaagc 360gtgttcccgc tagcccccag cagcaagagc accagcggcg gcacagccgc cctgggctgc 420ctggtgaagg actacttccc cgagcccgtg accgtgtcct ggaacagcgg agccctgacc 480tccggcgtgc acaccttccc cgccgtgctg cagagcagcg gcctgtacag cctgagcagc 540gtggtgaccg tgcccagcag cagcctgggc acccagacct acatctgtaa cgtgaaccac 600aagcccagca acaccaaggt ggacaagaga gtggagccca agagctgtga caagacccac 660acctgtcccc cctgcccagc ccccgagctg ctgggcggac ccagcgtgtt cctgttcccc 720cccaagccca aggacaccct gatgatcagc agaacccccg aggtgacctg tgtggtggtg 780gacgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggacgg cgtggaggtg 840cacaacgcca agaccaagcc cagagaggag cagtacaaca gcacctacag ggtggtgtcc 900gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg taaggtgtcc 960aacaaggccc tgccagcccc aatcgaaaag accatcagca aggccaaggg ccagccaaga 1020gagccccagg tgtacaccct gccacccagc agggaggaga tgaccaagaa ccaggtgtcc 1080ctgacctgtc tggtgaaggg cttctaccca agcgacatcg ccgtggagtg ggagagcaac 1140ggccagcccg agaacaacta caagaccacc cccccagtgc tggacagcga cggcagcttc 1200ttcctgtaca gcaagctgac cgtggacaag agcagatggc agcagggcaa cgtgttcagc 1260tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgtcc 1320ccaggcccgg tgggcctgat tggctgtgat ctgcctcaaa cccacagcct gggtagcagg 1380aggaccttga tgctcctggc acagatgagg aaaatctctc ttttctcctg cttgaaggac 1440agacatgact ttggatttcc ccaggaggag tttggcaacc agttccaaaa ggctgaaacc 1500atccctgtcc tccatgagat gatccagcag atcttcaatc tcttcagcac aaaggactca 1560tctgctgctt gggatgagac cctcctagac aaattctaca ctgaactcta ccagcagctg 1620aatgacctgg aagcctgtgt gatacagggg gtgggggtga cagagactcc cctgatgaag 1680gaggactcca ttctggctgt gaggaaatac ttccaaagaa tcactctcta tctgaaagag 1740aagaaataca gcccttgtgc ctgggaggtt gtcagagcag aaatcatgag atctttttct 1800ttgtcaacaa acttgcaaga aagtttaaga agtaaggaa 1839160613PRTArtificialheavy chain of 9G4-PVGLIG-IFNalpha (C-terminal fusion) 160Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ser Thr Ile Ser Ser Ser Arg Thr Tyr Thr Tyr Tyr Ser Ala Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gln Phe Ser His Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr145 150 155 160Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro225 230 235 240Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser305 310 315 320Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe385 390 395 400Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Pro Val Gly Leu Ile Gly 435 440 445Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 450 455 460Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp465 470 475 480Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 485 490 495Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 500 505 510Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 515 520 525Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 530 535 540Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys545 550 555 560Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 565 570 575Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 580 585 590Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 595 600 605Leu Arg Ser Lys Glu 6101611698DNAArtificialheavy chain of NHS76-PVGLIG-IL15 (C-terminal fusion) 161caggtgcagc tgcaggagtc cggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcgctg tctctggtta ctccatcagc agtggttact actggggctg gattcggcag 120cccccaggga aggggctgga gtggattggg agtatctatc atagtgggag cacctactac 180aacccgtccc tcaagagtcg agtcaccata tcagtagaca cgtccaagaa ccagttctcc 240ctgaagctga gctctgtgac cgccgcagac acggccgtgt attactgtgc aagagggaag 300tggtcgaagt ttgactattg gggccaaggc accctggtca ccgtctcttc agcgtccaca 360aagggcccaa gcgtgttccc gctagccccc agcagcaaga gcaccagcgg cggcacagcc 420gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgtc ctggaacagc 480ggagccctga cctccggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac 540agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcacccagac ctacatctgt 600aacgtgaacc acaagcccag caacaccaag gtggacaaga gagtggagcc caagagctgt 660gacaagaccc acacctgtcc cccctgccca gcccccgagc tgctgggcgg acccagcgtg 720ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcagaacccc cgaggtgacc 780tgtgtggtgg tggacgtgtc ccacgaggac ccagaggtga agttcaactg gtacgtggac 840ggcgtggagg tgcacaacgc caagaccaag cccagagagg agcagtacaa cagcacctac 900agggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 960tgtaaggtgt ccaacaaggc cctgccagcc ccaatcgaaa agaccatcag caaggccaag 1020ggccagccaa gagagcccca ggtgtacacc ctgccaccca gcagggagga gatgaccaag 1080aaccaggtgt ccctgacctg tctggtgaag ggcttctacc caagcgacat cgccgtggag 1140tgggagagca acggccagcc cgagaacaac tacaagacca cccccccagt gctggacagc 1200gacggcagct tcttcctgta cagcaagctg accgtggaca agagcagatg gcagcagggc 1260aacgtgttca gctgctccgt gatgcacgag gccctgcaca accactacac ccagaagagc 1320ctgagcctgt ccccaggccc ggtgggcctg attggcaact gggtgaatgt aataagtgat 1380ttgaaaaaaa ttgaagatct tattcaatct atgcatattg atgctacttt atatacggaa 1440agtgatgttc accccagttg caaagtaaca gcaatgaagt gctttctctt ggagttacaa 1500gttatttcac ttgagtccgg agatgcaagt attcatgata cagtagaaaa tctgatcatc 1560ctagcaaaca acagtttgtc ttctaatggg aatgtaacag aatctggatg caaagaatgt 1620gaggaactgg aggaaaaaaa tattaaagaa tttttgcaga gttttgtaca tattgtccaa 1680atgttcatca acacttct 1698162566PRTArtificialheavy chain of NHS76-PVGLIG-IL15 (C-terminal fusion) 162Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Lys Trp Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Pro Val 435 440 445Gly Leu Ile Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile 450 455 460Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu465 470 475 480Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu 485 490 495Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His 500 505 510Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser 515 520 525Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu 530 535 540Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln545 550 555 560Met Phe Ile Asn Thr Ser 5651631704DNAArtificialheavy chain of H16/L16-PVGLIG-IL15 (C-terminal fusion) 163caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggggac cctgtccctc 60acctgcgctg tctctggtgg ctccatcagc agtagtaact ggtggagttg ggtccgccag 120cccccaggga aggggctgga gtggattggg gaaatctatc atagtgggag caccaactac 180aacccgtccc tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc 240ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagatggacc 300gggcgtactg atgcttttga tatctggggc caagggacaa tggtcaccgt ctcaagcgcg 360tccacaaagg gcccaagcgt gttcccgcta gcccccagca gcaagagcac cagcggcggc 420acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 480aacagcggag ccctgacctc cggcgtgcac accttccccg ccgtgctgca gagcagcggc 540ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 600atctgtaacg tgaaccacaa gcccagcaac accaaggtgg acaagagagt ggagcccaag 660agctgtgaca agacccacac ctgtcccccc tgcccagccc ccgagctgct gggcggaccc 720agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 780gtgacctgtg tggtggtgga cgtgtcccac gaggacccag aggtgaagtt caactggtac 840gtggacggcg tggaggtgca caacgccaag accaagccca gagaggagca gtacaacagc 900acctacaggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960tacaagtgta aggtgtccaa caaggccctg ccagccccaa tcgaaaagac catcagcaag 1020gccaagggcc agccaagaga gccccaggtg tacaccctgc cacccagcag ggaggagatg 1080accaagaacc aggtgtccct gacctgtctg gtgaagggct tctacccaag cgacatcgcc 1140gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc cccagtgctg 1200gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag cagatggcag 1260cagggcaacg tgttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320aagagcctga gcctgtcccc aggcccggtg ggcctgattg gcaactgggt gaatgtaata 1380agtgatttga aaaaaattga agatcttatt caatctatgc atattgatgc tactttatat 1440acggaaagtg atgttcaccc cagttgcaaa gtaacagcaa tgaagtgctt tctcttggag 1500ttacaagtta tttcacttga gtccggagat gcaagtattc atgatacagt agaaaatctg 1560atcatcctag caaacaacag tttgtcttct aatgggaatg taacagaatc tggatgcaaa 1620gaatgtgagg aactggagga aaaaaatatt aaagaatttt tgcagagttt tgtacatatt 1680gtccaaatgt tcatcaacac ttct 1704164568PRTArtificialheavy chain of H16/L16-PVGLIG-IL15 (C-terminal fusion) 164Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Thr Gly Arg Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Pro Val Gly Leu Ile Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys 450 455 460Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr465 470 475 480Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys 485 490 495Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser 500 505 510Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu 515 520 525Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu 530 535 540Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile545 550 555 560Val Gln Met Phe Ile Asn Thr Ser 56516512DNAArtificiallinker 165tcaggcagat ct 121664PRTArtificiallinker 166Ser Gly Arg Ser116715DNAArtificiallinker 167tcaggcagat ctgcc 151685PRTArtificiallinker 168Ser Gly Arg Ser Ala1 516924DNAArtificiallinker 169ccgagcagcc ggagaagagt gaac 241708PRTArtificiallinker 170Pro Ser Ser Arg Arg Arg Val Asn1 5171447DNAHomo sapiens 171aaacccatca ccggcaccat caacgacctg aaccagcaag tgtggacact gcagggccag 60aatctggtgg ccgtgcctag aagcgatagc gtgacacctg tgaccgtggc cgtgatcaca 120tgcaagtacc ccgaggctct tgagcaaggc agaggcgatc ctatctacct gggcattcag 180aaccccgaga tgtgcctgta ctgcgagaaa gtgggagagc agcccacact gcagctgaaa 240gaacagaaaa tcatggacct gtacggccag cctgagcctg tgaagccctt cctgttttac 300agagccaaga ccggccggac cagcacactg gaatctgtgg cctttcctga ctggtttatc 360gccagcagca agcgggacca gcctatcatc ctgacaagcg agctgggcaa gagctacaac 420accgccttcg agctgaacat caacgat 447172149PRTHomo sapiens 172Lys Pro Ile Thr Gly Thr Ile Asn Asp Leu Asn Gln Gln Val Trp Thr1 5 10 15Leu Gln Gly Gln Asn Leu Val Ala Val Pro Arg Ser Asp Ser Val Thr 20 25 30Pro Val Thr Val Ala Val Ile Thr Cys Lys Tyr Pro Glu Ala Leu Glu 35 40 45Gln Gly Arg Gly Asp Pro Ile Tyr Leu Gly Ile Gln Asn Pro Glu Met 50 55 60Cys Leu Tyr Cys Glu Lys Val Gly Glu Gln Pro Thr Leu Gln Leu Lys65 70 75 80Glu Gln Lys Ile Met Asp Leu Tyr Gly Gln Pro Glu Pro Val Lys Pro 85 90 95Phe Leu Phe Tyr Arg Ala Lys Thr Gly Arg Thr Ser Thr Leu Glu Ser 100 105 110Val Ala Phe Pro Asp Trp Phe Ile Ala Ser Ser Lys Arg Asp Gln Pro 115 120 125Ile Ile Leu Thr Ser Glu Leu Gly Lys Ser Tyr Asn Thr Ala Phe Glu 130 135 140Leu Asn Ile Asn Asp1451731869DNAArtificialH16/L16-PVGLIG-IL36G heavy chain 173atggaaaccg acaccctgct gctgtgggtg ctgctgctct gggtcccagg ctctaccggc 60caggtgcagc tgcaggagtc gggaccagga ctggtgaagc cttcggggac cctgtccctc 120acctgcgctg tctctggtgg ctccatcagc agtagtaact ggtggagttg ggtccgccag 180cccccaggga aggggctgga gtggattggg gaaatctatc atagtgggag caccaactac 240aacccgtccc tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc 300ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagatggacc 360gggcgtactg atgcttttga tatctggggc caagggacaa tggtcaccgt ctcaagcgcg 420tccacaaagg gcccaagcgt gttcccgcta gcccccagca gcaagagcac cagcggcggc 480acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 540aacagcggag ccctgacctc cggcgtgcac accttccccg ccgtgctgca gagcagcggc 600ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 660atctgtaacg tgaaccacaa gcccagcaac accaaggtgg acaagagagt ggagcccaag 720agctgtgaca agacccacac ctgtcccccc tgcccagccc ccgagctgct gggcggaccc 780agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 840gtgacctgtg tggtggtgga cgtgtcccac gaggacccag aggtgaagtt caactggtac 900gtggacggcg tggaggtgca caacgccaag accaagccca gagaggagca gtacaacagc 960acctacaggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1020tacaagtgta aggtgtccaa caaggccctg ccagccccaa tcgaaaagac catcagcaag 1080gccaagggcc agccaagaga gccccaggtg tacaccctgc cacccagcag ggaggagatg 1140accaagaacc aggtgtccct gacctgtctg gtgaagggct tctacccaag cgacatcgcc 1200gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc cccagtgctg 1260gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag cagatggcag 1320cagggcaacg tgttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1380aagagcctga gcctgtcccc aggcccggtg ggcctgattg gcaaacccat caccggcacc 1440atcaacgacc tgaaccagca agtgtggaca ctgcagggcc agaatctggt ggccgtgcct 1500agaagcgata gcgtgacacc tgtgaccgtg gccgtgatca catgcaagta ccccgaggct 1560cttgagcaag gcagaggcga tcctatctac ctgggcattc agaaccccga gatgtgcctg 1620tactgcgaga aagtgggaga gcagcccaca ctgcagctga aagaacagaa aatcatggac 1680ctgtacggcc agcctgagcc tgtgaagccc ttcctgtttt acagagccaa gaccggccgg 1740accagcacac tggaatctgt ggcctttcct gactggttta tcgccagcag caagcgggac 1800cagcctatca tcctgacaag cgagctgggc aagagctaca acaccgcctt cgagctgaac 1860atcaacgat 1869174623PRTArtificialH16/L16-PVGLIG-IL36G heavy chain 174Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 20 25 30Lys Pro Ser Gly Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser 35 40 45Ile Ser Ser Ser Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr65 70 75 80Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys 85 90 95Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys Ala Arg Trp Thr Gly Arg Thr Asp Ala Phe Asp Ile 115 120 125Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly Pro Val Gly Leu Ile Gly Lys Pro Ile Thr Gly Thr465 470 475 480Ile Asn Asp Leu Asn Gln Gln Val Trp Thr Leu Gln Gly Gln Asn Leu 485 490 495Val Ala Val Pro Arg Ser Asp Ser Val Thr Pro Val Thr Val Ala Val 500 505 510Ile Thr Cys Lys Tyr Pro Glu Ala Leu Glu Gln Gly Arg Gly Asp Pro 515 520 525Ile Tyr Leu Gly Ile Gln Asn Pro Glu Met Cys Leu Tyr Cys Glu Lys 530 535 540Val Gly Glu Gln Pro Thr Leu Gln Leu Lys Glu Gln Lys Ile Met Asp545 550 555 560Leu Tyr Gly Gln Pro Glu Pro Val Lys Pro Phe Leu Phe Tyr Arg Ala 565 570 575Lys Thr Gly Arg Thr Ser Thr Leu Glu Ser Val Ala Phe Pro Asp Trp 580 585 590Phe Ile Ala Ser Ser Lys Arg Asp Gln Pro Ile Ile Leu Thr Ser Glu 595 600 605Leu Gly Lys Ser Tyr Asn Thr Ala Phe Glu Leu Asn Ile Asn Asp 610 615 6201751626DNAArtificial9G4-SGRS-hCXCL10 heavy chain 175atgaacttcg gactctccct gatcttcctt gttctggtgc ttaagggcgt gcagtgcgaa 60gtgatgttgg tagaaagcgg tggtgggctg gtgaaacctg gcgggtccct gaaactctcc 120tgtactgcta gtggattcac attcagcact tacgacatgt catgggtccg gcagacccca 180gagaagagac

tggagtgggt cagcactatc tcctcttctc ggacctatac gtattacagt 240gccagcgtca agggacgctt taccattagc agggataatg ccagaaacat actgtacctg 300cagatgtcaa gcctccgaag tgaggacacc gccatgtatt actgcgctag gcaattttca 360cactggggcc aggggacatt ggttaccgtg tctgcagcgt ccacaaaggg cccaagcgtg 420ttcccgctag cccccagcag caagagcacc agcggcggca cagccgccct gggctgcctg 480gtgaaggact acttccccga gcccgtgacc gtgtcctgga acagcggagc cctgacctcc 540ggcgtgcaca ccttccccgc cgtgctgcag agcagcggcc tgtacagcct gagcagcgtg 600gtgaccgtgc ccagcagcag cctgggcacc cagacctaca tctgtaacgt gaaccacaag 660cccagcaaca ccaaggtgga caagagagtg gagcccaaga gctgtgacaa gacccacacc 720tgtcccccct gcccagcccc cgagctgctg ggcggaccca gcgtgttcct gttccccccc 780aagcccaagg acaccctgat gatcagcaga acccccgagg tgacctgtgt ggtggtggac 840gtgtcccacg aggacccaga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 900aacgccaaga ccaagcccag agaggagcag tacaacagca cctacagggt ggtgtccgtg 960ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgtaa ggtgtccaac 1020aaggccctgc cagccccaat cgaaaagacc atcagcaagg ccaagggcca gccaagagag 1080ccccaggtgt acaccctgcc acccagcagg gaggagatga ccaagaacca ggtgtccctg 1140acctgtctgg tgaagggctt ctacccaagc gacatcgccg tggagtggga gagcaacggc 1200cagcccgaga acaactacaa gaccaccccc ccagtgctgg acagcgacgg cagcttcttc 1260ctgtacagca agctgaccgt ggacaagagc agatggcagc agggcaacgt gttcagctgc 1320tccgtgatgc acgaggccct gcacaaccac tacacccaga agagcctgag cctgtcccca 1380ggctcaggca gatctgtacc tctctctaga actgtacgct gtacctgcat cagcattagt 1440aatcaacctg ttaatccaag gtctttagaa aaacttgaaa ttattcctgc aagccaattt 1500tgtccacgtg ttgagatcat tgctacaatg aaaaagaagg gtgagaagag atgtctgaat 1560ccagaatcga aggccatcaa gaatttactg aaagcagtta gcaaggaaag gtctaaaaga 1620tctcct 1626176542PRTArtificial9G4-SGRS-hCXCL10 heavy chain 176Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30Pro Gly Gly Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe 35 40 45Ser Thr Tyr Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60Glu Trp Val Ser Thr Ile Ser Ser Ser Arg Thr Tyr Thr Tyr Tyr Ser65 70 75 80Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn 85 90 95Ile Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met 100 105 110Tyr Tyr Cys Ala Arg Gln Phe Ser His Trp Gly Gln Gly Thr Leu Val 115 120 125Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 130 135 140Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu145 150 155 160Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 165 170 175Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 180 185 190Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 195 200 205Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 210 215 220Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr225 230 235 240Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 245 250 255Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val305 310 315 320Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 355 360 365Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 370 375 380Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly385 390 395 400Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Arg 450 455 460Ser Val Pro Leu Ser Arg Thr Val Arg Cys Thr Cys Ile Ser Ile Ser465 470 475 480Asn Gln Pro Val Asn Pro Arg Ser Leu Glu Lys Leu Glu Ile Ile Pro 485 490 495Ala Ser Gln Phe Cys Pro Arg Val Glu Ile Ile Ala Thr Met Lys Lys 500 505 510Lys Gly Glu Lys Arg Cys Leu Asn Pro Glu Ser Lys Ala Ile Lys Asn 515 520 525Leu Leu Lys Ala Val Ser Lys Glu Arg Ser Lys Arg Ser Pro 530 535 5401771641DNAArtificialNHS76-SGRS-hCXCL10 heavy chain 177atggaaaccg acaccctgct gctgtgggtg ctgctgctct gggtcccagg ctctaccggc 60caggtgcagc tgcaggagtc cggcccagga ctggtgaagc cttcggagac cctgtccctc 120acctgcgctg tctctggtta ctccatcagc agtggttact actggggctg gattcggcag 180cccccaggga aggggctgga gtggattggg agtatctatc atagtgggag cacctactac 240aacccgtccc tcaagagtcg agtcaccata tcagtagaca cgtccaagaa ccagttctcc 300ctgaagctga gctctgtgac cgccgcagac acggccgtgt attactgtgc aagagggaag 360tggtcgaagt ttgactattg gggccaaggc accctggtca ccgtctcttc agcgtccaca 420aagggcccaa gcgtgttccc gctagccccc agcagcaaga gcaccagcgg cggcacagcc 480gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgtc ctggaacagc 540ggagccctga cctccggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac 600agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcacccagac ctacatctgt 660aacgtgaacc acaagcccag caacaccaag gtggacaaga gagtggagcc caagagctgt 720gacaagaccc acacctgtcc cccctgccca gcccccgagc tgctgggcgg acccagcgtg 780ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcagaacccc cgaggtgacc 840tgtgtggtgg tggacgtgtc ccacgaggac ccagaggtga agttcaactg gtacgtggac 900ggcgtggagg tgcacaacgc caagaccaag cccagagagg agcagtacaa cagcacctac 960agggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 1020tgtaaggtgt ccaacaaggc cctgccagcc ccaatcgaaa agaccatcag caaggccaag 1080ggccagccaa gagagcccca ggtgtacacc ctgccaccca gcagggagga gatgaccaag 1140aaccaggtgt ccctgacctg tctggtgaag ggcttctacc caagcgacat cgccgtggag 1200tgggagagca acggccagcc cgagaacaac tacaagacca cccccccagt gctggacagc 1260gacggcagct tcttcctgta cagcaagctg accgtggaca agagcagatg gcagcagggc 1320aacgtgttca gctgctccgt gatgcacgag gccctgcaca accactacac ccagaagagc 1380ctgagcctgt ccccaggctc aggcagatct gtacctctct ctagaactgt acgctgtacc 1440tgcatcagca ttagtaatca acctgttaat ccaaggtctt tagaaaaact tgaaattatt 1500cctgcaagcc aattttgtcc acgtgttgag atcattgcta caatgaaaaa gaagggtgag 1560aagagatgtc tgaatccaga atcgaaggcc atcaagaatt tactgaaagc agttagcaag 1620gaaaggtcta aaagatctcc t 1641178547PRTArtificialNHS76-SGRS-hCXCL10 heavy chain 178Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 20 25 30Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser 35 40 45Ile Ser Ser Gly Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr65 70 75 80Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys Ala Arg Gly Lys Trp Ser Lys Phe Asp Tyr Trp Gly 115 120 125Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala145 150 155 160Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys225 230 235 240Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr305 310 315 320Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu385 390 395 400Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460Pro Gly Ser Gly Arg Ser Val Pro Leu Ser Arg Thr Val Arg Cys Thr465 470 475 480Cys Ile Ser Ile Ser Asn Gln Pro Val Asn Pro Arg Ser Leu Glu Lys 485 490 495Leu Glu Ile Ile Pro Ala Ser Gln Phe Cys Pro Arg Val Glu Ile Ile 500 505 510Ala Thr Met Lys Lys Lys Gly Glu Lys Arg Cys Leu Asn Pro Glu Ser 515 520 525Lys Ala Ile Lys Asn Leu Leu Lys Ala Val Ser Lys Glu Arg Ser Lys 530 535 540Arg Ser Pro5451791647DNAArtificialH16L16-SGRS-hCXCL10 heavy chain 179atggaaaccg acaccctgct gctgtgggtg ctgctgctct gggtcccagg ctctaccggc 60caggtgcagc tgcaggagtc gggaccagga ctggtgaagc cttcggggac cctgtccctc 120acctgcgctg tctctggtgg ctccatcagc agtagtaact ggtggagttg ggtccgccag 180cccccaggga aggggctgga gtggattggg gaaatctatc atagtgggag caccaactac 240aacccgtccc tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc 300ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagatggacc 360gggcgtactg atgcttttga tatctggggc caagggacaa tggtcaccgt ctcaagcgcg 420tccacaaagg gcccaagcgt gttcccgcta gcccccagca gcaagagcac cagcggcggc 480acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 540aacagcggag ccctgacctc cggcgtgcac accttccccg ccgtgctgca gagcagcggc 600ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 660atctgtaacg tgaaccacaa gcccagcaac accaaggtgg acaagagagt ggagcccaag 720agctgtgaca agacccacac ctgtcccccc tgcccagccc ccgagctgct gggcggaccc 780agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 840gtgacctgtg tggtggtgga cgtgtcccac gaggacccag aggtgaagtt caactggtac 900gtggacggcg tggaggtgca caacgccaag accaagccca gagaggagca gtacaacagc 960acctacaggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1020tacaagtgta aggtgtccaa caaggccctg ccagccccaa tcgaaaagac catcagcaag 1080gccaagggcc agccaagaga gccccaggtg tacaccctgc cacccagcag ggaggagatg 1140accaagaacc aggtgtccct gacctgtctg gtgaagggct tctacccaag cgacatcgcc 1200gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc cccagtgctg 1260gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag cagatggcag 1320cagggcaacg tgttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1380aagagcctga gcctgtcccc aggctcaggc agatctgtac ctctctctag aactgtacgc 1440tgtacctgca tcagcattag taatcaacct gttaatccaa ggtctttaga aaaacttgaa 1500attattcctg caagccaatt ttgtccacgt gttgagatca ttgctacaat gaaaaagaag 1560ggtgagaaga gatgtctgaa tccagaatcg aaggccatca agaatttact gaaagcagtt 1620agcaaggaaa ggtctaaaag atctcct 1647180549PRTArtificialH16L16-SGRS-hCXCL10 heavy chain 180Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 20 25 30Lys Pro Ser Gly Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser 35 40 45Ile Ser Ser Ser Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr65 70 75 80Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys 85 90 95Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys Ala Arg Trp Thr Gly Arg Thr Asp Ala Phe Asp Ile 115 120 125Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly Ser Gly Arg Ser Val Pro Leu Ser Arg Thr Val Arg465 470 475 480Cys Thr Cys Ile Ser Ile Ser Asn Gln Pro Val Asn Pro Arg Ser Leu 485 490 495Glu Lys Leu Glu Ile Ile Pro Ala Ser Gln Phe Cys Pro Arg Val Glu 500 505 510Ile Ile Ala Thr Met Lys Lys Lys Gly Glu Lys Arg Cys Leu Asn Pro 515 520 525Glu Ser Lys Ala Ile Lys Asn Leu Leu Lys Ala Val Ser Lys Glu Arg 530 535 540Ser Lys Arg Ser

Pro5451811761DNAArtificialH16L16-SGRSA-hIL15 heavy chain 181atggaaaccg acaccctgct gctgtgggtg ctgctgctct gggtcccagg ctctaccggc 60caggtgcagc tgcaggagtc gggaccagga ctggtgaagc cttcggggac cctgtccctc 120acctgcgctg tctctggtgg ctccatcagc agtagtaact ggtggagttg ggtccgccag 180cccccaggga aggggctgga gtggattggg gaaatctatc atagtgggag caccaactac 240aacccgtccc tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc 300ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagatggacc 360gggcgtactg atgcttttga tatctggggc caagggacaa tggtcaccgt ctcaagcgcg 420tccacaaagg gcccaagcgt gttcccgcta gcccccagca gcaagagcac cagcggcggc 480acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 540aacagcggag ccctgacctc cggcgtgcac accttccccg ccgtgctgca gagcagcggc 600ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 660atctgtaacg tgaaccacaa gcccagcaac accaaggtgg acaagagagt ggagcccaag 720agctgtgaca agacccacac ctgtcccccc tgcccagccc ccgagctgct gggcggaccc 780agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 840gtgacctgtg tggtggtgga cgtgtcccac gaggacccag aggtgaagtt caactggtac 900gtggacggcg tggaggtgca caacgccaag accaagccca gagaggagca gtacaacagc 960acctacaggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1020tacaagtgta aggtgtccaa caaggccctg ccagccccaa tcgaaaagac catcagcaag 1080gccaagggcc agccaagaga gccccaggtg tacaccctgc cacccagcag ggaggagatg 1140accaagaacc aggtgtccct gacctgtctg gtgaagggct tctacccaag cgacatcgcc 1200gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc cccagtgctg 1260gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag cagatggcag 1320cagggcaacg tgttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1380aagagcctga gcctgtcccc aggctcaggc agatctgcca actgggtgaa tgtaataagt 1440gatttgaaaa aaattgaaga tcttattcaa tctatgcata ttgatgctac tttatatacg 1500gaaagtgatg ttcaccccag ttgcaaagta acagcaatga agtgctttct cttggagtta 1560caagttattt cacttgagtc cggagatgca agtattcatg atacagtaga aaatctgatc 1620atcctagcaa acaacagttt gtcttctaat gggaatgtaa cagaatctgg atgcaaagaa 1680tgtgaggaac tggaggaaaa aaatattaaa gaatttttgc agagttttgt acatattgtc 1740caaatgttca tcaacacttc t 1761182587PRTArtificialH16L16-SGRSA-hIL15 heavy chain 182Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 20 25 30Lys Pro Ser Gly Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser 35 40 45Ile Ser Ser Ser Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr65 70 75 80Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys 85 90 95Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys Ala Arg Trp Thr Gly Arg Thr Asp Ala Phe Asp Ile 115 120 125Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly Ser Gly Arg Ser Ala Asn Trp Val Asn Val Ile Ser465 470 475 480Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala 485 490 495Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala 500 505 510Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly 515 520 525Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn 530 535 540Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu545 550 555 560Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe 565 570 575Val His Ile Val Gln Met Phe Ile Asn Thr Ser 580 5851831914DNAArtificialH16L16-SGRSA-hIFNa heavy chain 183atggaaaccg acaccctgct gctgtgggtg ctgctgctct gggtcccagg ctctaccggc 60caggtgcagc tgcaggagtc gggaccagga ctggtgaagc cttcggggac cctgtccctc 120acctgcgctg tctctggtgg ctccatcagc agtagtaact ggtggagttg ggtccgccag 180cccccaggga aggggctgga gtggattggg gaaatctatc atagtgggag caccaactac 240aacccgtccc tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc 300ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagatggacc 360gggcgtactg atgcttttga tatctggggc caagggacaa tggtcaccgt ctcaagcgcg 420tccacaaagg gcccaagcgt gttcccgcta gcccccagca gcaagagcac cagcggcggc 480acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 540aacagcggag ccctgacctc cggcgtgcac accttccccg ccgtgctgca gagcagcggc 600ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 660atctgtaacg tgaaccacaa gcccagcaac accaaggtgg acaagagagt ggagcccaag 720agctgtgaca agacccacac ctgtcccccc tgcccagccc ccgagctgct gggcggaccc 780agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 840gtgacctgtg tggtggtgga cgtgtcccac gaggacccag aggtgaagtt caactggtac 900gtggacggcg tggaggtgca caacgccaag accaagccca gagaggagca gtacaacagc 960acctacaggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1020tacaagtgta aggtgtccaa caaggccctg ccagccccaa tcgaaaagac catcagcaag 1080gccaagggcc agccaagaga gccccaggtg tacaccctgc cacccagcag ggaggagatg 1140accaagaacc aggtgtccct gacctgtctg gtgaagggct tctacccaag cgacatcgcc 1200gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc cccagtgctg 1260gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag cagatggcag 1320cagggcaacg tgttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1380aagagcctga gcctgtcccc aggctcaggc agatctgcct gtgatctgcc tcaaacccac 1440agcctgggta gcaggaggac cttgatgctc ctggcacaga tgaggaaaat ctctcttttc 1500tcctgcttga aggacagaca tgactttgga tttccccagg aggagtttgg caaccagttc 1560caaaaggctg aaaccatccc tgtcctccat gagatgatcc agcagatctt caatctcttc 1620agcacaaagg actcatctgc tgcttgggat gagaccctcc tagacaaatt ctacactgaa 1680ctctaccagc agctgaatga cctggaagcc tgtgtgatac agggggtggg ggtgacagag 1740actcccctga tgaaggagga ctccattctg gctgtgagga aatacttcca aagaatcact 1800ctctatctga aagagaagaa atacagccct tgtgcctggg aggttgtcag agcagaaatc 1860atgagatctt tttctttgtc aacaaacttg caagaaagtt taagaagtaa ggaa 1914184638PRTArtificialH16L16-SGRSA-hIFNa heavy chain 184Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 20 25 30Lys Pro Ser Gly Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser 35 40 45Ile Ser Ser Ser Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr65 70 75 80Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys 85 90 95Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys Ala Arg Trp Thr Gly Arg Thr Asp Ala Phe Asp Ile 115 120 125Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly Ser Gly Arg Ser Ala Cys Asp Leu Pro Gln Thr His465 470 475 480Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Lys 485 490 495Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro 500 505 510Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val 515 520 525Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp 530 535 540Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu545 550 555 560Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val 565 570 575Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val 580 585 590Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr 595 600 605Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe 610 615 620Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu625 630 6351851923DNAArtificialH16L16-PSSRRRVN-hIFNa heavy chain 185atggaaaccg acaccctgct gctgtgggtg ctgctgctct gggtcccagg ctctaccggc 60caggtgcagc tgcaggagtc gggaccagga ctggtgaagc cttcggggac cctgtccctc 120acctgcgctg tctctggtgg ctccatcagc agtagtaact ggtggagttg ggtccgccag 180cccccaggga aggggctgga gtggattggg gaaatctatc atagtgggag caccaactac 240aacccgtccc tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc 300ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagatggacc 360gggcgtactg atgcttttga tatctggggc caagggacaa tggtcaccgt ctcaagcgcg 420tccacaaagg gcccaagcgt gttcccgcta gcccccagca gcaagagcac cagcggcggc 480acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 540aacagcggag ccctgacctc cggcgtgcac accttccccg ccgtgctgca gagcagcggc 600ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 660atctgtaacg tgaaccacaa gcccagcaac accaaggtgg acaagagagt ggagcccaag 720agctgtgaca agacccacac ctgtcccccc tgcccagccc ccgagctgct gggcggaccc 780agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 840gtgacctgtg tggtggtgga cgtgtcccac gaggacccag aggtgaagtt caactggtac 900gtggacggcg tggaggtgca caacgccaag accaagccca gagaggagca gtacaacagc 960acctacaggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1020tacaagtgta aggtgtccaa caaggccctg ccagccccaa tcgaaaagac catcagcaag 1080gccaagggcc agccaagaga gccccaggtg tacaccctgc cacccagcag ggaggagatg 1140accaagaacc aggtgtccct gacctgtctg gtgaagggct tctacccaag cgacatcgcc 1200gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc cccagtgctg 1260gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag cagatggcag 1320cagggcaacg tgttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1380aagagcctga gcctgtcccc aggcccgagc agccggagaa gagtgaactg tgatctgcct 1440caaacccaca gcctgggtag caggaggacc ttgatgctcc tggcacagat gaggaaaatc 1500tctcttttct cctgcttgaa ggacagacat gactttggat ttccccagga ggagtttggc 1560aaccagttcc aaaaggctga aaccatccct gtcctccatg agatgatcca gcagatcttc 1620aatctcttca gcacaaagga ctcatctgct gcttgggatg agaccctcct agacaaattc 1680tacactgaac tctaccagca gctgaatgac ctggaagcct gtgtgataca gggggtgggg 1740gtgacagaga ctcccctgat gaaggaggac tccattctgg ctgtgaggaa atacttccaa 1800agaatcactc tctatctgaa agagaagaaa tacagccctt gtgcctggga ggttgtcaga 1860gcagaaatca tgagatcttt ttctttgtca acaaacttgc aagaaagttt aagaagtaag 1920gaa 1923186641PRTArtificialH16L16-PSSRRRVN-hIFNa heavy chain 186Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 20 25 30Lys Pro Ser Gly Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser 35 40 45Ile Ser Ser Ser Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr65 70 75 80Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys 85 90 95Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys Ala Arg Trp Thr Gly Arg Thr Asp Ala Phe Asp Ile 115 120 125Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260

265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly Pro Ser Ser Arg Arg Arg Val Asn Cys Asp Leu Pro465 470 475 480Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln 485 490 495Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe 500 505 510Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr 515 520 525Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser 530 535 540Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe545 550 555 560Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile 565 570 575Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile 580 585 590Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu 595 600 605Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met 610 615 620Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys625 630 635 640Glu

Claims

1. A fusion protein comprising: (i) an antibody or antigen-binding fragment thereof fused to (ii) a cleavable peptide linker, and (iii) a cytokine, or functional fragments thereof.

2. The fusion protein of claim 1, wherein the cytokine is IL-15, CXCL10, IL-36, or IFN-.alpha..

3. The fusion protein of claim 1 or 2, wherein the cleavable peptide linker comprises a protease cleavage site.

4. The fusion protein of any one of claims 1 to 3, wherein the protease cleavage site is cleaved by a matrix metalloproteinase or by uPA.

5. The fusion protein of claim 4, wherein the matrix metalloproteinase is MMP-2, MMP-9.

6. The fusion protein of any one of claims 1 to 5, wherein the cleavable peptide linker has a sequence selected from the group consisting of: GPLGIAGQ, GPLGLWAQ, GPLGMLSQ, PLGLAG, PVGLIG, SGRS, SGRSA, and PSSRRRVN.

7. The fusion protein of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanised antibodies, scFv, single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv.

8. The fusion protein of any one of claims 1 to 7, wherein the cytokine is a murine or human, preferably a human cytokine, or functional fragment thereof.

9. The fusion protein of any one of claims 1 to 8, wherein: (i) the cytokine, or functional fragment thereof is fused to the cleavable peptide linker, and (ii) the cleavable peptide linker is used N-terminally or C-terminally to the antibody or antigen-binding fragment thereof.

10. The fusion protein of any one of claims 1 to 9, wherein: (i) the cytokine, or functional fragment thereof is fused to the cleavable peptide linker, and (ii) the cleavable peptide linker is fused N-terminally or C-terminally to the heavy chain of the antibody or antigen-binding fragment thereof.

11. The fusion protein of any one of claims 1 to 10 for use in therapy.

12. The fusion protein of any of claims 1 to 10 for use in the treatment of cancer in a mammal, preferably in a human.

13. The fusion protein for use according to claim 12, wherein said use comprises activation of immune cell, preferably T-cells or monocytes, of said mammal.

14. A pharmaceutical composition comprising at least one fusion protein according to any one of claims 1 to 9 and optionally a pharmaceutically acceptable excipient.

15. A method of selecting a cytokine or a variant thereof, said method comprising: (i) providing a fusion protein as claimed in any one of claims 1 to 10, said fusion protein comprising the cytokine or variant thereof to be tested; (ii) contacting said fusion protein with the relevant protease; and (iii) detecting the activity of said cytokine.

16. A method for identifying a cleavable peptide linker, said method comprising: (i) providing a fusion protein as claimed in any one of claims 1 to 10, said fusion protein comprising the peptide cleavable linker to be tested; (ii) contacting said fusion protein with the relevant protease; and (iii) detecting the cleavage of said fusion protein.

17. The method of claim 16, wherein step (iii) of said method comprises measuring the activity of the cytokine of said fusion protein.