WO2022015711A1

WO2022015711A1 - Fusion proteins of anti-pd-l1 and attenuated interferon, and compositions and therapeutic methods thereof

Info

Publication number: WO2022015711A1
Application number: PCT/US2021/041400
Authority: WO
Inventors: Yang-Xin Fu; Yang Wang
Original assignee: Immune Targeting Inc.
Priority date: 2020-07-14
Filing date: 2021-07-13
Publication date: 2022-01-20

Abstract

The invention provides novel fusion proteins and therapeutic uses thereof. More particularly, the invention provides novel heterodimer fusion proteins of programmed death- ligand 1 (PD-L1) binding moiety and attenuated Interferon (IFN), e.g., Interferon-α (IFNα), Interferon-β (ΙFΝβ), or prodrugs thereof, and compositions and methods of preparation thereof, useful in treating various diseases and disorders (e.g., hyperplasia, solid tumor or hematopoietic malignancy).

Description

FUSION PROTEINS OF ANTI-PD-L1 AND ATTENUATED INTERFERON, AND COMPOSITIONS AND THERAPEUTIC METHODS THEREOF

Priority Claims and Related Applications

[0001] This application claims the benefit to U.S. Provisional Application Serial No. 63/051,545, filed July 14, 2021, the entire content of which is incorporated herein by reference for all purposes.

Technical Field of the Invention

[0002] The invention generally relates to novel fusion proteins and therapeutic uses thereof.

More particularly, the invention provides novel Fc heterodimer fusion proteins of a programmed death-ligand 1 binding moiety and an attenuated ( e.g ., mutant or prodrug of) Interferon (IFN), and compositions and methods of preparation thereof, useful in treating various diseases and disorders (e.g., hyperplasia, solid tumor or hematopoietic malignancy).

Background of the Invention

[0003] The lack of innate sensing such as type I IFN inside tumor microenvironment has prevented effective T cell re-activation. IFNs are typically divided among three classes: Type I IFN, Type II IFN, and Type III IFN. Recently, the present inventors and others have found that endogenous IFNs play critical roles in tumor control by promoting dendritic cells mediated T cells reactivating. It has been suggested that the delivery of exogenous IFN may work as a “magic bullet” that impacts both immune responses and tumor cell proliferation/survival. However, high dose of IFN must be administered systemically to achieve high serum concentration for therapeutic effect. At therapeutic dose, severe toxicities were observed to limit its efficacy for most patients. In addition, IFNs are the most potent inducers of programmed death-ligand 1 (PD-L1), which serves as a negative feedback mechanism to prevent tissue damage but also dampens the antitumor effects. (Dubrot, etal. 2011 IntJ Cancer 128, 105-118; Liang, et al. 2018 Nat Commun 9, 4586; Sistigu, et al. 2014 Nature medicine 20, 1301-1309; Yang, etal. 2014 Cancer cell 25 , 37-48.)

[0004] The therapeutics and methods currently available for hyperplasia, solid tumor or hematopoietic malignancy are inadequate. There remains an urgent and ongoing need for novel and improved therapeutics to effectively treat such diseases and conditions. Summary of the Invention

[0005] The invention is based in part on novel fusion proteins disclosed herein. More particularly, the invention provides novel heterodimer fusion proteins comprising PD-L1 binding moiety and attenuated Interferon ( e.g ., Interferon-a (IFNa), Interferon-b (IFNβ)) where the interferon is characterized by reduced affinity via mutation or masking, their compositions and methods of preparation, as well as use in treating various diseases and disorders (e.g., hyperplasia, solid tumor or hematopoietic malignancy) with reduced off-target toxicities and side effects during treatment.

[0006] As disclosed herein, while both IFN-anti-PD-Ll heterodimer and homodimer retain intact PD-L1 binding ability and IFN bioactivity, the heterodimer showed much stronger anti-tumor effect in vivo than the homodimer did. Heterodimer had longer serum half-life and more tumor accumulation than homodimer in systemic administration, likely due to more stability of heterodimer than homodimer. Significantly, mutant IFN-anti-PD-Ll exhibited non-reduced anti-tumor effect but much less toxicity in humanized tumor model and much wider therapeutic window than wild type (WT) protein.

[0007] The data shown herein establishes that the disclosed heterodimer with low affinity IFNa allows both anti-PD-Ll and IFN targeting on the synapse of tumor associated dendritic cells that interact with anti-tumor T cells. Thus, these novel heterodimers allow IFN to reactivate dendritic cells while blocking PD-L1 -mediated suppression of T cells.

[0008] In one aspect, the invention generally relates to a fusion protein, comprising: a first structural unit comprising: (1) a moiety capable of specifically binding to programmed death-ligand 1 (PD-L1), and at its C-terminus a first fragment crystallizable (Fc) fragment; and (2) a second structural unit comprising: an attenuated Interferon (IFN) selected from a mutant or pro-interferon (pro-IFN) characterized by a reduced IFN activity, and at its C-terminus a second Fc fragment. The first Fc fragment comprises a knob and the second Fc fragment comprises a hole in the respective heavy chain 3 (C_H3) domains, or the first Fc fragment comprises a hole and the second Fc fragment comprises a knob in the respective C_H3 domains, so that the first structural unit and the second structural unit form a heterodimer fusion protein via disulfide bonds.

[0009] In another aspect, the invention generally relates to a prodrug comprising a fusion protein disclosed herein. For example, the linker is capable of being recognized and hydrolyzed by a proteolytic enzyme specifically expressed in a tumor microenvironment, e.g., MMP to restore to scFv or Fab, to increase affinity to PD-L1 or to restore activity of IFN. [0010] In yet another aspect, the invention generally relates to a substantially purified fusion protein or prodrug disclosed herein.

[0011] In yet another aspect, the invention generally relates to a polynucleotide encoding a fusion protein or prodrug disclosed herein.

[0012] In yet another aspect, the invention generally relates to an expression vector comprising the polynucleotide disclosed herein.

[0013] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising a fusion protein or prodrug disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0014] In yet another aspect, the invention generally relates to a method for treating a disease or condition. The method comprises administering to a patient in need thereof a therapeutically effective amount of a fusion protein or prodrug disclosed herein or a pharmaceutical composition disclosed herein, wherein the disease or condition is selected from hyperplasia, solid tumor or hematopoietic malignancy.

[0015] In yet another aspect, the invention generally relates to use of a fusion protein or prodrug disclosed herein for treating or reducing a disease or disorder.

[0016] In yet another aspect, the invention generally relates to use of a fusion protein or prodrug disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent, in preparation of a medicament for treating or reducing a disease or disorder.

[0017] In yet another aspect, the invention generally relates to a cell line comprising a polynucleotide encoding a fusion protein or prodrug disclosed herein.

[0018] In yet another aspect, the invention generally relates to a method for making a protein, comprising culturing the cell line disclosed herein.

[0019] In yet another aspect, the invention generally relates to a method for making a protein. The method comprises: providing an expression vector encoding a fusion protein or prodrug disclosed herein; introducing into a host cell the expression vector; culturing the host cell in media under conditions sufficient to express the protein; and purifying the protein from the host cell or media.

Brief Description of the Drawings

[0020] FIG. 1. Exemplary construction and characterization of IFN-armed anti-PD-Ll. (a) Schematic representations of IFNa-anti-PD-Ll fusion protein in homodimer or heterodimer format. scFv, single-chain variable fragment, (b) Fusion proteins were expressed in 293F cells and were analyzed by non-reducing (left) and reducing (right) SDS-PAGE after purification. M, molecular weight marker; 1, a-PDLl-Fc; 2, IFNa-Fc; 3, IFNa-anti-PD-Ll heterodimer; 4, homodimer; 5, IFN- a-HBs. (c) CE electropherograms of indicated proteins, (d) Exemplary constructions of anti-PD-Ll - pro-IFN.

[0021] FIG. 2. Exemplary construction and characterization of IFNα-armed anti-PD-Ll. (a-b) Flow cytometry histograms showing the binding of indicated protein in IFNARl^-/- A20 cells (a) and PD-L1 ^{- /} A20 cells (b) at the concentration of 80nM. Numbers indicate the mean fluorescent intensity (MFI). (c) Binding curves of a-PDLl-Fc, IFN-a-PDLl heterodimer or IFN-a-PDLl homodimer to the immobilized PDLl-Fc-biotin by BLI. The constants were determined by dynamic-analysis model, (d) The bioactivity of IFNa-anti-PD-Ll protein was measured by an antiviral infection biological assay. L929 cells were cultured with each protein overnight before being infected with VSV-GFP virus. After another 30 hours of culture, the percentage of virus-infected cells was determined by flow cytometry.

[0022] FIG. 3. Exemplary results of systemic administration with heterodimer showing better anti-tumor effect than homodimer, (a) Balb/c mice (n=5) were inoculated with 3x10⁶ A20 cells. After tumor established, mice were treated with 20 μg of control, anti-PD-Ll, IFNa-Fc, or fusion protein by i.t. (a, days 18 and 22) or i.v. (b, day 11 and 15) injection. Tumor size was measured twice per week, (b) C57BL/6 mice (n=4-8) were inoculated with 5xl0⁵ MC38 cells. Mice were treated i.v. with 25 μg control or fusion protein on days 14 and 18.

[0023] FIG. 4. Exemplary results showing heterodimer with longer serum half-life and more tumor accumulation than homodimer, (a-b) Mice were injected i.v. with 25 μg indicated protein. Protein concentrations in tumor tissues (h) or serum (i) at different time points were measured by ELISA. Data indicate mean ± SEM and are representative of at least two independent experiments. *, p<0.05; **, p<0.01.

[0024] FIG. 5. Exemplary results showing heterodimer with more in vitro stability than homodimer. To compare the stability, IFNa-anti-PD-Ll homodimer (a) or heterodimer (b) were added into mouse serum and incubate at 37°C for indicated time. The activity of proteins was detected by IFN anti-viral infection bioassay. Data represent two independent experiments.

[0025] FIG. 6. Potential peripheral distribution of IFN-anti-PD-Ll caused by IFNAR expressed in peripheral tissue. (A-C) A20, TC-1 or CT-26 tumor bearing mice were sacrificed and collect tissues (heart, liver, spleen, lung, kidney, tumor). mRNA of tissues were extracted and reversed into cDNA. The mRNA level of IFNAR2 in the indicated tissues were compared by RT-PCR.

[0026] FIG. 7. Exemplary results of mutated IFNa-anti-PD-Ll heterodimer protein showing reduced bioactivity in untargeted A20 (PDLl^{- /}) cells, but recovered bioactivity in targeted A20 (PDL1^+/+) cells. The bioactivity of indicated mIFNa-anti-PD-Ll fusion protein mutants were determined by cell proliferation assay. PD-L1 deficient A20 cells (as non-targeted cells) or PD-L1 expressing A20 cells (as targeted cells) were cultured with indicated each protein. Three days later, the cell viability was determined by CCK8 coloring.

[0027] FIG. 8. Exemplary results showing of mutant IFNa-anti-PD-Ll heterodimer showing higher binding specificity with IFNAR expressing cells. The bioactivity IC50 of mutant IFNa-anti- PDL1 fusion proteins were determined as in FIG. 3. The targeting ability were calculated using the formula below.

Mutant IFN-Fc relative activity = IC50_mutant-iFN-Fc / IC50wt miF\-Fc

[0028] FIG. 9. Exemplary results showing mutantIFNa-anti-PD-Ll homodimer protein showing lower targeting ability in vitro. Serial diluted fusion protein samples was added into 96-well plates and incubated with WT or PDL1-KO A20 cells for 72 hours. Then, each well was added

with CCK8 reagent for 2 hours at 37oC, then OD450 was detected to reflect the cell number of each well.

[0029] FIG. 10. Exemplary results showing attenuated IFNa-anti-PD-Ll homodimer protein showing lower targeting ability in vitro. Serial diluted fusion protein samples was added into 96-well plates and incubated with

WT or PDL1-KO A20 cells for 72 hours. Then, each well was added with CCK8 reagent for 2 hours at 37oC, then OD450 was detected to reflect the cell number of each well.

[0030] FIG. 11. Exemplary results showing mutant IFN(R144A)-anti-PD-Ll heterodimer showing better anti-tumor effect in low dose (5 μg). BALB/c mice were s.c. implanted with 3X10e6 A20 cells. When the tumor size reached 70mm³(dayl2), mice were started to treat with 20ug of indicated IFN-aPDLl protein (i.v.) every three days for twice. The tumor growth curve and mice survive was recorded and showed.

[0031] FIG. 12. Exemplary results showing low dose of mutant IFN (R144A)-aPDLl still inhibiting tumor growth. B6 mice were s.c. implanted with 8X10e5 MC38 cells. When the tumor size reached 30-50mm³ (day 10), mice were treated with indicated doses of mutant IFN-anti-PD-Ll protein (i.v.) every three days for third times. The tumor growth curve and mice survive was recorded and showed.

[0032] FIG. 13. Exemplary results showing mutant IFNa-anti-PD-Ll leading to much less toxicity in tumor-bearing mice. To determine the in vivo toxicity of IFNa-anti-PD-Ll protein, A20 bearing mice were treated with large dose of indicated proteins. The mice body weight change and survive were recorded.

[0033] FIG. 14. Exemplary results showing high dose of WT, but not mutant, IFNa-anti-PD-Ll inducing systemic platelet reduction and leukopenia in tumor-bearing mice. To determine the in vivo toxicity of IFNa-anti-PD-Ll protein, A20 bearing mice were treated with large dose of indicated proteins. The blood was collected at day 6 after first treatment, and platelet, myeloid cells and lymphocytes within blood were quantified.

[0034] FIG. 15. Exemplary results showing mutant human IFNa-anti-PD-Ll lead to comparable tumor control in humanized mice, but less peripheral lymphocyte reduction. 4-week old NSG-SGM3 mice were reconstituted with 8X10e4 hu-CD34+ cells after 1.3Gy IR. Human cell populations ( hCD45+, hCD3+, hCD19+) in the blood were detected at day 6 after reconstitution. Recovered for 14 weeks, Hu-NSGmice were s.c. implanted with 3X10e6 A549 cells. When the tumor size reached ~40mm³ size (day 17), mice were started to treat with 30ug indicated mutant human IFN-anti-PD-Ll protein (i.v.) every three days for three times. .

[0035] FIG. 16. Table with summary of activity data with IFNa mutants in human or mouse cells.

Definitions

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following terms, unless indicated otherwise according to the context wherein the terms are found, are intended to have the following meanings.

[0037] When trade names are used herein, the trade name includes the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.

[0038] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,

47, 48, 49, or 50.

[0039] As used herein, “at least” a specific value is understood to be that value and all values greater than that value.

[0040] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference, unless the context clearly dictates otherwise.

[0041] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

[0042] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.

[0043] The term “comprising”, when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. The term “consisting essentially of’, when used to define compositions and methods, shall mean that the compositions and methods include the recited elements and exclude other elements of any essential significance to the compositions and methods. For example, “consisting essentially of’ refers to administration of the pharmacologically active agents expressly recited and excludes pharmacologically active agents not expressly recited. The term consisting essentially of does not exclude pharmacologically inactive or inert agents, e.g., pharmaceutically acceptable excipients, carriers or diluents. The term “consisting of’, when used to define compositions and methods, shall mean excluding trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0044] As used herein, the term “antibody” refers to molecules that are capable of binding an epitope or antigenic determinant. The term is meant to include whole antibodies and antigen-binding fragments thereof. The term encompasses polyclonal, monoclonal, chimeric, Fabs, Fvs, single-chain antibodies and single or multiple immunoglobulin variable chain or CDR domain designs as well as bispecific and multi-specific antibodies. Antibodies can be from any animal origin. Preferably, the antibodies are mammalian, e.g., human, murine, rabbit, goat, guinea pig, camel, horse and the like, or other suitable animals. Antibodies may recognize polypeptide or polynucleotide antigens. The term includes active fragments, including for example, an antigen binding fragment of an immunoglobulin, a variable and/or constant region of a heavy chain, a variable and/or constant region of a light chain, a complementarity determining region (cdr), and a framework region. The terms include polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, chimeric antibodies, hybrid antibody molecules, F(ab)2 and F(ab) fragments; Fv molecules (for example, noncovalent heterodimers), dimeric and trimeric antibody fragment constructs; mini bodies, humanized antibody molecules, and any functional fragments obtained from such molecules, wherein such fragments retain specific binding.

[0045] As used herein, the term “antigen” as used herein is meant any substance that causes the immune system to produce antibodies or specific cell-mediated immune responses against it. A disease associated antigen is any substance that is associated with any disease that causes the immune system to produce antibodies or a specific-cell mediated response against it. An antigen is capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. An antigen can have one or more epitopes (B- and/or T-cell epitopes). An antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens. Antigens as used herein may also be mixtures of several individual antigens.

[0046] As used herein, the term “biologically active” entity, or an entity having “biological activity,” is one having structural, regulatory, or biochemical functions of a naturally occurring molecule or any function related to or associated with a metabolic or physiological process. A biologically active polypeptide or fragment thereof includes one that can participate in a biological process or reaction and/or can produce a desired effect. The biological activity can include an improved desired activity, or a decreased undesirable activity. For example, an entity demonstrates biological activity when it participates in a molecular interaction with another molecule, when it has therapeutic value in alleviating a disease condition, when it has prophylactic value in inducing an immune response, or when it has diagnostic and/or prognostic value in determining the presence of a molecule. A biologically active protein or polypeptide can be naturally-occurring or it can be synthesized from known components, e.g., by recombinant or chemical synthesis and can include heterologous components.

[0047] As used herein, the terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, sarcoma, blastoma and leukemia. More particular examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer. As used herein, the term "tumor" is used interchangeably with the term cancer and refers to any malignant or neoplastic cell. It is noted that the term tumor is typically used to refer to uncontrolled growth that occurs in solid tissue such as an organ, muscle, or bone.

[0048] As used herein, the term “cell” refers to any prokaryotic, eukaryotic, primary cell or immortalized cell line, any group of such cells as in, a tissue or an organ. Preferably the cells are of mammalian ( e.g ., human) origin and can be infected by one or more pathogens.

[0049] As used herein, the term “co-administer” refers to the presence of two pharmacological agents in the blood at the same time. The two pharmacological agents can be administered concurrently or sequentially.

[0050] As used herein, the terms “disease” or “disorder” refer to a pathological condition, for example, one that can be identified by symptoms or other identifying factors as diverging from a healthy or a normal state. The term “disease” includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.

[0051] As used herein, the term “effective amount” of an active agent refers to an amount sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the patient.

[0052] As used herein, the term "expression of a nucleic acid molecule" refers to the conversion of the information contained in the nucleic acid molecule into a gene product. The gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a peptide or polypeptide produced by translation of an mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing; and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation. [0053] As used herein, the term “host cell” refers to an individual cell or a cell culture that can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide(s). A host cell can be a transfected, transformed, transduced or infected cell of any origin, including prokaryotic, eukaryotic, mammalian, avian, insect, plant or bacteria cells, or it can be a cell of any origin that can be used to propagate a nucleic acid described herein. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell that comprises a recombinant vector of the invention may be called a “recombinant host cell.”

[0054] Host cells include, without limitation, the cells of mammals, plants, insects, fungi and bacteria. Bacterial cells include, without limitation, the cells of Gram-positive bacteria such as species of the genus Bacillus, Streptomyces and Staphylococcus and cells of Gram-negative bacteria such as cells of the genus Escherichia and Pseudomonas. Fungal cells include, preferably, yeast cells such as Saccharomyces, Pichia pastoris and Hansenula polymorpha. Insect cells include, without limitation, cells of Drosophila and Sf9 cells. Plant cells include, among others, cells from crop plants such as cereals, medicinal or ornamental plants or bulbs. Suitable mammal cells for the present invention include epithelial cell lines (porcine, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc.), glial cells (murine, etc.), liver cell lines (monkey, etc.). CHO cells (Chinese Hamster Ovary), COS cells, BHK cells, cells HeLa, 911, AT1080, A549, 293 or PER.C6, human ECCs NTERA-2 cells, D3 cells of the line of mESCs, human embryonic stem cells such as HS293 and BGV01, SHEFl, SHEF2 and HS181, cells NIH3T3, 293T, REH and MCF-7 and hMSCs cells.

[0055] As used herein, the term “Fc” refers to a molecule or sequence comprising the sequence of a non-antigen-binding fragment of whole antibody, whether in monomeric or multimeric form.

The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins ( e.g ., IgGl, IgG2). Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, IgGA2).

[0056] As used herein, the terms “Fc domain” or “Fc region” is meant to refer to the immunoglobulin heavy chain “fragment crystallizable” region. Generally, an Fc domain is capable of interacting with a second Fc domain to form a dimeric complex. The Fc domain may be capable of binding cell surface receptors called Fc receptors and/or proteins of the complement system or may be modified to reduce or augment these binding activities. The Fc domain may be derived from IgG, IgA, IgD, IgM or IgE antibody isotypes and effect immune activity including opsonization, cell lysis, degranulation of mast cells, basophils, and eosinophils, and other Fc receptor-dependent processes; activation of the complement pathway; and protein stability in vivo.

[0057] “Fc domain” encompasses native Fc and Fc variant molecules and sequences as defined herein. As with Fc variants and native Fc’s, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by recombinant gene expression or by other means.

[0058] Fc Fusion proteins have been reported to combine the Fc regions of IgG with the domains of another protein, such as various cytokines and soluble receptors. ( e.g ., Capon et al. 1989 Nature 337:525-531; Chamow etal. 1996 Trends Biotechnol. 14:52-60; U.S. Pat. Nos. 5,116,964 and 5,541,087).

[0059] The use of Fc fusions is known in the art (e.g., U.S. Pat. Nos. 7,754,855; 5,480,981; 5,808,029; W07/23614; W098/28427 and references cited therein. Fc fusion proteins can include variant Fc molecules (e.g., as described in U.S. Pat. No. 7,732,570). Fc fusion proteins can be soluble in the plasma or can associate to the cell surface of cells having specific Fc receptors.

[0060] As used herein, the term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97/34631 (published Sep. 25, 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference. Thus, the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, in certain embodiments, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.

[0061] As used herein, the term “fusion protein” refers to polypeptides comprising two or more regions from different or heterologous proteins covalently linked (i.e., “fused”) by recombinant, chemical or other suitable method. If desired, the fusion molecule can be fused at one or several sites through a peptide or other linker segment or sequence. For example, one or more peptide linkers may be used to assist in construction of a fusion protein.

[0062] As used herein, the term “high dosage” is meant at least 5% ( e.g ., at least 10%, 20%, 50%, 100%, 200%, or even 300%) more than the highest standard recommended dosage of a particular compound for treatment of any human disease or condition.

[0063] As used herein, the term “immune response” refers to a process whereby immune cells are stimulated and/or recruited from the blood to lymphoid as well as non-lymphoid tissues via a multifactorial process that involves distinct adhesive and/or activation steps. Activation conditions cause the release of cytokines, growth factors, chemokines and other factors, upregulate expression of adhesion and other activation molecules on the immune cells, promote adhesion, morphological changes, and/or extravasation concurrent with chemotaxis through the tissues, increase cell proliferation and cytotoxic activity, stimulate antigen presentation and provide other phenotypic changes including generation of memory cell types. Immune response is also meant to refer to the activity of immune cells to suppress or regulate inflammatory or cytotoxic activity of other immune cells. Immune response refers to the activity of immune cells in vivo or in vitro.

[0064] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., of an IFNa sequence), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to, or can be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and oftentimes over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length or over the full-length of an amino acid or nucleic acid sequences. [0065] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0066] A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST algorithms, which are described in Altschul et al. 1977 Nuc. Acids Res. 25:3389-3402 and Altschul et al. 1990 J. Mol. Biol. 215:403-410, respectively. BLAST software is publicly available through the National Center for Biotechnology Information on the worldwide web at ncbi.nlm.nih.gov/. Both default parameters or other non-default parameters can be used. The BLASTN program (for nucleotide sequences) uses as defaults a word- length (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. [0067] As used herein, the term "inhibit" refers to any measurable reduction of biological activity. Thus, as used herein, "inhibit" or "inhibition" may be referred to as a percentage of a normal level of activity.

[0068] As used herein, the term “Interferon-a” or “IFNa” refers to a family of proteins that include some of the main effectors of innate immunity. There are at least 15 known subtypes of human IFNa. The major subtypes identified are IFNal, IFNa2, IFNa8, IFNalO, IFNal4 and IFNa21.

[0069] As used herein, the term “PD-L1” refers to programmed cell death ligand 1 (see, for example, Freeman et al. (2000) J. Exp. Med. 192:1027). Representative amino acid sequence of human PD-L1 is disclosed under the NCBI accession number: NP 054862.1, and the representative nucleic acid sequence encoding the human PD-L1 is shown under the NCBI accession number: NM_014143.3. PD-L1 is expressed in placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells. PD-L1 binds to its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells and myeloid cells. The binding of PD-L1 and its receptor induces signal transduction to suppress TCR-mediated activation of cytokine production and T cell proliferation. Accordingly, PD-L1 plays a major role in suppressing immune system during particular events such as pregnancy, autoimmune diseases, tissue allografts, and is believed to allow tumor or cancer cells to circumvent the immunological checkpoint and evade the immune response. [0070] As used herein, the term “anti-PD-Ll” refers to a moiety that is capable of specific binding to PD-L1 (e.g., human PD-L1) with an affinity which is sufficient to provide for diagnostic and/or therapeutic use.

[0071] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind human and/or monkey PD-L1 with abinding affinity (K_D) of

to

. K_D as used herein refers to the ratio of the dissociation rate to the association rate (k_0ff/k_0n), may be determined using surface plasmon resonance methods for example using instrument such as Biacore.

[0072] As used herein, the terms an “isolated” molecule (such as a polypeptide or polynucleotide) is one that has been manipulated to exist in a higher concentration than in nature or has been removed from its native environment. For example, a subject antibody is isolated, purified, substantially isolated, or substantially purified when at least 10%, or 20%, or 40%, or 50%, or 70%, or 90% of non-subject-antibody materials with which it is associated in nature have been removed. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated.” Further, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Isolated RNA molecules include in vivo or in vitro RNA replication products of DNA and RNA molecules. Isolated nucleic acid molecules further include synthetically produced molecules. Additionally, vector molecules contained in recombinant host cells are also isolated. Thus, not all “isolated” molecules need be “purified.”

[0073] As used herein, the terms “linker” or “linking segment” refer to a molecule or group that connects two other molecules or groups. A peptide linker may allow the connected molecules or groups to acquire a functional configuration. The linker peptide preferably comprises at least two amino acids, at least three amino acids, at least five amino acids, at least ten amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, or at least 50 amino acids.

[0074] Components of a fusion protein, such as cytokines or other bioactive molecules and any peptide linkers, can be organized in nearly any fashion provided that the fusion protein has the function for which it was intended. In particular, each component of a fusion protein can be spaced from another component by at least one suitable peptide linker segment or sequence if desired. Additionally, the fusion protein may include tags, e.g., to facilitate modification, identification and/or purification of the fusion protein. More specific fusion proteins are in the examples described below. [0075] As used herein, the term “low dosage” refers to at least 5% less (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) than the lowest standard recommended dosage of a particular compound formulated for a given route of administration for treatment of any human disease or condition. For example, a low dosage of an agent that is formulated for administration by inhalation will differ from a low dosage of the same agent formulated for oral administration.

[0076] As used herein, the term “medium” or “media” includes any culture medium, solution, solid, semi-solid, or rigid support that may support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli, or Pseudomonas host cells, and cell contents. Thus, the term may encompass medium in which the host cell has been grown, e.g., medium into which a polypeptide has been secreted, including medium either before or after a proliferation step. The term also may encompass buffers or reagents that contain host cell lysates, such as in the case where a polypeptide is produced intracellularly and the host cells are lysed or disrupted to release the polypeptide.

[0077] As used herein, the term “pharmaceutically acceptable” excipient, carrier, or diluent refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymer as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0078] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” “nucleotide,” “oligonucleotide,” and "nucleic acid" are used interchangeably herein to refer to polymeric forms of nucleotides, including ribonucleotides as well as deoxyribonucleotides, of any length. They can include both double-, single-stranded or triple helical sequences and include, but are not limited to, cDNA from viral, prokaryotic, and eukaryotic sources; mRNA; genomic DNA sequences from viral ( e.g ., DNA viruses and retroviruses) or prokaryotic sources; RNAi; cRNA; antisense molecules; recombinant polynucleotides; ribozymes; and synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA. Nucleotides can be referred to by their commonly accepted single-letter codes.

[0079] Polynucleotides are not limited to polynucleotides as they appear in nature, and also include polynucleotides where unnatural nucleotide analogues and inter-nucleotide bonds appear. A nucleic acid molecule may comprise modified nucleic acid molecules (e.g., modified bases, sugars, and/or intemucleotide linkers). Non-limitative examples of this type of unnatural structures include polynucleotides wherein the sugar is different from ribose, polynucleotides wherein the phosphodiester bonds 3'-5' and 2'-5' appear, polynucleotides wherein inverted bonds (3 '-3' and 5'-5') appear and branched structures. Also, the polynucleotides of the invention include unnatural internucleotide bonds such as peptide nucleic acids (PNA), locked nucleic acids (LNA), C1-C4 alkylphosphonate bonds of the methylphosphonate, phosphoramidate, C1-C6 alkylphosphotriester, phosphorothioate and phosphorodithioate type. In any case, the polynucleotides of the invention maintain the capacity to hybridise with target nucleic acids in a similar way to natural polynucleotides.

[0080] Unless otherwise indicated or obvious from context, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. (Batzer et al. 1991 Nucleic Acid Res. 19:5081; Ohtsuka et al. 1985 J. Biol. Chem. 260:2605-2608; Rossolini et al. 1994 Mol. Cell. Probes 8:91-98.) [0081] As used herein, the term “promoter” refers to a DNA regulatory region capable of binding RNA polymerase in a mammalian cell and initiating transcription of a downstream (3' direction) coding sequence operably linked thereto. A promoter sequence includes the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence may be a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Promoters include those that are naturally contiguous to a nucleic acid molecule and those that are not naturally contiguous to a nucleic acid molecule. Additionally, the term "promoter" includes inducible promoters, conditionally active promoters such as a cre-lox promoter, constitutive promoters, and tissue specific promoters.

[0082] As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like. Furthermore, a polypeptide may refer to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate or may be accidental. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0083] As used herein, the term “purified” refers to a protein that may be substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e. a native cell, or host cell in the case of a recombinantly produced protein. A protein that may be substantially free of cellular material includes preparations of protein having less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein(s). When a protein or variant thereof is recombinantly produced by the host cells, the protein may be present at about 30%, at about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. When a protein or variant thereof is recombinantly produced by the host cells, the protein may be present in the culture medium at about 5 g/L, about 4 g/L, about 3 g/L, about 2 g/L, about 1 g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about 100 mg/L, about 50 mg/L, about 10 mg/L, or about 1 mg/L or less of the dry weight of the cells. Thus, a “substantially purified” protein may have a purity level of at least at least about 80%, specifically, a purity level of at least about 85%, and more specifically, a purity level of at least about 90%, a purity level of at least about 95%, a purity level of at least about 99% or greater as determined by appropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0084] Proteins and prodrugs of the present invention are, subsequent to their preparation, preferably isolated and/or purified to obtain a composition containing an amount by weight equal to or greater than 80% (“substantially pure”), which is then used or formulated as described herein. In certain embodiments, the compounds of the present invention are more than 95% pure.

[0085] As used herein, the term “receptor” refers to proteins, including glycoproteins or fragments thereof, capable of interacting with another molecule, called the ligand. The ligand may belong to any class of biochemical or chemical compounds. The ligand is usually an extracellular molecule which, upon binding to the receptor, usually initiates a cellular response, such as initiation of a signal transduction pathway. The receptor need not necessarily be a membrane-bound protein. [0086] As used herein, the term “recombinant,” with respect to a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant", as used with respect to a protein or polypeptide, means a polypeptide produced by expression of a recombinant polynucleotide. The term “recombinant” as used with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced.

[0087] As used herein, the term “sample” refers to a sample from a human, animal, or to a research sample, e.g., a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid, or coagulated material. The “sample” may be tested in vivo, e.g., without removal from the human or animal, or it may be tested in vitro. The sample may be tested after processing, e.g., by histological methods. “Sample” also refers, e.g., to a cell comprising a fluid or tissue sample or a cell separated from a fluid or tissue sample. “Sample” may also refer to a cell, tissue, organ, or fluid that is freshly taken from a human or animal, or to a cell, tissue, organ, or fluid that is processed or stored.

[0088] As used herein, the terms “stimulate” or “stimulating” refer to increase, to amplify, to augment, to boost a physiological activity, e.g., an immune response. Stimulation can be a positive alteration. For example, an increase can be by 5%, 10%, 25%, 50%, 75%, or even 90-100%. Other exemplary increases include 2-fold, 5 -fold, 10-fold, 20-fold, 40-fold, or even 100-fold.

[0089] As used herein, the terms “subject” and “patient” are used interchangeably herein to refer to a living animal (human or non-human). The subject may be a mammal. The terms “mammal” or “mammalian” refer to any animal within the taxonomic classification mammalia. A mammal may be a human or a non-human mammal, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice. The term "subject" does not preclude individuals that are entirely normal with respect to a disease or condition, or normal in all respects.

[0090] As used herein, the terms “suppress” or “suppressing” refer to decrease, to attenuate, to diminish, to arrest, or to stabilize a physiological activity, e.g., an immune response. Suppression can be a negative alteration. For example, a decrease can be by 5%, 10%, 25%, 50%, 75%, or even 90- 100%. Exemplary decreases include 2-fold, 5 -fold, 10-fold, 20-fold, 40-fold, or even 100-fold.

[0091] As used herein, the term “therapeutically effective amount” refers to the dose of a therapeutic agent or agents sufficient to achieve the intended therapeutic effect with minimal or no undesirable side effects. A therapeutically effective amount can be readily determined by a skilled physician, e.g., by first administering a low dose of the pharmacological agent(s) and then incrementally increasing the dose until the desired therapeutic effect is achieved with minimal or no undesirable side effects.

[0092] As used herein, the term “transfected” means possessing introduced DNA or RNA, with or without the use of any accompanying facilitating agents such as lipofectamine. Methods for transfection that are known in the art include, for example, calcium phosphate transfection, DEAE dextran transfection, protoplast fusion, electroporation, and lipofection.

[0093] As used herein, the terms “treatment” or “treating” a disease or disorder refers to a method of reducing, delaying or ameliorating such a condition, or one or more symptoms of such disease or condition, before or after it has occurred. Treatment may be directed at one or more effects or symptoms of a disease and/or the underlying pathology. The treatment can be any reduction and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. As compared with an equivalent untreated control, such degree of reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.

[0094] As used herein, the term “vector” refers to a nucleic acid molecule that is able to transmit genetic material to a host cell or organism. A vector may be composed of either DNA or RNA. A vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases which can be used for the insertion of foreign DNA, and usually selectable markers such as genes coding for antibiotic resistance, and often recognition sequences ( e.g ., promoter) for the expression of the inserted DNA. Common vectors include plasmid vectors and phage vectors. [0095] Any compositions or methods disclosed herein can be combined with one or more of any of the other compositions and methods provided herein.

Detailed Description of the Invention

[0096] The invention provides novel fusion proteins and therapeutic uses thereof. More particularly, the invention provides novel Fc heterodimers of a PD-L1 binding moiety and an attenuated (reduced affinity) Interferon, or prodrugs thereof, and compositions and methods of their preparation and use in treating various diseases and disorders, e.g., hyperplasia, solid tumor or hematopoietic malignancy, with reduced off-target toxicities and side effects during treatment.

[0097] PD-L1 is highly expressed in many tumors and more on dendritic cells inside tumor tissues but not in normal tissue. (Tang, et al. 2018 J Clin Investig 128, 580-588; Yang, et al. 2014 Cancer cell 25, 37-48; Zou, et al. 2016 Science Translat Med 8, 328rv324.) Anti-PD-Ll is utilized to specifically target IFNa into tumor tissues.

[0098] Disclosed herein two formats of IFN-anti-PD-Ll fusion proteins: a homodimer and a heterodimer. The homodimer was a commonly used format for antibody-cytokine fusion proteins. Although the homodimer showed higher IFN-receptor-binding affinity and more potent anti-viral activity in vitro, the heterodimer exhibited much better tumor-targeting, longer serum half-life, and better anti-tumor effect in vivo.

[0099] In the case of IFNa, for example, the receptor of IFNa (IFNAR) is widely expressed in peripheral tissues. IFNAR includes two subunits, IFNAR1 and IFNAR2. IFNa bind to IFNAR2 first due to high affinity among them, and then interact with IFNAR1 for signaling. The intensity of signaling depends on the binding affinity and the binding duration. To greatly reduce the potential off-tumor target side-effect, a further mutant IFNa was constructed that had greatly reduced its binding to its receptors and linked the mutant IFNa to anti-PD-Ll.

[00100] Several IFNa mutants were reported, which have decreased binding affinity resulting in attenuated activity. These homodimeric IFNa mutants Fc fusion protein exhibited decreased side- effect but also decreased anti-tumor efficacy when administered in vivo. However, heterodimeric mutant IFNa-anti-PD-Ll disclosed herein showed not only low toxicity, but also enhanced antitumor activity in vivo, with highly possibility that the arm of anti-PD-Ll in fusion protein facility the targeting of mutant IFN on dendritic cells inside tumors since tumor dendritic cells expressed higher level ofPD-Ll. (Piehler, etal. 2000 J Biol Chem 275, 40425-40433; Tang, etal. 2018 J Clin Investig 128, 580-588.)

[00101] The heterodimer molecule with mutant IFN disclosed herein showed much stronger antitumor effect than anti-PD-Ll with wild type (WT) IFNa at very low doses (0.04 μg to 1 μg per injection) without detectable toxicity. In fact, WT IFNa has lost efficacy at this low dose (clinically relevant dose < 5 μg). In vivo administration of even high dose (100 μg) of mutant IFNa-anti-PD-Ll did not lead to toxicity, such as no significant reduction of platelet and lymphocytes in the peripheral blood, no detectable inflammation cytokines increase or no body weight loss, while WT IFN led to severe toxicity. More remarkable was that extremely low dose (0.04 μg) of heterodimer anti-PD-Ll with mutated IFNa remained to have anti-tumor effect while homodimer lost its activity when the dose was reduced from 100 μg to 5 μg.

[00102] The data shown herein establishes that the disclosed heterodimer antibody with low affinity IFNa allows both anti-PD-Ll and IFNa targeting on the synapse of tumor associated dendritic cells that interact with anti-tumor T cells. Thus, these novel heterodimers allow IFN to reactivate dendritic cells while blocking PD-L1 -mediated suppression of T cells.

[00103] In certain embodiments, instead of employing a mutant IFN as an attenuated IFN, the invention contemplates employing a WT IFN in a prodrug form (a “pro-IFN”) where the activity of the IFN is blocked or attenuated via a covalently linked masking moiety such as an IFN-a/b receptor (IFNAR) domain. In certain embodiments, a pro-IFN includes: an IFNAR domain that retains IFN binding activity ( e.g ., the extracellular domain (ECD) of either natural IFNAR1 or IFNR2), an IFN domain that retains IFN activity when not engaged by the IFNAR domain, an Fc domain, a first linker fused at one end to the N-terminus of the IFN and fused at the other end to the IFNAR, wherein the first linker is protease cleavable, and a second linker fused at one end to the C-terminus of the IFN and fused at the other end to the N-terminus of the Fc domain. Various constructs for pro- IFN can be found in WO 2018/236701 (PCT/US2018/037982), the entire content of which is expressly incorporated hereby by reference for all purposes.

[00104] In one aspect, the invention generally relates to a fusion protein, comprising: a first structural unit comprising: (1) a moiety capable of specifically binding to programmed death-ligand 1 (PD-L1), and at its C-terminus a first fragment crystallizable (Fc) fragment; and (2) a second structural unit comprising: an attenuated Interferon (IFN) selected from a mutant or pro-interferon (pro-IFN) characterized by a reduced IFN activity, and at its C-terminus a second Fc fragment. The first Fc fragment comprises a knob and the second Fc fragment comprises a hole in the respective heavy chain 3 (C_H3) domains, or the first Fc fragment comprises a hole and the second Fc fragment comprises a knob in the respective C_H3 domains, so that the first structural unit and the second structural unit form a heterodimer fusion protein via disulfide bonds.

[00105] In certain embodiments, the moiety capable of specifically binding to PD-L1 is a singlechain variable fragment (scFv) or antigen-binding fragment (Fab) of an anti-PD-Ll antibody, the scFv comprising a heavy chain (V_H), a light chain (V_L), and a first linker peptide connecting the N- terminus of the V_H with the C-terminus of the V_L, or vice versa, or the Fab comprising one constant and one variable domain of the heavy chain (V_H C_H ¹) and of the light chain (V_LC_L), and a linker peptide connecting N-terminus of the C_H ¹ with the C-terminus of the VH, or N-terminus of the C_L with the C-terminus of the V_L.

[00106] In certain embodiments, the cleavable linker is used to link between VL and VH of scfv (a-PDLl) and/or between IFNAR and IFN, where the linker is capable of being recognized and hydrolyzed by a proteolytic enzyme specifically expressed in a tumor microenvironment, e.g., matrix metalloproteinase (MMP), to restore activity of scFv or Fab to PD-L1, or IFN exhibits anti-tumor activity locally with further reduced the toxicity.

[00107] In certain embodiments, the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloproteinase.

[00108] Any suitable linkers may be adopted. Exemplary peptide linker sequences include those having from about 7 to 20 amino acids, e.g., having from about 8 to 16 amino acids. The linker sequence is preferably flexible so as not hold the biologically active polypeptide or effector molecule in a single undesired conformation. The linker sequence can be used, e.g., to space the recognition site from the fused molecule. Specifically, the peptide linker sequence can be positioned so as to provide molecular flexibility. The linker preferably predominantly comprises amino acids with small side chains, such as glycine, alanine and serine, to provide for flexibility.

[00109] In certain embodiments, the scFv or Fab is that of a human or humanized anti-PD-Ll antibody.

[00110] In certain embodiments, the attenuated IFN is a mutant IFN.

[00111] In certain embodiments, the mutant IFN is mutant IFNa.

[00112] In certain embodiments, the mutant IFNa is a mutant human IFNa. [00113] In certain embodiments, the mutant IFNa is characterized by a reduced binding affinity to IFNa receptor. In certain embodiments, the reduction in binding affinity to IFNa receptor is by 80% or more.

[00114] In certain embodiments, the mutant IFNa comprises a mutation selected from: R144A, R149A, Q124R, S152A and A145G.

[00115] In certain embodiments, the mutant IFN is mutant IFNβ.

[00116] In certain embodiments, the mutant IFNa is a mutant human IFNβ.

[00117] In certain embodiments, the mutant IFNβ is characterized by a reduced binding affinity to IFNβ receptor. In certain embodiments, the reduction in binding affinity to IFNβ receptor is by 80% or more.

[00118] In certain embodiments, the attenuated IFN is a pro-IFN.

[00119] In certain embodiments, the attenuated IFN comprises an IFN-a/b receptor (IFNAR) domain fused at one end to the N-terminus of the IFN via a second linker peptide.

[00120] In certain embodiments, the IFNAR domain is the extracellular domain (ECD) of natural IFNAR1.

[00121] In certain embodiments, the IFNAR domain is the extracellular domain (ECD) of natural IFNR2

[00122] In certain embodiments, the first linker peptide and/or the second linker peptide is capable of being recognized and hydrolyzed by a proteolytic enzyme specifically expressed in a tumor microenvironment.

[00123] In certain embodiments, the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloproteinase (MMP).

[00124] In certain embodiments, the MMP is selected from MMPl, MMP3, MMP9, MMP 10, MMPll, MMP12, MMP13 and MMP14.

[00125] In certain embodiments, the moiety capable of specifically binding to PD-L1 comprises the amino acid sequence set forth in SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 25, 27, 29, 31, 33, 35, 37, 39, 41, 45 or 47.

[00126] In certain embodiments, the mutant IFN comprises the amino acid sequence set forth in SEQ ID Nos. 17, 19, 21, 23, 49, 51, 53, or 55.

[00127] In certain embodiments, the first Fc fragment comprises a knob and the second Fc fragment comprises a hole in the respective C_H3 domains. [00128] In certain embodiments, the first Fc fragment comprises a hole and the second Fc fragment comprises a knob in the respective C_H3 domains.

[00129] Any suitable antibody Fc fragment may be employed. In certain embodiments of the fusion protein, the antibody Fc fragment comprises a human Fc-knob or human Fc-hole. In certain embodiments, the antibody Fc fragment comprises the amino acid sequence set forth in SEQ ID No.

5. In certain embodiments, the antibody Fc fragment comprises a human IgGl. In certain embodiments, the antibody Fc fragment comprises the amino acid sequence set forth in SEQ ID No.

6

[00130] In another aspect, the invention generally relates to a prodrug comprising a fusion protein disclosed herein. For example, the linker is capable of being recognized and hydrolyzed by a proteolytic enzyme specifically expressed in a tumor microenvironment, e.g., MMP to restore to scFv or Fab, to increase affinity to PD-L1.

[00131] In yet another aspect, the invention generally relates to a substantially purified fusion protein or prodrug disclosed herein.

[00132] In yet another aspect, the invention generally relates to a polynucleotide encoding a fusion protein or prodrug disclosed herein.

[00133] In yet another aspect, the invention generally relates to an expression vector comprising the polynucleotide disclosed herein.

[00134] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising a fusion protein or prodrug disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[00135] In yet another aspect, the invention generally relates to a method for treating a disease or condition. The method comprises administering to a patient in need thereof a therapeutically effective amount of a fusion protein or prodrug disclosed herein or a pharmaceutical composition disclosed herein, wherein the disease or condition is selected from hyperplasia, solid tumor or hematopoietic malignancy.

[00136] In certain embodiments, the subject being treated is further administered one or more of chemotherapy, radiotherapy, targeted therapy, immunotherapy or hormonal therapy.

[00137] In certain embodiments, the method disclosed herein is in combination with one or more of immune check point blockade, co-signaling of T cells, and tumor targeting antibody therapies. [00138] In certain embodiments, the method further comprises administering a chemotherapeutic agent to the subject. [00139] In certain embodiments, the method further comprises administering a radiotherapy to the subject. In certain embodiments, the method further comprises administering a targeted therapy to the subject. In certain embodiments, the method further comprises administering an immunotherapy to the subject. In certain embodiments, the method further comprises administering hormonal therapy to the subject.

[00140] As used herein, the term "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Erlotinib (TARCEVA®, Genentech/OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA®, Novartis),

Imatinib mesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs), and Gefitimb (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1- TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (Angew Chem. Inti. Ed. Engl. (1994) 33: 183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, ADRIAMYCIN^® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esonibicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamniprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frobnic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophylinic acid; 2-ethylhydrazide; procarbazine; PSK^® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL^® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE^® (doxetaxel; Rhone- Poulenc Rorer, Antony, France); chloranmbucil; GEMZAR^® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE^® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA^®); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

[00141] Examples of the second (or further) agent or therapy may include, but are not limited to, immunotherapies (e.g. PD-1 inhibitors (pembrolizumab, nivolumab, cemiplimab), PD-L1 inhibitors (atezolizumab, avelumab, durvalumab), CTLA4 antagonist, cell signal transduction inhibitors (e.g., imatinib, gefitinib, bortezomib, erlotinib, sorafenib, sunitinib, dasatinib, vorinostat, lapatinib, temsirolimus, nilotinib, everolimus, pazopanib, trastuzumab, bevacizumab, cetuximab, ranibizumab, pegaptanib, panitumumab and the like), mitosis inhibitors (e.g., paclitaxel, vincristine, vinblastine and the like), alkylating agents (e.g., cisplatin, cyclophosphamide, chromabucil, carmustine and the like), anti-metabolites (e.g., methotrexate, 5-FU and the like), intercalating anticancer agents, (e.g., actinomycin, anthracycline, bleomycin, mitomycin-C and the like), topoisomerase inhibitors (e.g., irinotecan, topotecan, teniposide and the like), immunotherapic agents (e.g., interleukin, interferon and the like) and antihormonal agents (e.g., tamoxifen, raloxifene and the like).

[00142] In yet another aspect, the invention generally relates to use of a fusion protein or prodrug disclosed herein for treating or reducing a disease or disorder.

[00143] In yet another aspect, the invention generally relates to use of a fusion protein or prodrug disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent, in preparation of a medicament for treating or reducing a disease or disorder.

[00144] In certain embodiments, the disease or disorder is selected from acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, bladder urothelial carcinoma, breast ductal carcinoma, breast lobular carcinoma, carcinomas of the esophagus, castration-resistant prostate cancer (CRPC), cervical carcinoma, cholangiocarcinoma, chronic myelogenous leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal carcinoma, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, Hodgkin's lymphoma/primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC), kidney chromophobe carcinoma, kidney clear cell carcinoma, kidney papillary cell carcinoma, lower grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma & pheochromocytoma, prostate adenocarcinoma, renal cell carcinoma (RCC), sarcoma, skin cutaneous melanoma, squamous cell carcinoma of the head and neck, T-cell lymphoma, thymoma, thyroid papillary carcinoma, uterine carcinosarcoma, uterine corpus endometrioid carcinoma and uveal melanoma. In certain embodiments, the anticancer drug is effective for treating B-cell lymphoma or anti-colorectal cancer. [00145] In certain embodiments, the use disclosed herein is in combination with one or more of immune check point blockade, co-signaling of T cells, and tumor targeting antibody therapies.

[00146] In yet another aspect, the invention generally relates to a cell line comprising a polynucleotide encoding a fusion protein or prodrug disclosed herein.

[00147] In yet another aspect, the invention generally relates to a method for making a protein, comprising culturing the cell line disclosed herein. [00148] In certain embodiments, the method further comprises purifying or isolating a produced protein.

[00149] In yet another aspect, the invention generally relates to a method for making a protein. The method comprises: providing an expression vector encoding a fusion protein or prodrug disclosed herein; introducing into a host cell the expression vector; culturing the host cell in media under conditions sufficient to express the protein; and purifying the protein from the host cell or media.

[00150] In certain embodiments, the method comprises: constructing an expression vector comprising the coding gene encoding a fusion protein or prodrug; constructing the host cell comprising the expression vector by transiently transfection; culturing the host cell; collecting the cell supernatant; and purifying the fusion protein or prodrug.

General Discussions

[00151] Fusion proteins, prodrugs and compositions thereof disclosed herein may be useful in treating or reducing one or more diseases or disorder, for example, selected from head and neck cancer, endometrial cancer, colorectal cancer, ovarian cancer, breast cancer, melanoma, lung cancer, renal cancer, liver cancer, anal cancer, sarcoma, lymphoma, leukemia, brain tumors, gastric cancer, testicular cancer, pancreatic cancer, and thyroid cancer.

[00152] In general, preparation of the fusion proteins of the invention can be accomplished by procedures disclosed herein and by recognized recombinant DNA techniques involving, e.g., polymerase chain amplification reactions (PCR), preparation of plasmid DNA, cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of the DNA into a suitable cell, transformation or transfection of a host, culturing of the host. Additionally, the fusion molecules can be isolated and purified using chaotropic agents and well known electrophoretic, centrifugation and chromatographic methods. (Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd ed. (1989); and Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989) for disclosure relating to these methods.)

[00153] The invention further provides nucleic acid sequences and DNA sequences that encode the present fusion proteins. The DNA sequence may be carried by a vector suited for extrachromosomal replication such as a phage, virus, plasmid, phagemid, cosmid, YAC, or episome. For example, a DNA vector that encodes a desired fusion protein can be used to facilitate preparative methods described herein and to obtain significant quantities of the fusion protein or components thereof. The DNA sequence can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. A variety of host- vector systems may be utilized to express the protein-coding sequence. These may include mammalian cell systems infected with virus ( e.g ., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. (Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd ed. (1989); and Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989) for disclosure relating to these methods.)

[00154] Fusion protein components encoded by the DNA vector can be provided in a cassette format. By the term “cassette” is meant that each component can be readily substituted for another component by standard recombinant methods. In particular, a DNA vector configured in a cassette format is particularly desirable when the encoded fusion complex is to be used against pathogens that may have or have capacity to develop serotypes.

[00155] To make the vector coding for a fusion protein complex, the sequence coding for the biologically active polypeptide is linked to a sequence coding for the effector peptide by use of suitable ligases. DNA coding for the presenting peptide can be obtained by isolating DNA from natural sources such as from a suitable cell line or by known synthetic methods, e.g. the phosphate triester method. (Oligonucleotide Synthesis, IRL Press, M. J. Gait, ed., 1984). Synthetic oligonucleotides also may be prepared using commercially available automated oligonucleotide synthesizers. Once isolated, the gene coding for the biologically active polypeptide can be amplified by PCR or other means known in the art. Suitable PCR primers to amplify the biologically active polypeptide gene may add restriction sites to the PCR product. The PCR product preferably includes splice sites for the effector peptide and leader sequences necessary for proper expression and secretion of the biologically active polypeptide-effector fusion complex. The PCR product also preferably includes a sequence coding for the linker sequence, or a restriction enzyme site for ligation of such a sequence.

[00156] The fusion proteins described herein may be produced by standard recombinant DNA techniques. For example, once a DNA molecule encoding the biologically active polypeptide is isolated, sequence can be ligated to another DNA molecule encoding the effector polypeptide. The nucleotide sequence coding for a biologically active polypeptide may be directly joined to a DNA sequence coding for the effector peptide or, more typically, a DNA sequence coding for the linker sequence as discussed herein may be interposed between the sequence coding for the biologically active polypeptide and the sequence coding for the effector peptide and joined using suitable ligases. The resultant hybrid DNA molecule can be expressed in a suitable host cell to produce the fusion protein complex. The DNA molecules are ligated to each other in a 5' to 3' orientation such that, after ligation, the translational frame of the encoded polypeptides is not altered (i.e., the DNA molecules are ligated to each other in-frame). The resulting DNA molecules encode an in-frame fusion protein. [00157] Other nucleotide sequences also can be included in the gene construct. For example, a promoter sequence, which controls expression of the sequence coding for the biologically active polypeptide fused to the effector peptide, or a leader sequence, which directs the fusion protein to the cell surface or the culture medium, can be included in the construct or present in the expression vector into which the construct is inserted.

[00158] A host cell can be used for preparative purposes to propagate nucleic acid encoding a desired fusion protein or a component thereof. A host cell can include a prokaryotic or eukaryotic cell in which production of the fusion protein is specifically intended. Thus, host cells specifically include yeast, fly, worm, plant, frog, mammalian cells and organs that are capable of propagating nucleic acid encoding the fusion. Non-limiting examples of mammalian cell lines which can be used include CHO dhfr-cells (Urlaub and Chasm, 1980 Proc. Natl. Acad. Sci. USA, 77:4216), 293 cells (Graham et al. 1977 J. Gen. Virol., 36:59 ()) or myeloma cells like SP2 or NSO (Galfre and Milstein, 1981 Meth. Enzymol, 73(B):3).

[00159] Host cells capable of propagating nucleic acid encoding a desired fusion protein complexs encompass non-mammalian eukaryotic cells as well, including insect (e.g., Sp . frugiperdd), yeast (e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha; as generally reviewed by Fleer, R., 1992 Current Opinion in Biotechnology, 3(5):486496), fungal and plant cells. Also contemplated are certain prokaryotes such as E. coli and Bacillus.

[00160] Nucleic acid encoding a desired fusion protein can be introduced into a host cell by standard techniques for transfecting cells. The term “transfecting” or “transfection” is intended to encompass all conventional techniques for introducing nucleic acid into host cells, including calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, viral transduction and/or integration.

[00161] Various promoters (transcriptional initiation regulatory region) may be used according to the invention. The selection of the appropriate promoter is dependent upon the proposed expression host. Promoters from heterologous sources may be used as long as they are functional in the chosen host.

[00162] Promoter selection is also dependent upon the desired efficiency and level of peptide or protein production. Inducible promoters such as tac are often employed in order to dramatically increase the level of protein expression in E. coli. Overexpression of proteins may be harmful to the host cells. Consequently, host cell growth may be limited. The use of inducible promoter systems allows the host cells to be cultivated to acceptable densities prior to induction of gene expression, thereby facilitating higher product yields.

[00163] It is preferred that the fusion proteins of the present invention be substantially pure. That is, the fusion proteins have been isolated from cell substituents that naturally accompany it so that the fusion proteins are present preferably in at least 80% or 90% to 95% homogeneity (w/w). Fusion proteins having at least 98 to 99% homogeneity (w/w) are most preferred for many pharmaceutical, clinical and research applications. Once substantially purified the fusion protein should be substantially free of contaminants for therapeutic applications. Once purified partially or to substantial purity, the soluble fusion proteins can be used therapeutically, or in performing in vitro or in vivo assays as disclosed herein. Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis.

[00164] The invention also provides a pharmaceutical preparation comprising a therapeutically effective amount of a composition, a fusion protein, a polynucleotide, a gene construct, a vector or a host cell according to the invention and a pharmaceutically acceptable excipient or vehicle.

[00165] Preferred excipients for use in the present invention include sugars, starches, celluloses, gums and proteins. In a preferred embodiment, the pharmaceutical composition of the invention is formulated in a pharmaceutical form for administration as a solid (for example tablets, capsules, lozenges, granules, suppositories, crystalline or amorphous sterile solids that can be reconstituted to provide liquid forms, etc.), liquid (for example solutions, suspensions, emulsions, elixirs, lotions, unguents, etc.) or semi-solid (gels, ointments, creams and similar). The pharmaceutical compositions of the invention can be administered by any route, including, without limitation, oral, intravenous, intramuscular, intraarterial, intramedullary, intratecal, intraventricular, transdermic, subcutaneous, intraperitoneal, intranasal, enteric, topical, sublingual or rectal route. A revision of the different forms of administration of active principles, the excipients to be used and their manufacturing procedures can be found in Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 20^th edition, Williams & Wilkins PA, USA (2000) Examples of pharmaceutically acceptable vehicles are known in the state of the technique and include saline solutions buffered with phosphate, water, emulsions, such as oil/water emulsions, different types of humidifying agents, sterile solutions, etc. The compositions comprising said vehicles can be formulated by conventional procedures known in the state of the technique.

[00166] In the case of the pharmaceutical composition of the invention comprising nucleic acids (the polynucleotides of the invention, vectors or gene constructs), the invention contemplates specially prepared pharmaceutical compositions for administering said nucleic acids. The pharmaceutical compositions can comprise said nucleic acids in naked form, in other words, in the absence of compounds protecting the nucleic acids from degradation by the organism's nucleases, which entails the advantage of eliminating the toxicity associated to the reagents used for transfection. Suitable routes of administration for the naked compounds include intravascular, intratumoral, intracraneal, intraperitoneal, intrasplenic, intramuscular, subretinal, subcutaneous, mucous, topical and oral route (Templeton, 2002 DNA Cell Biol., 21:857-867). Alternatively, the nucleic acids can be administered forming part of liposomes, conjugated to cholesterol or conjugated to compounds capable of promoting translocation through cell membranes such as the Tat peptide derived from the TAT protein of HIV-1, the third helix of the homeodomain of the Antennapedia protein of D. melanogaster, the VP22 protein of the herpes simplex virus, oligomers of arginine and peptides such as those described in W007069090 (Lindgren, et al. 2000 Trends Pharmacol. Sci 21:99-103; Schwarze, etal. 2000 Trends Pharmacol. Sci. 21:45-48; Lundberg, etal. 2003 Mol. Therapy 8:143-150; and Snyder, etal. 2004 Pharm. Res. 21:389-393). Alternatively, the polynucleotide can be administered forming part of a plasmidic vector or of a viral vector, preferably vectors based on an adenovirus, in adeno-associated viruses or in retroviruses, such as viruses based on the virus of murine leukaemia (MLV) or on lentiviruses (HIV, FIV, EIAV).

[00167] The compositions of the invention can be administered at doses of less than 10 mg per kilogram of body weight, preferably less than 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per each kg of body weight and less than 200 nmol of agent, in other words, approximately 4.4xl0¹⁶ copies per kg of body weight or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15 or 0.075 nmol per Kg of body weight. The unitary dose can be administered by injection, by inhalation or by topical administration. The bifunctional polynucleotides and compositions of the invention can be administered directly into the organ in which the target mRNA is expressed in which case doses will be administered of between 0.00001 mg and 3 mg per organ, or preferably between 0.0001 and 0.001 mg per organ, about 0.03 and 3.0 mg per organ, about 0.1 and 3.0 mg per organ or between 0.3 and 3.0 mg per organ.

[00168] The dose will depend on the severity and response to the condition to be treated and may vary between several days and several months or until the condition is seen to remit. The optimum dose can be determined by periodically measuring the agent's concentrations in the patient's organism. The optimum dose can be determined from the EC50 values obtained through previous in vitro or in vivo tests in animal models. The unitary dose can be administered once a day or less than once a day, preferably, less than once every 2, 4, 8 or 30 days. Alternatively, it is possible to administer an initial dose followed by one or several maintenance doses, generally in a lesser amount that the initial dose. The maintenance regime may involve treating the patient with doses ranging between 0.01 μg and 1.4 mg/kg of body weight per day, for example 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of body weight per day. Maintenance doses are administered, preferably, at most once every 5, 10 or 30 days. The treatment must continue for a time that will vary according to the type of alteration suffered by the patient, its severity and the patient's condition. Following treatment, the patient's evolution must be monitored in order to determine whether the dose ought to be increased in the case of the disease not responding to the treatment or whether the dose ought to be decreased in the case of observing an improvement in the disease or unwanted secondary effects.

[00169] The daily dose can be administered in a single dose or in two or more doses according to the particular circumstances. If a repeated administration or frequent administrations are required, it is advisable to implant an administration device, such as a pump, a semi-permanent catheter (intravenous, intraperitoneal, intracistemal or intracapsular) or a reservoir.

[00170] The compositions of the invention are administered according to methods known to an expert in the art, including, without limitation, intravenous, oral, nasal, parenteral, topical, transdermic, rectal and similar.

[00171] The following examples are meant to be illustrative of the practice of the invention and not limiting in any way.

Examples

[00172] The below Examples describe certain exemplary embodiments of compounds prepared according to the disclosed invention. It will be appreciated that the following general methods, and other methods known to one of ordinary skill in the art, can be applied to compounds and subclasses and species thereof, as disclosed herein. [00173] Fusion proteins were generated that have a single chain variable fragment (scFv) of anti- PD-L1 antibody (PD-L1) and IFNa in either homodimer or heterodimer format (FIG. la). Sodium dodecyl sulfate polyacrylamide gel electrophoresis and capillary electrophoresis (CE) results showed that the purified proteins had a purity >95% (FIG. lb and lc). To evaluate the binding of the resulting IFNa-anti-PD-Ll fusion protein, their affinities for either PD-L1 or IFNAR were tested. A20 cells were positive for both PD-L1 and IFNAR1. One receptor was knocked out and tested binding to the other. In PD-L1 -expressing IFNAR1-/- A20 cells, the fusion protein bound with a similar affinity to that of the anti-PD-Ll antibody (FIG. 2a). In IFNAR expressing PD-L1-/- cells, the binding of the heterodimer was reduced in comparison to that of IFNa-Fc or to that of the homodimer (FIG. 2b). The binding affinities to PD-L1 determined by Biolayer interferometry (BLI) showed that heterodimer and homodimer have KD values in the nM range (2 nM or 0.2 nM) (FIG. 2c). The type I IFN bioactivity in the fusion proteins was identified through anti-viral infection biological assay. IFNa-Fc protein could inhibit VSV-GFP (vesicular stomatitis virus expressing green fluorescent protein) infecting L929 cells in a dose-dependent manner. While at the same efficient concentration, both homodimer and heterodimer protein showed higher inhibition efficiency compared to that of the control IFNa-Fc protein (FIG. 2d). Collectively, these data suggest that both the homodimer and heterodimer formats of IFNa-anti-PD-Ll fusion protein can bind to PD-L1 while maintaining robust IFNa bioactivity.

[00174] Mice bearing advanced A20 tumors were treated with fusion protein intratumorally (i.t). While the anti-PD-Ll antibody failed to control tumor growth, both the heterodimer and homodimer forms of the IFNa-anti-PD-Ll fusion protein overcame anti-PD-Ll resistance and induced complete tumor regression in most of the treated mice (FIG. 3a). To test the targeting effects of the fusion protein, A20 tumor-bearing mice were treated with fusion protein systemically. Surprisingly, although the homodimer showed higher binding affinity to IFN-receptor and more potent anti-viral activity in vitro (FIG. 2b and 2d), only the heterodimer was able to control tumor growth in vivo (FIG. 3a). Similar effects were observed in the MC38 model (FIG. 3b). It was determined that this difference was due to the drug’s kinetics in vivo. The tumor tissues contained a much higher concentration of the drug in its heterodimer form than in its homodimer form (FIG. 4a); moreover, the serum half-life of the heterodimer was significantly longer (FIG. 4b). Heterodimer exhibited more bioactivity stability than homodimer, when incubated in the serum at 37°C in vitro (FIG. 5a- 5b). Taken together, these data show that targeted delivery of IFNa by anti-PD-Ll induces potent antitumor effects, leading to improved tumor control. The heterodimer format is much superior to homodimer.

[00175] IFNAR was widely expressed in peripheral tissues (FIG. 6A-6C). To reduce the potential off-target side-effect, a mutant IFNa-anti-PD-Ll heterodimer protein was constructed. The IFN mutant has the reduced activity in the un-targeted (PD-L1 negative) cells (FIG. 7a). However, it had much recovered activity in the targeted (PD-L1 positive) cells (FIG. 7b). During the murine IFNa4 mutants, R144A mutant based heterodimer protein had highest targeting specificity index (FIG. 8). WT IFNa-anti-PD-Ll homodimer had much higher activity in un-targeted (PD-L1 negative) cells than heterodimer (FIG. 9), Indicate the binding ability of homodimer with IFNAR is much stronger than heterodimer, which may result in more binding with peripheral cells and poor tumor targeting. Consistently, the targeting specificity of attenuated homodimer is much lower than heterodimer (FIG. 10)

[00176] Next, the in vivo anti-tumor effect of attenuated IFNa-anti-PD-Ll heterodimer was tested. With the high administration dose (20 μg), mutant IFNa(R144A)-anti-PD-Ll heterodimer had similar anti-tumor effect as WT, and both of them significantly inhibit the tumor growing. Surprisingly, in a lower dose (5 μg) mutant IFNa(R144A)-anti-PD-Ll but not WT protein still keep its effective anti -tumor effect (FIG. lib). Further dose titration results showed that administration of lug or lower dose of mutant IFNa(R144A)-anti-PD-Ll is effective to inhibit tumor growing (FIG. 12). To compare the protein’s toxicity in vivo, a large dose (200 μg) was used. IFN(WT)-anti-PDLl treatment induced significant body weight lost and eventually mice death. Mutant IFN(R144A)-anti- PDL1 treatment showed no toxicity (FIG. 13). The platelet and lymphocytes decrease are more sensitive indicator of IFN toxicity. IFN(WT)-anti-PDLl treatment induced significant peripheral platelet and lymphocytes decrease, but mutant IFN(R144A)-anti-PDLl treatment did not change the peripheral platelet or lymphocytes compared with non-treated group (FIG. 14).

[00177] Finally, to explore the potential application in clinic, a mutant human IFNa-anti-PDLl protein was constructed. Mutant human IFNa-anti-PDLl protein showed similar anti-tumor effect as WT IFN in humanized mice tumor model (FIG. 15a). However, while WT IFNa-anti-PDLl lead to significant peripheral lymphocytes decrease, mutant IFN only showed slightly decrease (FIG. 15b). This suggests that mutant IFNa-anti-PDLl will have much less peripheral toxicity, while preserve its anti-tumor effect.

[00178] Applicant’s disclosure is described herein in preferred embodiments with reference to the Figures, in which like numbers represent the same or similar elements. Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[00179] The described features, structures, or characteristics of Applicant’s disclosure may be combined in any suitable manner in one or more embodiments. In the description, herein, numerous specific details are recited to provide a thorough understanding of embodiments of the invention.

One skilled in the relevant art will recognize, however, that Applicant’s composition and/or method may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the disclosure.

[00180] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.

Incorporation by Reference

[00181] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.

Equivalents

[00182] The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

SEQ Listings

1. 2.14H9 clone for Anti-PD-Ll (AstraZeneca)

Amino acid sequence encoding the variable region of the light chain

2. 2.14H9 clone for Anti-PD-Ll (AstraZeneca)

Nucleotide sequence encoding the variable region of the light chain

3. 2.14H9 clone for Anti-PD-Ll (AstraZeneca)

Amino acid sequence encoding the variable region of the heavy chain

4. 2.14H9 clone for Anti-PD-Ll (AstraZeneca)

Nucleotide sequence encoding the variable region of the heavy chain

cactatgata gtagtggtta tcttgactac tggggccagg gaaccctggt caccgtctcc 360 tea

5. MEDI4736 for Anti-PD-Ll (AstraZeneca) from WO 2011/066389A1 Amino acid sequence encoding the variable region of the light chain

6. MEDI4736 for Anti-PD-Ll (AstraZeneca) from WO 2011/066389A1 Nucleotide sequence encoding the variable region of the light chain

7. MEDI4736 for Anti-PD-Ll (AstraZeneca) from WO 2011/066389A1 Amino acid sequence encoding the variable region of the heavy chain

8. MEDI4736 for Anti-PD-Ll (AstraZeneca) from WO 2011/066389A1 Nucleotide sequence encoding the variable region of the heavy chain

9. BMS-936559 sequences: anti-PDLl 12A4 clone in US 9,580,507 B2:

Amino acid sequence encoding the variable region of the light chain

10. BMS-936559 sequences: anti-PDLl 12A4 clone in US 9,580,507 B2:

Nucleotide sequence encoding the variable region of the light chain

11. BMS-936559 sequences: anti-PDLl 12A4 clone in US 9,580,507 B2:

Amino acid sequence encoding the variable region of the heavy chain

12. BMS-936559 sequences: anti-PDLl 12A4 clone in US 9,580,507 B2:

Nucleotide sequence encoding the variable region of the heavy chain

13. Anti-PD-Ll(Genentech, YW243.55.S70; US 8,217,149 B2) Amino acid sequence encoding the variable region of the light chain

14. Anti-PD-Ll (Genentech, YW243.55.S70; US 8,217,149 B2)

Nucleotide sequence encoding the variable region of the light chain

15. Anti-PD-Ll (Genentech, YW243.55.S70; US 8,217,149 B2)

Amino acid sequence encoding the variable region of the heavy chain

16. Anti-PD-Ll (Genentech, YW243.55.S70; US 8,217,149 B2) nucleotide acid sequence encoding the variable region of the heavy chain

17. Human IFNa2 amino acid sequence

18. Human IFNa2 nucleotide sequence

19. Human IFNa2(Q124R) amino acid sequence

20. Human IFNa2(Q124R) nucleotide sequence

21. hIFNa2(R144A) amino acid sequence

22. hIFNa2(R144A) nucleotide sequence

23, hIFNa2(R149A) amino acid sequence

24. hIFNa2(R149A) nucleotide sequence

25. Anti-PDLl(ScFv)-Fc-knob

2.14H9 clone for Anti-PD-Ll (AstraZeneca), amino acid sequence

26. Anti-PDLl(ScFv)-Fc-knob

2.14H9 clone for Anti-PD-Ll (AstraZeneca), nucleotide sequence

27. Anti-PDLl(ScFv)-Fc-knob

MEDI4736 for Anti-PD-Ll (AstraZeneca) from WO2011/066389A1, amino acid sequence

28. Anti-PDLl(ScFv)-Fc-knob

MEDI4736 for Anti-PD-Ll (AstraZeneca) from WO2011/066389A1, nucleotide sequence

29, Anti-PDLl(ScFv)-Fc-knob

BMS-936559 sequences: anti-PDLl 12A4 clone in US 9,580,507 B2, amino acid sequence

30. Anti-PDLl(ScFv)-Fc-knob

BMS-936559 sequences: anti-PDLl 12A4 clone in US 9,580,507 B2, nucleotide sequence

31. Anti-PD-Ll(ScFv)-Fc-knob

(Genentech, YW243.55.S70; US8217149 B2), amino acid sequence

32. Anti-PD-Ll(ScFv)-Fc-knob_> nucleotide sequence (Genentech, YW243.55.S70; US 8,217,149 B2)

33, Anti-PDLIVL-KappaCL, 2.14H9 clone for Anti-PD-Ll (AstraZeneca), amino acid sequence

34, Anti-PDLIVL-KappaCL

2.14H9 clone for Anti-PD-Ll (AstraZeneca), nucleotide sequence

35. mPDLlVH-CHl-Fc(knob), 2.14H9 clone for Anti-PD-Ll (AstraZeneca), amino acid sequence

36. mPDLlVH-CHl-Fc(knob)

2.14H9 clone for Anti-PD-Ll (AstraZeneca), nucleotide sequence

37. Anti-PDLIVL-KappaCL, MED 14736 for Anti-PD-Ll (AstraZeneca) from WO2011/066389A1, amino acid sequence

38. Anti-PDLIVL-KappaCL, MEDI4736 for Anti-PD-Ll (AstraZeneca) from WO2011/066389A1, nucleotide sequence

39. mPDLlVH-CHl-Fc(knob) , MEDI4736 for Anti-PD-Ll (AstraZeneca) from WO2011/066389A1, amino acid sequence

40. mPDLlVH-CHl-Fc(knob), MEDI4736 for Anti-PD-Ll (AstraZeneca) from WO2011/066389A1, nucleotide sequence

41. Anti-PDLIVL-KappaCL, BMS-936559 sequences: anti-PDLl 12A4 clone in US 9,580,507 B2, amino acid sequence

42. Anti-PDLIVL-KappaCL, BMS-936559 sequences: anti-PDLl 12A4 clone in US 9,580,507 B2, nucleotide sequence

43. mPDLlVH-CHl-Fc(knob), BMS-936559 sequences: anti-PDLl 12A4 clone in US 9,580,507 B2, amino acid sequence

44. mPDLlVH-CHl-Fc(knob), BMS-936559 sequences: anti-PDLl 12A4 clone in US 9,580,507 B2, nucleotide sequence

45. Anti-PDLIVL-KappaCL (Genentech, YW243.55.S70; US 8,217,149 B2) , amino acid sequence

46. Anti-PDLIVL-KappaCL (Genentech, YW243.55.S70; US 8,217,149 B2), nucleotide sequence

47. mPDLlVH-CHl-Fc(knob), (Genentech, YW243.55.S70; US 8,217,149 B2), amino acid sequence

48. mPDLlVH-CHl-Fc(knob), (Genentech, YW243.55.S70; US 8,217,149 B2), nucleotide sequence

49. hIFNa2 (wt)-Fc-hole, amino acid sequence

50, hIFNa2 (wt)-Fc-hole, nucleotide sequence

51. hIFNa2 (Q124R)-Fc-hole, amino acid sequence

52. hIFNa2 (Q124R)-Fc-hole, nucleotide sequence

53. hIFNa2 (R144A)-Fc-hole, amino acid sequence

54, hIFNa2 (R144A)-Fc-hole, nucleotide sequence

55, hIFNa2 (R149A)-Fc-hole, amino acid sequence

56. hIFNa2 (R149A)-Fc-hole, nucleotide sequence

57, MMP14 Substrates

58, MMP14 Substrate Mutants

59, Human Pro-IFN sequence

60, Mouse IFNARl-ECD:

61. Mouse IFNAR2-ECD:

62, Human IFNARl-ECD:

63. Human IFNAR2-ECD:

64, Exemplary MMP Substrates

The entire content of each of WO 2018/236701A1, WO 2011/066389A1, US Pat. No. 9,580,507 B2, US Pat. No. 8,217,149 B2 is expressly incorporated herein by references for all purposes.

Claims

What is claimed is: CLAIMS

1. A fusion protein, comprising: a first structural unit comprising: a moiety capable of specifically binding to programmed death-ligand 1

(PD-L1), and at its C-terminus a first fragment crystallizable (Fc) fragment; and a second structural unit comprising: an attenuated Interferon (IFN) selected from a mutant or pro-interferon (pro-IFN) characterized by a reduced IFN activity, and at its C-terminus a second Fc fragment; wherein the first Fc fragment comprises a knob and the second Fc fragment comprises a hole in the respective heavy chain 3 (C_H3) domains, or the first Fc fragment comprises a hole and the second Fc fragment comprises a knob in the respective C_H3 domains, so that the first structural unit and the second structural unit form a heterodimer fusion protein via disulfide bonds.

2. The fusion protein of claim 1, wherein the moiety capable of specifically binding to PD- L1 is a single-chain variable fragment (scFv) or antigen-binding fragment (Fab) of an anti-PD-Ll antibody, the scFv comprising a heavy chain (V_H), a light chain (VL), and a first linker peptide connecting the N-terminus of the V_H with the C-terminus of the VL, or vice versa , or the Fab comprising one constant and one variable domain of the heavy chain (V_H CH¹) and of the light chain (VLCL), and a linker peptide connecting N-terminus of the CH¹ with the C-terminus of the VH, or N-terminus of the CL with the C-terminus of the VL.

3. The fusion protein of claim 1 or 2, wherein the scFv or Fab is that of a human or humanized anti-PD-Ll antibody.

4. The fusion protein of any one of claims 1-3, wherein the attenuated IFN is a mutant IFN.

5. The fusion protein of claim 4, wherein the mutant IFN is a mutant IFNa.

6. The fusion protein of claim 5, wherein the mutant IFNa is a mutant human IFNa.

7. The fusion protein of claim 6, wherein the mutant IFNa is characterized by a reduced binding affinity to IFNa receptor.

8. The fusion protein of claim 7, wherein the reduction in binding affinity is by more than 80%.

9. The fusion protein of claim 7 or 8, wherein the mutation comprises one or more mutations selected from: R144A, R149A, Q124R, S152A and A145G.

10. The fusion protein of any one of claims 1-3, wherein the mutant IFN is a mutant IFNp.

11. The fusion protein of claim 10, wherein the mutant IFNβ is a mutant human IFNp.

12. The fusion protein of claim 11, wherein the mutant IFNβ is characterized by a reduced binding affinity to IFNβ receptor.

13. The fusion protein of claim 12, wherein the reduction in binding affinity is by more than 80%.

14. The fusion protein of any one of claims 1-3, wherein the attenuated IFN is a pro-IFN.

15. The fusion protein of claim 14, wherein the attenuated IFN comprises an IFN-a/b receptor (IFNAR) domain fused at one end to the N-terminus of the IFN via a second linker peptide.

16. The fusion protein of claim 15, wherein the IFNAR domain is the extracellular domain (ECD) of natural IFNARl .

17. The fusion protein of claim 15, wherein the IFNAR domain is the extracellular domain (ECD) of natural IFNR2.

18. The fusion protein of any one of claims 2-17, wherein the first linker peptide and/or the second linker peptide is capable of being recognized and hydrolyzed by a proteolytic enzyme specifically expressed in a tumor microenvironment.

19. The fusion protein of claim 18, wherein the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloproteinase (MMP).

20. The fusion protein of claim 19, wherein the MMP is selected from MMPl, MMP3, MMP9, MMPIO, MMP11, MMP12, MMP13 and MMP14.

21. The fusion protein of any one of claims 1-20, wherein the moiety capable of specifically binding to PD-L1 comprises the amino acid sequence set forth in SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 25, 27, 29, 31, 33, 35, 37, 39, 41, 45 or 47.

22. The fusion protein of any one of claims 1-21, wherein the mutant IFN comprises the amino acid sequence set forth in SEQ ID Nos. 17, 19, 21, 23, 49, 51, 53, or 55.

23. The fusion protein of any one of claims 1-22, wherein the first Fc fragment comprises a knob and the second Fc fragment comprises a hole in the respective C_H3 domains.

24. The fusion protein of any one of claims 1-22, wherein the first Fc fragment comprises a hole and the second Fc fragment comprises a knob in the respective C_H3 domains.

25. A prodrug comprising a fusion protein of any one of claims 1-24.

26. A substantially purified fusion protein or prodrug of any one of claims 1-25.

27. A polynucleotide encoding a fusion protein or prodrug of any of claims 1-26.

28. An expression vector comprising the polynucleotide of claim 27.

29. A pharmaceutical composition comprising a fusion protein or prodrug of any one of claims 1-25 and a pharmaceutically acceptable excipient, carrier, or diluent.

30. A method for treating a disease or condition, comprising: administering to a patient in need thereof a therapeutically effective amount of a fusion protein or prodrug of any one of claims 1-25 or a pharmaceutical composition of claim 29, wherein the disease or condition is selected from hyperplasia, solid tumor or hematopoietic malignancy.

31. The method of claim 30, further comprising administering one or more of chemotherapy and radiotherapy on the subject.

32. Use of a fusion protein or prodrug of any one of claims 1-25 for treating or reducing a disease or disorder.

33. Use of a fusion protein or prodrug of any one of claims 1-25 and a pharmaceutically acceptable excipient, carrier, or diluent, in preparation of a medicament for treating or reducing a disease or disorder.

34. The method of claim 30 or 31 or the use of claim 32 or 33, wherein the disease or disorder is selected from acute myeloid leukemia, adrenocortical carcinoma. B-cell lymphoma, bladder urothelial carcinoma, breast ductal carcinoma, breast lobular carcinoma, carcinomas of the esophagus, castration-resistant prostate cancer (CRPC), cervical carcinoma, cholangiocarcinoma, chronic myelogenous leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal carcinoma, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, Hodgkin's lymphoma/primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC), kidney chromophobe carcinoma, kidney clear cell carcinoma, kidney papillary cell carcinoma, lower grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma & pheochromocytoma, prostate adenocarcinoma, renal cell carcinoma (RCC), sarcoma, skin cutaneous melanoma, squamous cell carcinoma of the head and neck, T-cell lymphoma, thymoma, thyroid papillary carcinoma, uterine carcinosarcoma, uterine corpus endometrioid carcinoma and uveal melanoma.

35. The method or use of any one of claims 30-33, in combination with one or more of immune check point blockade, co-signaling of T cells, and tumor targeting antibody therapies.

36. A cell line comprising a polynucleotide encoding a fusion protein or prodrug of any of claims 1-25.

37. A method for making a protein, comprising culturing the cell line of claim 36.

38. The method of claim 37, further comprising purifying or isolating a produced protein.

39. A method for making a fusion protein or prodrug, comprising: providing an expression vector encoding a fusion protein or prodrug of any of claims 1-25; introducing into a host cell the expression vector; culturing the host cell in media under conditions sufficient to express the protein; and purifying the protein from the host cell or media.

40. The method of claim 39, comprising: constructing an expression vector comprising the coding gene encoding a fusion protein or prodrug; constructing the host cell comprising the expression vector by transiently transfection; culturing the host cell; collecting the cell supernatant; and purifying the fusion protein or prodrug.