WO2024193705A1 - Tnf superfamily member immunocytokine and uses thereof - Google Patents

Tnf superfamily member immunocytokine and uses thereof Download PDF

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Publication number
WO2024193705A1
WO2024193705A1 PCT/CN2024/083357 CN2024083357W WO2024193705A1 WO 2024193705 A1 WO2024193705 A1 WO 2024193705A1 CN 2024083357 W CN2024083357 W CN 2024083357W WO 2024193705 A1 WO2024193705 A1 WO 2024193705A1
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seq
amino acid
acid sequence
set forth
heavy chain
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PCT/CN2024/083357
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French (fr)
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Qianting ZHAI
Maiyun YANG
Mingjian FEI
Runzi SUN
Xiaodong Huang
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Analytical Biosciences Shanghai Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present disclosure relates to the technical field of biomedicine or biopharmacy, and relates to antibody-cytokine fusion proteins, in particular, TNF superfamily member fusion proteins (immunocytokines) and uses thereof.
  • antibody-cytokine fusion proteins in particular, TNF superfamily member fusion proteins (immunocytokines) and uses thereof.
  • Cytokines are a class of small proteins (5-20 kDa) that act as cell signaling molecules to regulate inflammation and modulate cellular activities. They belong to different families and can have different functions, such as attracting and stimulating immune cells, inducing antibody production, and promoting lymph node formation. Cytokines are a vast and varied group of pro-inflammatory or anti-inflammatory factors, classified into families based on structural similarities or those of their receptors. Cytokines encompass chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, hormones, growth factors, and others.
  • TNFSF14 TNF superfamily member 14
  • LIGHT homologous to Lymphotoxin, exhibits inducible expression and competes with Herpes Simplex Virus glycoprotein D for Herpes Virus Entry Mediator, a receptor expressed by T cells
  • TNFRSF14 TNF receptor superfamily member 14
  • HVEM TNFRSF14
  • LT ⁇ R lymphotoxin beta receptor
  • DcR3 decoy receptor DcR3
  • HVEM is expressed on the surface of various immune cells, such as T cells, B cells, NKcells, and dendritic cells. LIGHT can bind to HVEM and subsequently stimulate T cells and promote inflammation.
  • TLS lymphoid organs and tertiary lymphoid structures
  • cytokine-based immunotherapy has shown promising efficacies in treating cancer, it suffers from a lack of specificity in targeting tumor tissues, which may result in systemic immune responses and severe side effects.
  • a potential solution is to conjugate cytokines with antibodies that have tumor-targeting specificity. For example, three hmLIGHT copies were fused to the N-terminal of Fc and then assembled with an anti-EGFR half-body (Tang et al., 2016) .
  • LIGHT was conjugated to anti-fibronectin F8 antibodies in a variety of formats. For example, tandem-linked three murine LIGHT copies were fused to the C-terminal of the heavy chain or the light chain of the mAbs or scFv.
  • the present disclosure provides an antibody-cytokine fusion protein platform for tumor-specific cytokine delivery.
  • the platform achieves correct assembly and molecules generated from the platform show robust formation of T cell and B cell zones in the tumor microenvironment and lead to promising antitumor efficacies.
  • a fusion protein including:
  • first cytokine fragment linked to the C-terminal of a first CH2-CH3 fragment including a first cytokine molecule, or a first cytokine molecule and a third cytokine molecule that are tandem linked
  • second cytokine fragment linked to the C-terminal of a second CH2-CH3 fragment including a second cytokine molecule, or a second cytokine molecule and a fourth cytokine molecule that are tandem linked
  • the first CH2-CH3 fragment and the second CH2-CH3 fragment form a dimer, such as a homodimer, or a heterodimer, totally three cytokine molecules are linked to the dimer.
  • the first CH2-CH3 fragment and the second CH2-CH3 fragment are modified to improve the correct assembly of the fusion protein.
  • the first cytokine fragment comprises the first cytokine molecule
  • the second cytokine fragment comprises the second cytokine molecule and the fourth cytokine molecule that are tandem linked.
  • the first cytokine fragment comprises the first cytokine molecule and a third cytokine molecule that are tandem linked
  • the second cytokine fragment comprises the second cytokine molecule.
  • the first antigen-binding fragment is an scFv that specifically recognizes a first antigen
  • the second antigen-binding fragment is an scFv or a Fab that specifically recognizes a second antigen.
  • the first antigen-binding fragment is a Fab that specifically recognizes a first antigen
  • the second antigen-binding fragment is a Fab or an scFv that specifically recognizes a second antigen.
  • the fusion protein provided herein includes a first heavy chain, from the N-terminus to the C-terminus, of which the first antigen-binding fragment, the first CH2-CH3 fragment, and the first cytokine fragment are operably linked; and a second heavy chain, from the N terminus to the C terminus, of which the second antigen-binding fragment, the second CH2-CH3 fragment, and the second cytokine fragment are operably linked.
  • the fusion protein provided herein includes a first light chain, which pairs with the first antigen-binding fragment to form a heterodimer; and/or a second light chain, which pairs with the second antigen-binding fragment to form a heterodimer.
  • an isolated polynucleotide encoding the first heavy chain, the first light chain, the second heavy chain or the second light chain of the fusion protein according to the present disclosure.
  • the present disclosure further provides a set of isolated polynucleotides, including the polynucleotide encoding the first heavy chain, the polynucleotide encoding the first light chain, the polynucleotide encoding the second heavy chain and the polynucleotide encoding the second light chain of the fusion protein according to the present disclosure.
  • an isolated vector including the isolated polynucleotide according to the present disclosure.
  • a host cell line harboring the isolated polynucleotide, the set of isolated polynucleotides or the isolated vector according to the present disclosure.
  • a pharmaceutical composition including the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector or the host cell line according to the present disclosure, and a pharmaceutically acceptable carrier.
  • kits including the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector or the host cell line or the pharmaceutical composition according to the present disclosure.
  • the fusion protein according to the present disclosure or the isolated polynucleotide according to the present disclosure or the set of isolated polynucleotides according to the present disclosure or the isolated vector according to the present disclosure or the host cell line according to the present disclosure or the pharmaceutical composition according to the present disclosure in the manufacture of a drug for preventing or treating a disease, or in the manufacture of a kit for diagnosing a disease.
  • a method of preventing or treating a disease in a subject in need thereof including administrating to the subject a therapeutically effective amount of the fusion protein according to the present disclosure or the isolated polynucleotide according to the present disclosure or the set of isolated polynucleotides according to the present disclosure or the isolated vector according to the present disclosure or the host cell according to the present disclosure or the pharmaceutical composition according to the present disclosure.
  • a method for the production of the fusion protein according to the present disclosure including introducing a first expression vector encoding the first heavy chain, a second expression vector encoding the first light chain, a third expression vector encoding the second heavy chain, and a fourth expression vector encoding the second light chain together into one host cell line, or into separate host cell lines, and expressing the first, second, third and fourth expression vectors under proper conditions.
  • Figure 1 illustrates the different formats of LIGHT-based immunocytokines that have been evaluated.
  • Figure 2 shows the SDS-PAGE analysis of LIGHT-based immunocytokines in Format D. The analysis was performed under reducing (R) and non-reducing (NR) conditions and a protein marker (M) was included for comparison.
  • Figure 3A and 3B show the HPLC analysis of LIGHT-based immunocytokines ABC233 and ABC234 after one-step protein A purification, respectively.
  • Figure 4A and 4B present the schematic format of LIGHT-based immunocytokines and the extended designs of LIGHT-based immunocytokines, respectively.
  • Figure 5 shows the SDS-PAGE analysis of LIGHT-based immunocytokines (A-D) under reducing (R) and non-reducing (NR) conditions, along with the protein marker (M) .
  • Figure 6 shows the SEC-HPLC analysis of LIGHT-based immunocytokines ABC538 and ABC539 after purification.
  • Figure 7 shows the SEC-HPLC analysis of LIGHT-based immunocytokines ABC770-ABC773 after purification.
  • Figures 10A-D show the ELISA analysis of LIGHT-based immunocytokines binding to the human HVEM extracellular domain
  • Figures 10E-H show the ELISA analysis of LIGHT-based immunocytokines binding to the mouse HVEM extracellular domain.
  • Figure 11 shows the ELISA analysis of LIGHT-based immunocytokines (A-F) binding to the human DcR3.
  • Figure 12 illustrates the binding of LIGHT-based immunocytokines (A-G) to 293T cells overexpressing human LT ⁇ R.
  • the mean fluorescence intensity (MFI) and concentration (Conc. ) are indicated.
  • Figure 13 shows the binding of LIGHT-based immunocytokines (A-G) to 293T cells overexpressing mouse LT ⁇ R.
  • MFI mean fluorescence intensity.
  • Figure 14 shows the binding of LIGHT-based immunocytokines (A-D) to 293T cells overexpressing human HVEM.
  • MFI mean fluorescence intensity.
  • Figure 15 shows the binding of LIGHT-based immunocytokines (A-D) to 293T cells overexpressing mouse HVEM.
  • MFI mean fluorescence intensity.
  • Figure 16 illustrates the NF- ⁇ B activities induced by LIGHT-based immunocytokines on HeLa-NF- ⁇ B cells in the absence ( Figures 16A-D) and presence ( Figures 16E-H) of 293T cells overexpressing FAP (293T-FAP) . Relative luciferase activity (RLU) is reported.
  • Figure 17 shows the NF- ⁇ B activities induced by LIGHT-based immunocytokines (A-C) on 293T-NF- ⁇ B cells overexpressing human HVEM. Relative luciferase activity (RLU) is reported.
  • Figure 18 demonstrates the NF- ⁇ B activities induced by LIGHT-based immunocytokines (A-C) on 293T-NF- ⁇ B cells overexpressing mouse HVEM. Relative luciferase activity (RLU) is reported.
  • Figure 19 shows the NF- ⁇ B activities induced by LIGHT-based immunocytokines (A-D) on HeLa-NF- ⁇ B cells in the absence and presence of 293T-FAP cells.
  • Figure 20 demonstrates the impact of DcR3 on the activities of LIGHT-based immunocytokines (A-D) .
  • Figure 21 shows the binding of LIGHT-based immunocytokines to BALB/c-3T3 cells (Figure 21A) and BALB/c-3T3 cells overexpressing FAP ( Figure 21B) .
  • Figure 22 shows the binding of LIGHT-based immunocytokines to primary human CD4 + T cells (Figure 22A) , primary human CD8 + T cells (Figure 22B) , primary mouse CD4 + T cells (Figure 22C) , and primary mouse CD8 + T cells ( Figure 22D) .
  • Figure 23 shows the induction of CCL2 by LIGHT-based immunocytokines in BALB/c-3T3-WT (wild type) ( Figure 23A) , and BALB/c-3T3-FAP cells (Figure 23B) .
  • Figure 23C presents the same results that are grouped by different molecules.
  • Figure 24 shows the transactivation of LT ⁇ R on 3T3 cells in the presence of CT26-FAP or CT26-WT upon addition of ABC233 (Figure 24A) , ABC538 ( Figure 24B) , or ABC539 ( Figure 24C) .
  • Figure 25 demonstrates the in vivo anti-tumor activity of anti-FAP ⁇ LIGHT immunocytokines in the CT26-FAP model, presenting the treatment timeline (Figure 25A) , tumor volume (Figure 25B) , survival curve (Figure 25C) , and the body weight (Figure 25D) .
  • Figure 26 shows the detected ratio of LIGHT to hIgG1 upon immunocytokine administration, which indicates the cleavage that releases LIGHT moieties from the immunocytokines in vivo.
  • Figure 27 demonstrates the in vivo anti-tumor activity of anti-FAP ⁇ LIGHT immunocytokines in the KPC model.
  • Figure 27A outlines the treatment timeline for Example 13;
  • Figure 27B shows individual tumor volumes of each group;
  • Figure 27C displays the average tumor volumes of each group;
  • Figure 27D indicates the tumor growth inhibition (TGI) of each group.
  • Figures 28A-28B show the NF- ⁇ B activities induced by ABC890 and ABC892, respectively, on HeLa-NF- ⁇ B cells in the absence or presence of 293T-FAP cells.
  • Figures 28C-28E show the NF- ⁇ B activities induced by ABC890, ABC892, and ABC538, respectively, on HEK293T-NF- ⁇ B cells overexpressing human HVEM in the absence or presence of 293T-FAP cells.
  • Figure 29 demonstrates HeLa-NF- ⁇ B activities induced by ABC653.
  • Figure 30 shows in vivo anti-tumor activity of ABC653 in the KPC0826 model.
  • fusion refers to connecting a protein with another biological material, such as a protein, a peptide, a nucleic acid molecule, or any other biological molecule or part thereof.
  • protein protein
  • peptide and “polypeptide” are used interchangeably herein to refer to a biological polymer including units derived from amino acids linked by peptide bonds.
  • a protein can be composed of one or more chains.
  • domain refers to a part of a molecule or structure that shares common physical, chemical or structural characteristics, such as similar hydrophobic or polar properties.
  • the exemplary domain may include a protein binding domain, a DNA binding domain, an ATP binding domain, or a similar folding structure such as globular or helical features.
  • the domains can be identified according to their homology with conserved structural or functional motifs.
  • antibody is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies) , full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.
  • the term “antibody” may refer to a full-length antibody or an antigen-binding fragment thereof.
  • a full-length antibody includes two heavy chains and two light chains. The variable regions/domains of the light and heavy chains are responsible for antigen binding.
  • the “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • variable domains of the heavy chain and light chain may be referred to as “VH” and “VL” , respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding sites.
  • VH variable domain of heavy chain/domain/region
  • CH3 constant heavy domains
  • CL constant light
  • the term “antigen-binding fragment” refers to an antibody fragment including a diabody, a Fab, a Fab’ , a F (ab’ ) 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv’ ) , a disulfide stabilized diabody (ds diabody) , a single-chain Fv (scFv) , an scFv dimer (bivalent diabody) , a multi-specific antibody formed from a portion of an antibody including one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not include a complete antibody structure.
  • An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a
  • a Fab fragment corresponds to one of the two identical arms of an antibody molecule and contains the complete light chain paired with the VH and CH1 domains of a heavy chain.
  • “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in a tight, non-covalent association. The folding of these two domains emanates six hypervariable loops (3 loops each from the heavy and light chain) thatprovide a conformation for antigen binding and confer antigen binding specificity to the antibody.
  • single-chain variable fragment is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH: : VL heterodimer.
  • the heavy (VH) and light chains (VL) are either joined directly or by a peptide-encoding linker or spacer, which connects the N-terminal of the VH with the C-terminal of the VL, or the C-terminal of the VH with the N-terminal of the VL.
  • CH2-CH3 fragment refers to a portion of the constant region and at least encompasses CH2 and CH3 domains.
  • two CH2-CH3 fragments can form an antibody Fc domain.
  • Fc domain refers to a pair of associated Fc regions. The two Fc regions dimerize to create the Fc domain. The two Fc regions within the Fc domain can be the same or different from one another.
  • Fc region or “Fc chain” as used herein is meant the polypeptide comprising the CH2-CH3 domains of an IgG molecule, and in some cases, inclusive of the hinge.
  • the CH2-CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to 230.
  • Human IgG Fc regions are of particular use in the present disclosure, and can be the Fc region from human IgG1, IgG2, IgG3 or IgG4.
  • the term “specifically binds” refers to measurable and reproducible interactions, such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules.
  • an antibody that specifically recognizes a target is an antibody that binds this target with greater affinity, avidity, more readily interaction, and/or with greater duration than its bindings to other targets.
  • Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to Octet, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM -tests and peptide scans.
  • linker means a polypeptide used to couple two molecules.
  • the linker typically is a stretch of amino acids, e.g., predominantly glycine and/or serine.
  • the linker may have a length of up to 100 amino acids, such as from about 1, 2, 5, 7, 10, 15 amino acid (s) up to about 15, 20, 25, 30, 35, 50, 75, or 100 amino acids.
  • VH-CH1 linker refers to a polypeptide connecting VH and CH1 in a naturally occurring antibody, and derivatives thereof.
  • VL-CL linker refers to a polypeptide connecting VL and CL in a naturally occurring antibody, and derivatives thereof.
  • CH2-CH3 linker refers to a polypeptide connecting CH2 and CH3 in a naturally occurring antibody, and derivatives thereof.
  • IgM tail linker refers to a polypeptide in a naturally occurring IgM antibody, and derivatives thereof.
  • the term “derivative” refers to a linker with an amino acid sequence that differs from a naturally occurring sequence by one or more amino acids, for example, containing one or more amino acid insertions, deletions, or substitutions relative to a naturally occurring sequence.
  • cytokine is understood to mean any protein, analog or functional fragment thereof, which is capable of stimulating or inducing a biological response against a preselected cell type. Accordingly, it is contemplated that a variety of cytokines can be incorporated into the present application.
  • cytokines include, for example, tumor necrosis factors (TNFs) , interleukins (ILs) , lymphokines (Ls) , colony stimulating factors (CSFs) , interferons (IFNs) , chemokine, growth factors including species variants, truncated analogs thereof which are capable of stimulating or inducing such biological responses, TNF family members such as LIGHT, lymphotoxin ⁇ , lymphotoxin ⁇ , 4-1BBL and the like.
  • TNFs tumor necrosis factors
  • ILs interleukins
  • Ls lymphokines
  • CSFs colony stimulating factors
  • IFNs interferons
  • chemokine growth factors including species variants, truncated analogs thereof which are capable of stimulating or inducing such biological responses
  • TNF family members such as LIGHT, lymphotoxin ⁇ , lymphotoxin ⁇ , 4-1BBL and the like.
  • cytokine is also understood to encompass any derivative of
  • wild-type (WT) refers to a cytokine with an amino acid sequence that is naturally occurring and encoded by a germline genome of a given species.
  • a species can have one or more wild-type sequences (for example, with one canonical wild-type sequence and one or more non-canonical wild-type sequences) .
  • a wild-type cytokine sequence can include a sequence that is truncated at the N and/or the C terminus relative to the sequence encoded by an open reading frame.
  • a wild-type cytokine sequence can be a mature form of a cytokine that has been processed to remove the N-terminal and/or the C-terminal residues.
  • a wild-type cytokine can lack a signal peptide or can include a signal peptide (e.g., a signal peptide can be added to the N-terminus of the wild-type cytokine) .
  • cytokine When describing a cytokine, the term “derivative” “variant” “mutein” and “mutant” are used interchangeably herein to refer to a cytokine with an amino acid sequence that differs from a wild-type sequence by one or more amino acids, for example, containing one or more amino acid insertions, deletions, or substitutions relative to a wild-type sequence.
  • naturally occurring refers to a sequence of natural origin which means that the whole or parts thereof are not synthetic and exist or are produced in nature. More preferably, the term “naturally occurring” as used herein refers to a sequence of natural origin which means that the whole sequence is not synthetic and exists or is produced in nature.
  • LIGHT has its general meaning in the art and refers to a protein transiently expressed on activated T cells, dendritic cells (DCs) , monocytes, natural killer cells (NK) , and others. LIGHT can stimulate HVEM-expressing cells, such as T cells, B cells, NK cells, and dendritic cells, leading to cell activation and inflammation. LIGHT binding to another receptor LT ⁇ R, which is expressed on stromal cells, myeloid cells and some epithelial cells, can lead to the development of lymphoid organs and tertiary lymphoid structures (TLS) . The term “LIGHT” is also understood to encompass WT-LIGHT and any derivative of a WT-LIGHT.
  • knocks-into-holes is used in its broadest sense and encompasses various situations, such as the CH1 domain of one heavy chain with the knob mutations and the CH1 domain of the other heavy chain with the hole mutations, the CH2 domain of one heavy chain with the knob mutations and the CH2 domain of the other heavy chain with the hole mutations, and/or the CH3 domain of one heavy chain with the knob mutations and the CH3 domain of the other heavy chain with the hole mutations.
  • “knobs-into-holes” may refer to an intra-interface modification between two antibody heavy chains in the CH3 domains: i) in the CH3 domain of one heavy chain (first CH3 domain) , an amino acid residue is substituted with another amino acid residue bearing a large side chain, thereby creating a protrusion ( “knob” ) in the interface in the first CH3 domain; ii) in the CH3 domain of the other heavy chain (second CH3 domain) , an amino acid residue is substituted with another amino acid residue bearing a smaller side chain, thereby creating a cavity ( “hole” ) within the interface in the second CH3 domain, in which a protrusion ( “knob” ) in the first CH3 domain can be placed.
  • DDKK refers to a modification mediating electrostatic steering effect that is used to enhance antibody Fc heterodimer formation as inter alia described by Gunasekaran et al., (J. Biol. Chem. 2010, 19637-19646) .
  • the Fc part of one heavy chain includes the mutations K392D and K409D (termed DD mutations) and the Fc part of the other heavy chain includes the mutations E356K and D399K (termed KK mutations) .
  • the numbering is according to the EU index.
  • An “isolated” polynucleotide encoding a protein, or portion or fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced.
  • the isolated polynucleotide is free of association with all components associated with the production environment.
  • the isolated polynucleotide encoding the protein, or portion or fragment thereof described herein is in a form other than in the form or setting in which it is found in nature. Isolated polynucleotides, therefore, are distinguished from polynucleotides encoding the protein, or portion or fragment thereof s described herein existing naturally in cells.
  • An isolated polynucleotide includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extra chromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors. ”
  • host cells or “host cell line” refers to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells.
  • Host cells include “transfected cells” , which include the primary transfected cells and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, and (or) may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transfected cell are included herein.
  • the host cell may be a mammalian cell, plant cell, yeast cell, and (or) a bacterial cell.
  • the host cell may contain the nucleic acid molecule or vector of the present disclosure as an extra-chromosomally (episomal) replicating molecule, or more preferably, includes the nucleic acid molecule or vector of the present disclosure integrated into the genome of the host cell.
  • transfected or “transfection” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” cell is one which has been transfected with exogenous nucleic acids. The cell includes the primary subject cell and its progeny.
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient (s) to be effective, and which contains no additional components which are unacceptably toxic to an individual to which the formulation would be administered.
  • Such formulations may be sterile.
  • a “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together includes a “pharmaceutical composition” for administration to an individual.
  • a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.
  • Cancer and “tumor” are used interchangeably herein to refer to any of several diseases that are characterized by an uncontrolled and abnormal proliferation of cells, the ability of the affected cells to spread locally or through the bloodstream and the lymphatic system to other parts of the body (metastasize) , as well as any of several characteristic structural and/or molecular features.
  • cancers that can be treated using the fusion protein of the present disclosure include solid tumors and hematologic cancers. Additional examples of cancers that can be treated using the fusion protein of the present disclosure include breast, lung, brain, bone, liver, kidney, colon, head and neck, ovarian, hematopoietic (e.g., leukemia) and prostate cancers.
  • cancers that can be treated using multivalent and multi-specific antibodies include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cancer cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, carcinoma of the salivary glands, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma and various types of cancers of the head and neck.
  • Other cancers and tumors that can be treated using the fusion protein of the present disclosure are described herein or are otherwise known in the art.
  • the term “agent” encompasses the fusion protein according to the present disclosure, the isolated polynucleotide according to the present disclosure, the set of isolated polynucleotides according to the present disclosure, the isolated vector according to the present disclosure, the host cell according to the present disclosure, and the pharmaceutical composition according to the present disclosure.
  • an “effective amount” of an agent of the present disclosure is an amount sufficient to achieve a specifically indicated purpose in order to cause an observable change in the level of one or more biological activities related to the target cell to which the fusion protein binds.
  • the change can increase the activity level of the target.
  • the change can reduce the activity level of the target.
  • An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
  • therapeutically effective amount refers to an amount of an agent of the present disclosure or another drug effective to “treat” a disease or disorder in a patient or mammal.
  • the therapeutically effective amount of the agent of the present disclosure can reduce angiogenesis and neovascularization; reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to some extent or stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow down to a certain point or stop) tumor metastasis; inhibit, to some extent, tumor growth or the incidence of tumors; stimulate immune responses against cancer cells and/or alleviate to some extent one or more of the symptoms associated with cancer. See the definition in this document of “treat.
  • a “therapeutically effective amount” may also refer to an effective amount, at the dosages and for the periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of the agent of the present disclosure may vary depending on factors such as the pathology, age, sex and weight of the individual and the ability of the composition to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which the toxic or detrimental effects of the therapeutic composition are overcome by the therapeutically beneficial effects.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival.
  • treatment is a reduction of pathological consequences of cancer (such as, for example, tumor volume) .
  • the methods of the application contemplate any one or more of these aspects of
  • treating includes any or all of the inhibiting growth of cancer cells, inhibiting replication of cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease.
  • inhibitors refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic.
  • To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to that of a reference.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20%or greater.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
  • the term “preventing” includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual that may be predisposed to the disease but has not yet been diagnosed with the disease.
  • the present disclosure provides the immunocytokines, which are antibody-cytokine fusion proteins.
  • the fusion protein includes a first cytokine fragment and a second cytokine fragment, wherein the first cytokine fragment and the second cytokine fragment are linked to the C-terminal of a first CH2-CH3 fragment and a second CH2-CH3 fragment, respectively.
  • the first CH2-CH3 fragment and the second CH2-CH3 fragment form a dimer, such as a homodimer or a heterodimer.
  • the first CH2-CH3 fragment and the second CH2-CH3 fragment form an Fc domain.
  • the Fc domain is derived from IgG, such as human IgG molecules, which encompass IgG1, IgG2, IgG3, and IgG4 subclass.
  • the first CH2-CH3 fragment and the second CH2-CH3 fragment form a human IgG1 Fc domain or IgG4 Fc domain.
  • the first CH2-CH3 fragment and the second CH2-CH3 fragment form a human IgG1 Fc with mutation N297A, which refers to the amino acid N (Asparagine) at position 297 numbering by EU numbering is mutated to amino acid (Alanine) .
  • mutations like this have a similar instruction.
  • the amino acid in the present disclosure refers to an organic compound containing amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain specific to each amino acid.
  • the amino acid in the present disclosure is natural amino acids, such as Alanine (A) , Cysteine (C) , Aspartic Acid (D) , Glutamic Acid (E) , Phenylalanine (F) , Glycine (G) , Histidine (H) , Isoleucine (I) , Lysine (K) , Leucine (L) , Methionine (M) , Asparagine (N) , Proline (P) , Glutamine (Q) , Arginine (R) , Serine (S) , Threonine (T) , Valine (V) , Tryptophan (W) , Tyrosine (Y) .
  • the Fc domain is modified to improve the correct assembly of the fusion protein when needed.
  • the first CH2-CH3 fragment and the second CH2-CH3 fragment include one or more modifications selected from the group consisting of knobs-into-holes, electrostatic steering of CH3 (e.g., DDKK) , DuoBody, SEEDbodies, cFAE, XmAb, Azymetric, and
  • the first CH2-CH3 fragment and the second CH2-CH3 fragment include one or more modifications selected from the group consisting of knobs-into-holes, and DDKK.
  • the first CH2-CH3 fragment includes knob mutations
  • the second CH2-CH3 fragment includes hole mutations
  • the first CH2-CH3 fragment includes hole mutations
  • the second CH2-CH3 fragment includes knob mutations
  • the first CH2-CH3 fragment includes DD mutations, and the second CH2-CH3 fragment includes the KK mutations. In some embodiments, the first CH2-CH3 fragment includes the KK mutations, and the second CH2-CH3 fragment includes the mutations DD mutations.
  • one of the first CH2-CH3 fragment and the second CH2-CH3 fragment includes one or more mutations selected from the group consisting of Y349C, T366S, L368A, and Y407V; and the other one includes mutations S354C and/or T366W, defined by EU numbering.
  • the first CH2-CH3 fragment includes mutations Y349C, T366S, L368A, and Y407V to form a “hole”
  • the second CH2-CH3 fragment includes mutations S354C and T366W to form a “knob”
  • the first CH2-CH3 fragment includes mutations S354C and T366W to form a “knob”
  • the second CH2-CH3 fragment includes mutations Y349C, T366S, L368A, and Y407V to form a “hole” .
  • the first cytokine fragment includes at least one wild type, truncated or mutated cytokine molecule. In some embodiments, the first cytokine fragment includes one or two wild type, truncated or mutated cytokine molecules. In some embodiments, the second cytokine fragment includes at least one wild type, truncated or mutated cytokine molecule. In some embodiments, the second cytokine fragment includes one or two wild type, truncated or mutated cytokine molecules.
  • the first cytokine fragment includes a first cytokine molecule. In some embodiments, the second cytokine fragment includes a second cytokine molecule. In some embodiments, the first cytokine fragment includes a tandem linked first cytokine molecule and a third cytokine molecule. In some embodiments, the second cytokine fragment includes a tandem linked second cytokine molecule and a fourth cytokine molecule.
  • the first cytokine fragment includes the first cytokine molecule
  • the second cytokine fragment includes the second cytokine molecule and the fourth cytokine molecule that are tandem linked.
  • the first cytokine fragment includes the first cytokine molecule and a third cytokine molecule that are tandem linked
  • the second cytokine fragment includes the second cytokine molecule.
  • the cytokine molecule (s) in the first cytokine fragment and the cytokine molecule (s) in the second cytokine fragment are the same. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the third cytokine molecule are the same. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the fourth cytokine molecule are the same.
  • the cytokine molecule (s) in the first cytokine fragment and the cytokine molecule (s) in the second cytokine fragment are different from each other. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the third cytokine molecule are different from each other. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the fourth cytokine molecule are different from each other.
  • the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other are selected from the group consisting of tumor necrosis factor, interleukin, lymphokine, interferon, colony stimulating factor, chemokine and growth factor.
  • the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other are selected from the group consisting of LIGHT, lymphotoxin ⁇ , lymphotoxin ⁇ , and 4-1BBL, for example, wild type LIGHT (preferably human LIGHT) , truncated LIGHT or mutein thereof.
  • the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other are truncated LIGHT (i.e., the amino acid sequence is short than wild type) .
  • Exemplary truncated LIGHT is WT-LIGHT (74-240) (SEQ ID NO: 68) , WT-LIGHT (91-240) (SEQ ID NO: 78) .
  • the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other include a truncated LIGHT amino acid sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 78.
  • the first cytokine molecule, second cytokine molecule and the third cytokine include or have amino acid sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 78. In some embodiments, the first cytokine molecule, second cytokine molecule and the fourth cytokine include or have amino acid sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 78.
  • the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other are LIGHT mutein.
  • Exemplary LIGHT mutein are LIGHT MUTEIN-1 (74-240) , LIGHT MUTEIN-2 (74-240) , LIGHT MUTEIN-9 (74-240) , LIGHT MUTEIN-11 (74-240) , LIGHT MUTEIN-18 (74-240) , LIGHT MUTEIN-22 (74-240) , LIGHT MUTEIN-29 (74-240) , LIGHT MUTEIN-37 (74-240) , LIGHT MUTEIN-52 (74-240) , (SEQ ID NOs: 69-77) ; LIGHT MUTEIN-1 (59-240) , LIGHT MUTEIN-1 (91-240) , LIGHT MUTEIN-1 (87-240) , LIGHT MUTEIN-90 (74-240) , LIGHT MUTEIN-63 (74-240)
  • the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other comprise a mutated LIGHT amino acid sequence set forth in SEQ ID NO: 25, 26, 58, 59, 69-77, 126-141, 158, 177, 178 and 180.
  • the first cytokine molecule, the second cytokine molecule and the third cytokine include or have amino acid sequence set forth in SEQ ID NO: 25, 26, 58, 59, 69-77, 126-141, 158, 177, 178 and 180.
  • the first cytokine molecule, second cytokine molecule and the fourth cytokine include or have amino acid sequence set forth in SEQ ID NO: 25, 26, 58, 59, 69-77, 126-141, 158, 177, 178 and 180.
  • the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are wild type 4-1BBL (preferably human 4-1BBL) , truncated 4-1BBL or mutein thereof.
  • the wild type 4-1BBL, truncated 4-1BBL or mutein thereof includes amino acid sequence of SEQ ID NO: 179.
  • the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are wild, truncated lymphotoxin ⁇ or lymphotoxin ⁇ , or mutein thereof.
  • the wild, truncated lymphotoxin ⁇ or lymphotoxin ⁇ , or mutein thereof includes amino acid sequence of SEQ ID NO: 181, 182.
  • first cytokine molecule, the second cytokine molecule, and the third cytokine molecule have the same amino acid sequence. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the fourth cytokine molecule have the same amino acid sequence.
  • the first cytokine fragment links to the C-terminal of the first CH2-CH3 fragment directly or via a linker.
  • the second cytokine fragment link to the C-terminal of the second CH2-CH3 fragment directly or via a linker.
  • the C terminal of the CH3 domain in the first CH2-CH3 fragment is fused to the N terminal of a first cytokine fragment directly or via a first linker A
  • the C terminal of the CH3 domain in the second CH2-CH3 fragment is fused to N terminal of a second cytokine fragment directly or via a first linker B.
  • the first cytokine molecule is fused to a third cytokine molecule directly or via a second linker A.
  • the second cytokine molecule is fused to a fourth cytokine molecule directly or via a second linker B.
  • the C terminal of the first cytokine molecule is fused to the N terminal of a third cytokine molecule directly or via a second linker A. In some embodiments, the C terminal of the second cytokine molecule is fused to the N terminal of a fourth cytokine molecule directly or via a second linker B.
  • first linker A and the first linker B independently of each other are absent or include the amino acid sequence as set forth in any one of SEQ ID NOs: 81-90. In some embodiments, the first linker A and the first linker B independently of each other are absent or have the amino acid sequence as set forth in any one of SEQ ID NOs: 81-90.
  • first linker A and the first linker B are the same. In some embodiments, the first linker A and the first linker B are different.
  • the second linker A and the second linker B independently of each other are absent or include the amino acid sequence of G or the amino acid sequence as set forth in any one of SEQ ID NOs: 85-88. In some embodiments, the second linker A and the second linker B independently of each other are absent or have the amino acid sequence of G or the amino acid sequence as set forth in any one of SEQ ID NOs: 85-89.
  • the fusion protein provided herein includes a first antigen-binding fragment and a second antigen-binding fragment, which are connected to N terminal of the first CH2-CH3 fragment and the second CH2-CH3 fragment, respectively.
  • the first antigen-binding fragment and the second antigen-binding fragment independently of each other are selected from the group consisting of a diabody, a Fab, a Fab’ , a F (ab’ ) 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv’ ) , a disulfide stabilized diabody (ds diabody) , a single-chain Fv (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody formed from a portion of an antibody including one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment.
  • the first antigen-binding fragment is an scFv that specifically binds to a first antigen.
  • the second antigen-binding fragment is an scFv that specifically binds to a second antigen.
  • the scFv includes a heavy chain variable region (VH) and a light chain variable region (VL) which can pair with each other to form an Fv fragment that specifically binds to an antigen.
  • VH heavy chain variable region
  • VL light chain variable region
  • the N-terminal of the VH is linked to the C-terminal of the VL directly or via a linker
  • the C-terminal of the VH is linked to the N-terminal of the VL directly or via a linker.
  • the linker between the VH and the VL is (GGGGS) 4 (SEQ ID NO: 88) .
  • the first antigen-binding fragment is a Fab that specifically binds to a first antigen.
  • the second antigen-binding fragment is a Fab that specifically binds to a second antigen.
  • the first antigen-binding fragment is an scFv that specifically binds to a first antigen; and the second antigen-binding fragment is an scFv or a Fab that specifically binds to a second antigen.
  • the first antigen-binding fragment is a Fab that specifically binds to a first antigen; and the second antigen-binding fragment is a Fab or an scFv that specifically binds to a second antigen.
  • the first antigen-binding fragment is an scFv or a Fab that specifically binds to a first antigen; and the second antigen-binding fragment is a Fab that specifically binds to a second antigen.
  • the first antigen-binding fragment and the second antigen-binding fragment are Fab. In some embodiments, the first antigen-binding fragment is an scFv and the second antigen-binding fragment is Fab. In some embodiments, the first antigen-binding fragment is a Fab and the second antigen-binding fragment is an scFv.
  • the first antigen and the second antigen independently of each other are selected from the group consisting of FAP, HER2, PDL-1, PD-1, EGFR, VEGFR, VEGF, CCR8, OX-40, 41BB, Angiopoietin-2, IL-4R ⁇ , BCMA, Blys, BTNO2, C5, CD122, CD13, CD133, CD137, CD138, CD16a, CD19, CD20, CD22, CD27, CD28, CD3, CD30, CD33, CD38, CD40, CD47, CD-8, CEA, CGPR/CGRPR, CSPGs, CTLA4, CTLA-4, DLL-4, EpCAM, factor IXa, factor X, GITR, GP130, Her3, HSG, ICOS, IGF1, IGF1/2, IGF-1R, IGF2, IGFR, IL-1, IL-12, IL-12p40, IL-13, IL-17A, IL-1 ⁇ , IL-23, IL
  • the first antigen and the second antigen are different antigens or different epitopes on the same antigen. In some embodiments, the first antigen and the second antigen are the same or the same epitope on the same antigen.
  • the fusion protein herein is monovalent or bivalent, monospecific or bispecific.
  • both of the first antigen and the second antigen are FAP, or both of the first antigen and the second antigen are HER2, or both of the first antigen and the second antigen are EGFR, or both of the first antigen and the second antigen are PDL-1, or the first antigen is HER2 and the second antigen are FAP.
  • the fusion protein provided herein includes: a first heavy chain, from the N terminal to the C terminal, including the operably linked first antigen-binding fragment, the first CH2-CH3 fragment, and the first cytokine fragment; and a second heavy chain, from the N terminal to the C terminal, including the operably linked second antigen-binding fragment, the second CH2-CH3 fragment, and the second cytokine fragment.
  • the fusion protein is with the “Y” shape.
  • the first heavy chain used herein is referred to the heavy chain of the left arm of “Y” shape, and the second heavy chain used herein is referred to the heavy chain of the right arm of “Y” shape.
  • first heavy chain and the second heavy chain are the same. In some embodiments, the first heavy chain and the second heavy chain are different.
  • the fusion protein provided herein includes a first light chain, which pairs with the first antigen-binding fragment to form a heterodimer. In some embodiments, the fusion protein provided herein includes a second light chain, which pairs with the second antigen-binding fragment to form a heterodimer.
  • first light chain and the second light chain are the same, under which circumstance they are collectively referred to as “light chain” . In some embodiments, the first light chain and the second light chain are different.
  • the first antigen-binding fragment is an scFv, in this case, the first light chain is absent.
  • the second antigen-binding fragment is an scFv, in this case, the second light chain is absent.
  • the first light chain and the second light chain independently of each other include the amino acid sequence as set forth in any one of SEQ ID NOs: 1, 29, 91 and 94. In some embodiments, the first light chain and the second light chain independently of each other have the amino acid sequence as set forth in any one of SEQ ID NOs: 1, 29, 91 and 94. In some embodiments, both the first light chain and the second light chain have the amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, both the first light chain and the second light chain have the amino acid sequence as set forth in SEQ ID NO: 29. In some embodiments, both the first light chain and the second light chain have the amino acid sequence as set forth in SEQ ID NO: 91.
  • both the first light chain and the second light chain have the amino acid sequence as set forth in SEQ ID NO: 94.
  • the first light chain has the amino acid sequence as set forth in SEQ ID NO: 1
  • the second light chain has the amino acid sequence as set forth in SEQ ID NO: 29.
  • the second light chain has the amino acid sequence as set forth in SEQ ID NO: 1
  • the first light chain has the amino acid sequence as set forth in SEQ ID NO: 29.
  • the first light chain has the amino acid sequence as set forth in SEQ ID NO: 1
  • the second light chain has the amino acid sequence as set forth in SEQ ID NO: 91.
  • the second light chain has the amino acid sequence as set forth in SEQ ID NO: 1, and the first light chain has the amino acid sequence as set forth in SEQ ID NO: 91.
  • the first light chain has the amino acid sequence as set forth in SEQ ID NO: 1
  • the second light chain has the amino acid sequence as set forth in SEQ ID NO: 94.
  • the second light chain has the amino acid sequence as set forth in SEQ ID NO: 1, and the first light chain has the amino acid sequence as set forth in SEQ ID NO: 94.
  • the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 24, 27, 30, 33, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 60, 62, 64, 66, 92, 95, 97, 101, 103, 105, 106, 108, 110, 112, 115, 116, 118, 120, 122, 124, 142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163, 165, 167, 169, 171, 173 or 175.
  • the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 23, 28, 31, 32, 35, 37, 39, 41, 43, 45, 47, 49, 51, 52, 53, 55, 57, 61, 63, 65, 67, 93, 96, 98, 99, 100, 102, 104, 107, 109, 111, 113, 114, 117, 119, 121, 123, 125, 143, 145, 147, 149, 151, 153, 157, 155, 160, 162, 164, 166, 168, 170, 172, 174 or 176.
  • the fusion protein is select from the following:
  • both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 3, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 3; (2) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 4, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 2; (3) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 6, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 5; (4) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or
  • the present disclosure provides an isolated polynucleotide encoding the first heavy chain, the first light chain, the second heavy chain or the second light chain of the fusion protein according to the present disclosure.
  • the isolated polynucleotide encodes the first heavy chain of the fusion protein according to the present disclosure.
  • the isolated polynucleotide encodes the second heavy chain of the fusion protein according to the present disclosure.
  • the present disclosure provides a set of isolated polynucleotides, including the polynucleotide encoding the first heavy chain, the polynucleotide encoding the first light chain, the polynucleotide encoding the second heavy chain and the polynucleotide encoding the second light chain of the fusion protein according to the present disclosure.
  • the polynucleotide is polymers of DNA, RNA, DNA/RNA hybrids, or modifications thereof. In some embodiments, the polynucleotide is polymers of DNA.
  • the polynucleotide is polymers of RNA.
  • DNA or RNA encoding an anti-SIRP ⁇ antibody or antigen binding fragment thereof or a bi-specific molecule described above is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) .
  • the encoding DNA or RNA may also be obtained by synthetic methods.
  • the present disclosure provides an isolated vector including the isolated polynucleotide.
  • the present disclosure further provides a host cell including the isolated polynucleotide, the set of insolated polynucleotides or the isolated vector according to the present disclosure.
  • the present disclosure further provides a method for the production of the fusion protein according to the present disclosure, including introducing a first expression vector encoding the first heavy chain, a second expression vector encoding the first light chain, a third expression vector encoding the second heavy chain and a fourth expression vector encoding the second light chain together into one host cell, or into separate host cells, and expressing the first, second, third and fourth expression vectors under a proper condition.
  • the host cell includes prokaryotic cell and eukaryotic cell.
  • the host cells are eukaryotic cells.
  • the host cells are mammalian cells, such as CHO, COS, HEK293 cells, or Bowes melanoma cells, or a combination thereof.
  • the present disclosure provides a pharmaceutical composition including the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector or the host cell according to the present disclosure, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be administered in a suitable manner according to the specific applicable form, physicochemical characteristics, etc. of the pharmaceutically acceptable excipient.
  • the pharmaceutical composition may be formulated in a form of a freeze-dried formulation or a liquid formulation, which may contain appropriate formulation additives in the art.
  • the above pharmaceutical composition typically contains more than one pharmaceutical carrier, for example, sterile liquid such as water and oil (including petroleum, oils of animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, and sesame oil, etc. ) .
  • water is a more representative carrier.
  • saline solution aqueous glucose, and glycerol solution may also be used as a liquid carrier, especially for an injectable solution.
  • Suitable pharmaceutical excipients are known in the art.
  • the above pharmaceutical composition may also contain a trace amount of wetting agent, emulsifier, or pH buffering agent as required.
  • the administration mode of the pharmaceutical composition is usually parenteral administration, which can be intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous injection, but is not limited thereto, for example, the pharmaceutical composition may be administered by infusion or bolus injection. See, e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A. H.
  • Kibbe (Pharmaceutical Press, London, UK, 2000) , which is incorporated by reference in its entirety. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) , which is incorporated by reference in its entirety.
  • the active agent can be administered to the subject via any suitable route of administration.
  • the active agent can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration.
  • routes of administration is merely provided to illustrate various embodiments and should not be construed as limiting the scope in any way.
  • kits including the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector, the host cell or the pharmaceutical composition described above.
  • kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers etc., as will be readily apparent to a person skilled in the art.
  • Instructions, either as inserts or a labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
  • the present disclosure provides use of the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector, the host cell or the pharmaceutical composition according to the present disclosure in the manufacture of a drug for preventing or treating a disease, or in the manufacture of a kit for diagnosing a disease.
  • the present disclosure provides a method of preventing or treating a disease in a subject in need thereof, including administrating to the subject a therapeutically effective amount of the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector, the host cell or the pharmaceutical composition according to the present disclosure.
  • subject , “individual, ” and “patient” are used interchangeably herein to refer to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.
  • the disease includes tumor or cancer.
  • the tumor is a solid tumor.
  • LIGHT-based immunocytokine (antibody-LIGHT fusion proteins) formats were developed for tumor-specific delivery. The designs incorporated the extracellular domain (residues 59-240, residues 74-240, residues 87-240 and residue 91-240) of wild-type LIGHT (Uniprot, #O43557) and an anti-FAP antibody clone 3F2 (patent No. WO 2012020006A) . Some formats linked three LIGHT subunits with polypeptide linkers and connected to the N-terminal or the C-terminal of the heavy chain or light chain of the antibody (Format A, E) .
  • Some formats fused two subunits of LIGHT to the N-terminal or the C-terminal of the heavy chain or the light chain connected with “G” or “GGGGS” linker (s) , with the remaining LIGHT subunit connected to the corresponding site on the opposite chain (Format B, C, D) .
  • Format C the heavy chain CH1 and light chain CL were switched.
  • the codon-optimized genes for LIGHT-based immunocytokines were synthesized by Genewiz and subcloned into the pCI vector (Promega #E1731) and co-transfected into Expi293F cells (ThermoFisher A14527) for protein production.
  • the cells were grown in suspension culture for about six days after transfection and then harvested by centrifugation (7, 000 RPM) at 4°C for 20 minutes. The supernatant was filtered through a 0.22 ⁇ m filter, and affinity-purified using protein A resins.
  • the protein was then eluted by elution buffer (1 M Glycine pH 3.8, 10%glycerol) and neutralized to pH 6.0 with 1M Tris (pH 7.5) .
  • the antibody-LIGHT fusion proteins were subjected to electrophoresis on 4-20%Tris-glycine gels (Bio-Rad) under reducing (R) and non-reducing (NR) conditions, by mixing with sample dye with or without DTT (Dithiothreitol) respectively and heated at 95 °C for 5 min.
  • the purity of the antibody-LIGHT fusion proteins was further characterized by High Performance Liquid Chromatography (HPLC) .
  • Anti-FAP monoclonal antibody ABC002 had the light chain as shown in SEQ ID NO: 79 and the heavy chain as shown in SEQ ID NO: 80.
  • Example 3 Binding affinities of LIGHT-based immunocytokines to LT ⁇ R, HVEM and DcR3 proteins
  • LIGHT binding to its cell surface receptor LT ⁇ R leads to downstream NF-kB signaling.
  • LIGHT-based immunocytokines to the human-LT ⁇ R-Fc (Novoprotein #CX78) and mouse-LT ⁇ R-Fc proteins (Acrobiosystems #LTR-H5251) .
  • 96-well ELISA plates (Thermo Fisher) were coated with the LT ⁇ R proteins at a concentration of 0.5 ⁇ g/mL, and then blocked with 2%BSA-PBS buffer for one hour. Serial dilutions of the LIGHT-based immunocytokines were added with a maximum concentration of 10 nM.
  • the plates were incubated for one hour, washed for four times with PBST, and then incubated with a biotinylated mouse anti-human Ig light chain ⁇ antibody (Biolegend, #316504) .
  • HRP-conjugated streptavidin (Sino Biological) was added, and the plates were further incubated before being washed for three times with PBST and treated with TMB substrate (#34029, Thermo Fisher) .
  • the plates were read at 450 nM on a SpectraMax M5 microplate reader (Molecule Devices) .
  • the ELISA results were analyzed using GraphPad Prism 9.0 software and the EC50s (half maximal effective concentration) were summarized in Table 1 and Table 2.
  • Table 1 EC50s of LIGHT-based immunocytokines binding to the human LT ⁇ R.
  • the LIGHT-based immunocytokines show specific dose-dependent binding to both human LT ⁇ R and mouse LT ⁇ R proteins, as shown in Figures 8A-8E and Figures 9A-9E.
  • Fc heterodimerization technologies such as knobs-into-holes and electronic steering, can facilitate the production and boost activities of LIGHT-based immunocytokine.
  • the holes-LIGHT/knobs-LIGHT-LIGHT and knobs-LIGHT/holes-LIGHT-LIGHT (knobs and holes exchange) designs show similar affinities to LT ⁇ R.
  • the results suggest that the binding affinities to LT ⁇ R were comparable across different linker-1 and linker-2 options, including no linker, G4S, (G4S) 2, (G4S) 3, (G4S) 4, GGGGSGGGGSGGGGSGGGGT, ISKAKGQPREPQ, ASTKGPSVFPLAPS, RTVAAPSVFIFPPS, and KSTGKPTLYNVSLVMSDTAGTCY. Additionally, the binding can be fine-tuned by using different linker-1 and linker-2 options.
  • Binding affinities to HVEM and DcR3 protein measured by ELISA Binding affinities to HVEM and DcR3 protein measured by ELISA
  • Recombinant HVEM proteins were produced by fusing the C-terminal of human HVEM (referred to as hHVEM) residues 39-202 or mouse HVEM (referred to as mHVEM) residues 38-207 with human Fc, while the recombinant DcR3 protein was generated by linking human DcR3 residues 33-300 to the N-terminal of rabbit Fc.
  • the recombinant proteins were expressed in expi293F and affinity-purified using protein A resins and polished using size exclusion column.
  • Human HVEM, mouse HVEM or human DcR3 proteins were immobilized onto maxiSorp 96-well ELISA plates (Thermo Fisher) at a concentration of 0.5 ⁇ g/mL and then blocked with 2%BSA-PBS buffer for one hour.
  • the LIGHT-based immunocytokines were added to the plates at various dilutions, with a maximum concentration of 10 nM.
  • the plates were incubated for an hour, washed four times with PBST, and further incubated with a biotinylated mouse anti-human Ig light chain ⁇ antibody (Biolegend, #316504) .
  • LIGHT-based immunocytokines The binding of LIGHT-based immunocytokines to the other two LIGHT receptors, HVEM and DcR3, was also evaluated. As described in patent application PCT/CN2022/097735, compared to wild-type LIGHT, the LIGHT-1 (SEQ ID NO: 69) homotrimer, and the LIGHT-2 (SEQ ID NO: 70) homotrimer have lower affinities with HVEM. Consistently, the anti-FAP ⁇ LIGHT-1 (ABC538) and the anti-FAP ⁇ LIGHT-2 (ABC539) immunocytokines also displayed reduced binding capabilities to HVEM ( Figure 10B and 10F) .
  • DcR3 is a soluble protein expressed in humans and cynomolgus macaques but is absent in mice. DcR3 negatively regulates the abilities of LIGHT to activate LT ⁇ R and HVEM (Liu et al., 2021) and is upregulated in certain autoinflammatory diseases and cancer patients (Fuchsberger et al., 2021) . Consistent with the lower binding affinities of DcR3 to LIGHT-1 or LIGHT-2 than wild-type LIGHT, DcR3 had significantly weaker binding to ABC538 and ABC539 than to ABC233 ( Figures 11A-11F) .
  • Full-length human or mouse HVEM or LT ⁇ R (listed in Table 4) was transfected into HEK-293T cells (ATCC) using lipofectamine 3000 (Thermo Fisher, L3000001) . Three days after transfection, the cells were treated with hygromycin B (Millipore Sigma) for fourteen days to obtain stable cell lines. The stable cells were used to evaluate the binding of LIGHT-based immunocytokines to HVEM or LT ⁇ R.
  • the stably transfected HEK-293T cells expressing LT ⁇ R or HVEM were used to evaluate the binding activities of LIGHT-based immunocytokines.
  • the cells were incubated with serially diluted fusion proteins for 30 mins on ice, with a maximum concentration of 50 nM in flow cytometry buffer (PBS, 0.5%BSA, 1 mM EDTA) .
  • the cells were then washed three times with PBS, and the bound immunocytokines were stained with FITC-conjugated mouse anti-human IgG antibody for 15 minutes on ice.
  • Table 5 EC50s of LIGHT-based immunocytokines binding to 293T cells expressing human LT ⁇ R
  • Table 7 EC50s of LIGHT-based immunocytokines binding to 293T cells expressing human or mouse HVEM
  • LIGHT-based immunocytokines exhibited dose-dependent binding to cells overexpressing human or mouse LT ⁇ R ( Figures 12A-12G and Figures 13A-13G) . Additionally, the binding affinities of these immunocytokines to cells expressing HVEM were dominated by the LIGHT mutein conjugated to the antibodies ( Figures 14A-14D and Figures 15A-15D) .
  • Some LIGHT-based immunocytokines, such as ABC477 and ABC543 exhibited similar binding to human or mouse HVEM-expressing cells as that of the wild-type immunocytokine (ABC233) , while others, such as ABC539, had minimal binding to both types of cells.
  • Example 5 Pathway activation of anti-FAP ⁇ LIGHT immunocytokines in the absence or presence of FAP-expressing cells in trans
  • the 293T-FAP cell line was generated by transfecting full-length human FAP (Table 4) into HEK-293T cells using lipofectamine 3000 following standard protocols. After three days, the cells were treated with hygromycin (Millipore Sigma) for 14 days to generate stable cell lines.
  • HeLa-NF-kB and HEK-293T-NF-kB cells were generated by transfecting the cells with the pNL3.2.
  • NF- ⁇ B-RE [NlucP/NF- ⁇ B-RE/Hygro] vector Promega #N1111
  • the cells were treated with hygromycin B (Sigma) and cultured for 14 days in 37°C, 5%CO 2 .
  • the resulting HeLa-NF- ⁇ B reporter cells were used to evaluate the downstream signaling of LT ⁇ R activation by the treatment of LIGHT-based immunocytokines.
  • the HEK293T-NF- ⁇ B cells were further transfected with full-length human or mouse HVEM (listed in Table 4) using lipofectamine 3000. After fourteen-day incubation in the presence of hygromycin B and puromycin (Millipore Sigma) , the resulting HEK293T-HVEM-NF- ⁇ B stable cells were collected for further analysis.
  • the reporter cells were either cultured alone or co-cultured with 293T-FAP cells and treated with indicated molecules. Twenty-four hours later, cells were lysed with lysis buffer (Promega #E397A) , and luciferase activity was measured (Promega #E4500) using SpectraMax M5 microplate reader (Molecule Devices) .
  • LIGHT-based immunocytokines were able to activate NF- ⁇ B signaling in HeLa cells harboring endogenous LT ⁇ R ( Figures 16A-16H and Table 8) .
  • Immunocytokines equipped with the same LIGHT mutein also showed similar capabilities to activate LT ⁇ R.
  • Coculturing with 293T-FAP cells enhanced LT ⁇ R activation for most immunocytokines (Table 8) , except for ABC543 ( Figures 19A-19D) .
  • ABC538 and ABC539 had weaker induction capabilities for NF- ⁇ B signaling than ABC233 with the wild-type LIGHT ( Figures 16A-16D, 19 and 28A-28B, Table 8) .
  • Table 9 EC50s of the LIGHT-based immunocytokines to activate HEK293T-HVEM-NF- ⁇ B cells.
  • DcR3 decoy receptor 3
  • HVEM and LT ⁇ R LIGHT also interacts with a decoy receptor, DcR3, which lacks the transmembrane and cytoplasmic segments. This interaction has the potential to disrupt the signaling pathways by sequestering LIGHT away from HVEM and LTbR. While DcR3 expression is typically low in healthy human tissues, it is often significantly upregulated in cancer patients (Wu et al., 2003; Yoo et al., 2022) .
  • Example 7 Binding of anti-FAP ⁇ LIGHT immunocytokines to endogenous mouse LT ⁇ R in the presence or absence of coexpressed FAP
  • BALB/c-3T3-FAP LIGHT muteins to activate endogenous mouse LT ⁇ R.
  • BALB/c-3T3-FAP cell lines were generated by transfecting BALB/c-3T3 cells (ATCC) with mouse Fap (referred to as mFAP) and screened for mFAP expression.
  • the immunocytokines were incubated with 0.1 million purified cells for 30 mins on ice in FACS buffer (PBS, 0.5%BSA, and 1 mM EDTA) .
  • Human and mouse T cells were utilized to evaluate the interaction between anti-FAP ⁇ LIGHT immunocytokines and h/mHVEM.
  • the T cells were negative for LT ⁇ R and FAP but positive for HVEM.
  • Human CD3 + T cells were purified from human PBMC with a purity above 97%through negative selection following the manufacturer's protocol (480134, Biolegend) .
  • Mouse CD3 + T cells were purified from pooled splenocytes of BALB/c mice with a purity above 97%by negative selection following the manufacturer's protocol (480031, Biolegend) .
  • the cells were incubated with twelve 3-fold serial dilutions of LIGHT-based immunocytokines on ice for 30mins in FACS buffer (PBS, 0.5%BSA, and 1 mM EDTA) .
  • FACS buffer PBS, 0.5%BSA, and 1 mM EDTA
  • the cells were washed with FACS buffer for 3 times, and live/dead cell discrimination was performed using the Live/Dead Fixable Near-IR Dead Cell Stain Kit (Life Technologies) .
  • human T cells were stained with either anti-hCD4 (RPA-T4, Biolegend) or anti-hCD8a (RPA-T8, Biolegend) along with 1 ⁇ g/mL goat anti-human IgG Fc (FITC) (ab97224, Abcam) on ice for 30 mins in FACS buffer.
  • Mouse T cells were stained with either anti-mCD4 (RM4-5, Biolegend) or anti-mCD8a (53-6.7, Biolegend) along with 1 ⁇ g/mL goat anti-human IgG Fc (FITC) (ab97224, Abcam) on ice for 30 mins in FACS buffer. Cells were then washed for three times with FACS buffer, and the cytometry data was collected using a Cytek Aurora cytometer (Cytek) and analyzed using FlowJo. The GraphPad Prism 9.0 software was utilized to determine the EC50, which refers to the concentration of protein required to achieve half of the maximum binding.
  • FITC goat anti-human IgG Fc
  • Example 9 Activation of the LT ⁇ R pathway by anti-FAP ⁇ LIGHT immunocytokines in the absence or presence of co-expressed FAP
  • BALB/c-3T3-FAP (LT ⁇ R + HVEM - FAP + ) and BALB/c-3T3 (LT ⁇ R + HVEM - FAP - ) were used to investigate the effect of LIGHT muteins to activate endogenous LT ⁇ R.
  • the cells were plated at 3, 000 cells per well in a 96-well tissue culture plate and incubated with different concentrations of anti-FAP ⁇ LIGHT immunocytokines for 24 hours at 37°C. After centrifugation, supernatants were collected to quantify the level of CCL2 protein by ELISA (Cat#432704, Biolegend) with a Tecan Spark reader.
  • Figure 23A showed that in the absence of FAP expression in 3T3 cells, the activation of the LT ⁇ R pathway and the induction of CCL2 protein were primarily determined by the binding affinity of the immunocytokines, with ABC233 being the most effective followed by ABC538 and ABC539. However, the co-expressed FAP significantly increased the capabilities of all three molecules to induce CCL2 expression, particularly at lower concentrations ( Figure 23B and 23C) .
  • cancer-associate fibroblasts (CAFs) in the tumor stroma play an important role in tumor biology and exhibit abundant expression of both FAP and LT ⁇ R
  • the anti-FAP ⁇ LIGHT immunocytokines are designed to preferentially deliver FAP-mediated avidity-driven LT ⁇ R stimulation to CAFs in the tumor microenvironment.
  • Example 10 Activation of the LT ⁇ R pathway by anti-FAP ⁇ LIGHT immunocytokines in the absence or presence of trans-expressed FAP
  • CT26-FAP cell line was generated by stably transfecting CT26 cells (ATCC) with a plasmid containing mouse FAP gene using lipofectamine 3000 and antibiotic selection.
  • ATCC stably transfecting CT26 cells
  • plasmid containing mouse FAP gene using lipofectamine 3000 and antibiotic selection.
  • CT26-WT or CT26-FAP cells were plated at 2.5K/well in a 96-well tissue culture plate and incubated with serially diluted LIGHT-based immunocytokines for one hour at 37°C, 5%CO 2 . Then 2.5K BALB/c-3T3 cells were added to the culture as responder cells.
  • ELISA assay (Cat#432704, Biolegend) was performed to measure the CCL2 protein level in the media, as a surrogate of the NF- ⁇ B pathway activation.
  • Example 11 Assessment of in vivo anti-tumor activity of anti-FAP ⁇ LIGHT immunocytokines in the CT26-FAP tumor model
  • ABC538 is equivalent to 200 ⁇ g of protein, while 1.5 nanomoles are equal to 300 ⁇ g of protein.
  • ABC002’s equivalent protein for 1 nanomole and 1.5 nanomoles is 144 ⁇ g and 200 ⁇ g, respectively.
  • Tumor growth was monitored by measuring the tumor volume using calipers at specific time points until day 22. The tumor volume was calculated using the formula: length ⁇ width 2 /2. The mouse’s body weight was also recorded at the indicated time points.
  • mice survival was calculated using the Kaplan-Meier method. The mice were considered expired if one of the following criteria was met: (1) the tumor volume reached ⁇ 2000 mm 3 , (2) it experienced a body weight loss of 20%, or (3) it died. The results showed that ABC538 had single-agent anti-tumor activity, as indicated by both tumor growth inhibition and mouse survival, as demonstrated in Figure 25.
  • Example 12 Assessment of in vivo cleavage of LIGHT-based immunocytokines
  • MMPs metalloprotease family members
  • inhibition of MMPs leads to an increased LIGHT expression on activated T cells (Morel et al., 2000) .
  • the LIGHT moiety in the immunocytokines was further truncated to residues 91-240 to generate cleavage-resistant immunocytokines, such as ABC890, ABC892, ABC1054, ABC1056, ABC1032 and ABC1048.
  • cleavage-resistant immunocytokines maintain their binding specificity and functionality.
  • ABC890 and ABC892 which share the same mutations as ABC538 (LIGHT1, residues 74-240) , exhibit similar binding affinities to human and mouse LT ⁇ R, and reduced binding to human and mouse HVEM and human DcR3 ( Figure 8D, 9D, 10B, 10F) .
  • the Live/Dead Fixable Near-IR Dead Cell Stain Kit (Life Technologies) was used to determine the live and dead cells, which were then stained with a mixture of 1 ⁇ g/mL goat anti-human IgG Fc (FITC) (ab97224, Abcam) and 1 ⁇ g/mL anti-hLIGHT (PE) (T5-39, Biolegend) for 30 mins on ice. After washing three times with FACS buffer, the cells were analyzed using flow cytometry on a Cytek Aurora cytometer (Cytek) and the results were analyzed by FlowJo software. Twelve 3-fold serial dilutions of the indicated antibodies were used with the 4-parameter logistics curve-fitting algorithm to generate standard curves. The ratio of LIGHT concentration to hIgG1 concentration was used to evaluate cleavage, with a ratio of 1 indicating no cleavage.
  • FITC goat anti-human IgG Fc
  • PE anti-hLIGHT
  • Example 13 In vivo anti-tumor activity of anti-FAP ⁇ LIGHT immunocytokines in the KPC0826 tumor model
  • the KPC0826 cell line was derived from a genetically modified KPC mouse model using a previously published protocol (Beatty et al., 2011) .
  • To establish a subcutaneous KPC0826 tumor model 2.5 million KPC0826 cells in 50 ⁇ l HBSS were mixed with 50 ⁇ l of matrigel and implanted into the flank of BALB/c mice.
  • the established subcutaneous KPC0826 tumor model showed a relatively high presence of FAP + CAF cells, around 1-5%of total dissociated tumor cells (data not shown) .
  • mice On day 0, when tumor volumes reached 70-102 mm 3 with an average ⁇ 87 mm 3 , the mice were divided into 6 groups based on body weight and tumor volume: G1_ABC002 (anti-FAP, negative control) , G2_ABC233 (anti-FAP ⁇ LIGHT-wt) , G3_ABC538 (anti-FAP ⁇ LIGHT-1) , G4_ABC539 (anti-FAP ⁇ LIGHT-2) , G5_ABC890 and G6_ABC892, with eight mice per group. Mice were treated twice a week for 6 doses with 1.5 nanomoles of the respective molecules, corresponding to a dose of 200 ⁇ g for ABC002 and 300 ⁇ g for the other molecules.
  • Tumor growth inhibition was compared to the control group (G1_ABC002) and calculated using the DRAP R package (J Transl Med 17, 39, 2019) .
  • the platform can also be applied to other TNFSF14 superfamily member.
  • 4-1BBL is another TNF superfamily member and its interaction with CD137/TNFRSF9/4-1BB can promote T lymphocyte activation and proliferation.
  • Antibody-4-1BBL fusion protein (ABC676) was successfully expressed using the format D. Lymphotoxin- ⁇ , similar to engineered LIGHT1, binds to LT ⁇ R without engaging HVEM and DcR3, potentially offering enhanced safety and efficacy in providing an anti-tumor effect. With Format D, an immunocytokine equipped with Lymphotoxin- ⁇ can also be generated (ABC653) .
  • HeLa-NF- ⁇ B reporter cells were utilized to assess the downstream signaling upon LT ⁇ R activation following treatment with Lymphotoxin-based immunocytokines.
  • the Lymphotoxin-based immunocytokine (ABC653) demonstrated the ability to activate NF- ⁇ B signaling in HeLa cells containing endogenous LT ⁇ R, with an EC50 of 0.135 nM ( Figure 29) .
  • ABC653 was evaluated for anti-tumor efficacy in genetically modified KPC mice. When tumor volumes reached 70-102 mm 3 , mice were treated twice a week for 8 doses with 1.5 nanomoles of ABC653 and the mAb control (ABC002) .
  • CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans. Science 331, 1612-1616. 10. 1126/science. 1198443.
  • HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160. J Exp Med 218. 10. 1084/jem. 20211112.

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Abstract

Disclosure relates to antibody-cytokine fusion proteins, in particular, TNF superfamily member fusion proteins (immunocytokines) and uses thereof. The fusion proteins exhibit anti-tumor activity.

Description

TNF SUPERFAMILY MEMBER IMMUNOCYTOKINE AND USES THEREOF TECHNICAL FIELD
The present disclosure relates to the technical field of biomedicine or biopharmacy, and relates to antibody-cytokine fusion proteins, in particular, TNF superfamily member fusion proteins (immunocytokines) and uses thereof.
BACKGROUND
The statements in this section merely provide background information related to the present disclosure and do not necessarily constitute prior art.
Cytokines are a class of small proteins (5-20 kDa) that act as cell signaling molecules to regulate inflammation and modulate cellular activities. They belong to different families and can have different functions, such as attracting and stimulating immune cells, inducing antibody production, and promoting lymph node formation. Cytokines are a vast and varied group of pro-inflammatory or anti-inflammatory factors, classified into families based on structural similarities or those of their receptors. Cytokines encompass chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, hormones, growth factors, and others.
TNFSF14 (TNF superfamily member 14) , also known as LIGHT (homologous to Lymphotoxin, exhibits inducible expression and competes with Herpes Simplex Virus glycoprotein D for Herpes Virus Entry Mediator, a receptor expressed by T cells) is an inducible inflammatory cytokine that can bind to TNFRSF14 (TNF receptor superfamily member 14, also known as HVEM) and LTβR (lymphotoxin beta receptor) , and the decoy receptor DcR3. HVEM is expressed on the surface of various immune cells, such as T cells, B cells, NKcells, and dendritic cells. LIGHT can bind to HVEM and subsequently stimulate T cells and promote inflammation. Another receptor, LTβR is expressed on the surface of epithelial, stromal, immature DC, and other myeloid cells but not on lymphocytes. Activation of LTβR signaling is critical for the recruitment and organization of immune cells, leading to the development of lymphoid organs and tertiary lymphoid structures (TLS) . TLS consists of B and T cell zones, dendritic cells, and other immune cells, and their presence in tumor tissues has been associated with improved clinical outcomes in a variety of cancer indications, including breast, lung, and colon cancer, suggesting that TLS may play an important role in the immune response against cancer.
Although cytokine-based immunotherapy has shown promising efficacies in treating cancer, it suffers from a lack of specificity in targeting tumor tissues, which may result in systemic immune responses and severe side effects. A potential solution is to conjugate cytokines with antibodies that have tumor-targeting specificity. For example, three hmLIGHT copies were fused  to the N-terminal of Fc and then assembled with an anti-EGFR half-body (Tang et al., 2016) . In a second study, LIGHT was conjugated to anti-fibronectin F8 antibodies in a variety of formats. For example, tandem-linked three murine LIGHT copies were fused to the C-terminal of the heavy chain or the light chain of the mAbs or scFv. Robust protein expression without significant aggregation was observed only when the tandem-linked murine LIGHTs were connected to a single-chain diabody, despite several antibody-LIGHT formats being evaluated (Stringhini et al., 2021) . In addition, a murine LIGHT fused with a vascular targeting peptide (VTP) has been shown to induce lymphoid neogenesis in solid tumors (Johansson-Percival et al., 2017) . However, many of these formats have limitations in clinical practice due to lackluster efficacies or challenging manufacturing processes. Therefore, there is an ongoing need to provide an antibody-cytokine fusion protein platform for robust manufacture and tumor-specific cytokine delivery to mitigate potential toxicity.
SUMMARY
The present disclosure provides an antibody-cytokine fusion protein platform for tumor-specific cytokine delivery. The platform achieves correct assembly and molecules generated from the platform show robust formation of T cell and B cell zones in the tumor microenvironment and lead to promising antitumor efficacies.
In the first aspect of the present disclosure, a fusion protein is provided, including:
a first cytokine fragment linked to the C-terminal of a first CH2-CH3 fragment, including a first cytokine molecule, or a first cytokine molecule and a third cytokine molecule that are tandem linked, and a second cytokine fragment linked to the C-terminal of a second CH2-CH3 fragment, including a second cytokine molecule, or a second cytokine molecule and a fourth cytokine molecule that are tandem linked, the first CH2-CH3 fragment and the second CH2-CH3 fragment form a dimer, such as a homodimer, or a heterodimer, totally three cytokine molecules are linked to the dimer.
In some embodiments, the first CH2-CH3 fragment and the second CH2-CH3 fragment are modified to improve the correct assembly of the fusion protein.
In some embodiments, the first cytokine fragment comprises the first cytokine molecule, and the second cytokine fragment comprises the second cytokine molecule and the fourth cytokine molecule that are tandem linked. In some embodiments, the first cytokine fragment comprises the first cytokine molecule and a third cytokine molecule that are tandem linked, and the second cytokine fragment comprises the second cytokine molecule.
In some embodiments, the first antigen-binding fragment is an scFv that specifically recognizes a first antigen, and/or the second antigen-binding fragment is an scFv or a Fab that specifically recognizes a second antigen. In some embodiments, the first antigen-binding  fragment is a Fab that specifically recognizes a first antigen; and/or the second antigen-binding fragment is a Fab or an scFv that specifically recognizes a second antigen.
The fusion protein provided herein includes a first heavy chain, from the N-terminus to the C-terminus, of which the first antigen-binding fragment, the first CH2-CH3 fragment, and the first cytokine fragment are operably linked; and a second heavy chain, from the N terminus to the C terminus, of which the second antigen-binding fragment, the second CH2-CH3 fragment, and the second cytokine fragment are operably linked.
The fusion protein provided herein includes a first light chain, which pairs with the first antigen-binding fragment to form a heterodimer; and/or a second light chain, which pairs with the second antigen-binding fragment to form a heterodimer.
In the second aspect of the present disclosure, there is provided an isolated polynucleotide encoding the first heavy chain, the first light chain, the second heavy chain or the second light chain of the fusion protein according to the present disclosure. The present disclosure further provides a set of isolated polynucleotides, including the polynucleotide encoding the first heavy chain, the polynucleotide encoding the first light chain, the polynucleotide encoding the second heavy chain and the polynucleotide encoding the second light chain of the fusion protein according to the present disclosure.
In the third aspect of the present disclosure, there is provided an isolated vector including the isolated polynucleotide according to the present disclosure.
In the fourth aspect of the present disclosure, there is provided a host cell line harboring the isolated polynucleotide, the set of isolated polynucleotides or the isolated vector according to the present disclosure.
In the fifth aspect of the present disclosure, there is provided a pharmaceutical composition including the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector or the host cell line according to the present disclosure, and a pharmaceutically acceptable carrier.
In the sixth aspect of the present disclosure, there is provided a kit including the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector or the host cell line or the pharmaceutical composition according to the present disclosure.
In the seventh aspect of the present disclosure, there is provided use of the fusion protein according to the present disclosure or the isolated polynucleotide according to the present disclosure or the set of isolated polynucleotides according to the present disclosure or the isolated vector according to the present disclosure or the host cell line according to the present disclosure or the pharmaceutical composition according to the present disclosure in the manufacture of a drug for preventing or treating a disease, or in the manufacture of a kit for diagnosing a disease.
In the eighth aspect of the present disclosure, there is provided a method of preventing or treating a disease in a subject in need thereof, including administrating to the subject a therapeutically effective amount of the fusion protein according to the present disclosure or the isolated polynucleotide according to the present disclosure or the set of isolated polynucleotides according to the present disclosure or the isolated vector according to the present disclosure or the host cell according to the present disclosure or the pharmaceutical composition according to the present disclosure.
In the ninth aspect of the present disclosure, there is provided a method for the production of the fusion protein according to the present disclosure, including introducing a first expression vector encoding the first heavy chain, a second expression vector encoding the first light chain, a third expression vector encoding the second heavy chain, and a fourth expression vector encoding the second light chain together into one host cell line, or into separate host cell lines, and expressing the first, second, third and fourth expression vectors under proper conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.
Figure 1 illustrates the different formats of LIGHT-based immunocytokines that have been evaluated.
Figure 2 shows the SDS-PAGE analysis of LIGHT-based immunocytokines in Format D. The analysis was performed under reducing (R) and non-reducing (NR) conditions and a protein marker (M) was included for comparison.
Figure 3A and 3B show the HPLC analysis of LIGHT-based immunocytokines ABC233 and ABC234 after one-step protein A purification, respectively.
Figure 4A and 4B present the schematic format of LIGHT-based immunocytokines and the extended designs of LIGHT-based immunocytokines, respectively.
Figure 5 shows the SDS-PAGE analysis of LIGHT-based immunocytokines (A-D) under reducing (R) and non-reducing (NR) conditions, along with the protein marker (M) .
Figure 6 shows the SEC-HPLC analysis of LIGHT-based immunocytokines ABC538 and ABC539 after purification.
Figure 7 shows the SEC-HPLC analysis of LIGHT-based immunocytokines ABC770-ABC773 after purification.
Figure 8 demonstrates ELISA analysis of LIGHT-based immunocytokines (A-E) binding to the human LTβR extracellular domain, n=2.
Figure 9 demonstrates ELISA analysis of LIGHT-based immunocytokines (A-E) binding to the mouse LTβR extracellular domain, n=2.
Figures 10A-D show the ELISA analysis of LIGHT-based immunocytokines binding to the human HVEM extracellular domain; Figures 10E-H show the ELISA analysis of LIGHT-based immunocytokines binding to the mouse HVEM extracellular domain.
Figure 11 shows the ELISA analysis of LIGHT-based immunocytokines (A-F) binding to the human DcR3.
Figure 12 illustrates the binding of LIGHT-based immunocytokines (A-G) to 293T cells overexpressing human LTβR. The mean fluorescence intensity (MFI) and concentration (Conc. ) are indicated.
Figure 13 shows the binding of LIGHT-based immunocytokines (A-G) to 293T cells overexpressing mouse LTβR. MFI: mean fluorescence intensity.
Figure 14 shows the binding of LIGHT-based immunocytokines (A-D) to 293T cells overexpressing human HVEM. MFI: mean fluorescence intensity.
Figure 15 shows the binding of LIGHT-based immunocytokines (A-D) to 293T cells overexpressing mouse HVEM. MFI: mean fluorescence intensity.
Figure 16 illustrates the NF-κB activities induced by LIGHT-based immunocytokines on HeLa-NF-κB cells in the absence (Figures 16A-D) and presence (Figures 16E-H) of 293T cells overexpressing FAP (293T-FAP) . Relative luciferase activity (RLU) is reported.
Figure 17 shows the NF-κB activities induced by LIGHT-based immunocytokines (A-C) on 293T-NF-κB cells overexpressing human HVEM. Relative luciferase activity (RLU) is reported.
Figure 18 demonstrates the NF-κB activities induced by LIGHT-based immunocytokines (A-C) on 293T-NF-κB cells overexpressing mouse HVEM. Relative luciferase activity (RLU) is reported.
Figure 19 shows the NF-κB activities induced by LIGHT-based immunocytokines (A-D) on HeLa-NF-κB cells in the absence and presence of 293T-FAP cells.
Figure 20 demonstrates the impact of DcR3 on the activities of LIGHT-based immunocytokines (A-D) .
Figure 21 shows the binding of LIGHT-based immunocytokines to BALB/c-3T3 cells (Figure 21A) and BALB/c-3T3 cells overexpressing FAP (Figure 21B) .
Figure 22 shows the binding of LIGHT-based immunocytokines to primary human CD4+ T cells (Figure 22A) , primary human CD8+ T cells (Figure 22B) , primary mouse CD4+ T cells (Figure 22C) , and primary mouse CD8+ T cells (Figure 22D) .
Figure 23 shows the induction of CCL2 by LIGHT-based immunocytokines in BALB/c-3T3-WT (wild type) (Figure 23A) , and BALB/c-3T3-FAP cells (Figure 23B) . Figure 23C presents the same results that are grouped by different molecules.
Figure 24 shows the transactivation of LTβR on 3T3 cells in the presence of CT26-FAP or CT26-WT upon addition of ABC233 (Figure 24A) , ABC538 (Figure 24B) , or ABC539 (Figure 24C) .
Figure 25 demonstrates the in vivo anti-tumor activity of anti-FAP×LIGHT immunocytokines in the CT26-FAP model, presenting the treatment timeline (Figure 25A) , tumor volume (Figure 25B) , survival curve (Figure 25C) , and the body weight (Figure 25D) .
Figure 26 shows the detected ratio of LIGHT to hIgG1 upon immunocytokine administration, which indicates the cleavage that releases LIGHT moieties from the immunocytokines in vivo.
Figure 27 demonstrates the in vivo anti-tumor activity of anti-FAP×LIGHT immunocytokines in the KPC model. Figure 27A outlines the treatment timeline for Example 13; Figure 27B shows individual tumor volumes of each group; Figure 27C displays the average tumor volumes of each group; Figure 27D indicates the tumor growth inhibition (TGI) of each group.
Figures 28A-28B show the NF-κB activities induced by ABC890 and ABC892, respectively, on HeLa-NF-κB cells in the absence or presence of 293T-FAP cells. Figures 28C-28E show the NF-κB activities induced by ABC890, ABC892, and ABC538, respectively, on HEK293T-NF-κB cells overexpressing human HVEM in the absence or presence of 293T-FAP cells.
Figure 29 demonstrates HeLa-NF-κB activities induced by ABC653.
Figure 30 shows in vivo anti-tumor activity of ABC653 in the KPC0826 model.
DETAILED DESCRIPTION
The present disclosure is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which does not depart from the instant invention. Hence, the following description is intended to illustrate some particular embodiments of the present disclosure, and not to exhaustively specify all permutations, combinations and variations thereof.
As used herein, the singular forms “a, ” “an, ” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
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 the disclosure pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present disclosure, the preferred materials and  methods are described herein. In describing and claiming the present disclosure, the following terminology will be used.
[term]
As used herein, the term “fusion” refers to connecting a protein with another biological material, such as a protein, a peptide, a nucleic acid molecule, or any other biological molecule or part thereof.
The term “protein” , “peptide” and “polypeptide” are used interchangeably herein to refer to a biological polymer including units derived from amino acids linked by peptide bonds. A protein can be composed of one or more chains.
As used herein, the term “domain” refers to a part of a molecule or structure that shares common physical, chemical or structural characteristics, such as similar hydrophobic or polar properties. The exemplary domain may include a protein binding domain, a DNA binding domain, an ATP binding domain, or a similar folding structure such as globular or helical features. The domains can be identified according to their homology with conserved structural or functional motifs.
As used herein, the term “antibody” is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies) , full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term “antibody” may refer to a full-length antibody or an antigen-binding fragment thereof. A full-length antibody includes two heavy chains and two light chains. The variable regions/domains of the light and heavy chains are responsible for antigen binding. The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL” , respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding sites. In some embodiments, from the N-to the C-terminus, the variable domain of heavy chain/domain/region, was followed by three constant heavy domains (CH1, CH2, and CH3) . Similarly, from the N-to the C-terminus, the variable domain of light chain/domain/region, was followed by a constant light (CL) domain.
As used herein, the term “antigen-binding fragment” refers to an antibody fragment including a diabody, a Fab, a Fab’ , a F (ab’ ) 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv’ ) , a disulfide stabilized diabody (ds diabody) , a single-chain Fv (scFv) , an scFv dimer (bivalent diabody) , a multi-specific antibody formed from a portion of an antibody including one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that  binds to an antigen but does not include a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds.
A Fab fragment corresponds to one of the two identical arms of an antibody molecule and contains the complete light chain paired with the VH and CH1 domains of a heavy chain.
“Fv” is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in a tight, non-covalent association. The folding of these two domains emanates six hypervariable loops (3 loops each from the heavy and light chain) thatprovide a conformation for antigen binding and confer antigen binding specificity to the antibody.
As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH: : VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or by a peptide-encoding linker or spacer, which connects the N-terminal of the VH with the C-terminal of the VL, or the C-terminal of the VH with the N-terminal of the VL.
As used herein, the term “CH2-CH3 fragment” refers to a portion of the constant region and at least encompasses CH2 and CH3 domains. In some embodiments, two CH2-CH3 fragments can form an antibody Fc domain. “Fc domain” refers to a pair of associated Fc regions. The two Fc regions dimerize to create the Fc domain. The two Fc regions within the Fc domain can be the same or different from one another.
The term “Fc region” or “Fc chain” as used herein is meant the polypeptide comprising the CH2-CH3 domains of an IgG molecule, and in some cases, inclusive of the hinge. In EU numbering for human IgG1, the CH2-CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to 230. Human IgG Fc regions are of particular use in the present disclosure, and can be the Fc region from human IgG1, IgG2, IgG3 or IgG4.
As used herein, the term “specifically binds” refers to measurable and reproducible interactions, such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically recognizes a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily interaction, and/or with greater duration than its bindings to other targets. Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to Octet, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORETM -tests and peptide scans.
As used herein, the term “linker” means a polypeptide used to couple two molecules. The linker typically is a stretch of amino acids, e.g., predominantly glycine and/or serine. The linker may have a length of up to 100 amino acids, such as from about 1, 2, 5, 7, 10, 15 amino acid (s) up to about 15, 20, 25, 30, 35, 50, 75, or 100 amino acids.
As used herein, the term “VH-CH1 linker” refers to a polypeptide connecting VH and CH1 in a naturally occurring antibody, and derivatives thereof.
As used herein, the term “VL-CL linker” refers to a polypeptide connecting VL and CL in a naturally occurring antibody, and derivatives thereof.
As used herein, the term “CH2-CH3 linker” refers to a polypeptide connecting CH2 and CH3 in a naturally occurring antibody, and derivatives thereof.
As used herein, the term “IgM tail linker” refers to a polypeptide in a naturally occurring IgM antibody, and derivatives thereof.
When describing a linker, the term “derivative” refers to a linker with an amino acid sequence that differs from a naturally occurring sequence by one or more amino acids, for example, containing one or more amino acid insertions, deletions, or substitutions relative to a naturally occurring sequence.
As used herein, the term “cytokine” is understood to mean any protein, analog or functional fragment thereof, which is capable of stimulating or inducing a biological response against a preselected cell type. Accordingly, it is contemplated thata variety of cytokines can be incorporated into the present application. Useful cytokines include, for example, tumor necrosis factors (TNFs) , interleukins (ILs) , lymphokines (Ls) , colony stimulating factors (CSFs) , interferons (IFNs) , chemokine, growth factors including species variants, truncated analogs thereof which are capable of stimulating or inducing such biological responses, TNF family members such as LIGHT, lymphotoxin α, lymphotoxin β, 4-1BBL and the like. The term “cytokine” is also understood to encompass any derivative of a wild-type cytokine that includes modification and maintains at least a significant portion (such as at least about 50%) of any of its desired functions.
When describing a cytokine, the term “wild-type (WT) ” refers to a cytokine with an amino acid sequence that is naturally occurring and encoded by a germline genome of a given species. A species can have one or more wild-type sequences (for example, with one canonical wild-type sequence and one or more non-canonical wild-type sequences) . A wild-type cytokine sequence can include a sequence that is truncated at the N and/or the C terminus relative to the sequence encoded by an open reading frame. A wild-type cytokine sequence can be a mature form of a cytokine that has been processed to remove the N-terminal and/or the C-terminal residues. A wild-type cytokine can lack a signal peptide or can include a signal peptide (e.g., a signal peptide can be added to the N-terminus of the wild-type cytokine) .
When describing a cytokine, the term “derivative” “variant” “mutein” and “mutant” are used interchangeably herein to refer to a cytokine with an amino acid sequence that differs from a wild-type sequence by one or more amino acids, for example, containing one or more amino acid insertions, deletions, or substitutions relative to a wild-type sequence.
As used herein, the term “naturally occurring” refers to a sequence of natural origin which means that the whole or parts thereof are not synthetic and exist or are produced in nature. More preferably, the term “naturally occurring” as used herein refers to a sequence of natural origin which means that the whole sequence is not synthetic and exists or is produced in nature.
As used herein, the term “LIGHT” has its general meaning in the art and refers to a protein transiently expressed on activated T cells, dendritic cells (DCs) , monocytes, natural killer cells (NK) , and others. LIGHT can stimulate HVEM-expressing cells, such as T cells, B cells, NK cells, and dendritic cells, leading to cell activation and inflammation. LIGHT binding to another receptor LTβR, which is expressed on stromal cells, myeloid cells and some epithelial cells, can lead to the development of lymphoid organs and tertiary lymphoid structures (TLS) . The term “LIGHT” is also understood to encompass WT-LIGHT and any derivative of a WT-LIGHT.
As used herein, the term “knobs-into-holes” is used in its broadest sense and encompasses various situations, such as the CH1 domain of one heavy chain with the knob mutations and the CH1 domain of the other heavy chain with the hole mutations, the CH2 domain of one heavy chain with the knob mutations and the CH2 domain of the other heavy chain with the hole mutations, and/or the CH3 domain of one heavy chain with the knob mutations and the CH3 domain of the other heavy chain with the hole mutations. For example, and generally, “knobs-into-holes” may refer to an intra-interface modification between two antibody heavy chains in the CH3 domains: i) in the CH3 domain of one heavy chain (first CH3 domain) , an amino acid residue is substituted with another amino acid residue bearing a large side chain, thereby creating a protrusion ( “knob” ) in the interface in the first CH3 domain; ii) in the CH3 domain of the other heavy chain (second CH3 domain) , an amino acid residue is substituted with another amino acid residue bearing a smaller side chain, thereby creating a cavity ( “hole” ) within the interface in the second CH3 domain, in which a protrusion ( “knob” ) in the first CH3 domain can be placed.
As used herein, the term “DDKK” refers to a modification mediating electrostatic steering effect that is used to enhance antibody Fc heterodimer formation as inter alia described by Gunasekaran et al., (J. Biol. Chem. 2010, 19637-19646) . The Fc part of one heavy chain includes the mutations K392D and K409D (termed DD mutations) and the Fc part of the other heavy chain includes the mutations E356K and D399K (termed KK mutations) . The numbering is according to the EU index.
An “isolated” polynucleotide encoding a protein, or portion or fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant  nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated polynucleotide is free of association with all components associated with the production environment. The isolated polynucleotide encoding the protein, or portion or fragment thereof described herein is in a form other than in the form or setting in which it is found in nature. Isolated polynucleotides, therefore, are distinguished from polynucleotides encoding the protein, or portion or fragment thereof s described herein existing naturally in cells. An isolated polynucleotide includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extra chromosomally or at a chromosomal location that is different from its natural chromosomal location.
As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors. ”
As used herein, the term “host cells” or “host cell line” refers to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transfected cells” , which include the primary transfected cells and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, and (or) may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transfected cell are included herein. The host cell may be a mammalian cell, plant cell, yeast cell, and (or) a bacterial cell. The host cell may contain the nucleic acid molecule or vector of the present disclosure as an extra-chromosomally (episomal) replicating molecule, or more preferably, includes the nucleic acid molecule or vector of the present disclosure integrated into the genome of the host cell.
As used herein, the term “transfected” or “transfection” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” cell is one which has been transfected with exogenous nucleic acids. The cell includes the primary subject cell and its progeny.
As used herein, the terms “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of the active ingredient (s) to be effective, and which contains no additional components which are unacceptably toxic to an individual to which the formulation would be administered. Such formulations may be sterile.
A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for  use with a therapeutic agent that together includes a “pharmaceutical composition” for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.
“Cancer” , and “tumor” are used interchangeably herein to refer to any of several diseases that are characterized by an uncontrolled and abnormal proliferation of cells, the ability of the affected cells to spread locally or through the bloodstream and the lymphatic system to other parts of the body (metastasize) , as well as any of several characteristic structural and/or molecular features. Examples of cancers that can be treated using the fusion protein of the present disclosure include solid tumors and hematologic cancers. Additional examples of cancers that can be treated using the fusion protein of the present disclosure include breast, lung, brain, bone, liver, kidney, colon, head and neck, ovarian, hematopoietic (e.g., leukemia) and prostate cancers. Additional examples of cancers that can be treated using multivalent and multi-specific antibodies include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Among the most particular examples of such cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cancer cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, carcinoma of the salivary glands, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma and various types of cancers of the head and neck. Other cancers and tumors that can be treated using the fusion protein of the present disclosure are described herein or are otherwise known in the art.
As used herein, the term “agent” encompasses the fusion protein according to the present disclosure, the isolated polynucleotide according to the present disclosure, the set of isolated polynucleotides according to the present disclosure, the isolated vector according to the present disclosure, the host cell according to the present disclosure, and the pharmaceutical composition according to the present disclosure.
An “effective amount” of an agent of the present disclosure is an amount sufficient to achieve a specifically indicated purpose in order to cause an observable change in the level of one or more biological activities related to the target cell to which the fusion protein binds. The change can increase the activity level of the target. The change can reduce the activity level of the target. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
The term “therapeutically effective amount” refers to an amount of an agent of the present disclosure or another drug effective to “treat” a disease or disorder in a patient or mammal. In the case of cancer, the therapeutically effective amount of the agent of the present disclosure can reduce angiogenesis and neovascularization; reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to some extent or stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow down to a certain point or stop) tumor metastasis; inhibit, to some extent, tumor growth or the incidence of tumors; stimulate immune responses against cancer cells and/or alleviate to some extent one or more of the symptoms associated with cancer. See the definition in this document of “treat. ” A “therapeutically effective amount” may also refer to an effective amount, at the dosages and for the periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the agent of the present disclosure may vary depending on factors such as the pathology, age, sex and weight of the individual and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which the toxic or detrimental effects of the therapeutic composition are overcome by the therapeutically beneficial effects.
As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequences of cancer (such as, for example, tumor volume) . The methods of the application contemplate any one or more of these aspects of treatment.
In the context of cancer, the term “treating” includes any or all of the inhibiting growth of cancer cells, inhibiting replication of cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease.
The terms “inhibition” or “inhibit” refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to that of a reference. In certain embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20%or greater. In another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
As used herein, the term “preventing” includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual that may be predisposed to the disease but has not yet been diagnosed with the disease.
[Antibody-cytokine Fusion Protein]
The present disclosure provides the immunocytokines, which are antibody-cytokine fusion proteins. The fusion protein includes a first cytokine fragment and a second cytokine fragment, wherein the first cytokine fragment and the second cytokine fragment are linked to the C-terminal of a first CH2-CH3 fragment and a second CH2-CH3 fragment, respectively.
In some embodiments, the first CH2-CH3 fragment and the second CH2-CH3 fragment form a dimer, such as a homodimer or a heterodimer.
In some embodiments, the first CH2-CH3 fragment and the second CH2-CH3 fragment form an Fc domain. Optionally, the Fc domain is derived from IgG, such as human IgG molecules, which encompass IgG1, IgG2, IgG3, and IgG4 subclass. In some embodiments, the first CH2-CH3 fragment and the second CH2-CH3 fragment form a human IgG1 Fc domain or IgG4 Fc domain.
In some embodiments, the first CH2-CH3 fragment and the second CH2-CH3 fragment form a human IgG1 Fc with mutation N297A, which refers to the amino acid N (Asparagine) at position 297 numbering by EU numbering is mutated to amino acid (Alanine) . Other mutations like this have a similar instruction.
The amino acid in the present disclosure refers to an organic compound containing amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain specific to each amino acid. Preferably, the amino acid in the present disclosure is natural amino acids, such as Alanine (A) , Cysteine (C) , Aspartic Acid (D) , Glutamic Acid (E) , Phenylalanine (F) , Glycine (G) , Histidine (H) , Isoleucine (I) , Lysine (K) , Leucine (L) , Methionine (M) , Asparagine (N) , Proline (P) , Glutamine (Q) , Arginine (R) , Serine (S) , Threonine (T) , Valine (V) , Tryptophan (W) , Tyrosine (Y) .
Optionally, the Fc domain is modified to improve the correct assembly of the fusion protein when needed. In some embodiments, the first CH2-CH3 fragment and the second CH2-CH3 fragment include one or more modifications selected from the group consisting of knobs-into-holes, electrostatic steering of CH3 (e.g., DDKK) , DuoBody, SEEDbodies, cFAE, XmAb, Azymetric, andIn some embodiments, the first CH2-CH3 fragment and the second CH2-CH3 fragment include one or more modifications selected from the group consisting of knobs-into-holes, and DDKK.
In some embodiments, the first CH2-CH3 fragment includes knob mutations, and the second CH2-CH3 fragment includes hole mutations. In some embodiments, the first CH2-CH3 fragment includes hole mutations, and the second CH2-CH3 fragment includes knob mutations.
In some embodiments, the first CH2-CH3 fragment includes DD mutations, and the second CH2-CH3 fragment includes the KK mutations. In some embodiments, the first CH2-CH3 fragment includes the KK mutations, and the second CH2-CH3 fragment includes the mutations DD mutations.
In some embodiments, one of the first CH2-CH3 fragment and the second CH2-CH3 fragment includes one or more mutations selected from the group consisting of Y349C, T366S, L368A, and Y407V; and the other one includes mutations S354C and/or T366W, defined by EU numbering.
In some embodiments, the first CH2-CH3 fragment includes mutations Y349C, T366S, L368A, and Y407V to form a “hole” , and the second CH2-CH3 fragment includes mutations S354C and T366W to form a “knob” . In some embodiments, the first CH2-CH3 fragment includes mutations S354C and T366W to form a “knob” , and the second CH2-CH3 fragment includes mutations Y349C, T366S, L368A, and Y407V to form a “hole” .
The C-terminal of the dimer is totally linked to three cytokine molecules. In some embodiments, the first cytokine fragment includes at least one wild type, truncated or mutated cytokine molecule. In some embodiments, the first cytokine fragment includes one or two wild type, truncated or mutated cytokine molecules. In some embodiments, the second cytokine fragment includes at least one wild type, truncated or mutated cytokine molecule. In some embodiments, the second cytokine fragment includes one or two wild type, truncated or mutated cytokine molecules.
In some embodiments, the first cytokine fragment includes a first cytokine molecule. In some embodiments, the second cytokine fragment includes a second cytokine molecule. In some embodiments, the first cytokine fragment includes a tandem linked first cytokine molecule and a third cytokine molecule. In some embodiments, the second cytokine fragment includes a tandem linked second cytokine molecule and a fourth cytokine molecule.
In some embodiments, the first cytokine fragment includes the first cytokine molecule, and the second cytokine fragment includes the second cytokine molecule and the fourth cytokine molecule that are tandem linked. In some embodiments, the first cytokine fragment includes the first cytokine molecule and a third cytokine molecule that are tandem linked, and the second cytokine fragment includes the second cytokine molecule.
In some embodiments, the cytokine molecule (s) in the first cytokine fragment and the cytokine molecule (s) in the second cytokine fragment are the same. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the third cytokine molecule are the  same. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the fourth cytokine molecule are the same.
In some embodiments, the cytokine molecule (s) in the first cytokine fragment and the cytokine molecule (s) in the second cytokine fragment are different from each other. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the third cytokine molecule are different from each other. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the fourth cytokine molecule are different from each other.
The first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other are selected from the group consisting of tumor necrosis factor, interleukin, lymphokine, interferon, colony stimulating factor, chemokine and growth factor. In some embodiments, the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other are selected from the group consisting of LIGHT, lymphotoxin α, lymphotoxin β, and 4-1BBL, for example, wild type LIGHT (preferably human LIGHT) , truncated LIGHT or mutein thereof.
In some embodiments, the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other are truncated LIGHT (i.e., the amino acid sequence is short than wild type) . Exemplary truncated LIGHT is WT-LIGHT (74-240) (SEQ ID NO: 68) , WT-LIGHT (91-240) (SEQ ID NO: 78) .
In some embodiments, the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other include a truncated LIGHT amino acid sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 78.
In some embodiments, the first cytokine molecule, second cytokine molecule and the third cytokine include or have amino acid sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 78. In some embodiments, the first cytokine molecule, second cytokine molecule and the fourth cytokine include or have amino acid sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 78.
In some embodiments, the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other are LIGHT mutein. Exemplary LIGHT mutein are LIGHT MUTEIN-1 (74-240) , LIGHT MUTEIN-2 (74-240) , LIGHT MUTEIN-9 (74-240) , LIGHT MUTEIN-11 (74-240) , LIGHT MUTEIN-18 (74-240) , LIGHT MUTEIN-22 (74-240) , LIGHT MUTEIN-29 (74-240) , LIGHT MUTEIN-37 (74-240) , LIGHT MUTEIN-52 (74-240) , (SEQ ID NOs: 69-77) ; LIGHT MUTEIN-1 (59-240) , LIGHT MUTEIN-1 (91-240) , LIGHT MUTEIN-1 (87-240) , LIGHT MUTEIN-90 (74-240) , LIGHT MUTEIN-63 (74-240) , LIGHT MUTEIN-86 (74-240) , LIGHT MUTEIN-88 (74-240) , LIGHT MUTEIN-60 (74-240) , LIGHT MUTEIN-92 (74-240) , (SEQ ID NOs: 126-134) ; LIGHT MUTEIN-93 (74-240) , LIGHT MUTEIN-41 (74-240) , (SEQ ID NOs: 25-26) ; LIGHT MUTEIN-42 (74-240) , LIGHT MUTEIN-2 (91-240) , (SEQ ID NOs: 58-59) ; LIGHT MUTEIN- 95 (91-240) , LIGHT MUTEIN-96 (91-240) , LIGHT MUTEIN-97 (91-240) , LIGHT MUTEIN-98 (91-240) , LIGHT MUTEIN-99 (91-240) , LIGHT MUTEIN-100 (91-240) , LIGHT1 (91-240) (M150V) , (SEQ ID NOs: 135-141) ; LIGHT MUTEIN-101 (91-240) (SEQ ID NO: 158) .
In some embodiments, the first cytokine molecule, second cytokine molecule, third cytokine molecule and fourth cytokine molecule independently of each other comprise a mutated LIGHT amino acid sequence set forth in SEQ ID NO: 25, 26, 58, 59, 69-77, 126-141, 158, 177, 178 and 180.
In some embodiments, the first cytokine molecule, the second cytokine molecule and the third cytokine include or have amino acid sequence set forth in SEQ ID NO: 25, 26, 58, 59, 69-77, 126-141, 158, 177, 178 and 180. In some embodiments, the first cytokine molecule, second cytokine molecule and the fourth cytokine include or have amino acid sequence set forth in SEQ ID NO: 25, 26, 58, 59, 69-77, 126-141, 158, 177, 178 and 180.
In some embodiments, the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are wild type 4-1BBL (preferably human 4-1BBL) , truncated 4-1BBL or mutein thereof. In some embodiments, the wild type 4-1BBL, truncated 4-1BBL or mutein thereof includes amino acid sequence of SEQ ID NO: 179.
In some embodiments, the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are wild, truncated lymphotoxin α or lymphotoxin β, or mutein thereof. In some embodiments, the wild, truncated lymphotoxin α or lymphotoxin β, or mutein thereof includes amino acid sequence of SEQ ID NO: 181, 182.
In some the first cytokine molecule, the second cytokine molecule, and the third cytokine molecule have the same amino acid sequence. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the fourth cytokine molecule have the same amino acid sequence.
In some embodiments, the first cytokine fragment links to the C-terminal of the first CH2-CH3 fragment directly or via a linker. In some embodiments, the second cytokine fragment link to the C-terminal of the second CH2-CH3 fragment directly or via a linker.
In some embodiments, the C terminal of the CH3 domain in the first CH2-CH3 fragment is fused to the N terminal of a first cytokine fragment directly or via a first linker A, and the C terminal of the CH3 domain in the second CH2-CH3 fragment is fused to N terminal of a second cytokine fragment directly or via a first linker B.
In some embodiments, the first cytokine molecule is fused to a third cytokine molecule directly or via a second linker A. In some embodiments, the second cytokine molecule is fused to a fourth cytokine molecule directly or via a second linker B.
In some embodiments, the C terminal of the first cytokine molecule is fused to the N terminal of a third cytokine molecule directly or via a second linker A. In some embodiments, the C terminal of the second cytokine molecule is fused to the N terminal of a fourth cytokine molecule directly or via a second linker B.
In some embodiments, the first linker A, the first linker B, the second linker A, and the second linker B independently of each other are absent or are selected from the group consisting of VH-CH1 linker ASTKGPSVFPLAPS (SEQ ID NO: 82) ; VL-CL linker RTVAAPSVFIFPPS (SEQ ID NO: 83) ; CH2-CH3 linker ISKAKGQPREPQ (SEQ ID NO: 81) ; IgM tail linker KSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 84) ; GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 89) , G; and (GGGGS) n, n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the first linker A, the first linker B, the second linker A, and the second linker B independently of each other are absent or are selected from the group consisting of VH-CH1 linker; VL-CL linker; CH2-CH3 linker; IgM tail linker; G; and (GGGGS) n, wherein n=1, 2, 3, 4, 5, or 6. In some embodiments, the first linker A, the first linker B, the second linker A, and the second linker B independently of each other are absent or are selected from the group consisting of VH-CH1 linker; VL-CL linker; CH2-CH3 linker; IgM tail linker; GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 89) , G; and (GGGGS) n, n=1, 2, 3, or 4.
In some embodiments, the first linker A and the first linker B independently of each other are absent or are selected from the group consisting of VH-CH1 linker; VL-CL linker; CH2-CH3 linker; IgM tail linker; and (GGGGS) n, n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the first linker A and the first linker B independently of each other are absent or are selected from the group consisting of VH-CH1 linker; VL-CL linker; CH2-CH3 linker; IgM tail linker; and (GGGGS) n, n=1, 2, 3, 4, 5, or 6. In some embodiments, the first linker A and the first linker B independently of each other are absent or are selected from the group consisting of VH-CH1 linker; VL-CL linker; CH2-CH3 linker; IgM tail linker; GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 89) , G, and (GGGGS) n, n=1, 2, 3, or 4.
In some embodiments, the first linker A and the first linker B independently of each other are absent or include the amino acid sequence as set forth in any one of SEQ ID NOs: 81-90. In some embodiments, the first linker A and the first linker B independently of each other are absent or have the amino acid sequence as set forth in any one of SEQ ID NOs: 81-90.
In some embodiments, the first linker A and the first linker B are the same. In some embodiments, the first linker A and the first linker B are different.
In some embodiments, the second linker A and the second linker B independently of each other are absent or are selected from the group consisting of G; and (GGGGS) n, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the second linker A and the second linker B independently of each other are absent or are selected from the group consisting of G; and  (GGGGS) n, wherein n=1, 2, 3, 4, 5, or 6. In some embodiments, the second linker A and the second linker B independently of each other are absent or are selected from the group consisting of G; and (GGGGS) n, wherein n=1, 2, 3, or 4.
In some embodiments, the second linker A and the second linker B independently of each other are absent or include the amino acid sequence of G or the amino acid sequence as set forth in any one of SEQ ID NOs: 85-88. In some embodiments, the second linker A and the second linker B independently of each other are absent or have the amino acid sequence of G or the amino acid sequence as set forth in any one of SEQ ID NOs: 85-89.
In some embodiments, the fusion protein provided herein includes a first antigen-binding fragment and a second antigen-binding fragment, which are connected to N terminal of the first CH2-CH3 fragment and the second CH2-CH3 fragment, respectively.
In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment independently of each other are selected from the group consisting of a diabody, a Fab, a Fab’ , a F (ab’ ) 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv’ ) , a disulfide stabilized diabody (ds diabody) , a single-chain Fv (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody formed from a portion of an antibody including one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment.
In some embodiments, the first antigen-binding fragment is an scFv that specifically binds to a first antigen. In some embodiments, the second antigen-binding fragment is an scFv that specifically binds to a second antigen.
In some embodiments, the scFv includes a heavy chain variable region (VH) and a light chain variable region (VL) which can pair with each other to form an Fv fragment that specifically binds to an antigen. In the scFv, the N-terminal of the VH is linked to the C-terminal of the VL directly or via a linker, or the C-terminal of the VH is linked to the N-terminal of the VL directly or via a linker. Those skilled in the art would be able to select the appropriate linker for use in the present disclosure, for example, the linker between the VH and the VL is (GGGGS) n, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker between the VH and the VL is (GGGGS) 4 (SEQ ID NO: 88) .
In some embodiments, the first antigen-binding fragment is a Fab that specifically binds to a first antigen. In some embodiments, the second antigen-binding fragment is a Fab that specifically binds to a second antigen.
In some embodiments, the first antigen-binding fragment is an scFv that specifically binds to a first antigen; and the second antigen-binding fragment is an scFv or a Fab that specifically binds to a second antigen. In some embodiments, the first antigen-binding fragment is a Fab that specifically binds to a first antigen; and the second antigen-binding fragment is a Fab or an scFv  that specifically binds to a second antigen. In some embodiments, the first antigen-binding fragment is an scFv or a Fab that specifically binds to a first antigen; and the second antigen-binding fragment is a Fab that specifically binds to a second antigen. In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment are Fab. In some embodiments, the first antigen-binding fragment is an scFv and the second antigen-binding fragment is Fab. In some embodiments, the first antigen-binding fragment is a Fab and the second antigen-binding fragment is an scFv.
In some embodiments, the first antigen and the second antigen independently of each other are selected from the group consisting of FAP, HER2, PDL-1, PD-1, EGFR, VEGFR, VEGF, CCR8, OX-40, 41BB, Angiopoietin-2, IL-4Rα, BCMA, Blys, BTNO2, C5, CD122, CD13, CD133, CD137, CD138, CD16a, CD19, CD20, CD22, CD27, CD28, CD3, CD30, CD33, CD38, CD40, CD47, CD-8, CEA, CGPR/CGRPR, CSPGs, CTLA4, CTLA-4, DLL-4, EpCAM, factor IXa, factor X, GITR, GP130, Her3, HSG, ICOS, IGF1, IGF1/2, IGF-1R, IGF2, IGFR, IL-1, IL-12, IL-12p40, IL-13, IL-17A, IL-1β, IL-23, IL-5, IL-6, IL-6R, Lag-3, LAG3, MAG, Met, NgR, NogoA, OMGp, OX40, PDGFR, PSMA, RGMA, RGMB, SARS-CoV-2, Te38, TIM-3, TNF, TNFα, TROP-2, and TWEAK. In some embodiments, the first antigen and the second antigen independently of each other are selected from the group consisting of FAP, HER2, PDL-1, and EGFR.
In some embodiments, the first antigen and the second antigen are different antigens or different epitopes on the same antigen. In some embodiments, the first antigen and the second antigen are the same or the same epitope on the same antigen.
The fusion protein herein is monovalent or bivalent, monospecific or bispecific.
In some embodiments, both of the first antigen and the second antigen are FAP, or both of the first antigen and the second antigen are HER2, or both of the first antigen and the second antigen are EGFR, or both of the first antigen and the second antigen are PDL-1, or the first antigen is HER2 and the second antigen are FAP.
In some embodiments, the fusion protein provided herein includes: a first heavy chain, from the N terminal to the C terminal, including the operably linked first antigen-binding fragment, the first CH2-CH3 fragment, and the first cytokine fragment; and a second heavy chain, from the N terminal to the C terminal, including the operably linked second antigen-binding fragment, the second CH2-CH3 fragment, and the second cytokine fragment.
As Figure 4A shows, the fusion protein is with the “Y” shape. The first heavy chain used herein is referred to the heavy chain of the left arm of “Y” shape, and the second heavy chain used herein is referred to the heavy chain of the right arm of “Y” shape.
In some embodiments, the first heavy chain and the second heavy chain are the same. In some embodiments, the first heavy chain and the second heavy chain are different.
In some embodiments, the fusion protein provided herein includes a first light chain, which pairs with the first antigen-binding fragment to form a heterodimer. In some embodiments, the fusion protein provided herein includes a second light chain, which pairs with the second antigen-binding fragment to form a heterodimer.
In some embodiments, the first light chain and the second light chain are the same, under which circumstance they are collectively referred to as “light chain” . In some embodiments, the first light chain and the second light chain are different.
In some embodiments, the first antigen-binding fragment is an scFv, in this case, the first light chain is absent. In some embodiments, the second antigen-binding fragment is an scFv, in this case, the second light chain is absent.
In some embodiments, the first light chain and the second light chain independently of each other include the amino acid sequence as set forth in any one of SEQ ID NOs: 1, 29, 91 and 94. In some embodiments, the first light chain and the second light chain independently of each other have the amino acid sequence as set forth in any one of SEQ ID NOs: 1, 29, 91 and 94. In some embodiments, both the first light chain and the second light chain have the amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, both the first light chain and the second light chain have the amino acid sequence as set forth in SEQ ID NO: 29. In some embodiments, both the first light chain and the second light chain have the amino acid sequence as set forth in SEQ ID NO: 91. In some embodiments, both the first light chain and the second light chain have the amino acid sequence as set forth in SEQ ID NO: 94. In some embodiments, the first light chain has the amino acid sequence as set forth in SEQ ID NO: 1, and the second light chain has the amino acid sequence as set forth in SEQ ID NO: 29. In some embodiments, the second light chain has the amino acid sequence as set forth in SEQ ID NO: 1, and the first light chain has the amino acid sequence as set forth in SEQ ID NO: 29. In some embodiments, the first light chain has the amino acid sequence as set forth in SEQ ID NO: 1, and the second light chain has the amino acid sequence as set forth in SEQ ID NO: 91. In some embodiments, the second light chain has the amino acid sequence as set forth in SEQ ID NO: 1, and the first light chain has the amino acid sequence as set forth in SEQ ID NO: 91. In some embodiments, the first light chain has the amino acid sequence as set forth in SEQ ID NO: 1, and the second light chain has the amino acid sequence as set forth in SEQ ID NO: 94. In some embodiments, the second light chain has the amino acid sequence as set forth in SEQ ID NO: 1, and the first light chain has the amino acid sequence as set forth in SEQ ID NO: 94.
In some embodiments, the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 24, 27, 30, 33, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 60, 62, 64, 66, 92, 95, 97, 101, 103, 105, 106, 108, 110, 112, 115, 116, 118,  120, 122, 124, 142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163, 165, 167, 169, 171, 173 or 175.
In some embodiments, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 23, 28, 31, 32, 35, 37, 39, 41, 43, 45, 47, 49, 51, 52, 53, 55, 57, 61, 63, 65, 67, 93, 96, 98, 99, 100, 102, 104, 107, 109, 111, 113, 114, 117, 119, 121, 123, 125, 143, 145, 147, 149, 151, 153, 157, 155, 160, 162, 164, 166, 168, 170, 172, 174 or 176.
In some embodiments, the fusion protein is select from the following:
(1) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 3, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 3; (2) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 4, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 2; (3) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 6, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 5; (4) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 8, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 7; (5) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 10, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 9; (6) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 12, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 11; (7) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 14, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 13; (8) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 16, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 15; (9) both of the first light chain and the second light chain include or have the amino acid  sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 18, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 17; (10) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 20, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 19; (11) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 22, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 21; (12) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 23, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 24; (13) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 27, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 28; (14) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 29, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 30, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 31; (15) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 32, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 33; (16) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 34, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 35; (17) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 37, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 36; (18) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 39, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 38; (19) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 41, and the second heavy chain includes or has the amino acid sequence as set  forth in SEQ ID NO: 40; (20) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 29, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 42, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 43; (21) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 45, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 44; (22) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 47, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 46; (23) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 49, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 48; (24) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 51, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 50; (25) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 52, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 2; (26) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 53, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 2; (27) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 55, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 54; (28) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 57, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 56; (29) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 61, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 60; (30) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid  sequence as set forth in SEQ ID NO: 63, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 62; (31) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 65, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 64; (32) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 67, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 66; (33) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 91, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 92, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 93; (34) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 94, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 95, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 96; (35) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 98, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 97; (36) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 99, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 97; (37) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 100, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 10; (38) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 102, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 103; (39) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 104, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 105; (40) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 107, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 106; (41) both of the first light chain and the second  light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 109, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 108; (42) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 111, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 110; (43) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 113, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 112; (44) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 114, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 115; (45) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 117, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 116; (46) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 119, and the second heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 118; (47) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 121, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 120; (48) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 123, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 122; (49) the second light chain includes or has the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 124, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 4; (50) the second light chain includes or has the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 2, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 125; (51) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 143, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO:  142; (52) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 145, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 144; (53) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 147, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 146; (54) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 149, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 148; (55) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 151, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 152; (56) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 153, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 154; (57) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 155, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 154; (58) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 157, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 156; (59) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 160, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 159; (60) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 162, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 161; (61) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 164, and the second heavy chain includes t or has he amino acid sequence as set forth in SEQ ID NO: 163; (62) both of the first light chain and the second light chain include or have the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes or has the amino acid  sequence as set forth in SEQ ID NO: 166, and the second heavy chain includes or has the amino acid sequence as set forth in SEQ ID NO: 165; (63) both of the first light chain and the second light chain include the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 168, and the second heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 167; (64) both of the first light chain and the second light chain include the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 170, and the second heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 169; (65) both of the first light chain and the second light chain include the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 172, and the second heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 171; (66) both of the first light chain and the second light chain include the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 174, and the second heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 173; (67) both of the first light chain and the second light chain include the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 176, and the second heavy chain includes the amino acid sequence as set forth in SEQ ID NO: 175.
[Polynucleotide]
The present disclosure provides an isolated polynucleotide encoding the first heavy chain, the first light chain, the second heavy chain or the second light chain of the fusion protein according to the present disclosure. In some embodiments, the isolated polynucleotide encodes the first heavy chain of the fusion protein according to the present disclosure. In some embodiments, the isolated polynucleotide encodes the second heavy chain of the fusion protein according to the present disclosure.
In another aspect, the present disclosure provides a set of isolated polynucleotides, including the polynucleotide encoding the first heavy chain, the polynucleotide encoding the first light chain, the polynucleotide encoding the second heavy chain and the polynucleotide encoding the second light chain of the fusion protein according to the present disclosure.
The polynucleotide is polymers of DNA, RNA, DNA/RNA hybrids, or modifications thereof. In some embodiments, the polynucleotide is polymers of DNA. The polynucleotide is polymers of RNA. DNA or RNA encoding an anti-SIRPα antibody or antigen binding fragment thereof or a bi-specific molecule described above is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) . The encoding DNA or RNA may also be obtained by synthetic methods.
The present disclosure provides an isolated vector including the isolated polynucleotide.
The present disclosure further provides a host cell including the isolated polynucleotide, the set of insolated polynucleotides or the isolated vector according to the present disclosure.
The present disclosure further provides a method for the production of the fusion protein according to the present disclosure, including introducing a first expression vector encoding the first heavy chain, a second expression vector encoding the first light chain, a third expression vector encoding the second heavy chain and a fourth expression vector encoding the second light chain together into one host cell, or into separate host cells, and expressing the first, second, third and fourth expression vectors under a proper condition. The host cell includes prokaryotic cell and eukaryotic cell.
In some embodiments, the host cells are eukaryotic cells. In some embodiments, the host cells are mammalian cells, such as CHO, COS, HEK293 cells, or Bowes melanoma cells, or a combination thereof.
[Pharmaceutical Composition]
The present disclosure provides a pharmaceutical composition including the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector or the host cell according to the present disclosure, and a pharmaceutically acceptable carrier.
The pharmaceutical composition can be administered in a suitable manner according to the specific applicable form, physicochemical characteristics, etc. of the pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition may be formulated in a form of a freeze-dried formulation or a liquid formulation, which may contain appropriate formulation additives in the art. For example, the above pharmaceutical composition typically contains more than one pharmaceutical carrier, for example, sterile liquid such as water and oil (including petroleum, oils of animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, and sesame oil, etc. ) ) . In the case of intravenous administration of the above pharmaceutical composition, water is a more representative carrier. In addition, saline solution, aqueous glucose, and glycerol solution may also be used as a liquid carrier, especially for an injectable solution. Suitable pharmaceutical excipients are known in the art. The above pharmaceutical composition may also contain a trace amount of wetting agent, emulsifier, or pH buffering agent as required. The administration mode of the pharmaceutical composition is usually parenteral administration, which can be intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous injection, but is not limited thereto, for example, the pharmaceutical composition may be administered by infusion or bolus injection. See, e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000) , which is incorporated by reference in its entirety. Remington’s Pharmaceutical Sciences,  Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) , which is incorporated by reference in its entirety.
With regard to the present disclosure, the active agent, or pharmaceutical composition comprising the same, can be administered to the subject via any suitable route of administration. For example, the active agent can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following discussion on routes of administration is merely provided to illustrate various embodiments and should not be construed as limiting the scope in any way.
[Kit]
The present disclosure provides kits including the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector, the host cell or the pharmaceutical composition described above. Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers etc., as will be readily apparent to a person skilled in the art. Instructions, either as inserts or a labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
[Method Of Uses]
The present disclosure provides use of the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector, the host cell or the pharmaceutical composition according to the present disclosure in the manufacture of a drug for preventing or treating a disease, or in the manufacture of a kit for diagnosing a disease.
The present disclosure provides a method of preventing or treating a disease in a subject in need thereof, including administrating to the subject a therapeutically effective amount of the fusion protein, the isolated polynucleotide, the set of isolated polynucleotides, the isolated vector, the host cell or the pharmaceutical composition according to the present disclosure.
The terms “subject” , “individual, ” and “patient” are used interchangeably herein to refer to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.
In some embodiments, the disease includes tumor or cancer. In some embodiments, the tumor is a solid tumor.
Examples
Example 1: Design and verification of antibody-LIGHT immunocytokine formats
A variety of LIGHT-based immunocytokine (antibody-LIGHT fusion proteins) formats (Figure 1) were developed for tumor-specific delivery. The designs incorporated the extracellular  domain (residues 59-240, residues 74-240, residues 87-240 and residue 91-240) of wild-type LIGHT (Uniprot, #O43557) and an anti-FAP antibody clone 3F2 (patent No. WO 2012020006A) . Some formats linked three LIGHT subunits with polypeptide linkers and connected to the N-terminal or the C-terminal of the heavy chain or light chain of the antibody (Format A, E) . Some formats fused two subunits of LIGHT to the N-terminal or the C-terminal of the heavy chain or the light chain connected with “G” or “GGGGS” linker (s) , with the remaining LIGHT subunit connected to the corresponding site on the opposite chain (Format B, C, D) . In Format C, the heavy chain CH1 and light chain CL were switched.
The codon-optimized genes for LIGHT-based immunocytokines were synthesized by Genewiz and subcloned into the pCI vector (Promega #E1731) and co-transfected into Expi293F cells (ThermoFisher A14527) for protein production. The cells were grown in suspension culture for about six days after transfection and then harvested by centrifugation (7, 000 RPM) at 4℃ for 20 minutes. The supernatant was filtered through a 0.22 μm filter, and affinity-purified using protein A resins. The protein was then eluted by elution buffer (1 M Glycine pH 3.8, 10%glycerol) and neutralized to pH 6.0 with 1M Tris (pH 7.5) . After size exclusion chromatography (SEC) was performed on AKTA equipment, concentrations of the refined products were measured using NanoDrop at 280 nm. The antibody-LIGHT fusion proteins were subjected to electrophoresis on 4-20%Tris-glycine gels (Bio-Rad) under reducing (R) and non-reducing (NR) conditions, by mixing with sample dye with or without DTT (Dithiothreitol) respectively and heated at 95 ℃ for 5 min. The purity of the antibody-LIGHT fusion proteins was further characterized by High Performance Liquid Chromatography (HPLC) .
Among all the tested formats, only Format D (ABC232, ABC233) successfully generated high-quality wild-type LIGHT (residues 74-240) fusion proteins using either the “GGGGS-GGGGSGGGGSGGGGSGGGGT” or “G” linker (Figure 2) , as seen in the HPLC-SEC analysis (Figure 3A) . In contrast, ABC243, the same molecular format expressing a shorter version of LIGHT (residues 87-240) , exhibited poorer expression and higher aggregation than ABC233 on HPLC-SEC (Figure 3B) .
Example 2: Extended design of LIGHT-based immunocytokines
Once the molecular format of the antibody-LIGHT fusion proteins (ABC232 and ABC233) identified, more constructs were generated to test different parameters (Figures 4A-4B) : (1) the linker-1 between CH3 and cytokine was substituted with ISKAKGQPREPQ, ASTKGPSVFPLAPS, RTVAAPSVFIFPPS, KSTGKPTLYNVSLVMSDTAGTCY, G4S, (G4S) 2 (SEQ ID NO: 86) , (G4S) 3, GGGGSGGGGSGGGGSGGGGT or without linker; (2) the linker-2 between the two cytokine units conjugated on the same heavy chain, was substituted with GGGGSGGGGSGGGGSGGGGSGGGGT, G, (G4S) 2; (3) different LIGHT variants, including human WT-LIGHT, human LIGHT mutants, were tested; (4) different IgG Fc hetero- dimerization technologies, such as knobs-into-holes (hereinafter referred to as KIH) and electrostatic steering of CH3 were evaluated; (5) different antibodies, such as anti-FAP, anti-HER2, anti-EGFR, and anti-PDL1 were assessed; (6) bispecific antibodies such as anti-FAP×anti-HER2 were examined; (7) wild-type and a glycosylated IgG1 with N297A mutation were investigated; (8) various IgG subclasses, such as IgG1 and IgG4, were tested. As a result, LIGHT-based immunocytokines ABC475, ABC476, ABC477, ABC538, ABC539, ABC540, ABC541, ABC542, ABC543, ABC580, ABC584, ABC581, ABC639, ABC582, ABC640, ABC641, ABC677, ABC678, ABC679, ABC680, ABC232, ABC233, ABC649, ABC651, ABC650, ABC652, ABC566, ABC578, ABC567, ABC653, ABC568, ABC579, ABC569, ABC583, ABC690, ABC691, ABC770, ABC771, ABC772, ABC773, ABC890, ABC892, ABC687, ABC688, ABC689, ABC985, ABC986, ABC987, ABC988, ABC1054, ABC1056, ABC234, ABC548, ABC549, ABC676, ABC831, ABC893, ABC894, ABC1209, ABC1210, ABC1030, ABC1031, ABC1032, ABC1033, ABC1034, ABC1035, ABC1047, ABC1048, and ABC1056 were expressed and purified, and the amino acid sequences of these LIGHT-based immunocytokines are listed in the table below.
The formats of the immunocytokines produced above were listed as D1, D2, D3, D4, D5 and D6. The schematic diagram of these constructs was shown in Figure 4B.



Anti-FAP monoclonal antibody ABC002 had the light chain as shown in SEQ ID NO: 79 and the heavy chain as shown in SEQ ID NO: 80.
The above LIGHT-based immunocytokines were expressed and purified using similar protocols described in Example 1.
Successful expression and correct assembly of all the LIGHT-based immunocytokines were demonstrated by the presence of a dominant band in the non-reducing gel that corresponds to the molecular weight of correctly assembled molecules. This was further supported by SDS-PAGE analysis of reduced proteins, which showed two heavy chains and one light chain, providing additional evidence of correct assembly (Figures 5A-5D) . Additionally, after one-step protein A affinity purification and size-exclusion chromatography (SEC) polishing, each immunocytokine displayed as a single peak upon SEC-HPLC analysis, such as ABC538, ABC539, ABC770, ABC771, ABC772, and ABC773 (Figure 6 and Figure 7) . Thus, this platform presents a promising approach for generating a variety of LIGHT-based immunocytokines.
Example 3: Binding affinities of LIGHT-based immunocytokines to LTβR, HVEM and DcR3 proteins
Binding affinity to LTβR protein measured by ELISA
LIGHT binding to its cell surface receptor LTβR leads to downstream NF-kB signaling. To evaluate the binding of LIGHT-based immunocytokines to the human-LTβR-Fc (Novoprotein #CX78) and mouse-LTβR-Fc proteins (Acrobiosystems #LTR-H5251) , 96-well ELISA plates (Thermo Fisher) were coated with the LTβR proteins at a concentration of 0.5 μg/mL, and then blocked with 2%BSA-PBS buffer for one hour. Serial dilutions of the LIGHT-based immunocytokines were added with a maximum concentration of 10 nM. The plates were incubated for one hour, washed for four times with PBST, and then incubated with a biotinylated mouse anti-human Ig light chain κ antibody (Biolegend, #316504) . HRP-conjugated streptavidin (Sino Biological) was added, and the plates were further incubated before being washed for three times with PBST and treated with TMB substrate (#34029, Thermo Fisher) . The plates were read at 450 nM on a SpectraMax M5 microplate reader (Molecule Devices) . The ELISA results were analyzed using GraphPad Prism 9.0 software and the EC50s (half maximal effective concentration) were summarized in Table 1 and Table 2.
Table 1: EC50s of LIGHT-based immunocytokines binding to the human LTβR.

Table 2. EC50s of LIGHT-based immunocytokines binding to the mouse LTβR.
The LIGHT-based immunocytokines show specific dose-dependent binding to both human LTβR and mouse LTβR proteins, as shown in Figures 8A-8E and Figures 9A-9E. Fc heterodimerization technologies, such as knobs-into-holes and electronic steering, can facilitate the production and boost activities of LIGHT-based immunocytokine. The holes-LIGHT/knobs-LIGHT-LIGHT and knobs-LIGHT/holes-LIGHT-LIGHT (knobs and holes exchange) designs show similar affinities to LTβR.
The results also suggest that the binding affinities to LTβR were comparable across different linker-1 and linker-2 options, including no linker, G4S, (G4S) 2, (G4S) 3, (G4S) 4, GGGGSGGGGSGGGGSGGGGT, ISKAKGQPREPQ, ASTKGPSVFPLAPS, RTVAAPSVFIFPPS, and KSTGKPTLYNVSLVMSDTAGTCY. Additionally, the binding can be fine-tuned by using different linker-1 and linker-2 options.
Binding affinities to HVEM and DcR3 protein measured by ELISA
Recombinant HVEM proteins were produced by fusing the C-terminal of human HVEM (referred to as hHVEM) residues 39-202 or mouse HVEM (referred to as mHVEM) residues 38-207 with human Fc, while the recombinant DcR3 protein was generated by linking human DcR3 residues 33-300 to the N-terminal of rabbit Fc. The recombinant proteins were expressed in expi293F and affinity-purified using protein A resins and polished using size exclusion column.
Human HVEM, mouse HVEM or human DcR3 proteins were immobilized onto maxiSorp 96-well ELISA plates (Thermo Fisher) at a concentration of 0.5 μg/mL and then blocked with 2%BSA-PBS buffer for one hour. The LIGHT-based immunocytokines were added to the plates at various dilutions, with a maximum concentration of 10 nM. The plates were incubated for an hour, washed four times with PBST, and further incubated with a biotinylated mouse anti-human Ig light chain κ antibody (Biolegend, #316504) . After three washes with PBST, the plates were further incubated with HRP-conjugated streptavidin (Sino Biological) and washed again with PBST. The TMB substrate (#34029, Thermo Fisher) was added to the plates, which were read at 450 nM on a SpectraMax M5 microplate reader (Molecule Devices) . The ELISA results were analyzed using GraphPad Prism 9.0 software and the EC50s (half maximal effective concentration) were summarized in Table 3.
Table 3. EC50s for LIGHT-based immunocytokines binding to human and mouse HVEM and human DcR3.

The binding of LIGHT-based immunocytokines to the other two LIGHT receptors, HVEM and DcR3, was also evaluated. As described in patent application PCT/CN2022/097735, compared to wild-type LIGHT, the LIGHT-1 (SEQ ID NO: 69) homotrimer, and the LIGHT-2 (SEQ ID NO: 70) homotrimer have lower affinities with HVEM. Consistently, the anti-FAP×LIGHT-1 (ABC538) and the anti-FAP×LIGHT-2 (ABC539) immunocytokines also displayed reduced binding capabilities to HVEM (Figure 10B and 10F) .
DcR3 is a soluble protein expressed in humans and cynomolgus macaques but is absent in mice. DcR3 negatively regulates the abilities of LIGHT to activate LTβR and HVEM (Liu et al., 2021) and is upregulated in certain autoinflammatory diseases and cancer patients (Fuchsberger et al., 2021) . Consistent with the lower binding affinities of DcR3 to LIGHT-1 or LIGHT-2 than wild-type LIGHT, DcR3 had significantly weaker binding to ABC538 and ABC539 than to ABC233 (Figures 11A-11F) .
Example 4: Cell binding affinities of LIGHT-based immunocytokines Cell line generation:
Full-length human or mouse HVEM or LTβR (listed in Table 4) was transfected into HEK-293T cells (ATCC) using lipofectamine 3000 (Thermo Fisher, L3000001) . Three days after transfection, the cells were treated with hygromycin B (Millipore Sigma) for fourteen days to obtain stable cell lines. The stable cells were used to evaluate the binding of LIGHT-based immunocytokines to HVEM or LTβR.
Table 4. Protein sequence accession/source.
Cell binding analysis by FACS:
The stably transfected HEK-293T cells expressing LTβR or HVEM were used to evaluate the binding activities of LIGHT-based immunocytokines. The cells were incubated with serially diluted fusion proteins for 30 mins on ice, with a maximum concentration of 50 nM in flow cytometry buffer (PBS, 0.5%BSA, 1 mM EDTA) . The cells were then washed three times with PBS, and the bound immunocytokines were stained with FITC-conjugated mouse anti-human IgG antibody for 15 minutes on ice. The cells were further washed with PBS three times, and the  fluorescence was measured using a Cytek Aurora cytometer (Cytek) and analyzed using FlowJo and GraphPad Prism 9.0 software to determine the EC50. Results were summarized in Tables 5, 6, and 7.
Table 5: EC50s of LIGHT-based immunocytokines binding to 293T cells expressing human LTβR
Table 6: EC50s of LIGHT-based immunocytokines binding to 293T cells expressing mouse LTβR

Table 7: EC50s of LIGHT-based immunocytokines binding to 293T cells expressing human or mouse HVEM

FACS analysis suggested that the LIGHT-based immunocytokines exhibited dose-dependent binding to cells overexpressing human or mouse LTβR (Figures 12A-12G and Figures 13A-13G) . Additionally, the binding affinities of these immunocytokines to cells expressing HVEM were dominated by the LIGHT mutein conjugated to the antibodies (Figures 14A-14D and Figures 15A-15D) . Some LIGHT-based immunocytokines, such as ABC477 and ABC543, exhibited similar binding to human or mouse HVEM-expressing cells as that of the wild-type immunocytokine (ABC233) , while others, such as ABC539, had minimal binding to both types of cells.
Example 5: Pathway activation of anti-FAP×LIGHT immunocytokines in the absence or presence of FAP-expressing cells in trans
The 293T-FAP cell line was generated by transfecting full-length human FAP (Table 4) into HEK-293T cells using lipofectamine 3000 following standard protocols. After three days, the cells were treated with hygromycin (Millipore Sigma) for 14 days to generate stable cell lines.
HeLa-NF-kB and HEK-293T-NF-kB cells were generated by transfecting the cells with the pNL3.2. NF-κB-RE [NlucP/NF-κB-RE/Hygro] vector (Promega #N1111) using lipofectamine 3000. After three days, the cells were treated with hygromycin B (Sigma) and cultured for 14 days in 37℃, 5%CO2. The resulting HeLa-NF-κB reporter cells were used to evaluate the downstream signaling of LTβR activation by the treatment of LIGHT-based immunocytokines.
To evaluate the ability of LIGHT-based immunocytokines to activate HVEM, the HEK293T-NF-κB cells were further transfected with full-length human or mouse HVEM (listed in Table 4) using lipofectamine 3000. After fourteen-day incubation in the presence of hygromycin B and puromycin (Millipore Sigma) , the resulting HEK293T-HVEM-NF-κB stable cells were collected for further analysis.
To evaluate the effect of FAP on LIGHT-induced pathway activation, the reporter cells were either cultured alone or co-cultured with 293T-FAP cells and treated with indicated molecules. Twenty-four hours later, cells were lysed with lysis buffer (Promega #E397A) , and luciferase activity was measured (Promega #E4500) using SpectraMax M5 microplate reader (Molecule Devices) .
LIGHT-based immunocytokines were able to activate NF-κB signaling in HeLa cells harboring endogenous LTβR (Figures 16A-16H and Table 8) . Immunocytokines equipped with the same LIGHT mutein also showed similar capabilities to activate LTβR. Coculturing with 293T-FAP cells enhanced LTβR activation for most immunocytokines (Table 8) , except for ABC543 (Figures 19A-19D) . For instance, when cultured alone, ABC538 and ABC539 had weaker induction capabilities for NF-κB signaling than ABC233 with the wild-type LIGHT (Figures 16A-16D, 19 and 28A-28B, Table 8) . However, they were able to activate NF-κB signaling similarly to the wild-type molecule when co-cultured with HeLa-NF-κB reporter cells and 293T-FAP cells. These results suggest that immunocytokines such as ABC538 and ABC539 can be selectively activated in the tumor microenvironment when FAP-expressing fibroblasts are present.
Table 8: EC50s of LIGHT-based immunocytokines to activate HeLa-NF-κB cells
The abilities of LIGHT-based immunocytokines to activate the HVEM downstream signaling were evaluated with 293T-NF-κB cells overexpressing human or mouse HVEM. The results showed that while the wild-type LIGHT (ABC233) effectively activated downstream NF- κB signaling (Figures 17A-17C, 18A-18C and 28C-28E, Table 9) , ABC538 ABC539, ABC890, and ABC892 exhibited reduced activities to activate the pathway, consistent with their reduced binding to HVEM.
Table 9: EC50s of the LIGHT-based immunocytokines to activate HEK293T-HVEM-NF-κB cells.
Example 6: Characterization of DcR3’s impact on the activity of LIGHT-based
immunocytokines
In addition to HVEM and LTβR, LIGHT also interacts with a decoy receptor, DcR3, which lacks the transmembrane and cytoplasmic segments. This interaction has the potential to disrupt the signaling pathways by sequestering LIGHT away from HVEM and LTbR. While DcR3 expression is typically low in healthy human tissues, it is often significantly upregulated in cancer patients (Wu et al., 2003; Yoo et al., 2022) .
To minimize the binding of LIGHT-based immunocytokines to DcR3, ABC538 and ABC539 were developed from LIGHT-1 and LIGHT-2, two muteins with considerably weaker affinities to DcR3. To investigate the impact of DcR3 on LIGHT-induced LTβR activation, HeLa-NF-κB reporter cells were exposed to 200 μg/ml soluble DcR3-Fc protein, when treated  with LIGHT-based immunocytokines. After 24 hours, the cells were lysed (Promega #E397A) and the luciferase activity was measured with a SpectraMax M5 microplate reader (Molecule Devices) using Promega #E4500.
The addition of DcR3-Fc protein reduced the ABC233-induced LTβR activation by 12 to 26 folds, regardless of the presence or absence of FAP-expressing cells, whereas DcR3 had a minimum effect on ABC538-induced LTβR activation (Figures 20A-20D) . These results suggest that ABC538 is less likely to be affected by soluble DcR3, leading to potentially improved pharmacokinetics (PK) properties and potentially higher levels of active molecules in the tumor microenvironment.
Example 7: Binding of anti-FAP×LIGHT immunocytokines to endogenous mouse LTβR in the presence or absence of coexpressed FAP
Two murine cell lines, BALB/c-3T3-FAP (LTβR+ HVEM-FAP+) and BALB/c-3T3 (LTβR+ HVEM-FAP-) were used to investigate the effect of LIGHT muteins to activate endogenous mouse LTβR. BALB/c-3T3-FAP cell lines were generated by transfecting BALB/c-3T3 cells (ATCC) with mouse Fap (referred to as mFAP) and screened for mFAP expression. The immunocytokines were incubated with 0.1 million purified cells for 30 mins on ice in FACS buffer (PBS, 0.5%BSA, and 1 mM EDTA) . Then the cells were washed with FACS buffer for 3 times, and live/dead cell discrimination was performed using Live/Dead Fixable Near-IR Dead Cell Stain Kit (Life Technologies) . Cells then were stained with 1 μg/mL goat anti-human IgG Fc (FITC) (ab97224, Abcam) on ice for 30 mins in FACS buffer. Cells were then washed with FACS buffer for 3 times before the final flow cytometric analysis. All flow cytometry data were acquired on a Cytek Aurora cytometer (Cytek) and analyzed by FlowJo software (v. 10.5.3, Treestar, Inc. ) . GraphPad Prism 9.0 software was used to determine the EC50.
In the absence of FAP, the binding affinities of all three LIGHT-based immunocytokines to endogenous mLTβR were relatively weak, with EC50 values exceeding 10 nM, despite ABC538 and ABC233 showing better binding than ABC539. However, co-expression of FAP and LTβR on the same cells resulted in a significantly decreased in EC50 values of all three molecules to the nanomolar range (Figures 21A-21B) . Therefore, anti-FAP×LIGHT immunocytokines allowed for the preferential targeting of FAP-expressing cells over those not expressing FAP.
Example 8: Binding of LIGHT-based immunocytokines to endogenous mouse HVEM
Primary human and mouse T cells were utilized to evaluate the interaction between anti-FAP×LIGHT immunocytokines and h/mHVEM. The T cells were negative for LTβR and FAP but positive for HVEM. Human CD3+ T cells were purified from human PBMC with a purity above 97%through negative selection following the manufacturer's protocol (480134, Biolegend) . Mouse CD3+ T cells were purified from pooled splenocytes of BALB/c mice with a purity above 97%by negative selection following the manufacturer's protocol (480031,  Biolegend) . The cells were incubated with twelve 3-fold serial dilutions of LIGHT-based immunocytokines on ice for 30mins in FACS buffer (PBS, 0.5%BSA, and 1 mM EDTA) . The cells were washed with FACS buffer for 3 times, and live/dead cell discrimination was performed using the Live/Dead Fixable Near-IR Dead Cell Stain Kit (Life Technologies) . Afterward, human T cells were stained with either anti-hCD4 (RPA-T4, Biolegend) or anti-hCD8a (RPA-T8, Biolegend) along with 1 μg/mL goat anti-human IgG Fc (FITC) (ab97224, Abcam) on ice for 30 mins in FACS buffer. Mouse T cells were stained with either anti-mCD4 (RM4-5, Biolegend) or anti-mCD8a (53-6.7, Biolegend) along with 1 μg/mL goat anti-human IgG Fc (FITC) (ab97224, Abcam) on ice for 30 mins in FACS buffer. Cells were then washed for three times with FACS buffer, and the cytometry data was collected using a Cytek Aurora cytometer (Cytek) and analyzed using FlowJo. The GraphPad Prism 9.0 software was utilized to determine the EC50, which refers to the concentration of protein required to achieve half of the maximum binding.
The results in Figure 22 showed that ABC538 displayed weaker binding to endogenous HVEM on primary CD4 and CD8 T cells than ABC233, whereas ABC539 exhibited minimal binding to HVEM. These results suggest that ABC538, with its reduced binding to HVEM, may predominantly activate the LTBR rather than the HVEM pathway within the tumor microenvironment.
Example 9: Activation of the LTβR pathway by anti-FAP×LIGHT immunocytokines in the absence or presence of co-expressed FAP
BALB/c-3T3-FAP (LTβR+ HVEM-FAP+) and BALB/c-3T3 (LTβR+ HVEM-FAP-) were used to investigate the effect of LIGHT muteins to activate endogenous LTβR. The cells were plated at 3, 000 cells per well in a 96-well tissue culture plate and incubated with different concentrations of anti-FAP×LIGHT immunocytokines for 24 hours at 37℃. After centrifugation, supernatants were collected to quantify the level of CCL2 protein by ELISA (Cat#432704, Biolegend) with a Tecan Spark reader.
Figure 23A showed that in the absence of FAP expression in 3T3 cells, the activation of the LTβR pathway and the induction of CCL2 protein were primarily determined by the binding affinity of the immunocytokines, with ABC233 being the most effective followed by ABC538 and ABC539. However, the co-expressed FAP significantly increased the capabilities of all three molecules to induce CCL2 expression, particularly at lower concentrations (Figure 23B and 23C) . Since cancer-associate fibroblasts (CAFs) in the tumor stroma play an important role in tumor biology and exhibit abundant expression of both FAP and LTβR, the anti-FAP×LIGHT immunocytokines are designed to preferentially deliver FAP-mediated avidity-driven LTβR stimulation to CAFs in the tumor microenvironment.
Example 10: Activation of the LTβR pathway by anti-FAP×LIGHT immunocytokines in the absence or presence of trans-expressed FAP
To study the effect of trans-expressed FAP on the activation of endogenous LTβR pathway in BALB/c-3T3-WT (LTβR+ HVEM-FAP-) cells, a CT26-FAP cell line was generated by stably transfecting CT26 cells (ATCC) with a plasmid containing mouse FAP gene using lipofectamine 3000 and antibiotic selection. We confirmed that only 3T3 cells, but not CT26-WT or CT26-FAP, could induce CCL2 after stimulation by LIGHT-based immunocytokines.
CT26-WT or CT26-FAP cells were plated at 2.5K/well in a 96-well tissue culture plate and incubated with serially diluted LIGHT-based immunocytokines for one hour at 37℃, 5%CO2. Then 2.5K BALB/c-3T3 cells were added to the culture as responder cells. ELISA assay (Cat#432704, Biolegend) was performed to measure the CCL2 protein level in the media, as a surrogate of the NF-κB pathway activation.
In the presence of CT26-FAP cells, ABC233, ABC538 and ABC539 enhanced the activation of LTβR signaling and the production of CCL2 from 3T3 cells at low concentrations, suggesting that the immunocytokine’s anti-FAP arm binding to the CT26-FAP cells could assist LIGHT-LTβR signaling in trans (Figure 24) .
Example 11: Assessment of in vivo anti-tumor activity of anti-FAP×LIGHT immunocytokines in the CT26-FAP tumor model
To assess the anti-tumor activity of the immunocytokines, 0.4 million CT26-FAP cells in 50 μl of HBSS were mixed with 50 μl of matrigel and implanted subcutaneously into the flank of BALB/c mice. When tumor volumes reached 60-93 mm3 on day 8, mice were grouped based on body weight and tumor volume to ensure similar distribution. Mice were divided into three groups: G1_buffer (negative control) , G2_ABC002 (anti-FAP, negative control) and G3_ABC538, n=9/group. Mice were treated twice a week at 1 nanomole/mouse for the first 3 doses, and 1.5 nanomole/mouse for the next 2 doses. One nanomole of ABC538 is equivalent to 200 μg of protein, while 1.5 nanomoles are equal to 300 μg of protein. ABC002’s equivalent protein for 1 nanomole and 1.5 nanomoles is 144 μg and 200 μg, respectively. Tumor growth was monitored by measuring the tumor volume using calipers at specific time points until day 22.The tumor volume was calculated using the formula: length × width2/2. The mouse’s body weight was also recorded at the indicated time points.
The survival of the mice was calculated using the Kaplan-Meier method. The mice were considered expired if one of the following criteria was met: (1) the tumor volume reached ～2000 mm3, (2) it experienced a body weight loss of 20%, or (3) it died. The results showed that ABC538 had single-agent anti-tumor activity, as indicated by both tumor growth inhibition and mouse survival, as demonstrated in Figure 25.
Example 12: Assessment of in vivo cleavage of LIGHT-based immunocytokines
It has been shown that the metalloprotease family members (MMPs) can cleave the surface LIGHT expressed on activated T cells. Indeed, inhibition of MMPs leads to an increased LIGHT expression on activated T cells (Morel et al., 2000) . For enhanced the efficacy and half-life of the LIGHT-based immunocytokines, the LIGHT moiety in the immunocytokines was further truncated to residues 91-240 to generate cleavage-resistant immunocytokines, such as ABC890, ABC892, ABC1054, ABC1056, ABC1032 and ABC1048.
These cleavage-resistant immunocytokines maintain their binding specificity and functionality. For instance, ABC890 and ABC892, which share the same mutations as ABC538 (LIGHT1, residues 74-240) , exhibit similar binding affinities to human and mouse LTβR, and reduced binding to human and mouse HVEM and human DcR3 (Figure 8D, 9D, 10B, 10F) .
These cleavage-resistant immunocytokines can activate LTβR with comparable EC50s as shown in Table 8, and their activities of activating LTβR were augmented by clustering with cells expressing the antigen. However, since all three molecules have reduced binding for HVEM, their activities of stimulating HVEM were only observed in the presence of cells expressing the antigen, such as 293T-hFAP cells (Figures 28A-28E) . These results suggest that molecules such as ABC538 and ABC539 can be selectively activated in the tumor microenvironment when FAP-expressing fibroblasts are present.
To evaluate the in vivo cleavage of the anti-FAP×LIGHT immunocytokines, 6-week-old female C57BL/6J mice were given a single dose (i.p. ) of 1.5 nanomoles of ABC538, ABC890, or ABC892. Plasma samples were collected 24h and 96h after the injection and then analyzed for cleavage. The plasma cells were diluted 100-fold and incubated with 0.2 million 293T-mFAP cells for 30 mins on ice, followed by washing three times with FACS buffer. The Live/Dead Fixable Near-IR Dead Cell Stain Kit (Life Technologies) was used to determine the live and dead cells, which were then stained with a mixture of 1 μg/mL goat anti-human IgG Fc (FITC) (ab97224, Abcam) and 1 μg/mL anti-hLIGHT (PE) (T5-39, Biolegend) for 30 mins on ice. After washing three times with FACS buffer, the cells were analyzed using flow cytometry on a Cytek Aurora cytometer (Cytek) and the results were analyzed by FlowJo software. Twelve 3-fold serial dilutions of the indicated antibodies were used with the 4-parameter logistics curve-fitting algorithm to generate standard curves. The ratio of LIGHT concentration to hIgG1 concentration was used to evaluate cleavage, with a ratio of 1 indicating no cleavage.
The results showed that ABC538 underwent partial cleavage in vivo, resulting in a 50%ratio of detected LIGHT to detected hIgG1 in mouse plasma at both 24 and 96 hours. In contrast, ABC890 and ABC892, with shorter LIGHT subunit, exhibited higher ratios, suggesting less cleavage, improved stability, and increased bioavailability in vivo (Figure 26) .
Example 13: In vivo anti-tumor activity of anti-FAP×LIGHT immunocytokines in the KPC0826 tumor model
The KPC0826 cell line was derived from a genetically modified KPC mouse model using a previously published protocol (Beatty et al., 2011) . To establish a subcutaneous KPC0826 tumor model, 2.5 million KPC0826 cells in 50 μl HBSS were mixed with 50 μl of matrigel and implanted into the flank of BALB/c mice. The established subcutaneous KPC0826 tumor model showed a relatively high presence of FAP+ CAF cells, around 1-5%of total dissociated tumor cells (data not shown) . On day 0, when tumor volumes reached 70-102 mm3 with an average ～87 mm3, the mice were divided into 6 groups based on body weight and tumor volume: G1_ABC002 (anti-FAP, negative control) , G2_ABC233 (anti-FAP×LIGHT-wt) , G3_ABC538 (anti-FAP×LIGHT-1) , G4_ABC539 (anti-FAP×LIGHT-2) , G5_ABC890 and G6_ABC892, with eight mice per group. Mice were treated twice a week for 6 doses with 1.5 nanomoles of the respective molecules, corresponding to a dose of 200 μg for ABC002 and 300 μg for the other molecules. Tumor growth was monitored by measuring tumor volumes using calipers at indicated time points until day 21. The tumor volume was calculated using the formula: length x width2/2. Tumor growth inhibition (TGI) was compared to the control group (G1_ABC002) and calculated using the DRAP R package (J Transl Med 17, 39, 2019) .
The results demonstrated that all tested anti-FAP×LIGHT immunocytokines exhibited single-agent anti-tumor activity in the KPC0826 model. Among them, ABC890 and ABC892 showed the highest tumor growth inhibition (TGI) , followed by ABC538, ABC233, and ABC539. The cleavage-resistant derivatives, ABC890 and ABC892, demonstrated superior anti-tumor activity compared to ABC538 (shown in Figures 27A-27D) .
Example 14: other TNFSF14 superfamily member immunocytokine
The platform can also be applied to other TNFSF14 superfamily member. 4-1BBL is another TNF superfamily member and its interaction with CD137/TNFRSF9/4-1BB can promote T lymphocyte activation and proliferation. Antibody-4-1BBL fusion protein (ABC676) was successfully expressed using the format D. Lymphotoxin-αββ, similar to engineered LIGHT1, binds to LTβR without engaging HVEM and DcR3, potentially offering enhanced safety and efficacy in providing an anti-tumor effect. With Format D, an immunocytokine equipped with Lymphotoxin-αββ can also be generated (ABC653) .
HeLa-NF-κB reporter cells were utilized to assess the downstream signaling upon LTβR activation following treatment with Lymphotoxin-based immunocytokines. The Lymphotoxin-based immunocytokine (ABC653) demonstrated the ability to activate NF-κB signaling in HeLa cells containing endogenous LTβR, with an EC50 of 0.135 nM (Figure 29) . ABC653 was evaluated for anti-tumor efficacy in genetically modified KPC mice. When tumor volumes reached 70-102 mm3, mice were treated twice a week for 8 doses with 1.5 nanomoles of ABC653 and the mAb control (ABC002) . Tumor growth was monitored by measuring tumor volumes using calipers, and the tumor volume was calculated using the formula: length x  width2/2. The results demonstrated that anti-FAP×Lymphotoxin (ABC653) immunocytokines exhibited single-agent anti-tumor activity in the KPC0826 model (Figure 30) .
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Morel, Y., Schiano de Colella, J.M., Harrop, J., Deen, K.C., Holmes, S.D., Wattam, T.A., Khandekar, S.S., Truneh, A., Sweet, R.W., Gastaut, J.A., et al. (2000) . Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor. J Immunol 165, 4397-4404. 10.4049/jimmunol.165.8.4397.

Claims (48)

  1. A fusion protein, comprising:
    a first cytokine fragment linked to the C-terminal of a first CH2-CH3 fragment, comprising a first cytokine molecule, or a first cytokine molecule and a third cytokine molecule that are tandem linked, and
    a second cytokine fragment linked to the C-terminal of a second CH2-CH3 fragment, comprising a second cytokine molecule, or a second cytokine molecule and a fourth cytokine molecule that are tandem linked,
    the first CH2-CH3 fragment and the second CH2-CH3 fragment form a dimer, totally three cytokine molecules are linked to the dimer.
  2. The fusion protein according to claim 1, wherein the first cytokine fragment comprises the first cytokine molecule, and the second cytokine fragment comprises the second cytokine molecule and the fourth cytokine molecule that are tandem linked, or
    the first cytokine fragment comprises the first cytokine molecule and a third cytokine molecule that are tandem linked, and the second cytokine fragment comprises the second cytokine molecule.
  3. The fusion protein according to claim 1 or 2, wherein the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are selected from the group consisting of tumor necrosis factor, interleukin, lymphokine, interferon, colony stimulating factor, chemokine and growth factor.
  4. The fusion protein according to any one of claims 1-3, wherein the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are LIGHT, lymphotoxin α, lymphotoxin β, or 4-1BBL.
  5. The fusion protein according to any one of claims 1-4, wherein the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are wild type LIGHT (preferably human LIGHT) , truncated LIGHT or mutein thereof; optionally, wild type LIGHT, truncated LIGHT or mutein thereof comprises amino acid sequence of any one of SEQ ID NOs: 25, 26, 58, 59, 68-78, 126-141, 158, 177, 178 and 180;
    the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are wild type 4-1BBL (preferably human 4-1BBL) , truncated 4-1BBL or mutein thereof; optionally, wild type 4-1BBL, truncated 4-1BBL or mutein thereof comprises amino acid sequence of SEQ ID NO: 179;
    the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are wild, truncated lymphotoxin α or lymphotoxin β, or mutein thereof; optionally, the wild, truncated lymphotoxin α or lymphotoxin β, or mutein thereof comprises amino acid sequence of SEQ ID NO: 181, 182.
  6. The fusion protein according to any one of claims 1-5, wherein the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are truncated LIGHT, optionally, the truncated LIGHT includes an amino acid sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 78.
  7. The fusion protein according to any one of claims 1-6, wherein the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule independently are LIGHT mutein; optionally, the LIGHT mutein includes an amino acid sequence set forth in one of SEQ ID NOs: 25-26, 58-59, 69-77, 126-141, 158, 177, 178 and 180.
  8. The fusion protein according to any one of claims 1-7, wherein the dimer is an Fc domain, which is modified to improve the correct assembly of the fusion protein.
  9. The fusion protein according to claim 8, wherein the Fc domain is derived from IgG, which includes IgG1, IgG2, IgG3, and IgG4, optionally the Fc domain is derived from human IgG1 or IgG4 Fc domain.
  10. The fusion protein according to any one of claims 1-9, wherein the first CH2-CH3 fragment and the second CH2-CH3 fragment comprise one or more modifications selected from the group consisting of knobs-into-holes, electrostatic steering of CH3 (such as DDKK) , DuoBody, SEEDbodies, cFAE, XmAb, Azymetric, and
    optionally, the modifications comprise knobs-into-holes, and/or DDKK.
  11. The fusion protein according to any one of claims 1-10, wherein the first CH2-CH3 fragment and the second CH2-CH3 fragment form a human IgG1 Fc domain with mutation N297A.
  12. The fusion protein according to any one of claims 1-11, wherein the first CH2-CH3 fragment or the second CH2-CH3 fragment further comprises one or more mutations selected from the group consisting of Y349C, T366S, L368A, and Y407V; and the other one comprises mutations S354C and/or T366W.
  13. The fusion protein according to any one of claims 1-12, wherein the first cytokine fragment links to the C-terminal of the first CH2-CH3 fragment directly or via a linker; and/or
    the second cytokine fragment link to the C-terminal of the second CH2-CH3 fragment directly or via a linker.
  14. The fusion protein according to any one of claims 1-13, wherein the C terminal of the CH3 domain in the first CH2-CH3 fragment is fused to the N terminal of the first cytokine fragment directly or via a first linker A; and/or the C terminal of the CH3 domain in the second CH2-CH3 fragment is fused to the N-terminal of the second cytokine fragment directly or via a first linker B.
  15. The fusion protein according to any one of claims 1-14, wherein the first cytokine molecule is fused to the third cytokine molecule directly or via a second linker A; and/or
    the second cytokine molecule is fused to the fourth cytokine molecule directly or via a second linker B.
  16. The fusion protein according to any one of claims 1-15, wherein the C terminal of the first cytokine molecule is fused to the N terminal of the third cytokine molecule directly or via a second linker A; and/or the C terminal of the second cytokine is fused to the N terminal of the fourth cytokine directly or via a second linker B.
  17. The fusion protein according to any one of claims 1-16, wherein the first linker A, the first linker B, the second linker A, and the second linker B independently are absent or are selected from the group consisting of VH-CH1 linker (SEQ ID NO: 82) , VL-CL linker (SEQ ID NO: 83) , CH2-CH3 linker (SEQ ID NO: 81) , IgM tail linker (SEQ ID NO: 84) , GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 89) , G, and (GGGGS) n, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
    optionally, the first linker A and the first linker B independently are absent or are selected from the group consisting of SEQ ID NOs: 81, 82, 83, 84 and 89, G, and (GGGGS) n, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  18. The fusion protein according to any one of claims 1-17, wherein the first linker A and the first linker B independently are absent or comprise the amino acid sequence as set forth in any one of SEQ ID NOs: 81-90.
  19. The fusion protein according to any one of claims 1-18, wherein the first linker A and the first linker B are the same or different.
  20. The fusion protein according to any one of claims 1-19, wherein the second linker A and the second linker B independently are absent or are G, GGGGSGGGGSGGGGSGGGGT, or (GGGGS) n, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  21. The fusion protein according to any one of claims 1-20, wherein the second linker A and the second linker B independently of each other are absent or comprise the amino acid G or the amino acid sequence as set forth in any one of SEQ ID NOs: 85-89.
  22. The fusion protein according to any one of claims 1-21, comprising a first antigen-binding fragment and a second antigen-binding fragment, which are connected to the N-terminal of the first CH2-CH3 fragment and the second CH2-CH3 fragment, respectively.
  23. The fusion protein according to claim 22, wherein the first antigen-binding fragment and the second antigen-binding fragment independently are selected from the group consisting of a diabody, a Fab, a Fab’, a F (ab’) 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv’) , a disulfide stabilized diabody (ds diabody) , a single-chain Fv (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody formed from a portion of an antibody including one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment.
  24. The fusion protein according to claim 22 or 23, wherein the first antigen-binding fragment is an scFv or a Fab that specifically binds to a first antigen; and the second antigen-binding fragment is a Fab that specifically binds to a second antigen.
  25. The fusion protein according to any one of claims 22-24, comprising
    a first heavy chain, from N terminal to C terminal, including the operably linked first antigen-binding fragment, the first CH2-CH3 fragment, and the first cytokine fragment;
    a second heavy chain, from the N terminal to the C terminal, including the operably linked second antigen-binding fragment, the second CH2-CH3 fragment, and the second cytokine fragment.
  26. The fusion protein according to any one of claims 22-25, further comprising
    a first light chain, which pairs with the first antigen-binding fragment to form a heterodimer; and/or
    a second light, which pairs with the second antigen-binding fragment to form a heterodimer.
  27. The fusion protein according to any one of claims 22-26, wherein the first antigen and the second antigen independently of each other are selected from the group consisting of FAP, HER2, PDL-1, PD-1, EGFR, VEGFR, VEGF, CCR8, OX-40, 41BB, Angiopoietin-2, IL-4Rα, BCMA, Blys, BTNO2, C5, CD122, CD13, CD133, CD137, CD138, CD16a, CD19, CD20, CD22, CD27, CD28, CD3, CD30, CD33, CD38, CD40, CD47, CD-8, CEA, CGPR/CGRPR, CSPGs, CTLA4, CTLA-4, DLL-4, EpCAM, factor IXa, factor X, GITR, GP130, Her3, HSG, ICOS, IGF1, IGF1/2, IGF-1R, IGF2, IGFR, IL-1, IL-12, IL-12p40, IL-13, IL-17A, IL-1β, IL-23, IL-5, IL-6, IL-6R, Lag-3, LAG3 , MAG, Met, NgR, NogoA, OMGp, OX40, PDGFR, PSMA, RGMA, RGMB, SARS-CoV-2, Te38, TIM-3, TNF, TNFα, TROP-2, and TWEAK.
  28. The fusion protein according to any one of claims 22-27, wherein the first antigen and the second antigen independently of each other are selected from the group consisting of FAP, HER2, PDL-1, and EGFR.
  29. The fusion protein according to any one of claims 22-28, wherein both the first antigen and the second antigen are FAP, HER2, EGFR, or PDL-1, or the first antigen is HER2 and the second antigen is FAP.
  30. The fusion protein according to any one of claims 1-29, wherein the fusion protein is monovalent or bivalent, monospecific or bispecific.
  31. The fusion protein according to any one of claims 1-30, wherein the first cytokine molecule, the second cytokine molecule, and the third cytokine molecule have the same amino acid sequence; or
    the first cytokine molecule, the second cytokine molecule, and the fourth cytokine molecule have the same amino acid sequence.
  32. The fusion protein according to any one of claims 26-31, wherein the first light chain and the second light chain independently comprise an amino acid sequence as set forth in SEQ ID NO: 1, 29, 91 or 94.
  33. The fusion protein according to any one of claims 22-32, wherein the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 23, 28, 31, 32, 35, 37, 39, 41, 43, 45, 47, 49, 51, 52, 53, 55, 57, 61, 63, 65, 67, 93, 96, 98, 99, 100, 102, 104, 107, 109, 111, 113, 114, 117, 119, 121, 123, 125, 143, 145, 147, 149, 151, 153, 157, 155, 160, 162, 164, 166, 167, 169, 171, 173 or 175.
  34. The fusion protein according to any one of claims 22-33, wherein the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 24, 27, 30, 33, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 60, 62, 64, 66, 92, 95, 97, 101, 103, 105, 106, 108, 110, 112, 115, 116, 118, 120, 122, 124, 142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163, 165, 168, 170, 172, 174 or 176.
  35. The fusion protein according to any one of claims 22-34, wherein the fusion protein is select from the following:
    (1) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 3, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 2;
    (2) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 4, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 2;
    (3) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 6, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 5;
    (4) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 8, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 7;
    (5) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 10, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 9;
    (6) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 11, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 11;
    (7) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 14, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 13;
    (8) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 16, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 15;
    (9) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 18, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 17;
    (10) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 20, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 19;
    (11) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 22, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 21;
    (12) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 23, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 24;
    (13) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 27, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 28;
    (14) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 29, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 30, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 31;
    (15) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 32, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 33;
    (16) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 34, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 35;
    (17) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 37, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 36;
    (18) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 39, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 38;
    (19) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 41, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 40;
    (20) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 29, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 42, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 43; or
    (21) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 45, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 44;
    (22) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 47, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 46;
    (23) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 49, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 48;
    (24) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 51, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 50;
    (25) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 52, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 2;
    (26) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 53, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 2;
    (27) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 55, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 54;
    (28) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 57, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 56;
    (29) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 61, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 60;
    (30) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 63, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 62;
    (31) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 65, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 64;
    (32) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 67, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 66;
    (33) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 91, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 92, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 93;
    (34) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 94, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 95, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 96;
    (35) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 98, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 97;
    (36) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 99, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 97;
    (37) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 100, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 101;
    (38) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 102, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 103;
    (39) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 104, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 105;
    (40) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 107, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 106;
    (41) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 109, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 108;
    (42) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 111, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 110;
    (43) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 113, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 112;
    (44) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 114, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 115;
    (45) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 117, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 116;
    (46) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 119, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 118;
    (47) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 121, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 120;
    (48) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 123, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 122;
    (49) the second light chain comprises the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 124, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 4;
    (50) the second light chain comprises the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 2, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 125;
    (51) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set  forth in SEQ ID NO: 143, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 142;
    (52) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 145, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 144;
    (53) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 147, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 146;
    (54) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 149, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 148;
    (55) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 151, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 150;
    (56) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 153, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 152;
    (57) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 155, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 154;
    (58) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 157, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 156;
    (59) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 160, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 159;
    (60) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set  forth in SEQ ID NO: 162, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 161;
    (61) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 164, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 163;
    (62) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 166, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 165;
    (63) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 168, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 167;
    (64) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 170, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 169;
    (65) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 172, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 171;
    (66) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 174, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 173;
    (67) both of the first light chain and the second light chain comprise the amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 176, and the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 175.
  36. An isolated polynucleotide encoding the first heavy chain, the first light chain, the second heavy chain or the second light chain of the fusion protein according to any one of claims 1-35.
  37. A set of isolated polynucleotides, comprising the polynucleotide encoding the first heavy chain, the polynucleotide encoding first light chain, the polynucleotide encoding second  heavy chain and the polynucleotide encoding second light chain of the fusion protein according to any one of claims 1-35.
  38. An isolated vector comprising the isolated polynucleotide according to claim 36 or 37.
  39. A host cell comprising the isolated polynucleotide according to claim 36, the set of isolated polynucleotides according to claim 37 or the isolated vector according to claim 38.
  40. A pharmaceutical composition comprising the fusion protein according to any one of claims 1-35, the isolated polynucleotide according to claim 36, the set of isolated polynucleotides according to claim 37, the isolated vector according to claim 38 or the host cell according to claim 39, and a pharmaceutically acceptable carrier.
  41. A kit, comprising the fusion protein according to any one of claims 1-35, the isolated polynucleotide according to claim 36, the set of isolated polynucleotides according to claim 37, the isolated vector according to claim 38, the host cell according to claim 39 or the pharmaceutical composition according to claim 40.
  42. Use of the fusion protein according to any one of claims 1-35, the isolated polynucleotide according to claim 36, the set of isolated polynucleotides according to claim 37, the isolated vector according to claim 38, the host cell according to claim 39, the pharmaceutical composition according to claim 40 or the kit according to claim 41 in the manufacture of a drug for preventing or treating a disease, or in the manufacture of a kit for diagnosing a disease.
  43. The use according to claim 42, wherein the disease comprises tumor, optionally solid tumor.
  44. A method of diagnosing, preventing or treating a disease in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the fusion protein according to any one of claims 1-36, the isolated polynucleotide according to claim 36, the set of isolated polynucleotides according to claim 37, the isolated vector according to claim 38, the host cell according to claim 39, the pharmaceutical composition according to claim 40 or the kit of claim 41.
  45. The method according to claim 44, wherein the disease comprises tumor, optionally solid tumor.
  46. A method for production of the fusion protein according to any one of claims 1-35, comprising introducing a first expression vector encoding the first heavy chain, a second expression vector encoding the first light chain, a third expression vector encoding the second heavy chain and/or a fourth expression vector encoding the second light chain together into one host cell, or into separate host cells, and expressing the first, second, third and fourth expression vectors under a proper condition.
  47. The method according to claim 46, wherein the host cells are eukaryotic cells.
  48. The method according to claim 46 or 47, wherein the host cells are mammalian cells.
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