TNF superfamily member immune cytokines and uses thereof

Abstract

The present disclosure relates to antibody-cytokine fusion proteins, in particular TNF superfamily member fusion proteins (immunocytokines) and uses thereof. The fusion protein shows antineoplastic activity.

Description

Technical Field

[0001] This disclosure relates to the fields of biomedical or biopharmaceutical technology, and specifically to antibody-cytokine fusion proteins, particularly TNF superfamily member fusion proteins (immune cytokines) and their uses. Background Technology

[0002] The statements in this section are provided only as background information in relation to this disclosure and do not necessarily constitute prior art.

[0003] Cytokines are a class of small proteins (5kDa-20kDa) that act as cell signaling molecules to regulate inflammation and cellular activity. 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 broad and diverse group of pro-inflammatory or anti-inflammatory factors, classified into families based on structural similarity or the structural similarity of their receptors. Cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factor, hormones, and growth factors.

[0004] TNFSF14 (TNF superfamily member 14), also known as LIGHT (homogeneous with lymphotoxin, exhibiting inducible expression and competing with herpes simplex virus glycoprotein D for herpesvirus entry mediators, which are receptors 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 β receptor), as well as the decoy receptor DcR3. HVEM is expressed on the surface of various immune cells, such as T cells, B cells, NK cells, 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 cells, matrix cells, immature dendritic cells, and other bone marrow cells, but has not been found on lymphocytes. Activation of LTβR signaling is crucial for the recruitment and organization of immune cells, leading to the development of lymphoid organs and tertiary lymphoid structures (TLS). The immune stratum corneum (TLS) consists of B-cell and T-cell regions, dendritic cells, and other immune cells. Its presence in tumor tissue is 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.

[0005] While cytokine-based immunotherapies have shown promising efficacy in treating cancer, their lack of specificity in targeting tumor tissue can lead to systemic immune responses and severe side effects. One potential solution is to conjugate cytokines with antibodies that have tumor-targeting specificity. For example, three copies of hmLIGHT were fused to the N-terminus of an Fc antibody and then assembled with an anti-EGFR half-antibody (Tang et al., 2016). In a second study, LIGHT was conjugated to various forms of anti-fibronectin F8 antibodies. For example, three tandemly linked mouse LIGHT copies were fused to the C-terminus of either the heavy or light chain of a mAb or scFv. Although several antibody-LIGHT forms were evaluated, robust protein expression without significant aggregation was only observed when the tandemly linked mouse LIGHT was fused to a single-chain biantibody (Stringhini et al., 2021). Furthermore, mouse LIGHT fused with a vascular-targeting peptide (VTP) has been shown to induce lymphogenesis in solid tumors (Johansson-Percival et al., 2017). However, many of these forms have limitations in clinical practice due to a lack of efficacy or challenging manufacturing processes. Therefore, there remains a continued need for antibody-cytokine fusion protein platforms that provide robust manufacturing and mitigation of potential toxicity for tumor-specific cytokine delivery. Summary of the Invention

[0006] This disclosure provides an antibody-cytokine fusion protein platform for tumor-specific cytokine delivery. The platform enables correct assembly, and molecules generated by the platform exhibit robust formation in T-cell and B-cell regions within the tumor microenvironment, leading to promising antitumor efficacy.

[0007] In a first aspect of this disclosure, a fusion protein is provided, the fusion protein comprising:

[0008] A first cytokine fragment is attached to the C-terminus of a first CH2-CH3 fragment, the first cytokine fragment comprising a first cytokine molecule, or a first cytokine molecule and a third cytokine molecule connected in series; and a second cytokine fragment is attached to the C-terminus of a second CH2-CH3 fragment, the second cytokine fragment comprising a second cytokine molecule, or a second cytokine molecule and a fourth cytokine molecule connected in series, the first CH2-CH3 fragment and the second CH2-CH3 fragment forming a dimer, such as a homodimer or a heterodimer, with a total of three cytokine molecules attached to the dimer.

[0009] In some implementations, the first CH2-CH3 fragment and the second CH2-CH3 fragment are modified to improve the correct assembly of the fusion protein.

[0010] In some embodiments, the first cytokine fragment comprises a first cytokine molecule, and the second cytokine fragment comprises a second cytokine molecule and a fourth cytokine molecule connected in tandem. In some embodiments, the first cytokine fragment comprises a first cytokine molecule and a third cytokine molecule connected in tandem, and the second cytokine fragment comprises a second cytokine molecule.

[0011] In some embodiments, the first antigen-binding fragment is an scFv that specifically recognizes the first antigen, and / or the second antigen-binding fragment is an scFv or Fab that specifically recognizes the second antigen.

[0012] The fusion protein provided herein comprises a first heavy chain operatively linked from the N-terminus to the C-terminus to a first antigen-binding fragment, a first CH2-CH3 fragment, and a first cytokine fragment; and a second heavy chain operatively linked from the N-terminus to the C-terminus to a second antigen-binding fragment, a second CH2-CH3 fragment, and a second cytokine fragment.

[0013] The fusion protein provided herein comprises a first light chain that pairs with a first antigen-binding fragment to form a heterodimer; and / or a second light chain that pairs with a second antigen-binding fragment to form a heterodimer.

[0014] In a second aspect of this disclosure, a separate polynucleotide is provided that encodes a first heavy chain, a first light chain, a second heavy chain, or a second light chain of a fusion protein according to this disclosure. This disclosure further provides a set of separate polynucleotides comprising polynucleotides encoding a first heavy chain, a first light chain, a second heavy chain, and a second light chain of a fusion protein according to this disclosure.

[0015] In a third aspect of this disclosure, an isolated vector is provided that comprises an isolated polynucleotide according to this disclosure.

[0016] In a fourth aspect of this disclosure, a host cell line is provided containing isolated polynucleotides, a set of isolated polynucleotides, or isolated vectors according to this disclosure.

[0017] In a fifth aspect of this disclosure, a pharmaceutical composition is provided comprising a fusion protein according to this disclosure, isolated polynucleotides, a group of isolated polynucleotides, isolated vectors or host cell lines, and a pharmaceutically acceptable carrier.

[0018] In a sixth aspect of this disclosure, a kit is provided comprising a fusion protein according to this disclosure, isolated polynucleotides, a group of isolated polynucleotides, isolated vectors or host cell lines or pharmaceutical compositions.

[0019] In a seventh aspect of this disclosure, use is provided of a fusion protein according to this disclosure, or a polynucleotide isolated according to this disclosure, or a group of polynucleotides isolated according to this disclosure, or a vector isolated according to this disclosure, or a host cell line according to this disclosure, or a pharmaceutical composition according to this disclosure in the preparation of a medicament for the prevention or treatment of a disease, or in the preparation of a kit for the diagnosis of a disease.

[0020] In an eighth aspect of this disclosure, a method for preventing or treating a disease in a subject in need is provided, the method comprising administering to the subject a therapeutically effective amount of a fusion protein according to this disclosure, or a separated polynucleotide according to this disclosure, or a group of separated polynucleotides according to this disclosure, or a separated carrier according to this disclosure, or a host cell according to this disclosure, or a pharmaceutical composition according to this disclosure.

[0021] In a ninth aspect of this disclosure, a method for generating a fusion protein according to this disclosure is provided, the method comprising introducing a first expression vector encoding a first heavy chain, a second expression vector encoding a first light chain, a third expression vector encoding a second heavy chain, and a fourth expression vector encoding a second light chain together into a host cell line, or into a separate host cell line, and expressing the first expression vector, the second expression vector, the third expression vector, and the fourth expression vector under appropriate conditions. Attached Figure Description

[0022] The following is a description of the accompanying drawings, which are presented for the purpose of illustrating the exemplary embodiments disclosed herein and not for the purpose of limiting those exemplary embodiments.

[0023] Figure 1 This describes the different forms of LIGHT-based immune cytokines that have been evaluated.

[0024] Figure 2 An SDS-PAGE analysis of LIGHT-based immune cytokines in form D is shown. Analysis was performed under reducing (R) and non-reducing (NR) conditions, and protein markers (M) were included for comparison.

[0025] Figure 3A and Figure 3B HPLC analysis of LIGHT-based immune cytokines ABC233 and ABC234 after one-step protein A purification is shown.

[0026] Figure 4A and Figure 4BThe schematic form of LIGHT-based immune cytokines and the extended design of LIGHT-based immune cytokines are presented respectively.

[0027] Figure 5 The SDS-PAGE analysis of LIGHT-based immune cytokines (AD) under reducing (R) and non-reducing (NR) conditions, as well as under protein marker (M) conditions, is shown.

[0028] Figure 6 The SEC-HPLC analysis of purified LIGHT-based immune cytokines ABC538 and ABC539 is shown.

[0029] Figure 7 The SEC-HPLC analysis of purified LIGHT-based immune cytokines ABC770-ABC773 is shown.

[0030] Figure 8 An ELISA analysis of the binding of LIGHT-based immune cytokines (AE) to the extracellular domain of human LTβR is shown, n=2.

[0031] Figure 9 An ELISA analysis of the binding of LIGHT-based immune cytokines (AE) to the extracellular domain of mouse LTβR is shown, n=2.

[0032] Figure 10 A to Figure 10 D shows an ELISA analysis of the binding of LIGHT-based immune cytokines to the extracellular domain of human HVEM cells; Figure 10 E to Figure 10 H shows an ELISA analysis of the binding of LIGHT-based immune cytokines to the extracellular domain of mouse HVEM cells.

[0033] Figure 11 An ELISA analysis of the binding of LIGHT-based immune cytokines (AF) to human DcR3 is shown.

[0034] Figure 12 This study demonstrates the binding of LIGHT-based immune cytokines (AG) to 293T cells overexpressing human LTβR. Mean fluorescence intensity (MFI) and concentration (Conc.) are shown.

[0035] Figure 13 The binding of LIGHT-based immune cytokines (AG) to 293T cells overexpressing mouse LTβR is shown. MFI: Median fluorescence intensity.

[0036] Figure 14The binding of LIGHT-based immune cytokines (AD) to 293T cells overexpressing human HVEM is shown. MFI: Median fluorescence intensity.

[0037] Figure 15 The binding of LIGHT-based immune cytokines (AD) to 293T cells overexpressing mouse HVEM is shown. MFI: Median fluorescence intensity.

[0038] Figure 16 This indicates that FAP-overexpressing 293T cells (293T-FAP) do not contain ( Figure 16 A to Figure 16 D) and existence ( Figure 16 E to Figure 16 In the case of H), the NF-κB activity of HeLa-NF-κB cells induced by LIGHT-based immune cytokines was reported. The relative luciferase activity (RLU) was also reported.

[0039] Figure 17 This study demonstrates LIGHT-based immunocytokine (AC)-induced NF-κB activity in 293T-NF-κB cells overexpressing human HVEM. Relative luciferase activity (RLU) is also reported.

[0040] Figure 18 This study demonstrates LIGHT-based immunocytokine (AC)-induced NF-κB activity in 293T-NF-κB cells overexpressing mouse HVEM. Relative luciferase activity (RLU) is also reported.

[0041] Figure 19 The study demonstrates LIGHT-based immune cytokine (AD)-induced NF-κB activity in HeLa-NF-κB cells in the absence and presence of 293T-FAP cells.

[0042] Figure 20 The effect of DcR3 on the activity of LIGHT-based immune cytokines (AD) was demonstrated.

[0043] Figure 21 This demonstrates the interaction between LIGHT-based immune cytokines and BALB / c-3T3 cells ( Figure 21 A) and BALB / c-3T3 cells overexpressing FAP ( Figure 21 The combination of B).

[0044] Figure 22 This demonstrates the interaction between LIGHT-based immune cytokines and primary human CD4. + T cells ( Figure 22 A) Original CD8 + T cells (Figure 22 B) Primary mouse CD4 + T cells ( Figure 22 C) and primary mouse CD8 + T cells ( Figure 22 The combination of D).

[0045] Figure 23 The results show that in BALB / c-3T3-WT (wild type) Figure 23 A) and BALB / c-3T3-FAP cells ( Figure 23 B) LIGHT-based immune cytokines induce CCL2. Figure 23 C presents the same results when grouped by different molecules.

[0046] Figure 24 This shows the addition of ABC233 ( Figure 24 A), ABC538 ( Figure 24 B) or ABC539 Figure 24 Following C), LTβR transactivates 3T3 cells in the presence of CT26-FAP or CT26-WT.

[0047] Figure 25 The in vivo antitumor activity of anti-FAP×LIGHT immune cytokines in the CT26-FAP model was demonstrated, and the treatment timeline was presented. Figure 25 A) Tumor volume ( Figure 25 B) Survival curve ( Figure 25 C) and weight ( Figure 25 D).

[0048] Figure 26 The ratio of LIGHT to hIgG1 detected after administration of immune cytokines is shown, indicating the cleavage of LIGHT fractions released from immune cytokines in vivo.

[0049] Figure 27 illustrates the in vivo antitumor activity of anti-FAP×LIGHT immune cytokines in the KPC model. Figure 27A summarizes the treatment timeline of Example 13; Figure 27B The individual tumor volumes for each group are shown; Figure 27C shows the average tumor volume for each group. Figure 27D This demonstrates tumor growth inhibition (TGI) in each group.

[0050] Figure 28 A to Figure 28 B shows the NF-κB activity in HeLa-NF-κB cells induced by ABC890 and ABC892, respectively, in the absence or presence of 293T-FAP cells. Figure 28 C to Figure 28E shows the NF-κB activity induced by ABC890, ABC892 and ABC538 in HEK293T-NF-κB cells overexpressing human HVEM, in the absence or presence of 293T-FAP cells.

[0051] Figure 29 This demonstrates HeLa-NF-κB activity induced by ABC653.

[0052] Figure 30 The in vivo antitumor activity of ABC653 in the KPC0826 model was demonstrated. Detailed Implementation

[0053] This disclosure is explained in more detail below. This description is not intended to be a detailed list of all different ways in which the invention can be implemented or all features that can be added to the invention. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be removed from that embodiment. Furthermore, various variations and additions to the embodiments presented herein will be apparent to those skilled in the art without departing from this disclosure. Therefore, the following description is intended to illustrate some specific embodiments of the disclosure, rather than to exhaustively specify all its substitutions, combinations, and variations.

[0054] As used herein, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural referents. Thus, for example, a reference to “the method” includes one or more methods and / or steps of the type described herein, which will become apparent to those skilled in the art upon reading this disclosure, etc.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While similar or equivalent methods and substances may also be used in the practice of testing with respect to this disclosure, preferred substances and methods are described herein. The following terminology will be used in describing and claiming protection for this disclosure.

[0056] [TERMS]

[0057] As used in this article, the term "fusion" refers to the connection of a protein with another biological material, such as a protein, peptide, nucleic acid molecule, or any other biomolecule or a portion thereof.

[0058] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to biopolymers comprising units derived from amino acids linked by peptide bonds. Proteins can consist of one or more chains.

[0059] As used herein, the term "domain" refers to a portion of a molecule or structure that shares common physical, chemical, or structural properties, such as similar hydrophobic or polar properties. Exemplary domains may include protein-binding domains, DNA-binding domains, ATP-binding domains, or similar folded structures, such as globular or helical features. Domains can be identified based on their homology to conserved structural or functional motifs.

[0060] As used herein, the term "antibody" is used in its broadest sense and encompasses a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and their antigen-binding fragments, provided they exhibit the desired antigen-binding activity. The term "antibody" can refer to a full-length antibody or its antigen-binding fragment. A full-length antibody comprises two heavy chains and two light chains. 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 N-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. These domains are typically the most variable part of the antibody (relative to other antibodies of the same class) and contain the antigen-binding site. In some embodiments, following the variable domain of the heavy chain / domain / region from the N-terminus to the C-terminus are three constant heavy domains (CH1, CH2, and CH3). Similarly, following the variable domain of the light chain / domain / region from the N-terminus to the C-terminus are constant light (CL) domains.

[0061] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including biantibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibodies (ds biantibodies), single-chain Fv (scFv), scFv dimers (bivalent biantibodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camel-derived single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds an antigen but does not contain a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen that is bound to a parent antibody or a fragment of a parent antibody (e.g., a parental scFv).

[0062] The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains a complete light chain that pairs with the VH and CH1 domains of the heavy chain.

[0063] "Fv" is the smallest antibody fragment containing complete antigen recognition and antigen binding sites. This fragment consists of a dimer of a tightly non-covalently associated heavy chain variable region domain and a light chain variable region domain. The folding of these two domains produces six hypervariable rings (three rings each in the heavy and light chains), which provide the conformation for antigen binding and confer antigen-binding specificity to the antibody.

[0064] As used herein, the term "single-chain variable fragment" or "scFv" is a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin (e.g., mouse or human) are covalently linked to form a VH::VL heterodimer. The heavy chain (VH) and light chain (VL) are linked directly or via a peptide-coding linker or spacer region that links the N-terminus of the VH to the C-terminus of the VL, or vice versa.

[0065] As used herein, the term "CH2-CH3 fragment" refers to a portion of a constant region and encompasses at least the CH2 and CH3 domains. In some embodiments, two CH2-CH3 fragments may form an antibody Fc domain. An "Fc domain" refers to a pair of associated Fc regions. The two Fc regions dimerize to produce the Fc domain. The two Fc regions within the Fc domain may be identical or different from each other.

[0066] As used herein, the terms "Fc region" or "Fc chain" refer to a polypeptide containing the CH2-CH3 domain of an IgG molecule, and in some cases, include a hinge. In the EU designation of human IgG1, the CH2-CH3 domain contains amino acids 231 to 447, and the hinge is 216 to 230. The human IgG Fc region is particularly useful in this disclosure and can be an Fc region derived from human IgG1, IgG2, IgG3, or IgG4.

[0067] As used herein, the term "specific binding" refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically recognizes a target (which may be an epitope) is an antibody that binds to the target with greater affinity, stronger interaction, and / or longer duration compared to its binding to other targets. The binding specificity of an antibody or antigen-binding domain can be experimentally determined by methods known in the art. Such methods include, but are not limited to, Octet, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIACORE™ assays, and peptide scanning.

[0068] As used herein, the term "linker" refers to a polypeptide used to couple two molecules. A linker is typically a segment of amino acids, such as primarily glycine and / or serine. Linkers can have lengths of up to 100 amino acids, such as about 1, 2, 5, 7, 10, 15 amino acids, or up to about 15, 20, 25, 30, 35, 50, 75, or 100 amino acids.

[0069] As used herein, the term “VH-CH1 linker” refers to a polypeptide and its derivatives that link VH and CH1 in naturally occurring antibodies.

[0070] As used herein, the term “VL-CL linker” refers to a polypeptide and its derivatives that link VL and CL in naturally occurring antibodies.

[0071] As used herein, the term "CH2-CH3 linker" refers to a polypeptide and its derivatives that link the CH2 and CH3 molecules in naturally occurring antibodies.

[0072] As used in this article, the term "IgM tail adapter" refers to polypeptides and their derivatives in naturally occurring IgM antibodies.

[0073] When describing a linker, the term "derivative" refers to a linker having an amino acid sequence that differs from the naturally occurring sequence by one or more amino acids, for example, containing one or more amino acid insertions, deletions, or substitutions relative to the naturally occurring sequence.

[0074] As used herein, the term "cytokine" should be understood to mean any protein, its analogue, or functional fragment capable of stimulating or inducing a biological response against a preselected cell type. Therefore, a variety of cytokines are expected to be incorporated herein. Useful cytokines include, for example, tumor necrosis factor (TNF), interleukin (IL), lymphokines (L), colony-stimulating factor (CSF), interferon (IFN), chemokines, growth factors (including species variants and truncated analogues capable of stimulating or inducing such biological responses), members of the TNF family (such as LIGHT), lymphotoxin α, lymphotoxin β, 4-1BBL, etc. The term "cytokine" should also be understood to encompass any derivative of wild-type cytokines that includes at least a significant portion (such as at least about 50%) that modifies and maintains any desired function.

[0075] When describing cytokines, the term "wild-type (WT)" refers to a cytokine having a naturally occurring amino acid sequence encoded by the germline genome of a given species. A species may have one or more wild-type sequences (e.g., one canonical wild-type sequence and one or more non-canonical wild-type sequences). Wild-type cytokine sequences may include sequences truncated at the N-terminus and / or C-terminus relative to the sequence encoded by the open reading frame. Wild-type cytokine sequences may be mature forms of cytokines that have been processed to remove N-terminal and / or C-terminal residues. Wild-type cytokines may lack a signal peptide or may contain a signal peptide (e.g., a signal peptide may be added to the N-terminus of a wild-type cytokine).

[0076] When describing cytokines, the terms “derivative,” “variant,” “mutant protein,” and “mutant” are used interchangeably herein to refer to a cytokine having an amino acid sequence that differs from the wild-type sequence by one or more amino acids (e.g., having one or more amino acid insertions, deletions, or substitutions relative to the wild-type sequence).

[0077] As used herein, the term "naturally occurring" means a sequence of natural origin, which means that it is wholly or partially non-synthetic and exists or arises in nature. More preferably, as used herein, the term "naturally occurring" means a sequence of natural origin, which means that the entire sequence is non-synthetic and exists or arises in nature.

[0078] 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 (NKs), etc. LIGHT can stimulate cells expressing HVEM, such as T cells, B cells, NK cells, and dendritic cells, leading to cell activation and inflammation. Binding of LIGHT to another receptor, LTβR (expressed on stromal cells, bone marrow cells, and some epithelial cells), can lead to the development of lymphoid organs and tertiary lymphoid structures (TLS). The term "LIGHT" should also be understood to encompass WT-LIGHT and any derivatives of WT-LIGHT.

[0079] As used herein, the term "mortar and pestle structure" is used in its broadest sense and covers a variety of situations, such as a CH1 domain of one heavy chain having a pestle mutation and a CH1 domain of another heavy chain having a mortar mutation, a CH2 domain of one heavy chain having a pestle mutation and a CH2 domain of another heavy chain having a mortar mutation, and / or a CH3 domain of one heavy chain having a pestle mutation and a CH3 domain of another heavy chain having a mortar mutation. For example, and generally, "mortar and pestle structure" can refer to an intra-interfacial modification between two antibody heavy chains in the CH3 domain: i) in the CH3 domain of one heavy chain (first CH3 domain), an amino acid residue is substituted by another amino acid residue with a large side chain, thereby creating a protrusion ("pestle") at the interface in the first CH3 domain; ii) in the CH3 domain of another heavy chain (second CH3 domain), an amino acid residue is substituted by another amino acid residue with a smaller side chain, thereby creating a cavity ("mortar") at the interface in the second CH3 domain, in which the protrusion ("pestle") in the first CH3 domain can be placed.

[0080] As used herein, the term "DDKK" refers to a modification that mediates an electrostatic redirection effect, used to enhance antibody Fc heterodimer formation, particularly as described by Gunasekaran et al. (J. Biol. Chem. 2010, 19637:19646). The Fc portion of one heavy chain includes the mutations K392D and K409D (referred to as the DD mutation), and the Fc portion of the other heavy chain includes the mutations E356K and D399K (referred to as the KK mutation). Numbering is based on the EU index.

[0081] An "isolated" polynucleotide encoding the protein or a portion or fragment thereof described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminating nucleic acid molecule that is typically associated with the environment in which the nucleic acid molecule was produced. Preferably, the isolated polynucleotide does not associate with any components associated with the production environment. The isolated polynucleotide encoding the protein or a portion or fragment thereof described herein exists in forms or settings other than those found in nature. Therefore, isolated polynucleotides are distinguished from polynucleotides encoding the protein or a portion or fragment thereof that is naturally present in cells. Isolated polynucleotides include nucleic acid molecules contained in cells that typically contain nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0082] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which the vector has been introduced. Some vectors are capable of guiding the expression of nucleic acids to which they are efficiently linked. Such vectors are referred to herein as "expression vectors."

[0083] As used herein, the term "host cell" or "host cell line" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transfected cells," which include primary transfected cells and their derived progeny, regardless of passage number. Progeny may not be identical to parental cells in terms of nucleic acid content and / or may contain mutations. This includes mutant progeny that have the same function or biological activity as those screened or selected in the initially transfected cells. Host cells can be mammalian cells, plant cells, yeast cells, and / or bacterial cells. Host cells may contain nucleic acid molecules or vectors of this disclosure as extrachromosomal (attachment) replicators, or more preferably, nucleic acid molecules or vectors of this disclosure integrated into the host cell genome.

[0084] As used herein, the term "transfected" or "transfection" refers to the process of transferring or introducing exogenous nucleic acids into host cells. "Transfected" cells are cells that have been transfected with exogenous nucleic acids. Cells include primary subject cells and their progeny.

[0085] As used herein, the term "pharmaceutical composition" refers to a formulation which is present in a form that allows the bioactivity of the active ingredient to be effective and does not contain any additional components that would have unacceptable toxicity to the individual to whom the formulation will be administered. Such formulations may be sterile.

[0086] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation adjuvant, or carrier conventional in the art used with a therapeutic agent, which together constitute a "pharmaceutical composition" for individual administration. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation in which it is used.

[0087] The terms “cancer” and “tumor” are used interchangeably herein and refer to any of a number of diseases characterized by any of the following: uncontrolled and abnormal proliferation of cells, the ability of affected cells to spread locally or via the bloodstream and lymphatic system to other parts of the body (metastasis), and any of a number of characteristic structural and / or molecular features. Examples of cancers that can be treated with the fusion proteins of this disclosure include solid tumors and hematologic cancers. Additional examples of cancers that can be treated with the fusion proteins of this disclosure include breast cancer, lung cancer, brain cancer, bone cancer, liver cancer, kidney cancer, colon cancer, head and neck cancer, ovarian cancer, hematopoietic system cancers (e.g., leukemia), and prostate cancer. Additional examples of cancers that can be treated with multivalent and multispecific antibodies include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. The most specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, and various types of head and neck cancers. Other cancers and tumors that can be treated with the fusion protein of this disclosure are described herein or otherwise known in the art.

[0088] As used herein, the term "agent" encompasses a fusion protein according to the present disclosure, an isolated polynucleotide according to the present disclosure, a group of isolated polynucleotides according to the present disclosure, an isolated vector according to the present disclosure, a host cell according to the present disclosure, and a pharmaceutical composition according to the present disclosure.

[0089] The "effective amount" of the agent disclosed herein is an amount sufficient to achieve a specifically indicated purpose in order to induce an observable change in the level of one or more biological activities associated with target cells binding to the fusion protein. This change may increase the activity level of the target. This change may decrease the activity level of the target. The "effective amount" may be determined empirically and in a conventional manner, in relation to the stated purpose.

[0090] The term "therapeuticly effective amount" refers to the amount by which the agent of this disclosure or another drug effectively "treats" a disease or disorder in a patient or mammal. In the case of cancer, a therapeutically effective amount of the agent of this disclosure can reduce angiogenesis and neovascularization; reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., to a certain extent slow down or stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow down to a certain point or stop) tumor metastasis; to a certain extent inhibit tumor growth or tumor incidence; stimulate an immune response against cancer cells and / or to a certain extent alleviate one or more symptoms associated with cancer. See the definition of "treatment" herein. "Therapeuticly effective amount" can also refer to an effective amount that achieves the desired therapeutic outcome within the necessary dose and time period. The therapeutically effective amount of the agent of this disclosure can vary depending on factors such as an individual's pathology, age, sex, and weight, as well as the ability of the composition to elicit the desired response in an individual. Therapeuticly effective amount is also the amount by which the toxicity or adverse effects of the therapeutic composition outweigh the beneficial therapeutic effects.

[0091] As used herein, “treatment” is a method for obtaining a beneficial or desired outcome, including clinical outcomes. For the purposes of this application, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: relief of one or more symptoms caused by a disease, reduction of the severity of the disease, stabilization of the disease (e.g., prevention or delay of disease progression), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease status, provision of disease remission (partial or complete), reduction of the dosage of one or more other medications required to treat the disease, delay of disease progression, increase or improvement of quality of life, increase in weight gain and / or prolongation of survival. “Treatment” also encompasses a reduction in the pathological consequences of cancer, such as, for example, tumor volume. The methods of this application consider any or more of these therapeutic aspects.

[0092] In the context of cancer, the term "treatment" includes any or all of the following: inhibiting cancer cell growth, inhibiting cancer cell replication, reducing overall tumor burden, and improving one or more disease-related symptoms.

[0093] The term "inhibition" refers to a reduction or cessation of any phenotypic trait, or a reduction or cessation of the occurrence, extent, or likelihood of that trait. "Reduction" or "inhibition" means a reduction, decrease, or prevention of activity, function, and / or quantity compared to a reference. In some embodiments, "reduction" or "inhibition" means the ability to cause an overall reduction of 20% or greater. In some embodiments, "reduction" or "inhibition" means the ability to cause an overall reduction of 50% or greater. In yet another embodiment, "reduction" or "inhibition" means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or greater.

[0094] As used herein, the term “prevention” includes providing preventative measures for the onset or recurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with it.

[0095] [ANTIBODY- CYTOKINE FUSION PROTEINS]

[0096] This disclosure provides an immune cytokine, which is an antibody-cytokine fusion protein. The fusion protein comprises a first cytokine fragment and a second cytokine fragment, wherein the first cytokine fragment and the second cytokine fragment are respectively linked to the C-terminus of the first CH2-CH3 fragment and the second CH2-CH3 fragment.

[0097] In some implementations, the first CH2-CH3 fragment and the second CH2-CH3 fragment form a dimer, such as a homodimer or a heterodimer.

[0098] 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 a human IgG molecule, which encompasses IgG1, IgG2, IgG3, and IgG4 subclasses. In some embodiments, the first CH2-CH3 fragment and the second CH2-CH3 fragment form a human IgG1 Fc domain or an IgG4 Fc domain.

[0099] In some implementations, the first CH2-CH3 fragment and the second CH2-CH3 fragment form human IgG1 Fc with the mutation N297A, which refers to the mutation of amino acid N (asparagine) at position 297 (EU numbering) to amino acid (alanine). Other mutations like this have similar instructions.

[0100] The amino acids in this disclosure refer to organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups, as well as side chains specific to each amino acid. Preferably, the amino acids in this disclosure are 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), and tyrosine (Y).

[0101] Optionally, the Fc domain may be modified as needed to improve proper assembly of the fusion protein. In some embodiments, the first CH2-CH3 fragment and the second CH2-CH3 fragment contain one or more modifications selected from the group consisting of: mortar and pestle structures, electrostatic orientation of CH3 (e.g., DDKK), DuoBody, SEEDbodies, cFAE, XmAb, Azymetric, and BEAT. ® In some embodiments, the first CH2-CH3 segment and the second CH2-CH3 segment contain one or more modifications selected from the group consisting of a pestle and mortar structure and DDKK.

[0102] In some embodiments, the first CH2-CH3 fragment contains a mortar mutation, and the second CH2-CH3 fragment contains a mortar mutation.

[0103] In some embodiments, the first CH2-CH3 fragment contains the DD mutation, and the second CH2-CH3 fragment contains the KK mutation. In some embodiments, the first CH2-CH3 fragment contains the KK mutation, and the second CH2-CH3 fragment contains the DD mutation.

[0104] In some implementations, one of the first CH2-CH3 fragment and the second CH2-CH3 fragment contains one or more mutations selected from the group consisting of Y349C, T366S, L368A and Y407V; and the other contains the mutation S354C and / or the mutation T366W as defined by the EU number.

[0105] In some embodiments, the first CH2-CH3 fragment contains mutations Y349C, T366S, L368A, and Y407V to form a "mortar," and the second CH2-CH3 fragment contains mutations S354C and T366W to form a "pestle."

[0106] The C-terminus of the dimer is completely linked to three cytokine molecules. In some embodiments, the first cytokine fragment comprises at least one wild-type, truncated, or mutant cytokine molecule. In some embodiments, the first cytokine fragment comprises one or two wild-type, truncated, or mutant cytokine molecules. In some embodiments, the second cytokine fragment comprises at least one wild-type, truncated, or mutant cytokine molecule. In some embodiments, the second cytokine fragment comprises one or two wild-type, truncated, or mutant cytokine molecules.

[0107] 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 first cytokine molecule and a third cytokine molecule connected in tandem. In some embodiments, the second cytokine fragment includes a second cytokine molecule and a fourth cytokine molecule connected in tandem.

[0108] In some embodiments, the first cytokine fragment comprises a first cytokine molecule, and the second cytokine fragment comprises a second cytokine molecule and a fourth cytokine molecule connected in tandem. In some embodiments, the first cytokine fragment comprises a first cytokine molecule and a third cytokine molecule connected in tandem, and the second cytokine fragment comprises a second cytokine molecule.

[0109] In some embodiments, the cytokine molecules in the first cytokine fragment and the second cytokine fragment are identical. In some embodiments, the first, second, and third cytokine molecules are identical. In some embodiments, the first, second, and fourth cytokine molecules are identical.

[0110] In some embodiments, the cytokine molecules in the first cytokine fragment and the cytokine molecules 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.

[0111] The first, second, third, and fourth cytokine molecules are independently selected from the group consisting of tumor necrosis factor, interleukin, lymphokine, interferon, colony-stimulating factor, chemokine, and growth factor. In some embodiments, the first, second, third, and fourth cytokine molecules are independently selected from the group consisting of LIGHT, lymphotoxin α, lymphotoxin β, and 4-1BBL, such as wild-type LIGHT (preferably human LIGHT), truncated LIGHT, or mutant proteins thereof.

[0112] In some embodiments, the first cytokine molecule, the second cytokine molecule, the third cytokine molecule, and the fourth cytokine molecule are independently truncated LIGHTs (i.e., with amino acid sequences shorter than the wild type). Exemplary truncated LIGHTs are WT-LIGHT (74-240) (SEQ ID NO: 68) and WT-LIGHT (91-240) (SEQ ID NO: 78).

[0113] In some embodiments, the first cytokine molecule, the second cytokine molecule, the third cytokine molecule, and the fourth cytokine molecule independently comprise the truncated LIGHT amino acid sequence shown in SEQ ID NO: 68 or SEQ ID NO: 78.

[0114] In some embodiments, the first cytokine molecule, the second cytokine molecule, and the third cytokine comprise or have the amino acid sequence shown in SEQ ID NO: 68 or SEQ ID NO: 78. In some embodiments, the first cytokine molecule, the second cytokine molecule, and the fourth cytokine comprise or have the amino acid sequence shown in SEQ ID NO: 68 or SEQ ID NO: 78.

[0115] In some embodiments, the first cytokine molecule, the second cytokine molecule, the third cytokine molecule, and the fourth cytokine molecule are independently LIGHT mutant proteins. Exemplary LIGHT mutant proteins are LIGHT mutant protein-1 (74-240), LIGHT mutant protein-2 (74-240), LIGHT mutant protein-9 (74-240), LIGHT mutant protein-11 (74-240), LIGHT mutant protein-18 (74-240), LIGHT mutant protein-22 (74-240), LIGHT mutant protein-29 (74-240), LIGHT mutant protein-37 (74-240), and LIGHT mutant protein-52 (74-240), (SEQ ID NO: 69-77); LIGHT mutant protein-1 (59-240), LIGHT mutant protein-1 (91-240), LIGHT mutant protein-1 (87-240), LIGHT mutant protein-90 (74-240), LIGHT mutant protein-63 (74-240), LIGHT mutant protein-86 (74-240), LIGHT mutant protein-88 (74-240), LIGHT mutant protein-60 (74-240), LIGHT mutant protein-92 (74-240), (SEQ ID NO: 126-134); LIGHT mutant protein-93 (74-240), LIGHT mutant protein-41 (74-240), (SEQ ID NO: 25-26); LIGHT mutant protein-42 (74-240), LIGHT mutant protein-2 (91-240), (SEQ ID NO: 126-134); LIGHT mutant protein-93 (74-240), LIGHT mutant protein-41 (74-240), (SEQ ID NO: 126-134 ...42 (74-240), LIGHT mutant protein-2 (91-240), (SEQ ID NO: 126-134); LIGHT mutant protein-93 (74-240), LIGHT mutant protein-41 (74-240), (SEQ ID NO: 126-134); LIGHT mutant protein-42 (74-2 NO:58-59); LIGHT mutant protein-95 (91-240), LIGHT mutant protein-96 (91-240), LIGHT mutant protein-97 (91-240), LIGHT mutant protein-98 (91-240), LIGHT mutant protein-99 (91-240), LIGHT mutant protein-100 (91-240), LIGHT1 (91-240) (M150V), (SEQ ID NO: 135-141); LIGHT mutant protein-101 (91-240) (SEQ ID NO: 158).

[0116] In some embodiments, the first cytokine molecule, the second cytokine molecule, the third cytokine molecule, and the fourth cytokine molecule independently contain the mutated LIGHT amino acid sequence shown in SEQ ID NO: 25, 26, 58, 59, 69-77, 126-141, 158, 177, 178, and 180.

[0117] In some embodiments, the first cytokine molecule, the second cytokine molecule, and the third cytokine comprise or have the amino acid sequences shown 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 fourth cytokine comprise or have the amino acid sequences shown in SEQ ID NO: 25, 26, 58, 59, 69-77, 126-141, 158, 177, 178, and 180.

[0118] In some embodiments, the first cytokine molecule, the second cytokine molecule, the third cytokine molecule, and the fourth cytokine molecule are independently wild-type 4-1BBL (preferably human 4-1BBL), truncated 4-1BBL, or mutant proteins thereof. In some embodiments, wild-type 4-1BBL, truncated 4-1BBL, or mutant proteins thereof comprise the amino acid sequence of SEQ ID NO:179.

[0119] In some embodiments, the first cytokine molecule, the second cytokine molecule, the third cytokine molecule, and the fourth cytokine molecule are independently wild-type, truncated lymphotoxin α or lymphotoxin β, or mutant proteins thereof. In some embodiments, the wild-type, truncated lymphotoxin α or lymphotoxin β, or mutant proteins thereof comprise the amino acid sequences of SEQ ID NO: 181, 182.

[0120] In some embodiments, 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.

[0121] In some embodiments, the first cytokine fragment is attached directly or via a linker to the C-terminus of the first CH2-CH3 fragment. In some embodiments, the second cytokine fragment is attached directly or via a linker to the C-terminus of the second CH2-CH3 fragment.

[0122] In some embodiments, the C-terminus of the CH3 domain in the first CH2-CH3 fragment is fused directly or via a first linker A to the N-terminus of the first cytokine fragment, and the C-terminus of the CH3 domain in the second CH2-CH3 fragment is fused directly or via a first linker B to the N-terminus of the second cytokine fragment.

[0123] In some embodiments, the first cytokine molecule fuses with the third cytokine molecule directly or via a second connector A. In some embodiments, the second cytokine molecule fuses with the fourth cytokine molecule directly or via a second connector B.

[0124] In some embodiments, the C-terminus of the first cytokine molecule is fused directly to or via a second connector A to the N-terminus of the third cytokine molecule. In some embodiments, the C-terminus of the second cytokine molecule is fused directly to or via a second connector B to the N-terminus of the fourth cytokine molecule.

[0125] In some embodiments, the first connector A, the first connector B, the second connector A, and the second connector B are independently absent or selected from the group consisting of: VH-CH1 connector ASTKGPSVFPLAPS (SEQ ID NO: 82); VL-CL connector RTVAAPSVFIFPPS (SEQ ID NO: 83); CH2-CH3 connector ISKAKGQPREPQ (SEQ ID NO: 81); IgM tail connector 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 connector A, the first connector B, the second connector A, and the second connector B are independently absent from each other or selected from the group consisting of: VH-CH1 connector; VL-CL connector; CH2-CH3 connector; IgM tail connector; G; and (GGGGS)n, where n = 1, 2, 3, 4, 5, or 6. In some embodiments, the first connector A, the first connector B, the second connector A, and the second connector B are independently absent from each other or selected from the group consisting of: VH-CH1 connector; VL-CL connector; CH2-CH3 connector; IgM tail connector; GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 89), G; and (GGGGS)n, where n = 1, 2, 3, or 4.

[0126] In some embodiments, first connector A and first connector B are independently absent from each other or selected from the group consisting of: VH-CH1 connector; VL-CL connector; CH2-CH3 connector; IgM tail connector; and (GGGGS)n, n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, first connector A and first connector B are independently absent from each other or selected from the group consisting of: VH-CH1 connector; VL-CL connector; CH2-CH3 connector; IgM tail connector; and (GGGGS)n, n=1, 2, 3, 4, 5 or 6. In some embodiments, first connector A and first connector B are independently absent from each other or selected from the group consisting of: VH-CH1 connector; VL-CL connector; CH2-CH3 connector; IgM tail connector; GGGGSGGGGSGGGGSGGGT (SEQ ID NO: 89), G and (GGGGS)n, n=1, 2, 3 or 4.

[0127] In some embodiments, first connector A and first connector B are independently absent from each other or contain amino acid sequences as shown in any of SEQ ID NO: 81-90.

[0128] In some implementations, first connector A and first connector B are the same. In other implementations, first connector A and first connector B are different.

[0129] In some embodiments, the second linker A and the second linker B are independently absent from each other or are selected from the group consisting of: G; and (GGGGS)n, where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the second linker A and the second linker B are independently absent from each other or are selected from the group consisting of: G; and (GGGGS)n, where n = 1, 2, 3, 4, 5, or 6. In some embodiments, the second linker A and the second linker B are independently absent from each other or are selected from the group consisting of: G; and (GGGGS)n, where n = 1, 2, 3, or 4.

[0130] In some embodiments, the second connector A and the second connector B independently lack or contain an amino acid sequence of G or an amino acid sequence as shown in any of SEQ ID NO: 85-88. In some embodiments, the second connector A and the second connector B independently lack or contain an amino acid sequence of G or an amino acid sequence as shown in any of SEQ ID NO: 85-89.

[0131] In some embodiments, the fusion protein provided herein comprises a first antigen-binding fragment and a second antigen-binding fragment, which are respectively linked to the N-terminus of the first CH2-CH3 fragment and the second CH2-CH3 fragment.

[0132] In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment are independently selected from the group consisting of: biantibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibodies (ds biantibodies), single-chain Fv (scFv), scFv dimers (bivalent biantibodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camel-derived single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragments.

[0133] In some embodiments, the first antigen-binding fragment is an scFv that specifically binds to the first antigen. In some embodiments, the second antigen-binding fragment is an scFv that specifically binds to the second antigen.

[0134] In some embodiments, the scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL) that can pair with each other to form an Fv fragment that specifically binds to the antigen. In the scFv, the N-terminus of the VH is directly or via a linker to the C-terminus of the VL, or the C-terminus of the VH is directly or via a linker to the N-terminus of the VL. Those skilled in the art can select suitable linkers for use in this disclosure; for example, the linker between the VH and VL is (GGGGS)n, where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker between the VH and VL is (GGGGS)4 (SEQ ID NO: 88).

[0135] In some embodiments, the first antigen-binding fragment is a Fab that specifically binds to the first antigen. In some embodiments, the second antigen-binding fragment is a Fab that specifically binds to the second antigen.

[0136] In some embodiments, the first antigen-binding fragment is an scFv that specifically binds to the first antigen; and the second antigen-binding fragment is an scFv or Fab that specifically binds to the second antigen. In some embodiments, the first antigen-binding fragment is a Fab that specifically binds to the first antigen; and the second antigen-binding fragment is a Fab or scFv that specifically binds to the second antigen. In some embodiments, the first antigen-binding fragment is an scFv or Fab that specifically binds to the first antigen; and the second antigen-binding fragment is a Fab that specifically binds to the second antigen. In some embodiments, both the first and second antigen-binding fragments are Fab. In some embodiments, the first antigen-binding fragment is an scFv, and the second antigen-binding fragment is a Fab. In some embodiments, the first antigen-binding fragment is a Fab, and the second antigen-binding fragment is an scFv.

[0137] In some implementations, the first and second antigens are independently 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 and second antigens are independently selected from FAP, HER2, PDL-1, and EGFR.

[0138] 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.

[0139] The fusion proteins described in this article are monovalent or bivalent, and single- or double-specific.

[0140] In some implementations, the first antigen and the second antigen are both FAP, or both the first antigen and the second antigen are HER2, or both the first antigen and the second antigen are EGFR, or both the first antigen and the second antigen are PDL-1, or the first antigen is HER2 and the second antigen is FAP.

[0141] In some embodiments, the fusion protein provided herein comprises: a first heavy chain comprising, from the N-terminus to the C-terminus, a first antigen-binding fragment, a first CH2-CH3 fragment, and a first cytokine fragment operably linked together; and a second heavy chain comprising, from the N-terminus to the C-terminus, a second antigen-binding fragment, a second CH2-CH3 fragment, and a second cytokine fragment operably linked together.

[0142] like Figure 4A As shown, the fusion protein has a "Y" shape. The first heavy chain used in this paper refers to the heavy chain of the left arm of the "Y" shape, and the second heavy chain used in this paper refers to the heavy chain of the right arm of the "Y" shape.

[0143] In some implementations, the first heavy chain and the second heavy chain are the same. In other implementations, the first heavy chain and the second heavy chain are different.

[0144] In some embodiments, the fusion protein provided herein comprises a first light chain that pairs with a first antigen-binding fragment to form a heterodimer. In some embodiments, the fusion protein provided herein comprises a second light chain that pairs with a second antigen-binding fragment to form a heterodimer.

[0145] In some implementations, the first light chain and the second light chain are the same; in this case, they are collectively referred to as "light chains." In other implementations, the first light chain and the second light chain are different.

[0146] In some embodiments, the first antigen-binding fragment is scFv, in which case the first light chain is absent. In some embodiments, the second antigen-binding fragment is scFv, in which case the second light chain is absent.

[0147] In some embodiments, the first light chain and the second light chain independently comprise an amino acid sequence as shown in any one of SEQ ID NO: 1, 29, 91, and 94. In some embodiments, the first light chain and the second light chain independently have an amino acid sequence as shown in any one of SEQ ID NO: 1, 29, 91, and 94. In some embodiments, both the first light chain and the second light chain have an amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, both the first light chain and the second light chain have an amino acid sequence as shown in SEQ ID NO: 29. In some embodiments, both the first light chain and the second light chain have an amino acid sequence as shown in SEQ ID NO: 91. In some embodiments, both the first light chain and the second light chain have an amino acid sequence as shown in SEQ ID NO: 94. In some embodiments, the first light chain has an amino acid sequence as shown in SEQ ID NO: 1, and the second light chain has an amino acid sequence as shown in SEQ ID NO: 29. In some embodiments, the second light chain has an amino acid sequence as shown in SEQ ID NO: 1, and the first light chain has an amino acid sequence as shown in SEQ ID NO: 29. In some embodiments, the first light chain has the amino acid sequence shown in SEQ ID NO: 1, and the second light chain has the amino acid sequence shown in SEQ ID NO: 91. In some embodiments, the second light chain has the amino acid sequence shown in SEQ ID NO: 1, and the first light chain has the amino acid sequence shown in SEQ ID NO: 91. In some embodiments, the first light chain has the amino acid sequence shown in SEQ ID NO: 1, and the second light chain has the amino acid sequence shown in SEQ ID NO: 94. In some embodiments, the second light chain has the amino acid sequence shown in SEQ ID NO: 1, and the first light chain has the amino acid sequence shown in SEQ ID NO: 94.

[0148] In some embodiments, the second heavy chain comprises or has an amino acid sequence as shown 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.

[0149] In some embodiments, the first heavy chain comprises or has an amino acid sequence as shown 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.

[0150] In some implementations, the fusion protein is selected from the following:

[0151] (1) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 3, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 3; (2) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 4, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 2; (3) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 6, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 5; (4) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 8, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 3. (5) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 10, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 9; (6) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 12, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 11; (7) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 14, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 13; (8) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 16, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 13. The amino acid sequence shown in SEQ ID NO: 15; (9) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 18, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 17.(10) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 20, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 19; (11) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 22, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 21; (12) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 23, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 24; (13) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 27, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 28. (14) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 29, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 30, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 31; (15) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 32, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 33; (16) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 34, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 35; (17) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 29, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 30. (37) The first light chain contains or has the amino acid sequence shown in SEQ ID NO: 36; (18) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 39, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 38.(19) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 41, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 40; (20) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 29, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 42, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 43; (21) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 45, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 44; (22) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 47, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 48. (23) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 49, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 48; (24) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 51, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 50; (25) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 52, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 2; (26) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 53, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 54. The amino acid sequence shown in SEQ ID NO: 1; (27) the first light chain and the second light chain both contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 55, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 54.(28) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 57, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 56; (29) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 61, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 60; (30) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 63, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 62; (31) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 65, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 66. (32) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 67, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 66; (33) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 91, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 92, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 93; (34) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 94, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 95, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 96; (35) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 98, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 96. The amino acid sequence shown in SEQ ID NO: 97; (36) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 99, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 97.(37) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 100, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 10; (38) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 102, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 103; (39) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 104, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 105; (40) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 107, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 105. (41) The first light chain and the second light chain both contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 109, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 108; (42) The first light chain and the second light chain both contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 111, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 110; (43) The first light chain and the second light chain both contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 113, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 112; (44) The first light chain and the second light chain both contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 109, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 108; The first light chain contains or has the amino acid sequence shown in SEQ ID NO: 114, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 115; (45) the first light chain and the second light chain both contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 117, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 116.(46) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains the amino acid sequence shown in SEQ ID NO: 119, and the second heavy chain contains the amino acid sequence shown in SEQ ID NO: 118; (47) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 121, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 120; (48) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 123, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 122; (49) The second light chain contains or has the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 124, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 122. (50) The second light chain contains or has the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 2, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 125; (51) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 143, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 142; (52) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 145, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 144; (53) Both the first and second light chains contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 147, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 144. The amino acid sequence shown in NO: 146; (54) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 149, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 148.(55) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 151, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 152; (56) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 153, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 154; (57) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 155, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 154; (58) Both the first light chain and the second light chain contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 157, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 154. (59) The first light chain and the second light chain both contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 160, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 159; (60) The first light chain and the second light chain both contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 162, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 161; (61) The first light chain and the second light chain both contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 164, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 163; (62) The first light chain and the second light chain both contain or have the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 163, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 164, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 163; The first light chain contains the amino acid sequence shown in SEQ ID NO: 166, and the second heavy chain contains or has the amino acid sequence shown in SEQ ID NO: 165; (63) both the first light chain and the second light chain contain the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains the amino acid sequence shown in SEQ ID NO: 168, and the second heavy chain contains the amino acid sequence shown in SEQ ID NO: 167.(64) Both the first and second light chains contain the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains the amino acid sequence shown in SEQ ID NO: 170, and the second heavy chain contains the amino acid sequence shown in SEQ ID NO: 169; (65) Both the first and second light chains contain the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains the amino acid sequence shown in SEQ ID NO: 172, and the second heavy chain contains the amino acid sequence shown in SEQ ID NO: 171; (66) Both the first and second light chains contain the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains the amino acid sequence shown in SEQ ID NO: 174, and the second heavy chain contains the amino acid sequence shown in SEQ ID NO: 173; (67) Both the first and second light chains contain the amino acid sequence shown in SEQ ID NO: 1, the first heavy chain contains the amino acid sequence shown in SEQ ID NO: 176, and the second heavy chain contains the amino acid sequence shown in SEQ ID NO: 175.

[0152] [Polynucleotides]

[0153] This disclosure provides an isolated polynucleotide encoding a first heavy chain, a first light chain, a second heavy chain, or a second light chain of a fusion protein according to this disclosure. In some embodiments, the isolated polynucleotide encodes the first heavy chain of the fusion protein according to this disclosure. In some embodiments, the isolated polynucleotide encodes the second heavy chain of the fusion protein according to this disclosure.

[0154] In another aspect, this disclosure provides a set of isolated polynucleotides, including polynucleotides encoding a first heavy chain, a first light chain, a second heavy chain, and a second light chain of a fusion protein according to this disclosure.

[0155] Polynucleotides are polymers of DNA, RNA, DNA / RNA hybrids, or modifications thereof. In some embodiments, polynucleotides are polymers of DNA. Polynucleotides are polymers of RNA. DNA or RNA encoding anti-SIRPα antibodies or antigen-binding fragments thereof, or the aforementioned bispecific molecules, can be readily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of antibodies. The encoding DNA or RNA can also be obtained by synthetic methods.

[0156] This disclosure provides isolated vectors containing isolated polynucleotides.

[0157] This disclosure further provides a host cell comprising isolated polynucleotides, a set of isolated polynucleotides, or isolated vectors according to this disclosure.

[0158] This disclosure further provides a method for generating a fusion protein according to this disclosure, the method comprising introducing a first expression vector encoding a first heavy chain, a second expression vector encoding a first light chain, a third expression vector encoding a second heavy chain, and a fourth expression vector encoding a second light chain together into a host cell, or into a separate host cell, and expressing the first, second, third, and fourth expression vectors under appropriate conditions. The host cell includes prokaryotic cells and eukaryotic cells.

[0159] In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell, such as CHO, COS, HEK293 cells, or Bowes melanoma cells, or a combination thereof.

[0160] [Pharmaceutical Compositions]

[0161] This disclosure provides a pharmaceutical composition comprising a fusion protein according to this disclosure, isolated polynucleotides, a group of isolated polynucleotides, isolated vectors or host cells, and a pharmaceutically acceptable carrier.

[0162] Pharmaceutical compositions can be administered in a suitable manner depending on the specific applicable form, physicochemical properties, etc., of pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition can be formulated as a lyophilized or liquid formulation, which may contain appropriate formulation additives in the art. For example, the pharmaceutical compositions described above typically contain more than one drug carrier, such as a sterile liquid, such as water and oil (including petroleum, animal, vegetable, or synthetically derived oils (e.g., peanut oil, soybean oil, mineral oil, and sesame oil)). In the case of intravenous administration of the pharmaceutical compositions described above, water is a more representative carrier. In addition, saline solutions, glucose solutions, and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable drug excipients are known in the art. The pharmaceutical compositions described above may also contain trace amounts of wetting agents, emulsifiers, or pH buffers as needed. The pharmaceutical compositions are typically administered parenterally, and may be administered intradermally, intramuscularly, intraperitoneally, intravenously, or subcutaneously, but are not limited to these methods; for example, the pharmaceutical compositions may be administered by infusion or bolus injection. See, for example, the Handbook of Pharmaceutical Excipients, 3rd edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated in whole by reference. Remington's Pharmaceutical Sciences, 16th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated in whole by reference.

[0163] With respect to this disclosure, the active agent or pharmaceutical composition comprising it may be administered to a subject via any suitable route of administration. For example, the active agent may be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following discussion of routes of administration is provided for illustrative purposes only and should not be construed as limiting the scope in any way.

[0164] [Kit]

[0165] This disclosure provides a kit comprising the aforementioned fusion protein, isolated polynucleotides, a group of isolated polynucleotides, isolated vectors, host cells, or pharmaceutical compositions. If desired, such kits may further include one or more of various conventional pharmaceutical kit components, such as, for example, containers having one or more pharmaceutically acceptable carriers, additional containers, etc., as will be apparent to those skilled in the art. The kit may also include instructions, as inserts or labels, indicating the amount of components to be administered, administration guidelines, and / or guidelines for mixing components.

[0166] [Methods of Use]

[0167] This disclosure provides the use of fusion proteins, isolated polynucleotides, a group of isolated polynucleotides, isolated vectors, host cells, or pharmaceutical compositions according to this disclosure in the preparation of medicaments for the prevention or treatment of diseases, or in the preparation of kits for the diagnosis of diseases.

[0168] This disclosure provides a method for preventing or treating a disease in a subject in need, the method comprising administering to the subject a therapeutically effective amount of a fusion protein, isolated polynucleotide, a group of isolated polynucleotides, an isolated vector, a host cell, or a pharmaceutical composition according to this disclosure.

[0169] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to mammals, including but not limited to humans, cattle, horses, cats, dogs, rodents, or primates. In some implementations, the individual is a human.

[0170] In some implementations, the disease includes a tumor or cancer. In some implementations, the tumor is a solid tumor.

[0171] Examples

[0172] Example 1: Design and validation of antibody-LIGHT immunocytokine formats

[0173] Several LIGHT-based immune cytokine (antibody-LIGHT fusion protein) forms have been developed. Figure 1 For tumor-specific delivery, it was designed to incorporate the extracellular domains (residues 59-240, 74-240, 87-240, and 91-240) of wild-type LIGHT (Uniprot, #O43557) and the anti-FAP antibody clone 3F2 (patent number WO 2012020006A). Some forms link the three LIGHT subunits to a peptide linker and to the N-terminus or C-terminus of the heavy or light chain of the antibody (forms A, E). Some forms fuse two LIGHT subunits to the N-terminus or C-terminus of the heavy or light chain, which is linked to a "G" or "GGGGS" linker, with the remaining LIGHT subunit linked to the corresponding site on the opposing chain (forms B, C, D). In form C, the heavy chain CH1 and the light chain CL are exchanged.

[0174] Codon-optimized genes for LIGHT-based immune cytokines were synthesized by Genewiz and subcloned into the pCI vector (Promega#E1731), which was then co-transfected into Expi293F cells (ThermoFisher A14527) for protein production. Following transfection, cells were grown in suspension culture for approximately six days and then harvested by centrifugation (7,000 RPM) at 4°C for 20 minutes. The supernatant was filtered through a 0.22 µm filter and purified using protein A resin for affinity purification. Proteins were then eluted with elution buffer (1 M glycine, pH 3.8, 10% glycerol) and neutralized to pH 6.0 with 1 M Tris (pH 7.5). The concentration of the purified product was measured at 280 nm using NanoDrop after size exclusion chromatography (SEC) on an AKTA instrument. The antibody-LIGHT fusion protein was electrophoresed on a 4%–20% Tris-glycine gel (Bio-Rad) under reducing (R) and non-reducing (NR) conditions by mixing it separately with sample dyes containing or without DTT (dithiothreitol) and heating at 95°C for 5 minutes. The purity of the antibody-LIGHT fusion protein was further characterized by high-performance liquid chromatography (HPLC).

[0175] Of all the tested forms, only form D (ABC232, ABC233) successfully produced a high-quality wild-type LIGHT (residues 74-240) fusion protein using the "GGGGS-GGGGSGGGGSGGGGSGGGT" or "G" linker. Figure 2 ), as seen in HPLC-SEC analysis ( Figure 3A Conversely, on HPLC-SEC, the same molecular form ABC243 expressing the shorter form of LIGHT (residues 87-240) showed poorer expression and higher aggregation than ABC233. Figure 3B ).

[0176] Example 2: Extended design of LIGHT-based immunocytokines

[0177] Once the molecular forms of the antibody-LIGHT fusion proteins (ABC232 and ABC233) were identified, more constructs were generated to test different parameters. Figure 4A to Figure 4B(1) The linker-1 between CH3 and cytokines was replaced by ISKAKGQPREPQ, ASTKGPSVFPLAPS, RTVAAPSVFIFPPS, KSTGKPTLYNVSLVMSDTAGTCY, G4S, (G4S)2 (SEQ ID NO: 86), (G4S)3, GGGGSGGGGSGGGGSGGGT or no linker was present; (2) The linker-2 between two cytokine units conjugated on the same heavy chain was replaced by GGGGSGGGGSGGGGSGGGSGGGT, G, (G4S)2; (3) Different LIGHT variants, including human WT-LIGHT and human LIGHT mutants, were tested; (4) Different IgGs were evaluated. Fc heterodimerization techniques, such as the kilometreotype (KIH) and electrostatic reversal of CH3; (5) different antibodies, such as anti-FAP, anti-HER2, anti-EGFR and anti-PDL1, were evaluated; (6) bispecific antibodies, such as anti-FAP×anti-HER2, were detected; (7) wild-type and glycosylated IgG1 with N297A mutation were studied; (8) various IgG subclasses, such as IgG1 and IgG4, were tested. As a result, the following LIGHT-based immune cytokines were expressed and purified: 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, and ABC691. The amino acid sequences of these LIGHT-based immune cytokines are listed in the table below.

[0178] The forms of the immune cytokines produced above are listed as D1, D2, D3, D4, D5, and D6. Schematic diagrams of these constructs are shown below. Figure 4B As shown.

[0179]

[0180]

[0181]

[0182] The light chain of the anti-FAP monoclonal antibody ABC002 is shown in SEQ ID NO: 79, and the heavy chain is shown in SEQ ID NO: 80.

[0183] The above-described LIGHT-based immune cytokines were expressed and purified using a similar protocol as described in Example 1.

[0184] Successful expression and correct assembly of all LIGHT-based immune cytokines were demonstrated by dominant bands present in the non-reducing gel, corresponding to the molecular weight of the correctly assembled molecules. This was further supported by SDS-PAGE analysis of the reduced proteins, which showed two heavy chains and one light chain, providing additional evidence of correct assembly. Figure 5 A to Figure 5 D). Additionally, after one-step affinity purification and size exclusion chromatography (SEC) polishing of protein A, each immune cytokine showed a single peak in SEC-HPLC analysis, such as ABC538, ABC539, ABC770, ABC771, ABC772, and ABC773 (…). Figure 6 and Figure 7 Therefore, this platform provides a promising method for generating a variety of light-based immune cytokines.

[0185] Example 3: Binding affinity of LIGHT-based immunocytokines to LTβR, HVEM and DcR3 proteins

[0186] Binding affinity to LTβR proteins measured by ELISA

[0187] LIGHT binds to its cell surface receptor LTβR, leading to downstream NF-κB signaling. To evaluate the binding of LIGHT-based immunocytokines to human LTβR-Fc (Novoprotein #CX78) and mouse LTβR-Fc protein (Acrobiosystems #LTR-H5251), 96-well ELISA plates (Thermo Fisher) were coated with 0.5 μg / mL LTβR protein and blocked with 2% BSA-PBS buffer for one hour. Serial dilutions of LIGHT-based immunocytokines were added, up to a maximum concentration of 10 nM. The plates were incubated for one hour, washed four times with PBST, and then incubated with biotinylated mouse anti-human Ig light chain κ antibody (Biolegend, #316504). HRP-conjugated streptavidin (Sino Biological) was added, and the plates were further incubated, washed three times with PBST, and treated with TMB substrate (#34029, Thermo Fisher). Plate readings were taken at 450 nM using a SpectraMax M5 Molecule Device. ELISA results were analyzed using GraphPadPrism 9.0 software, and the EC50 (half-maximum effective concentration) is summarized in Tables 1 and 2.

[0188] Table 1: EC50 of LIGHT-based immunocytokines binding to human LTβR .

[0189]

[0190] Table 2. EC50 of LIGHT-based immunocytokines binding to mouse LTβR .

[0191]

[0192] LIGHT-based immune cytokines showed specific dose-dependent binding to human LTβR and mouse LTβR proteins, such as... Figure 8 A to Figure 8 E and Figure 9 A to Figure 9 As shown in E, Fc heterodimerization techniques, such as pestle-and-mortar structures and electronic manipulation, can promote the production and enhance the activity of LIGHT-based immune cytokines. The pestle-and-mortar / pestle-and-mortar-and-mortar-and-mortar-and-mortar (pestle and mortar exchange) designs exhibit similar affinity to LTβR.

[0193] The results also showed that the bonding affinity for LTβR was comparable across different connector-1 and connector-2 options (including connectorless, G4S, (G4S)2, (G4S)3, (G4S)4, GGGGSGGGGSGGGGSGGGT, ISKAKGQPREPQ, ASTKGPSVFPLAPS, RTVAAPSVFIFPPS, and KSTGKPTLYNVSLVMSDTAGTCY). Furthermore, the bonding could be fine-tuned by using different connector-1 and connector-2 options.

[0194] Binding affinity to HVEM and DcR3 proteins measured by ELISA

[0195] Recombinant HVEM proteins were generated by fusing the C-terminus of human HVEM (referred to as hHVEM) residues 39-202 or mouse HVEM (referred to as mHVEM) residues 38-207 to human Fc, while recombinant DcR3 proteins were generated by linking human DcR3 residues 33-300 to the N-terminus of rabbit Fc. The recombinant proteins were expressed in expi293F and purified using protein A resin for affinity extraction and size exclusion column purification.

[0196] Human HVEM, mouse HVEM, or human DcR3 protein were immobilized at a concentration of 0.5 μg / mL on maxiSorp 96-well ELISA plates (Thermo Fisher) and subsequently blocked with 2% BSA-PBS buffer for one hour. Light-based immunocytokines were added to the plates at various dilutions, with a maximum concentration of 10 nM. The plates were incubated for one hour, washed four times with PBST, and then further incubated with biotinylated mouse anti-human Ig light chain κ antibody (Biolegend, #316504). After washing three times with PBST, the plates were further incubated with HRP-conjugated streptavidin (Sino Biological) and washed again with PBST. TMB substrate (#34029, Thermo Fisher) was added to the plates and read at 450 nM using a SpectraMax M5 Molecular Devices microplate reader. The ELISA results were analyzed using GraphPad Prism 9.0 software, and the EC50 (half-maximum effective concentration) is summarized in Table 3.

[0197] Table 3. EC50 of LIGHT-based immunocytokines binding to human and mouse HVEM and human DcR3 .

[0198]

[0199]

[0200] The binding of LIGHT-based immune cytokines to two other LIGHT receptors, HVEM and DcR3, was also evaluated. As described in patent application PCT / CN2022 / 097735, the LIGHT-1 (SEQ ID NO: 69) homotrimer and the LIGHT-2 (SEQ ID NO: 70) homotrimer showed lower affinity for HVEM compared to wild-type LIGHT. Consistently, anti-FAP×LIGHT-1 (ABC538) and anti-FAP×LIGHT-2 (ABC539) immune cytokines also showed reduced binding affinity to HVEM. Figure 10 B and Figure 10 F).

[0201] DcR3 is a soluble protein expressed in humans and cynomolgus monkeys, but absent in mice. DcR3 negatively regulates the ability of LIGHT to activate LTβR and HVEM (Liu et al., 2021), and is upregulated in some autoinflammatory diseases and cancer patients (Fuchsberger et al., 2021). Consistent with the lower binding affinity of DcR3 to LIGHT-1 or LIGHT-2 compared to wild-type LIGHT, DcR3 binds significantly weaker to ABC538 and ABC539 than to ABC233. Figure 11 A to Figure 11 F).

[0202] Example 4: Cell binding affinity of LIGHT-based immunocytokines

[0203] Cell line generation :

[0204] Full-length human or mouse HVEM or LTβR (listed in Table 4) were transfected into HEK-293T cells (ATCC) using lipofectamine 3000 (Thermo Fisher, L3000001). Three days after transfection, cells were treated with hygromycin B (MilliporeSigma) for fourteen days to obtain stable cell lines. Stable cells were used to assess the binding of LIGHT-based immune cytokines to HVEM or LTβR.

[0205] Table 4. Protein sequence accession numbers / source .

[0206]

[0207] Cell binding analysis by FACS :

[0208] The binding activity of light-based immunocytokines was assessed using stably transfected HEK-293T cells expressing LTβR or HVEM. Cells were incubated on ice for 30 min with serially diluted fusion proteins at a maximum concentration of 50 nM in flow cytometry buffer (PBS, 0.5% BSA, 1 mM EDTA). Cells were then washed three times with PBS, and the bound immunocytokines were stained on ice for 15 min with FITC-conjugated mouse anti-human IgG antibody. Cells were further washed three times with PBS, and fluorescence was measured using a Cytek Aurora cytometer (Cytek). EC50 was determined using FlowJo and GraphPadPrism 9.0 software. Results are summarized in Tables 5, 6, and 7.

[0209] Table 5: EC50 of LIGHT-based immunocytokines binding to 293T cells expressing human LTβR

[0210]

[0211]

[0212] Table 6: EC50 of LIGHT-based immunocytokines binding to 293T cells expressing mouse LTβR

[0213]

[0214]

[0215] Table 7: EC50 of LIGHT-based immunocytokines binding to 293T cells expressing human or mouse HVEM

[0216]

[0217]

[0218] FACS analysis showed that LIGHT-based immune cytokines exhibited dose-dependent binding to cells overexpressing human or mouse LTβR. Figure 12 A to Figure 12 G and Figure 13 A to Figure 13 G). Furthermore, the binding affinity of these immune cytokines to cells expressing HVEM is dominated by the antibody-conjugated LIGHT mutant protein (G). Figure 14 A to Figure 14 D and Figure 15 A to Figure 15 D). Some LIGHT-based immune cytokines (such as ABC477 and ABC543) exhibit similar binding to cells expressing human or mouse HVEM as wild-type immune cytokines (ABC233), while other immune cytokines (such as ABC539) bind minimally to both cell types.

[0219] Example 5: Pathway activation of anti-FAP x LIGHT immunocytokines in the absence or presence of trans FAP-expressing cells Figure 16

[0220] The 293T-FAP cell line was generated by transfecting full-length human FAP (Table 4) into HEK-293T cells using lipofectamine 3000 according to a standard protocol. Three days later, the cells were treated with hygromycin (Millipore Sigma) for 14 days to generate a stable cell line.

[0221] HeLa-NF-κB and HEK-293T-NF-κB cells were generated by transfecting cells with the pNL3.2.NF-κB-RE[NlucP / NF-κB-RE / Hygro] vector (Promega # N1111) using lipofectamine 3000. Three days later, the cells were treated with hygromycin B (Sigma) and cultured at 37°C and 5% CO2 for 14 days. The resulting HeLa-NF-κB reporter cells were used to evaluate downstream signaling pathways induced by LIGHT-based immunocytokine treatment and activation of LTβR.

[0222] To assess the ability of LIGHT-based immune cytokines to activate HVEMs, HEK293T-NF-κB cells were further transfected with full-length human or mouse HVEMs (listed in Table 4) using lipofectamine 3000. After incubation for fourteen days in the presence of hygromycin B and puromycin (Millipore Sigma), the resulting stable HEK293T-HVEM-NF-κB cells were collected for further analysis.

[0223] To assess the effect of FAP on LIGHT-induced pathway activation, reporter cells were cultured alone or co-cultured with 293T-FAP cells and treated with the specified molecule. After 24 hours, cells were lysed with lysis buffer (Promega #E397A) and luciferase activity was measured using a SpectraMax M5 microplate reader (Promega #E4500).

[0224] LIGHT-based immune cytokines can activate NF-κB signaling in HeLa cells with endogenous LTβR. Figure 16 A to Figure 19 H and Table 8). Immune cytokines with the same LIGHT mutant protein also showed a similar ability to activate LTβR. Co-culturing with 293T-FAP cells enhanced LTβR activation of most immune cytokines (Table 8), except for ABC543 (H and Table 8). Figure 19 A to Figure 16D). For example, when cultured alone, ABC538 and ABC539 showed weaker ability to induce NF-κB signaling than ABC233 with wild-type LIGHT. Figure 16 A to Figure 19 D、 Figure 28 and Figure 28 A to Table 8: EC50 of LIGHT-based immunocytokines to activate HeLa-NF-κΒ cells (B, Table 8). However, when co-cultured with HeLa-NF-κB reporter cells and 293T-FAP cells, they were able to activate NF-κB signaling in a manner similar to that of wild-type molecules. These results suggest that immune cytokines such as ABC538 and ABC539 can be selectively activated in the tumor microenvironment when FAP-expressing fibroblasts are present.

[0225] Figure 17

[0226]

[0227] The ability of LIGHT-based immunocytokines to activate downstream HVEM signaling was assessed using 293T-NF-κB cells overexpressing human or mouse HVEM. Results showed that while wild-type LIGHT (ABC233) effectively activated downstream NF-κB signaling (…),… Figure 17 A to Figure 18 C Figure 18 A to Figure 28 C and Figure 28 C to Table 9: EC50 of LIGHT-based immunocytokines to activate HEK293T-HVEM-NF-κΒ cells (E, Table 9), but ABC538, ABC539, ABC890 and ABC892 exhibited reduced activity of the activation pathway, consistent with their reduced binding to HVEM.

[0228] Example 6: Characterization of the effect of DcR3 on the activity of LIGHT-based immunocytokines .

[0229]

[0230] Figure 20

[0231] In addition to HVEM and LTβR, LIGHT also interacts with the decoy receptor DcR3, which lacks both transmembrane and cytoplasmic segments. This interaction has the potential to disrupt signaling pathways by isolating LIGHT from HVEM and LTβR. Although DcR3 expression is typically low in healthy human tissues, it is frequently and significantly upregulated in cancer patients (Wu et al., 2003; Yoo et al., 2022).

[0232] To minimize the binding of LIGHT-based immunocytokines to DcR3, ABC538 and ABC539 were developed from LIGHT-1 and LIGHT-2, respectively, both of which exhibit relatively weak affinity for DcR3. To investigate the effect of DcR3 on LIGHT-induced LTβR activation, HeLa-NF-κB reporter cells were exposed to 200 μg / ml soluble DcR3-Fc protein while treated with LIGHT-based immunocytokines. After 24 hours, cells were lysed (Promega #E397A), and luciferase activity was measured using a Promega #E4500 microplate reader (SpectraMax M5 Molecule Devices).

[0233] Regardless of the presence of FAP-expressing cells, the addition of DcR3-Fc protein reduced ABC 233-induced LTβR activation by 11 / 12 to 25 / 26, while DcR3 had minimal effect on ABC 538-induced LTβR activation. Figure 20 A to Example 7: Binding of anti-FAP x LIGHT immunocytokines to endogenous mouse LTβR in the presence or absence of co-expressed FAP D). These results suggest that ABC538 is unlikely to be affected by soluble DcR3, thus leading to potentially improved pharmacokinetic (PK) properties and potentially higher levels of active molecules in the tumor microenvironment.

[0234] Figure 21 Figure 21

[0235] Two mouse cell lines, BALB / c-3T3-FAP (LTβR), were used. + HVEM - FAP + ) and BALB / c-3T3 (LTβR + HVEM - FAP -To investigate the role of the LIGHT mutant protein in activating endogenous mouse LTβR, we conducted an investigation. BALB / c-3T3-FAP cell lines were generated by transfecting BALB / c-3T3 cells (ATCC) with mouse Fap (referred to as mFAP), and mFAP expression was screened. Immunocytokines were administered to 100,000 purified cells in FACS buffer (PBS, 0.5% BSA, and 1 mM EDTA) on ice for 30 minutes. Cells were then washed three times with FACS buffer, and live / dead cell differentiation was performed using a Live / Dead Fixable Near-IR Cell Stain Kit (Life Technologies). Cells were then stained in FACS buffer with 1 μg / mL goat anti-human IgG Fc (FITC) (ab97224, Abcam) on ice for 30 minutes. Cells were then washed three times with FACS buffer before final flow cytometry analysis. All flow cytometry data were acquired on a Cytek Aurora cytometer (Cytek) and analyzed using FlowJo software (v.10.5.3, Treestar, Inc.). EC50 was determined using GraphPad Prism 9.0 software.

[0236] In the absence of FAP, all three LIGHT-based immune cytokines showed relatively weak binding affinity to endogenous mLTβR, with EC50 values ​​exceeding 10 nM, although ABC538 and ABC233 exhibited better binding than ABC539. However, co-expression of FAP and LTβR on the same cells resulted in a significant reduction in the EC50 values ​​of all three molecules to the nanomolar range. ​ A to ​ B). Therefore, the anti-FAP×LIGHT immune cytokine allows preferential targeting of cells that express FAP, rather than cells that do not express FAP.

[0237] Example 8: Binding of LIGHT-based immune cytokines to endogenous mouse HVEM

[0238] The interaction between anti-FAP×LIGHT immunocytokines and h / mHVEM was assessed using primary human and mouse T cells. T cells were negative for LTβR and FAP, but positive for HVEM. Human CD3 was purified from human PBMCs with a purity greater than 97% via negative selection according to the manufacturer's protocol (480134, Biolegend). + T cells. Mouse CD3 was purified from confluent spleen cells of BALB / c mice with a purity greater than 97% by negative selection according to the manufacturer's protocol (480031, Biolegend). +T cells. Cells were incubated together with twelve light-based immunocytokines in FACS buffer (PBS, 0.5% BSA, and 1 mM EDTA) at 3-fold serial dilutions on ice for 30 minutes. Cells were washed three times with FACS buffer and live / dead cells were distinguished using a Live / Dead Fixable Near-IR Cell Stain Kit (Life Technologies). Subsequently, human T cells were stained in FACS buffer with anti-hCD4 (RPA-T4, Biolegend) or anti-hCD8a (RPA-T8, Biolegend) along with 1 μg / mL goat anti-human IgG Fc (FITC) (ab97224, Abcam) at 30 minutes on ice. Mouse T cells were stained in FACS buffer with 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 minutes. Cells were then washed three times with FACS buffer, and cell quantification data were collected using a Cytek Aurora cytometer (Cytek) and analyzed using FlowJo. EC50, which is the protein concentration required to achieve half of the maximum binding, was determined using GraphPad Prism 9.0 software.

[0239] Figure 22 The results showed that ABC538, compared to ABC233, exhibited weaker binding to endogenous HVEM on primary CD4 T cells and CD8 T cells, while ABC539 showed minimal binding to HVEM. These results suggest that ABC538 (with reduced binding to HVEM) primarily activates the LTBR pathway within the tumor microenvironment rather than the HVEM pathway.

[0240] Example 9: Anti-FAP×LIGHT immune cytokines against LT in the absence or presence of co-expressed FAP Activation of the βR pathway

[0241] Use BALB / c-3T3-FAP (LTβR) + HVEM - FAP + ) and BALB / c-3T3 (LTβR + HVEM - FAP -To investigate the role of the LIGHT mutant protein in activating endogenous LTβR, cells were seeded at 3,000 cells / well in 96-well tissue culture plates and incubated with different concentrations of anti-FAP×LIGHT immune cytokines at 37°C for 24 hours. After centrifugation, the supernatant was collected to quantify CCL2 protein levels using a Tecan Spark reader via ELISA (catalog number 432704, Biolegend).

[0242] Figure 23 A showed that in the absence of FAP expression in 3T3 cells, activation of the LTβR pathway and induction of CCL2 protein were mainly determined by the binding affinity of immune cytokines, with ABC233 being the most effective, followed by ABC538 and ABC539. However, co-expression of FAP significantly increased the ability of all three molecules to induce CCL2 expression, especially at lower concentrations. Figure 23 B and Figure 23 C). Since cancer-associated fibroblasts (CAFs) in the tumor stroma play an important role in tumor biology and exhibit high expression of both FAP and LTβR, the anti-FAP×LIGHT immune cytokine was engineered to preferentially deliver FAP-mediated affinity-driven LTβR stimulation to CAFs in the tumor microenvironment.

[0243] Example 10: Immunotherapy with anti-FAP×LIGHT cytokines in the absence or presence of trans-expressed FAP Activation of the LTβR pathway under these conditions

[0244] To investigate the effect of trans-expressed FAP on BALB / c-3T3-WT (LTβR) + HVEM - FAP - The effect of endogenous LTβR pathway activation in cells was investigated by using lipofectamine 3000 and antibiotic selection to stably transfect CT26 cells (ATCC) with a plasmid containing the mouse FAP gene to generate the CT26-FAP cell line. We demonstrated that only 3T3 cells, not CT26-WT or CT26-FAP, could induce CCL2 after stimulation with LIGHT-based immunocytokines.

[0245] CT26-WT or CT26-FAP cells were seeded at 2.5K / well in 96-well tissue culture plates and incubated for one hour at 37°C and 5% CO2 with serially diluted LIGHT-based immunocytokines. Then, 2.5K BALB / c-3T3 cells were added to the culture as responders. An ELISA assay (catalog number 432704, Biolegend) was performed to measure CCL2 protein levels in the culture medium as an alternative to NF-κB pathway activation.

[0246] In the presence of CT26-FAP cells, ABC233, ABC538, and ABC539 at low concentrations enhanced the activation of LTβR signaling and the production of CCL2 from 3T3 cells, indicating that the binding of the anti-FAP arm of immune cytokines to CT26-FAP cells can facilitate trans-LIGHT-LTβR signaling. Figure 24 ).

[0247] Example 11: In vivo antitumor activity of anti-FAP×LIGHT immune cytokines in a CT26-FAP tumor model Assessment

[0248] To evaluate the antitumor activity of immune cytokines, 400,000 CT26-FAP cells from 50 μl of HBSS were mixed with 50 μl of Masturbation Gel and subcutaneously implanted into the flank of BALB / c mice. When the tumor volume reached 60 mm² on day 8... 3 -93mm 3 Mice were grouped based on body weight and tumor volume to ensure a similar distribution. Mice were divided into three groups: G1_buffered saline (negative control), G2_ABC002 (anti-FAP, negative control), and G3_ABC538, n=9 / group. Mice were treated twice weekly, with the first three doses at 1 nanomolar / mouse and the next two doses at 1.5 nanomolar / mouse. One nanomolar of ABC538 is equivalent to 200 μg of protein, while 1.5 nanomolars is equivalent to 300 μg of protein. The equivalent protein amounts of 1 nanomolar and 1.5 nanomolars of ABC002 are 144 μg and 200 μg, respectively. Tumor growth was monitored by measuring tumor volume using calipers at specific time points until day 22. Tumor volume was calculated using the following formula: length × width. 2 / 2. The weight of the mice was also recorded at specified time points.

[0249] The survival rate of mice was calculated using the Kaplan-Meier method. Mice were considered ineffective if one of the following criteria was met: (1) the tumor volume reached approximately 2000 mm. 3 (2) They experienced a 20% weight loss; or (3) They died. Results showed that ABC538 possessed single-agent antitumor activity, as demonstrated by tumor growth inhibition and mouse survival, as shown in [examples of other drugs]. Figure 25 As shown.

[0250] Example 12: Evaluation of in vivo lysis of LIGHT-based immune cytokines

[0251] It has been shown that members of the metalloproteinase family (MMPs) can cleave surface light expressed on activated T cells. In fact, inhibition of MMPs leads to increased light expression on activated T cells (Morel et al., 2000). To enhance the efficacy and half-life of light-based immunocytokines, the light moiety in immunocytokines has been further truncated to residues 91-240 to produce anti-cleavage immunocytokines such as ABC890, ABC892, ABC1054, ABC1056, ABC1032, and ABC1048.

[0252] These cleavage-resistant immune cytokines maintain their binding specificity and functionality. For example, ABC890 and ABC892, which share the same mutation as ABC538 (LIGHT1, residues 74-240), exhibit similar binding affinity to human and mouse LTßR, and reduced binding to human HVEM, mouse HVEM, and human DcR3. Figure 8 D、 Figure 9 D、 Figure 10 B. Figure 10 F).

[0253] These anti-cleavage immune cytokines can activate LTβR at comparable EC50 levels, as shown in Table 8, and their LTβR-activating activity is enhanced by aggregation with antigen-expressing cells. However, since the binding of all three molecules to HVEM is reduced, their HVEM-stimulating activity was only observed in the presence of antigen-expressing cells (such as 293T-hFAP cells). Figure 28 A to Figure 28 E). These results indicate that molecules such as ABC538 and ABC539 can be selectively activated in the tumor microenvironment when fibroblasts expressing FAP are present.

[0254] To assess in vivo lysis of the anti-FAP×LIGHT immunocytokine, 6-week-old female C57BL / 6J mice were administered a single dose (ip) of 1.5 nanomolar of ABC538, ABC890, or ABC892. Plasma samples were collected at 24 and 96 h post-injection for analysis of lysis. Plasma cells were diluted 100-fold and incubated with 200,000 293T-mFAP cells on ice for 30 min, followed by washing three times with FACS buffer. Live and dead cells were determined using a Live / Dead Fixable Near-IR Dead Cell Stain Kit (Life Technologies) and stained on ice for 30 min 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). After washing three times with FACS buffer, cells were analyzed by flow cytometry on a Cytek Aurora cytometer (Cytek), and results were analyzed using FlowJo software. A standard curve was generated by using twelve 3-fold serial dilutions of the indicated antibody with a 4-parameter logic curve fitting algorithm. The ratio of LIGHT concentration to hIgG1 concentration was used to assess lysis, where a ratio of 1 indicated no lysis.

[0255] The results showed that ABC538 underwent partial cleavage in vivo, resulting in a 50% ratio of LIGHT to hIgG1 detected in mouse plasma at 24 and 96 hours. Conversely, ABC890 and ABC892, with their shorter LIGHT subunits, exhibited a higher ratio, indicating less cleavage, improved stability, and increased in vivo bioavailability. Figure 26 ).

[0256] Example 13: In vivo antitumor activity of anti-FAP×LIGHT immune cytokines in the KPC0826 tumor model

[0257] 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 from 50 μl of HBSS were mixed with 50 μl of matrix gel and implanted into the flank of BALB / c mice. The established subcutaneous KPC0826 tumor model showed relatively high FAP (free-adjusted cellularity). + The presence of CAF cells was approximately 1%-5% of the total dissociated tumor cells (data not shown). On day 0, when the tumor volume reached 70 mm... 3 -102mm 3 87mm on average 3Mice were randomly divided into six 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 in each group. Mice were treated twice weekly with 1.5 nanomolar doses of the corresponding molecules for six consecutive weeks, with a dose of 200 μg for ABC002 and 300 μg for the other molecules. Tumor growth was monitored by measuring tumor volume using calipers at specified time points until day 21. Tumor volume was calculated using the following formula: length x width. 2 / 2. Tumor growth inhibition (TGI) was compared with the control group (G1_ABC002) and calculated using the DRAPR package (J Transl Med 17, 39, 2019).

[0258] The results showed that all tested anti-FAP×LIGHT immune cytokines exhibited single-agent antitumor activity in the KPC0826 model. Among them, ABC890 and ABC892 showed the highest tumor growth inhibition (TGI), followed by ABC538, ABC233, and ABC539. Compared with ABC538, the anti-cleavage derivatives ABC890 and ABC892 showed superior antitumor activity (e.g., Figure 27A to Figure 27D (As shown).

[0259] Example 14: Other TNFSF14 superfamily members of immune cytokines

[0260] This platform can also be applied to other members of the TNFSF14 superfamily. 4-1BBL is another member of the TNF superfamily, and its interaction with CD137 / TNFRSF9 / 4-1BB can promote T lymphocyte activation and proliferation. The antibody-4-1BBL fusion protein (ABC676) was successfully expressed using form D. Lymphotoxin-αββ (similar to engineered LIGHT1) binds to LTβR without binding to HVEM and DcR3, potentially providing enhanced safety and efficacy in delivering antitumor effects. Using form D, an immune cytokine equipped with lymphotoxin-αββ (ABC653) can also be generated.

[0261] HeLa-NF-κB reporter cells were used to evaluate downstream signaling following LTβR activation after treatment with a lymphotoxin-based immunocytokine. 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 ).

[0262] The antitumor efficacy of ABC653 was evaluated in genetically modified KPC mice. When the tumor volume reached 70 mm... 3 -102mm 3 Mice were treated twice weekly for eight weeks with 1.5 nanomolars of ABC653 and a mAb control (ABC002). Tumor growth was monitored by measuring tumor volume with calipers, and tumor volume was calculated using the following formula: length × width. 2 / 2. The results demonstrated that the anti-FAP×lymphotoxin (ABC653) immune cytokine exhibited single-agent antitumor activity in the KPC0826 model. Figure 30 ).

[0263] References :

[0264] Beatty, GL, Chiorean, EG, Fishman, MP, Saboury, B., Teitelbaum, UR, Sun, W., Huhn, RD, Song, W., Li, D., Sharp, LL, et al. (2011). CD40agonists alter tumor stroma and show efficacy against pancreatic carcinoma inmice and humans. Science 331, 1612-1616. 10.1126 / science.1198443.

[0265] Fuchsberger, FF, Kim, D., Baranova, N., Kagelmacher, M., Wawrzinek, R., and Rademacher, C. (2021). 10.1101 / 2021.05.10.443458.

[0266] Liu, W., Chou, T.F., Garrett-Thomson, S.C., Seo, G.Y., Fedorov, E.,Ramagopal, U.A., Bonanno, J.B., Wang, Q., Kim, K., Garforth, S.J., et al.(2021). HVEM structures and mutants reveal distinct functions of binding toLIGHT and BTLA / CD160. J Exp Med 218. 10.1084 / jem.20211112.

[0267] 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 virusentry 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

1. A fusion protein comprising: a first cytokine fragment linked to the C-terminus of a first CH2-CH3 fragment, the first cytokine fragment comprising a first cytokine molecule, or a first cytokine molecule and a third cytokine molecule linked in tandem, and a second cytokine fragment linked to the C-terminus of a second CH2-CH3 fragment, the second cytokine fragment comprising a second cytokine molecule, or a second cytokine molecule and a fourth cytokine molecule linked in tandem, the first CH2-CH3 fragment and the second CH2-CH3 fragment form a dimer, a total of three cytokine molecules 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 linked in tandem, or the first cytokine fragment comprises the first cytokine molecule and a third cytokine molecule linked in tandem 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 are independently 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 to 3, wherein the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule are independently LIGHT, lymphotoxin alpha, lymphotoxin beta or 4-1BBL.

5. The fusion protein according to any one of claims 1 to 4, wherein the first cytokine molecule, the second cytokine molecule, the third cytokine molecule and the fourth cytokine molecule are independently wild-type LIGHT (preferably human LIGHT), truncated LIGHT or muteins thereof; optionally, the wild-type LIGHT, truncated LIGHT or muteins thereof comprise the 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 are independently wild-type 4-1BBL (preferably human 4-1BBL), truncated 4-1BBL or muteins thereof; optionally, the wild-type 4-1BBL, truncated 4-1BBL or muteins thereof comprise the amino acid sequence of SEQ ID NO: 179; The first, second, third, and fourth cytokine molecules are independently wild-type, truncated lymphotoxin alpha or lymphotoxin beta, or muteins thereof; optionally, the wild-type, truncated lymphotoxin alpha or lymphotoxin beta, or muteins thereof comprise the amino acid sequence of SEQ ID NO: 181, 182.

6. The fusion protein of any one of claims 1-5, wherein the first, second, third, and fourth cytokine molecules are independently truncated LIGHT, optionally the truncated LIGHT comprises the amino acid sequence set forth in SEQ ID NO: 68 or SEQ ID NO:

78.

7. The fusion protein of any one of claims 1-6, wherein the first, second, third, and fourth cytokine molecules are independently LIGHT muteins; optionally, the LIGHT muteins comprise the amino acid sequence set forth in one of SEQ ID NO: 25-26, 58-59, 69-77, 126-141, 158, 177, 178, and 180.

8. The fusion protein of any one of claims 1-7, wherein the dimer is an Fc domain, which is modified to improve correct assembly of the fusion protein.

9. The fusion protein of claim 8, wherein the Fc domain is derived from an IgG, including IgGl, IgG2, IgG3, and IgG4, optionally the Fc domain is derived from a human IgGl or IgG4 Fc domain.

10. The fusion protein according to any one of claims 1 to 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 BEAT ® , Optionally, the modifications include a knob-in-hole and / or DDKK.

11. The fusion protein of any one of claims 1-10, wherein the first and second CH2-CH3 fragments form a human IgGl Fc domain with a mutation N297A.

12. The fusion protein of any one of claims 1-11, wherein the first or second CH2-CH3 fragment further comprises one or more mutations selected from the group consisting of Y349C, T366S, L368A, and Y407V; and the other comprises mutations S354C and / or T366W.

13. The fusion protein of any one of claims 1-12, wherein the first cytokine fragment is connected to the C-terminal end of the first CH2-CH3 fragment, directly or via a linker; and / or the second cytokine fragment is connected to the C-terminal end of the second CH2-CH3 fragment, directly or via a linker.

14. The fusion protein of any one of claims 1 to 13, wherein the C-terminus of a CH3 domain in the first CH2-CH3 fragment is fused directly or via a first linker A to the N- terminus of the first cytokine fragment; and / or the C-terminus of a CH3 domain in the second CH2-CH3 fragment is fused directly or via a first linker B to the N-terminus of the second cytokine fragment.

15. The fusion protein of any one of claims 1 to 14, wherein the first cytokine molecule is fused directly or via a second linker A to the third cytokine molecule; and / or the second cytokine molecule is fused directly or via a second linker B to the fourth cytokine molecule.

16. The fusion protein of any one of claims 1 to 15, wherein the C-terminus of the first cytokine molecule is fused directly or via a second linker A to the N-terminus of the third cytokine molecule; and / or the C-terminus of the second cytokine is fused directly or via a second linker B to the N-terminus of the fourth cytokine. Optionally, the first linker A and the first linker B are independently absent or 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.

17. The fusion protein according to any one of claims 1 to 16, wherein the first linker A, the first linker B, the second linker A and the second linker B are independently absent or 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 18. The fusion protein of any one of claims 1 to 17, wherein the first linker A and the first linker B are independently absent or comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 81-90.

19. The fusion protein of any one of claims 1 to 18, wherein the first linker A and the first linker B are the same or different.

20. The fusion protein of any one of claims 1 to 19, wherein the second linker A and the second linker B are independently 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 of any one of claims 1 to 20, wherein the second linker A and the second linker B are independently of each other absent or comprise the amino acid G or an amino acid sequence as set forth in any one of SEQ ID NOs: 85-89.

22. The fusion protein of any one of claims 1 to 21, comprising a first antigen binding fragment and a second antigen binding fragment, which are linked to the N-terminus of the first CH2-CH3 fragment and the second CH2-CH3 fragment, respectively. ​ 23. The fusion protein of claim 22, wherein the first and second antigen binding fragments are independently selected from the group consisting of a diabody, a Fab, a Fab', a F(ab')2, a 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), a scFv dimer (bivalent diabody), a multi-specific antibody formed from a portion of an antibody comprising one or more CDRs, a camelid-derived single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment.

24. The fusion protein of claim 22 or 23, wherein the first antigen binding fragment is a 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 of any one of claims 22-24, comprising a first heavy chain comprising, from the N-terminus to the C-terminus, the operably linked first antigen binding fragment, the first CH2-CH3 fragment, and the first cytokine fragment; a second heavy chain comprising, from the N-terminus to the C-terminus, the operably linked second antigen binding fragment, the second CH2-CH3 fragment, and the second cytokine fragment.

26. The fusion protein of any one of claims 22-25, further comprising a first light chain paired with the first antigen binding fragment to form a heterodimer; and / or a second light chain paired with the second antigen binding fragment to form a heterodimer.

27. The fusion protein of any one of claims 22-26, wherein the first antigen and the second antigen are each independently selected from the group consisting of FAP, HER2, PDL-1, PD-1, EGFR, VEGFR, VEGF, CCR8, OX-40, 41BB, Angiopoietin-2, IL-4Ra, 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-1b, 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, TNFa, TROP-2, and TWEAK.

28. The fusion protein of any one of claims 22-27, wherein the first antigen and the second antigen are each independently selected from the group consisting of FAP, HER2, PDL-1, and EGFR.

29. The fusion protein of any one of claims 22-28, wherein the first antigen and the second antigen are both FAP, HER2, EGFR, or PDL-1, or the first antigen is HER2 and the second antigen is FAP.

30. The fusion protein of any one of claims 1-29, wherein the fusion protein is monovalent or bivalent, monospecific or bispecific.

31. The fusion protein of 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 of any one of claims 26-31, wherein the first light chain and the second light chain independently comprise the amino acid sequence set forth as SEQ ID NO: 1, 29, 91, or 94. ​ 33. The fusion protein of any one of claims 22-32, wherein the first heavy chain comprises an 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 of any one of claims 22-33, wherein the second heavy chain comprises an 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 of any one of claims 22-34, wherein the fusion protein is selected from the following: (1) both the first and second light chains comprise an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 2; (2) both the first and second light chains comprise an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 4, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 2; (3) both the first and second light chains comprise an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 6, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 5; (4) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 8, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 7; (5) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 10, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9; (6) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 11, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 11; (7) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 14, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 13; (8) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 15; (9) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 18, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 17; (10) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 20, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 19; (11) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 22, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 21; (12) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 23, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 24; (13) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 27, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28; (14) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 29, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 31; (15) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 33; (16) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 35; (17) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 37, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 36; (18) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 39, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 38; (19) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 41, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 40; (20) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 29, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 42, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 43; or (21) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 45, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 44; (22) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 47, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 46; (23) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 49, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 48; (24) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 51, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 50; (25) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 52, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 2; (26) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 53, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 2; (27) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 55, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 54; (28) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 57, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 56; (29) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 61, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 60; (30) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 63, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 62; (31) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 65, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 64; (32) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 67, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 66; (33) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 91, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 92, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 93; (34) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 94, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 95, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 96; (35) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 98, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 97; (36) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 99, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 97; (37) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 100, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 101; (38) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 102, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 103; (39) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 104, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 105; (40) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 107, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 106; (41) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 109, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 108; (42) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 111, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 110; (43) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 113, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 112; (44) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 114, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 115; (45) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 117, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 116; (46) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 119, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 118; (47) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 121, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 120; (48) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 123, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 122; (49) the second light chain comprises an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 124, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 4; (50) the second light chain comprises an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 2, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 125; (51) the first and second light chains both comprise an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 143, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 142; (52) the first and second light chains both comprise an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 145, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 144; (53) the first and second light chains both comprise an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 147, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 146; (54) the first and second light chains both comprise an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 149, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 148; (55) the first and second light chains both comprise an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 151, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 150; (56) the first and second light chains both comprise an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 153, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 152; (57) the first and second light chains both comprise an amino acid sequence as set forth in SEQ ID NO: 1, the first heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 155, and the second heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 154; (58) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 157, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 156; (59) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 160, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 159; (60) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 162, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 161; (61) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 164, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 163; (62) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 166, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 165; (63) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 168, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 167; (64) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 170, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 169; (65) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 172, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 171; (66) the first and second light chains both comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 174, and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 173; (67) both the first and second light chains comprise the amino acid sequence set forth in SEQ ID NO: 1, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 176, and the second heavy chain comprises the amino acid sequence 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 of any one of claims 1 to 35.

37. A set of isolated polynucleotides comprising polynucleotides encoding the first heavy chain, the first light chain, the second heavy chain, and the second light chain of the fusion protein of any one of claims 1 to 35.

38. An isolated vector comprising the isolated polynucleotide of claim 36 or 37.

39. A host cell comprising the isolated polynucleotide of claim 36, the set of isolated polynucleotides of claim 37, or the isolated vector of claim 38.

40. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 35, the isolated polynucleotide of claim 36, the set of isolated polynucleotides of claim 37, the isolated vector of claim 38, or the host cell of claim 39, and a pharmaceutically acceptable carrier.

41. A kit comprising the fusion protein of any one of claims 1 to 35, the isolated polynucleotide of claim 36, the set of isolated polynucleotides of claim 37, the isolated vector of claim 38, the host cell of claim 39, or the pharmaceutical composition of claim 40.

42. Use of the fusion protein of any one of claims 1 to 35, the isolated polynucleotide of claim 36, the set of isolated polynucleotides of claim 37, the isolated vector of claim 38, the host cell of claim 39, the pharmaceutical composition of claim 40, or the kit of claim 41 in the manufacture of a medicament for the prevention or treatment of a disease, or in the manufacture of a kit for the diagnosis of a disease.

43. The use of claim 42, wherein the disease comprises a tumor, optionally a solid tumor.

44. A method of diagnosing, preventing, or treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the fusion protein of any one of claims 1 to 36, the isolated polynucleotide of claim 36, the set of isolated polynucleotides of claim 37, the isolated vector of claim 38, the host cell of claim 39, the pharmaceutical composition of claim 40, or the kit of claim 41.

45. The method of claim 44, wherein the disease comprises a tumor, optionally a solid tumor.

46. A method for producing the fusion protein of any one of claims 1 to 35, the method 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 into one host cell, or into separate host cells, and expressing the first expression vector, the second expression vector, the third expression vector, and the fourth expression vector under appropriate conditions.

47. The method of claim 46, wherein the host cell is a eukaryotic cell.

48. The method of claim 46 or 47, wherein, The host cell is a mammalian cell.

Images