FcεRI-binding proteins

Abstract

The present application provides binding proteins, which contain an IgG Fc region or a fragment thereof and are capable of binding to FcεRI. The present application also provides nucleic acid molecules encoding the binding proteins, vectors and cells containing the nucleic acid molecules, pharmaceutical compositions containing the binding proteins, and uses of the binding proteins in the preparation of medicaments.

Description

[Technical Field]

[0001] The present application relates to the field of biomedical science, and specifically to FcεRI binding proteins. [Background technology]

[0002] The neonatal Fc receptor (FcRn) belongs to a broad and functionally distinct family of MHC molecules. In contrast to classical MHC family members, FcRn exhibits little diversity and is unable to present antigens. Instead, FcRn regulates the serum half-life of IgG and albumin through its ability to bind these two proteins with high affinity at low pH. The serum half-life of IgG is significantly longer than that of uniformly sized globular proteins, including IgE, which does not bind FcRn (approximately 21 days for IgG and approximately <2 days for IgE). In addition, FcRn plays an important role in mucosal and systemic immunity through its ability to influence the longevity of IgG and its involvement in both innate and adaptive immune responses.

[0003] The IgE receptor FcεRI is involved in IgE-mediated activation of a wide range of immune cells, leading to various immune diseases, including allergic diseases. Blocking the binding of IgE to the FcεRI receptor with FcεRI binding proteins has the potential to treat immune diseases. In addition, mobilizing relevant immune cells by targeting the FcεRI receptor can also be used to treat tumors. Therefore, FcεRI binding proteins have various therapeutic potential.

[0004] Although natural IgE antibodies have strong FcεRI binding activity, they still have many drawbacks for their development as pharmaceuticals. For example, natural IgE antibodies have approximately 14 potential glycosylation sites (see Figure 1A), which is not conducive to quality control after large-scale production. IgE Fc cannot bind to protein A or G, and therefore cannot be purified on a large scale using existing purification methods for well-established IgG-based drugs. IgE Fc does not have the ability to bind to FcRn, resulting in a short plasma half-life of natural IgE antibodies. In addition, the affinity of recombinant IgE for FcεRI is too high (10-10 M) (Metzger H. The receptor with high affinity for IgE. Immunol Rev. 1992;125:37-48), which may cause severe allergic reactions, especially when administered as a pharmaceutical. In summary, proteins with improved properties compared to both IgE and IgG are needed. Summary of the Invention

[0005] The present application provides binding proteins that have the ability to bind to FcεRIa while maintaining the good druggability properties associated with IgG Fc. The binding proteins of the present application have one or more of the following properties: (1) It can bind to FcεRIa, (2) it can bind to FcRn, (3) it has good expression quality, (4) it can be directly purified with protein A or G, and (5) it has reduced potential glycosylation sites compared to IgE Fc.

[0006] In one aspect, the application provides a binding protein capable of binding to FcεRI, the binding protein comprising an IgG Fc region or a fragment thereof, wherein the IgG Fc region or fragment thereof comprises a BC loop (BC-LOOP), a DE-turn and / or an FG loop (FG-LOOP) derived from CH3 in an IgE Fc region, or a fragment thereof, and wherein a region in CH2 in the IgG Fc region corresponding to the BC loop, DE turn and / or FG loop in CH3 in the IgE Fc region, or a fragment thereof, is substituted or replaced by the BC loop, DE turn and / or FG loop in CH3 in the IgE Fc region, or a fragment thereof.

[0007] In some embodiments, the IgG is human IgG.

[0008] In some embodiments, the IgG is an IgG1, IgG2, IgG3, or IgG4.

[0009] In some embodiments, the CH2 in the wild-type IgG Fc region comprises the amino acid sequence set forth in any one of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0010] In some embodiments, the fragment of an IgG Fc region comprises at least CH2 of an IgG Fc region or a fragment thereof.

[0011] In some embodiments, the BC loop, DE turn and / or FG loop from CH3 in an IgE Fc region, or a fragment thereof, is capable of binding to FcεRI.

[0012] In some embodiments, the BC loop in CH2 of an IgG Fc region comprises the amino acid sequence of positions 27 to 38 in CH2 of an IgG Fc region.

[0013] In some embodiments, the BC loop in CH2 in an IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

[0014] In some embodiments, the DE turn in CH2 of the IgG Fc region comprises the amino acid sequence of positions 84.1 to 84.4 and 85.4 to 85.1 in CH2 of the IgG Fc region.

[0015] In some embodiments, the DE turn in CH2 in an IgG Fc region comprises the amino acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:39, and SEQ ID NO:40.

[0016] In some embodiments, the FG loop in CH2 of the IgG Fc region comprises the amino acid sequence of positions 105 to 117 in CH2 of the IgG Fc region.

[0017] In some embodiments, the FG loop in CH2 in an IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0018] In some embodiments, the BC loop in CH3 of the IgE Fc region comprises the amino acid sequence of positions 27 to 38 in CH3 of the IgE Fc region.

[0019] In some embodiments, the BC loop in CH3 in the IgE Fc region comprises the amino acid sequence set forth in SEQ ID NO:8.

[0020] In some embodiments, the DE turn in CH3 of the IgE Fc region comprises the amino acid sequence of positions 84.1 to 84.4 and 85.4 to 85.1 in CH3 of the IgE Fc region, and the DE turn in CH3 of the IgE Fc region comprises the amino acid sequence of positions 84.1 to 84.4 and 85.4 to 85.1 in CH3 of the IgE Fc region.

[0021] In some embodiments, the DE turn in CH3 in the IgE Fc region comprises the amino acid sequence set forth in SEQ ID NO:9.

[0022] In some embodiments, the FG loop in CH3 of the IgE Fc region comprises the amino acid sequence of positions 105 to 117 in CH3 of the IgE Fc region.

[0023] In some embodiments, the FG loop in CH3 in the IgE Fc region comprises the amino acid sequence set forth in SEQ ID NO:10.

[0024] In some embodiments, the location of the region of amino acids is determined by the IMGT unique numbering for C domain coding.

[0025] In some embodiments, the CH2 in the IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:41.

[0026] In another aspect, the present application provides a binding protein comprising an IgG Fc region or a fragment thereof, wherein the IgG Fc region or fragment thereof comprises one or more amino acid mutations compared to a wild-type IgG Fc region, and wherein the mutant IgG Fc region or fragment thereof is capable of binding to FcεRI.

[0027] In some embodiments, the IgG comprises human IgG.

[0028] In some embodiments, IgG comprises IgG1, IgG2, IgG3, or IgG4.

[0029] In some embodiments, the CH2 of the wild-type IgG Fc region comprises the amino acid sequence set forth in any one of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0030] In some embodiments, the IgG Fc region comprises at least CH2 within the IgG Fc region.

[0031] In some embodiments, the BC loop in CH2 in the IgG Fc region has one or more amino acid mutations.

[0032] In some embodiments, the BC loop in CH2 in the IgG Fc region has amino acid mutations at one or more sites selected from the group consisting of V28, S29, H30, E31, D34, P35, and / or K / Q38.

[0033] In some embodiments, the BC loop in CH2 in the IgG Fc region has one or more amino acid mutations selected from the group consisting of V28L, S29A, H30P, E31S, D34K, P35G, E36T, and / or K / Q38N.

[0034] In some embodiments, the BC loop in CH2 in the mutated IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:8.

[0035] In some embodiments, the DE turn in CH2 in the IgG Fc region has amino acid mutations at one or more sites selected from the group consisting of E84.1, Y / F84.3, S85.4, Y / F85.2, and / or R85.1.

[0036] In some embodiments, the DE turn in CH2 in the IgG Fc region has one or more amino acid mutations selected from the group consisting of E84.1K, Y / F84.3R, S85.4G, Y / F85.2L, and / or R85.1T.

[0037] In some embodiments, the DE turn in CH2 in the mutant IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:9.

[0038] In some embodiments, the FG loop in CH2 in the IgG Fc region has amino acid mutations at one or more sites selected from the group consisting of K105, S107, N108, K109, A / G110, A / S115, P / S116, and / or I117.

[0039] In some embodiments, the FG loop in CH2 in the IgG Fc region has one or more amino acid mutations selected from the group consisting of K105R, S107T, N108H, K109P, A / G110H, A / S115R, P / S116A, and / or I117L.

[0040] In some embodiments, the FG loop in CH2 in the mutated IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:10.

[0041] In some embodiments, the IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:11, SEQ ID NO:38, or SEQ ID NO:41.

[0042] In some embodiments, the N-terminus of CH2 in the IgG Fc region has amino acid mutations at one or more sites selected from the group consisting of P1.5, E1.4, L / P / F1.3, L / V1.2, G / A1.1, G1, and / or P2.

[0043] In some embodiments, the N-terminus of CH2 in the IgG Fc region has one or more amino acid mutations selected from the group consisting of P1.5D, E1.4S, L / P / F1.3N, L / V1.2P, G / A1.1R, and / or P2V.

[0044] In some embodiments, the location of the region of amino acids is determined by the IMGT unique numbering for C domain coding.

[0045] In some embodiments, the CH2 in the mutated IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:41.

[0046] In some embodiments, the binding protein further comprises a CH3 within an IgG Fc region.

[0047] In some embodiments, the binding protein comprises the amino acid sequence set forth in SEQ ID NO:29 or SEQ ID NO:38.

[0048] In some embodiments, the binding protein further comprises a CH2 within the constant region of IgE or a fragment thereof.

[0049] In some embodiments, the IgG Fc region of the binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:30, and SEQ ID NO:31.

[0050] In some embodiments, the binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:30, and SEQ ID NO:31.

[0051] In some embodiments, the binding protein has one or more of the following properties: (1) capable of binding to FcεRIa; (2) capable of binding to FcRn; (3) have good expression quality; (4) can be directly purified with Protein A or G; (5) The number of potential glycosylation sites is reduced compared to IgE Fc.

[0052] In another aspect, the present application provides a polypeptide comprising a binding protein.

[0053] In another aspect, the present application provides a fusion protein comprising the binding protein.

[0054] In some embodiments, the fusion protein comprises one or more other functionally active proteins.

[0055] In some embodiments, the other functionally active protein is one or more selected from the group consisting of a Fab, a scFv, a VHH, a cytokine, a soluble receptor or ligand, and a pharmaceutical polypeptide.

[0056] In some embodiments, the fusion protein is an IgG-like molecule.

[0057] In some embodiments, the fusion protein is capable of specifically binding to a tumor-associated antigen.

[0058] In some embodiments, the tumor-associated antigen comprises Her2.

[0059] In some embodiments, the heavy chain of the IgG-like molecule comprises the amino acid sequence set forth in SEQ ID NO:20 or SEQ ID NO:21.

[0060] In some embodiments, the light chain of the IgG-like molecule comprises the amino acid sequence set forth in SEQ ID NO:22.

[0061] In some embodiments, the tumor-associated antigen comprises GPC3.

[0062] In some embodiments, the heavy chain of the IgG-like molecule comprises the amino acid sequence set forth in SEQ ID NO:32 or SEQ ID NO:33.

[0063] In some embodiments, the light chain of the IgG-like molecule comprises the amino acid sequence set forth in SEQ ID NO:34.

[0064] In some embodiments, the tumor-associated antigen comprises CD20.

[0065] In some embodiments, the heavy chain of the IgG-like molecule comprises the amino acid sequence set forth in SEQ ID NO:35 or SEQ ID NO:36.

[0066] In some embodiments, the light chain of the IgG-like molecule comprises the amino acid sequence set forth in SEQ ID NO:37.

[0067] In another aspect, the present application also provides a drug molecule comprising a binding protein, polypeptide, or fusion protein.

[0068] In another aspect, the present application also provides one or more isolated nucleic acid molecules encoding the binding protein, polypeptide, or fusion protein.

[0069] In another aspect, the present application also provides a vector comprising the nucleic acid molecule.

[0070] In another aspect, the present application also provides a cell comprising the nucleic acid molecule or vector.

[0071] In another aspect, the present application also provides a pharmaceutical composition comprising the binding protein, polypeptide, fusion protein, drug molecule, nucleic acid molecule, vector or cell, and optionally a pharmaceutically acceptable carrier.

[0072] In another aspect, the application also provides a method of preparing a binding protein, polypeptide, or fusion protein, comprising culturing cells under conditions that allow expression of the binding protein, polypeptide, or fusion protein.

[0073] In another aspect, the present application also provides the use of the binding protein, polypeptide, fusion protein, drug molecule, nucleic acid molecule, vector, cell or pharmaceutical composition in the preparation of a medicament for preventing or treating a disease and / or disorder.

[0074] In some embodiments, the disease and / or disorder comprises a tumor.

[0075] In some embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0076] Those skilled in the art will readily recognize other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the contents of the present application will enable those skilled in the art to make modifications to the particular embodiments disclosed without departing from the spirit and scope of the invention(s) involved in the present application. Correspondingly, the drawings and descriptions in the specification of the present application are merely illustrative and not restrictive.

[0077] Specific features of the invention involved in this application are set forth in the appended claims. The characteristics and advantages of the invention involved in this application can be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings, a brief description of which follows. [Brief explanation of the drawings]

[0078] [Figure 1] Schematic diagrams of the structures of IgE and IgG and potential glycosylation modification sites are shown. [Figure 2] A schematic diagram of the binding protein structure and potential glycosylation sites is shown. [Figure 3] A schematic diagram of the structure of the binding protein fused to the Fab and potential glycosylation sites is shown. [Figure 4A] Figure 1 shows the IMGT coding and domain division of the constant region CH3 of the heavy chain of human IgE (coding mode is the IMGT specific numbering for the C domains). [Figure 4B] Figure 1 shows the IMGT coding and domain division of the constant region CH2 of the heavy chain of human IgG (coding mode is the IMGT unique numbering for the C domains). [Figure 5] The amino acid sequences of different binding proteins and the sequence alignment results are shown. [Figure 6] 1 shows the results of affinity assays of various binding proteins for recombinant human FcεRIa. [Figure 7]1 shows the results of SEC-HPLC detection of binding proteins DB177, DB364, and DB365. [Figure 8] 1 shows the results of SEC-HPLC detection of binding proteins DB366, DB-B63, and DB-B64. [Figure 9] FIG. 1 shows the results of SEC-HPLC detection of Her2-specific binding proteins DB871 and DB872. [Figure 10] 1 shows the results of SEC-HPLC detection of GPC3-specific binding proteins DB-B229 and DB-B230. [Figure 11] FIG. 1 shows the results of SEC-HPLC detection of CD20-specific binding proteins DB-B232 and DB-B233. [Figure 12A] 1 shows the results of ELISA detection of the blocking effect of the binding proteins of the present application in blocking the binding of human IgE to the receptor FcεRIa. [Figure 12B] 1 shows the results of ELISA detection of the blocking effect of the binding proteins of the present application in blocking the binding of human IgE to the receptor FcεRIa. [Figure 12C] 1 shows the results of ELISA detection of the blocking effect of the binding proteins of the present application in blocking the binding of human IgE to the receptor FcεRIa. [Figure 13A] 1 shows the results of detecting the effect of binding proteins mediated activated mast cells expressing a luciferase reporter gene. 2 shows the results of detecting the effect of Her2-specific binding proteins DB871 and DB872 mediated NCI-N87 cell activated mast cells expressing a luciferase reporter gene. [Figure 13B] 1 shows the results of detecting the effects of binding proteins mediated by activated mast cells expressing a luciferase reporter gene. 2 shows the results of detecting the effects of GPC3-specific binding proteins DB-B229 and DB-B230 mediated HepG2 cell activation of mast cells expressing a luciferase reporter gene. [Figure 13C]1 shows the results of detecting the effects of binding protein-mediated activated mast cells expressing a luciferase reporter gene. 2 shows the results of detecting the effects of CD20-specific binding proteins DB-B232 and DB-B233-mediated Raji cell-activated mast cells expressing a luciferase reporter gene. [Figure 14] 1 shows the results of detecting the binding of a control antibody and a binding protein to recombinant human FcRn. DETAILED DESCRIPTION OF THE INVENTION

[0079] Implementation of the present application is illustrated in the following specific examples, and other benefits and advantages of the present application will be readily apparent to those skilled in the art from the disclosure herein.

[0080] Definition of Terms In this application, the term "amino acid mutation" generally encompasses amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, as long as the final construct possesses the desired properties. A specific amino acid mutation is an amino acid substitution. For example, to alter the binding characteristics of the Fc region, non-conservative amino acid substitutions can be selected, i.e., one amino acid can be replaced with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with unnatural amino acids or natural amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for altering amino acid side groups other than genetic engineering, such as chemical modification, are also available.

[0081] In the present application, "Xn" refers to residue X corresponding to position n in the amino acid sequence of a wild-type IgG Fc region, where n is a positive integer and X is an abbreviation for any amino acid residue, and the sequence position is determined by the IMGT unique numbering for C domain numbering scheme, a commonly used numbering scheme known to those skilled in the art. For example, "K105" represents amino acid K at position 105, which corresponds to the sequence of CH2 in the IgG Fc region, using the IMGT unique numbering for C domain numbering scheme.

[0082] In this application, an amino acid substitution of "X / YnZ" means that residue X or Y at position n in an amino acid sequence is substituted with amino acid residue Z, according to the numbering scheme in this application, where n is a positive integer, and X, Y, and Z are each independently an abbreviation of any amino acid residue.

[0083] In the present application, the terms "BC loop," "DE turn," and "FG loop" generally refer to segments of the amino acid sequence of the Fc region of an immunoglobulin molecule, which can be determined by the IMGT unique numbering system for coding the C domain. Such definitions are clear to those skilled in the art. For example, the BC loop in CH2 of an IgG Fc region comprises the amino acid sequence at positions 27 to 38 in CH2 of the IgG Fc region. For example, the DE turn in CH2 of an IgG Fc region comprises the amino acid sequence at positions 84.1 to 84.4 and 85.4 to 85.1 in CH2 of the IgG Fc region. For example, the FG loop in CH2 of an IgG Fc region comprises the amino acid sequence at positions 105 to 117 in CH2 of the IgG Fc region. For example, the BC loop in CH3 of an IgE Fc region comprises the amino acid sequence at positions 27 to 38 in CH3 of the IgE Fc region. For example, the DE turn in CH3 of the IgE Fc region comprises the amino acid sequence of positions 84.1 to 84.4 and 85.4 to 85.1 in CH3 of the IgE Fc region, and the FG loop in CH3 of the IgE Fc region comprises the amino acid sequence of positions 105 to 117 in CH3 of the IgE Fc region.

[0084] In this application, the term "polypeptide" generally refers to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The term can also encompass amino acid polymers that are modified, for example, by the addition of sugar residues to form glycoproteins or by phosphorylation. Polypeptides can be naturally occurring and produced from non-recombinant cells, or from genetically engineered or recombinant cells, and can include molecules with the amino acid sequence of a native protein or molecules with deletions, additions, and / or substitutions of one or more amino acids from the native sequence. The term "polypeptide" can include antigen-binding fragments, antibodies, or sequences with deletions, additions, and / or substitutions of one or more amino acids of the antigen-binding fragment. The term "polypeptide" can refer to polypeptides with amino-terminal deletions, carboxy-terminal deletions, and / or internal deletions compared to the full-length protein. Such fragments can also contain modified amino acids compared to the full-length protein.

[0085] In the present application, the term "VHH" generally refers to an antibody comprising the variable antigen-binding domain of a heavy chain antibody. VHHs may also be called nanobodies (Nbs) and / or single domain antibodies.

[0086] The term "Fab" refers to a molecule comprising a variable region domain of the heavy chain (VH) and the first constant domain of the heavy chain (C H 1) together with the complete light chain ((V L and C L ) refers to an antibody fragment composed of the light chain fragment (L) and the heavy chain fragment (H) of an intact antibody. Papain digestion of an intact antibody can be used to generate two Fab fragments, each of which contains a single antigen-binding site. Generally, the light chain and heavy chain fragments of the Fab generated by papain digestion are linked by an interchain disulfide bond.

[0087] In this application, the term "scFv" generally refers to a VL and VH linked together using recombinant methods or by a synthetic linker that allows them to be produced as a single protein chain, where the VL and VH domains pair to form a monovalent molecule (called a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci USA 85:5879-5883 (1988)).

[0088] In this application, the term "FcεRI" generally refers to the high-affinity receptor for the Fc region of immunoglobulin E (IgE) (FcεRI, also known as Fc epsilon RI). FcεRI is a tetrameric receptor complex composed of one α chain (i.e., FcεRIα), one β chain (FcεRIβ), and two γ chains (FcεRIγ) that can bind to the Fc region of the ε heavy chain of IgE. Generally, the α chain can act as the binding site for antibodies (e.g., IgE), the γ chain can act as the initiation site for downstream signals, and the β chain can act to amplify downstream signals. FcεRI is expressed on epidermal Langerhans cells, eosinophils, mast cells, and basophils. Due to its cellular distribution, this receptor plays a major role in allergic reactions. FcεRI can also be expressed on antigen-presenting cells and is involved in the production of important immune mediators that promote inflammation, such as cytokines, interleukins, leukotrienes, and prostaglandins.

[0089] As used herein, the terms "specifically bind to" or "specific" generally refer to a measurable and reproducible interaction, e.g., binding between a target and an antibody, that allows the presence of the target to be determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) is one that binds to the target with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In some embodiments, an antibody specifically binds to an epitope on a protein, where the epitope is conserved among proteins of different species. In some embodiments, specific binding includes, but is not required to be, exclusive binding.

[0090] In this application, the term "nucleic acid" generally refers to a polymer of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) and can include naturally occurring nucleic acids (adenine, guanine, cytosine, uracil, and thymidine), non-naturally occurring nucleic acids, and modified nucleic acids. The term "nucleic acid" can include genes, cDNAs, or mRNAs. For example, nucleic acid molecules can be synthetic (e.g., chemically synthesized) or recombinant. Nucleic acids can include nucleic acids containing analogs or derivatives of naturally occurring nucleotides, which have similar binding properties to those of the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Nucleic acid sequences can also include conservatively modified variants thereof (e.g., with degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly designated sequences. The term is not limited by the length of the polymer. Nucleic acids can be single- or double-stranded and generally contain 5'-3' phosphodiester linkages, although nucleotide analogs may have other linkages.

[0091] In the present application, the term "cell" generally refers to an individual cell, cell line, or cell culture that may contain or have contained a plasmid or vector containing a nucleic acid molecule of the present application, or that is capable of expressing a fusion protein or antigen-binding fragment thereof of the present application. A cell may include the progeny of a single cell. Due to natural, accidental, or deliberate mutation, progeny cells may not necessarily be exactly identical in morphology or genome to the original parent cell, so long as they are capable of expressing a fusion protein or antigen-binding fragment thereof of the present application. Cells can be obtained by transfecting cells in vitro with a vector of the present application. Cells may be prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, or myeloma cells).

[0092] In this application, the term "vector" generally refers to a nucleic acid molecule that can autonomously replicate in a suitable host and transfer an inserted nucleic acid molecule into and / or between cells (e.g., a host cell). Vectors may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and vectors primarily for expressing DNA or RNA by transcription and / or translation. Vectors also include vectors having more than one of the above functions. A vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into an appropriate cell. Typically, a vector can produce a desired expression product by culturing an appropriate cell containing the vector. In this application, a vector may be a plasmid.

[0093] In the present application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, which may be a human patient. For example, a pharmaceutical composition of the present application may include an antigen-binding protein of the present application, an immunoconjugate of the present application, a nucleic acid molecule of the present application, a vector of the present application, and / or a cell of the present application, and optionally a pharmaceutically acceptable carrier. In addition, a pharmaceutical composition may also include one or more of a (pharmaceutically effective) carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, and / or preservative, and other suitable formulation agents. Acceptable components of a composition may be non-toxic to a recipient at the dosages and concentrations used. Pharmaceutical compositions of the present application may include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0094] In this application, the term "subject" generally refers to a human or non-human animal (including, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey).

[0095] In this application, the term "IgG" generally refers to immunoglobulin G. IgG is one of the human immunoglobulins, the others being IgA, IgM, IgD, and IgE. Human IgG has four subtypes: IgG1, IgG2, IgG3, and IgG4, based on antigenic differences in the γ chain in the IgG molecule. In this application, the term "IgG1" generally refers to the most abundant IgG subtype, which has high affinity for Fc receptors. For example, the IgG may be human IgG. Furthermore, for example, the IgG may be selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0096] In this application, the term "IgG-like molecule" generally refers to a molecule having a structure similar to that of an IgG-like molecule. For example, an IgG-like molecule may include a constant region and a variable region. For example, an IgG-like molecule may include a light chain and a heavy chain. An IgG-like molecule essentially includes various domains of an IgG molecule (not limited to a target), but each domain may be modified to provide the IgG-like molecule with new functions or to change its action.

[0097] In this application, the term "fusion protein" generally refers to a chimeric protein containing the amino acid sequences of two or more different proteins. Typically, fusion proteins can be produced by in vitro recombination strategies well known in the art.

[0098] In this application, the term "drug molecule" generally refers to a molecule having a desired biological effect. A drug may be prophylactic or therapeutic. Drug molecules may include, but are not limited to, protein molecules (including, but not limited to, peptides, polypeptides, proteins including post-translationally modified proteins, fusion proteins, antibodies, etc.), small molecules (including inorganic or organic compounds), and nucleic acid molecules (including, but not limited to, double-stranded or single-stranded DNA or double-stranded or single-stranded RNA (e.g., antisense molecules, RNAi, etc.), intron sequences, triple-helical nucleic acid molecules, and aptamers), or vaccines.

[0099] It should be understood that the proteins, polypeptides, and / or amino acid sequences involved in this application also include variants or homologs having the same or similar function as the protein or polypeptide, at least to the extent that:

[0100] In the present application, a variant may be, for example, a protein or polypeptide having one or more amino acid substitutions, deletions, or additions in the amino acid sequence of the protein and / or polypeptide. For example, a functional variant may include a protein or polypeptide having at least one, e.g., 1 to 30, 1 to 20, or 1 to 10, and further, e.g., 1, 2, 3, 4, or 5, amino acid substitutions, deletions, and / or insertions. A functional variant can substantially retain the biological properties of the protein or polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, a functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding ability) of the protein or polypeptide prior to the alteration. For example, the substitution may be a conservative substitution.

[0101] In the present application, a homolog can be a protein or polypeptide that has at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or polypeptide.

[0102] In this application, homology generally refers to the similarity, similarity, or relationship between two or more sequences. A "percentage of sequence identity" can be calculated by comparing and aligning two sequences within a comparison window, determining the number of positions (where the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is present in both sequences to obtain the number of matching positions), dividing the number of matching positions by the total number of positions within the comparison window (i.e., window size), and multiplying the result by 100 to generate a percentage of sequence identity. Alignment to determine the percentage of sequence homology can be achieved in various ways known in the art, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the full-length sequence range compared to or within the region of the target sequence. Homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85:2444-2448, 1988, and D.J. Lipman and W.R. Pearson, "Rapid and Sensitive Protein Similarity Searches," Science, 227:1435-1441, 1989.A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, EW Myers and D. Lipman, "Basic Local Alignment Search Tool," Journal of Molecular Biology, 215:403-410, 1990.

[0103] In this application, the term "comprise" generally means the inclusion of features explicitly specified but without excluding other elements.

[0104] In this application, the term "about" generally refers to a variation of 0.5% to 10% above and below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0105] Detailed Description of the Invention The binding proteins involved in this application can be obtained by grafting amino acids located in the BC loop, DE turn, and FG loop regions of the IgE Fc CH3 domain to replace the corresponding amino acids in the BC loop, DE turn, and FG loop regions of the IgG Fc CH2 domain (the above regions and amino acid positions are based on the IMGT-specific coding of antibody constant regions, i.e., C domains, and the IMGT-specific numbering of strand, turn, and loop length ranges in the C domain and C-like domains, https: / / www.imgt.org / ). The modified IgG Fc CH2 was obtained by graft replacement. The binding proteins of this application can bind to FcεRIa.

[0106] The binding proteins involved in the present application can be obtained by transplanting some of the amino acids located in the BC loop, DE turn, and FG loop regions of the IgE Fc CH3 domain and replacing the corresponding amino acids in the corresponding BC loop, DE turn, and FG loop regions of the IgG Fc CH2 domain. For example, a modified IgG Fc CH2 was obtained by replacing the amino acids in the BC loop, DE turn, and FG loop regions of the IgG Fc CH2 domain with some of the amino acids in the corresponding BC loop, DE turn, and FG loop regions of the IgE Fc CH3 domain.

[0107] In this application, the binding protein may also be mutated within the grafted region.

[0108] The binding proteins involved in this application can also be obtained by mutating amino acids located in the BC loop, DE turn and FG loop regions of the IgG Fc CH2 domain.

[0109] The binding protein of the present application has the ability to bind to FcεRIa while maintaining the good druggability properties associated with IgG Fc: 1. good expression quality, with up to 90% or more monomer purity after a single purification step; 2. removal of excess glycosylation sites in IgE native antibodies, facilitating quality control; 3. easy purification, suitable for direct purification using Protein A or G; 4. capable of binding to FcRn with a long half-life; 5. low molecular weight and good tissue permeability; 6. can be easily fused to other proteins to construct IgG-like molecules such as Fab, scFv, VHH, cytokines, soluble receptors or ligands, polypeptides, etc.

[0110] In one aspect, the application provides a binding protein capable of binding to FcεRI, wherein the binding protein comprises an IgG Fc region or a fragment thereof, wherein the IgG Fc region or fragment thereof comprises the BC loop, DE turn and / or FG loop from CH3 in an IgE Fc region, or a fragment thereof, and wherein a region in CH2 in the IgG Fc region corresponding to the BC loop, DE turn and / or FG loop in CH3 in the IgE Fc region, or a fragment thereof, is substituted or replaced by the BC loop, DE turn and / or FG loop in CH3 in the IgE Fc region, or a fragment thereof.

[0111] In the present application, the IgG may be human IgG.

[0112] In the present application, IgG can be IgG1, IgG2, IgG3, or IgG4.

[0113] In the present application, the CH2 in the human IgG Fc region before grafting / replacement may comprise the amino acid sequence set forth in any one of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. In the present application, the sequence of CH2 in different IgG Fc regions may vary, but all may correspond to the amino acids identified according to the numbering scheme set forth in the present application.

[0114] In the present application, a fragment of an IgG Fc region may comprise at least CH2 of the IgG Fc region or a fragment thereof. In the present application, the main region for graft replacement is centered on CH2 of the IgG Fc region.

[0115] In the present application, the BC loop, DE turn and / or FG loop from CH3 in an IgE Fc region or a fragment thereof is capable of binding to FcεRI.

[0116] In the present application, the BC loop in CH2 of an IgG Fc region comprises the amino acid sequence of positions 27 to 38 in CH2 of the IgG Fc region. For example, the BC loop in CH2 of an IgG Fc region may comprise the amino acid sequence set forth in DVSHEDPEV(K / Q) (SEQ ID NO: 1 or SEQ ID NO: 2).

[0117] In the present application, the DE turn in CH2 of an IgG Fc region may comprise the amino acid sequence of positions 84.1 to 84.4 and positions 85.4 to 85.1 in CH2 of an IgG Fc region. For example, the DE turn in CH2 of an IgG Fc region may comprise the amino acid sequence set forth in EQ(Y / F)NST(Y / F)R (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 39, or SEQ ID NO: 40).

[0118] In the present application, the FG loop in CH2 of the IgG Fc region comprises the amino acid sequence of positions 105 to 117 in CH2 of the IgG Fc region. For example, the FG loop in CH2 of the IgG Fc region may comprise the amino acid sequence set forth in KVSNK(A / G)LP(A / S)(P / S)I (SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7).

[0119] In the present application, the BC loop, DE turn, and / or FG loop, or portions thereof, in CH2 in an IgG Fc region may be replaced by the corresponding BC loop, DE turn, and / or FG loop, or portions thereof, in CH3 in an IgE Fc region.

[0120] In the present application, the BC loop in CH3 of the IgE Fc region may comprise the amino acid sequence of positions 27 to 38 in CH3 of the IgE Fc region. For example, the BC loop in CH3 of the IgE Fc region may comprise the amino acid sequence set forth in DLAPSKGTVN (SEQ ID NO: 8).

[0121] In the present application, the DE turn in CH3 of the IgE Fc region may comprise the amino acid sequence of positions 84.1 to 84.4 and positions 85.4 to 85.1 in CH3 of the IgE Fc region. For example, the DE turn in CH3 of the IgE Fc region may comprise the amino acid sequence set forth in KQRNGTLT (SEQ ID NO: 9).

[0122] In the present application, the FG loop in CH3 of the IgE Fc region may comprise the amino acid sequence of positions 105 to 117 in CH3 of the IgE Fc region. For example, the FG loop in CH3 of the IgE Fc region may comprise the amino acid sequence set forth in RVTHPHLPRAL (SEQ ID NO: 10).

[0123] In the present application, the CH2 in the grafted / replaced IgG Fc region may comprise the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 41.

[0124] In another aspect, the binding proteins of the present application can be obtained by amino acid mutations on the CH2 fragment in the IgG Fc region. The binding proteins provided in the present application comprise an IgG Fc region or a fragment thereof that contains one or more amino acid mutations compared to a wild-type IgG Fc region, and the mutant IgG Fc region or fragment thereof is capable of binding to FcεRI.

[0125] In the present application, FcεRI may include FcεRIa.

[0126] In the present application, IgG may include human IgG.

[0127] In the present application, IgG includes IgG1, IgG2, IgG3, or IgG4.

[0128] In the present application, the CH2 in a wild-type IgG Fc region may comprise the amino acid sequence set forth in any one of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0129] In the present application, an IgG Fc region may include at least CH2 within the IgG Fc region.

[0130] In the present application, the BC loop in CH2 in the IgG Fc region has one or more amino acid mutations.

[0131] In the present application, the BC loop in CH2 in the IgG Fc region may have amino acid mutations at V28, S29, H30, E31, D34, P35, and / or K / Q38, where K / Q38 means that the amino acid at position 38 before mutation may correspond to the amino acid K or Q, depending on the different IgG Fc CH2 sequences.

[0132] In the present application, the BC loop in CH2 in the IgG Fc region may have the following amino acid mutations: V28L, S29A, H30P, E31S, D34K, P35G, E36T, and / or K / Q38N.

[0133] In the present application, the BC loop in CH2 in the IgG Fc region may have the following amino acid mutations: V28L, S29A, H30P, E31S, D34K, P35G, E36T, and K / Q38N.

[0134] In the present application, the BC loop in CH2 in the IgG Fc region may have the mutations listed in the table below: [Table 1]

[0135] In the present application, the BC loop in CH2 in the mutant IgG Fc region may comprise the amino acid sequence set forth in SEQ ID NO:8.

[0136] In the present application, the DE turn in CH2 in the IgG Fc region may have amino acid mutations E84.1, Y / F84.3, S85.4, Y / F85.2 and / or R85.1.

[0137] In the present application, the DE turn in CH2 in the IgG Fc region may have amino acid mutations at E84.1, Y / F84.3, S85.4, Y / F85.2, and R85.1.

[0138] In the present application, the DE turn in CH2 in the IgG Fc region may have the amino acid mutations E84.1K, Y / F84.3R, S85.4G, Y / F85.2L and / or R85.1T.

[0139] In the present application, the DE turn in CH2 in the IgG Fc region may have the following amino acid mutations: E84.1K, Y / F84.3R, S85.4G, Y / F85.2L, and R85.1T.

[0140] In the present application, the DE turn in CH2 in the IgG Fc region may have the mutations listed in the table below: [Table 2]

[0141] In the present application, the DE turn in CH2 in the mutant IgG Fc region may comprise the amino acid sequence set forth in SEQ ID NO:9.

[0142] In the present application, the DE turn in CH2 in the IgG Fc region may have the amino acid mutations E84.1K, Y / F84.3R, S85.4G, Y / F85.2L and / or R85.1T.

[0143] In the present application, the FG loop in CH2 in the IgG Fc region may have amino acid mutations at K105, S107, N108, K109, A / G110, A / S115, P / S116 and / or I117.

[0144] In the present application, the FG loop in CH2 in the IgG Fc region may have amino acid mutations at K105, S107, N108, K109, A / G110, A / S115, P / S116, and I117.

[0145] In the present application, the FG loop in CH2 in the IgG Fc region may have the following amino acid mutations: K105R, S107T, N108H, K109P, A / G110H, A / S115R, P / S116A, and / or I117L.

[0146] In the present application, the FG loop in CH2 in the IgG Fc region may have the following amino acid mutations: K105R, S107T, N108H, K109P, A / G110H, A / S115R, P / S116A, and I117L.

[0147] In the present application, the FG loop in CH2 in the IgG Fc region may have the mutations listed in the table below: [Table 3]

[0148] In the present application, the FG loop in CH2 in the mutant IgG Fc region may comprise the amino acid sequence set forth in SEQ ID NO:10.

[0149] In the present application, the N-terminus of CH2 in the IgG Fc region has amino acid mutations at P1.5, E1.4, L / P / F1.3, L / V1.2, G / A1.1, G1 and / or P2.

[0150] In the present application, the N-terminus of CH2 in the IgG Fc region may have amino acid mutations at P1.5, E1.4, L / P / F1.3, L / V1.2, G / A1.1, G1, and P2.

[0151] In the present application, the N-terminus of CH2 in the IgG Fc region may have the amino acid mutations P1.5D, E1.4S, L / P / F1.3N, L / V1.2P, G / A1.1R, and / or P2V.

[0152] In the present application, the N-terminus of CH2 in the IgG Fc region may have the following amino acid mutations: P1.5D, E1.4S, L / P / F1.3N, L / V1.2P, G / A1.1R, and P2V.

[0153] In the present application, the N-terminus of CH2 in the IgG Fc region may have the amino acid mutations listed in the table below: [Table 4]

[0154] In the present application, the CH2 in the mutant IgG Fc region may comprise the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 41.

[0155] In the present application, the binding protein can also comprise a CH3 within an IgG Fc region. For example, the binding protein can comprise the amino acid sequence set forth in SEQ ID NO:29 or SEQ ID NO:38.

[0156] In the present application, the binding protein may also comprise the CH2 in the constant region of IgE or a fragment thereof.

[0157] In the present application, the IgG Fc region may comprise the amino acid sequence set forth in any one of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:30, and SEQ ID NO:31.

[0158] In another aspect, the present application also provides a polypeptide comprising the binding protein.

[0159] In another aspect, the present application also provides a fusion protein comprising the binding protein. In the present application, the fusion protein may also comprise one or more other functionally active molecules. For example, the other functionally active protein may be one or more selected from the group consisting of Fab, scFv, VHH, cytokine, soluble receptor or ligand, and pharmaceutical polypeptide.

[0160] For example, the fusion protein can be an IgG-like molecule. For example, the fusion protein can include a binding protein and a Fab molecule.

[0161] In the present application, the fusion protein is capable of specifically binding to a tumor-associated antigen.

[0162] For example, the tumor-associated antigen may comprise Her2. For example, the fusion protein may comprise a light chain and a heavy chain. For example, the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 22. For example, the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21. For example, the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 22, and the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 20. For example, the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 22, and the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 21.

[0163] For example, the tumor-associated antigen may comprise GPC3. For example, the fusion protein may comprise a light chain and a heavy chain. For example, the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 34. For example, the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 33. For example, the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 34, and the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 32. For example, the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 34, and the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 33.

[0164] For example, the tumor-associated antigen may comprise CD20. For example, the fusion protein may comprise a light chain and a heavy chain. For example, the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 37. For example, the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 36. For example, the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 37, and the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 35. For example, the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 37, and the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 36.

[0165] In another aspect, the present application provides a drug molecule comprising a binding protein, polypeptide, or fusion protein.

[0166] In another aspect, the application provides one or more isolated nucleic acid molecules encoding the binding protein, polypeptide, or fusion protein.

[0167] In another aspect, the present application provides one or more isolated nucleic acid molecules encoding a binding protein, polypeptide, or fusion protein. The one or more isolated nucleic acid molecules of the present application can be isolated nucleotides, deoxyribonucleotides, or ribonucleotides, or analogs thereof, of any length, isolated from a natural environment or artificially synthesized, but capable of encoding a binding protein of the present application.

[0168] In another aspect, the present application also provides vectors that can contain the nucleic acid molecules of the present application. A vector can transform, transduce, or transfect a host cell, thereby enabling expression of the genetic material elements carried within the host cell. For example, vectors can include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as lambda phage or M13 phage, and animal viruses. Examples of animal virus species that can be used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Furthermore, for example, vectors can contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors can also contain an origin of replication. Moreover, vectors may also include components that aid in entry into cells, such as, but not limited to, virions, lipidosomes, or protein membranes.

[0169] In another aspect, the present application also provides cells that can contain the nucleic acid molecules of the present application or the vectors of the present application. Cells can include the progeny of a single cell. Due to natural, accidental, or deliberate mutations, the progeny may not necessarily be identical (in terms of the morphology of the total DNA complement or in terms of the genome) to the original parent cell. In some embodiments, cells can also include cells transfected in vitro with vectors of the present application. In some embodiments, the cells can be bacterial cells (e.g., E. coli), yeast cells, or other eukaryotic cells.

[0170] In another aspect, the present application also provides pharmaceutical compositions, which can include a binding protein of the present application, a polypeptide of the present application, a fusion protein of the present application, a nucleic acid molecule of the present application, a vector of the present application, and / or a cell of the present application, and optionally a pharmaceutically acceptable carrier.

[0171] In some embodiments, the pharmaceutical composition may also include one or more of a (pharmaceutically effective) carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, and / or preservative, and other suitable formulation agents. Acceptable components of the composition may preferably be non-toxic to recipients at the dosages and concentrations used. Pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0172] In some embodiments, pharmaceutically acceptable adjuvants may include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are generally safe, non-toxic, and not biologically or otherwise undesirable and compatible with the pharmaceutical agent.

[0173] In some embodiments, the pharmaceutical composition may be administered parenterally, transdermally, intraperitoneally, intraarterially, intrathecally, and / or intranasally, or may be injected directly into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, administration of the pharmaceutical composition may be accomplished in different ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the pharmaceutical composition may be administered continuously. Continuous (or continuous) administration may be achieved by a miniature pump system worn by the patient that meters the flow of therapeutic agent to the patient, as described in WO 2015 / 036583.

[0174] In another aspect, the present application also provides methods for preparing the binding proteins, polypeptides, and fusion proteins of the present application, which may include culturing the cells of the present application under conditions that allow expression of the binding proteins of the present application.

[0175] In another aspect, the application provides the use of the binding protein, polypeptide, fusion protein, drug molecule, nucleic acid molecule, vector, cell, and / or pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease and / or disorder.

[0176] In another aspect, the present application provides binding proteins, polypeptides, fusion proteins, drug molecules, nucleic acid molecules, vectors, cells, and / or pharmaceutical compositions for use in preventing and / or treating diseases and / or disorders.

[0177] In another aspect, the present application provides methods for preventing and / or treating diseases and / or disorders, comprising administering to a subject in need thereof an effective amount of a binding protein, polypeptide, fusion protein, drug molecule, nucleic acid molecule, vector, cell, and / or pharmaceutical composition.

[0178] In this application, administration can be done in different ways, such as intravenously, intratumorally, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally.

[0179] In some embodiments, the disease and / or disorder comprises a tumor.

[0180] In some embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0181] Without intending to be limited by any theory, the following examples are merely illustrative of the binding proteins, preparation methods, and uses of the present application, and are not intended to limit the scope of the present application. [Example]

[0182] Example 1. Molecular construction, protein expression, purification and purity analysis 1.1 Molecular construction The Fc region of the binding protein was designed by transplanting amino acids located in the BC loop, DE turn, and FG loop regions of the IgE Fc CH3 domain to replace the corresponding amino acids in the BC loop, DE turn, and FG loop regions of the IgG Fc CH2 domain, or by replacing amino acids in the BC loop, DE turn, and FG loop regions of the IgG Fc CH2 domain with some of the amino acids in the BC loop, DE turn, and FG loop regions located in the IgE Fc CH3 domain. Here, the CH2 of the IgG Fc region after transplantation / replacement can comprise the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 41, or SEQ ID NO: 42. Genes encoding the binding proteins were synthesized by Genewiz (Suzhou) and Tsingke (Nanjing). For secretory expression in mammalian cells, a signal peptide with the following sequence was added to the N-terminus of the binding protein: MGWSCIILFLVATATGVHS (SEQ ID NO: 28). Using standard molecular biology techniques, the gene was cloned into a eukaryotic expression vector (e.g., pCDNA3.1 purchased from Miaoling Bio). After sequence verification, a plasmid Midiprep kit (NucleoBond Xtra Midi Plus, Macherey-Nagel) was used to prepare the plasmid for protein expression (see the manufacturer's instructions for specific procedures). Figure 1 shows a schematic diagram of the IgE and IgG structures and potential glycosylation sites. Figure 2 shows a schematic diagram of the structural design of the binding proteins and potential glycosylation sites. The diagram shows that graft replacement was performed in the IgG Fc CH2 domain, replacing some amino acids with the corresponding amino acids in the IgE Fc CH3 domain. According to the structure of binding protein 1 in Figure 2, the IgG Fc region after graft replacement may further comprise the CH2 domain of IgE or a fragment thereof; in Figure 2, binding proteins DB177 and DB364 have the structure of binding protein 1, and binding proteins DB365, DB366, DB-B63, and DB-B64 have the structure of binding protein 2.Figure 3 shows a schematic diagram of the structure of binding proteins fused to Fab and potential glycosylation sites, where binding proteins DB871, DB-B229, and DB-B232 have the structure of binding protein 3, and binding proteins DB872, DB-B230, and DB-B233 have the structure of binding protein 4. Figure 4A shows the IMGT coding and domain division of the constant region CH3 of the heavy chain of human IgE, and Figure 4B shows the IMGT coding and domain division of the constant region CH2 of the heavy chain of human IgG, where the coding mode is the IMGT unique numbering for the C domain. The amino acid sequences of the binding proteins constructed according to the present application are shown in Table 1.

[0183] 1.2 Protein Expression, Purification, and Purity Analysis Protein expression was achieved by transient transfection of Expi293 cells (purchased from Thermofisher). The prepared heavy and light chain plasmids were co-transfected into Expi293 cells using PEI-MW40000 (PolySciences) to transiently express the desired proteins. Instructions for the preparation and use of PEI can be found in the PolySciences PEI MAX manual (see also Jager, V., et al., BMC Biotechnol 13, 52, 2013). Procedures for culturing and transfecting Expi293 cells can be found in the Thermofisher Expi293 Expression System manual. Five to seven days after transient transfection, the cell supernatant was collected by centrifugation and filtered through a 0.45 μm filter for subsequent protein purification.

[0184] The transiently expressed proteins were purified using a column pre-packed with Protein A (GE Lifesciences) (the procedure can be found in the manufacturer's instructions). The collected eluted protein sample was exchanged into a PBS buffer system by dialysis or ultrafiltration, and then the sample was filtered through a 0.22 μm filter to remove bacteria, obtaining the protein sample to be tested. The protein sample was quantified using a NanoDrop™ spectrophotometer (Thermo Scientific) in combination with the theoretical extinction coefficient of the protein. The protein expression and purification results showed that the binding protein was well expressed and could be directly purified using Protein A, retaining expression and purification properties similar to those of IgG Fc or IgG-like antibodies.

[0185] Protein purity was analyzed using size-exclusion chromatography (SEC) by applying purified samples in PBS to a TSKgel SuperSW3000 300 x 4.6 mm, 5 μm column (TOSOH). SEC was performed using a U3000 HPLC instrument (DIONEX). All proteins were determined using a UV assay at 280 nm and 214 nm. Elution was isocratic at a flow rate of 0.25 mL / min. The analytical results showed that all binding proteins purified in one step using Protein A had good monomer purity in solution (Figures 7-11 and Table 1). The purities of DB364 (SEQ ID NO: 17), DB365 (SEQ ID NO: 18), and DB366 (SEQ ID NO: 19) were all greater than 90%, indicating that selective optimization of the nonbinding region sequences of binding proteins can significantly improve protein purity. Furthermore, based on DB177 and DB366, DB871 and DB872 fused with the Her2 antibody pertuzumab Fab, DB-B229 and DB-B230 fused with the GPC3 antibody codrituzumab Fab, and DB-B229 and DB-B230 fused with the CD20 antibody rituximab Fab all had monomer purity of over 95% after one-step purification using Protein A (Figures 7-11 and Table 1), indicating that the binding proteins fused with Fab also had excellent purity.

[0186] [Table 5]

[0187] Example 2: Affinity assay of binding proteins to FcεRIa (BioLayer Interferometry, BLI) To verify the binding ability of the binding protein to FcεRIa, biolayer interferometry (BLI, GatorBio) was used to detect the affinity of the binding protein to FcεRIa. The detailed affinity assay method can be found in the GatorBio device's instruction manual for use or application, which is briefly described as follows: Recombinantly expressed FcεRIa-mG2Ahis protein (40 nM, recombinant human FcεRIa extracellular region-mouse IgG2a Fc-6xHis fusion protein, produced in-house, protein number DB967; see SEQ ID NO: for amino acid sequence) was loaded with an Anti-HIS probe (20-5066, GatorBio) (loading, 120 s), then affinity bound with a gradient of binding protein at dilutions of 200, 50, 12.5, and 0 (baseline) nM (association, 300 s), followed by dissociation in PBST buffer (dissociation, 500 s). After data collection was completed, affinity was calculated using the instrument's software.

[0188] The affinity assay results showed that all binding proteins effectively bound to recombinant human FcεRIa protein, with a relatively consistent binding affinity of approximately 1E-09 M (Figure 6). The length of the non-binding region and the presence or absence of fusion with Fab did not affect affinity. In another study, the affinity of the binding protein DB-B64 was slightly lower, indicating that the amino acids at positions 1.1-1.6 and 2 of the N-terminal CH2 of the fusion protein affected affinity.

[0189] Example 3: Assaying the effect of binding proteins to block FcεRIa To verify the effect of binding proteins on blocking FcεRIa, ELISA was used to detect the effect of binding proteins on blocking the binding of FcεRIa to its ligand IgE. Specifically, recombinantly expressed human FcεRIa-Fc protein was diluted to 1 μg / mL in PBS, coated onto an ELISA plate (Corning, part number 42592) at 100 μL / well, and incubated overnight at 4°C. The next day, the coating solution was discarded, and the plate was blocked with PBS containing 2.5% nonfat milk at 25°C for 1 hour. After blocking, the plate was washed three times with PBST (PBS + 0.05% Tween 20). Next, a premix containing 0.5 nM human IgE or IgE Fc (labeled with biotin) and a gradient dilution of the binding protein to be tested (premixed for 1 h) were added and incubated at 25°C for 1 h, where the final concentrations of the binding protein to be tested were serially diluted 3- or 4-fold starting from 50, 200, or 1200 nM, plus a 0 nM point. After incubation, the plate was washed three times with PBST. A 1:2000 dilution of streptavidin-HRP (Sangon Biotech, Shanghai) was added and incubated for 30 min. Unbound secondary antibody was washed away with PBST buffer. TMB was then added for color development, and the reaction was stopped by adding stop solution once color development was complete. OD 450 IC values ​​were measured and analyzed using a four-parameter regression model to determine the antibody IC 50 was calculated.

[0190] The assay results showed that all binding proteins were able to effectively block the binding of FcεRIa to its ligand IgE (Figures 12A-C), indicating that all binding proteins had blocking activity. In addition, when the blocking activity of the binding proteins was compared with that of DB366 as a control, the blocking activity of the binding proteins was essentially the same except for DB-B64. Although there were some differences between the different experimental groups, the experimental results showed that the blocking activity of DB-B64 was slightly reduced, which was consistent with the results of the affinity assay.

[0191] Example 4 Assay of the ability of binding proteins to activate the FcεRI receptor To verify whether the binding proteins have the effect of mediating activation of the FcεRI receptor, they were detected using a rat mast cell line RBL-2H3 luciferase reporter gene assay.

[0192] A human FcεRIα-expressing, NFAT-inducible luciferase reporter cell line (RBL-2H3-FcεRIα-NFATLuc cells) was constructed using the rat basophil RBL-2H3 cell line (purchased from the Cell Bank of the Chinese Academy of Sciences). This cell line was designated RBL-2H3-FcεRIα-NFATLuc. For details of the construction process, see Analytical and Bioanalytical Chemistry, volume 412, pages 1901-1914 (2020); Allergy 2010;65:1266-1273; J Immunol, July 1, 1996, 157(1)221-230.

[0193] To verify whether the binding proteins of the present application have the ability to activate the FcεRI receptor, RBL-2H3-FcεRIα-NFATLuc cells were mixed with antigen-expressing cells, and then gradient dilutions of antigen-specific binding proteins were added separately and co-incubated for about 20 hours. Then, a luciferase activity assay was performed to detect the ability of each binding protein to mediate FcεRI receptor activation. Specifically, 40,000 RBL-2H3-FcεRIα-NFATLuc cells (25 μL) were homogeneously mixed with 4,000 antigen-expressing cells (25 μL, Her2-expressing cells were NCI-N87, GPC3-expressing cells were HepG2, and CD20-expressing cells were Raji, all purchased from the Cell Bank of the Chinese Academy of Sciences) in a 96-well plate. 50 μL of gradient dilutions of antigen-specific binding proteins (Her2-specific binding proteins DB871 and DB872, 5-fold diluted starting from 5 nM; GPC3-specific binding proteins DB-B229 and DB-B230, 5-fold diluted starting from 10 nM; CD20-specific binding proteins DB-B232 and DB-B233, 5-fold diluted starting from 10 nM) were added to the mixture, and the mixture was incubated at 37°C and 5% CO2 for 20 hours in an incubator. The results were then analyzed by a microplate reader (Molecular For detection using Vazyme Devices, luciferase substrate (DD1203-03, Vazyme) was added at 100 μL / well.

[0194] The assay results showed that all antigen-specific binding proteins were able to efficiently mediate FcεRI receptor activation, with EC50 values ​​ranging from 16 to 40 pM (Figures 13A-C), demonstrating potent activation. The experimental data for DB871 and DB872, DB-B229 and DB-B230, and DB-B232 and DB-B233 indicate that the structure-activity relationships of binding proteins 3 and 4 were not significantly different.

[0195] Example 5 Assay for binding to FcRn To verify whether the binding proteins of the present application retain the ability to bind IgG Fc to FcRn, an assay was performed using biolayer interferometry (BLI) with pertuzumab (see recommended INN list R51 for specific sequence) as a positive control and human IgE as a negative control. The assay method was briefly described as follows: recombinantly expressed human FcRn protein (100 nM, 6xHis-tagged, FCN-H52W7, ACROBiosystems) was loaded (loading, 120 seconds) using an anti-HIS probe (20-5066, GatorBio), followed by affinity binding (association, 300 seconds) with a control gradient dilution or binding protein at dilutions of 9000, 3000, 1000, 333.33, and 0 (baseline) nM under pH 6.0 buffer conditions, followed by dissociation in pH 6.0 buffer (dissociation, 300 seconds). After data collection was completed, the software provided with the instrument was used for graphing and analysis.

[0196] The results of the assay showed that both binding proteins DB366 and DB872 of the present application had the ability to bind to FcRn similar to the positive control pertuzumab, while human IgE was essentially unable to bind to FcRn, indicating that the binding proteins of the present application retain the ability to bind IgG to FcRn, regardless of whether a Fab is fused thereto or not ( FIG. 14 ).

Claims

1. A binding protein capable of binding to FcεRI, wherein the binding protein comprises an IgG Fc region or a fragment thereof, the IgG Fc region or the fragment thereof comprises a BC loop (BC-LOOP), a DE turn (DE-TURN) and / or an FG loop (FG-LOOP) derived from CH3 in an IgE Fc region, or a fragment thereof, and a region in CH2 in the IgG Fc region corresponding to the BC loop, DE turn and / or FG loop, or a fragment thereof, in CH3 in the IgE Fc region is substituted or replaced by the BC loop, DE turn and / or FG loop in CH3 in the IgE Fc region, or a fragment thereof.

2. 2. The binding protein of claim 1, wherein the IgG is a human IgG.

3. 3. The binding protein of claim 1 or 2, wherein the IgG is IgG1, IgG2, IgG3, or IgG4.

4. 4. The binding protein of any one of claims 1 to 3, wherein the CH2 in the wild-type IgG Fc region comprises the amino acid sequence set forth in any one of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:

27.

5. The binding protein of any one of claims 1 to 4, wherein the fragment of the IgG Fc region comprises at least CH2 of the IgG Fc region or fragment thereof.

6. 6. The binding protein of any one of claims 1 to 5, wherein the BC loop, DE turn and / or FG loop from CH3 in the IgE Fc region, or a fragment thereof, is capable of binding to FcεRI.

7. The binding protein of any one of claims 1 to 6, wherein the BC loop in CH2 in the IgG Fc region comprises the amino acid sequence of positions 27 to 38 in CH2 in the IgG Fc region.

8. The binding protein of any one of claims 1 to 7, wherein the BC loop in CH2 in the IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:

2.

9. The binding protein of any one of claims 1 to 8, wherein the DE turn in CH2 of the IgG Fc region comprises the amino acid sequence of positions 84.1 to 84.4 and 85.4 to 85.1 in CH2 of the IgG Fc region.

10. 10. The binding protein of any one of claims 1 to 9, wherein the DE turn in CH2 in the IgG Fc region comprises the amino acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:39, and SEQ ID NO:

40.

11. The binding protein of any one of claims 1 to 10, wherein the FG loop in CH2 of the IgG Fc region comprises the amino acid sequence of positions 105 to 117 in CH2 of the IgG Fc region.

12. 12. The binding protein of any one of claims 1 to 11, wherein the FG loop in CH2 of the IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:

7.

13. The binding protein of any one of claims 1 to 12, wherein the BC loop in CH3 in the IgE Fc region comprises the amino acid sequence of positions 27 to 38 in CH3 in the IgE Fc region.

14. The binding protein of any one of claims 1 to 13, wherein the BC loop in CH3 in the IgE Fc region comprises the amino acid sequence set forth in SEQ ID NO:

8.

15. 15. The binding protein of any one of claims 1 to 14, wherein the DE turn in CH3 of the IgE Fc region comprises the amino acid sequence of positions 84.1 to 84.4 and 85.4 to 85.1 in CH3 of the IgE Fc region.

16. 16. The binding protein of any one of claims 1 to 15, wherein the DE turn in CH3 in the IgE Fc region comprises the amino acid sequence set forth in SEQ ID NO:

9.

17. The binding protein of any one of claims 1 to 16, wherein the FG loop in CH3 of the IgE Fc region comprises the amino acid sequence of positions 105 to 117 in CH3 of the IgE Fc region.

18. 18. The binding protein of any one of claims 1 to 17, wherein the FG loop in CH3 within the IgE Fc region comprises the amino acid sequence set forth in SEQ ID NO:

10.

19. 19. The binding protein of any one of claims 1 to 18, wherein the position of said region of amino acids is determined by the IMGT unique numbering for C domain coding.

20. 20. The binding protein of any one of claims 1 to 19, wherein CH2 within the IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO:

41.

21. A binding protein comprising an IgG Fc region or fragment thereof, wherein the IgG Fc region or fragment thereof comprises one or more amino acid mutations compared to a wild-type IgG Fc region, and wherein the mutant IgG Fc region or fragment thereof is capable of binding to FcεRI.

22. 22. The binding protein of claim 21 , wherein the IgG comprises human IgG.

23. 23. The binding protein of claim 21 or 22, wherein the IgG comprises IgG1, IgG2, IgG3, or IgG4.

24. 24. The binding protein of any one of claims 21 to 23, wherein CH2 in the wild-type IgG Fc region comprises the amino acid sequence set forth in any one of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:

27.

25. 25. The binding protein of any one of claims 21-24, wherein the IgG Fc region comprises at least CH2 of the IgG Fc region.

26. 26. The binding protein of claim 25, wherein the BC loop in CH2 in the IgG Fc region has one or more amino acid mutations.

27. The binding protein of claim 25 or 26, wherein the BC loop in CH2 of the IgG Fc region has amino acid mutations at one or more sites selected from the group consisting of V28, S29, H30, E31, D34, P35 and / or K / Q38.

28. 28. The binding protein of any one of claims 25 to 27, wherein the BC loop in CH2 in the IgG Fc region has one or more amino acid mutations selected from the group consisting of V28L, S29A, H30P, E31S, D34K, P35G, E36T, and / or K / Q38N.

29. The binding protein of any one of claims 25 to 28, wherein the BC loop in CH2 in the mutant IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:

8.

30. The binding protein of any one of claims 25 to 29, wherein the DE turn in CH2 in the IgG Fc region has amino acid mutations at one or more sites selected from the group consisting of E84.1, Y / F84.3, S85.4, Y / F85.2, and / or R85.

1.

31. 31. The binding protein of any one of claims 25 to 30, wherein the DE turn in CH2 in the IgG Fc region has one or more amino acid mutations selected from the group consisting of E84.1K, Y / F84.3R, S85.4G, Y / F85.2L, and / or R85.1T.

32. 32. The binding protein of any one of claims 25 to 31, wherein the DE turn in CH2 in the mutant IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:

9.

33. The binding protein of any one of claims 25 to 32, wherein the FG loop in CH2 of the IgG Fc region has amino acid mutations at one or more sites selected from the group consisting of K105, S107, N108, K109, A / G110, A / S115, P / S116, and / or I117.

34. 34. The binding protein of any one of claims 25 to 33, wherein the FG loop in CH2 of the IgG Fc region has one or more amino acid mutations selected from the group consisting of K105R, S107T, N108H, K109P, A / G110H, A / S115R, P / S116A, and / or I117L.

35. The binding protein of any one of claims 25 to 34, wherein the FG loop in CH2 of the mutant IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO:

10.

36. 36. The binding protein of any one of claims 1 to 35, wherein the N-terminus of CH2 in the IgG Fc region has amino acid mutations at one or more sites selected from the group consisting of P1.5, E1.4, L / P / F1.3, L / V1.2, G / A1.1, G1, and / or P2.

37. 37. The binding protein of claim 36, wherein the N-terminus of CH2 in the IgG Fc region has one or more amino acid mutations selected from the group consisting of P1.5D, E1.4S, L / P / F1.3N, L / V1.2P, G / A1.1R, and / or P2V.

38. 38. The binding protein of claim 37, wherein the CH2 in the mutant IgG Fc region comprises the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO:

41.

39. 39. The binding protein of any one of claims 21 to 38, wherein the position of the region of amino acids is determined by the IMGT unique numbering for C domain coding.

40. 39. The binding protein of any one of claims 1 to 38, further comprising a CH3 within the IgG Fc region.

41. 41. The binding protein of claim 40, comprising the amino acid sequence set forth in SEQ ID NO:29 or SEQ ID NO:

38.

42. 42. The binding protein of any one of claims 1 to 41, further comprising a constant region fragment of IgE.

43. 43. The binding protein of any one of claims 1 to 42, further comprising CH2 of the constant region of IgE or a fragment thereof.

44. 44. The binding protein of any one of claims 1 to 43, wherein the constant region fragment of IgE comprises the amino acid sequence set forth in any one of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:

15.

45. 45. The binding protein of any one of claims 1 to 44, comprising the amino acid sequence set forth in any one of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:30, and SEQ ID NO:

31.

46. The following characteristics: (1) capable of binding to FcεRIa; (2) capable of binding to FcRn; (3) have good expression quality; (4) It can be directly purified with Protein A or G. (5) The number of potential glycosylation sites is reduced compared to IgE Fc.

46. ​​The binding protein of any one of claims 1 to 45, having one or more of:

47. A polypeptide comprising a binding protein according to any one of claims 1 to 46.

48. A fusion protein comprising a binding protein according to any one of claims 1 to 47.

49. 49. The fusion protein of claim 48, further comprising one or more other functionally active proteins.

50. The fusion protein of claim 47, wherein the other functionally active protein is one or more selected from the group consisting of Fab, scFv, VHH, cytokine, soluble receptor or ligand, and pharmaceutical polypeptide.

51. The fusion protein of any one of claims 48 to 50, which is an IgG-like molecule.

52. A fusion protein according to any one of claims 48 to 51, capable of specifically binding to a tumor-associated antigen.

53. 53. The fusion protein of claim 52, wherein the tumor-associated antigen comprises Her2.

54. 54. The fusion protein of any one of claims 51 to 53, comprising a heavy chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:20 or SEQ ID NO:

21.

55. 55. The fusion protein of any one of claims 51 to 54, comprising a light chain, and wherein said light chain comprises the amino acid sequence set forth in SEQ ID NO:

22.

56. 53. The fusion protein of claim 52, wherein the tumor-associated antigen comprises GPC3.

57. 57. The fusion protein of claim 56, comprising a heavy chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO:

33.

58. 58. The fusion protein of claim 56 or 57, comprising a light chain, and wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:

34.

59. 53. The fusion protein of claim 52, wherein the tumor-associated antigen comprises CD20.

60. 60. The fusion protein of claim 59, comprising a heavy chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:35 or SEQ ID NO:

36.

61. 61. The fusion protein of claim 59 or 60, comprising a light chain, and wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:

37.

62. 62. A drug molecule comprising a binding protein according to any one of claims 1 to 46, a polypeptide according to claim 47, or a fusion protein according to any one of claims 48 to 61.

63. 62. One or more isolated nucleic acid molecules encoding the binding protein of any one of claims 1 to 46, the polypeptide of claim 47, or the fusion protein of any one of claims 48 to 61.

64. A vector comprising the nucleic acid molecule of claim 63.

65. 65. A cell comprising the nucleic acid molecule of claim 63 or the vector of claim 64.

66. 66. A pharmaceutical composition comprising a binding protein according to any one of claims 1 to 46, a polypeptide according to claim 47, a fusion protein according to any one of claims 48 to 61, a drug molecule according to claim 62, a nucleic acid molecule according to claim 63, a vector according to claim 64 or a cell according to claim 65, and optionally a pharmaceutically acceptable carrier.

67. 62. A method for preparing a binding protein according to any one of claims 1 to 46, a polypeptide according to claim 47 or a fusion protein according to any one of claims 48 to 61, the method comprising culturing a cell according to claim 59 under conditions that allow expression of said binding protein, said polypeptide or said fusion protein.

68. 67. Use of a binding protein according to any one of claims 1 to 46, a polypeptide according to claim 47, a fusion protein according to any one of claims 48 to 61, a drug molecule according to claim 62, a nucleic acid molecule according to claim 63, a vector according to claim 64, a cell according to claim 65 or a pharmaceutical composition according to claim 66 in the preparation of a medicament for the prevention or treatment of a disease and / or disorder.

69. 69. The use of claim 68, wherein the disease and / or disorder comprises a tumor.

70. 70. The use according to any one of claims 68 to 69, wherein the tumor comprises a solid tumor and / or a hematological tumor.

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