Heavy chain-based fusion protein

Abstract

The present invention relates to a heavy chain-based fusion protein and, specifically, to a fusion protein comprising a CH1 domain of IgG and one or more Fc regions selected from IgG and IgE, and to a method for producing same. The fusion protein according to the present invention is capable of forming a structure consisting solely of heavy chains by a light chain constant region variant, and can be produced stably and at a high yield. In addition, the fusion protein of the present invention is capable of forming constructs in combination with various known or newly developed antibodies or fragments thereof. Such constructs may be implemented in various forms, such as monospecific antibodies, bispecific antibodies or heterodimeric antibodies, and may be modified and produced according to the purpose or use. Furthermore, the fusion protein of the present invention may be utilized for the treatment of autoimmune diseases, immunological analysis or as a reference antibody, etc., by leveraging the immunoregulatory functions mediated by IgG or IgE heavy chain constant regions.

Description

heavy chain-based fusion protein

[0001] The present invention relates to a heavy chain-based fusion protein, specifically to a fusion protein comprising a CH1 domain of IgG; and one or more Fc regions selected from IgG and IgE; and a method for preparing the same.

[0002] Immunoglobulin G (IgG) is the most abundant antibody in the human body and plays a key role not only in defending against infections but also in regulating various immune responses and inflammatory and autoimmune diseases. IgG antibodies consist of two heavy chains and two light chains, with each heavy chain containing a variable region and a constant region. Among these, the constant region of the heavy chain is known to play a crucial role in the antibody's effector function, including structural stability, secretion processes, regulation of half-life, and interactions with immune receptors.

[0003] The IgG heavy chain constant domain consists of CH1, CH2, and CH3 domains, among which the CH1 domain plays a role in regulating antibody biosynthesis and extracellular secretion. The CH1 domain binds to BiP (Immunoglobulin heavy chain binding protein), a molecular chaperone within the endoplasmic reticulum, to regulate the folding state of the heavy chain; folding is not completed until a disulfide bond is formed with the light chain constant domain. Consequently, extracellular secretion may be restricted if the heavy chain is not bound to the light chain. While the CH2 and CH3 domains allow for the formation of dimers between heavy chains, the assembly with the light chain within the entire heavy chain constant domain affects structural completeness and secretion potential. Therefore, expressing the heavy chain constant domain or a portion thereof alone without the light chain is disadvantageous for stable structural formation and efficient secretion, and most antibody designs are based on the premise of covalent bonding between the heavy and light chains.

[0004] Meanwhile, the Fc (fragment crystallizable) fragment, composed of CH2 and CH3 domains within the IgG heavy chain constant region, is known as a key region that mediates immune regulatory functions through interactions with complement (C1q) binding, Fcγ receptors (FcγR), and neonatal Fc receptors (FcRn). A representative therapeutic method utilizing these Fc-mediated functions is intravenous immunoglobulin (IVIg) therapy, which involves the intravenous administration of high doses of polyclonal plasma-derived IgG; this is used to treat various autoimmune diseases such as idiopathic thrombocytopenic purpura, Kawasaki disease, and myasthenia gravis.

[0005] Along with this, therapeutic strategies based on Fc fragments rather than whole IgG antibodies are also being continuously studied. For example, Fc fragment multiplexing, the introduction of mutations that increase FcRn affinity, or protein-based approaches targeting FcRn are being researched and developed, and recently, Vyvgart, an Fc fragment variant ® With the approval of Fc as a treatment for autoimmune diseases, the clinical efficacy of Fc-based treatment strategies has been demonstrated. However, these approaches still have room for improvement in terms of structural design constraints, production efficiency, and functional scalability.

[0006] Meanwhile, immunoglobulin E (IgE) is an antibody primarily involved in allergic reactions and immediate hypersensitivity reactions, and is known to induce a potent immune response through binding to high-affinity IgE receptors (FcεRI) expressed on mast cells and basophils. IgE also contains a heavy chain constant domain (Cε), which plays a key role in receptor binding and signal transduction. However, compared to IgG, IgE has a lower blood concentration, and due to its structural characteristics, it is difficult to produce and handle, which has limited its application in the development of therapeutic agents.

[0007] Accordingly, there is a continuous demand for new types of fusion proteins that go beyond existing approaches limited to whole antibodies or Fc fragments, utilizing the structural and functional characteristics of heavy chain constant regions while offering potential improvements in terms of structural stability and production efficiency.

[0008] The inventors completed the present invention by conducting research to develop a new type of fusion protein and confirming that a fusion protein with a structure consisting only of heavy chains can be produced in high yields by using a light chain constant region variant.

[0009] Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0010] Furthermore, terms not specifically defined in this specification should be understood to have the meanings commonly used in the technical field to which the present invention pertains. Additionally, unless specifically defined in the context, the singular includes the plural, and the plural includes the singular.

[0011]

[0012] One aspect of the present invention provides a fusion protein comprising: a CH1 domain of IgG; and one or more Fc regions selected from IgG and IgE.

[0013] The fusion protein according to the present invention is characterized by comprising a CH1 domain of IgG. As long as the fusion protein comprises a CH1 domain of IgG, the Fc region may be derived from an antibody of the same type as the CH1 domain or from antibodies of different types. Specifically, the fusion protein may comprise a CH1 domain of IgG and an Fc region of IgG, or may comprise a CH1 domain of IgG and an Fc region of IgE.

[0014] The above CH1 domain can perform the role of regulating the intracellular synthesis and extracellular secretion of the fusion protein according to the present invention, and specifically, can promote the extracellular secretion of the fusion protein in a structure containing only a heavy chain. In addition, the above Fc region can perform the function of binding to an Fc receptor.

[0015] As used herein, the term "fusion protein" refers to a protein in which part or all of two or more different proteins, antibodies, antibody fragments, or antibody domain fragments are combined. The fusion protein may further include additional amino acids for inter-domain linkage, protein expression, stability, or extracellular secretion. Examples of such additional amino acids include, but are not limited to, signal peptides, linkers, hinges, etc.

[0016] As used herein, the term "antibody" refers to an immunoglobulin molecule that is immunologically reactive with a specific antigen, or a protein molecule that acts as a receptor specifically recognizing an antigen. In the present invention, the term "antibody" is used in a broad sense and is interpreted to include polyclonal antibodies, monoclonal antibodies, whole antibodies (antibodies consisting of at least two heavy chains and two light chains interconnected by disulfide bonds), and antibody fragments. The whole antibodies include IgA, IgD, IgE, IgM, and IgG. Additionally, the IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes.

[0017] As used herein, the term "antibody fragment" refers to a protein molecule consisting of a part of an antibody derived from or designed based on a full-length antibody. The antibody fragment may consist of a single domain or multiple domains and may include, for example, Fc, Fab, Fab', F(ab')₂, Fv, scFv, full-length heavy chain (HC), full-length light chain (LC), a fragment of the full-length heavy chain, a fragment of the full-length light chain, a heavy chain constant region fragment (CH), a light chain constant region fragment (CL), a heavy chain variable region fragment (VH), a light chain variable region fragment (VL), a single domain, a combination thereof, or a variant thereof, but is not limited thereto.

[0018] As used herein, the term "heavy chain" refers to a full-length heavy chain and a fragment thereof comprising a heavy chain constant region (CH) and a heavy chain variable region (VH) of an antibody. The heavy chain may include gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε), each corresponding to IgG, IgM, IgA, IgD, and IgE. Additionally, the heavy chain may include γ1, γ2, γ3, and γ4, each corresponding to IgG1, IgG2, IgG3, and IgG4. Specifically, the heavy chain constant region may include a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain in the case of IgG; and may include a Cμ1 domain, a Cμ2 domain, a Cμ3 domain, and a Cμ4 domain in the case of IgM; In the case of IgA, it may include a Cα1 domain, a hinge region, a Cα2 domain, and a Cα3 domain; in the case of IgD, it may include a Cδ1 domain, a hinge region, a Cδ2 domain, and a Cδ3 domain; and in the case of IgE, it may include a Cε1 domain, a Cε2 domain, a Cε3 domain, and a Cε4 domain. Additionally, the heavy chain variable region may have a VH domain that is common to IgG, IgM, IgA, IgD, and IgE.

[0019] As used herein, the term "Fc region (fragment crystallizable region)" refers to a portion of the heavy chain constant region of an antibody that does not directly participate in antigen binding, and means a protein molecule composed of one or more domains constituting the heavy chain constant region. Specifically, the Fc region may include one or more selected from the group consisting of the CH2 domain of IgG, the CH3 domain of IgG, the Cε2 domain of IgE, the Cε3 domain of IgE, and the Cε4 domain of IgE, but is not limited thereto. For example, the Fc region may include the CH2 domain and CH3 domain derived from IgG, but is not limited thereto. Additionally, the Fc region may include the Cε2 domain, Cε3 domain, and Cε4 domain derived from IgE, but is not limited thereto.

[0020] As used herein, the term "light chain" refers to a full-length light chain and a fragment thereof comprising a light chain constant region (CL) and a light chain variable region (VL) of an antibody. The light chain may include kappa (κ) and lambda (λ), which may be commonly used in IgG, IgM, IgA, IgD, and IgE. Specifically, the light chain constant region may include a Cκ domain or a Cλ domain commonly in IgG, IgM, IgA, IgD, and IgE. Additionally, the light chain variable region may include a Vκ domain or a Vλ domain commonly in IgG, IgM, IgA, IgD, and IgE.

[0021] In the present invention, the fusion protein may include a heavy chain and not include a light chain. Additionally, the fusion protein may include a heavy chain constant region and a heavy chain variable region, and not include a light chain constant region and a light chain variable region. Additionally, the fusion protein may include a heavy chain constant region and not include a light chain constant region and a light chain variable region.

[0022] Specifically, the fusion protein may include a domain derived from one or more heavy chains selected from IgG and IgE, and may not include a domain derived from a light chain. Additionally, the fusion protein may include a domain derived from one or more heavy chain constant regions and heavy chain variable regions selected from IgG and IgE, and may not include a domain derived from a light chain constant region and a light chain variable region. Additionally, the fusion protein may include a domain derived from one or more heavy chain constant regions selected from IgG and IgE, and may not include a domain derived from a light chain constant region and a light chain variable region.

[0023] In one embodiment according to the present invention, the fusion protein may comprise a CH1 domain of IgG; and a CH2 domain of IgG and a CH3 domain of IgG.

[0024] In another embodiment according to the present invention, the fusion protein may comprise the CH1 domain of IgG; and the Cε2 domain of IgE, the Cε3 domain of IgE, and the Cε4 domain of IgE.

[0025] In the present invention, the fusion protein may include a structure in which CH1, a hinge region, CH2, and CH3 are sequentially connected from the N-terminus to the C-terminus. Additionally, the fusion protein may include a structure in which CH1, CH2, and CH3 are sequentially connected from the N-terminus to the C-terminus. Additionally, the fusion protein may include a structure in which CH1, a hinge region, Cε2, Cε3, and Cε4 are sequentially connected from the N-terminus to the C-terminus. Additionally, the fusion protein may include a structure in which CH1, Cε2, Cε3, and Cε4 are sequentially connected from the N-terminus to the C-terminus.

[0026] In the present invention, the fusion protein may be in the form of a monomer consisting of two heavy chains. In addition, the fusion protein may include cases where the two heavy chains have the same sequence as well as cases where they have different sequences.

[0027] The above term, "monomer," means a single antibody structural unit formed by two heavy chains being joined together by disulfide bonds and / or non-covalent interactions.

[0028] Specifically, the monomer composed of two heavy chains may be a monomer that does not include a light chain. Additionally, the monomer composed of two heavy chains may include a heavy chain constant region and a heavy chain variable region. Additionally, the monomer composed of two heavy chains may include a heavy chain constant region but not a heavy chain variable region.

[0029] In this specification, the designation for each domain may be indicated by omitting the term "domain," and such notation is interpreted as being used interchangeably. For example, "CH1 domain" may be used interchangeably with "CH1" in this specification.

[0030] Additionally, in this specification, the designation for each region may be indicated by omitting the term "region," and such notation is interpreted as being used interchangeably. For example, "Fc region" may be used interchangeably with "Fc" in this specification, and "hinge region" may be used interchangeably with "hinge" in this specification.

[0031]

[0032] Another aspect of the present invention provides a nucleic acid molecule encoding the fusion protein.

[0033] In nucleic acid molecules according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0034] As used in this specification, the term "nucleic acid molecule" has a meaning that comprehensively includes DNA and RNA molecules, and the nucleotides, which are the basic constituent units of said nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base sites are modified. The sequence of the nucleic acid molecule encoding the fusion protein of the present invention may be modified, and said modification includes the addition, deletion, non-conservative substitution, or conservative substitution of nucleotides.

[0035] In addition, all sequences used in the present invention, including nucleic acid sequences and amino acid sequences, are interpreted to include sequences that exhibit substantial identity with the sequences listed in the sequence list, provided that variations having biologically equivalent activity are taken into account. The term "substantial identity" means a sequence that exhibits at least 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology when any other sequence is aligned with the sequence of the present invention to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0036] Accordingly, sequences having high homology with the sequences represented by SEQ ID NOs 1 to 35 of the present invention, for example, sequences having high homology of 70% or more, specifically 80% or more, and more specifically 90% or more, should also be interpreted as being included within the scope of the present invention.

[0037]

[0038] Another aspect of the present invention provides an expression vector comprising the nucleic acid molecule.

[0039] In the expression vector according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0040] As used herein, the term "vector" means a plasmid, virus, or other medium known in the art capable of inserting or introducing a nucleic acid molecule encoding the fusion protein into a host cell as a means for expressing the fusion protein of the present invention. The vector may be constructed as a vector for cloning or as a vector for expression. Specific examples may include viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, and more specific examples may include plasmids (e.g., pcDNA series such as pcDNA3 or pcDNA3.1, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4·λB, λ-Charon, λΔz1 and M13, etc.) or viruses (e.g., SV40, etc.), but are not limited thereto.

[0041] The vector of the present invention may be such that a nucleic acid molecule encoding the fusion protein is operably linked to a promoter. The term "operably linked" implies a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, thereby allowing the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0042] When the vector of the present invention is an expression vector and the host is a eukaryotic cell, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rhoese's sarcoma virus (RSV) promoter) may be used, and may include a polyadenylation sequence as a transcription termination sequence. For example, the recombinant vector of the present invention may include a CMV promoter, but is not limited thereto.

[0043] When the vector of the present invention is an expression vector and a prokaryotic cell is used as the host, it may include a potent promoter capable of proceeding transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. For example, when Escherichia coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator sites of the Escherichia coli tryptophan biosynthesis pathway, or the left-leaning promoter of phage λ (pLλ promoter) can be used as a regulatory site, and when Bacillus is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis or any promoter that can be expressed in Bacillus can be used as a regulatory site.

[0044] The recombinant vector system of the present invention can be constructed through various methods known in the art and may include antibiotic resistance genes commonly used in the art as selection markers (e.g., resistance genes to ampicillin, gentamicin, cabbageillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline).

[0045]

[0046] Another aspect of the present invention provides an isolated host cell into which the expression vector is introduced.

[0047] In the host cell according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0048] As used herein, the term "host cell" refers to a cell comprising the expression vector and capable of stably and continuously cloning and expressing the fusion protein of the present invention. For example, Bacillus strains such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, and prokaryotic host cells such as Streptomyces, Pseudomonas, Proteus mirabilis, or Staphylococcus; The host cells may be fungi such as Aspergillus species, eukaryotic host cells such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, or Neurospora crassa; lower eukaryotic cells; higher eukaryotic cells such as insect-derived cells; plant cells; or cells derived from mammals such as COS7 cells (monkey kidney cells), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cells, HuT 78 cells, or HEK293 cells, but are not limited thereto, and any host cells commonly used in the art may be used without limitation.

[0049]

[0050] Another aspect of the present invention provides a method for producing the fusion protein, comprising the step of introducing the expression vector into an isolated host cell.

[0051] In the method for preparing a fusion protein according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0052] Specifically, the method for producing the fusion protein of the present invention is,

[0053] (a) a step of introducing the expression vector of the present invention into isolated host cells;

[0054] (b) a step of culturing the host cells; and

[0055] (c) a step of obtaining a fusion protein from the host cell; may be included.

[0056] In one embodiment according to the present invention, the method for producing a fusion protein of the present invention may further include the step of introducing an expression vector comprising a nucleic acid molecule encoding Cκ or Cλ into an isolated host cell. In this case, the method comprises:

[0057] (a) a step of introducing the expression vector of the present invention into isolated host cells;

[0058] (a') A step of introducing an expression vector comprising a nucleic acid molecule encoding Cκ or Cλ into the isolated host cell;

[0059] (b) a step of culturing the host cells; and

[0060] (c) a step of obtaining a fusion protein from the host cell; may be included.

[0061] The step (a) of introducing the expression vector of the present invention into an isolated host cell may be a step of expressing a fusion protein containing a heavy chain. The fusion protein containing a heavy chain may not contain a light chain.

[0062] Additionally, the step (a') of introducing an expression vector comprising a nucleic acid molecule encoding Cκ or Cλ according to the present invention into an isolated host cell may be a step of expressing a fusion protein comprising a light chain constant region (Cκ or Cλ). The fusion protein comprising the light chain constant region may not include a light chain variable region and a heavy chain.

[0063] Specifically, the above Cκ or Cλ may include a mutation and may be a light chain invariant region including a mutation. In this specification, "light chain invariant region including a mutation" may be used interchangeably with "light chain invariant region variant," "kappa light chain invariant region including a mutation" may be used interchangeably with "kappa light chain invariant region variant" and "Cκ variant," and "lambda light chain invariant region including a mutation" may be used interchangeably with "lambda light chain invariant region variant" and "Cλ variant."

[0064] Through steps (a) and (a') above, the fusion protein containing the heavy chain can be expressed together with a light chain constant region variant. By being expressed together with the fusion protein containing the heavy chain, the light chain constant region variant can promote the extracellular secretion of the fusion protein in a structure containing only the heavy chain. Accordingly, the fusion protein intended for the present invention can be efficiently produced.

[0065] In one embodiment according to the present invention, the mutation included in Cκ may be a substitution or deletion of cysteine ​​at position 107. For example, the mutation included in Cκ may be a substitution of cysteine ​​at position 107 with serine (Ser), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gln), histidine (His), lysine (Lys), arginine (Arg), glycine (Gly), proline (Pro), or threonine (Thr), but is not limited thereto. Here, the position of the amino acid into which the mutation is introduced refers to a position based on Kabat numbering and / or EU numbering.

[0066] In another embodiment according to the present invention, the mutation included in Cλ may be one in which cysteine ​​at position 105 is substituted or deleted. For example, the mutation included in Cλ may be one in which cysteine ​​at position 105 is substituted with serine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, glycine, proline, or threonine, but is not limited thereto. Here, the position of the amino acid into which the mutation is introduced refers to a position based on Kabat numbering and / or EU numbering.

[0067] Specifically, the above Cκ may be represented by the amino acid sequence of SEQ ID NO. 4, but is not limited thereto. SEQ ID NO. 4 is an amino acid sequence in which the cysteine ​​at position 107 of SEQ ID NO. 2, which represents the amino acid sequence of wild-type Cκ, is substituted with serine. In SEQ ID NO. 4, the amino acid at position 107 may be substituted with any amino acid other than cysteine.

[0068] Additionally, the nucleic acid molecule encoding the above Cκ may be represented by the DNA sequence of SEQ ID NO. 3, but is not limited thereto. SEQ ID NO. 3 is a DNA sequence in which the codons encoding cysteine ​​at positions 319 to 321 of SEQ ID NO. 1, which represents the DNA sequence encoding wild-type Cκ, are substituted with codons encoding serine. In SEQ ID NO. 3, the codons at positions 319 to 321 may be substituted with codons encoding any amino acid other than cysteine.

[0069] Additionally, the above Cλ may be represented by the amino acid sequence of SEQ ID NO. 8, but is not limited thereto. SEQ ID NO. 8 is an amino acid sequence in which the cysteine ​​at position 105 of SEQ ID NO. 6, which represents the amino acid sequence of wild-type Cλ, is substituted with serine. In SEQ ID NO. 8, the amino acid at position 105 may be substituted with any amino acid other than cysteine.

[0070] Additionally, the nucleic acid molecule encoding the above Cλ may be represented by the DNA sequence of SEQ ID NO. 7, but is not limited thereto. SEQ ID NO. 7 is a DNA sequence in which the codons encoding cysteine ​​at positions 313 to 315 of SEQ ID NO. 5, which represents the DNA sequence encoding wild-type Cλ, are substituted with codons encoding serine. In SEQ ID NO. 7, the codons at positions 313 to 315 may be substituted with codons encoding any amino acid other than cysteine.

[0071] Additionally, steps (a) and (a') may be steps for preparing a transformant containing each expression vector, and the step of preparing the transformant may be performed by transforming or transfecting a host cell.

[0072] The term "transformer" refers to an organism in which artificial genetic changes have occurred by introducing external DNA into a cell, such that the DNA becomes capable of replication as a chromosomal factor or through the completion of chromosomal integration by introducing DNA into a host.

[0073] Transforming the aforementioned host cells can be performed using any transformation or transfection method and can be easily performed according to conventional methods in the art. For example, it may include, but is not limited to, the CaCl2 precipitation method, the Hanahan method with increased efficiency by using DMSO (dimethyl sulfoxide) in the CaCl2 method, electroporation, calcium phosphate precipitation method, protoplasmic fusion method, stirring method using silicon carbide fibers, Agrobacterium-mediated transformation method, PEG-mediated transformation method, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation method, and transformation or transfection methods conventionally used in the art may be used without limitation.

[0074] The step (b) of culturing the host cells of the present invention may be performed according to media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Depending on the cell growth method, suspension culture or attachment culture may be used; depending on the culture method, batch, fed-batch, or continuous culture methods may be used.

[0075] In animal cell culture, the medium may contain various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, or cellulose; fats such as soybean oil, sunflower oil, castor oil, or coconut oil; fatty acids such as palmitic acid, stearic acid, or linoleic acid; alcohols such as glycerol or ethanol; or organic acids such as acetic acid, and these carbon sources may be used alone or in combination. Examples of nitrogen sources may include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquid, or soybean meal; or inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, or ammonium nitrate, and these nitrogen sources may be used alone or in combination. Examples of trace components include phosphorus, such as potassium dihydrogen phosphate or dipotassium hydrogen phosphate; It may include metal salts such as magnesium sulfate or iron sulfate. In addition, it may include amino acids, vitamins, or suitable precursors.

[0076] In addition, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, or sulfuric acid may be added to the culture in an appropriate manner during cultivation to adjust the pH of the culture, antifoaming agents such as fatty acid polyglycol esters may be used during cultivation to suppress bubble formation, and oxygen or an oxygen-containing gas (e.g., air) may be injected into the culture to maintain an aerobic state of the culture.

[0077] In addition, the culture may be cultured by maintaining the temperature of the culture medium at 20°C to 45°C, specifically 25°C to 40°C, but is not limited thereto, and a person skilled in the art may select an appropriate temperature range.

[0078] The step (c) of expressing the fusion protein in the host cell of the present invention may be a step of recovering, purifying, and concentrating the fusion protein of the present invention from the culture medium obtained through the step (b).

[0079] The above fusion protein may be used in an unpurified state, or it may be used after further recovery, purification, and concentration. Specifically, methods commonly used in the industry (e.g., dialysis, salt precipitation, chromatography, etc.) may be used, thereby enabling simultaneous recovery, purification, and concentration. More specifically, it may be recovered, purified, and concentrated using chromatography (e.g., ion exchange chromatography, size exclusion chromatography, or affinity chromatography), and the type and order of columns used may be appropriately selected according to the characteristics of the fusion protein and the culture method according to the present invention.

[0080]

[0081] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of an autoimmune disease comprising the fusion protein.

[0082] In the pharmaceutical composition according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0083] The fusion protein according to the present invention can regulate the action of autoantibodies involved in the pathogenesis of autoimmune diseases through interaction with Fc receptors (FcγR and / or FcRn). Specifically, the fusion protein can alleviate or suppress autoimmune responses by influencing the in vivo half-life of autoantibodies or by competitively inhibiting the interaction of autoantibodies with Fc receptors. While this mechanism of action may be similar to the therapeutic mechanisms reported by existing intravenous immunoglobulins (IgG, IVIg) or Fc fragments, the fusion protein according to the present invention simplifies the production process, improves production efficiency, and enhances therapeutic effects due to its heavy chain-based structure that does not include light chains.

[0084] As used in this invention, the term "treatment" refers to all instances where an autoimmune disease improves, reverses, or is completely cured by the administration of the composition according to this invention.

[0085] The term "prevention" as used in this invention refers to all actions that suppress, delay, or prevent the occurrence or recurrence of an autoimmune disease by administering the composition according to this invention.

[0086] In the present invention, the autoimmune disease may be one or more selected from the group consisting of generalized severe myasthenia gravis, idiopathic thrombocytopenic purpura, Kawasaki disease, bullous pemphigus, chronic inflammatory demyelinating polyneuropathy, immune thrombocytopenia, autoimmune myositis and pemphigus, but is not limited thereto.

[0087] The pharmaceutical composition of the present invention contains the fusion protein in an effective amount and can be administered to a subject requiring prevention or treatment of an autoimmune disease.

[0088] As used herein, the term "administration" means the physical introduction of a composition to a subject using any of the various methods and delivery systems known to those skilled in the art. Such administration may be performed, for example, by oral administration, or by intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration, such as by injection or infusion, but is not limited thereto. Such administration may be performed, for example, as a single dose, multiple doses, or over one or more extended periods.

[0089] As used herein, the term “subject” includes a human or any non-human animal, and said non-human animal may be a vertebrate, e.g., a primate, a dog, a cow, a horse, a pig, a rodent, e.g., a mouse, a rat, a guinea pig, etc. In this specification, said “subject” is used interchangeably with “individual” and “patient”.

[0090] Additionally, the above effective dose may be a "therapeutic effective dose" or a "preventive effective dose." The term "therapeutic effective dose" refers to any amount that, when a drug or therapeutic agent is used alone or in combination with other therapeutic agents, can result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or the prevention of damage or disability caused by disease suffering. The term "preventive effective dose" refers to any amount that suppresses the onset or recurrence of an autoimmune disease in the subject. The level of the above effective dose may be determined based on factors including the subject's severity, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0091] In addition, the above pharmaceutical composition may vary depending on the age, gender, and weight of the subject. Specifically, depending on the symptoms of the subject, 0.1 to 100 mg / kg of the composition of the present invention may be administered once or several times a day, or at intervals of several days to several months. In addition, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, gender, weight, age, etc.

[0092] Additionally, the pharmaceutical composition may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. Carriers, excipients, and diluents that may be included in the composition may be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto.

[0093] In addition, the above pharmaceutical composition may be administered in combination with other therapeutic agents. In this case, the pharmaceutical composition of the present invention and the other therapeutic agents may be administered simultaneously, sequentially, or individually. The other therapeutic agents may be drugs, such as compounds or proteins, having effects of preventing, treating, and improving autoimmune diseases, but are not limited thereto.

[0094] In addition, the above pharmaceutical composition may be formulated to be administered simultaneously, sequentially, or individually with other therapeutic agents. For example, the above pharmaceutical composition may be administered simultaneously with other therapeutic agents as a single formulation, or may be administered simultaneously, sequentially, or individually as separate formulations. To administer simultaneously, sequentially, or individually, the pharmaceutical composition of the present invention and other therapeutic agents may be formulated separately in individual containers, or formulated together in the same container. Furthermore, the pharmaceutical effective amount, administration time, administration interval, administration route, treatment duration, etc., of the pharmaceutical composition of the present invention and other therapeutic agents may be the same or different from each other.

[0095]

[0096] Another aspect of the present invention provides a method for preventing or treating an autoimmune disease, comprising the step of administering the fusion protein.

[0097] In the prevention or treatment method according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0098] In addition, in the method for preventing or treating an autoimmune disease according to the present invention, the fusion protein may be administered to a subject simultaneously, sequentially, or individually with other therapeutic agents.

[0099] The above "simultaneous" administration means administering the fusion protein and other therapeutic agents at once as a single preparation, or administering the fusion protein and other therapeutic agents at once as separate preparations, in which case the administration route of the fusion protein and the administration route of other therapeutic agents may be different from each other.

[0100] The above "sequential" administration means administering the fusion protein and other therapeutic agents relatively continuously, allowing for the minimum possible time consumed in the administration interval.

[0101] The above "individual" administration refers to administering the fusion protein and other therapeutic agents at regular time intervals. The method of administration of the fusion protein and other therapeutic agents may be appropriately selected by a physician or expert in the art, taking into consideration the therapeutic efficacy and side effects of the subject.

[0102] The fusion protein according to the present invention can form a structure composed solely of heavy chains by means of a light chain constant region variant, and can be manufactured with stable and high yields. Furthermore, the fusion protein of the present invention can form constructs by combining it with various antibodies or fragments thereof that are previously known or newly developed. Such constructs can be implemented in various forms, such as monospecific antibodies, bispecific antibodies, or heteromeric antibodies, and can be produced by modifying them according to the purpose or application. Moreover, the fusion protein of the present invention can be utilized for the treatment of autoimmune diseases, immunological analysis, or as a reference antibody by leveraging the immune regulatory function mediated by the IgG or IgE heavy chain constant region.

[0103] Figure 1A relates to the structure of a typical IgG antibody composed of a heavy chain and a kappa (κ) light chain, and Figure 1B relates to the structure of a typical IgG antibody composed of a heavy chain and a lambda (λ) light chain.

[0104] FIG. 2 illustrates a mechanism for manufacturing a fusion protein containing a heavy chain according to the present invention, relating to the case where a mutation is introduced into the kappa light chain constant region (Cκ). Here, P represents a promoter and L represents a leader sequence. FIG. 2A relates to the case of expressing a heavy chain (HC) containing a constant region (CH) and a variable region (VH). FIG. 2B relates to a heavy chain (HC) containing a constant region (CH) and a variable region (VH), and a kappa light chain constant region variant (Cκ) containing a variable region (Vκ) and a C107S mutation. -C107SThis relates to the case where ) are expressed together. Fig. 2C is a heavy chain (HC) containing an invariant region (CH) and a variable region (VH), and a kappa light chain invariant region variant (Cκ -C107S This relates to the case where ) is expressed together. Fig. 2D shows a heavy chain fragment containing an invariant region (CH) and a kappa light chain invariant region variant (Cκ -C107S This concerns the case where ) is expressed together.

[0105] FIG. 3 illustrates a mechanism for manufacturing a fusion protein containing a heavy chain according to the present invention, relating to the case where a mutation is introduced into the lambda light chain constant region (Cλ). Here, P represents a promoter and L represents a leader sequence. FIG. 3A relates to the case where a heavy chain (HC) containing a constant region (CH) and a variable region (VH) is expressed. FIG. 3B relates to a heavy chain (HC) containing a constant region (CH) and a variable region (VH), and a variant of the lambda light chain constant region (Cλ) and a variable region (Vλ). -C105S This relates to the case where ) are expressed together. Fig. 3C shows a heavy chain (HC) containing an invariant region (CH) and a variable region (VH), and a lambda light chain invariant region variant (Cλ -C105S This relates to the case where ) is expressed together. Fig. 3D shows a heavy chain fragment containing an invariant region (CH) and a lambda light chain invariant region variant (Cλ -C105S This concerns the case where ) is expressed together.

[0106] FIG. 4 shows the results of SEC-HPLC analysis performed on a fusion protein containing a heavy chain constant region expressed together with light chain constant region variants according to the present invention. FIG. 4A is a fusion protein (CH + Cκ) containing a heavy chain constant region (CH) expressed together with kappa (κ) light chain constant region variants. -C107S , CH + Cκ -C107A , CH + Cκ -C107D , CH + Cκ -C107K , CH + Cκ -C107FThis relates to ). FIG. 4B is a fusion protein (CH + Cλ) containing a heavy chain constant region (CH) expressed together with lambda (λ) light chain constant region variants. -C105S , CH + Cλ -C105A , CH + Cλ -C105D , CH + Cλ -C105K , CH + Cλ -C105F It is about ).

[0107] Figure 5 shows the immunoblot results for analyzing the intracellular expression and extracellular secretion of a fusion protein containing a heavy chain according to the present invention. CH + Cκ is a fusion protein containing a heavy chain constant region and a light chain constant region, CH is a fusion protein containing a heavy chain constant region, Fc is a fusion protein containing an Fc region (CH2 and CH3), CH + Cκ -C107S means a fusion protein containing a heavy chain constant region expressed together with a light chain constant region variant.

[0108] FIG. 6 shows the SDS-PAGE results for analyzing the structural integrity of a fusion protein containing a heavy chain according to the present invention. FIG. 6A is a fusion protein containing a heavy chain invariant region (CH + Cκ) expressed together with a light chain invariant region variant. -C107S It relates to the ), and Fc regions. Fig. 6B shows a fusion protein (CH + Cλ) containing a heavy chain constant region expressed together with a light chain constant region variant. -C105S It concerns the regions of ), and Fc.

[0109] FIG. 7 shows the results of expression and structural analysis of a fusion protein comprising a heavy chain constant region and a heavy chain variable region expressed together with a light chain constant region variant according to the present invention. The heavy chain (HC) and an unmutated kappa light chain constant region (Cκ) or a kappa light chain constant region variant (Cκ -C107S This relates to the results of performing SDS-PAGE and immunoblot analysis on the culture supernatant and purified protein under reducing and non-reducing conditions after co-expressing ).

[0110] FIG. 8 shows the results of expression and structural analysis of a fusion protein comprising a heavy chain constant region and / or a heavy chain variable region expressed together with a light chain constant region variant according to the present invention. Heavy chain (HC or CH) and lambda light chain constant region variant (Cλ -C105S This relates to the results of performing SDS-PAGE and immunoblot analysis on the culture supernatant and purified protein under reducing and non-reducing conditions after co-expressing ).

[0111] FIG. 9 shows the heavy chain-based fusion protein according to the present invention, Vyvgart, which is an Fc fusion protein. ® Examples of fusion proteins prepared to include [the following] and the results of their structural analysis are shown. FIG. 9A relates to the structure of each fusion protein. FIG. 9B relates to the results of SDS-PAGE analysis performed under reducing and non-reducing conditions for each fusion protein exemplified in FIG. 9A. FIG. 9C relates to the results of SEC-HPLC analysis performed on each fusion protein exemplified in FIG. 9A.

[0112] FIG. 10 shows a heavy chain-based fusion protein according to the present invention, based on the known Eylea ® This shows examples of fusion proteins prepared to include the D2 domain of derived VEGFR1 and the D3 domain of VEGFR2, as well as the results of their structural analysis. Figure 10A relates to the structure of each fusion protein, where D2 is Eylea ® It refers to the D2 domain of the derived VEGFR1, and D3 is Eylea ® It refers to the D3 domain of the derived VEGFR2. Fig. 10B relates to the results of SDS-PAGE analysis performed under reducing and non-reducing conditions for each fusion protein exemplified in Fig. 10A. Fig. 10C relates to the results of SEC-HPLC analysis performed on each fusion protein exemplified in Fig. 10A.

[0113] FIG. 11 shows examples of bispecific fusion proteins to which heavy chain-based fusion proteins according to the present invention are applied, and the results of their structural analysis. FIG. 11A relates to the structure of each fusion protein, and D2 is Eylea ® It refers to the D2 domain of the derived VEGFR1, and D3 is Eylea ® It refers to the D3 domain of derived VEGFR2. Fig. 11B relates to the results of SDS-PAGE analysis performed under reducing and non-reducing conditions for each fusion protein exemplified in Fig. 11A. Fig. 11C relates to the results of SEC-HPLC analysis performed on each fusion protein exemplified in Fig. 11A.

[0114] FIG. 12 shows examples of bispecific and heteromeric fusion proteins to which heavy chain-based fusion proteins according to the present invention are applied, and the results of their structural analysis. FIG. 12A relates to the structure of each fusion protein, and D2 is Eylea ® It refers to the D2 domain of the derived VEGFR1, and D3 is Eylea ® It refers to the D3 domain of the derived VEGFR2. The circle indicated in the CH3 domain represents a mutation introduced to induce heteromer formation and indicates a knob-in-hole mutation designed for the selective binding of different heavy chains. Fig. 12B relates to the results of SDS-PAGE analysis performed under reducing and non-reducing conditions for each fusion protein exemplified in Fig. 12A. Fig. 12C relates to the results of SEC-HPLC analysis performed on each fusion protein exemplified in Fig. 12A.

[0115] FIG. 13 shows examples of fusion proteins containing IgE constant regions (Cε2, Cε3, Cε4) to which heavy chain-based fusion proteins according to the present invention are applied, and the results of structural analysis thereof. FIG. 13A relates to the structure of each fusion protein. FIG. 13B relates to the results of SDS-PAGE analysis performed under reducing and non-reducing conditions for each fusion protein exemplified in FIG. 13A. FIG. 13C relates to the results of SEC-HPLC analysis performed on each fusion protein exemplified in FIG. 13A.

[0116] FIG. 14 shows a fusion protein (HC + Cκ) expressed together with a light chain constant region variant according to the present invention. -C107S This shows examples of fusion proteins (HC) expressed alone without ) and light chain constant region variants, and the results of their analysis. Fig. 14A relates to the structure of each fusion protein, and D2 is Eylea ® It refers to the D2 domain of the derived VEGFR1, and D3 is Eylea ® It refers to the D3 domain of derived VEGFR2. Fig. 14B relates to the results of SDS-PAGE analysis performed under reducing and non-reducing conditions for each fusion protein exemplified in Fig. 14A. Fig. 14C relates to the results of SEC-HPLC analysis performed on each fusion protein exemplified in Fig. 14A.

[0117] The present invention will be explained in more detail below by way of examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited by these examples.

[0118]

[0119] Example 1. Preparation of a fusion protein containing a heavy chain

[0120] Example 1-1. Preparation of a light chain invariant region containing a mutation

[0121] A fusion protein containing a heavy chain (hereinafter referred to as "heavy chain-based fusion protein") was prepared using a fragment containing a light chain constant region of an antibody. Specifically, a kappa light chain constant region (Cκ) or a lambda light chain constant region (Cλ) was used as the light chain constant region, and a mutation was introduced and used.

[0122] A mutation in which cysteine ​​at position 107 was substituted into Cκ was introduced. Specifically, cysteine ​​(Cys; C) located at position 107 of the wild-type Cκ amino acid sequence represented by SEQ ID NO. 1 was substituted with serine (Ser; S), alanine (Ala; A), aspartic acid (Asp; D), lysine (Lys; K), or phenylalanine (Phe; F), respectively. Among the amino acids used for the above substitution, serine was selected to represent a polar non-charged amino acid, alanine a non-polar aliphatic amino acid, aspartic acid an acidic amino acid, lysine a basic amino acid, and phenylalanine an aromatic amino acid, respectively, to encompass amino acids with different physicochemical properties.

[0123] In addition, each kappa light chain constant region (hereinafter referred to as "light chain constant region variant") in which cysteine ​​at position 107 of Cκ is substituted with serine, alanine, aspartic acid, lysine, or phenylalanine is "Cκ -C107S ", "CH + Cκ -C107S ", "CH + Cκ -C107A ", "CH + Cκ -C107D ", "CH + Cκ -C107K ", "CH + Cκ -C107F It was named as "

[0124] A mutation in which cysteine ​​at position 105 was substituted was introduced into Cλ. Specifically, cysteine ​​at position 105 of the wild-type Cλ amino acid sequence denoted by SEQ ID NO. 1 was substituted with serine, alanine, aspartic acid, lysine, or phenylalanine, respectively. Among the amino acids used for the above substitution, serine was selected to represent a polar non-charged amino acid, alanine a non-polar aliphatic amino acid, aspartic acid an acidic amino acid, lysine a basic amino acid, and phenylalanine an aromatic amino acid, respectively, to encompass amino acids with different physicochemical properties.

[0125] In addition, each of the kappa light chain constant region variants in which the cysteine ​​at position 105 of Cλ is substituted with serine, alanine, aspartic acid, lysine, or phenylalanine is "Cλ -C105S ", "CH + Cλ -C105S ", "CH + Cλ -C105A ", "CH + Cλ -C105D ", "CH + Cλ -C105K ", "CH + Cλ -C105F It was named as "

[0126] The sequence numbers of wild-type Cκ, Cκ variant, wild-type Cλ, and Cλ variant are shown in Table 1, and a schematic diagram of an IgG antibody containing wild-type Cκ or wild-type Cλ is shown in Figure 1.

[0127] Classification DNA Sequence (Sequence Number) Amino Acid Sequence (Sequence Number) Wild Type CκCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC(Sequence Number 1) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2)Cκ VariantsCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGA GAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCT (SEQ ID NO: 3) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES (SEQ ID NO: 4) Wild typeCλGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA(서열번호 5)GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(서열번호 6)Cλ 변이체GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAAAGTTCA(서열번호 7)GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS(서열번호 8)

[0128] SEQ ID NO. 3 is a DNA sequence in which the codons encoding cysteine ​​at positions 319 to 321 of SEQ ID NO. 1 are substituted with codons encoding serine. Additionally, the codons at positions 319 to 321 may be substituted with codons encoding any amino acid other than cysteine. SEQ ID NO. 4 is an amino acid sequence in which the cysteine ​​at position 107 of SEQ ID NO. 2 is substituted with serine. Additionally, the amino acid at position 107 may be substituted with any amino acid other than cysteine. SEQ ID NO. 7 is a DNA sequence in which the codons encoding cysteine ​​at positions 313 to 315 of SEQ ID NO. 5 are substituted with codons encoding serine. Additionally, the codons at positions 313 to 315 may be substituted with codons encoding any amino acid other than cysteine. SEQ ID NO. 8 is an amino acid sequence in which the cysteine ​​at position 105 of SEQ ID NO. 6 is substituted with serine. In addition, the amino acid at position 105 can be substituted with any amino acid other than cysteine.

[0129] Here, the position of the amino acid where the mutation is introduced refers to a position based on Kabat numbering and / or EU numbering.

[0130]

[0131] Example 1-2. Preparation of a fusion protein using a light chain invariant region containing a mutation

[0132] The light chain constant region variant prepared in Example 1-1 above does not form covalent bonds with the CH1 domain via disulfide bonds, but forms only non-covalent interactions. These non-covalent interactions induce structural changes in the CH1 domain, and accordingly, the binding site of BiP (Immunoglobulin heavy chain binding protein) may be modified.

[0133] This reduces the binding affinity of BiP to the CH1 domain, thereby inducing the spontaneous dissociation of BiP. Since BiP typically binds to unfolded proteins to inhibit secretion from the endoplasmic reticulum (ER), the dissociation of BiP from the CH1 domain allows the CH1 domain to undergo normal folding, enabling the folding and secretion of the heavy chain. In other words, the light chain constant region variant can promote the folding and secretion of the heavy chain by inducing the dissociation of BiP from the CH1 domain.

[0134] The mechanism of production of such fusion proteins is shown in Figures 2 and 3.

[0135] As shown in FIG. 2A and FIG. 3A, when a heavy chain (HC) containing a constant region (CH) and a variable region (VH) is expressed alone, the heavy chain is expressed within the cell but is not secreted outside the cell.

[0136] On the other hand, as shown in FIG. 2B and FIG. 3B, when a heavy chain (HC) containing a constant region (CH) and a variable region (VH) and a light chain constant region variant containing a constant region (CL; Cκ or Cλ) and a variable region (VL; Vκ or Vλ) are expressed together, the heavy chain and light chain fragments are secreted outside the cell but form a modified IgG antibody structure. This modified IgG antibody structure is believed to be formed by a non-covalent bond between VH and VL, in which case no disulfide bond is formed between CH1 and CL.

[0137] Meanwhile, as shown in FIG. 2C and FIG. 3C, when a heavy chain (HC) containing a constant region (CH) and a variable region (VH) and a light chain constant region variant containing only a constant region (Cκ or Cλ) are expressed together, the heavy chain and light chain fragments are secreted outside the cell. However, binding between the heavy chain and the light chain fragments does not occur, and accordingly, a heavy chain-based fusion protein according to the present invention is produced.

[0138] In addition, as shown in Fig. 2D and Fig. 3D, even when a heavy chain fragment containing only an invariant region (CH) and a light chain invariant region variant containing only an invariant region (Cκ or Cλ) are expressed together, the heavy chain fragment and the light chain fragment are secreted outside the cell, and no binding occurs between the heavy chain fragment and the light chain fragment, and accordingly, a heavy chain-based fusion protein according to the present invention is produced.

[0139] In addition, the results of the SEC-HPLC analysis performed to evaluate the preparation results and structural uniformity of the fusion protein are shown in Fig. 4. The method for preparing the fusion protein and the SEC-HPLC analysis method are described in detail in the following examples.

[0140] As shown in Fig. 4A, the fusion protein (CH + Cκ) containing a heavy chain constant region expressed with wild-type Cκ exhibited a single main peak of approximately 93.8%, demonstrating high structural uniformity. The fusion protein (CH + Cκ) prepared by co-expressing with light chain constant region variants in which the cysteine ​​at position 107 of Cκ was substituted with serine, alanine, aspartic acid, lysine, or phenylalanine, respectively -C107S , CH + Cκ -C107A , CH + Cκ -C107D , CH + Cκ -C107K , CH + Cκ -C107F In addition, it formed a main peak at an elution time similar to that of CH + Cκ above, and showed a single peak ratio of about 81.9% to 84.7%.

[0141] In addition, as shown in Fig. 4B, the fusion protein (CH + Cλ) containing a heavy chain constant region expressed together with wild-type Cλ exhibited a single main peak of approximately 96.8%, demonstrating high structural uniformity. The fusion protein (CH + Cλ) prepared by co-expressing with light chain constant region variants in which the cysteine ​​at position 105 of Cλ was substituted with serine, alanine, aspartic acid, lysine, or phenylalanine, respectively-C105S , CH + Cλ -C105A , CH + Cλ -C105D , CH + Cλ -C105K , CH + Cλ -C105F In addition, it formed a main peak at an elution time similar to that of CH + Cλ above, and showed a single peak ratio of about 76.2% to 78.9%.

[0142] The above results indicate that when a variant in which cysteine ​​is substituted in the light chain constant region is used, a heavy chain-based fusion protein containing only the heavy chain can be stably produced. In other words, unlike the perception of conventional technology, which has held that the production and stability of antibodies or antibody-derived proteins containing only the heavy chain depend heavily on disulfide bonds, the present invention confirms that a stable structure of a fusion protein based on non-covalent interactions can be formed through the use of a light chain constant region variant.

[0143] In addition, serine, alanine, aspartic acid, lysine, and phenylalanine used in this embodiment are amino acids representing different physical and chemical properties. It was confirmed that the amino acid at the corresponding position does not depend on the properties of a specific amino acid, as the structural formation and stability of the fusion protein are maintained even when substituted with amino acids of these various properties. Therefore, the use of a light chain constant region variant in which cysteine ​​is substituted with another amino acid also falls under the present invention. This allows the heavy chain-based fusion protein of the present invention to exhibit structural stability at a level identical or similar to that of a natural antibody fragment containing a heavy chain and a light chain, without forming disulfide bonds that appear in natural antibodies.

[0144]

[0145] Example 2. Expression and secretion of a fusion protein containing a heavy chain

[0146] Intracellular expression of the heavy chain-based fusion protein according to Example 1, extracellular secretion, and the effect of increasing secretion efficiency by the light chain constant region variant were confirmed.

[0147] Specifically, the nucleotide sequences of the light chain constant region variants listed in Table 1 above and the nucleotide sequences of the heavy chains listed in Table 2 below were used. Here, for the heavy chains, sequences including a constant region and a variable region (HC), sequences including a constant region alone (CH1, CH2 and CH3; CH), and sequences including an Fc region among the constant regions (CH2 and CH3; Fc) were used.

[0148] Classification DNA Sequence (Sequence Number) Amino Acid Sequence (Sequence Number) HCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA(Sequence Number 9)EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGYGSRSAMDYWGQ GTSVTVSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:10)CHGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA(서열번호11)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(서열번호12)FcGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA(서열번호 13)EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(서열번호 14)

[0149] Subsequently, each sequence was cloned into an expression vector and transfected into the HEK293f cell line (FreeStyle™ 293-F). HEK293f cells numbered 1×10⁶ 24 hours prior to transfection. 6 They were inoculated and cultured at a concentration of cells / mL, and the cell density at transfection was 2×10⁶ 6 The cell / mL ratio was adjusted. Each expression vector was mixed with polyethyleneimine (PEI) to perform transient transfection, and the transfected cells were cultured with shaking at 37°C under 8% CO₂ conditions. On day 7 after transfection, the culture supernatant was collected, centrifuged, and filtered, after which the expressed proteins were purified using a Protein A column. Subsequently, the proteins were eluted with glycine-HCl solution, and the final proteins were recovered through ultrafiltration concentration. In addition, immunoblotting was performed to confirm the expression and extracellular secretion of each protein in HEK293f cells. Cells and culture supernatants were collected on days 1, 3, 5, and 7 after transfection, respectively, to prepare SDS-PAGE samples. The samples were separated by SDS-PAGE on a 4-20% gradient gel, transferred to a PVDF membrane, and each expressed and secreted fusion protein was detected using an anti-human IgG Fc antibody.

[0150] As a result, as shown in Figure 5, the amount of protein expressed within the cell of the fusion protein (CH + Cκ) containing the heavy chain constant region and the light chain constant region increased as the number of culture days increased, and the amount of protein secreted outside the cell and present in the supernatant also increased as the number of culture days increased.

[0151] However, in the case of fusion proteins containing only the heavy chain constant region (CH) or fusion proteins containing only the Fc region (including CH2 and CH3; Fc) among the constant regions, the amount of protein expressed within the cell increased with the number of culture days, whereas the amount of protein secreted outside the cell and present in the supernatant decreased relatively.

[0152] On the other hand, when a fusion protein containing a heavy chain constant region is expressed together with a light chain constant region variant (CH + Cκ -C107S As the number of culture days increased, not only the amount of protein expressed within the cell but also the amount of protein secreted outside the cell and present in the supernatant increased.

[0153] The above results indicate that the extracellular secretion efficiency of heavy chain-based fusion proteins can be increased by the presence of light chain constant region variants. Furthermore, the results suggest that light chain constant region variants are involved in the folding and secretion processes of the heavy chain constant region and can improve the yield of antibody-derived proteins even without including the entire light chain.

[0154]

[0155] Example 3. Structural integrity of a fusion protein containing a heavy chain

[0156] Example 3-1. Fusion protein containing a heavy chain constant region

[0157] It was confirmed whether the heavy chain-based fusion protein according to Example 1 above can form and maintain a monomer structure consisting of two heavy chains. Specifically, it was confirmed whether a fusion protein containing a heavy chain constant region can exist in the form of a monomer consisting of two heavy chains, regardless of whether a disulfide bond is formed, when expressed together with a light chain constant region variant.

[0158] Each fusion protein was expressed and purified using the same method as described above. Subsequently, 2 μg of the purified protein was loaded onto a gradient gel with a 4-20% concentration gradient under reducing and non-reducing conditions and subjected to SDS-PAGE. Afterward, the gel was stained with Coomassie blue staining solution and repeatedly destained with destaining buffer to detect proteins separated by size.

[0159] As a result, as shown in Figures 6A and 6B, a fusion protein (CH + Cκ) comprising a heavy chain invariant region expressed together with a mutated kappa light chain invariant region or a mutated lambda light chain invariant region -C107S or CH + Cλ -C105S It was detected in the increased molecular weight region under non-reducing conditions compared to reducing conditions, indicating that it exists in a form consisting of two heavy chains. In addition, each fusion protein was detected as a single band under both reducing and non-reducing conditions, confirming that no high molecular weight aggregates or low molecular weight degradation products were formed.

[0160] In addition, the above fusion protein (CH + Cκ -C107S or CH + Cλ -C105S ) was detected in a larger molecular weight region compared to a fusion protein in which the Fc region was expressed alone (Fc containing CH2 and CH3), and through this, it was found that the fusion protein contains not only the Fc region but also the CH1 domain.

[0161] The above results mean that the fusion protein containing the heavy chain constant region according to the present invention contains a CH1 domain and can also form and stably maintain a structure consisting of two heavy chains.

[0162]

[0163] Example 3-2. Fusion protein comprising a heavy chain constant region and a heavy chain variable region

[0164] It was confirmed whether the heavy chain-based fusion protein according to Example 1 above could form and maintain a structure consisting of two complete heavy chains. Specifically, it was confirmed whether a fusion protein including a heavy chain constant region and a heavy chain variable region could exist in a form consisting of two heavy chains regardless of whether a disulfide bond was formed when expressed together with a light chain constant region variant.

[0165] Each fusion protein was expressed in cells using the same method as described above, and the culture supernatant was collected. SDS-PAGE was performed under reducing and non-reducing conditions, and detection was performed using an anti-Fc antibody (α-hFc) binding to the Fc region of the heavy chain constant, an anti-kappa light chain antibody (α-hCκ) binding to the kappa light chain variable region, or an anti-lambda light chain antibody (α-hCλ) binding to the lambda light chain variable region. Additionally, each fusion protein was purified from the culture supernatant and SDS-PAGE analysis was performed under the same conditions. As a control, the culture supernatant of HEK293f cells that had not undergone transfection was used.

[0166]

[0167] Example 3-2-1. Kappa light chain invariant region

[0168] The types of fusion proteins used in this example are listed below.

[0169] 1. A fusion protein prepared by co-expressing a heavy chain (HC) containing a heavy chain constant region (CH1, CH2, and CH3) and a heavy chain variable region (VH) with an unmutated kappa light chain constant region (Cκ) (hereinafter, "HC + Cκ")

[0170] 2. A heavy chain (HC) comprising a heavy chain invariant region (CH1, CH2, and CH3) and a heavy chain variable region (VH), and a modified kappa light chain invariant region (HC + Cκ -C107S A fusion protein prepared by co-expressing ) (hereinafter, "HC + Cκ -C107S ")

[0171] As shown in Fig. 7, under reducing conditions, HC + Cκ and HC + Cκ -C107S In both cases, a band in the molecular weight region corresponding to the heavy chain was detected by α-hFc, and a band in the molecular weight region corresponding to the light chain was detected by α-hCκ. The molecular weights of the heavy and light chains detected in the two fusion proteins were identical, and through this, the HC + Cκ and HC + Cκ -C107S It was found that the two fusion proteins formed the same structure.

[0172] Under non-reducing conditions, HC + Cκ was detected by α-hFc as a band in the molecular weight region corresponding to a structure containing two heavy chains and two light chains (H2L2) and a band in the molecular weight region corresponding to a structure containing only two heavy chains (H2). Additionally, by α-hCκ, a band in the molecular weight region corresponding to a structure containing two heavy chains and two light chains (H2L2) was detected. Through this, it was found that the fusion protein of HC + Cκ forms a structure containing two heavy chains and two light chain fragments.

[0173] On the other hand, under non-reducing conditions, HC + Cκ -C107S In the case of α-hFc, only the band corresponding to the structure (H2) containing only two heavy chains was detected, and no band was detected by α-hCκ. Through this, the above HC + Cκ -C107S It was found that the fusion protein did not contain a light chain fragment and formed a structure containing only two heavy chains.

[0174] In particular, the aforementioned detection pattern appeared identically in both the culture supernatant and the purified protein. This indicated that the structure of the fusion protein is already formed during the extracellular secretion stage and is not artificially generated or modified by the purification process.

[0175] The above results show that when the light chain constant region variant is used for the expression of a heavy chain-based fusion protein, the heavy chain-based fusion protein is produced without forming disulfide bonds with the light chain and remains unbound to the light chain, and that a structure consisting solely of the heavy chain is maintained even during the purification process.

[0176]

[0177] Example 3-2-2. Lambda light chain invariant region

[0178] The types of fusion proteins analyzed in this example are listed below.

[0179] 1. A heavy chain (HC) comprising a heavy chain invariant region (CH1, CH2, and CH3) and a heavy chain variable region (VH), a lambda light chain variable region (VL), and a mutated lambda light chain invariant region (Cλ -C105S ) are co-expressed to form a fusion protein (hereinafter referred to as "HC + LCλ") -C105S ")

[0180] 2. A heavy chain (HC) comprising a heavy chain invariant region (CH1, CH2, and CH3) and a heavy chain variable region (VH), and a modified lambda light chain variable region (Cλ -C105S ) are co-expressed to form a fusion protein (hereinafter referred to as "HC + LCλ") -C105S ")

[0181] 3. A fusion protein comprising a heavy chain constant region (CH1, CH2, and CH3; CH) and a mutated lambda light chain constant region (hereinafter, "CH + Cλ -C105S ")

[0182] As shown in Fig. 8, under reduction conditions, HC + LCλ -C105S , HC + Cλ -C105S and CH + Cλ -C105SIn all cases, bands in the molecular weight region corresponding to the heavy chain (HC or CH) were detected by α-hFc. This detection pattern was confirmed identically in both the culture supernatant and the purified protein, which indicated that the structure of the fusion protein was already formed during the extracellular secretion stage and was not artificially created or modified by the purification process.

[0183] Meanwhile, for purified antibodies, HC + LCλ by α-hCλ under reducing conditions -C105S A band in the molecular weight region corresponding to the light chain (Cλ) was detected only in [the sample]. This means that although no disulfide bond is formed between the heavy chain and the light chain, when the entire light chain is included, the light chain may exist in a state where it is partially bonded to the heavy chain by non-covalent interactions between VH and VL, and / or between CH1 and CL.

[0184] On the other hand, under non-reducing conditions, HC + LC λ-C105S and HC + C λ-C105S In the case of, only the band corresponding to the structure (HC2) containing two heavy chains encompassing both the invariant and variable regions was detected, and CH + C λ-C105S In the case of, only the band corresponding to the structure (CH2) containing two heavy chains that include only the invariant region was detected. Through this, HC + C λ-C105S and CH + C λ-C105S It was found that the fusion protein did not contain a light chain fragment and formed a structure containing only two heavy chains.

[0185] The above results demonstrate that when a light chain constant region variant is used for the expression of a heavy chain-based fusion protein, the heavy chain-based fusion protein is produced without forming disulfide bonds with the light chain and remains unbound to it, and that a structure consisting solely of the heavy chain is maintained even during the purification process. Furthermore, this trend was confirmed equally not only when the heavy chain contains only the constant region but also when it contains both the constant and variable regions. Accordingly, it was confirmed that a heavy chain-based fusion protein can be formed and maintained without binding to the light chain, regardless of the heavy chain composition.

[0186]

[0187] Example 4. Production quantity of fusion protein containing heavy chain

[0188] The production volume of the heavy chain-based fusion protein according to Example 1 above was confirmed.

[0189] The protein concentration of each purified fusion protein was calculated using the absorbance (A280) at 280 nm and the extinction coefficient calculated from the amino acid sequence, and the purification yield was calculated based on this. The extinction coefficient was calculated from the amino acid sequence of each fusion protein via the webpage (http: / web.expasy.org / protparam / ), and the protein concentration was obtained using a formula that applied the molecular weight and the extinction coefficient to the A280 value. In addition, the average production of fusion proteins containing both CH and Cκ, or both CH and Cλ, was set as a reference value (1), and the relative production of each fusion protein was calculated based on this. The results for Cκ are shown in Table 3 below, and the results for Cλ are shown in Table 4 below.

[0190] Fusion Protein Production (mg / L) Relative Production (mg / L) CH + Cκ 18.40 - 18.90 1.00 CH 0.60 - 0.90 0.06 Fc 2.10 - 2.20 0.23 CH + Cκ -C107S 10.20 - 14.200.85HC + Cκ -C107S11.00 - 12.000.62

[0191] Fusion Protein Production (mg / L) Relative Production (mg / L) CH + Cλ 18.40 - 18.90 1.00 CH 0.60 - 0.90 0.06 Fc 2.10 - 2.20 0.23 CH + Cλ -C105S 10.00 - 14.300.85HC + Cλ -C105S 11.00 - 12.000.62

[0192] As can be seen in Tables 3 and 4 above, the production of CH + Cκ or CH + Cλ was approximately 18.4–18.9 mg / L, whereas in the case of CH-only expression, the production decreased significantly to approximately 0.6–0.9 mg / L, and Fc-only expression also showed a relatively low production of approximately 2.1–2.2 mg / L. On the other hand, CH is a light chain constant region variant (Cκ -C107S or Cλ -C105S When expressed together with ), it showed a production of about 10.0-14.3 mg / L, which was a significant increase compared to CH expression alone, and the average production also showed a level close to CH + Cκ or CH + Cλ (about 0.85).

[0193] In addition, HC is a light chain invariant region variant (Cκ -C107S or Cλ -C105S Even when expressed together with ), a production yield of approximately 11.0–12.2 mg / L was observed, confirming that the effect of increasing production occurs not only when the heavy chain contains only the constant region but also when it contains both the constant and variable regions. This trend was observed equally in both the kappa and lambda light chain constant regions.

[0194] The above results demonstrate that the light chain constant region variant significantly increases the production of heavy chain-based fusion proteins during expression, despite not forming disulfide bonds with the heavy chain. In particular, compared to the case where the heavy chain was expressed alone, the production of heavy chain-based fusion proteins was distinctly improved when the light chain constant region variant was used in combination, implying that the light chain constant region variant can contribute to the expression, secretion, and stable production of heavy chain-based fusion proteins.

[0195]

[0196] Example 5. Utility of a fusion protein containing a heavy chain

[0197] It was confirmed whether the heavy chain-based fusion protein according to the present invention can be manufactured in a form that includes the existing antibody structure as is, and furthermore, whether it can be manufactured in a form that includes various domains of the existing antibody or a separately designed antibody.

[0198]

[0199] Example 5-1. Fusion protein containing Vyvgart

[0200] The heavy chain-based fusion protein according to the present invention is Vyvgart ® It was confirmed whether it is possible to manufacture a structure that includes the exact structure of an existing Fc-based therapeutic fusion protein, such as the ingredient name efgartigimod.

[0201] Specifically, fusion proteins containing the Fc structure of Viebergart were prepared. The structure of each fusion protein is shown in Fig. 9A, and the sequence number is shown in Table 5.

[0202] 구분아미노산 서열 (서열번호)FA0380DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(서열번호 15)FA0381ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(서열번호 16)FA0382ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(서열번호17)FA0383ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(서열번호 18)FA0384ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(서열번호 19)

[0203] Subsequently, each fusion protein was expressed and purified according to the method described above, and its molecular weight and structure were confirmed through SDS-PAGE analysis under reducing and non-reducing conditions, and its structural uniformity was evaluated through SEC-HPLC analysis. As a result, as shown in Fig. 9B, the heavy chain-based fusion proteins (FA0382 and FA0384) according to the present invention were detected as a single band in the molecular weight region corresponding to a monomer consisting of two heavy chains in SDS-PAGE analysis under non-reducing conditions, and bands corresponding to the heavy chains of each fusion protein were detected under reducing conditions. In addition, FA0382 and FA0384 exhibited similar band patterns under non-reducing conditions when compared to fusion proteins containing light chains (FA0381 or FA0383) or Wiebgard (FA0380), and no non-specific binding, degradation, or polymer aggregates were detected.

[0204] In addition, as shown in Figure 9C, FA0382 and FA0384 were detected as major molecular species corresponding to monomers consisting of two heavy chains, with more than 88% of the total peaks being detected, and no aggregates or low molecular weight degradation products were significantly identified.

[0205] Accordingly, it was confirmed that the above FA0382 and FA0384 exist in the form of monomers consisting of two heavy chains even in a structure that does not include a light chain.

[0206]

[0207] Example 5-2. Fusion protein containing an Eylea fragment

[0208] It was confirmed whether the fusion protein according to the present invention can also be manufactured in a form including components of other therapeutic antibodies as needed.

[0209] Specifically, Eylea ®A heavy chain-based fusion protein was prepared containing the D2 domain of VEGFR1 and the D3 domain of VEGFR2, which are VEGFR-derived binding domains used in aflibercept. The structure of each fusion protein is shown in Figure 10A, and the sequence numbers are shown in Table 6.

[0210] 구분아미노산 서열 (서열번호)FA0366SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(서열번호20)FA0367SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(서열번호21)FA0374SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(서열번호22)FA0375SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(서열번호23)(Sequence No. 24)

[0211] Subsequently, each fusion protein was expressed and purified according to the method described above, and then SDS-PAGE analysis and SEC-HPLC analysis were performed. As a result, as shown in Fig. 10B, the heavy chain-based fusion proteins (FA0374 and FA0376) according to the present invention were detected as a single band in the molecular weight region corresponding to a monomer consisting of two heavy chains in SDS-PAGE analysis under non-reducing conditions, and bands corresponding to the heavy chains of each fusion protein were detected under reducing conditions. In particular, FA0374 and FA0376 exhibited similar band patterns under non-reducing conditions even when compared to fusion proteins containing light chains (FA0367 or FA0375) or the Eylea antibody (FA0366), and no non-specific binding, degradation, or polymer aggregates were detected.

[0212] In addition, as shown in Figure 10C, FA0374 and FA0376 were detected as major molecular species corresponding to monomers consisting of two heavy chains, with more than 88% and up to about 99% of the total peaks being detected, and no aggregates or low molecular weight degradation products were significantly identified.

[0213] Accordingly, it was confirmed that the above FA0374 and FA0376 exist in the form of monomers consisting of two heavy chains even in a structure that does not include a light chain.

[0214]

[0215] Example 5-3. Fusion protein containing a bispecific binding site

[0216] It was confirmed whether the heavy chain-based fusion protein according to the present invention can be manufactured not only as a single-specific antibody but also as a bispecific antibody structure comprising multiple binding domains having different binding specificities.

[0217] Specifically, a fusion protein comprising multiple binding domains having different binding specificities was prepared. For example, Eylea ® A fusion protein was prepared comprising the D2 domain of derived VEGFR1 and the D3 domain of VEGFR2; and an scFv that binds to mesothelin (MSLN). The structure of each fusion protein is shown in Figure 11A, and the sequence number is shown in Table 7.

[0218] 구분아미노산 서열 (서열번호)FA0368SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTAVDTAVYYCARDKDWGGFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCSGTSSDIGRYDRVSWYQHHPGRAPKLIIFDVNVRPSGVSNRFSGSKSGNSASLTISGLQPEDEADYYCSSYGYLGTPGTLFYVFGTGTKVTVL(서열번호25)FA0369SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTAVDTAVYYCARDKDWGGFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCSGTSSDIGRYDRVSWYQHHPGRAPKLIIFDVNVRPSGVSNRFSGSKSGNSASLTISGLQPEDEADYYCSSYGYLGTPGTLFYVFGTGTKVTVL(서열번호26)FA0377SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTAVDTAVYYCARDKDWGGFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCSGTSSDIGRYDRVSWYQHHPGRAPKLIIFDVNVRPSGVSNRFSGSKSGNSASLTISGLQPEDEADYYCSSYGYLGTPGTLFYVFGTGTKVTVL(서열번호27)FA0378SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTAVDTAVYYCARDKDWGGFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCSGTSSDIGRYDRVSWYQHHPGRAPKLIIFDVNVRPSGVSNRFSGSKSGNSASLTISGLQPEDEADYYCSSYGYLGTPGTLFYVFGTGTKVTVL(서열번호28)FA0379SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTAVDTAVYYCARDKDWGGFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCSGTSSDIGRYDRVSWYQHHPGRAPKLIIFDVNVRPSGVSNRFSGSKSGNSASLTISGLQPEDEADYYCSSYGYLGTPGTLFYVFGTGTKVTVL(서열번호 29)

[0219] Subsequently, each fusion protein was expressed and purified according to the method described above, and then SDS-PAGE analysis and SEC-HPLC analysis were performed. As a result, as shown in Fig. 11B, the heavy chain-based fusion proteins (FA0377 and FA0379) according to the present invention were detected as a single band in the molecular weight region corresponding to a monomer consisting of two heavy chains in SDS-PAGE analysis under non-reducing conditions, and bands corresponding to the heavy chains of each fusion protein were detected under reducing conditions. In addition, FA0377 and FA0379 exhibited similar band patterns under non-reducing conditions when compared to fusion proteins containing light chains (FA0369 or FA0377) or a reference antibody (FA0368), and no non-specific binding, degradation, or polymer aggregates were detected.

[0220] In addition, as shown in Figure 11C, FA0377 and FA0379 were detected as major molecular species corresponding to monomers consisting of two heavy chains, with more than 92% of the total peaks being detected, and no aggregates or low molecular weight degradation products were significantly identified.

[0221] Accordingly, it was confirmed that the above FA0377 and FA0379 exist in the form of monomers consisting of two heavy chains even in a structure that does not include a light chain.

[0222]

[0223] Example 5-4. Fusion protein containing a heteromeric antibody

[0224] It was confirmed whether the heavy chain-based fusion protein according to the present invention can be prepared not only in the structure of a single-specific antibody or a homodimeric antibody, but also in the structure of a heterodimeric antibody having different binding specificities.

[0225] Specifically, a fusion protein containing different heavy chains was prepared. For example, one heavy chain is Eylea ®A fusion protein was prepared containing the D2 domain of derived VEGFR1 and the D3 domain of VEGFR2, and the other heavy chain not containing said domains. The structure of each fusion protein is shown in Fig. 12A, and the sequence number is shown in Table 8.

[0226] 구분아미노산 서열 (서열번호)FA0387HC1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(서열번호 30)HC2SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(서열번호31)FA0390HC1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(서열번호 32)HC2SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(서열번호 33)

[0227] Subsequently, each fusion protein was expressed and purified according to the method described above, and then SDS-PAGE and SEC-HPLC analyses were performed. As a result, as shown in Fig. 12B, the heavy chain-based fusion protein (FA0390) according to the present invention was detected as a single band in the molecular weight region corresponding to the heteromer in SDS-PAGE analysis under non-reducing conditions, while bands corresponding to each constituent heavy chain were detected under reducing conditions. In the case of the fusion protein containing a light chain (FA0387), a band corresponding to a monomer consisting of two heavy chains was also detected, but it exhibited a band pattern different from that of FA0390. Furthermore, as shown in Fig. 12C, more than 95% of the total peaks of FA0390 were detected as major molecular species corresponding to the heteromer, and no high molecular weight aggregates or low molecular weight degradation products were significantly identified.

[0228]

[0229] Example 5-5. Fusion protein containing an IgE antibody

[0230] It was confirmed whether the heavy chain-based fusion protein according to the present invention can be manufactured with a structure including the constant region of an IgE antibody.

[0231] Specifically, fusion proteins containing Cε2, Cε3, and Cε4 of the heavy chain constant region of an IgE antibody were prepared. The structure of each fusion protein is shown in Figure 13A, and the sequence number is shown in Table 9.

[0232] 구분아미노산 서열 (서열번호)FA0073ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK(서열번호 34)FA0389ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK(서열번호 35)

[0233] Subsequently, each fusion protein was expressed and purified according to the method described above, and then SDS-PAGE and SEC-HPLC analyses were performed. As a result, as shown in Fig. 13B, the IgE-based fusion protein containing a heavy chain according to the present invention (FA0389) was detected as a major band in the molecular weight region corresponding to a monomer consisting of two heavy chains in SDS-PAGE analysis under non-reducing conditions, and a band corresponding to the heavy chain was detected under reducing conditions. In addition, as shown in Fig. 13C, more than about 78% of the total peaks of FA0389 were detected as major molecular species corresponding to a monomer consisting of two heavy chains, and high molecular weight aggregates or low molecular weight degradation products were identified only to a limited extent. Accordingly, it was confirmed that the heavy chain-based fusion protein exists in the form of a monomer consisting of two heavy chains even in a structure containing an IgE constant region.

[0234] Through the results of Example 5 above, it was confirmed that the heavy chain-based fusion protein according to the present invention can be manufactured not only in a form containing the existing antibody structure as is, but also in a form containing various domains of separately designed antibodies. In particular, it was confirmed that it can be manufactured in the structures of monospecific antibodies, bispecific antibodies, and heterodimer antibodies, and that it can be manufactured in a structure containing the constant region of an IgE antibody as well as an IgG antibody.

[0235]

[0236] Example 6. Increase in the production yield of fusion proteins containing heavy chains

[0237] In this example, the production yield of heavy chain-based fusion proteins, the formation of monomers consisting of two heavy chains, and structural uniformity were confirmed by the light chain constant region variant.

[0238] Specifically, FA0382, FA0374, and FA0377 fusion proteins were prepared by expressing only the heavy chain constant region alone (HC) or by expressing variants of the heavy chain constant region and light chain constant region together (HC + Cκ -C107S The outline of each antibody structure is shown in Figure 14A, and the sequence numbers are shown in Table 10.

[0239] 구분아미노산 서열 (서열번호)FA0382ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(서열번호 17)FA0374SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(서열번호22)FA0377SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTAVDTAVYYCARDKDWGGFDVWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCSGTSSDIGRFDRVSWYQHHPGRAPKLIIFDVNVRPSGVSNRFSGSKSGNSASLTISGLQPEDEADYYCSSVGYLGTPGTLFSVFGTGTKVTVL(서열번호 27)

[0240] Subsequently, each fusion protein was expressed and purified according to the aforementioned method, and then SDS-PAGE and SEC-HPLC analyses were performed. As a result, as shown in Fig. 14B, when the fusion protein was expressed as a heavy chain alone (HC), numerous molecular species other than the monomer consisting of two heavy chains were identified in SDS-PAGE and SEC-HPLC analyses under non-reducing conditions. On the other hand, when the heavy chain was expressed together with a light chain constant region variant (HC + Cκ -C107S In ), a single band was detected in the molecular weight region corresponding to a monomer consisting of two heavy chains under non-reducing conditions, and only the band corresponding to the heavy chain was detected under reducing conditions.

[0241] Furthermore, as shown in Fig. 14C, in the case of heavy chain-only expression (HC), the proportion of major molecular species corresponding to monomers composed of two heavy chains was approximately 30-50%, whereas in the case where the heavy chain was expressed together with a light chain constant region variant (HC + Cκ -C107S In ), more than 88-93% of the total peaks were detected as major molecular species corresponding to monomers composed of two heavy chains, and no aggregates or low molecular weight degradation products were significantly identified.

[0242] In particular, when the above fusion protein was expressed with the heavy chain alone, FA0382 showed a low production level of about 2.6 mg / L, FA0374 showed about 60.4 mg / L, and FA0377 showed about 25.3 mg / L. On the other hand, when the heavy chain was expressed together with the light chain constant region variant, FA0382 showed a high production level of about 76 mg / L, FA0374 showed about 143 mg / L, and FA0377 showed about 137 mg / L, and the production level increased significantly from about 2.4 times to about 29 times compared to the expression of the heavy chain alone.

[0243] The above results indicate that when a heavy chain is expressed together with a light chain invariant variant to produce a heavy chain-based fusion protein according to the present invention, the production yield of the fusion protein can be significantly increased.

Claims

1. A fusion protein comprising: a CH1 domain of IgG; and one or more Fc regions selected from IgG and IgE.

2. In Paragraph 1, A fusion protein wherein the above Fc region comprises one or more selected from the group consisting of the CH2 domain of IgG, the CH3 domain of IgG, the Cε2 domain of IgE, the Cε3 domain of IgE, and the Cε4 domain of IgE.

3. In Paragraph 1, The above fusion protein is a fusion protein comprising the CH1 domain of IgG; and the CH2 domain of IgG and the CH3 domain of IgG.

4. In Paragraph 1, The above fusion protein is a fusion protein comprising the CH1 domain of IgG; and the Cε2 domain of IgE, the Cε3 domain of IgE and the Cε4 domain of IgE.

5. In Paragraph 1, The above fusion protein is a fusion protein that does not contain the light chain of an antibody.

6. In Paragraph 1, The above fusion protein is a fusion protein that is a monomer composed of two heavy chains.

7. A nucleic acid molecule encoding the fusion protein of paragraph 1.

8. An expression vector comprising the nucleic acid molecule of claim 7.

9. A method for producing the fusion protein of claim 1, comprising the step of introducing the expression vector of claim 8 into isolated host cells.

10. In Paragraph 9, The above method further comprises the step of introducing an expression vector containing a nucleic acid molecule encoding Cκ or Cλ into an isolated host cell.

11. In Paragraph 10, A method in which the above Cκ or Cλ includes a mutation.

12. In Paragraph 11, A method in which the mutation included in the above Cκ is a substitution or deletion of cysteine ​​at position 107.

13. In Paragraph 11, A method in which the mutation included in the above Cλ is a substitution or deletion of cysteine ​​at position 105.

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