Linker conjugated to j chain and j chain complex comprising same

Abstract

The present invention relates to a linker conjugated to a J chain and a J chain complex comprising the linker and the J chain. The linker according to the present invention can be used to conjugate various biologically active molecules to a J chain within an antibody-based structure and can increase the activity of the J chain complex comprising the linker and the biologically active molecules. In addition, the effect of the linker can appear regardless of the type of biologically active molecule conjugated to the linker.

Description

A linker connected to a J chain and a J chain complex comprising the same

[0001] The present invention relates to a linker connected to a J chain, and a J chain complex comprising the linker and the J chain.

[0002] A wide variety of biologically active molecules, including antibodies, antibody fragments, compounds, enzymes, and hormones, are being used to treat diseases. Because the activity of biologically active molecules is directly related to therapeutic efficacy, various approaches are being pursued to enhance their activity.

[0003] For example, one way to enhance the therapeutic efficacy of a biologically active molecule is to increase its affinity or avidity for its target receptor. This allows receptor activation even at low concentrations within the organism, thereby enhancing the therapeutic efficacy of the active molecule.

[0004] Another approach is to introduce two or more biologically active molecules with different activities into an antibody-based material. This can promote the transport of the active molecules to the target tissue, engage cells such as cytotoxic T cells, or activate cellular costimulation. This approach is primarily utilized in IgG antibodies or IgG antibody-based constructs. A single IgG antibody can exhibit two or more activities by linking different antigen-binding fragments (Fabs) to each antigen-binding site of the IgG antibody, or by linking additional antigen-binding fragments to the terminal of the crystal fragment (Fc).

[0005] Currently, these biologically active molecules are linked to IgG antibodies, etc., via a flexible linker represented by (GGGGS)n. However, it has been reported that, depending on the type of biologically active molecule linked by a flexible linker, it may not be expressed during the production process, the expression level may decrease, or even if it is expressed normally, its original biological activity may decrease or disappear (Y. Maeda, et. al, Engineering of functional chimeric protein G-Vargula luciferase, Anal. Biochem. 249 (1997) 147-152.). In addition, although a prior patent suggesting an IgA antibody, etc. in which an active molecule is linked via a flexible linker has been published (Korean Patent Publication No. 10-2380402), the prior patent does not disclose the expression level of IgA antibodies, differences in biological activity of IgA antibodies, etc., depending on the type of linker other than the flexible linker.

[0006] Therefore, in order to enhance the biological activity of antibody-based materials, continuous research is needed on methods that can maintain or enhance the activity of biologically active molecules linked to antibodies.

[0007] The present inventors have diligently researched methods to enhance the biological activity of antibody-based materials. As a result, they confirmed that when biologically active molecules are linked to the J chain in an IgM antibody-based structure, the activity of the IgM antibody containing them varies depending on the type of linker. The inventors have developed a linker that can maximize this activity, leading to the completion of the present invention.

[0008] One aspect of the present invention provides a linker connected to at least one selected from the group consisting of an N-terminus and a C-terminus of a J chain.

[0009] As used herein, the term "J chain" refers to a polypeptide that is a component of an IgM or IgA antibody and links IgM or IgA monomers to form a multimer. Specifically, the J chain links IgM monomers or IgA monomers to each other and polymerizes each monomer, and IgM forms a pentamer and IgA forms a dimer through the J chain. The amino acid sequence constituting the J chain and the base sequence encoding it are known in existing databases (NCBI Reference Sequence: NP_653247, NM_144646, etc.).

[0010] The J chain according to the present invention may be wild type or contain a mutation.

[0011] Specifically, the wild-type J chain may include, but is not limited to, the amino acid sequence of SEQ ID NO: 1, the amino acid sequence encoded by the base sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 2.

[0012] In addition, the J chain comprising the mutation may be one in which one or more of the amino acids at positions 105, 106, and 107 in the amino acid sequence of SEQ ID NO: 1 are deleted or substituted. More specifically, the J chain comprising the mutation may be one in which the amino acid at position 105 in the amino acid sequence of SEQ ID NO: 1 has any one mutation selected from the group consisting of D105A and D105K. In addition, the J chain comprising the mutation may have any one mutation selected from the group consisting of R106A, R106D, R106E, R106F, R106G, R106H, R106I, R106K, R106L, R106M, R106N, R106P, R106Q, R106S, R106T, R106V, R106W, and R106Y in the amino acid sequence of SEQ ID NO: 1. In addition, the J chain comprising the mutation may have any one mutation selected from the group consisting of N107A, N107E, N107F, and N107K in the amino acid sequence of SEQ ID NO: 1 in the amino acid sequence of SEQ ID NO: 1.

[0013] The term "linker" as used herein refers to an amino acid present between each domain of a protein, and can play a role in connecting each domain within a J chain complex or fusion protein in which multiple domains are connected.

[0014] In the present invention, the linker may serve to connect the J chain and the biologically active molecule. Specifically, the linker may be connected to the N-terminus or the C-terminus of the J chain, or to both the N-terminus and the C-terminus, and may be connected to the N-terminus or the C-terminus of the biologically active molecule, or to both the N-terminus and the C-terminus, thereby serving to connect the two. More specifically, the linker may be connected to the N-terminus of the J chain and to the C-terminus of the biologically active molecule, thereby serving to connect the two.

[0015] The linker according to the present invention may be a rigid linker or a semi-rigid linker.

[0016] As used herein, the term "rigid linker" refers to a linker characterized by rigidity, stiffness, and stability. Rigid linkers are primarily used when a fixed distance between domains must be maintained, or when each domain must maintain independent activity without interaction. Rigid linkers can secure stiffness by including a high proportion of proline (Pro; P), which can provide rigidity.

[0017] Specifically, the rigid linker may be (XP)n.

[0018] The (XP)n may be a proline-rich linker containing a high proportion of proline. The X may be alanine, glutamine, or lysine. The n is an integer from 1 to 10, and the (XP)n may include 2 to 20 amino acids selected from the group consisting of alanine, glutamine, lysine, and proline, but is not limited thereto. In addition, the (XP)n may include the amino acid sequence of SEQ ID NO: 7, or may include the amino acid sequence encoded by the base sequence of SEQ ID NO: 8, or may consist of the amino acid sequence, but is not limited thereto.

[0019] As used herein, the term "semi-rigid linker" refers to a linker characterized by a fusion of a rigid linker and a flexible linker. The semi-rigid linker has properties intermediate between those of a rigid linker and a flexible linker, thereby ensuring flexibility and mobility while also maintaining a certain distance between each domain.

[0020] Specifically, the semi-rigid linker may be {(GGGGS)(EAAAK)(GGGGS)}n. Wherein n is an integer from 1 to 10, and {(GGGGS)(EAAAK)(GGGGS)}n may include 10 to 100 amino acids selected from the group consisting of alanine, glutamine, lysine, glycine (Gly; G), and serine (Ser; S), but is not limited thereto. In addition, {(GGGGS)(EAAAK)(GGGGS)}n may include an amino acid sequence of SEQ ID NO: 5, include an amino acid sequence encoded by a base sequence of SEQ ID NO: 6, or consist of the amino acid sequence, but is not limited thereto.

[0021] Here, the term "flexible linker" refers to a linker that is mainly used when interaction between domains is required or when mobility of domains is required. It is composed of relatively small-sized amino acids such as glycine (Gly; G), serine (Ser; S), and threonine (Thr; T), and provides flexibility of the linker and mobility of the connected domains.

[0022] Specifically, the flexible linker may be a GS linker, and may be at least one selected from the group consisting of (GS)n, (SG)n, (GGGS)n, (GGGGS)n, GCGS(GGGS)n, and GCGGS(GGGGS)n. Wherein n is an integer of 1 to 10, for example, 2 to 6, and may include 2 to 55 amino acids selected from the group consisting of glycine and serine, but is not limited thereto. In addition, the flexible linker may include 4 to 35 amino acids composed of glycine, serine, and cysteine ​​(Cys; C) in order to increase stability through a disulfide bond, but is not limited thereto. In addition, the flexible linker may include an amino acid sequence of SEQ ID NO: 3, an amino acid sequence encoded by a base sequence of SEQ ID NO: 4, or may be composed of the amino acid sequence, but is not limited thereto.

[0023]

[0024] Another aspect of the present invention provides a J chain complex or fusion protein comprising the linker and the J chain.

[0025] In the J chain complex or fusion protein according to the present invention, unless specifically stated otherwise, the related terms are understood to have the same meaning as the terms described above.

[0026] As used herein, the term "J chain complex" refers to a protein complex comprising a J chain and a linker. Preferably, in the J chain complex, the linker is bound to the J chain.

[0027] In the present invention, the J chain complex can be represented by the following structural formula:

[0028] (X)a-(Y)b-(Z)-(Y)c-(X)d

[0029] At this time, in the above structural formula,

[0030] The above X is a biologically active molecule,

[0031] The above Y is a linker,

[0032] The above Z is a J chain,

[0033] The above a, b, c and d are independently 0 or 1, and at least one of the above b and c is 1.

[0034] When b and c are 1, the linkers Y may be the same or different from each other. When a and d are 1, the biologically active molecules X may be the same or different from each other.

[0035] In one embodiment, a may be 1, b may be 1, c may be 0, and d may be 0. In this case, the structural formula is represented by (X)-(Y)-(Z), and the J chain complex may be in a form in which a biologically active molecule is linked to the N-terminus of the J chain via a linker.

[0036] In another embodiment, a may be 0, b may be 0, c may be 1, and d may be 1. In this case, the structural formula is represented by (Z)-(Y)-(X), and the J chain complex may be in a form in which a biologically active molecule is linked to the C-terminus of the J chain via a linker.

[0037] In another embodiment, a may be 1, b may be 1, c may be 1, and d may be 1. In this case, the structural formula is represented by (X)-(Y)-(Z)-(Y)-(X), and the J chain complex may be in a form in which two biologically active molecules are connected to the N-terminus and C-terminus of the J chain, respectively, via a linker.

[0038] The term "biologically active molecule" as used herein refers to a substance that can exhibit a specific action or effect within a living organism, for example, a substance that can perform a function such as promoting or inhibiting the expression of a specific gene by activating or inactivating a specific signaling pathway, enhancing or inhibiting the function of a specific protein, or inducing or inhibiting cell death. Specifically, the biologically active molecule may exhibit immune regulation and / or anticancer activity.

[0039] In the present invention, the biologically active molecule can serve as a functional group or active moiety so that the J chain complex or fusion protein containing it can exhibit the desired effect.

[0040] Specifically, the biologically active molecule may be at least one selected from the group consisting of antibodies, antigen-binding fragments of antibodies, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, compounds, soluble proteins, membrane-bound proteins, ligands, receptors, virus-like particles, protein toxins, chemokines, cytokines, and enzymes, but is not limited thereto. In this case, the receptor may be, but is not limited to, a cytokine receptor and / or an immune checkpoint receptor.

[0041] As a specific example, the antigen-binding fragment of the antibody may be at least one selected from the group consisting of Fab, F(ab')2, F(ab)2, Fab', Fab2, Fab3, Fv, scFv, Bis-scFv, Minibody, Triabody, Diabody, Tandem Diabody (TandAb), Nanobody, and Tetrabody, but is not limited thereto.

[0042] In addition, the antigen-binding fragment of the antibody may bind to HLA-G (human leukocyte antigen G). HLA-G is a type of type 1 HLA, expressed in the placenta of pregnant women and involved in the immune tolerance of the fetus. HLA-G binds to ILT2, ILT4, and KIR2DL4, which are inhibitory receptors of NK (natural killer) cells, thereby inhibiting NK cells and regulatory T cells (Treg ) plays a role in promoting differentiation. In addition, when cancer cells overexpress HLA-G on the surface, the activity of cytotoxic T cells, helper T cells, and NK cells involved in cytotoxicity is inhibited, thereby reducing the effect of immunotherapy for cancer treatment. Therefore, an antigen-binding fragment that selectively binds to HLA-G and inhibits HLA-G from binding to an inhibitory receptor can be utilized as an anticancer therapeutic agent. For example, the antigen-binding fragment of the antibody may be a binding fragment of an antibody that binds to HLA-G, and may be a Fab of the antibody that binds to HLA-G. Specifically, the Fab of the antibody that binds to HLA-G may include, but is not limited to, a light chain (VL domain and CL domain) and a portion of a heavy chain (VH domain and CH1 domain). At this time, the light chain included in the Fab of the antibody binding to the HLA-G may include the amino acid sequence of SEQ ID NO: 15, include the amino acid sequence encoded by the base sequence of SEQ ID NO: 16, or consist of the amino acid sequence, but is not limited thereto. In addition, a portion of the heavy chain included in the Fab of the antibody binding to the HLA-G may include the amino acid sequence of SEQ ID NO: 17, include the amino acid sequence encoded by the base sequence of SEQ ID NO: 18, or consist of the amino acid sequence, but is not limited thereto.

[0043] In addition, the antigen-binding fragment of the antibody may bind to CD3 (cluster of differentiation 3). CD3 is a co-receptor that binds to the T cell antigen receptor (TCR) present on the surface of cytotoxic T cells (killer T cells; Tc) or helper T cells (Th) when the TCR is activated, and is composed of CD3γ, CD3δ, CD3ε, and CD3ζ molecules. The antigen-binding fragment of the antibody has an agonist property that binds to CD3ε to form a TCR and CD3 complex and activates T cells, and thus can be utilized to activate T cells, or can be utilized as a BiTE (Bispecific T cell engager) that engages cancer cells and cytotoxic T cells to kill cancer cells. For example, the antigen-binding fragment of the antibody can be a binding fragment of an antibody that binds to CD3, or can be an scFv of an antibody that binds to CD3. Specifically, the scFv of the antibody binding to CD3 may include the amino acid sequence of SEQ ID NO: 9, include the amino acid sequence encoded by the base sequence of SEQ ID NO: 10, or consist of the amino acid sequence, but is not limited thereto.

[0044] In addition, the antigen-binding fragment of the antibody may bind to CD28 (cluster of differentiation 28). CD28 is a receptor expressed on T cells, and it functions to provide co-stimulation by binding to CD80 and CD86, which are ligands overexpressed on the surface of licensed dendritic cells. Stimulation by binding of the TCR of cytotoxic T cells with the major histocompatibility complex class I (MHCI) presented with an antigen-specific peptide of the dendritic cell (signal 1), co-stimulation by binding of CD80 / CD86 - CD28 (signal 2), and antigen-specific cytotoxic T cells activated by cytokines (signal 3) can rapidly grow, proliferate, and be activated to effectively attack and kill target cells. The antigen-binding fragment of the antibody may be an agonist that binds to CD28 and activates CD28 similarly to CD80 / 86. For example, the antigen-binding fragment of the antibody may be a binding fragment of an antibody that binds to CD28, and may be an scFv of an antibody that binds to CD28. Specifically, the scFv of the antibody that binds to CD28 may include, but is not limited to, the amino acid sequence of SEQ ID NO: 11, the amino acid sequence encoded by the base sequence of SEQ ID NO: 12, or may be composed of the amino acid sequence.

[0045] In addition, the antigen-binding fragment of the antibody may bind to CD40 (cluster of differentiation 40). CD40 is a receptor expressed on dendritic cells (DC), B cells, macrophages, monocytes, etc., and mainly plays a role in providing co-stimulation by binding to the CD40 ligand (CD40L) of the helper T cell during the licensing process in which activated dendritic cells bind to activated helper T cells. Dendritic cells licensed by stimulation by TCR binding of helper T cells to major histocompatibility complex class II (MHCII) presenting antigen-specific peptides (signal 1), co-stimulation by CD40-CD40L binding (signal 2), and cytokines (signal 3) activate antigen-specific cytotoxic T cells, allowing the cytotoxic T cells to attack target infected cells or cancer cells. The antigen-binding fragment of the antibody may be an agonist that binds to CD40 and activates CD40 similarly to CD40L. For example, the antigen-binding fragment of the antibody may be a binding fragment of an antibody that binds to CD40, and may be an scFv of an antibody that binds to CD40. Specifically, the scFv of the antibody binding to CD40 may include the amino acid sequence of SEQ ID NO: 13, include the amino acid sequence encoded by the base sequence of SEQ ID NO: 14, or consist of the amino acid sequence, but is not limited thereto.

[0046] As another specific example, the ligand may be, but is not limited to, HLA (Human Leukocyte Antigen). HLA refers to a protein encoded by the human major histocompatibility complex (MHC) gene complex, and is an important immunological molecule that mainly exists in a membrane-bound form and is responsible for regulating the immune system. HLA presents peptide fragments of foreign or self-proteins from inside the cell to the cell surface and activates cytotoxic T cells by recognizing and binding to the T cell antigen receptor (TCR) of the cytotoxic T cell. HLA is divided into two types: type 1 HLA is present on the surface of all cells, while type 2 HLA is present only on specific antigen-presenting cells (APCs) such as NK cells, macrophages, and dendritic cells. The HLA gene has a highly polymorphic characteristic and expresses six different HLA protein α chains (two HLA-A, two HLA-B, and two HLA-C). Since the type 1 HLA can selectively activate cytotoxic T cells expressing TCR when a specific peptide fragment is loaded, a recombinant type 1 HLA with the membrane-bound domain portion removed can be used for the treatment of cancer, infectious diseases, etc. Specifically, the HLA-A may include the amino acid sequence of SEQ ID NO: 19, or may include the amino acid sequence encoded by the base sequence of SEQ ID NO: 20, or may be composed of the amino acid sequence, but is not limited thereto.

[0047] As used herein, the term "fusion protein" means a protein in which part or all of two or more different proteins are linked, and in particular, in the present specification, means a structure in which another protein molecule is linked to an antibody.

[0048] In one embodiment, the fusion protein may be represented by, for example, the following structural formula:

[0049] [(X) e -(Y) f -(IgG Fc region)-(IgM Fc region)]5-(W)

[0050] At this time, in the above structural formula,

[0051] The above X is a biologically active molecule,

[0052] The above Y is a linker,

[0053] The above IgG Fc region is a monomer,

[0054] The above IgM Fc region is a monomer,

[0055] The above W is a J chain complex according to the present invention,

[0056] The above e is 1 or 2, and the above f is 1 or 2.

[0057] In one embodiment, e may be 2 and f may be 2.

[0058] In another embodiment, e may be 1 and f may be 1.

[0059] The above fusion protein has 5 to 10 binding domains through the IgM Fc region to maximize the binding activity (avidity) of biologically active molecules, and solves the steric hindrance problem of existing IgM antibodies through a linker. In addition, the fusion protein maximizes FcRn recycling through the IgG Fc region. Mutations can be introduced into the IgM Fc region and / or J chain included in the above fusion protein, thereby suppressing or blocking the possibility of a reduction in blood half-life due to binding to an IgM-specific receptor, and suppressing or blocking the possibility of side effects due to non-specific binding.

[0060] As used herein, the term "IgG Fc region" refers to the Fc region (fragment crystallizable region) of immunoglobulin G (IgG), which is the remaining portion of IgG excluding the Fab region (fragment, antigen binding region). The IgG Fc region is indirectly linked to a biologically active molecule through a linker within a fusion protein, and here, the IgG Fc region can serve as a linker, supporter, carrier, etc. of the biologically active molecule so as to include one or two or more of the biologically active molecules. In the present specification, the 'IgG Fc region' may be used interchangeably with 'IgG Fc fragment' with the same meaning.

[0061] Specifically, the IgG Fc region monomer may be composed of two heavy chains. One heavy chain may include at least one selected from the group consisting of a CH1 domain, a CH2 domain, and a CH3 domain. For example, one heavy chain may include a CH2 domain and a CH3 domain, or may be composed of a CH2 domain and a CH3 domain.

[0062] In the present invention, the IgG Fc region may be derived from IgG1, IgG2, IgG3 or IgG4.

[0063] Additionally, the IgG Fc region may be derived from a wild-type IgG, and the wild-type IgG may include an amino acid sequence of SEQ ID NO: 21 or 22.

[0064] As used herein, the term "IgM Fc region" refers to the Fc region (fragment crystallizable region) of immunoglobulin M (IgM), and refers to the remaining portion of IgM excluding the Fab region (fragment, antigen binding region). The IgM Fc region is indirectly linked to a biologically active molecule within a fusion protein through an IgG Fc region and a linker, and wherein the IgM Fc region can serve as a linker, supporter, carrier, etc. of the biologically active molecule so as to include one or two or more of the biologically active molecules. In the present specification, the 'IgM Fc region' may be used interchangeably with 'IgM Fc fragment' with the same meaning.

[0065] Specifically, the IgM Fc region monomer may be composed of two heavy chains. One heavy chain may include at least one selected from the group consisting of a Cμ3 domain and a Cμ4 domain. For example, one heavy chain may include a Cμ3 domain and a Cμ4 domain, or may be composed of a Cμ3 domain and a Cμ4 domain.

[0066] Additionally, the IgM Fc region may be derived from wild-type IgM or mutant IgM.

[0067] Specifically, the wild-type IgM may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 23, or the amino acid sequence encoded by the base sequence of SEQ ID NO: 24, or may be composed of the amino acid sequence.

[0068] At this time, the mutant IgM may have one or more of the amino acids at positions 125 and 126 in the amino acid sequence of SEQ ID NO: 23 deleted or substituted. Specifically, the mutant IgM may have one or more mutations in the amino acid at position 125 in the amino acid sequence of SEQ ID NO: 23 selected from the group consisting of N125A, N125F, and N125K. In addition, the mutant IgM may have any one mutation selected from the group consisting of L126A, L126D, L126E, L126F, L126G, L126H, L126I, L126K, L126M, L126N, L126P, L126Q, L126R, L126S, L126T, L126V, L126W, and L126Y in the amino acid at position 126 in the amino acid sequence of SEQ ID NO: 23.

[0069]

[0070] One aspect of the present invention provides a nucleic acid molecule encoding the J chain complex or fusion protein.

[0071] In the 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.

[0072] As used herein, the term "nucleic acid molecule" has a comprehensive meaning including DNA and RNA molecules, and the nucleotides, which are the basic structural units of the nucleic acid molecule, include not only natural nucleotides but also analogues in which the sugar or base portion is modified. The sequence of a nucleic acid molecule encoding the J chain complex or fusion protein of the present invention may be modified, and the modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0073] In addition, all sequences used in the present invention, including nucleic acid sequences and amino acid sequences, are interpreted to include sequences that show substantial identity with the sequences listed in the sequence listing, considering mutations that have biologically equivalent activity. The term, 'substantial identity', means a sequence that shows 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 the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0074] Accordingly, it should be interpreted that sequences having high homology with the sequences represented by SEQ ID NOs: 1 to 24 of the present invention, for example, sequences having high homology of 70% or more, specifically 80% or more, and more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, are also included in the scope of the present invention.

[0075]

[0076] Another aspect of the present invention provides a vector for expressing a J chain complex or fusion protein, comprising the nucleic acid molecule.

[0077] In the vector for expressing the J chain complex or fusion protein according to the present invention, unless specifically stated otherwise, the related terms are understood to have the same meaning as the terms described above.

[0078] The term "vector" as used herein refers to a plasmid, virus, or other vector known in the art that can insert or introduce a nucleic acid molecule encoding the J chain complex or fusion protein into a host cell, as a means for expressing the J chain complex or fusion protein of the present invention. The vector can be constructed as a vector for cloning or as a vector for expression. As a specific example, it may be a viral vector such as a plasmid vector, a cosmid vector, a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector, and as a more specific example, it may be a plasmid (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.), a phage (e.g., λgt4·λB, λ-Charon, λΔz1, and M13, etc.) or a virus (e.g., SV40, etc.), but is not limited thereto.

[0079] The vector of the present invention may be one in which a nucleic acid molecule encoding the J chain complex or fusion protein is operably linked to a promoter. The term "operably linked" refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription regulatory element binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0080] When the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine 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, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used, and can include a polyadenylation sequence as a transcription termination sequence. For example, the recombinant vector of the present invention can include a CMV promoter, but is not limited thereto.

[0081] When the vector of the present invention is an expression vector and uses a prokaryotic cell as a host, it may include a strong promoter capable of initiating 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, 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 region of the E. coli tryptophan biosynthesis pathway, or the left-hand promoter of phage λ (pLλ promoter) may be used as a regulatory region; When Bacillus bacteria are used as host cells, the promoter of the toxin protein gene of Bacillus thuringiensis or any promoter that can be expressed in Bacillus bacteria can be used as a regulatory region.

[0082] The recombinant vector system of the present invention can be constructed through various methods known in the art, and can include an antibiotic resistance gene commonly used in the art as a selection marker (e.g., a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline).

[0083]

[0084] Another aspect of the present invention provides a host cell into which a vector for expressing the J chain complex or fusion protein is introduced.

[0085] 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.

[0086] As used herein, the term "host cell" refers to a cell that includes a vector for expressing the J chain complex or fusion protein, and can stably and continuously clone and express the J chain complex or fusion protein of the present invention. For example, a prokaryotic host cell such as a Bacillus strain such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, Streptomyces, Pseudomonas, Proteus mirabilis, or Staphylococcus; The host cell may be, but is not limited to, a fungus such as an Aspergillus species, a eukaryotic host cell such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces or Neurospora crassa; a lower eukaryotic cell; a higher eukaryotic cell such as an insect-derived cell; a plant cell; or a cell derived from a mammal such as a COS7 cell (monkey kidney cell), NSO cell, SP2 / 0, Chinese hamster ovary (CHO) cell, W138, baby hamster kidney (BHK) cell, MDCK, a myeloma cell line, HuT 78 cell or 293 cell, and any host cell commonly used in the art may be used without limitation.

[0087]

[0088] Another aspect of the present invention provides a method for producing the J chain complex or fusion protein, comprising the step of introducing a vector for expressing the J chain complex or fusion protein into a host cell.

[0089] In the method for producing a J chain complex or 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.

[0090] Specifically, the method for producing the J chain complex or fusion protein of the present invention comprises:

[0091] (a) a step of introducing the vector of the present invention into a host cell;

[0092] (b) a step of culturing the host cell; and

[0093] (c) may include a step of obtaining a J chain complex or fusion protein from the host cell.

[0094] The step (a) of introducing the vector of the present invention into a host cell may be a step of producing a transformant including the vector for expressing the J chain complex or fusion protein, and the step of producing the transformant may be transforming a host cell.

[0095] The above term, "transformant", refers to an organism in which genetic changes have been artificially introduced into cells by introducing external DNA into the host, thereby making the DNA replicable as a chromosomal element or by completing chromosomal integration.

[0096] Transformation of the aforementioned host cells can be performed using any transformation method, and can be easily performed according to a conventional method in the art. For example, the method may include, but is not limited to, CaCl2 precipitation method, Hanahan method with increased efficiency by using dimethyl sulfoxide (DMSO) in the CaCl2 method, electroporation, calcium phosphate precipitation method, protoplast fusion method, stirring method using silicon carbide fiber, Agrobacterium-mediated transformation method, PEG-based transformation method, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation method, and the like, and any transformation or transfection method conventionally used in the art can be used without limitation.

[0097] The above step (b) of culturing the host cell of the present invention can be performed according to media and culture conditions known in the art. Those skilled in the art can easily adjust and use this culture process according to the selected strain. Depending on the cell growth pattern, suspension culture or attachment culture may be used; depending on the culture method, batch, fed-batch, or continuous culture methods may be used.

[0098] In animal cell culture, the medium may include various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources 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 include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor, 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 elements include phosphorus sources such as potassium dihydrogen phosphate or dipotassium hydrogen phosphate; It may contain metal salts such as magnesium sulfate or iron sulfate. In addition, it may contain amino acids, vitamins, or suitable precursors.

[0099] In addition, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid or sulfuric acid can be added to the culture in an appropriate manner during cultivation to adjust the pH of the culture, an antifoaming agent such as fatty acid polyglycol ester can be used during cultivation to suppress bubble formation, and oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture to maintain an aerobic state of the culture.

[0100] Additionally, the culture can be cultured by maintaining the temperature of the culture at 20°C to 45°C, specifically 25°C to 40°C.

[0101] The step (c) of expressing the J chain complex or fusion protein in the host cell of the present invention may be a step of recovering, purifying, and concentrating the J chain complex or fusion protein of the present invention from the culture solution obtained through the step (b).

[0102] The above J chain complex or fusion protein may be used in an unpurified state, or may be further recovered, purified, and concentrated for use. Specifically, a method commonly used in the art (e.g., dialysis, salt precipitation, chromatography, etc.) may be used, through which recovery, purification, and concentration can be performed simultaneously. 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 therefor may be appropriately selected depending on the characteristics of the J chain complex or fusion protein, the culture method, etc.

[0103]

[0104] Another aspect of the present invention provides the use of the J chain complex or fusion protein for the manufacture of a diagnostic or therapeutic medicament.

[0105] Another aspect of the present invention provides a pharmaceutical composition comprising the J chain complex or fusion protein.

[0106] In the uses and pharmaceutical compositions according to the present invention, unless specifically stated otherwise, the related terms are understood to have the same meaning as the terms described above.

[0107] In one embodiment according to the present invention, the biologically active molecule included in the J chain complex or fusion protein may exhibit immune regulation activity or anticancer activity, but is not limited thereto.

[0108] In addition, the J chain complex or fusion protein includes a biologically active molecule that exhibits effects such as immunoregulation and anticancer effects, and thus can be used for the purpose of prevention, treatment, or diagnosis of diseases caused by a decrease or loss of immunoregulatory function, or diseases such as cancer.

[0109] The linker according to the present invention can be used to link various biologically active molecules to the J chain within an antibody-based structure, and can increase the activity of a J chain complex comprising the linker and the biologically active molecule. Furthermore, the effect of this linker can occur regardless of the type of biologically active molecule linked to the linker.

[0110] Figure 1 is an image showing the structure of a fusion protein comprising a J chain complex comprising a J chain, a linker connected to the J chain, and a biologically active molecule (scFv) connected to the linker; and an antibody fragment to which the J chain complex and the biologically active molecule (IgG Fab) are connected.

[0111] Figure 2 is an image showing the results of reducing SDS-PAGE analysis of fusion proteins according to the present invention. '1' represents A2014.eP-J-linker1-anti-CD3 scFv fusion protein; '2' represents A2014.eP-J-linker3-anti-CD3 scFv fusion protein; '3' represents A2014.eP-anti-CD3 scFv-linker1-J fusion protein; '4' represents A2014.eP-anti-CD3 scFv-linker3-J fusion protein; and '5' represents A2014.eP-J-linker2-anti-CD3 scFv fusion protein.

[0112] Figure 3 is a histogram showing the binding activity of the fusion protein according to the present invention and the control protein to CD3 expressing cells.

[0113] Figure 4 is a graph showing the activity of TCR / CD3 reporter cells by a fusion protein according to the present invention and a control protein.

[0114] Figure 5 is a graph showing the activity of CD40 reporter cells by a fusion protein according to the present invention and a control protein.

[0115] Figure 6 is a graph showing the activity of CD28 effector cells by the fusion protein according to the present invention and the control protein.

[0116] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are intended to exemplify the present invention, and the scope of the present invention is not limited by these examples.

[0117]

[0118] Example 1. Preparation of linker

[0119] A linker for use in the production of a J chain complex or fusion protein was prepared. Specifically, a linker was prepared that can be used to link various biologically active molecules to a J chain or to link various biologically active molecules to antibody fragments within a fusion protein.

[0120] First, a flexible linker having the form of (GGGGS)n, a semi-rigid linker having the form of {(GGGGS)(EAAAK)(GGGGS)}n, and a rigid linker having the form of (XP)n were prepared.

[0121] The flexible linker is named 'linker 1', its amino acid sequence is represented by SEQ ID NO: 3, and its base sequence is represented by SEQ ID NO: 4. The semi-rigid linker is named 'linker 2', its amino acid sequence is represented by SEQ ID NO: 5, and its base sequence is represented by SEQ ID NO: 6. The rigid linker is named 'linker 3', its amino acid sequence is represented by SEQ ID NO: 7, and its base sequence is represented by SEQ ID NO: 8.

[0122]

[0123] Example 2. Preparation of a J chain complex containing a linker

[0124] A J chain complex containing the linker prepared in Example 1 was prepared.

[0125] Specifically, J chain complexes were prepared by linking various biologically active molecules to the J chain via a linker. In this example, anti-CD3 scFv, anti-CD40 scFv, or anti-CD28 scFv were used as examples as biologically active molecules linked to the J chain. The prepared J chain complexes are shown in Table 1 below.

[0126] For example, among the J chain complexes described in Table 1 below, 'J-linker1-anti-CD3 scFv' refers to a J chain complex in which anti-CD3 scFv is linked to the C-terminus of the J chain via linker 1. In addition, 'anti-CD3 scFv-linker1-J' refers to a J chain complex in which anti-CD3 scFv is linked to the N-terminus of the J chain via linker 1. In addition, 'anti-CD40 scFv-linker3-J' refers to a J chain complex in which anti-CD40 scFv is linked to the N-terminus of the J chain via linker 3.

[0127] J chain complex Linker Biologically active molecule Position J-linker 1-anti-CD3 scFv linker 1 C-terminus of J chain J-linker 2-anti-CD3 scFv linker 2 C-terminus of J chain J-linker 3-anti-CD3 scFv linker 3 C-terminus of J chain Anti-CD3 scFv linker 1-J linker 1 N-terminus of J chain Anti-CD3 scFv linker 3-J linker 3 N-terminus of J chain Anti-CD40 scFv linker 3-J linker 3 N-terminus of J chain Anti-CD28 scFv linker 3-J linker 3 N-terminus of J chain

[0128]

[0129] Example 3. Preparation of a fusion protein containing a J chain complex

[0130] A fusion protein containing the J chain complex prepared in Example 2 above was prepared.

[0131] Specifically, the fusion protein comprises an antibody fragment and a J chain complex prepared in Example 2 (i.e., a linker, a J chain, and a biologically active molecule linked to the J chain via the linker).

[0132] The antibody fragment includes an IgG Fc region comprising an IgG hinge, an IgG CH2 domain, and an IgG CH3 domain; and an IgM Fc region comprising an IgM cμ3 domain and an IgM cμ4 domain, to which a biologically active molecule is linked. In this embodiment, anti-HLA-G IgG Fab or HLA-A was used as an example as the biologically active molecule linked to the antibody fragment. For example, 'A2014.eP' is a fusion protein comprising an IgG Fc region and an IgM Fc region, to which anti-HLA-G IgG Fab is linked as the biologically active molecule. In addition, 'STAM.eP' is a fusion protein comprising an IgG Fc region and an IgM Fc region, to which HLA-A is linked as the biologically active molecule.

[0133] As described above, fusion proteins were prepared with different types of linkers, different positions of biologically active molecules linked to the J chain, different types of biologically active molecules linked to the J chain, and different types of biologically active molecules linked to the antibody fragment, and these are shown in Table 1 below.

[0134] For example, among the fusion proteins described in Table 2 below, 'A2014.eP-J-linker1-anti-CD3 scFv' refers to a fusion protein in which the J chain complex of 'J-linker1-anti-CD3 scFv' is linked to the antibody fragment of 'A2014.eP'. In addition, 'A2014.eP-anti-CD3 scFv-linker1-J' refers to a fusion protein in which the J chain complex of 'anti-CD3 scFv-linker1-J' is linked to the antibody fragment of 'A2014.eP'. In addition, 'STAM.eP-anti-CD40 scFv-linker3-J' refers to a fusion protein in which the J chain complex of 'anti-CD40 scFv-linker3-J' is linked to the antibody fragment of 'STAM.eP'.

[0135] Fusion protein linker biologically active molecule location A2014.eP-J-linker1-anti-CD3 scFv linker1 C-terminus of chain J A2014.eP-J-linker2-anti-CD3 scFv linker2 C-terminus of chain J A2014.eP-J-linker3-anti-CD3 scFv linker3 C-terminus of chain J A2014.eP-anti-CD3 scFv linker1-J-linker1 N-terminus of chain A2014.eP-anti-CD3 scFv linker3-J-linker3 N-terminus of chain J STAM.eP-anti-CD40 scFv linker3-J-linker3 N-terminus of chain J STAM.eP-anti-CD28 scFv linker3-J-linker3

[0136]

[0137] Example 3-1. Construction of recombinant plasmids

[0138] To produce the fusion protein, a recombinant plasmid was first constructed.

[0139] Specifically, for gene cloning, each of the above materials was codon optimized, and then the codon-optimized nucleotide molecules were synthesized by Cosmogene Tech (Seoul, Korea).

[0140] Using the optimized nucleotides and primers above, insert DNA was prepared by PCR so that overlap PCR could be performed on the MCS (multiple cloning site) of the pcDNA 3.1 (manufacturer: Thermo Scientific) vector. After overlap PCR of each obtained insert DNA and pcDNA 3.1 vector, the template was removed by treatment with DpnI restriction enzyme, and each vector with each substance inserted was constructed.

[0141] The above vector is Stella ® After inserting into competent cells (manufacturer: Clontech), colonies were selected by spreading on agar plates containing ampicillin antibiotics matching the antibiotic resistance gene in the vector, and the base sequence was confirmed through DNA sequencing using Applied biosystems 3730xl DNA analyzer (manufacturer: Thermo Scientific).

[0142]

[0143] Example 3-2. Establishment of an expression system

[0144] A transient expression system was established to quickly secure the fusion protein of the present invention by introducing the recombinant plasmid vector obtained through gene cloning in Example 3-1 into Expi293 (Thermo Scientific) animal cells.

[0145] Specifically, one day before transformation, 3.0 x 10 6 After dividing the cells at a concentration of 10 cells / ml, they were cultured for 24 hours. On the day of transfection, Expi293 ® Add expression medium to 3.0 x 10 6 cells / ml concentration. Afterwards, 1 μg / ml of each recombinant plasmid vector was added to Opti-MEM ®Mix the badge and ExpiFectamine ® Reagents and Opti-MEM ® After mixing the medium to the appropriate volume, it was left at room temperature for 5 minutes. Then, DNA and ExpiFectamine were added for 15 minutes. ® The reagents were reacted and added to the flask, enhancer1 and 2 were treated after 16 to 22 hours, and when the viability was 75% or higher on the 4th to 5th day, they were checked and harvested.

[0146]

[0147] Example 3-3. Purification and concentration of fusion protein

[0148] The expressed fusion protein was purified using IgM affinity chromatography.

[0149] Specifically, the culture solution obtained from the expression system of Example 3-2 was filtered through a PES filtration membrane with a pore size of 0.2 μm to remove impurities. The recovered filtrate was subjected to an IgM affinity matrix POROS™ CaptureSelect™ IgM Affinity Matrix. ® The fusion protein was purified by loading it onto (Thermo Scientific). IgM affinity chromatography was performed under the following conditions.

[0150] <Chromatography conditions>

[0151] - Resin: POROS™ CaptureSelect™ IgM Affinity Matrix ®

[0152] - Flow rate: 480 cm / h

[0153] - Equilibrium: PBS (Phosphate-buffered saline) buffer

[0154] - Loading: Up to 6 g protein / L resin volume

[0155] - Regeneration and sterilization: 0.01 M NaOH solution

[0156] - Dissolution: 0.1 M glycine-HCl, pH 2.7 buffer

[0157] - Storage: 20% EtOH

[0158] After performing IgM affinity chromatography as described above, 1 / 10 of the volume of the eluate was neutralized with 0.1 M Tris-HCl, pH 8.5 buffer. The recovered fusion protein was then concentrated using ultrafiltration, dialyzed against TBS pH 7.4, and stored at -20°C.

[0159]

[0160] Example 3-4. Confirmation of the production of fusion proteins

[0161] Using reducing SDS-PAGE, it was confirmed whether the fusion proteins manufactured through Examples 3-1 to 3-3 were properly manufactured to have each structure.

[0162] Specifically, 2 μg of each fusion protein was mixed with 5x reducing-PAGE sample buffer, heated at 100°C for 5 minutes, and then left at room temperature for 20 minutes. After inserting a polyamide gel (3–8%, Invitrogen) into the gel kit and loading the sample, electrophoresis was performed at 125 V for 2 hours, and the gel was separated from the kit, staining solution (Coomassie Brilliant Blue staining solution) was added, and left for 1 hour. The left gel was transferred to a destaining buffer (70% triple-distilled water, 20% methanol, 10% acetic acid), incubated overnight, and the results were confirmed.

[0163] As a result, as shown in Fig. 2, all manufactured fusion proteins had sizes corresponding to each substance. This indicates that each fusion protein was well manufactured to have the desired structure.

[0164]

[0165] Example 4. Activity analysis of fusion proteins

[0166] In order to confirm the effect of the linker manufactured in Example 1, the activity of the fusion protein manufactured in Example 3 was analyzed.

[0167] Specifically, when the biologically active molecule linked to the J chain via a linker is an anti-CD3 scFv, the activity of the fusion protein containing it was analyzed in terms of binding activity to CD3-expressing cells and activity of TCR / CD3 reporter cells by the fusion protein.

[0168] In addition, when the biologically active molecule linked to the J chain via a linker is an anti-CD40 scFv, the activity of the fusion protein containing it was analyzed as the activity of CD40 reporter cells by the fusion protein.

[0169] In addition, when the biologically active molecule linked to the J chain via a linker is an anti-CD28 scFv, the activity of the fusion protein containing it was analyzed as the activity of CD28 reporter cells by the fusion protein.

[0170]

[0171] Example 4-1. Binding activity to CD3 expressing cells

[0172] The binding activity of the fusion protein manufactured in Example 3 above to CD3-expressing cells was analyzed through FACS.

[0173] At this time, 'anti-CD3 IgG', 'A2014.eP', and 'A2014.eP-anti-CD40 scFv-linker3-J' were used as control proteins. The anti-CD3 IgG is an antibody that binds to CD3 and serves as a positive control. The A2014.eP is a fusion protein that does not contain a linker and a biologically active molecule linked to the J chain via the linker and serves as a negative control. The A2014.eP-anti-CD40 scFv-linker3-J is a negative control in that the biologically active molecule linked to the J chain is an antibody fragment (anti-CD40 scFv) that binds to CD40, not an antibody fragment (anti-CD3 scFv) that binds to CD3.

[0174] Specifically, 3.5x10 Jurkat cells (ATCC) naturally expressing CD3 5 Serial dilutions of each fusion protein or control protein prepared in Example 3 were added to 100 μl of cells and incubated at 4°C for 1 hour. Afterwards, the cells were washed with FACS buffer (5% FBS in PBS), treated with anti-IgM-PE (ebioscience) or anti-IgG Fc-FITC (Biolegend), and incubated at 4°C for 30 minutes. Finally, after washing with FACS buffer, the binding activity of each fusion protein or control protein to Jurkat cells was confirmed using a flow cytometer (BD bioscience, FACSLyric™).

[0175] As a result, as shown in Fig. 3, the negative control groups A2014.eP and A2014.eP-anti-CD40 scFv-linker3-J did not show binding activity.

[0176] The binding activity of the fusion proteins differed depending on the type of linker used to connect the J chain and the biologically active molecule (anti-CD3 scFv) within the J chain complex. The fusion protein containing the rigid linker, linker 3, exhibited higher binding activity toward CD3-expressing cells than the fusion protein containing the flexible linker, linker 1, and this was concentration-dependent.

[0177] In addition, the binding activity of the fusion proteins differed depending on which part of the J chain the biologically active molecule (anti-CD3 scFv) was linked to within the J chain complex. Specifically, the fusion protein in which the biologically active molecule (anti-CD3 scFv) was linked to the N-terminus of the J chain via a linker within the J chain complex exhibited higher binding activity toward CD3-expressing cells than the fusion protein in which the biologically active molecule (anti-CD3 scFv) was linked to the C-terminus of the J chain via a linker.

[0178]

[0179] Example 4-2. TCR / CD3 reporter cell activation

[0180] The activity of TCR / CD3 reporter cells by the fusion protein manufactured in Example 3 was analyzed using a luciferase assay. At this time, 'anti-CD3 IgG' and 'A2014.eP-anti-CD40 scFv-linker3-J' were used as control proteins.

[0181] Specifically, TCR / CD3 reporter cells (Promega) were cultured in RPMI-1640 (10% FBS). 1x10 5After dispensing cells / well, serial dilutions of each fusion protein or control protein prepared in Example 3 were added to each well, and the plates were incubated at 37°C for 6 hours using a 5% CO2 incubator. Thereafter, luminescence was measured using the BIO_GLO luciferase assay system (Promega) according to the manufacturer's protocol.

[0182] As a result, as shown in Fig. 4, the activity of TCR / CD3 reporter cells (i.e., CD3 signal) was high with the positive control, anti-CD3 IgG, whereas no cell activity was observed with the negative control, A2014.eP-anti-CD40 scFv-linker3-J.

[0183] Differences in the activity of the fusion protein were observed depending on the type of linker used to connect the J chain and the biologically active molecule (anti-CD3 scFv) within the J chain complex.

[0184] Specifically, in the case of fusion proteins in which a biologically active molecule (anti-CD3 scFv) is linked to the C-terminus of the J chain within the J chain complex, cellular activity was hardly detected by the fusion protein containing the flexible linker, Linker 1. On the other hand, cellular activity was detected by the fusion proteins containing the semi-rigid linker, Linker 2, and the rigid linker, Linker 3, and in particular, cellular activity was increased the most by the fusion protein containing Linker 3.

[0185] In addition, the cellular activity of the fusion proteins differed depending on which part of the J chain the biologically active molecule (anti-CD3 scFv) was linked to within the J chain complex. Specifically, the cellular activity was further increased by the fusion protein in which the biologically active molecule (anti-CD3 scFv) was linked to the N-terminus of the J chain via a linker than by the fusion protein in which the biologically active molecule (anti-CD3 scFv) was linked to the C-terminus of the J chain within the J chain complex.

[0186] It was observed that the fusion protein containing the linker 3 and the biologically active molecule (anti-CD3 scFv) linked to the N-terminus of the J chain within the J chain complex increased cell activity. It is interpreted that when the biologically active molecule is linked to the N-terminus of the J chain within the J chain complex via the rigid linker, the activity of the fusion protein can be maximized.

[0187]

[0188] Example 4-3. CD40 reporter cell activity

[0189] The activity of CD40 reporter cells by the fusion protein manufactured in Example 3 was analyzed using a luciferase assay. At this time, 'STAM.eP' was used as a control protein. STAM.eP is a fusion protein that does not contain a linker and a biologically active molecule (anti-CD40 scFv) that binds to the J chain through the linker, and serves as a negative control.

[0190] Specifically, CD40 reporter cells (Promega) were cultured in MCCOY's 5A medium (10% FBS). One day before the assay, CD40 reporter cells were added at a density of 1x10 4 After dispensing cells / 100 ㎕ / well into a 96-well plate, they were cultured in a CO2 incubator at 37°C. Each fusion protein or control protein prepared in Example 3 was serially diluted 1 / 10 from 200 nM and treated at 8 concentrations, and after reacting for 6 hours at 37°C, luminescence was measured using Promega's BIO_GLO luciferase assay system.

[0191] As a result, as shown in Fig. 5, the activity of CD40 reporter cells was increased in a concentration-dependent manner by a fusion protein in which a biologically active molecule (anti-CD40 scFv) was linked to the N-terminus of the J chain via a rigid linker within the J chain complex. This effect by the linker was observed regardless of the type of biologically active molecule linked to the linker.

[0192]

[0193] Example 4-4. CD28 reporter cell activity

[0194] The activity of CD28 reporter cells by the fusion protein manufactured in Example 3 was analyzed using a luciferase assay. At this time, 'STAM.eP' was used as the control protein.

[0195] Specifically, TCR / CD3 (IL-2) reporter cells (CD28 effector cells) were cultured in RPMI 1640 medium (10% FBS). First, 20 μl / well of anti-CD3 was dispensed to a concentration of 500 pM. Subsequently, each fusion protein or control protein prepared in Example 3 was serially diluted and treated. Afterwards, 1x10 CD28 effector cells were cultured. 5 After dispensing cells / 40 ㎕ / well into a 96-well plate, the cells were reacted in a CO2 incubator at 37℃ for 5 hours. Finally, luminescence was measured using Promega's BIO_GLO luciferase assay system.

[0196] As a result, as shown in Fig. 6, the activity of CD28 effector cells was increased in a concentration-dependent manner by a fusion protein in which a biologically active molecule (anti-CD28 scFv) was linked to the N-terminus of the J chain via a rigid linker within the J chain complex. This linker-induced effect was observed regardless of the type of biologically active molecule linked to the linker.

[0197]

[0198] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are exemplary in all respects and are not limiting. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. In a linker connected to at least one selected from the group consisting of the N-terminus and C-terminus of the J chain, The above linker is a rigid linker or a semi-rigid linker.

2. In paragraph 1, The linker wherein the J chain is wild type or contains a mutation.

3. In paragraph 1, The semi-rigid linker is {(GGGGS)(EAAAK)(GGGGS)}n, wherein n is an integer from 1 to 10.

4. In paragraph 1, The above rigid linker is (XP)n, wherein n is an integer from 1 to 10 and X is an amino acid.

5. In paragraph 4, A linker wherein X is alanine (Ala; A), glutamine (Glu; E), or lysine (Lys, K).

6. A J chain complex comprising the linker and J chain of the first clause.

7. In paragraph 6, The J chain complex is a J chain complex further comprising a biologically active molecule.

8. In paragraph 6, The above J chain complex is represented by the following structural formula: (X) a -(AND) b -(Z)-(Y) c -(X) d At this time, in the above structural formula, The above X is a biologically active molecule, The above Y is a linker, The above Z is a J chain, The above a, b, c and d are 0 or 1, and at least one of the above b and c is 1.

9. In paragraph 7, The J chain complex, wherein the biologically active molecule is at least one selected from the group consisting of an antibody, an antigen-binding fragment of an antibody, an antibody-drug conjugate, an antibody-like molecule, an antigen-binding fragment of an antibody-like molecule, a compound, a soluble protein, a membrane-bound protein, a ligand, a receptor, a virus-like particle, a protein toxin, an enzyme, a co-stimulatory receptor, a cytokine, and a cell engager.

10. A nucleic acid molecule encoding the J chain complex of paragraph 6.

11. A vector for expressing a J chain complex, comprising the nucleic acid molecule of item 10.

12. A host cell into which a vector for expressing the J chain complex of item 11 has been introduced.

13. A method for producing the J chain complex of claim 6, comprising the step of introducing the vector for expressing the J chain complex of claim 11 into a host cell.

Images