KR20130110577A - Optimization method of tnfr2 - Google Patents

Abstract

The present invention removes the unnecessary portion from the water-soluble receptor TNFR2-Fc, a drug for treating autoimmune diseases, and prepares an optimized TNFR2-Fc, while having the same therapeutic effect as that of TNFR2-Fc, and with other target drugs. It relates to an optimized TNFR2-Fc that is easy to fuse and can function as a dual antagonist. Specifically, the present invention confirms that the O-glycosylation site and the N-glycosylation site are unnecessary in TNFR2-Fc, and removes the O-glycosylation site, the N-glycosylation site, or the O-glycosylation site and the N-glycosylation site. By making the A, the size of the full-length TNFR2 protein can be minimized to minimize the side effect (drug effect), and has a high affinity with TNF-α, which is the main etiology of autoimmune arthritis, Provided are optimized TNFR-Fc that have the effect of stability and ease of making fusions with other drugs.

Description

Optimization method of ＴＮＦＲ２ {Optimization Method Of TNFR2}

The present invention relates to an improved TNFR2 protein by minimizing the size of TNFR2 to improve stability in the body and to facilitate fusion with other drugs.

The immune system protects the body from harmful foreign antigens. The types of antigens include bacteria, viruses, toxins, cancer cells and blood and tissues to other humans or animals. The immune system produces antibodies to destroy these harmful substances. However, when autoimmune disorders occur, the immune system cannot distinguish between organs of its healthy body and harmful antigens and destroys normal tissues. This reaction is called autoimmune disease. These reactions are allergic to hypersensitivity reactions. In the case of allergies, the reaction is to external substances that are not harmful to the body. However, autoimmune abnormalities cause reactions to normal tissues of the body.

One type of autoimmune disease, autoimmune arthritis (RA), is a disease of unknown origin, despite a high prevalence of 21 million people, or about 1% of the world's population. Autoimmune arthritis is a systemic chronic inflammatory finding in which the pathological symptoms are mainly symmetric in the diarthrodial joint and, in severe cases, are serious diseases leading to joint failure. In addition to inflammation and pain in the joints due to abnormalities of autoimmunity, it also penetrates the joints, the tissues around them, and also many organs, and leads to osteoporosis, lungs, skin, and eyes. It causes poor quality and makes life impossible for normal people.

In the past, treatment of autoimmune arthritis has focused on slowing or reducing the incidence, reducing the pain, suppressing and treating infections, and improving joint function, such as lifestyle improvement, surgery, and treatment. In recent years, he has been focusing on the functional recovery and cure of joints. This is made possible by the development of anti-TNF antagonists, a breakthrough in the treatment of autoimmune arthritis.

Tumor necrosis factor (TNF) is a pleiotropic cytokine, which plays an important role in the immune system as well as an inflammatory response, and in patients with joint and Crohn's disease of autoimmune arthritis. It is found in the colon and has been reported to play an important role in the process of osteoclast.

Thus, therapeutic agents are designed to bind to ligands belonging to the TNF superfamily to inhibit signal transduction or to interfere with the binding between TNF ligands and receptors in order to inhibit the action of TNF acting as a etiology.

The development has been done by using a single antibody against TNF ligand and using a synthetic protein to suppress TNF signal transduction, using a single antibody such as Remicade and Humira. Therapeutics and therapeutic agents using synthetic proteins such as CTLA-4 Ig and Enbrel. Among these, Enbrel is a fusion protein composed of the extracellular domain of p75 TNF receptor and the hinge and Fc domain of human IgG1. In addition, it is the first biological treatment certified as an autoimmune arthritis therapeutic agent, and has been used as a therapeutic agent with high efficacy in clinical practice for the past 10 years.

The development of therapeutic agents targeting other cytokines other than TNF is under development.Disease-modifying antirheumatic drugs (DMARDs) that inhibit interleukin are under development for targeting IL-6 or IL-1. have.

TWEF (TNF-related weak inducer of apoptosis), a derivative of apoptosis, is a type of ligand belonging to the TNF superfamily, which regulates various cellular responses such as anti-inflammatory response, angiogenesis and cell division. It is a cytokine. TWEAK has been shown to reduce joint inflammation, synovial angiogenesis, and joint and bone erosion when TWEAK antibodies are treated with collagen-induced arthritis (CIA) animal models prior to onset and inhibited signaling. (J Immunol. 2006 Aug 15; 177 (4): 2610-20.).

Interleukin (IL) is a type of cytokine that acts as a chemical signal between red blood cells, among which IL-2, when approved by the FDA for the treatment of liver cancer in late 1992, is used to treat cancer. It was the first single immunotherapy used and was later used for metastatic melanoma. IL-2 helps the immune system to grow and divide cells faster. IL-2 has been used to treat cancer and has been used in combination with vaccines. Treatment with IL-2 has side effects such as colds, chills, fever, fatigue and confusion. In addition, IL-15 and IL-21, which belong to the IL-2 family, are being actively studied as a single agent or adjuvant as a cancer treatment agent.

IL-21 is a type of cytokine with alpha-helix structure, which causes an inflammatory response through signaling through the receptor and gamma chain of IL-21. IL-21 has been reported to induce maturation of NK cell precursors from bone marrow (Parrish-Novak J., et al., Nature, 408: 57-63, 2000), in particular cytokine production and apoptosis of NK cells Has been reported to enhance effector functions such as (Strengell M, et al., J Immunol., 170: 5464-5469, 2003; Brady J, et al., J Immunol., 172: 2048-2058). , 2004), it has also been reported to promote anticancer responses of intrinsic and adaptive immune systems by increasing the effect function of CD8 + T cells (Takaki R., et al., J Immunol., 175: 2167-2173, 2005; Moroz A. , et al., J Immunol, 173: 900-909, 2004). It also activates NK cells isolated from human peripheral blood (Parrish-Novak J., et al., Nature, 408: 57-63, 2000) and induces mature NK cells from hematopoietic stem cells isolated from umbilical cord blood. An important role has been reported (Sonia A. P, et al., Int. Immunol., 18: 49-58, 2006).

The effects of autoimmune arthritis drugs do not appear in all patients, but in about two-thirds of patients treated, one-third do not work, depending on the patient's history or heredity. This means that there is a limit to treatment depending on the specific factors. In addition to the pain caused by the disease, the side effects and safety issues that may occur when the treatment is given are important issues that need to be addressed.In the case of pregnant women with autoimmune arthritis, There is a need for follow-up studies to improve the stability and treatment effect in the body, such as safety issues are discussed.

In order to solve the above problems, the present inventors remove the unnecessary portion of the water-soluble receptor TNFR2-Fc well known as a drug for treating autoimmune diseases, and by producing an optimized TNFR2-Fc, the same therapeutic effect as TNFR2-Fc The present invention was completed by preparing an optimized TNFR2-Fc having the advantages of enhancing the stability in the body and facilitating fusion with other target drugs and functioning as a dual antagonist.

Accordingly, an object of the present invention is to provide a method for improving the efficiency of a Tumor necrosis factor receptor type 2 (TNFR2) protein comprising the step of removing the O-glycosylation site, the N-glycosylation site, or the O-glycosylation site and the N-glycosylation site. To provide.

Another object of the present invention is to provide a fragment of TNFR2 represented by SEQ ID NO: 2 by removing the O-glycosylation site from the amino acid sequence of Tumor necrosis factor receptor type 2 (TNFR2) described by SEQ ID NO: 1.

It is another object of the present invention to provide a fragment of TNFR2 represented by SEQ ID NO: 3, wherein the N-glycosylation site is removed from the amino acid sequence of Tumor necrosis factor receptor type 2 (TNFR2) described by SEQ ID NO: 1.

Still another object of the present invention is a fragment of TNFR2 represented by SEQ ID NO: 4, in which an O-glycosylation site and an N-glycosylation site are removed from an amino acid sequence of Tumor necrosis factor receptor type 2 (TNFR2) described in SEQ ID NO: 1 To provide.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases comprising the fragment as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object of the present invention, the present invention is a method for improving the efficiency of the Tumor necrosis factor receptor type 2 (TNFR2) protein, the method is O-glycosylation site, N-glycosylation site, or O-glycosylation It provides a method comprising the step of removing the site and the N-glycosylation site.

In one embodiment of the present invention, the O-glycosylation site is characterized in that the amino acid site 202 to 252 from the N- terminal of TNFR2.

In another embodiment of the present invention, the N-glycosylation site is characterized in that the 28, 31 or 34 positions from the N-terminus of TNFR2.

In another embodiment of the present invention, the N-glycosylation site is characterized in that to replace the proline No. 34 Proline (Asparagine).

The present invention also provides a fragment of TNFR2 represented by SEQ ID NO: 2, wherein the O-glycosylation site is removed from the amino acid sequence of Tumor necrosis factor receptor type 2 (TNFR2) described by SEQ ID NO: 1.

In one embodiment of the present invention, the O-glycosylation site is characterized in that the amino acid site 202 to 252 from the N- terminal of TNFR2.

The present invention also provides a fragment of TNFR2 represented by SEQ ID NO: 3, wherein the N-glycosylation site is removed from the amino acid sequence of Tumor necrosis factor receptor type 2 (TNFR2) described by SEQ ID NO: 1.

In one embodiment of the present invention, the N-glycosylation site is characterized in that the proline No. 34 (Proline) is replaced with asparagine (Asparagine).

The present invention also provides a fragment of TNFR2 represented by SEQ ID NO: 4, in which all O-glycosylation sites and N-glycosylation sites are removed from the amino acid sequence of Tumor necrosis factor receptor type 2 (TNFR2) described in SEQ ID NO: 1.

In one embodiment of the present invention, the O-glycosylation site is characterized in that the amino acid site 202 to 252 from the N- terminal of TNFR2.

In another embodiment of the present invention, the N-glycosylation site is characterized in that the proline No. 34 (Proline) is replaced with asparagine (Asparagine).

In addition, the present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases, including any one of the fragments as an active ingredient.

In one embodiment of the present invention, the autoimmune disease is characterized in that the disease is selected from the group consisting of pernicious anemia, type 1 diabetes, autoimmune arthritis, lupus, multiple sclerosis, reactive arthritis, and dermatitis.

The present invention confirms that the O-glycosylation site and the N-glycosylation site are unnecessary in TNFR2-Fc and removes them, thereby minimizing side effects on drugs because they are smaller than the full-length TNFR2 protein. And has a high affinity with TNF-α, which is a major etiology of autoimmune arthritis, and has an excellent effect of providing high stability in the body and optimized TNFR-Fc that is easy to prepare fusions with other drugs. In addition, in terms of protein production, the production rate is increased by two to three times compared to the conventional TNFR2-Fc due to the removal of the o-glycosylation site, thereby improving economics and increasing productivity due to cost reduction.

Figure 1 shows a cleavage map of the TNFR2-Fc mammalian expression vector (pYK602) produced in one embodiment of the present invention.
Figure 2 shows the DNA base sequence (SEQ ID NO: 15) of TNFR2.
Figure 3 shows the amino acid sequence of the TNFR2 protein (SEQ ID NO: 1).
4 shows Western blot results (left) and purification results (right) of TNFR2 protein.
Figure 5 shows a cleavage map of the Opti-TNFR2-Fc mammalian expression vector (pYK602) prepared in one embodiment of the present invention.
Figure 6 shows the DNA base sequence (SEQ ID NO: 16) of Opti-TNFR2-Fc.
Figure 7 shows the amino acid sequence of the Opti-TNFR2-Fc protein (SEQ ID NO: 2).
8 shows Western blot results (left) and purification results (right) of Opti-TNFR2-Fc protein.
Figure 9 shows a cleavage map of the TNFR2-M2 mammalian expression vector (pYK602) produced in one embodiment of the present invention.
Figure 10 shows the DNA base sequence (SEQ ID NO: 17) of TNFR2-M2.
11 shows the amino acid sequence of the TNFR2-M2 protein (SEQ ID NO: 3).
12 shows Western blot results (left) and purification results (right) of TNFR2-M2 protein.
Figure 13 shows a cleavage map of the Opti-TNFR2-M2 mammalian expression vector (pYK602) prepared in one embodiment of the present invention.
Figure 14 shows the DNA base sequence (SEQ ID NO: 18) of Opti-TNFR2-M2.
15 shows the amino acid sequence of the Opti-TNFR2-M2 protein (SEQ ID NO: 4).
Figure 16 shows Western blot results (left) and purification results (right) of Opti-TNFR2-M2 protein.
Figure 17 shows a cleavage map of the Opti-TNFR2 / TWEAKR mammalian expression vector (pYK602) produced in an embodiment of the present invention.
Figure 18 shows the DNA base sequence (SEQ ID NO: 19) of Opti-TNFR2 / TWEAKR.
Figure 19 shows the amino acid sequence of the Opti-TNFR2 / TWEAKR protein (SEQ ID NO: 5).
Figure 20 shows Western blot results (left) and purification results (right) of Opti-TNFR2 / TWEAKR protein.
Figure 21 shows a cleavage map of the IL21R / Opti-TNFR2 mammalian expression vector (pYK602) prepared in one embodiment of the present invention.
Figure 22 shows the DNA base sequence (SEQ ID NO: 20) of IL21R / Opti-TNFR2.
Figure 23 shows the amino acid sequence of the IL21R / Opti-TNFR2 protein (SEQ ID NO: 6).
Figure 24 shows Western blot results (left) and purification results (right) of IL21R / Opti-TNFR2 protein.
25 shows a cleavage map of the anti-IL6R A7 HC / Opti-TNFR2 mammalian expression vector (pNATABH) prepared in one embodiment of the present invention.
Figure 26 shows the DNA base sequence (SEQ ID NO: 21) of anti-IL6R A7 HC / Opti-TNFR2.
FIG. 27 shows amino acid sequence of anti-IL6R A7 HC / Opti-TNFR2 protein (SEQ ID NO: 7). FIG.
Figure 28 shows a cleavage map of the anti-IL6R A7 LC mammalian expression vector (pNATABL) prepared in one embodiment of the present invention.
Figure 29 shows the DNA base sequence (SEQ ID NO: 22) of anti-IL6R A7 LC.
Figure 30 shows the amino acid sequence of the anti-IL6R A7 LC protein (SEQ ID NO: 8).
Figure 31 shows Western blot results (left) and purification results (right) of anti-IL6R A7 / Opti-TNFR2 protein.
32 shows a cleavage map of the anti-IL6R B10 HC / Opti-TNFR2 mammalian expression vector (pNATABH) prepared in one embodiment of the present invention.
33 shows the DNA base sequence (SEQ ID NO: 23) of anti-IL6R B10 HC / Opti-TNFR2.
34 shows the amino acid sequence of the anti-IL6R B10 HC / Opti-TNFR2 protein (SEQ ID NO: 9).
Figure 35 shows a cleavage map of the anti-IL6R B10 LC mammalian expression vector (pNATABL) prepared in one embodiment of the present invention.
36 shows the DNA sequence of the anti-IL6R B10 LC (SEQ ID NO: 24).
37 shows the amino acid sequence of the anti-IL6R B10 LC protein (SEQ ID NO: 10).
38 shows Western blot results (left) and purification results (right) of anti-IL6R B10 / Opti-TNFR2 protein.
Figure 39 shows a cleavage map of the anti-IL6R F2 HC / Opti-TNFR2 mammalian expression vector (pNATABH) prepared in one embodiment of the present invention.
40 shows the DNA base sequence (SEQ ID NO: 25) of anti-IL6R F2 HC / Opti-TNFR2.
Figure 41 shows the amino acid sequence of the anti-IL6R F2 HC / Opti-TNFR2 protein (SEQ ID NO: 11).
Figure 42 shows a cleavage map of the anti-IL6R F2 LC mammalian expression vector (pNATABL) prepared in one embodiment of the present invention.
Figure 43 shows the DNA sequence of the anti-IL6R F2 LC (SEQ ID NO: 26).
Figure 44 shows the amino acid sequence of the anti-IL6R F2 LC protein (SEQ ID NO: 12).
45 shows Western blot results (left) and purification results (right) of anti-IL6R F2 / Opti-TNFR2 protein.
46 shows a cleavage map of the anti-IL6R D2 HC / Opti-TNFR2 mammalian expression vector (pNATABH) prepared in one embodiment of the present invention.
Figure 47 shows the DNA base sequence (SEQ ID NO: 27) of anti-IL6R D2 HC / Opti-TNFR2.
Figure 48 shows the amino acid sequence of the anti-IL6R D2 HC / Opti-TNFR2 protein (SEQ ID NO: 13).
Figure 49 shows a cleavage map of the anti-IL6R D2 LC mammalian expression vector (pNATABL) prepared in one embodiment of the present invention.
Figure 50 shows the DNA sequence of the anti-IL6R D2 LC (SEQ ID NO: 28).
51 shows the amino acid sequence of the anti-IL6R D2 LC protein (SEQ ID NO: 14).
Figure 52 shows Western blot results (left) and purification results (right) of anti-IL6R D2 / Opti-TNFR2 protein.
Figure 53 shows the binding force of TNFR2-Fc, Opti-TNFR2-Fc, TNFR2-M2-Fc, Opti-TNFR2-M2-Fc to TNF-α.
54 shows the binding force of Opti-TNFR2 / TWEAKR, IL21R / Opti-TNFR2 to TNF-α.
55 shows avidity of anti-IL6R A7 / Opti-TNFR2 antibody, anti-IL6R B10 / Opti-TNFR2 antibody, anti-IL6R F2 / Opti-TNFR2 antibody, anti-IL6R D2 / Opti-TNFR2 antibody to TNF-α. It is shown.
56 shows Agilent analysis results (left) and pharmacokinetic analysis results (right) of TNFR2-Fc, Opti-TNFR2-Fc, TNFR2-M2-Fc, and Opti-TNFR2-M2-Fc.

The inventors of the present invention, while studying the optimization model of TNFR2-Fc that has the same function as TNFR2-Fc, which is well known as an autoimmune arthritis treatment agent, but can minimize the protein size and increase the stability in the body, the O-glycosylation site and N The inventors have found that TNFR2-Fc can be optimized by removing the -glycosylation site and completed the present invention.

Accordingly, the present invention provides a method for improving the efficiency of a Tumor necrosis factor receptor type 2 (TNFR2) protein, the method comprising removing an O-glycosylation site, an N-glycosylation site, or an O-glycosylation site and an N-glycosylation site. It can provide a method comprising a.

TNFR2-Fc is a fusion protein consisting of the extracellular domain of the p75 TNF receptor, the hinge of human IgG1 and the Fc domain. The extracellular domain consists of four TNFR regions and an O-glycosylation region, and the O-glycosylation region consists of 51 amino acids from amino acids 202 (Serine, S) to 252 (Glutamic acid, E). It consists of amino acids. In addition, there are three N-glycosylation sites in the TNFR region, which are located at No. 28 (Alanine, A), No. 31 (Proline, P), and No. 34 (Proline, P).

We named the O-glycosylation site from TNFR2-Fc as Opti-TNFR2-Fc, and changed the N-glycosylation site from proline (P), amino acid 34 from the N terminus to asparagine (A). The removal was named TNFR2-M2-Fc and the removal of O-glycosylation site and N-glycosylation site was named Opti-TNFR2-M2-Fc.

Opti-TNFR2-Fc, TNFR2-M2-Fc and / or Opti-TNFR2-M2-Fc according to the present invention are more than twice as productive as TNFR2-Fc. That is, according to one embodiment of the present invention In order to obtain each protein, cloned into a mammalian expression vector, expressed and purified in mammalian cells, the protein was secured and the productivity was compared under the same conditions. As a result, the productivity of TNFR2-Fc optimized compared to TNFR2-Fc was doubled. It was found to increase over.

In addition, to determine whether the optimized TNFR2-Fc is functionally different from TNFR2-Fc, as a result of confirming the binding ability with the inflammation-induced cytokine TNF-a, TNF-α ligands are TNFR2-M2-Fc, Opti- TNFR2-Fc, Opti-TNFR2-M2-Fc all showed a lower Kd value than the control TNFR-Fc, it can be seen that it has a high binding force.

From the above, it can be seen that even if the O-glycosylation site or N-glycosylation site of TNFR2 is removed, the binding ability to TNF-α is not affected at all.

Accordingly, when the drug efficacy is similar, since the small size is useful because it can reduce the purity or the side effects thereof during protein purification, it can be inferred that the optimized TNFR2-Fc of the present invention can be used more effectively as a drug. .

In addition, the present inventors conducted an in vivo half-life analysis to determine whether there is a difference in stablity, which is the basis of drug efficacy evaluation, when the O-glycosylation site and / or N-glycosylation site of TNFR2-Fc is removed. According to one embodiment of the present invention, the optimized TNFR-Fc of the present invention was shown to be significantly superior in vivo stability than the control TNFR-Fc.

The above results indicate that O-glycosylation sites and / or N-glycosylation sites inhibit the stability of TNFR2 in the body. Accordingly, when using the optimized TNFR-Fc of the present invention as a drug, it can be seen that the stability in vivo will be superior to the conventional drug.

On the other hand, in the recent drug development flow, the development of drugs capable of dual targeting rather than the drug for one target is being actively made. In the case of the dual-target drug, there may be a binding between the water-soluble receptor and the water-soluble receptors, or the antibody and the water-soluble receptor, which is advantageous as the size of the protein is smaller. Therefore, according to one embodiment of the present invention, fusion of different target drugs of water-soluble receptors (inflammation-related IL21R, TWEAKR) and Opti-TNFR2, antibodies of different target drugs (anti-IL6R A7 / Opti-TNFR2 antibody, anti -IL6R B10 / Opti-TNFR2 antibody, anti-IL6R F2 / Opti-TNFR2 antibody, anti-IL6R D2 / Opti-TNFR2 antibody) and fusion with Opti-TNFR2 to facilitate fusion with other antibodies As a result of confirming the binding ability with TNF-α whether it can act as a double antagonist inhibiting the dual target, it was found that there is no difference with the binding capacity of TNFR2-Fc.

From the above, the optimized TNFR2-Fc of the present invention is smaller in size than the existing drug TNFR2-Fc can reduce drug side effects, has a high affinity with TNF-α, has a high stability in the human body as well as other drugs It can be seen that the fusion with them has all of the advantages.

Therefore, the present invention can provide a fragment from which the O-glycosylation site, the N-glycosylation site, or the O-glycosylation site and the N-glycosylation site of TNFR2 are removed, and the prevention of autoimmune diseases including the fragment as an active ingredient. Alternatively, a therapeutic pharmaceutical composition may be provided. The fragment from which the O-glycosylation site is removed may be preferably SEQ ID NO: 2, the fragment from which the N-glycosylation site is removed may be preferably SEQ ID NO: 3, and the fragment from which the O-glycosylation site and the N-glycosylation site is removed Preferably SEQ ID NO: 4.

In addition, the fragment from which the O-glycosylation site, the N-glycosylation site, or the O-glycosylation site and the N-glycosylation site of the TNFR2 of the present invention is removed has an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 It may be a functional equivalent for each polypeptide. The 'functional equivalent' is at least 60%, preferably 70%, more preferably 80, of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 as a result of the addition, substitution, or deletion of the amino acid. It refers to a polypeptide having a sequence homology of at least%, and exhibits substantially homogeneous activity with fragments from which the O-glycosylation site, the N-glycosylation site, or the O-glycosylation site and the N-glycosylation site of the TNFR2 of the present invention are removed. Here, "substantially homogeneous activity" means the activity of the optimized TNFR2 described above. Such functional equivalents may include, for example, amino acid sequence variants in which some of the amino acids of the amino acid sequence have been substituted, deleted or added. Substitution of amino acids may preferably be conservative substitutions, examples of conservative substitutions of amino acids present in nature are as follows; (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids may preferably be located at portions not directly involved in the optimized TNFR2 activity of the present invention. The functional equivalent may also include polypeptide derivatives in which some chemical structures of the polypeptide have been modified while maintaining the optimized backbone of TNFR2 and its physiological activity. For example, fusion proteins made by fusion with other proteins while maintaining structural changes and physiological activities for altering the stability, storage, volatility or solubility of the polypeptide of the present invention may be included.

In the present invention, the gene of the O-glycosylation site, the N-glycosylation site, or the fragment from which the O-glycosylation site and the N-glycosylation site of the TNFR2 is removed may be an O-glycosylation site, an N-glycosylation site, or an O-glycosylation site of the TNFR2. And a base sequence encoding a protein having N-glycosylation sites removed or a functional equivalent thereof, the base sequence may include all DNA, cDNA, and RNA sequences, preferably SEQ ID NO: 2, SEQ ID NO: 3 It may be a base sequence encoding the amino acid sequence of SEQ ID NO.

The optimized TNFR2-Fc protein according to the present invention may bind to the cytokine TNF-α to prevent or treat autoimmune diseases caused by TNF-α.

One of the most important characteristics of all normal individuals is their ability to recognize and react to and eliminate non-self antigens, while not harmfully reacting to the antigenic substances that make up self. Have Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance.

However, when there is a problem in inducing or maintaining such self-tolerance, an immune response occurs to autoantigens, thereby attacking one's own tissues. It is called.

The type of autoimmune disease may include, but is not limited to, pernicious anemia, type 1 diabetes, autoimmune arthritis, lupus, multiple sclerosis, reactive arthritis or dermatitis.

Therefore, the composition according to the present invention can be used as a pharmaceutical composition capable of preventing or treating autoimmune diseases.

Refers to reversing, alleviating, inhibiting, or preventing the disease or disorder to which the term applies, or one or more symptoms of the disease or disorder, unless otherwise stated And the term " treatment ", as used herein, refers to an act of treating when " treating " is defined as above. Thus, "treatment" or "therapy" of an autoimmune disease in a mammal may include one or more of the following:

(1) inhibits the growth of an autoimmune disease, ie inhibits its development,

(2) preventing the spread of autoimmune diseases, ie preventing metastasis,

(3) alleviates autoimmune diseases.

(4) prevent recurrence of autoimmune diseases, and

(5) alleviating the symptoms of autoimmune diseases

Pharmaceutical compositions for the prophylaxis or treatment of autoimmune diseases according to the invention may comprise a pharmaceutically effective amount of an optimized TNFR2-Fc protein alone or may comprise one or more pharmaceutically acceptable carriers, excipients or diluents. . As used herein, “pharmaceutically effective amount” refers to an amount sufficient to prevent, ameliorate, and treat the symptoms of an autoimmune disease.

The pharmaceutically effective amount of optimized TNFR2-Fc according to the invention is 0.5-100 mg / day / kg body weight, preferably 0.5-5 mg / day / kg body weight, more preferably twice a week for adults. 50 mg / kg body weight, 0.4 mg / kg body weight twice a week for children. However, the pharmaceutically effective amount may be appropriately changed depending on the degree of symptoms of autoimmune disease, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

In addition, "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, optimized TNFR-Fc according to the present invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, the dosage of active ingredient being determined by the route of administration, age, gender, weight and patient The pharmaceutical composition for the prevention or treatment of autoimmune diseases according to the present invention may be appropriately selected according to various factors such as the severity of the present invention, in parallel with known compounds having the effect of preventing, ameliorating or treating the symptoms of autoimmune diseases. Can be administered.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

TNFR2 , Opti - TNFR2 , TNFR2 - M2  And Opti - TNFR2 - M2  Protein acquisition

The present inventors performed the following experiments to prepare an optimized TNFR2-Fc by removing unnecessary portions from TNFR2-Fc, a water-soluble receptor known as a drug for treating autoimmune diseases.

<1-1> polymerase chain reaction PCR ) Perform

The inventors performed cloning to obtain proteins of inflammation-related water-soluble receptors TNFR2, Opti-TNFR2, TNFR2-M2, Opti-TNFR2-M2. At this time, Opti-TNFR2 removes 51 amino acids from N-terminal, which is the O-glycosylation site of TNFR2, from No. 202 (Serine: S) to No. 252 (Glutamic acid: E), and TNFR2-M2 is N of TNFR2. N-glycosylation site was removed by replacing amino acid P (Proline: CCC), which is 34 amino acid, from N-terminal -glycosylation site with A (asparagine: AAC), and Opti-TNFR2-M2 is N-glycosylation of Opti-TNFR2. The amino acid P (Proline: CCC) 34 from the N-terminus of the site was replaced with A (asparagine: AAC) to eliminate the N-glycosylation site.

First, the polymerase chain reaction (PCR) is a cDNA library mix (Kidney, placenta, pancrease, liver) as a template to secure the extracellular domain (primary cellular) as shown in Table 1 below Designed and carried out under the conditions of <Table 3> with the composition of <Table 2> to obtain the amplified product.

primer  order Amplified gene Sequence name Primer sequence SEQ ID NO: TNFR2 TNFR2-F (SfiI) Forward 5'-agggggccgtgggggccttgcccgcccaggtgg-3 ' 29 TNFR2-R (SfiI) Reverse 5'-tagcggccgacgcggccaattcagctggggggct-3 ' 30 Opti-TNFR2 TNFR2-F (SfiI) Forward 5'-agggggccgtgggggccttgcccgcccaggtgg-3 ' 29 Opti-TNFR2-R (SfiI) Reverse 5'-tagcggccgacgcggccaacgtgcagactgcatcc-3 ' 31 TNFR2-M2 TNFR2-M2-F (SfiI) Forward 5'-agggggccgtgggggccttgcccgcccaggtggcatttacaccctacgccccggagaac-3 ' 32 TNFR2-R (SfiI) Reverse 5'-tagcggccgacgcggccaattcagctggggggct-3 ' 30 Opti-TNFR2-M2 TNFR2-M2-F (SfiI) Forward 5'-agggggccgtgggggccttgcccgcccaggtggcatttacaccctacgccccggagaac-3 ' 32 Opti-TNFR2-R (SfiI) Reverse 5'-tagcggccgacgcggccaacgtgcagactgcatcc-3 ' 31

PCR  Furtherance Template 100 ng pfu DNA polymerase 2.5 unit Prime forward 10 pmol / total 50 μl reaction Primer reverse 10 pmol / total 50 μl reaction

PCR  Condition Initially Denatured 2 min at 94 ℃ Denatured 30 sec 94 ℃
30 cycle
Annealing 30 sec at 58 ℃ Extension 1 min at 72 ℃ Final Extension 10 min 72 ℃

<1-2> Cleavage using restriction enzyme Digestion ) And connections ( Ligation )

The amplified product was cut with restriction enzyme SfiI (# R033S, Enzynomics, Korea) and inserted into agarose (# 9002-18-0, BIO BASIC INC, USA) gel for insertion into pYK602-Fc, a mammalian expression vector. Next, a gel purification kit (# 1014876, QIAGEN, USA) was used to secure the insert to be inserted into the vector. Then, the ligation of the insert with the vector pYK602-Fc was performed. Recombination was performed using T4 DNA ligase (# M001S, Enzynomics, korea). Restriction enzyme reaction was based on cutting 1μg DNA. After mixing 1μl SfiI (10 unit) and 5μl 10x reaction buffer, the total volume was added 50 μl was allowed to react for 6 hours in a 37 ° C. warmer, and then purified from agarose gel. Then, the ratio of the vector and the insertion portion was 1: 3, and the 150ng insertion portion was mixed in the 50ng vector, and incubated for 16 hours at 16 ° C with 1μl ligation enzyme (10unit) and 1ul 10x reaction buffer.

<1-3> Transformation ( Transformation ) And transfection ( Transfection )

After 16 hours of incubation, the mixture was transformed into E. coli cells, and the resulting colonies were sequenced to obtain clones correctly inserted into the pYK602-Fc expression vector. The obtained clone was midi prep (# 740 410.100, MACHERY-NAGEL, Germany) and transfected into mammalian cells 293E to confirm the expression rate. For transfection, 293E cells were plated on a 150 mm plate (# 430599 Corning USA) at 70-80% confluency, then the DNA to PEI ratio was 1: 2, i.e. 20 μg DNA, PEI (# 23966, Polysciences , USA) 40μg was mixed, left at room temperature for 20 minutes and dropped on the plate. After 16 to 20 hours, the supernatant was removed once every 2 to 3 days by replacing the medium with a serum-free medium (No FBS DMEM, # SH30243.01 Hyclone Thermo. USA). The supernatant (1 L total) after 2, 5, 7, and 9 days was purified, respectively. To this end, the supernatant was first filtered with a 0.22 μm top-filter (# PR02890 Millipore USA), followed by a 5 ml column. 500 μl Protein A beads (# 17-1279-03 GE healthcare Sweden) filled were bound. At this time, binding was performed overnight at 4 ° C. at a rate of 0.9 ml / min using a Peri-start pump, washed with 100 ml or more of PBS (Phosphate Buffered Saline: # 70011 Gibco USA) and 0.1M Glycine-HCl (# G7126, Sigma, USA) was eluted into six fractions and neutralized with 1M Tris (# T-1503-5KG. Sigma. USA) (pH 9.0). Then, the obtained protein was quantified, 2-3 fractions containing the protein were collected, concentrated to amicon ultra (# UFC805024, Millipore, USA), and the buffer was replaced about 10 times with 1X PBS (# 70011 Gibco USA).

TNFR2 protein (SEQ ID NO: 1), Opti-TNFR2 (SEQ ID NO: 2), TNFR2-M2 (SEQ ID NO: 3), Opti-TNFR2-M2 (SEQ ID NO: 4), which are water-soluble receptors, were obtained in the same manner as above. Protein concentrations were TNFR2-Fc 1.5 μg / μl (1.5 mg total), Opti-TNFR2-Fc 3 μg / μl (3 mg total), TNFR2-M2-Fc 1.9 μg / μl (1.9 mg total), Opti-TNFR2-M2 -Fc 3.2μg / μl (total 3.2mg), it can be seen that the productivity of the optimized structure is superior to the control TNFR2-Fc.

TNF for -α TNFR2 - Fc  Binding force measurement of optimized structures

In order to determine whether the antagonistic action can be performed by binding to the inflammation-inducing cytokine and ligand TNF-α, TNFR2, TNFR2-M2, Opti-TNFR2 and Opti-TNFR2, which are the proteins obtained in Example 1 Only a purity of 90% or more at -M2 was selected, and the binding affinity test was performed using this. After coating overnight at 4 ° C. with 100 ng of TNF-α (# C001-1MG, enzynomics, Korea) using an ELISA plate (# 439454, Nunc, Denmark), 4% skim milk in 200 μl of PBS ( # 232100, Difco, France) was placed in an ELISA plate and blocked for approximately 1 hour at room temperature (blocking: 4% skim milk / 1 × PBS) to inhibit nonspecific reactions. After blocking, the blocking buffer was removed from the ELISA plate, and each purified protein was prepared in 100 μl of PBS solution containing 1% skim milk at 100 nM and serial dilution at 1/4 dilution. Then, the reaction was performed at room temperature for about 2 hours. After washing the plate five times with 200 μl PBST, 2 μl of a secondary antibody, anti-Human Fc-HRP (# 31413, Thermo, USA), was mixed with 4 ml of PBS containing 1% skim milk to form one of the ELISA plates. 200 μl per well was added and reacted at room temperature for 1 hour. After the reaction, the secondary antibody on the ELISA plate was removed, washed five times with 200 μl of PBS and then 10 μl of hydrogen peroxide solution (H 2 O 2, # H1009-100ML, Sigma, USA), 10 ml of PC buffer (5.1 g Citric) acid monohydrate, 7.3 g Sodium phosphate / L (pH5.0).), and one OPD tablet (# P8787-100TAB Sigma USA) were mixed and the reaction solution was added in a total volume of 100 μl. After 10 minutes of reaction in the dark at room temperature, the color was confirmed. To stop the reaction, the reaction was terminated by stopping the reaction by adding 50 μl of a stop buffer per well, using an ELISA reader to a wavelength of 490 nm. Kd value was measured at.

As a result, as shown in Figure 53, the TNF-α ligand and each protein shows a low Kd value, it can be seen that it has a high binding force. It can be seen that the binding capacity of TNFR2-Fc (control) to TNF-α shows a binding capacity of 6x10 ^-10 M. The Kd value of TNFR2-M2-Fc to TNF-α is 8.2x10 ^-11 M, Opti- The Kd value for TNF-α of TNFR2-Fc is 4 × ^{10 −10} M and the Kd value for TNF-α of Opti-TNFR2-M2-Fc is 6.8 × ^{10 −11} M. It can be seen that it is similar or superior to the control.

The above results indicate that removing the O-glycosylation site or N-glycosylation site of TNFR2 does not affect the binding capacity to TNF-α.

TNFR2 - Fc  Of optimized structures Pharmacokinetics ( pharmacokinetics ) evaluation

The present inventors to evaluate the drug efficacy of the fusion protein optimized by removing the N-glycosylation position of TNFR2-Fc of the present invention, or to remove the O-glycosylation site, in vivo (in In vivo half-life analysis and Agilent analysis were performed, and wild type TNFR2-Fc was used as a control. Seven-week-old male BALB / c mice (OrientBio) were purchased for in vivo half-life analysis and acclimated in animal kennels for 3-4 days. Proteins of TNFR2-Fc, TNFR2-M2-Fc, Opti-TNFR2-Fc, Opti-TNFR2-M2-Fc were adjusted to 1 mg / ml with 1xPBS and then tested using three experimental mice per protein drug, The dose was calculated as 10 mg (drug) / 1 kg (mouse body weight) was administered by 250μg. The method of administration was IP injection (injection peritoneal), and mouse blood samples were taken through orbital (eye) blood collection, and blood collection was performed immediately after administration (0 hour), 30 minutes after administration, 2 hours later, After 4 hours, 6 hours, 24 hours, 30 hours, and 48 hours, 50 μl of blood was collected from each drug administration group at each time interval. Only a serum was obtained from the blood collected using a low temperature centrifuge, and a 96 well-immunoplate coated with 100 ng TNF-α (# C001-1MG, enzynomics, Korea) per well prepared. Nunc, Denmark) was subjected to blocking for 1 hour by adding 200 μl / well of 4% skim milk (# 232100, Difco, France) / 1 × PBS. To make a standard curve one hour after blocking, prepare proteins of TNFR2-Fc, TNFR2-M2-Fc, Opti-TNFR2-Fc, and Opti-TNFR2-M2-Fc to set the starting concentration to 100 nM Diluted with, diluted gradually to the 12th well was added to 100μl in 96 well-immune plate coated with TNF-α. At the same time, the serum samples obtained at 0 (immediately after administration), 30 minutes, 2 hours, 4 hours, 6 hours, 24 hours, 30 hours, and 48 hours for each protein were 1/100, 1/1000, 1 using 1xPBS. Diluted with / 10000, 100μl each put in TNF-α-coated 96 well-immune plate and placed at room temperature for 2 hours. After 2 h, washed 5 times with 200 μl of 1 × PBST and attached secondary antibody. Anti-human Fc-HRP (# 31413, Thermo, USA) was diluted to 1/2000 and mixed with 1X PBST containing 1% skim milk, mixed, 200 μl of each well and placed at room temperature for 1 hour. After washing 5 times with 200 μl 1X PBST, two color developing solutions (OPD tablet (# P8787-100TAB Sigma USA), 20 ml PC solution (5.1 g Citric acid monohydrate, 7.3 g Sodium phosphate / L (pH5) .0) and 20μl H2O2 (# H1009-100ML, Sigma, USA) were mixed with 100μl per well, and then placed in a dark well for 30 minutes in a 96-well immunity plate and checked for color development every 10 minutes. When the color development was completed, 50 μl of stop solution (2.5M H2SO4) was added to each well to terminate the reaction, and Agilent protein analysis was performed, and the value at 490 nm was determined using an ELISA reader. For the values, the standard curves were calculated, the values outside the standard range were discarded, and only the values within the range were taken.

As a result, as shown in FIG. 56, the highest concentration value appears to be Opti-TNFR2-Fc> Opti-TNFR2-M2-Fc> TNFR2-M2-Fc> TNFR2-Fc. This shows the amount remaining in the body after 2 hours when each drug is injected into the body, it can be seen that Opti-TNFR2-Fc is the most stable, and then Opti-TNFR2-M2-Fc is stable. .

Therefore, when using the proteins of the present invention as a drug, Opti-TNFR2-Fc can be seen that the best in vivo stability.

As a result of Agilent analysis, as shown in FIG. 56, in the case of the second and third lines having O-glycosylation sites, the size of the protein after purification was confirmed in a wide range (95-120 KDa) due to glycosylation effects. On the other hand, lines 4 and 5 without the O-glycosylation site can be seen that the exact size of the protein is about 65KDa.

Accordingly, it can be seen that the absence of O-glycosylation site during protein purification can not only reduce the purification step but also ensure a high purity protein.

From the above, it can be seen that the in vivo stability of Opti-TNFR2 and Opti-TNFR2-M2 without O-glycosylation site is high, which may be a factor that inhibits the stability of TNFR2 in the body. Means.

Opti - TNFR2 - Fc  Preparation of Double Antagonists Using Structures

The present inventors confirmed from Example 3 that Opti-TNFR2 of the present invention has a longer half-life in vivo than TNFR2 and has the best stability among optimized structures, and is fused with IL21R and TWEAKR, which are soluble receptors related to inflammation. In addition, it has been confirmed that a single drug can act as a dual antagonist that inhibits the dual target by facilitating the fusion with anti-IL6R antibodies related to inflammation.

To this end, the gene was cloned into a mammalian expression vector, and the protein was obtained by expression and purification in 293E cells, which are mammalian cells. A fusion of a polypeptide comprising amino acids Nos. 28 to 49 from the N terminus, which is the full-length extracellular domain, and the fusion of IL21R and Opti-TNFR2 (Bitrap) is a combination of the Opti-TNFR2 extracellular domain and IL21R. As a fusion of a polypeptide including amino acids 20 to 213 from the N-terminal, which is the full-length extracellular domain, the protein securing process was performed in the same manner as in <Example 1>. The obtained Fc fusion protein concentration of Opti-TNFR2 / TWEAKR (SEQ ID NO: 5) was 3.5 μg / μl (3.5 mg total), and the Fc fusion protein concentration of IL21R / Opti-TNFR2 (SEQ ID NO 6) was 4 μg / μl (total) 4 mg).

The fusion antibody anti-IL6R (heavy chain, light chain) / Opti-TNFR2 (A7, B10, F2, D2) is an anti-IL6R prepared by the known method (Phage-display of Antibody. Immunological Reviews 1992, No. 130.) The extracellular region of Opti-TNFR2 was fused to the C terminus of heavy chains (A7, B10, F2, D2), and inserted into pNATABH, a heavy chain (HC) mammalian expression vector, to obtain a fusion antibody protein. In order to fuse the A7 heavy chain / Opti-TNFR2, B10 heavy chain / Opti-TNFR2, F2 heavy chain / Opti-TNFR2, D2 heavy chain / Opti-TNFR2 by the PCR technique described in Example 1-1, PCR conditions are shown in Tables 4 and 5 below.

primer  order Amplified gene Sequence name Primer sequence SEQ ID NO: Anti-IL6R A7 HC TNFR2-F Forward 5'-ttgcccgcccaggtgg-3 ' 33 Opti-TNFR-R (XhoI) Reverse 5'-ccagctcgagcgtgcagactgcatcc-3 ' 34 Anti-IL6R A7 LC NATVK1-1 (SfiI) Forward 5'-ttggtggccacagcggccgatgtccactcggatgtccactcgcagctcg-3 ' 35 NATJK-R4 (BglII) Reverse 5'-gaggagagatcttaggacggtgaccttgg-3 ' 36 Anti-IL6R B10 HC TNFR2-F Forward 5'-ttgcccgcccaggtgg-3 ' 33 Opti-TNFR-R (XhoI) Reverse 5'-ccagctcgagcgtgcagactgcatcc-3 ' 34 Anti-IL6R B10 LC NATVK5 (SfiI) Forward 5'-ttggtggccacagcggccgatgtccactcgcagctcgtgctgactcag-3 ' 37 NATJK-R4 (BglII) Reverse 5'-gaggagagatcttaggacggtgaccttgg-3 ' 36 Anti-IL6R F2 HC TNFR2-F Forward 5'-ttgcccgcccaggtgg-3 ' 33 Opti-TNFR-R (XhoI) Reverse 5'-ccagctcgagcgtgcagactgcatcc-3 ' 34 Anti-IL6R F2 LC NATVK6 (SfiI) Forward 5'-ttggtggccacagcggccgatgtccactcgcagctcgtgctaactcag-3 ' 37 NATJK-R4 (BglII) Reverse 5'-gaggagagatcttaggacggtgaccttgg-3 ' 36 Anti-IL6R D2 HC TNFR2-F Forward 5'-ttgcccgcccaggtgg-3 ' 33 Opti-TNFR-R (XhoI) Reverse 5'-ccagctcgagcgtgcagactgcatcc-3 ' 34 Anti-IL6R D2 LC NATVK5 (SfiI) Forward 5'-ttggtggccacagcggccgatgtccactcgcagctcgtgctgactcag-3 ' 37 NATJK-R4 (BglII) Reverse 5'-gaggagagatcttaggacggtgaccttgg-3 ' 36

PCR  Condition Initially Denatured 2 min at 94 ℃ Denatured 30 sec 94 ℃
30 cycle
Annealing 30 sec at 58 ℃ Extension 1 min at 72 ℃ Final Extension 5 min 72 ℃

In order to ligation the PCR product with the vector pNATABH, each was cut with restriction enzyme 1ul SfiI (10 units, # R033S, Enzynomics, Korea), 1ul NheI (# R016M, Enzynomics, Korea), and the following procedure was carried out as described above. Example 1-2> was linked to the vector, a clone was correctly inserted into the pNATABH heavy chain expression vector was secured. In addition, in order to insert into pNATABL which is a light chain (LC) mammalian expression vector, by performing PCR with the primers and conditions shown in Tables 4 and 5, A7 light chain, B10 light chain, and F2 Amplify the light chain and the D2 light chain, and cut each of them with restriction enzymes SfiI (# R033S, Enzynomics, Korea) and BglII (# R010M, Enzynomics, Korea), and then link to the vector in the same manner as in <Example 1-2>. Then, a clone was correctly inserted into the pNATABL light chain expression vector. The obtained heavy and light chain clones were co-transfected into mammalian cells 293E. For this purpose, the 293E cells were plated in a 100 mm plate (# 430599 Corning USA) at 80-90% confluency, and the ratio of DNA and PEI was 1 2: That is, 13 μg of DNA (heavy chain: light chain = 4: 6) and 40 μg of PEI (# 23966, Polysciences, USA) were mixed, left at room temperature for 20 minutes, and dropped on the plate. Example 1-3 was carried out in the same manner.

The total Fc fusion protein concentration of anti-IL6R A7 / Opti-TNFR2 obtained from the above was 1 mg / μl, and the total Fc fusion protein concentration of anti-IL6R B10 / Opti-TNFR2 was 700 μg / μl, and the anti-IL6R F2 / Opti-TNFR2 (Fc fusion protein concentration of the total is 400μg / μl, Fc fusion protein concentration of anti-IL6R D2 / Opti-TNFR2 is a total of 500μg / μl (mg total).

Opti - TNFR2 - Fc  Of dual antagonists using structures TNF Binding force measurement for -α

Fusion of Opti-TNFR2 prepared in Example 4: Opti-TNFR2 / TWEAKR, IL21R / Opti-TNFR2 and double antibody: anti-IL6R A7 / Opti-TNFR2 antibody, anti-IL6R B10 / Opti-TNFR2 antibody, Binding affinity test was performed to determine whether anti-IL6R F2 / Opti-TNFR2 antibody and anti-IL6R D2 / Opti-TNFR2 antibody can antagonize by binding to TNF-α. Experimental method was carried out in the same manner as in <Example 2>.

As a result, after confirming the binding ability of TNF-a by fusion of different target drugs, the water-soluble receptor and Opti-TNFR2, and fusion of the different target drugs, the antibody and Opti-TNFR2, as shown in Figure 54, Opti-TNFR2 / TWEAKR The binding capacity of -Fc fusion protein and TNF-a to IL21R / Opti-TNFR2-Fc fusion protein was 4.7x10 ^-10 M and 1.1x10 ^-10 M, respectively, similar to the control TNFR2-Fc. It can be seen that there is no. In addition, the anti -IL6R / Opti-TNFR2 antibody confirmed four kinds of TNF-a binding ability with the result, as shown in Figure 55, each of ^{7.8x10 -11 M, 7.2x10 -10 M,} 4.3x10 -10 M, 6x10 of ^{- 10} M showed similar binding capacity to TNFR2-Fc as a control.

From the above, TNFR2-Fc optimized according to the present invention has the advantage of smaller protein size than conventional TNFR2-Fc without affecting the ability to bind TNF-α even when fused with other drugs, It can be inferred that dual-targeted drugs with increased purity can reduce side effects and will be easier to use.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

<110> A & R THERAPEUTICS CO. LTD. <120> Optimization Method Of TNFR2 <130> PB12-10440 <160> 37 <170> Kopatentin 2.0 <210> 1 <211> 230 <212> PRT <213> Homo sapiens <400> 1 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser   1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys              20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr          35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu      50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser  65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys                  85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys             100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala         115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro     130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala                 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Ser Ala Pro Gly Ala Val             180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr         195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly     210 215 220 Pro Ser Pro Pro Ala Glu 225 230 <210> 2 <211> 179 <212> PRT <213> Artificial Sequence <220> O-glycosylation region deleted TNFR2 amino acid sequence <400> 2 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser   1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys              20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr          35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu      50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser  65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys                  85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys             100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala         115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro     130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala                 165 170 175 Val Cys Thr             <210> 3 <211> 230 <212> PRT <213> Artificial Sequence <220> N-glycosylation site deleted TNFR2 amino acid sequence <400> 3 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Asn Gly Ser   1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys              20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr          35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu      50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser  65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys                  85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys             100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala         115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro     130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala                 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Ser Ala Pro Gly Ala Val             180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr         195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly     210 215 220 Pro Ser Pro Pro Ala Glu 225 230 <210> 4 <211> 179 <212> PRT <213> Artificial Sequence <220> <223> O-glycosylation region and N-glycosylation site deleted TNFR2          amino acid sequence <400> 4 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Asn Gly Ser   1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys              20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr          35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu      50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser  65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys                  85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys             100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala         115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro     130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala                 165 170 175 Val Cys Thr             <210> 5 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> O-glycosylation region deleted TNFR2 / TWEAKR amino acid sequence <400> 5 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser   1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys              20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr          35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu      50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser  65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys                  85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys             100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala         115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro     130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala                 165 170 175 Val Cys Thr Glu Gln Ala Pro Gly Thr Ala Pro Cys Ser Arg Gly Ser             180 185 190 Ser Trp Ser Ala Asp Leu Asp Lys Cys Met Asp Cys Ala Ser Cys Arg         195 200 205 Ala Arg Pro His Ser Asp Phe Cys Leu Gly Cys Ala Ala Ala Pro Pro     210 215 220 Ala Pro Phe Arg 225 <210> 6 <211> 391 <212> PRT <213> Artificial Sequence <220> <223> IL21R / O-glycosylation region deleted TNFR2 amino acid sequence <400> 6 Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys   1 5 10 15 Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp              20 25 30 Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu          35 40 45 His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met      50 55 60 Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr  65 70 75 80 Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala                  85 90 95 Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser             100 105 110 Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala Phe         115 120 125 Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg     130 135 140 Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val Asp 145 150 155 160 Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser                 165 170 175 Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln             180 185 190 Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser         195 200 205 Glu Glu Leu Lys Glu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro     210 215 220 Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala 225 230 235 240 Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe                 245 250 255 Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr             260 265 270 Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser         275 280 285 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln     290 295 300 Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys 305 310 315 320 Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly                 325 330 335 Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys             340 345 350 Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile         355 360 365 Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala     370 375 380 Ser Met Asp Ala Val Cys Thr 385 390 <210> 7 <211> 637 <212> PRT <213> Artificial Sequence <220> <223> anti-IL6R A7 HC / O-glycosylation region deleted TNFR2 amino acid          sequence <400> 7 Asp Val His Ser Gln Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val   1 5 10 15 Gln Pro Gly Thr Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys              20 25 30 Phe Glu Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly          35 40 45 Leu Glu Trp Val Ser Gly Val Ser Trp Asn Ser Gly Thr Ile Ala Tyr      50 55 60 Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys  65 70 75 80 Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Asp Phe Thr Tyr Phe Tyr Glu Ser Ser Gly             100 105 110 Tyr Tyr Ala Phe Asp Leu Trp Gly Gln Gly Thr Met Val Thr Val Ser         115 120 125 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser     130 135 140 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 145 150 155 160 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr                 165 170 175 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr             180 185 190 Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln         195 200 205 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp     210 215 220 Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 225 230 235 240 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro                 245 250 255 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr             260 265 270 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn         275 280 285 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg     290 295 300 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 305 310 315 320 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser                 325 330 335 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys             340 345 350 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp         355 360 365 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe     370 375 380 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 385 390 395 400 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe                 405 410 415 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly             420 425 430 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr         435 440 445 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala     450 455 460 Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu 465 470 475 480 Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly                 485 490 495 Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp             500 505 510 Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu         515 520 525 Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln     530 535 540 Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp 545 550 555 560 Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu                 565 570 575 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr             580 585 590 Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr         595 600 605 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val     610 615 620 Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr 625 630 635 <210> 8 <211> 115 <212> PRT <213> Homo sapiens <400> 8 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Thr Asn Ser Asn              20 25 30 Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Val Pro Gly Thr          35 40 45 Thr Pro Lys Leu Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala  65 70 75 80 Ile Thr Gly Leu Gln Ala Glu Asp Gly Gly Asp Tyr Tyr Cys Gln Ser                  85 90 95 Phe Asp Ser Ser Leu Thr Gly Ser Val Phe Gly Thr Gly Thr Lys Val             100 105 110 Thr Val Leu         115 <210> 9 <211> 633 <212> PRT <213> Artificial Sequence <220> <223> anti-IL6R B10 HC / O-glycosylation region deleted TNFR2 amino acid          sequence <400> 9 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val   1 5 10 15 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile              20 25 30 Phe Asp Asp Tyr Ala Met Phe Trp Val Arg Gln Val Pro Gly Lys Gly          35 40 45 Leu Glu Trp Val Ala Gly Ile Asn Trp Asn Gly Asn Gly Ile Gly Tyr      50 55 60 Gly Asp Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Arg  65 70 75 80 Asn Ser Leu Asp Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Pro Ser Leu Tyr Gly Gly Asn Ser Glu Phe             100 105 110 Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr         115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser     130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His                 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser             180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys         195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu     210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys                 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val             260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp         275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr     290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu                 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg             340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys         355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp     370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser                 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser             420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser         435 440 445 Leu Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala Phe Thr Pro Tyr     450 455 460 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln 465 470 475 480 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys                 485 490 495 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp             500 505 510 Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys         515 520 525 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg     530 535 540 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 545 550 555 560 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg                 565 570 575 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val             580 585 590 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr         595 600 605 Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly     610 615 620 Asn Ala Ser Met Asp Ala Val Cys Thr 625 630 <210> 10 <211> 114 <212> PRT <213> Homo sapiens <400> 10 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Pro Thr Ile Gly              20 25 30 Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro          35 40 45 Lys Leu Leu Ile Tyr Gly Asn Leu Asn Arg Pro Ser Gly Val Pro Asp      50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr  65 70 75 80 Asp Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys His Thr Tyr Asp                  85 90 95 Ser Ser Leu Ser Gly Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr             100 105 110 Val Leu         <210> 11 <211> 632 <212> PRT <213> Artificial Sequence <220> <223> anti-IL6R F2 HC / O-glycosylation region deleted TNFR2 amino acid          sequence <400> 11 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys   1 5 10 15 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr              20 25 30 Phe Thr Asn Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly          35 40 45 Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Asn Thr Gly Tyr      50 55 60 Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Met Ser Thr  65 70 75 80 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Gly Leu Pro Trp Gly Glu Asn Gly Leu Asp             100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys         115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly     130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr                 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val             180 185 190 Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn         195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro     210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp                 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp             260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly         275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn     290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro                 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu             340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn         355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile     370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys                 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys             420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu         435 440 445 Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala     450 455 460 Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr 465 470 475 480 Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val                 485 490 495 Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser             500 505 510 Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly         515 520 525 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu     530 535 540 Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser 545 550 555 560 Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro                 565 570 575 Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys             580 585 590 Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp         595 600 605 Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn     610 615 620 Ala Ser Met Asp Ala Val Cys Thr 625 630 <210> 12 <211> 115 <212> PRT <213> Homo sapiens <400> 12 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Ser Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn              20 25 30 Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr          35 40 45 Ala Pro Lys Val Leu Ile Tyr Gly Asp Ser Asp Arg Pro Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Lys Ser Ala Thr Ser Ala Ser Leu Ala  65 70 75 80 Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser                  85 90 95 Tyr Asp Ser Ser Leu Ser Ala Tyr Val Phe Gly Thr Gly Thr Lys Val             100 105 110 Thr Val Leu         115 <210> 13 <211> 635 <212> PRT <213> Artificial Sequence <220> <223> anti-IL6R D2 HC / O-glycosylation region deleted TNFR2 amino acid          sequence <400> 13 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys   1 5 10 15 Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Gly Ser Gly Asp Thr              20 25 30 Phe Pro Asn Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly          35 40 45 Pro Glu Trp Met Gly Arg Ile Ile Pro Met Leu Gly Thr Ser Asp Tyr      50 55 60 Ala Glu Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr  65 70 75 80 Asn Thr Ala Tyr Met Gly Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Val Lys Gly Pro Arg Tyr Tyr Gly Thr Asp Ser Tyr             100 105 110 Tyr Leu Glu Lys Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala         115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Ser Ser Ser     130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly                 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu             180 185 190 Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr         195 200 205 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg     210 215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys                 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val             260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr         275 280 285 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu     290 295 300 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys                 325 330 335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln             340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu         355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro     370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu                 405 410 415 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val             420 425 430 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln         435 440 445 Lys Ser Leu Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala Phe Thr     450 455 460 Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr 465 470 475 480 Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His                 485 490 495 Ala Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys             500 505 510 Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu         515 520 525 Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys     530 535 540 Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys 545 550 555 560 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys                 565 570 575 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp             580 585 590 Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser         595 600 605 Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile     610 615 620 Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr 625 630 635 <210> 14 <211> 114 <212> PRT <213> Homo sapiens <400> 14 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Pro Thr Ile Gly              20 25 30 Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro          35 40 45 Lys Leu Leu Ile Tyr Gly Asn Leu Asn Arg Pro Ser Gly Val Pro Asp      50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr  65 70 75 80 Asp Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys His Thr Tyr Asp                  85 90 95 Ser Ser Leu Ser Gly Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr             100 105 110 Val Leu         <210> 15 <211> 690 <212> DNA <213> Homo sapiens <400> 15 ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc 60 agagaatact atgaccagac agctcagatg tgctgcagca agtgctcgcc gggccaacat 120 gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca 180 tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct 240 gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc 300 ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag 360 tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag 420 ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac 480 cagatctgta acgtggtggc catccctggg aatgcaagca tggatgcagt ctgcacgtcc 540 acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca 600 cgatcccaac acacgcagcc aactccagaa cccagcactg ctccaagcac ctccttcctg 660 ctcccaatgg gccccagccc cccagctgaa 690 <210> 16 <211> 537 <212> DNA <213> Artificial Sequence <220> <223> O-glycosylation region deleted TNFR2 DNA sequence <400> 16 ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc 60 agagaatact atgaccagac agctcagatg tgctgcagca agtgctcgcc gggccaacat 120 gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca 180 tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct 240 gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc 300 ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag 360 tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag 420 ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac 480 cagatctgta acgtggtggc catccctggg aatgcaagca tggatgcagt ctgcacg 537 <210> 17 <211> 690 <212> DNA <213> Artificial Sequence <220> <223> N-glycosylation site deleted TNFR2 DNA sequence <400> 17 ttgcccgccc aggtggcatt tacaccctac gccccggaga acgggagcac atgccggctc 60 agagaatact atgaccagac agctcagatg tgctgcagca agtgctcgcc gggccaacat 120 gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca 180 tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct 240 gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc 300 ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag 360 tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag 420 ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac 480 cagatctgta acgtggtggc catccctggg aatgcaagca tggatgcagt ctgcacgtcc 540 acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca 600 cgatcccaac acacgcagcc aactccagaa cccagcactg ctccaagcac ctccttcctg 660 ctcccaatgg gccccagccc cccagctgaa 690 <210> 18 <211> 537 <212> DNA <213> Artificial Sequence <220> <223> O-glycosylation region and N-glycosylation site deleted TNFR2 DNA          sequence <400> 18 ttgcccgccc aggtggcatt tacaccctac gccccggaga acgggagcac atgccggctc 60 agagaatact atgaccagac agctcagatg tgctgcagca agtgctcgcc gggccaacat 120 gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca 180 tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct 240 gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc 300 ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag 360 tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag 420 ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac 480 cagatctgta acgtggtggc catccctggg aatgcaagca tggatgcagt ctgcacg 537 <210> 19 <211> 684 <212> DNA <213> Artificial Sequence <220> <223> O-glycosylation region deleted TNFR2 / TWEAKR DNA sequence <400> 19 ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc 60 agagaatact atgaccagac agctcagatg tgctgcagca aatgctcgcc gggccaacat 120 gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca 180 tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct 240 gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc 300 ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag 360 tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag 420 ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac 480 cagatctgta acgtggtggc catccctggg aatgcaagca tggatgcagt ctgcacggag 540 caagcgccag gcaccgcccc ctgctcccgc ggcagctcct ggagcgcgga cctggacaag 600 tgcatggact gcgcgtcttg cagggcgcga ccgcacagcg acttctgcct gggctgcgct 660 gcagcacctc ctgccccctt ccgg 684 <210> 20 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> IL21R / O-glycosylation region deleted TNFR2 DNA sequence <400> 20 tgccccgacc tcgtctgcta caccgattac ctccagacgg tcatctgcat cctggaaatg 60 tggaacctcc accccagcac gctcaccctt acctggcaag accagtatga agagctgaag 120 gacgaggcca cctcctgcag cctccacagg tcggcccaca atgccacgca tgccacctac 180 acctgccaca tggatgtatt ccacttcatg gccgacgaca ttttcagtgt caacatcaca 240 gaccagtctg gcaactactc ccaggagtgt ggcagctttc tcctggctga gagcatcaag 300 ccggctcccc ctttcaacgt gactgtgacc ttctcaggac agtataatat ctcctggcgc 360 tcagattacg aagaccctgc cttctacatg ctgaagggca agcttcagta tgagctgcag 420 tacaggaacc ggggagaccc ctgggctgtg agtccgagga gaaagctgat ctcagtggac 480 tcaagaagtg tctccctcct ccccctggag ttccgcaaag actcgagcta tgagctgcag 540 gtgcgggcag ggcccatgcc tggctcctcc taccagggga cctggagtga atggagtgac 600 ccggtcatct ttcagaccca gtcagaggag ttaaaggaac ccgcccaggt ggcatttaca 660 ccctacgccc cggagcccgg gagcacatgc cggctcagag aatactatga ccagacagct 720 cagatgtgct gcagcaagtg ctcgccgggc caacatgcaa aagtcttctg taccaagacc 780 tcggacaccg tgtgtgactc ctgtgaggac agcacataca cccagctctg gaactgggtt 840 cccgagtgct tgagctgtgg ctcccgctgt agctctgacc aggtggaaac tcaagcctgc 900 actcgggaac agaaccgcat ctgcacctgc aggcccggct ggtactgcgc gctgagcaag 960 caggaggggt gccggctgtg cgcgccgctg cgcaagtgcc gcccgggctt cggcgtggcc 1020 agaccaggaa ctgaaacatc agacgtggtg tgcaagccct gtgccccggg gacgttctcc 1080 aacacgactt catccacgga tatttgcagg ccccaccaga tctgtaacgt ggtggccatc 1140 cctgggaatg caagcatgga tgcagtctgc acg 1173 <210> 21 <211> 1911 <212> DNA <213> Artificial Sequence <220> <223> anti-IL6R A7 HC / O-glycosylation region deleted TNFR2 DNA sequence <400> 21 gatgtccact cgcagatgca gctggtggag tctgggggag gcttggtaca gcctggcacg 60 tccctgagac tctcctgtgc agcctctgga ttcaagtttg aagattatgc catgcactgg 120 gtccggcaag ctccagggaa gggcctggag tgggtctcag gtgttagttg gaacagtggt 180 accatagcct atgtggactc tgtgaagggc cgattcacca tttccagaga caacgccaag 240 aactccctgt atctgcaaat gaacagtctg agagccgagg acacggccgt gtattactgt 300 gcgagagatt tcacatattt ttatgagagt agtggttact atgcttttga tctctggggc 360 caagggacaa tggtcaccgt ctcctcagct agcaccaagg gcccatcggt cttccccctg 420 gcaccctcct ccaagagcac ctctgggggc acagcggccc tgggctgcct ggtcaaggac 480 tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 540 accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 600 ccctccagca gcttgggcac ccagacctac atctgcaacg tgaatcacaa gcccagcaac 660 accaaggtgg acaagagagt tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 720 tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tctttccccc aaaacccaag 780 gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 840 gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 900 acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 960 ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1020 ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1080 tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 1140 gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1200 aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1260 aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1320 catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaaccc 1380 gcccaggtgg catttacacc ctacgccccg gagcccggga gcacatgccg gctcagagaa 1440 tactatgacc agacagctca gatgtgctgc agcaagtgct cgccgggcca acatgcaaaa 1500 gtcttctgta ccaagacctc ggacaccgtg tgtgactcct gtgaggacag cacatacacc 1560 cagctctgga actgggttcc cgagtgcttg agctgtggct cccgctgtag ctctgaccag 1620 gtggaaactc aagcctgcac tcgggaacag aaccgcatct gcacctgcag gcccggctgg 1680 tactgcgcgc tgagcaagca ggaggggtgc cggctgtgcg cgccgctgcg caagtgccgc 1740 ccgggcttcg gcgtggccag accaggaact gaaacatcag acgtggtgtg caagccctgt 1800 gccccgggga cgttctccaa cacgacttca tccacggata tttgcaggcc ccaccagatc 1860 tgtaacgtgg tggccatccc tgggaatgca agcatggatg cagtctgcac g 1911 <210> 22 <211> 345 <212> DNA <213> Homo sapiens <400> 22 gatgtccact cgcagctcgt gctgactcag ccgccctcag tgtctggggc cccagggcag 60 agggtcacca tctcctgcac tgggaccaac tccaacatcg gagcaggtta tgatgtccac 120 tggtaccagc aggttccagg aacaaccccc aaactcctca tctatggtaa caccaatcgg 180 ccctcagggg tccctgaccg attctctggc tccaagtctg gcacttcagc ctccctggcc 240 atcactggcc tccaggctga ggatgggggt gattattact gccagtcctt tgacagcagc 300 ctgactggtt cggtgttcgg aactgggacc aaggtcaccg tccta 345 <210> 23 <211> 1899 <212> DNA <213> Artificial Sequence <220> <223> anti-IL6R B10 HC / O-glycosylation region deleted TNFR2 DNA          sequence <400> 23 gatgtccact cgcaggtgca gctggtggag tctgggggag gcttggtaca gcctggaggg 60 tccctgagac tctcctgtgc agcctctgga ttcatctttg atgattatgc catgttttgg 120 gtccgacaag ttccagggaa gggcctggag tgggtcgcag gtattaattg gaatggtaat 180 ggtattgggt atggggactc tgtgaggggc cgattcatca tctctagaga caacgccagg 240 aactctctgg atttgcaaat gaacagtctg agagccgagg acacggccgt gtattactgt 300 gcgagaccaa gtctctacgg tggtaactcc gaatttgacc tctggggcca gggaaccctg 360 gtcaccgtct cctcagctag caccaagggc ccatcggtct tccccctggc accctcctcc 420 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540 gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 600 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660 aagagagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720 gaactcctgg ggggaccgtc agtcttcctc tttcccccaa aacccaagga caccctcatg 780 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960 tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1020 gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1080 ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140 tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1200 accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1260 gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaacccgc ccaggtggca 1380 tttacaccct acgccccgga gcccgggagc acatgccggc tcagagaata ctatgaccag 1440 acagctcaga tgtgctgcag caagtgctcg ccgggccaac atgcaaaagt cttctgtacc 1500 aagacctcgg acaccgtgtg tgactcctgt gaggacagca catacaccca gctctggaac 1560 tgggttcccg agtgcttgag ctgtggctcc cgctgtagct ctgaccaggt ggaaactcaa 1620 gcctgcactc gggaacagaa ccgcatctgc acctgcaggc ccggctggta ctgcgcgctg 1680 agcaagcagg aggggtgccg gctgtgcgcg ccgctgcgca agtgccgccc gggcttcggc 1740 gtggccagac caggaactga aacatcagac gtggtgtgca agccctgtgc cccggggacg 1800 ttctccaaca cgacttcatc cacggatatt tgcaggcccc accagatctg taacgtggtg 1860 gccatccctg ggaatgcaag catggatgca gtctgcacg 1899 <210> 24 <211> 342 <212> DNA <213> Homo sapiens <400> 24 gatgtccact cgcagctcgt gctgactcag ccgccctcag tgtctggggc cccaggccag 60 acggtcacca tctcctgcac tgggcccacc atcggggcag gatatgatgt ccactggtac 120 caacaacttc caggagcagc ccccaaactc ctcatctatg gtaatctcaa tcggccctca 180 ggtgtccctg accgattctc tggctccaag tctggcacct cagcctccct ggccatcacg 240 gacctccagg ctgaggatga ggctgactat tactgccata cctatgacag tagcctcagt 300 ggttcttggg tcttcggcgg agggaccaag ctgaccgtcc ta 342 <210> 25 <211> 1896 <212> DNA <213> Artificial Sequence <220> <223> anti-IL6R F2 HC / O-glycosylation region deleted TNFR2 DNA sequence <400> 25 gatgtccact cgcaggtgca gctggtagag tctggggctg aggtgaagaa gcctggggcc 60 tcagtgaagg tttcctgcaa ggcatctgga tacaccttca ccaactacta tatgcactgg 120 gtgcgacagg cccctggaca agggcttgag tggatgggaa taatcaaccc tagtggtggt 180 aacacgggct acgcacagaa gttccagggc agagtcacca tgaccaggga catgtccaca 240 agcacagcct acatggagct gagcagcctg agatctgagg acacggccgt gtattactgt 300 gcgagaggtc tgccctgggg tgaaaacggt ctggacgtct ggggccaggg gaccacggtc 360 accgtctcct cagctagcac caagggccca tcggtcttcc ccctggcacc ctcctccaag 420 agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 480 gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 540 ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 600 ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 660 agagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 720 ctcctggggg gaccgtcagt cttcctcttt cccccaaaac ccaaggacac cctcatgatc 780 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 840 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 900 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 960 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1020 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1080 tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1140 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1200 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1260 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1320 aaccactaca cgcagaagag cctctccctg tctccgggta aacccgccca ggtggcattt 1380 acaccctacg ccccggagcc cgggagcaca tgccggctca gagaatacta tgaccagaca 1440 gctcagatgt gctgcagcaa gtgctcgccg ggccaacatg caaaagtctt ctgtaccaag 1500 acctcggaca ccgtgtgtga ctcctgtgag gacagcacat acacccagct ctggaactgg 1560 gttcccgagt gcttgagctg tggctcccgc tgtagctctg accaggtgga aactcaagcc 1620 tgcactcggg aacagaaccg catctgcacc tgcaggcccg gctggtactg cgcgctgagc 1680 aagcaggagg ggtgccggct gtgcgcgccg ctgcgcaagt gccgcccggg cttcggcgtg 1740 gccagaccag gaactgaaac atcagacgtg gtgtgcaagc cctgtgcccc ggggacgttc 1800 tccaacacga cttcatccac ggatatttgc aggccccacc agatctgtaa cgtggtggcc 1860 atccctggga atgcaagcat ggatgcagtc tgcacg 1896 <210> 26 <211> 345 <212> DNA <213> Homo sapiens <400> 26 gatgtccact cgcagctcgt gctaactcag ccgtcctcag tgtctggggc cccagggcag 60 acagtcacca tatcatgcac tgggagcagc tccaacatcg gggcaggtta tgatgtacac 120 tggtaccagc agcttccagg aacagccccc aaagtcctca tctatggtga tagtgatcgg 180 ccctccgggg tccctgaccg attctctggt tccaagtctg ccacctcagc ctccctggcc 240 atcactgggc tccaggctga ggatgaggct gattattact gccagtccta tgacagcagc 300 ctgagtgctt atgtcttcgg aactgggacc aaggtcaccg tccta 345 <210> 27 <211> 1905 <212> DNA <213> Artificial Sequence <220> <223> anti-IL6R D2 HC / O-glycosylation region deleted TNFR2 DNA sequence <400> 27 gatgtccact cgcaggtgca gctggtggag tctggggctg aggtgaagaa gcctgggtcc 60 tcggtgaagg tctcctgcaa gggttctgga gacaccttcc ccaactatgc tatcaactgg 120 gtgcgacagg cccctggaca agggcctgag tggatgggaa gaatcatccc gatgcttgga 180 acatcagact acgcagagaa attccagggc agagtcacga tcaccgcgga caaatccacg 240 aacacagcct acatggggct gaacagcctg agatctgagg acacggccgt gtattactgt 300 gttaaaggcc caagatacta cggcacggac agttattatc ttgagaagtg gggccaggga 360 accatggtca ccgtctcctc agctagcacc aagggcccat cggtcttccc cctggcaccc 420 tcctccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480 cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540 ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600 agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660 gtggacaaga gagttgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca 720 gcacctgaac tcctgggggg accgtcagtc ttcctctttc ccccaaaacc caaggacacc 780 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 840 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 900 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 960 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1020 cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1080 ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 1140 ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1200 tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 1260 accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1320 gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa acccgcccag 1380 gtggcattta caccctacgc cccggagccc gggagcacat gccggctcag agaatactat 1440 gaccagacag ctcagatgtg ctgcagcaag tgctcgccgg gccaacatgc aaaagtcttc 1500 tgtaccaaga cctcggacac cgtgtgtgac tcctgtgagg acagcacata cacccagctc 1560 tggaactggg ttcccgagtg cttgagctgt ggctcccgct gtagctctga ccaggtggaa 1620 actcaagcct gcactcggga acagaaccgc atctgcacct gcaggcccgg ctggtactgc 1680 gcgctgagca agcaggaggg gtgccggctg tgcgcgccgc tgcgcaagtg ccgcccgggc 1740 ttcggcgtgg ccagaccagg aactgaaaca tcagacgtgg tgtgcaagcc ctgtgccccg 1800 gggacgttct ccaacacgac ttcatccacg gatatttgca ggccccacca gatctgtaac 1860 gtggtggcca tccctgggaa tgcaagcatg gatgcagtct gcacg 1905 <210> 28 <211> 342 <212> DNA <213> Homo sapiens <400> 28 gatgtccact cgcagctcgt gctgactcag ccgccctcag tgtctggggc cccaggccag 60 acggtcacca tctcctgcac tgggcccacc atcggggcag gatatgatgt ccactggtac 120 caacaacttc caggagcagc ccccaaactc ctcatctatg gtaatctcaa tcggccctca 180 ggtgtccctg accgattctc tggctccaag tctggcacct cagcctccct ggccatcacg 240 gacctccagg ctgaggatga ggctgactat tactgccata cctatgacag tagcctcagt 300 ggttcttggg tcttcggcgg agggaccaag ctgaccgtcc ta 342 <210> 29 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TNFR2-F (SfiI) Forward <400> 29 agggggccgt gggggccttg cccgcccagg tgg 33 <210> 30 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> TNFR2-R (SfiI) Reverse <400> 30 tagcggccga cgcggccaat tcagctgggg ggct 34 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Opti-TNFR2-R (SfiI) Reverse <400> 31 tagcggccga cgcggccaac gtgcagactg catcc 35 <210> 32 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> TNFR2-M2-F (SfiI) Forward <400> 32 agggggccgt gggggccttg cccgcccagg tggcatttac accctacgcc ccggagaac 59 <210> 33 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> TNFR2-F Forward <400> 33 ttgcccgccc aggtgg 16 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Opti-TNFR-R (XhoI) Reverse <400> 34 ccagctcgag cgtgcagact gcatcc 26 <210> 35 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> NATVK1-1 (SfiI) Forward <400> 35 ttggtggcca cagcggccga tgtccactcg gatgtccact cgcagctcg 49 <210> 36 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> NATJK-R4 (BglII) Reverse <400> 36 gaggagagat cttaggacgg tgaccttgg 29 <210> 37 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> NATVK5 (SfiI) Forward <400> 37 ttggtggcca cagcggccga tgtccactcg cagctcgtgc tgactcag 48

Claims (13)

A method of improving the efficiency of a Tumor necrosis factor receptor type 2 (TNFR2) protein, the method comprising removing an O-glycosylation site, an N-glycosylation site, or an O-glycosylation site and an N-glycosylation site. The method of claim 1,
Wherein the O-glycosylation site is an amino acid site 202 to 252 from the N-terminus of TNFR2. The method of claim 1,
Wherein the N-glycosylation site is 28, 31 or 34 from the N-terminus of TNFR2. The method of claim 3,
Wherein the N-glycosylation site, characterized in that to replace the proline No. 34 (Proline) with asparagine (Asparagine). A fragment of TNFR2 represented by SEQ ID NO: 2, with the O-glycosylation site removed from the amino acid sequence of Tumor necrosis factor receptor type 2 (TNFR2) described in SEQ ID NO: 1. The method of claim 5,
The O-glycosylation site is a fragment, characterized in that amino acid sites 202 to 252 from the N- terminal of TNFR2. A fragment of TNFR2 represented by SEQ ID NO: 3 with the N-glycosylation site removed from the amino acid sequence of Tumor necrosis factor receptor type 2 (TNFR2) described in SEQ ID NO: 1. The method of claim 7, wherein
The N-glycosylation site is a fragment, characterized in that for replacing proline No. 34 (Proline) with asparagine (Asparagine). A fragment of TNFR2 represented by SEQ ID NO: 4, wherein both the O-glycosylation site and the N-glycosylation site are removed from the amino acid sequence of Tumor necrosis factor receptor type 2 (TNFR2) described in SEQ ID NO: 1. 10. The method of claim 9,
The O-glycosylation site is a fragment, characterized in that amino acid sites 202 to 252 from the N- terminal of TNFR2. 10. The method of claim 9,
The N-glycosylation site is a fragment, characterized in that for replacing proline No. 34 (Proline) with asparagine (Asparagine). A pharmaceutical composition for preventing or treating autoimmune diseases, comprising the fragment of any one of claims 5 to 11 as an active ingredient. The method of claim 12,
Wherein said autoimmune disease is a disease selected from the group consisting of pernicious anemia, type 1 diabetes mellitus, autoimmune arthritis, lupus, multiple sclerosis, reactive arthritis, and dermatitis.

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