KR20120130478A - TNF- specific antibody and use thereof - Google Patents

Abstract

PURPOSE: A human TNF-alpha specific antibody containing a heavy chain constant region derived from human IgG2 and IgG4 is provided to minimize ADCC(Antibody-Dependent Cell-mediated Cytotoxicity) and CDC(Complement-Dependent Cytotoxicity). CONSTITUTION: A human TNF-alpha specific antibody contains: a light chain containing a light chain constant region and a light chain variable region with light chain CDR1 of sequence number 1, light chain CDR2 of sequence number 2, and light chain CDR3 of sequence number 3; and a heavy chain containing a heavy chain constant region and a heavy chain variable region. A method for preparing the human TNF-alpha specific antibody comprises: a step of culturing a transformant; and a step of purifying the human TNF-alpha specific antibody from a culture medium. A pharmaceutical composition for preventing or treating TNF-alpha-mediated diseases contains the human TNF-alpha specific antibody.

Description

TNP-α specific antibody and use thereof

The present invention provides a human TNF-α specific antibody comprising a heavy chain constant region derived from human IgG2 and IgG4, a polynucleotide encoding the heavy or light chain of the antibody, an expression vector comprising the polynucleotide, and a transformation with the expression vector. The present invention relates to a transformant, a method for producing the antibody, and a pharmaceutical composition for preventing or treating a TNF-α mediated disease comprising the antibody.

The immune system protects the body from harmful foreign antigens. These types of antigens include bacteria, viruses, toxins, cancer cells and blood and tissues from other people 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 hypersensitivity reactions, but in the case of allergies it reacts to foreign substances that are not harmful to the body, but when autoimmune abnormalities occur, the reaction occurs in the normal tissue of the body. It is not clear what causes the immune system to distinguish between organs and antigens in the body, but it is hypothesized that microorganisms or drugs, such as bacteria, cause illness in people born with genes susceptible to autoimmune diseases.

One type of autoimmune disease, autoimmune arthritis (RA), is an autoimmune 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, it is a serious disease leading to joint failure. In addition to the inflammation and pain of the joints as well as autoimmunity, the patients with massive organ invasion and pain that invade the joint and surrounding tissues and many organs and invade into the osteoporosis, lung, skin and eyes It leads to poor quality of life and makes life impossible for normal people.

Increased levels of TNF-α have been reported in both synovial and terminal blood of such autoimmune arthritis patients. TNF-α is a pro-inflammatory cytokine secreted by cells of the immune system and reacting with cells of the immune system. Thus, TNF-α is secreted by macrophages activated by lipopolysaccharide (LPS) of Gram-negative bacteria. Blocking agents for these TNF-α, when injected into patients with autoimmune arthritis can reduce inflammation, improve symptoms and delay joint damage.

Currently, blocking agents for TNF-α used for the treatment of autoimmune arthritis are available from Enbrel ^™ , marketed by Amgen. Remicade ^TM, marketed by Centocor Corporation and Cimzia ^™ , marketed by Union Chimique Belge.

Among them, Remicade ^™ is a chimeric antibody with a murine anti-TNF-α variable domain and a human IgG1 constant domain, and antibody-dependent cellularcytotoxicity (ADCC) and complement dependence when targeting TNF-α. It is essential to prevent complement-dependent cytotoxicity (CDC), but IgG1 causes ADCC and CDC. In addition, Cimzia ^TM As Fab that binds to human TNF-α, it is commercially available in PEGylated form, and it is known by animal experiments that PEGylated proteins or molecules accumulate in kidney cells to form vacuole. Disadvantages that can trigger PEG immune responses are also known by animal experiments.

Therefore, the inventors of the present invention, as antibodies that bind specifically to TNF-α with high affinity, do not induce ADCC and CDC, and as a result of diligent efforts to develop antibodies to solve the problems of PEGylated antibodies, It was confirmed that the antibody containing the heavy chain constant region of the fused form can solve the above problem, and completed the present invention.

An object of the present invention is a light chain comprising a light chain variable region and a light chain constant region comprising a light chain CDR1 of SEQ ID NO: 1, a light chain CDR2 of SEQ ID NO: 2 and a light chain CDR3 of SEQ ID NO: 3; And a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 5, and a heavy chain CDR3 of SEQ ID NO: 6, a CH1 derived from IgG2 represented by SEQ ID NO: 7, a hinge derived from IgG2 represented by SEQ ID NO: 8, It provides a human TNF-α specific antibody comprising a; heavy chain comprising a heavy chain constant region comprising IgG2 represented by SEQ ID NO: 9 and CH2 derived from IgG4 and CH3 derived from IgG4 represented by SEQ ID NO: 10.

Another object of the present invention is to provide a polynucleotide encoding the heavy chain of the antibody.

Another object of the present invention is to provide a polynucleotide encoding the light chain of the antibody.

Still another object of the present invention is to provide an expression vector comprising the polynucleotide.

Still another object of the present invention is to provide a transformant transformed with an expression vector comprising a polynucleotide encoding the light and heavy chains of the antibody.

Still another object of the present invention is the step of culturing the transformant; And it provides a method for producing a human TNF-α specific antibody comprising the step of purifying the antibody from the culture.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating TNF-α mediated disease comprising the antibody.

As one embodiment for achieving the above object, the present invention is a light chain comprising a light chain variable region and a light chain constant region comprising a light chain CDR1 of SEQ ID NO: 1, a light chain CDR2 of SEQ ID NO: 2 and a light chain CDR3 of SEQ ID NO: 3; And a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 5, and a heavy chain CDR3 of SEQ ID NO: 6, a CH1 derived from IgG2 represented by SEQ ID NO: 7, a hinge derived from IgG2 represented by SEQ ID NO: 8, A heavy chain comprising a heavy chain constant region comprising an IgG2 represented by SEQ ID NO: 9 and a CH3 derived from IgG4, and a CH3 derived from IgG4 represented by SEQ ID NO: 10; provides a human TNF-α specific antibody.

The antibody of the present invention is an antibody in which the heavy chain constant regions constituting the antibody molecule, CH1, hinge, CH2 and CH3 are composed of amino acid sequences derived from IgG2 and IgG4, and the antibody is composed of CH1 (SEQ ID NO: 7) and hinge (SEQ ID NO: 7). No. 8) consists of an amino acid sequence derived from IgG2, and CH3 (SEQ ID NO: 10) consists of an amino acid sequence derived from IgG4, while CH2 (SEQ ID NO: 9) consists of an amino acid sequence derived from IgG2 and IgG4.

Generally, one antibody molecule has two heavy chains and two light chains, each of which has a variable region at its N-terminus. Each variable region consists of three complementarity determining regions (CDRs) and four framework regions (FRs), which determine the antigen-binding specificity of an antibody and determine the structure of the variable region. There is a relatively short peptide sequence that is maintained at the sites.

The antibody of the present invention is also an antibody specifically binding to human TNF-α, and includes a light chain CDR1 (KASQNVGTNVA) of SEQ ID NO: 1, a light chain CDR2 (SASFLYS) of SEQ ID NO: 2, and a light chain CDR3 (QQYNIYPLT) of SEQ ID NO: 3 A heavy chain variable region comprising a variable region and a light chain constant region and a heavy chain CDR1 (DYGMN) of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 5 (WINTYIGEPIYADSVKG), and a heavy chain CDR3 of SEQ ID NO: 6 (GYRSYAMDY) and SEQ ID NO: 7 A heavy chain constant region comprising a CH1 derived from IgG2, a hinge derived from IgG2 represented by SEQ ID NO: 8, an IgG2 represented by SEQ ID NO: 9 and CH2 derived from IgG4, and a CH3 derived from IgG4 represented by SEQ ID NO: 10 It may include a heavy chain.

CH1 from IgG2 represented by SEQ ID NO: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV

Hinge derived from IgG2 represented by SEQ ID NO: 8 ERKCCVECPPCP

CH2 from IgG2 and IgG4 represented by SEQ ID NO: 9: APPVAGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK

CH3 from IgG4 represented by SEQ ID NO: 10: GQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Preferably, the light chain constant region may be represented by the amino acid sequence of SEQ ID NO: 11, the light chain variable region may be represented by the amino acid sequence of SEQ ID NO: 12. Also, preferably, the heavy chain variable region may be represented by the amino acid sequence of SEQ ID NO.

SEQ ID NO: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 12 DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYS GVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIK

SEQ ID NO: 13 EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYI GEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSS

In one embodiment of the present invention, the human TNF-α specific antibody (ABX902) of the present invention was prepared, and the binding force to human TNF-α (FIG. 2), binding affinity (FIG. 3), and TNF-α Inhibition of apoptosis (FIG. 4) and in vivo efficacy in the TNF-α-induced lethality model of the mouse (Table 2) confirmed that the binding ability to TNF-α is excellent, inhibiting TNF-α apoptosis and lethality It could be confirmed. Accordingly, the human TNF-α specific antibodies of the present invention can be usefully used for the prevention and treatment of TNF-α mediated diseases, and also accumulate in the kidney cells generated from conventional PEGylated antibodies to form vacuoles. There is an advantage that can solve the problem that can cause the anti-PEG immune response.

In addition, recent studies on the adverse effects of various anti-TNF-α antibody drugs revealed that the PEGylated Cimzia had more severe side effects of serious infections than other antibody drugs. This means that PEGylated antibodies can become infected when the infection occurs due to the faster and longer accumulation of macromolecules (eg, PEG) of more than 20 kDa at the inflammation site (EPR). It is thought that the early defense in the body is more incapacitated by blocking TNF α faster and longer at the infection site. In this regard, although the antibody of the present invention is also a polymer, unlike the pegylated antibody, it is mainly recycled in endothelial cells through the neonatal Fc receptor (FcRn) and thus less EPR occurs. There is an advantage that does not cause serious infection than the antibody that has been lengthened.

In another aspect, the invention provides a polynucleotide encoding the heavy chain of an antibody according to the invention or a polynucleotide encoding the light chain of an antibody according to the invention.

In still another aspect, the present invention provides an expression vector comprising the polynucleotide according to the present invention.

The polynucleotide of the present invention is a polymer of nucleotides in which nucleotide monomers are long chained by covalent bonds, and are DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) strands of a predetermined length or more, and the antibody according to the present invention. It is a polynucleotide encoding the heavy or light chain of.

The polynucleotide encoding the heavy or light chain of the antibody of the present invention is expressed from the coding region due to the degeneracy of the codon or in consideration of the codons preferred in the organism to express the heavy or light chain of the antibody. Various modifications may be made to the coding region within the range of not changing the amino acid sequence of the antibody, and various modifications or modifications may be made to the region except for the coding region without affecting the expression of the gene. It will be understood by those skilled in the art that it is also within the scope of the present invention. That is, as long as the polynucleotide of the present invention encodes a protein having equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, which are also included in the scope of the present invention.

The expression vector of the present invention is a means for introducing the DNA into the host cell to express the antibody of the present invention in a microorganism. When preparing the expression vector, a promoter (promoter) according to the type of the host cell to produce the antibody, Expression control sequences such as terminators, enhancers, and the like, sequences for membrane targeting or secretion, etc. may be appropriately selected and various combinations may be made depending on the purpose.

Expression vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Preferably, the expression vector of the present invention may be a vector capable of expressing the heavy chain and the light chain of the antibody according to the present invention by respective viral promoters. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers and can be prepared in various ways depending on the purpose. The promoter of the expression vector may be constitutive or inducible. As the signal sequence, MFα signal sequence, SUC2 signal sequence, etc. may be used when the host is a yeast, and insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. may be used when the host is an animal cell. It is not limited to this. In addition, the expression vector may include a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, may include a replication origin.

In another embodiment, the present invention provides a transformant transformed with an expression vector comprising a polynucleotide encoding the heavy and light chains of an antibody according to the present invention, and (1) culturing the transformant of the present invention. step; And (2) provides a method for producing a human TNF-α specific antibody comprising the step of purifying the antibody of the present invention from the culture of step (1).

The antibody of the present invention is transformed by transforming an expression vector comprising polynucleotides encoding the heavy and light chains of the antibody of the present invention into an appropriate host cell, for example, a yeast cell or an animal cell, and then culturing the transformed host cell. Can mass produce. In addition, an expression vector comprising a polynucleotide encoding the heavy chain of the antibody and an expression vector containing the polynucleotide encoding the light chain of the antibody are simultaneously transformed into appropriate host cells, followed by culturing the transformed host cell. The mass production of the antibody of this invention can be carried out by this.

As used herein, the term "transformation" refers to introducing a gene into a host cell so that the gene can be expressed in the host cell, and if the transformed gene can be expressed in the host cell, the insertion into the chromosome of the host cell or Anything located outside the chromosome is included without limitation. The gene also includes DNA and RNA as polynucleotides capable of encoding a polypeptide. The gene may be introduced in any form as long as it can be expressed by being introduced into a host cell. For example, the gene may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct containing all the elements necessary for its expression. The expression cassette typically includes a promoter, transcription termination signal, ribosomal binding site and translation termination signal operably linked to the gene.

The transformation method by introducing the expression vector into the host cell is a method known in the art, such as, but not limited to, transient transfection of the expression vector comprising the heavy or light chain DNA of the antibody of the present invention. ), Micro injection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene- It can be transformed by introducing into host cells by a known method such as mediated transfection (polybrene-mediated transfection), electroporation (electroporation).

The host cell may be a eukaryotic cell derived from yeast, insect cells, plant cells, animal cells, such as Saccharomyces cerevisiae, and the animal cell may be an autologous or allogeneic animal cell. Preferably, the animal cell may be a mammalian cell, more preferably a CHO cell.

Preferably, the transformant may be a cell having accession number KCLRF-BP-00263. The cell line having accession number KCLRF-BP-00263 is a cell line stably expressing a human TNF-α specific antibody of the present invention. It was deposited with the Korean Cell Line Research Foundation on April 27, 2011 and was given accession number KCLRF-BP-00263.

The method of culturing the transformant of the present invention may be appropriately selected and used in any method known in the art for producing the human antibody or fragment thereof of the present invention.

As the culture medium, it is preferable to select a medium suitable for the transformant from the culture medium known to those skilled in the art. The antibody purification method may be any purification method known to those skilled in the art.

In one embodiment of the present invention, the light chain DNA and heavy chain DNA of the antibody are introduced into one eukaryotic expression vector using different restriction endonuclease sites, and the expression vector is introduced into HEK293 cells. After transfection into, incubated for 7 days at a temperature of 37 ° C. in Free style 293 medium, the supernatant was taken only by centrifugation, and purified by using AKTApurifier (GE) equipment and Mabselect column. The size of the purified antibody was analyzed using SDS-PAGE and HPLC equipment. As a result, it was confirmed that an antibody having a molecular weight of 150 kDa was purified to a purity of 97% or more (Example 2 and FIG. 1).

As another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of TNF-α mediated disease comprising the antibody of the present invention.

In still another aspect, the present invention provides a method for preventing or treating a TNF-α mediated disease, comprising administering the pharmaceutical composition to a subject having or likely to develop a TNF-α mediated disease. do.

In the present invention, "prophylaxis" means any action that inhibits or delays the onset of TNF-α mediated disease by administration of the pharmaceutical composition of the present invention, and "treatment" refers to the administration of the pharmaceutical composition of the present invention. By all means the action to improve or beneficially change the symptoms caused by TNF-α mediated disease.

In the present invention, "individual" means any animal, including humans, who can develop or invent a TNF-α mediated disease.

Preferably, the TNF-α mediated disease is inflammatory bowel disease including an autoimmune arthritis, osteoarthritis, psoriasis, psoriatic arthritis, atopic dermatitis, ulcerative colitis and Crohn's disease, ankylosing spondylitis, multiple sclerosis, systemic lupus erythematosus (SLE) ), Chronic obstructive pulmonary disease (COPD), sepsis, endotoxin shock, hepatitis and type 1 diabetes, and more preferably autoimmune arthritis, osteoarthritis or Crohn's disease.

The route of administration of the pharmaceutical compositions of the invention can be administered via any general route so long as it can reach the desired tissue or cell. The pharmaceutical compositions of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, pulmonary, rectally, intracellularly or indirectly, as desired. To this end, the pharmaceutical compositions of the present invention may be administered by any device in which the active substance may migrate to the target cell.

The pharmaceutical composition of the present invention may comprise an acceptable carrier. The pharmaceutical composition comprising a pharmaceutically acceptable carrier may be in a variety of oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablet pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose in one or more compounds. ) And gelatin. Liquid preparations for oral administration include suspensions, solution solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories It can have any one formulation.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is determined by the type of subject and its severity, age, sex, activity of the drug, drug. Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the treatment of TNF-α mediated diseases.

The human TNF-α specific antibodies of the present invention can be expressed and purified in mammalian cells in the form of IgG2 / 4, which has the advantage of being able to produce uniformly.

In addition, the antibodies of the present invention have the advantage of minimizing antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).

In addition, the antibody of the present invention may cause pegylated proteins or molecules generated in pegylated antibodies to accumulate in kidney cells to form vacuoles, to cause anti-PEG immune responses, and to cause serious infections. There is an advantage that can solve the problem.

1 is a reduction (R) and non-reduction (NR) SDS-PAGE result (A) and size exclusion chromatography (B) showing that the TNF-α specific antibody (ABX902) is present in a single molecule form.
FIG. 2 shows ELISA results showing binding of TNF-α specific antibody (ABX902) to human TNF-α.
Figure 3 shows the results of analysis of the binding affinity of the TNF-α specific antibody (ABX902) to human TNF-α by SPR.
Figure 4 is a graph showing the cell death inhibition of TNF-α specific antibody (ABX902) by TNF-α.
Figure 5 is an ELISA result showing the binding capacity of human TNF-α of TNF-α specific antibody (ABX902) produced in the cell line of the present invention.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  One : TNF -α specific antibodies ( ABX902 Gene production

First, DNA corresponding to the variable regions (SEQ ID NOs: 12 and 13) of the TNF-α specific antibody (hereinafter referred to as ABX902 antibody) of the present invention was synthesized. The light chain variable region portion (VL) was PCR amplified using the primers of SEQ ID NOs: 14 and 15 of Table 1 as a template using the synthesized variable region DNA. For each PCR reaction, 200 ng of template DNA, 15 pmol of forward and reverse primers, 0.5 μl of Taq polymerase, 1.75 μl of 10 mM dNTP mixture, and 10 μl of 10 × PCR buffer were added. Add to μl. The mixture was repeated 25 minutes at 94 ° C. for 3 minutes, 94 ° C. at 45 seconds-56 ° C., 30 seconds at 72 ° C., 1 minute 30 seconds, and then left at 72 ° C. for 10 minutes. Amplified DNA was electrophoresed on 0.8% agarose gel and then purified using a gel extraction kit. The VL thus prepared was treated with restriction enzymes (Msc I, Rsr II, NEB) and inserted into a vector (pBluescript SK derived vector) containing a human kappa constant sequence to prepare light chain DNA. Except for using SEQ ID NO: 16 and 17, the DNA of the heavy chain variable region (VH) was PCR amplified and purified in the same manner as above.

In addition, DNA in the form in which the heavy chain constant region (CH) of human IgG2 and IgG4 was fused was synthesized by Genscript (USA). This is a fusion of the DNA of CH1, hinge and CH2 of IgG2 (1st to 29th amino acid) with a portion of CH2 (31st to 110th amino acid) of IgG4 and DNA of CH3. The synthesized heavy chain constant region DNA was inserted into a vector (pBluescript SK) treated with restriction enzymes (Apa I, BamHI). The heavy chain variable region DNA prepared above was treated with restriction enzymes (MscI, Apa I) and inserted into a vector containing CH to prepare heavy chain DNA.

Example  2 : ABX902  Expression and Purification of Antibodies

ABX902 produced in Example 1 The light and heavy chain DNAs of the antibody were introduced into one eukaryotic expression vector using different restriction endonuclease sites. The expression vectors are designed to express light and heavy chains by respective viral promoters. After transfection of the expression vector into HEK293 cells, the cells were cultured in a free style 293 medium at a temperature of 37 ° C. for 7 days, followed by centrifugation to take only the supernatant, followed by expression using an AKTApurifier (GE) device and a Mabselect column. The purified antibody was then purified, and the size of the purified antibody was analyzed using SDS-PAGE and HPLC equipment. As a result, it was confirmed that an antibody having a molecular weight of 150 kDa was purified to a purity of 97% or more.

Size exclusion chromatography (SEC) and reducing and non-reducing SDS-PAGE were performed to determine whether the purified antibody was present in solution or in the form of a single molecule or multi-molecule. As a result, as shown in FIG. 1, when the antibody was injected at 1.3 mg / ml, it could be seen that purely eluted as a single molecule, and there was no multimolecular form even in reducing and non-reducing SDS-PAGE (FIG. 1). ).

Example  3: ABX902  Affinity measurement of antibodies

The binding activity of the ABX902 antibody to human TNF-α was determined by indirect ELISA. To this end, the recombinant human TNF-α protein was coated on a 96 well plate, 0.1 pM to 1 μM of ABX902 antibody and HRP-conjugated anti-human Fc antibody were added sequentially, and the color development by ABTS was absorbed at 405 nm. Quantification by measurement. ELISA was performed at the same concentration range using Humira (component name: Adalimumab, Abbott), an antibody against the same antigen, as a control. As a result, as shown in Figure 2, it was confirmed that the antibody and Humira antibody of the present invention has a similar binding force (Fig. 2).

Binding affinity to human TNF-α was measured using Biacore X100 SPR. ABX902 After immobilizing the antibody to a CM5 chip (Carboxylmethylated dextran surface chip), the recombination results in the injection of human TNF-α quantification of interaction with the antibody, binding affinity kinetic rate constant was diluted at various concentrations (K _on and K _off ) and the equilibrium dissociation constant (K _D ) were obtained (FIG. 3).

Example  4 : ABX902  Antibody TNF Cell death by -α Inhibitory ability  Confirm

Inhibition of cell death of the ABX902 antibody was confirmed using mouse fibroblasts (L929 cell line). Flat-bottomed 96-well plates were seeded with 1.5 × 10 ⁴ L929 cells and incubated at 37 ° C. for 24 hours. After removing the medium, a mixture of recombinant human TNF-α 0.6 ng / ml and 1 μg / ml Actinomycin D was treated with antibodies, including ABX902 antibodies at various concentrations. After incubation at 37 ° C. for 16 hours, it was used for MTT analysis. As a control, the same assay was performed within the same concentration range using proteins (Humira, Remicade, Embrel, etc.) for the same antigen. The difference in absorbance at 550 nM and 650 nM indicates the survival of L929 cells.

As a result, as shown in FIG. 4, it was confirmed that the ABX902 antibody inhibited apoptosis by TNF-α in a concentration-dependent manner (FIG. 4).

Example  5: mouse TNF using a α-induced lethal model ABX902  Confirmation of In vivo Efficacy of Antibodies

Injecting human TNF-α with D-galactosamine in mice rapidly dies after some time. In this animal model, we tried to determine whether ABX902 antibody can suppress the death caused by TNF-α. To this end, several concentrations of ABX902 antibody were administered intraperitoneally, and 30 minutes later, a mixed solution of recombinant human TNF-α and D-galactosamine was administered intraperitoneally to evaluate survival after 24 hours. Survival evaluation was performed when the same concentration was treated using Humira, an antibody against the same antigen as a control.

As a result, as shown in Table 2, it was confirmed that the ABX902 antibody inhibited lethality caused by TNF-α above 10 ㎍ / mouse concentration (Table 2).

Example  6: ABX902  Construction of mammalian cell line stably expressing antibody

<6-1: ABX902  Construction of Antibody-producing Cell Lines>

The plasmid containing the DNA sequence encoding the ABX902 antibody was transfected into CHO cells by electroporation, and the cells were selected after 48 hours in selection medium (including L-glutamine, 500 μg / ml G418). Incubation began. When the survival rate of the cells was lowered to more than 90%, 5 x 10 ⁵ cells / ml of cells were placed in a 125 ml shake flask and 50 nM, 100 nM, and 250 nM MTX (methotrexate) were treated. Each concentration was incubated in a shaking incubator with 30 ml volume.

After survival of the cells cultured for 14-21 days by MTX concentration was improved to 90% or more, they were subjected to limiting dilution to allow one cell to enter 100 µl of medium in each well of a 96 well plate. . After culturing these colonies to grow to about 75% of the wells in a 5% CO ₂ incubator, the culture supernatant was taken and the candidate expression was selected by measuring the antibody expression by ELISA. Selected candidate clones were transferred to 48 well plates and 24 well plates in order to increase the number of cells, and then the same number of cells of each clone were placed in 24 well plates and cultured for a period of time, followed by ELISA. Excellent clones were selected by quantification.

Selected clones were transferred to T25 flasks and T75 flasks in turn, and then cultured in 125 ml shake flasks. The same number of cells of each candidate clone was placed in a 125 ml shake flask and cultured under the same conditions (batch culture) to quantify the amount of antibody in the culture supernatant using ELISA. As a result, the clone with the largest amount of antibody expression (M100-19, 50 mg / L) was selected as the final clone. The M100-19 was deposited with the Korean Cell Line Research Foundation on April 27, 2011 and was given accession number KCLRF-BP-00263.

<6-2: ABX902  Purified from Antibody-producing Cell Lines ABX902  Confirmation of Antibody Binding Capacity>

After purifying the antibody in the culture supernatant in the same manner as in Example 2, the binding capacity of the ABX902 antibody of the present invention to human TNF-α in the same manner as in Example 3 was confirmed through indirect ELISA. As a result, as shown in Figure 5, it was confirmed that the antibody of the present invention has a specific binding capacity to human TNF-α (Fig. 5).

Korea Cell Line Research Foundation KCLRFBP00263 20110427

<110> Abxign, Inc. <120> TNF-alpha specific antibody and use <130> PA100753KR <160> 17 <170> Kopatentin 1.71 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 of ABX902 <400> 1 Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala   1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 of ABX902 <400> 2 Ser Ala Ser Phe Leu Tyr Ser   1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 of ABX902 <400> 3 Gln Gln Tyr Asn Ile Tyr Pro Leu Thr   1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of ABX902 <400> 4 Asp Tyr Gly Met Asn   1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 of ABX902 <400> 5 Trp Ile Asn Thr Tyr Ile Gly Glu Pro Ile Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of ABX902 <400> 6 Gly Tyr Arg Ser Tyr Ala Met Asp Tyr   1 5 <210> 7 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> CH1 of ABX902 <400> 7 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg   1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr              20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser          35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser      50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr  65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                  85 90 95 Thr val         <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> hinge of ABX902 <400> 8 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro   1 5 10 <210> 9 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> CH2 of ABX902 <400> 9 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro   1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val              20 25 30 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val          35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln      50 55 60 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln  65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly                  85 90 95 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys             100 105 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CH3 of ABX902 <400> 10 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu   1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe              20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu          35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe      50 55 60 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly  65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr                  85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys             100 105 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CL of ABX902 <400> 11 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu   1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe              20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln          35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser      50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu  65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                  85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 12 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL of ABX902 <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn              20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile          35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Tyr Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Leu                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH of ABX902 <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Val Phe Thr Asp Tyr              20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met          35 40 45 Gly Trp Ile Asn Thr Tyr Ile Gly Glu Pro Ile Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Tyr Arg Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 14 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> forward primer for VL <400> 14 tggtggccac cgccaccggc gacattcaaa tgacccagag c 41 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for VL <400> 15 ccacggtccg tttgatttct actttagtac c 31 <210> 16 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> forward primer for VH <400> 16 tggtggccac cgccaccggc gaggttcagc tggtcgagt 39 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for VH <400> 17 aggggccctt ggtggaggct gaggagactg tgactagggt 40

Claims (14)

A light chain comprising a light chain variable region and a light chain constant region comprising a light chain CDR1 of SEQ ID NO: 1, a light chain CDR2 of SEQ ID NO: 2, and a light chain CDR3 of SEQ ID NO: 3; And
A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 5, and a heavy chain CDR3 of SEQ ID NO: 6, a CH1 derived from IgG2 represented by SEQ ID NO: 7, a hinge derived from IgG2 represented by SEQ ID NO: 8, and a sequence A human TNF-α specific antibody comprising a heavy chain comprising a heavy chain constant region comprising an IgG2 represented by No. 9 and an CH4 derived from IgG4 and a CH3 derived from IgG4 represented by SEQ ID NO: 10.
The antibody of claim 1, wherein the light chain constant region is represented by the amino acid sequence of SEQ ID NO: 11.
The antibody of claim 1, wherein the light chain variable region is represented by the amino acid sequence of SEQ ID NO. 12.
The antibody of claim 1, wherein the heavy chain variable region is represented by the amino acid sequence of SEQ ID NO: 13. 3.
Polynucleotide encoding the heavy chain of the antibody according to any one of claims 1 to 4.
A polynucleotide encoding the light chain of an antibody according to any one of claims 1 to 4.
An expression vector comprising the polynucleotide of claim 5.
An expression vector comprising the polynucleotide of claim 6.
A transformant transformed with an expression vector comprising the polynucleotide of claim 5 and claim 6.
The transformant of claim 9, wherein the transformant is a mammalian cell.
The transformant of claim 9, wherein the transformant is accession number KCLRF-BP-00263.
(1) culturing the transformant of claim 9; And
(2) A method for producing a human TNF-α specific antibody comprising the step of purifying the antibody of any one of claims 1 to 4 from the culture medium of step (1).
A pharmaceutical composition for preventing or treating a TNF-α mediated disease comprising the antibody of any one of claims 1 to 4.
The method of claim 13, wherein the TNF-α mediated disease is an autoimmune arthritis, osteoarthritis, psoriasis, psoriatic arthritis, atopic dermatitis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, multiple sclerosis, systemic lupus erythematosus A composition selected from the group consisting of lupus (SLE), chronic obstructive pulmonary disease (COPD), sepsis, endotoxin shock, hepatitis and type 1 diabetes.

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