WO2008141511A1

WO2008141511A1 - A HUMAN ANTI-TNFα MONOCLONAL ANTIBODY AND THE USE THEREOF

Info

Publication number: WO2008141511A1
Application number: PCT/CN2007/070044
Authority: WO
Inventors: Qingfa Liu
Original assignee: Human Antibodomics (Shanghai) Inc.
Priority date: 2007-05-22
Filing date: 2007-05-22
Publication date: 2008-11-27
Also published as: WO2008141511A8

Abstract

A protein specifically binding to human TNFα (tumor necrosis factor α) is disclosed, i.e. human anti-TNFα monoclonal antibody derived from human B cells. The light and heavy chain of the antibody have the amino acid sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Also disclosed are the encoded sequences of the human anti- TNFα antibody, its Fab antibody and the peglated Fab antibody. The present invention further provides the uses of the antibodies in the preparation of the medicament for treatment of TNFα-related inflammation.

Description

Anti-human TNFa monoclonal antibody and its application

TECHNICAL FIELD The present invention relates to the field of biotechnology, and in particular to TNFa binding proteins and uses thereof.

BACKGROUND OF THE INVENTION Regarding rheumatoid arthritis:

Rheumatoid arthritis (RA) is a common disease worldwide, which is characterized by red, swollen and painful joints of the limbs, which seriously affects the quality of life of patients. If not treated, it can cause joint rigidity within 2 years. Permanent sequelae such as deformity. The incidence of the disease in the United States is nearly 1%. There is no reliable statistics on the incidence of morbidity in the country, which is estimated to be close to the United States. It is a disease that seriously affects people's health. The damage caused to society is difficult to estimate.

At present, there are many clinical treatments for RA. It is preferred to use high-dose non-inflammatory anti-inflammatory drugs to control symptoms. If it is not effective, clinically preferred immunosuppressive drugs such as methotrexate. About one-fourth of the patients will not relapse after the first onset of the disease, and about one-half of the patients will be relieved temporarily after treatment, but will still recur, and some eventually develop into joint dysfunction; One-third of the patients were ineffective for all current routine treatments.

Foreign studies on the pathogenesis of RA have been carried out, and it is found that Tumor Necrosis Factor alpha (TNFa) plays a key role in the pathological process of the disease. The TNF level in the joint cavity of patients or animal models is abnormally elevated. high. TNF is a multifunctional immunomodulatory molecule in the body that binds to receptors on the cell membrane, often causing death of target cells (its name is derived from this) or attracting local aggregation of immune effector cells. Because it is not well understood, its content in the joint cavity is greatly increased. On the one hand, it binds to the receptor of the synovial cells of the joint to cause direct damage to the cell. On the other hand, it can recruit immune effector cells to gather here. Secrete more cytokines, resulting in a stronger and more sustained autoimmune response.

TNFa and TNFP are a class of homologous secretory proteins in mammalian cells that share high homology with each other, both of which can have multiple effects on many cell types. Both proteins can affect the biological function of the cell by interacting with receptors on the cell membrane.

Tumor necrosis factor TNFa can cause hemorrhage and necrosis of tumor tissue and has many functions. The mature molecule consists of 157 amino acids with two disulfide bonds and no glycosylation sites. Its precursor is a polypeptide consisting of 233 amino acids. The N-terminal 76 amino acid polypeptide is not a signal peptide, which immobilizes the precursor on the cell membrane, leaving the mature secretory form on the cell membrane. The length of the mature mRNA of the precursor is 1. 7 kb. It is mainly produced by mononuclear macrophages in the body. Monoclonal antibody, therapeutic monoclonal antibody and its development:

Monoclonal antibody drugs have grown tremendously over the past decade. Several products have been successfully developed in the fight against TNFa-related diseases and have achieved great success. Due to the huge market demand, these drugs are still under high-strength development.

At present, the methods for obtaining monoclonal antibodies are mainly hybridomas, mainly involving rodents (such as mice, rabbits), birds (such as chickens), primates (such as macaques, baboons, etc.) and humans. In addition, there have been more and more studies on camelid antibodies in recent years. Monoclonal antibodies have also been developed using mouse humanized antibodies.

Although the human immune system is capable of producing extremely large types of antibodies, human or whole-body antibody technology has encountered bottlenecks: the abundance of precursor cells that produce specific antibodies in humans is low, even in antibody-positive individuals, The precursor cells of sex antibodies are also much lower than the number of specific antibody cells required for human hybridoma technology. Therefore, isolation, purification, and enrichment of B cells that specifically secrete antibodies are one of the key technologies for establishing a fully human antibody platform.

The efficient isolation, purification and enrichment of B cells secreting specific antibodies in positive individuals is a prerequisite for the transformation of human B lymphocytes by Epstein-Barr virus. The characteristics of B cells also lay the foundation for the efficient isolation, purification and enrichment of certain antigen-specific B cells. B cells account for 10% to 15% of the total number of lymphocytes in the blood. Membrane surface immunoglobulin (mlg) is a receptor for B cell-specific recognition of antigens, and is also an important characteristic marker of B cells. Immature B cells express mlgM, mature B cells express both mlgM and mI _g D, while memory B cells express mI _g G, mlgA or mI _g E on the surface. These mlg have antigen specificity, especially on the surface of memory B cells. Mlg has undergone antigen selection, affinity maturation and type conversion (as shown in Figure 1).

The present invention establishes a technique for isolating, purifying, enriching and proliferating B cells of interest capable of secreting antibodies.

The specificity of antibodies secreted by subcloning of B cells can be screened and identified by means of ELISP0T, ELISA and the like. The gene sequence of the antibody of interest is obtained from the monoclonal cultured cell line to construct a prokaryotic or eukaryotic expression vector, and the activity of the antibody can be reconstructed after expression. In the present invention, the platform is used to obtain an anti-human TNFa monoclonal antibody from B cells of a rheumatoid arthritis patient.

There are many requirements for monoclonal antibodies as therapeutic drugs, where affinity, neutralization ability, specificity, and side effects are important considerations, and these three aspects are interrelated. In general, the affinity is high, the required dose is small, not only the manufacturing cost is lowered, but also the possibility of causing side effects is small; the specificity is high, the influence on other components in the body is small, and the side effect is low. Therefore, obtaining high-affinity monoclonals has always been an important aspect of antibody drug development. but Yes, not every method can obtain monoclonal antibodies with high affinity or neutralization ability that meet pharmaceutical requirements.

Although there are many ways to obtain monoclonal antibodies, it is not an easy task to obtain monoclonal antibodies that meet pharmaceutical requirements. Sometimes, specificity, neutralization, affinity, or other limitations make many monoclonal antibodies unusable for clinical treatment.

Another important requirement for being a candidate drug is whether it is easy to manufacture. This requires the production of antibodies to be produced on a large scale and at low cost. The most important indicator of this is the amount of antibody that can be produced per liter of culture medium, the so-called expression level.

In the past two decades, recombinant monoclonal antibody drugs have been greatly developed, and new products have emerged in an endless stream. They have become the fastest growing field of clinical treatment drugs, which has aroused widespread concern in the industry. Governments and pharmaceutical companies are actively investing heavily in research and development of monoclonal antibodies. However, at the same time as the rapid development of monoclonal antibody drugs, it has also encountered problems that are difficult to solve. Among them, it is more prominent that the monoclonal antibody drugs must be produced by recombinant mammalian cells, and the dosage is large, resulting in high production costs and the patient is overwhelmed. Therefore, how to reduce costs under the premise of ensuring efficacy has become one of the key breakthroughs in the development of monoclonal antibody drugs. The low-cost production of miniaturized antibodies by E. coli, and PEGylation and nano-modification, thereby greatly reducing the cost of monoclonal antibody research and development, and the development of monoclonal antibody drugs into a new stage.

The rapid increase in sales of marketed antibody drugs and the continued availability of new antibody drugs challenge the antibody production capacity of antibody manufacturers. Small molecule antibody technology is one of the best solutions to break through this bottleneck. The E. coli expression system has the advantages of mature method, simple process, high yield, short cycle and low production cost. Although Escherichia coli cannot catalyze glycosyltransferase on Fc due to its lack of glycosylation ability, it is not suitable for expression of intact antibodies, but can be used for expression of antibody fragments such as Fab, Fv, ScFv, etc., plus PEGylation technology. Artificial glycosylation modification to achieve or approximate the properties of natural antibodies. The upcoming CDP-870 is produced using E. coli at a cost that is reduced to one-quarter of the cost of other TNF inhibitors.

Physiological effects of TNFa:

It has a different effect on different cells: killing cells, inhibiting, promoting or not affecting cell growth. It inhibits hematopoiesis, causes red blood cell reduction, can cause macrophage activity and killing function, enhances macrophage's ability to promote immune response, and promotes local neutrophil activity and aggregation of inflammation. It also inhibits B-cell responses to EBV, inhibits B cell proliferation and secretes immunoglobulins. In terms of tumors, this factor can inhibit hemorrhagic necrosis of tumors by affecting the blood vessels in the tumor area, and enhance the body's anti-tumor ability by enhancing immune function. In addition, the factor also has important physiological effects on liver, bone, muscle, blood vessels and other systems. use.

TNFa is associated with diseases such as rheumatoid arthritis. Excessive TNFa expression in rheumatoid arthritis patients is one of the major factors contributing to swelling and pain in the affected area, and TNFa is the most abundant protein in the TNF family that is most closely related to the disease.

Antagonists of TNFa and its clinical application:

As mentioned above, TNFa is one of the main factors causing swelling and pain in the affected area of rheumatoid arthritis, and a large number of studies have shown that it also causes septic shock syndrome, ankylosing spondylitis, psoriasis and rheumatoid arthritis. The main medium. Elevated levels of TNFa in the serum of patients with septic shock syndrome and rheumatoid arthritis indicate an increase in mortality or disability. Passive input of TNFa antagonists can prevent shock and tissue damage caused by LPS and bacteria. Clinical application of TNFa antibodies or their receptors has obvious effects on septic shock syndrome, ankylosing spondylitis, psoriasis and rheumatoid arthritis.

So far, a variety of antibodies against the human TNFa and receptor-Fc fusion proteins have been successfully used in the clinical treatment of the above clinical indications, and have achieved great success in industrialization.

Humira is a monoclonal antibody drug developed by Abbott. The product was approved for marketing in 2002. The first indication is rheumatoid arthritis (RA). The target is TNFa. Due to its outstanding efficacy, global sales reached 20 in 2006. One hundred million U.S. dollars. The amino acid sequence of the variable region of the product and the corresponding DNA sequence are derived from a human antibody library developed by CAT, and the coding region of the Fc fragment is derived from human IgG1. Thus, the variable region sequence of the product is not directly from humans, but from an antibody library derived from human B cells.

This project is based on the B cells of patients with rheumatoid arthritis. Through a series of screening, a fully human antibody with neutralizing ability to human TNFa was obtained, and its heavy and light chain variable regions were determined by molecular biology techniques. The gene was cloned and the biologically active full length and Fab forms were successfully expressed in CH0 cells and E. coli. By PEGylation, a PEGylated Fab was successfully obtained. Therefore, the amino acid sequence of the anti-human TNFa antibody of the present invention and the corresponding DNA sequence are directly derived from humans. Compared with Humira, the anti-human TNFa antibody of the present invention has a significantly higher affinity than Humira, and the clinical dose of the same therapeutic effect is only 1/4 of that of Humira. Summary of the invention

The object of the present invention is to disclose an anti-human TNFa antibody and a gene encoding the same.

Another object of the present invention is to disclose a Fab antibody of the above anti-human TNFa antibody and a PEGylated product thereof. A third object of the present invention is to disclose the pharmaceutical use of the above anti-human TNFa antibody and its Fab antibody.

One aspect of the invention discloses an anti-human TNFa antibody having a light chain amino acid sequence of SEQ ID NO: 3, a heavy chain The amino acid sequence is SEQ ID NO: 4.

Another aspect of the invention discloses a polynucleotide encoding the light chain of the above anti-human TNFa antibody, preferably the sequence of the polynucleotide is SEQ ID NO: 1.

The third aspect of the present invention discloses a polynucleotide encoding the heavy chain of the above anti-human TNFa antibody, and preferably, the sequence of the polynucleotide is SEQ ID NO: 2.

In a fourth aspect of the invention, a polynucleotide encoding a full-length heavy chain of the anti-human TNFa antibody is disclosed. Preferably, the polynucleotide sequence is SEQ ID NO: 6.

In a fifth aspect of the invention, the Fab antibody against the human TNFa antibody is disclosed. Preferably, the above Fab antibody is PEGylated.

In a sixth aspect of the invention, the use of the above anti-human TNFa antibody or Fab antibody thereof for the preparation of a medicament for treating TNFa-related inflammation, such as a medicament for the treatment of rheumatoid arthritis, is disclosed. When the anti-human TNFa antibody or Fab antibody of the present invention is used for the treatment of TNFa-related inflammation such as rheumatoid arthritis, the conventional usage amount of the existing TNFa antibody can be referred to.

The beneficial effects of the invention:

Although existing TNF antibody drugs are considered to perform better than any other drug, they also have major drawbacks. First, they are all expressed and purified by mammalian cells. Due to their low expression levels and limited production capacity, they are far from meeting market demand. Even Amgen has invested 500 million US dollars to create the world's largest 20,000 liter cell culture biological response. This problem persists (according to the 2004 US biopharma report); second, the high cost of these antibodies has prevented a large proportion of patients from being excluded. It is estimated that conventional applications of Enbrel, Remicade and Humira for RA-years The costs are 11400, 14200, and 16776 dollars, respectively, and need to be used for a long time (according to the 2004 US biopharma report); Third, the Fc segment carried by these antibodies has activities such as binding to complement and ADCC, and in vivo application leads to the expression of TNF-expressing cells. Death, it will produce a series of side effects; Fourth, Remicade is a human-mouse chimeric monoclonal antibody containing 1/3 of the mouse protein component. Continuous injection can stimulate human anti-mouse protein antibody response in more than 10% of patients or Human anti-chimeric protein antibody response affects therapeutic effects and may lead to serious adverse reactions. The forthcoming CDP870 is a PEGylated humanized anti-TNF antibody Fab segment with a mouse protein component down to 10% but still immunogenic.

The invention adopts the human hybridoma technology, and obtains the anti-human TNFa whole human antibody. The preliminary analysis of the physical and chemical activity and biological activity of the antibody indicates that the antibody has good affinity with TNF, and can effectively neutralize TNF to L929 in vitro. Cell killing. On the one hand, since all of its amino acid sequences are completely identical to human antibodies, its immunogenicity to the human body is minimized. Secondly, the expression of Fab small molecule antibody is expressed by the E. coli expression system, which greatly reduces the production cost. Low, and no side effects caused by effects such as complement binding and ADCC. Third, the use of PEGylated surface modification technology to reduce the probability of phagocytosis by the endothelial system, prolong the time in the blood circulation system, and at the same time achieve the purpose of sustained release in vivo, avoiding the shortcomings of short-term half-life of small molecule antibodies While maintaining its anti-TNFa activity, it is more suitable for in vivo applications. Compared with the existing monoclonal antibody drugs, the frequency of administration can be greatly reduced. This not only further reduces the patient's financial burden, but also greatly reduces the pain of patient injection and reduces labor losses. DRAWINGS

Figure 1: Pre-B cells and their conversion to memory B cells.

Figure 2: Comparison of the protective activities of 8H6, 8H6-Fab, 8H6-Fab-PEG and Humira on L929.

detailed description

The invention adopts the following experimental ideas:

Obtaining human B cells secreting anti-human TNFa antibodies:

Peripheral blood was drawn from patients with active rheumatoid arthritis and leukocytes were separated using lymphocyte separation fluid. The isolated B cells were cultured, and positive clones were identified based on the ELISA results.

The total RNA of the positive clone was isolated and reverse transcribed to obtain cDNA. PCR amplification was carried out by the method of SE. Dohmen et al. (Journal of Immunological Methods 298 (2005) 9-20, Production of recombinant Ig molecules from antigen-selected single B cells and restricted usage of Ig-gene segments by anti-D antibodies) The coding sequences of the heavy and light chain variable regions of the anti-human TNFa antibody were obtained by cloning and sequencing. From this, the corresponding amino acid sequence can be inferred.

1. Construct a full-length heavy chain coding region and a light chain coding region, respectively, into the expression vector pcDNA3.1 (+) (Invitrogen), and co-transfect the vector into CH0 cells to express anti-human TNFa antibody. Increased antibody expression by MTX screening.

2. Cloning and expression of anti-human TNFa-Fab, PEGylation of anti-human TNFa_Fab.

3. To study the antagonism of anti-human TNFa antibody, Fab and Fab-PEG against TNFa, and compare it with the existing TNFa antibody Humira. The invention is further illustrated by the following examples. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions and the reagents which do not specify the formulation are It is carried out or configured according to the conditions described in the conventional conditions such as Sambrook et al., Molecular Cloning: Test Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Example 1 Acquisition of B cells secreting anti-human TNFa antibody 5 ml of peripheral blood of patients with active rheumatoid arthritis was taken, and leukocytes were separated by lymphocyte separation solution, cultured, and positive clones were identified based on ELISA results.

1. Select a blood sample

In order to obtain human B cells secreting anti-human TNFa, 96-well plates were conventionally coated with human TNFa (purchased from Shenzhen Jingmei Co., Ltd.), and 250 ng of the protein was used per well, and coated overnight. Then, it was blocked with 5% skim milk powder for 2 hours at room temperature, and the milk powder was prepared with pH 7.2 PBS. After washing, 100 μl of serum from different patients was added and incubated for 1 hour at room temperature. Peroxidase-labeled goat anti-human IgG was then added and allowed to stand at room temperature for 1 hour. After washing at least 5 times, add TMD or other developer, and treat at room temperature or 37 ° for 20 minutes. Finally, a stop solution is added. After adding the peroxide chromogenic substrate for 10 minutes, the reaction was terminated with 50 μl of IN sulfuric acid, and the 450 nm optical density value was read. Serum with positive and high 0D values was selected as the selected blood sample.

2. Separation of human B-cells

The selected blood samples were diluted with PBS (pH 7.2, 50 mM) and superimposed on the Ficol l separation layer (usually 20 ml of Ficol l solution per 50 ml separation tube). The blood suspension was centrifuged at 4 ° C, 3000 rpm for 30 minutes. Subsequently, a top solution containing mainly peripheral blood mononuclear cel l (PBMC) was collected and placed in a new tube. PBMC were washed thoroughly with PBS (pH 7.2, 50 mM) and washed at least three times. cell counts.

Human B cell enrichment

In PBMC, multiple clones of human B cells have previously been exposed to various immunogens. Specific B cell clones can be screened from the obtained PBMC mixture. The bio panning method was used here, which proved to be an efficient method for obtaining 1⁄21 ⁶ cells per unit of blood.

Bio pinning method: A 24-well plate (GREINER, GERMANY) coated with 2 μ _β tumor necrosis factor per well, incubated at 4 ° C overnight or 37 ° C for 2 hours. The TNF supernatant was removed, and 2 ml of isolated PBMC was added to each well of the coated 24-well plate, and incubated at 37 ° C for 2 hours. Enrichment of B cells secreting anti-TNF antibodies, the detailed steps are as follows:

1) 500 μΐ TRIS-HCL (pH 9. 5 ) solution, in which rabbit anti-human IgG 5 μ _β /ιιι1 was coated in a 24-well plate (NUNC) at 4 ° C overnight.

2) The next day, the aforementioned PBMC were cultured in 10% FBS-RPMI-1640 based on suspension at 37 ° C, C0 ₂ 5%, and cultured in a 75 ml culture flask for 2 hours. 3) The supernatant was removed in a 24-well plate and washed once with PBS (pH 7.2, 50 mM) containing 5% fetal bovine serum (FBS, fetal bovine serum). After the monocytes were removed, the washed enriched cells were placed in the aforementioned 24-well plates at 1 ° C for 1 hour with intermittent stirring.

4) Subsequently, the cells attached to the wells were microscopically examined. Resuspend the cells.

5) After washing the suspended cells with 5% FBS_RPMI-1640 medium, the attached cells were washed from the wells and collected.

6) Count the microscope and adjust the number of cells.

3. Serving the preparation of cells

The microenvironment in which human B cells proliferate in vitro is required. The human B cell proliferation microenvironment established here is initially composed of human bone marrow stromal cell layer and/or murine peritoneal exudate cells, including macrophages and dendritic cells. Isolated human or murine spheroid cells form unique monolayers that produce a variety of growth factors, including hematopoietic factors, that maintain the proliferation and differentiation of human B cells. The initial concentration of the spheroid cells consisting of human or murine macrophages and dendritic cells was 3× ^{10 5} /ml, and cultured at 37 °C. With RPMI_1640 (containing 10% fetal bovine serum, 100 units per liter _100μ β penicillin and streptomycin) medium, C0 ₂ 5%, the cells to a cell culture collection of 80-90%.

4. EBV (chronic Burkitt's lymphoma virus) transformation and long-term culture

With RPMI-1640 (containing 10% FBS, 100 units per liter penicillin and streptomycin _100μ β) media in 96 well plates, so raising servo monolayer cells covered. Sputum cells were added to wells in which monolayer spheroid cells were grown. The amount of sputum cells added was about 3-5 cells per well, each well containing 200 μl of complete medium, co-cultured at 37 ° C, and ΙΟΟμΙ EBV from Β958 cells (purchased from the Department of Pathophysiology, Chongqing Medical University) Add to a 24-well plate containing a single layer of spheroid cells. Every 4 days, half of the supernatant was replaced with fresh complete medium. After 2 days of culture, the morphology of the cell colonies under the microscope was visible.

5. Antibody-response ELISA screening

The secretion of anti-TNFa IgG was detected by ELISA. Samples of human B cell culture supernatants transformed by the EBV immortalization method were screened. Among them, the positive clone numbered 6D4 had the best binding ability (0D value was 0.175, negative control reading was 0. 036), and it was IgGl. This cell line was named HAI6D4. Example 2 Acquisition of anti-human TNFa antibody gene

5,000-10000 of the above HAI6D4 cells were taken, total RNA was isolated using Trizol (GIBC0), and cDNA was obtained using MMLV reverse transcriptase (Promega product). The above operations are carried out in accordance with the manufacturer's instructions. PCR was carried out using the following primers and conditions, and the amplification enzyme used was ClonTech's Pfu to ensure that possible mutations were reduced during amplification. Light chain upstream primer: GACATCGAGCTGACCCAGTC (SEQ ID NO: 7) Light chain downstream primer: CTAACACTCTCCCCTGTTGAAGC (SEQ ID NO: 8)

Heavy chain upstream primer: GAGGTGCAGCTGGTGGAGTC (SEQ ID NO: 9)

Heavy chain downstream primer: CTAGCATGTGTGAGTTTTGTCACAAG (SEQ ID NO: 10)

PCR conditions:

a) The composition of the reaction system:

Quantity of ingredients ( μΐ )

10X PCR Reaction Buffer 5

25 mM Mg ₂ S0 ₄ 5

Pfu DNA polymerase (5 units / microliter) 1

Upstream primer, downstream primer (2 μg / 1 μL each, total 25 μL)

cDNA 1. 5μ1

Sterilize three distilled water to 50 μl b) Reaction conditions:

Pre-denaturation: 94 degrees 2 minutes

Main cycle: 94 degrees 1 minute, 55 degrees 1 minute, 72 degrees 3 minutes.

Number of cycles: 20

After extension: 72 degrees 5 minutes

The PCR process was performed on a PROGENE Genium thermal cycler.

c) Electrophoresis identification and glue recovery

PCR was carried out in accordance with the above conditions. After completion, the products were identified on 1% agarose gel electrophoresis. The products of the two PCRs were 650 bp and 670 bp, respectively, which were consistent with the theoretical size. The fragment was recovered using Promega's gel recovery kit and operated according to the manufacturer's instructions, each giving approximately 2 micrograms of DNA.

Cloning and sequencing:

The above product was routinely cloned into PUC57, and the correct clone was identified and sequenced. The cloning method was carried out using the MBI kit, and the operation was carried out according to the instructions. The positive clones were sequenced and the results showed that the two clones were the genes encoding the heavy and light chain variable regions of the human IgG1 antibody, respectively. The sequencing results are as follows: GGGCTCCGTGGGCGACCGCGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTT TGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTACAGCCT GAACCCAAGCTGCTGATCTACAAGGCCTCCTCCCTGGAGTCCGGCGTGACTTTTGGCCAGGGGACCAAGCTTATCGAGAT CAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTG TGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTA CGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCSACGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGTTAG (SEQ ID NO: 1)

V2H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCCGGCAGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATT CACCTTTGCCTCCGGCGGCACCTTCAACGTCCGGCAAGCTCCAGGGAAGGGCCTGGAATGGGTCTCAGCTATGGGCCGCT ACGCCCAGCACATAGACTATGCGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTAT CTGCAAATGAACAGTCTGAGAGCTGAGGATACGGCCGTATATTACTGTGCGGCCCCCCTGGACTACTGGGGCCAGGGCAC CCTTGACTATTGGGGCCAAGGTACCCTGGTCACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTG TCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCTAG (SEQ ID NO: 2) encoding the amino acid sequence of the polypeptide are:

2L DIQMTQSPSYLSASVGDRVTITCRASQGSVGDRAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQP EPKLLIYKASSLESGVTFGQGTKLIEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSTPVTKSFNRGEC. ( SEQ ID NO: 3)

2H EVQLVESGGGLVQPGRSLRLSCAASGFTFASGGTFNVRQAPGKGLEWVSAMGRYAQHIDYADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAAPLDYWGQGTLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC. (SEQ ID NO: 4) Example 3 Preparation of anti-human TNFa antibody obtained embodiment 1. The human IgGl heavy chain Fc fragment coding sequence for full-length antibody gene expression, we synthesized heavy chain human IgGl The Fc sequence is such that it constitutes a complete anti-human TNFa antibody gene. According to the literature, the DNA coding sequence of the human IgG1 heavy chain Fc fragment is as follows: DNA sequence of human IgG1 Fc fragment (5, →3') (SEQ ID NO: 5)

The above sequences were synthesized according to conventional gene synthesis methods. 2. Acquisition of full-length anti-human TNF mAb gene and expression product The above Fc fragment was ligated with the heavy chain variable region coding region according to the conventional Overlapping PCR method. The length of the ligated full length fragment was: 1424 bp. This fragment is a full length heavy chain coding region.

SEQ ID NO: 6

CCGGGTAAATAG

The full-length coding region (SEQ ID NO: 6) of the above heavy chain and the aforementioned coding region (SEQ ID NO: 1) of the light chain were cloned into the pOptiVec (Invitrogen product) vector, respectively, according to a conventional method (Reference: Yves Durocher, Sylvie Perret and Amine Kamen, 2002, High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cel ls, Nucleic Acid Research, 2002, 30 (2): E9) Transfected CH0 cells.

3. Transfection and screening of CH0 cells In this study, CH0 cells were transfected by liposome method (LipoFemine 2000, Invitrogen). The transfection kit was purchased from Invitrogene, and the above purified ones were each weighted. 100 μg of the plasmid of the strand and the light chain was transfected into CH0 cells as a DNA sample, and the transfection procedure was carried out according to the manufacturer's instructions. The transfected CH0 cells were screened by methotrexate (MTX) for 3 consecutive months, and the concentration was from 0. 05μΜ to 10μΜ, and the concentration was increased every two weeks. The amount of each sputum was about twice that of the previous one. Depending on the growth situation. The cell culture was carried out as usual, and the medium was: 15% fetal bovine serum (Gibco) + RPM1640/DEME. Incubate in a 37 degree 5% CO ₂ incubator. Monoclonalization is then carried out according to the conventional extreme dilution method. The expression levels of the antibodies were detected by ELISA, and a total of 87 clones were obtained.

4. Study of gene expression level The expression intensity of some of the above clones was studied by ELISA. The specific data are as follows:

MTX Level 2θ6ηΜ ΙμΜ 5μΜ ΙΟμΜ

Average expression level ο. 29+0. 23 0. 47+0. 31 0. 34+0. 19 1. 29+0. 42

(mg/L supernatant) According to the expression level, it is determined that 8Η6 (number) clones have good growth characteristics and expression levels, as a continuing study Object.

The above-mentioned expression cell line 8H6 was cultured and subjected to affinity chromatography using Protein A-Sepharose to obtain an antibody protein having a purity of 90% or more, and the obtained antibody was named 8H6. Example 4 Cloning and expression of anti-human TNFa-Fab

l (gg the above DNA fragments SEQ ID NO: 1 and SEQ ID NO: 2 were cloned into the pC0M3H vector according to a conventional method (Wu SC, Lin YJ, Chou JW, Lin CW. 2004, Construction and characterization of a Fab recombinant protein for Japanese) Encephal itis virus neutral ization. Vaccine. 25;23(2): 163-71), after electroporation of 5ml E. coli DH5a (from New England Biolabs or Takara) cells, the product was initially screened on IPTG/X-gal plates. Identification: 20 plaques were inoculated into liquid LB medium containing ampicillin for amplification. After IPTG induction and ELISA identification, a small amount of Fab antibody preparation was performed, which was called 8H6_Fab. The prepared Fab antibody was L929 cells (ECACC). 85011425) A protective test was performed and it was found that 8H6_Fab has a good protective effect (see Figure 1 for the results). Example 5 Anti-human TNFa-Fab PEGylation

The 8H6_Fab obtained in Example 4 was PEGylated, and the PEGylation and purification method of Fab was carried out by the method of AP Champman et al. [Reference: Therapeutic antibody fragments with prolonged in vivo half life. Nature Biotech. V17 1999 August, Page 780-783]. , PEGylated anti-human TNFa-Fab 8H6-Fab-PEG was obtained. Example 6 Study of TNFa antagonism by 8H6, 8H6-Fab and 8H6-Fab_PEG The above purified antibody was tested for its ability to neutralize TNFa by the following method.

1. Take L929 cells in logarithmic growth phase, dilute the cells thoroughly with digestive juice, and dilute to 2× ^{10 5} cells/ml with cell culture medium.

2. Take four 96-well plates, each well was added 100 microliters of cell suspension, 5% (: 0 ₂ incubator at 37 ° C for 24 hours.

3. Dilute the test sample to 10 μg/ml, 1.0 μg/ml with a cell culture medium of 2 g/ml actinomycin D and 0.001 μg/ml TNFa standard (Phamacia). 00001微克/毫升。 0. 1 μg / ml, 0. 001 μg / ml, 0. 0001 μg / ml and 0. 00001 μg / ml.

4. Add 100 μl of each dilution of sample to each well, 3 wells per dilution. Negative control only included A cell culture medium of 2 g/ml actinomycin D. High doses of TNFa (2 g/ml) cell culture medium were added to the positive control.

5. 5% (: 0 ₂ incubator cultured at 37 degrees for 24 hours or longer. The culture time was determined based on the total number of cells in the positive control wells.

6. After reading with MTT, read the optical density data on the reader. Plot the data. (The results are shown in Figure 1). It can be seen from the experimental data that 8H6 is roughly equivalent to Humira, and its 8H6_Fab is significantly lower than the full length, and the difference between H6-Fab-PEG and its full length is not significant.

Claims

Rights request

An anti-human TNFa antibody characterized in that the light chain amino acid sequence thereof is SEQ ID NO: 3 and the heavy chain amino acid sequence is SEQ ID NO: 4.

2. A polynucleotide, which encodes the light chain of the anti-human TNFa antibody of claim 1.

3. The polynucleotide according to claim 2, wherein the sequence of the polynucleotide is SEQ ID NO: 1.

A polynucleotide characterized by encoding the heavy chain of the anti-human TNFa antibody of claim 1.

The polynucleotide according to claim 4, wherein the sequence of the polynucleotide is SEQ ID NO: 2.

A polynucleotide characterized by encoding the full-length heavy chain of the anti-human TNFa antibody of claim 1.

The polynucleotide according to claim 6, wherein the polynucleotide sequence is SEQ ID NO: 6.

8. The Fab antibody against human TNFa antibody of claim 1.

The Fab antibody according to claim 8, wherein the Fab antibody is PEGylated.

10. The anti-human TNFa antibody of claim 1 or the Fab antibody of claim 8 or 9 for use in the manufacture of a medicament for the treatment of inflammation associated with TNFa.