WO2009105934A1

WO2009105934A1 - Monoclonal antibody anti-human rankl

Info

Publication number: WO2009105934A1
Application number: PCT/CN2008/070366
Authority: WO
Inventors: 刘庆法; 庄贤韩; 朱化星
Original assignee: 上海先导药业有限公司
Priority date: 2008-02-27
Filing date: 2008-02-27
Publication date: 2009-09-03

Abstract

Provided are antibodies specially binding to human receptor activator of nuclear factor kappa B ligand (RANKL), which have the light chain variable region represented by SEQ ID NO: 2 and the heavy chain variable region represented by SEQ ID NO: 4. Also provided are the polynucleotides encoding anti-human RANKL antibodies, Fab antibody and pegylated Fab antibody. These antibodies can be used for the manufacture of medicaments for treating osteoporosis or skeletal damage.

Description

Anti-human RANKL monoclonal antibody

TECHNICAL FIELD The present invention relates to drug development using recombinant antibodies for the treatment of human osteoporosis and bone damage.

BACKGROUND OF THE INVENTION The formation and maintenance of bone is a very complex biological process. Molecular biology studies have shown that in bone metabolism, there is a group of nuclear factor-κΒ receptor activating factors (abbreviated as RANK, also known as osteoclast differentiation factor or osteoclast differentiation), which is mainly composed of stromal cells/osteoblasts. Activated receptor), its receptor (abbreviated as RANKL), and osteoprotegerin

The biological process (abbreviated as 0PG) is extremely important to maintain bone growth balance. RANK and its ligand RANKL are key components of osteoclast differentiation, maturation and function [1, 2, 3, 4, 5]. RANKL, when combined with RANK, stimulates precursor osteoclast differentiation, activates mature osteoclasts, and causes bone resorption [3, 4, 5, 6]. This was originally the normal mechanism by which the body maintains the balance of bone metabolism. However, if the bone resorption process is excessive, it may cause osteoporosis. In order to regulate the action of RANK, the body produces osteoprotegerin 0PG in combination with the ligand, so that the number of osteoclasts is maintained in an appropriate equilibrium state, which does not lead to excessive bone resorption. Studies have confirmed that in osteoporosis, RANKL exceeds the level of osteoprotegerin causing excessive bone resorption [5, 7, 8].

Due to the discovery of the function of RANK and its ligands, the mechanism of osteoporosis and bone damage has been clearly elucidated. Osteoporosis and bone damage involve the balance and regulation of 0PG/RAM/RAML [1, 3, 6].

The adjustment system involves three components: RANK, RANKL and 0PG. RANK (receptor activator of nuclear factor-KB), a functional receptor of RANKL, is mainly expressed on the surface of osteoclast precursors and mature osteoclasts. RANK is a type 1 transmembrane protein containing 616 amino acids, of which 184 amino acids constitute the extracellular domain and 383 amino acids constitute the intracellular domain. RANKL (receptor activator of nuclear factor-KB igand) is a key cytokine for bone resorption that stimulates osteoclast differentiation and exerts bone resorption [1, 3]. Osteogenic/stromal cells

( osteoblast / stromal cel ls ), activated lymphocytes and other expression. RAML belongs to the TNF ligand superfamily and is available in both membrane-bound (mRANKL) and soluble (sRANKL) forms. mRANKL is a type II transmembrane glycoprotein composed of 316 amino acids with a short N-terminal intracellular In the region, sRANKL is formed by proteolytic hydrolysis of the extracellular domain of mRANKL. Both types of RAML stimulate osteoclast differentiation and bone resorption, but mRANKL is more active than sRANKL. OPG (steoprotegerin) is a natural soluble inducer of RANKL, which can be expressed in a variety of cells. OPG, including osteogenic/stromal cells, dendritic cells, B lymphocytes, and endothelial cell precursors and fibroblast precursors. 0PG is a member of the TNF receptor superfamily and differs from other TNF receptor superfamily members in that it lacks a hydrophobic transmembrane sequence and is a soluble glycoprotein consisting of 401 amino acids. 0PG has two forms of dimer and monomer outside the cell, and the two forms of 0PG have the same physiological activity [1, 3, 4, 5, 6, 7].

In the process of bone resorption, RANKL, RANK, and OPG are involved in regulating the differentiation and function of osteoclasts. When multiple stimulating bone resorption factors act on osteogenesis/stromal cells, osteogenesis/stromal cells express RANKL on their surface, specifically recognize osteoclasts, and bind to the functional receptor RANK on their membranes. In the presence of CSF, the signal is transmitted to the cells, and the osteoclast precursor is stimulated to develop into mature osteoclasts. At the same time, RANKL can also rapidly form an actin ring in mature osteoclasts through the RANK pathway, and activate mature osteoclasts to exert bone resorption. The role of RAML/RAM can be suppressed by 0PG [ 1, 3, 6].

0PG can be directly combined with RAML to competitively inhibit the binding of RAML to RAM, thereby inhibiting the differentiation and function of osteoclasts. At the same time, 0PG can also form a trimer with the RANKL/RANK combination to directly suppress the effect of RAML/RAM [14]. Abnormal bone resorption occurred in mice with RAML deletion or RAM deletion, and severe bone sclerosis occurred. While 0PG-deficient mice develop osteoporosis after birth, the addition of recombinant 0PG can increase bone density. It can be seen that RANKL/RANK/0PG together constitute a key loop regulation system that regulates osteoclast differentiation and exerts bone resorption function [1, 2, 5, 6].

Osteoporosis is quite common in middle-aged and elderly people, especially menopausal women, due to the above-mentioned mechanism of action. The market growth potential for osteoporosis at home and abroad is very strong. The main reasons are twofold: First, the population is aging, and second, the current diagnosis and treatment rate of osteoporosis is still low, even in developed countries (in the world seven Less than 25% of women with osteoporosis in the major pharmaceutical markets are diagnosed and treated).

In 1999, the world's seven major pharmaceutical markets (US, France, Italy, Spain, the United Kingdom, and Japan) had approximately 35 million osteoporosis patients and are now considered by the World Health Organization to be the primary health care after cardiovascular disease. The second disease of the problem. According to a survey by Datamonitor, the market for osteoporosis and hormone replacement therapy (HRT) in 2003 reached $8.3 billion, and the market is expected to increase to $17.9 billion by 2014. According to a new study by Decision Resources, sales of osteoporosis therapeutics in the world's major markets will more than triple, from $2.8 billion in 1999 to $9.3 billion in 2009, an average annual increase of 13%. The growth of this market is mainly due to the significant increase in the number of elderly populations worldwide and the advancement of diagnostic methods.

There are 10 million osteoporosis patients and 18 million low-bone people in the United States, and the number of fractures caused by osteoporosis is 1.5 million per year. One-half and one-eighth of women over the age of 50 and men in the United States have a risk of osteoporosis-related fractures. In Europe, as the population ages faster, the more osteoporotic fractures The more common it is. British statisticians estimate that there are 3 million osteoporosis patients in the UK, and one-third and one-twelfth of women and men over the age of 50 are developing this disease. The onset of osteoporosis is not limited to a particular race, culture, age or gender, although there is no corresponding large epidemiological survey in the country. In the United States, the annual cost of medical expenses for the treatment of osteoporosis and its fractures is about $14 billion, and the latter is estimated to triple by 2030 to reach an annual cost of $60 billion. Some researchers predict that osteoporosis will affect 41 million Americans in 2015. The cost of osteoporosis-related care in Canada is currently around $1.3 billion, most of which is spent on long-term effects, hospitalization, and subsequent care. The cost of treating osteoporotic fractures in Canada over the next 25 years is estimated to be $32 billion. The current annual cost of treating osteoporosis in the UK exceeds £1.7 billion, which is equivalent to a daily cost of £5 million. The social burden of osteoporosis and its consequences cannot be ignored.

According to the results of a sample survey conducted by the Ministry of Health in China, the incidence of osteoporosis in different age groups in China is: 21% for 50-60 years old, 58% for 60-70 years old, and nearly 100% for those over 70 years old. China is a country with a large population and the country with the largest population. According to statistics, the total number of potential patients with osteoporosis (including low bones) in China is estimated to be about 87 million, accounting for nearly 7% of the country's total population. Considering that the number of people over the age of 60 in China has accounted for about 9% of the total population, and the degree of aging of the society is still rising, the number of patients suffering from such diseases is expanding. According to statistics, the number of new patients is 7 million per year. There are many. It is estimated that by 2010, the number of patients with underlying osteoporosis will increase to 144 million people; by 2025, it will reach 1.5 billion people. Obviously, the market potential of domestic osteoporosis prevention and treatment drugs is extremely huge. According to statistics, China's osteoporosis treatment drugs (excluding health care products) had a market capacity of about 1.37 billion in 1999, and the market capacity in 2000 was about 1.68 billion yuan, with an annual growth rate of about 22%. As in the case of rheumatoid arthritis, this market will continue to expand dramatically with the emergence of a new generation of therapeutics such as those with outstanding therapeutic antibodies. It is expected that the market will be expanded 10 times in five years to reach 15 billion yuan. Therefore, the market demand for such drugs is very clear and will continue to grow at a high rate.

In addition to the above-mentioned large number of patients with osteoporosis, bone damage can also occur due to the above mechanisms in the development and treatment of certain malignant tumors and autoimmune diseases, such as periodontitis, rheumatoid arthritis, bone cancer, and Bone cancer due to metastasis of malignant tumors has a high proportion of bone damage. Osteoporosis or bone damage can also be caused by the use of drugs such as hormones to treat certain diseases.

Monoclonal antibody, therapeutic monoclonal antibody and its development: Monoclonal antibody drugs have been greatly developed in the past decade. Several products have been successfully developed in terms of anti-TNFa-related diseases and have achieved great success. Due to huge market demand, these drugs are still in high-intensity development [9, 10, 11, 12] 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, research on camelid antibodies has increased 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.

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 production is reduced, but also the possibility of 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. However, it is not a monoclonal antibody that can achieve high affinity or neutralization ability to meet pharmaceutical requirements in every method [9, 10, 11, 12]

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 [9, 10].

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 therapeutic 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 by itself. 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 the low cost on the premise of ensuring the efficacy has become one of the key breakthrough directions for the development of monoclonal antibody. 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. E. coli Due to lack of glycosylation ability, it can not catalyze glycosyl transfer on Fc, and 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, through this artificial glycosylation Modifications 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 a quarter of other TNF inhibitor drugs.

The antibody library technology is a method for obtaining antibodies that can be obtained from the hybridoma technology at the end of the last century. It has been greatly developed in the past two decades and has gradually become an indispensable important technology for antibody engineering. Evolution, antibody modification, and antibody maturation have played an important role [13, 14, 15].

RANKL antagonists and their clinical application: As mentioned above, RANKL is one of the main factors causing osteoporosis and bone damage, and there are numerous studies showing that it is ankylosing spondylitis, psoriasis and rheumatoid arthritis. The main mediators of bone damage in patients and osteoporosis in the elderly and postmenopausal women [16, 17, 18, 19, 20].

In view of the high disability rate of osteoporosis and bone damage and the large number of morbidity groups, more and more institutions are developing globally to develop related preventive and therapeutic drugs this year. So far, a variety of antibodies against the human RANKL and receptor-Fc fusion proteins have entered the clinical trials in the world, and promising results have been obtained, including recombinant human 0PG and recombinant anti-RAML monoclonal antibodies [21 ].

This project is based on the whole human Na'ive combinatorial antibody library. Through a series of screening (Panning), a fully human antibody with neutralizing ability to human RANKL was obtained, and its heavy and light chains were used by molecular biology techniques. The variable region 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. references

1. Dougal l WC, Glaccum M, Chat Tier K, et al. RANK is essential for osteoclast and lymphonode development . Genes Dev, 1999. 13 ( 18 ) : 2 412—2 424

2. Bucay N, Sarosi l . Dunstan CR _? et al OPG - deficient mice develop early onset osteoporosis and arterial calcification . Genes Dev, 1998. 12 ( 9) : 1 260 —1 268

3. Liu D, Xu JK, Figl iomeni L, et al . Expression of RANKL and OPG mRNA in periodontal disease: possible involvement in bone destruction. Int J Mol Med, 2003, 11: 17-21 Crotti T, Smith MD, Hirsch R, et al. Receptor activator NF-KB l igand ( RANKL) and osteoprotegerin ( OPG ) protein expression in periodontitis . J Periodont Res , 2003 , 38 : 380—387 Mogi M, Otogoto ], Ota N, et al. Diferential expression of RANKL and osteoprotegerin in gingival crevicular fluid of patients with

Periodontitis J Dent Res , 2004, 83 ( 2 ) : 166-169 Hasegawa T, Yoshimura Y , Kikuiri T, et al . Expression of receptor activator of NF - KB l igand and osteoprotegerin in culture of human periodontal l iganment cel ls . J Periodont Res, 2002 37: 405 — 411 Wittrant Y, Theoleyre S, Coui l laud S, et al . Relevance in in osteosynthesis osteophysis lastogenesis system to study receptor activator of NF-kB l igand and osteoprotegerin biological activities . Exp Cel l Res , 2004, 293: 292- 301 Nakashima T. Protein expression and function difference of membrane bound and soluble receptor activator of NF-KB l igand: modulation of expression by osteotmpic factors an d cytokines . Biochem Biophys Res Commun, 2000, 275 ( 3 ) : 768-775 Antibody Engineering, Edited by Benny KC Lo, in Met hods in Molecular Bi logy, V248, Humana Press. , Beyond borders: Global Giotechnology Report 2005, Ernest & Young, 2005. Progessions 2005, The Ernest & Young Annual Global Pharmaceutical Report, Ernest & Young, 2005. Monoclonal Antibody Successes in the clonic, Janice M Reichert, et al, 2005, Nature Biotech., 23 ( 9) 1073-1078· A strategy for the Generation of specific human antibodies by directed evolution and phage display-An example of a single-chain antibody fragment that neutral izes a major component of scorpion venom, Lidia RiarT o-Umbarila, Victor Rivelino Jua'rez-Gonza' lez, Timoteo Olamendi-Portugal, Mauricio OrtT z-Leo' n, Lourival Domingos Possani and Baltazar Becerr, 2005, FEBS Journal 272: 2591 - 2601. Directed evolution, phage display and Combination of evolved mutants: a strategy to recover the neutralization properties of the scFv version of BCF2, a neutralizing monoclonal antibody specific to scorpion toxin Cn2, Juarez-Gonzalez VR, Riafio-Umbarila L, Quintero-Herndndez V,

Olamendi-Portugal T, Ortiz-Leon M, Ortiz E, Possani LD, Becerril B., 2005, J Mol Biol. 2005 Mar 11 ;346 (5) :1287-97. Phage display as a tool for the directed evolution of enzymes Ana Fernandez-Gacio, Marilyne Uguen and Jacques Fastrez, 2003, Trends in Biotechnology, 21 (9) :408-414· 0PGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis, Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, Morony S, 01iveira-dos-Santos AJ, Van G, Itie A, Khoo W, Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ, Penninger JM, 1999, 28;397

(6717): 315-23. RANK is essential for osteoclast and lymph node development, William C. Dougall, Moira Glaccum, Keith Charrier, et al, 1999, Genes & Dev., 13: 2412-2424. RANKL/RANK/0PG The Role of Regulatory Systems in the Pathogenesis of Periodontitis, Zhang Jing, E1 Cavity Medicine Research, August 2005, Vol. 21, No. 4. The molecular mechanism of osteoclastogenesis in rheumatoid arthritis, Nobuyuki Udagawal, Shigeru Kotake2, Naoyuki Kamatan, et al , 2003, Arthritis Res., 4:281-289. Role of RANK ligand in mediating increased bone resorption in early postmenopausal women, Guitty Eghbali-Fatourechi, 1 Sundeep Khosla, 1 Arunik Sanyal, et al., 2003, J. Clin. Invest. 111 : 1221 - 1230.

21. Cl inical development of anti-RANKL therapy, Edward M Schwarz and

Christopher T Ritchl in, 2007, Arthri tis Research & Therapy , 9 ( Suppl 1.

Summary of the invention

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

Another object of the present invention is to disclose a Fab antibody of the above anti-human RANKL antibody and a PEGylated product thereof.

A third object of the present invention is to disclose the pharmaceutical use of the above anti-human RANKL antibody and its Fab antibody.

In one aspect, the invention features an anti-human RANKL antibody comprising a light chain amino acid sequence comprising SEQ ID NO: 2 and a heavy chain amino acid sequence comprising SEQ ID NO: 4.

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

A third aspect of the invention discloses a polynucleotide encoding the heavy chain of the above anti-human RANKL antibody. Preferably, the sequence of the polynucleotide comprises SEQ ID NO: 3.

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

In a fifth aspect of the invention, the Fab antibody of the above anti-human RANKL antibody is disclosed. Preferably, the above Fab antibody is PEGylated.

In a sixth aspect of the invention, the use of the above anti-human RANKL antibody or Fab antibody thereof for the preparation of a medicament for treating osteoporosis and bone damage associated with RANKL, such as preparation of senile osteoporosis, bone mass after menopause in women, is disclosed. A drug that causes bone damage caused by diseases such as loose rheumatoid arthritis. The anti-human RANKL antibody or Fab antibody of the present invention can be used for treating osteoporosis or bone damage in rheumatoid arthritis, certain cancers, postmenopausal women, the elderly, and the like.

The beneficial effects of the invention:

Although existing drugs for treating osteoporosis and bone damage have a good effect, they also have the following problems: s But they also have great drawbacks. Currently, treatment and prevention are mainly based on phosphate drugs, and global market opportunities are dominated by Merak's Fosamax. The drug of the present invention has a completely different mechanism of action than Fosamax, Fosamax requires at least one dose per week, and this product is once every six months; and unlike Fosamax, it can cause gastrointestinal side effects.

The invention adopts the human hybridoma technology, obtains an anti-human RANKL whole human antibody, and physicochemical and biological activity of the antibody Preliminary analysis of the sex indicates that the antibody has a good affinity with human RANKL, and can effectively neutralize the physiological role of RANKL in promoting osteoclast formation in vitro. 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 antibodies by the E. coli expression system is greatly reduced, and there is no side effect 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-human RANKL 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: Schematic representation of the structure of a mammalian cell expression vector. The ampicillin resistance gene AMP and origin of replication 0 are from PUC57, the eukaryotic promoter Pcmv is derived from human cytomegalovirus, and the SV40 Ori and SV40 PolyA signals are derived from the SV40 virus. Its resistance gene Neo (R) is derived from pCDNA3.1 (Invitrogen). Figure 2: HPLC detection of target antibodies. The figure shows that there is only one target band, and the integral shows that its purity is over 98%. Figure 3: Effect of anti-human RANKL antibody on osteoclast formation. The data in the figure is the result of counting TRAP stained cells. The ordinate is the number of osteoclasts formed; the abscissa is the antibody concentration, and the range is as described in the text.

detailed description

The basic strategy of the present invention is to obtain the variable regions of the heavy and light chains of the anti-human RANKL antibody from the antibody library, and obtain the Fab and the full-length forms thereof based on the same, in order to evaluate the medicinal value thereof. .

Example 1: Screening of anti-human RANKL antibody gene variable region from antibody library

(1) Construction of Na'ive human source combination antibody library

Blood samples from nearly 3,000 healthy young people from 22 provinces or municipalities including 15 nationalities (1 ml per person) were used to extract total RNA with TRIZ0L (GIBC0 BRL), and the heavy chain and light were amplified according to the following literature. The coding region of the variable region of the chain, and the construction of an antibody library.

1. Hoogenboom and Winter, By-passing immunisation: human antibodies from synthetic repertoires of germin ine VH gene segments rearranged in vitro. J. Mol. Biol., 227, 381-388

2. Griffiths, AD, Williams, SC, Hartley, 0., Toml inson, IM, Waterhouse, P., Crosby, WL, Kontermann, RE, Jones, PT, Low, NM, Al l ison, TJ, Prospero , TD, Hoogenboom, HR, Nissim, A., Cox, JPL, Harrison, JL, Zaccolo, M., Gherardi, E. & Winter, G. (1994). Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J., 13, 3245-3260.

3. Nissim, A., Hoogenboom, HR, Toml inson, IM, Flynn, G., Midgley, C., Lane, D. & Winter, G. (1994) . Antibody fragments from a ' single pot' phage display l Ibrary as immunochemical reagents. EMBO J. , 13, 692-698.

4. Marks et al. By-passing immunization: human antibodies from V-gene l ibraries displayed on phage. J. Mol. Biol. , 222, 581-597

5. Haidaris CG, Malone J, Sherri ll LA, Bl iss JM, Gaspari AA, Insel RA, Sul l ivan MA., Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens. J Immunol Methods. 2001 Nov 1; 257

( 1-2 ) : 185-202.

The constructed antibody library was placed in -80C for storage.

(2) Screening steps:

1. The frozen antibody library strain 1 ml, after thawing at 37 degrees, add 14 ml of fresh LB medium and incubate at 37 °C for 16 hours in a 50 ml triangle flask.

2. Centrifuge at 12,000 rpm for 10 minutes at high speed, transfer the supernatant to a sterile 50 ml centrifuge tube and store for use. Its titer should be above 2X10E11.

3. Purified human RANKL protein (product of Orbigen) was used as an antigen, and a 25 ml cell culture flask was coated by a conventional method.

4. Add not less than 3X10E10 phage particles to the coated cell vial and incubate for 37 hours at 37 degrees.

5. Pour off the liquid in the bottle and wash the flask 10 times with 10 ml of PBS supplemented with 1% Tween-20.

6. Add 1 ml of log phase TG1 cells to the flask and incubate for 37 hours at 37 °C.

7. Repeat 6 steps for a total of 4 repeat cycles.

8. The cells obtained above were diluted to 100,000 cells/ml and cultured on a 1.5% agar plate to which 0.1% ampicillin was added to obtain a monoclonal antibody. 9. The clones on the above plates were cultured on 96-well deep well plates, one clone per well, and a total of 960 clones (10 96-well plates) were prepared.

10. Centrifuge the deep well plate at 5000 RPM for 20 minutes on a 96-well plate centrifuge, transfer the supernatant to a new sterile deep well plate, and store at 4 degrees after sealing.

11. Take 10 pieces of 96-well plate, add human RANKL ( lOug/ml) to each well and add 10 μl of conventional coating. Add 10 μl of the above-preserved supernatant, and incubate for 1 hour at 37 degrees. Tween-20 was washed 20 times with PBS.

12. Add 1 μl of HRP-labeled goat anti-M13 mAb (purchased from GE), incubate at 37 degrees for 30 minutes, and wash 10 times with 1% T _wee n_20 in PBS.

13. Add 200 μl of PBS containing 0.025% DAB developer and 1 μl of 1% 3⁄40 ₂ , incubate for 30 minutes at 37 degrees, and read the light absorption at 595 nm on a plate reader.

14. Determine the wells with strong color reaction based on the light absorption reading. The corresponding clones of these wells are the affinity clones with strong affinity. In this study, 762 positive clones were screened by the above procedure, and 8 of the most affinity clones were initially determined based on the readings.

15. A small sample preparation was performed on the above 8 clones and used for the study of affinity determination. It was finally determined that the affinity of clone 2G8 was the highest, reaching 3. 2X10E-9o affinity was determined by Scatchard analysis (Munson et al, 1980). , Anal. BioChem, 107: 220), the results showed that the affinity of 2G8 reached 3. 2X10E_9.

(3) Cloning of the antibody variable region coding sequence into an expression vector

1. The above 2G8 strain was amplified in 100 ml of LB medium, and plasmid DNA was purified using Promega's plasmid DNA extraction and purification kit.

2. The above plasmid was digested with DNA, and the fragment was digested on a 1.5% agarose gel electrophoresis, and a band of about 360 bp was taken for gel recovery, and the resulting fragment was a heavy chain variable region.

3. The above plasmid DNA was separated on a 1.5% agarose gel electrophoresis, and a band of about 320 bp was taken for gel recovery, and the resulting fragment was a light chain variable region.

Then using the overlapping PCR method [Two-step total gene synthesis method, Lei Young* and Qihan Dong, Nucleic Acids Research, Vol. 32 No. 7; Assembly PCR ol igo maker: a tool for designing ol igo deoxynuc- leotides for constructing long DNA molecules for RNA production, Roman Rydzanicz, X. Sharon Zhao and Phi l ip E. Johnson, Nucleic Acids Research, 2005, Vol. 33], respectively, the heavy chain variable region and the light chain variable region are respectively correspondingly constant The regions (including the signal peptide coding region) are spliced to form a coding region for the full-length heavy chain and the full-length light chain. The sequences of the constant regions of the heavy and light chains are as follows:

(1) light chain constant region

tctagaccaccatggaaaccccagcgcagcttctcttcctcctgctactctggctcccagataccaccgga [V region] cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgt gtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaact cccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagca gactacgagaaacacaaagtctacgcctgcgaagtcaccccatcagggcctgagctcgcccgtcacaaagagcttca acaggggagagtgttgataagtcgac (SEQ ID NO: 5)

(2) heavy chain constant region

aaggttgaccaccatggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgt [V region] cctccaccaagggcccatcggtcttccccc tggcgccctgctccaggagcacctccgagagcacagcggccctggg ctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcaca ccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggc acccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttg tgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggaca ccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccg tgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaag gcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgccc ccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgc cgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctcct tcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatg Ctccgtgatgcat gaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataagtcgac (SEQ ID

NO : 6)

4. The coding regions of the above full-length heavy and light chains are inserted into an expression vector to constitute an expression vector for the full-length antibody. The expression vector used in this experiment used phCMV-I I (Fig. 1). In this example, a vector such as pcDNA3.1 of Provigen and pCI-Neo of Promega can also be used. Example 2: Transient expression of recombinant antibodies in CHO cells and preparation of small samples

1. The expression vector carrying the antibody gene constructed above was amplified by inoculating Escherichia coli DH5a strain in 100 ml of LB medium, and the plasmid DNA was extracted with Qiagen's Ultrapure Plasmid DNA Purification Kit. There are many similar products on the market to choose from.

2. The purified plasmid DNA was transfected into CH0 cells using Invitrogen's Lipofamine kit, and the manufacturer's instructions were followed. PEI25000 or other companies' transfection reagents or kits are also available.

2. Transfection conditions: CHO DHFR (-) cells (ATCC No. CRL-9096) cultured in 24 hours were added to 5 ml of fresh E302 medium to achieve a seeding density of 2x10e5 cells/ml; after 37 hours of culture at 37 degrees The cells were collected at 450Xg and transferred to 1 ml of fresh DMEM medium. After careful reselection of the cells, co-transfection was performed according to the manufacturer's instructions. After co-transfected CH0 cells were cultured for 48 hours, 50 μl of the supernatant was taken for detection of antibody expression by ELISA.

3. Add 45 ml of fresh E302 medium to the above culture system, add G418 to a final concentration of 50 ug/ml, and continue to culture for 96 hours at 37 degrees.

4. Purification of monoclonal antibody: The purification of the monoclonal antibody was directly isolated and purified from the above cell culture supernatant by Protein A affinity chromatography column, and the purity of the obtained product was more than 90% by SDA-PAGE electrophoresis.

5. The product of the above affinity chromatography was again subjected to molecular sieve chromatography to obtain a sample with a purity of > 98% (Fig. 2). These samples can be used for further analysis and research as follows.

Example 3: Neutralizing activity and affinity of monoclonal antibodies

1. Neutralizing activity of anti-human RANKL antibody

(1) Inhibition of osteoclast formation

RAW264. 7 (ATCC No. TIB-71, Manassas, Va.) is a tumor-derived macrophage cell line induced by Abelson mouse leukemia virus, which can differentiate into osteoclasts in the presence of RANKL. Simonet et al. (1997, Cell 89 p. 309) and Lacey et al. (1998, Cell 93 p. 165) describe in detail the detection of osteoclasts in RAW cell culture in the presence of RANKL. RAW cells can be stimulated by ligands to differentiate into osteoclasts, and their differentiation can be understood by detecting the activity of TRAP (Tartrate resistant acid phosphatase (TRAP). Therefore, anti-human RANKL antibody pairs The effects of osteoporosis or bone damage can also be detected.

In the presence of a certain amount (50 ng/ml) of RAML, 10 ng/ml to 150 ng/ml of the anti-human RANKL antibody prepared by the present invention was added, and RAW cells were cultured for 4 days, and treated with ara-nitrophosphate. After 5 minutes, the medium was aspirated from the cells, and 120 ul of 0.1 M citrate buffer (containing 1 mL of triton X-100) was added to each well, and then treated at room temperature for 5 minutes. Using the QUIDEL Metra TRAP5b ELISA kit (QUIDEL product number: 8036), add 100 μl of PNPP solution (157. 8 mg acid phosphatase reagent (Sigma 104-100), 7. 2 ml tartrate solution (Sigma cat. no) 387-3) and 22. 8 ml citrate buffer), treat at room temperature for 3 to 5 minutes. Finally, the reaction was stopped by adding 50 ul of 0.5 M NaOH solution.

TRAP converts para-nitrophosphate to para-nitrophenol, which can be quantified at 405 nm. The activity of TRAP is a surrogate for osteoclast development and is associated with an optical density of 405 nm. A set of data on the optical density and anti-RANKL antibody concentration reflects the inhibition of osteoclast formation by the antibody.

Neutralization test: Tartrate acid phosphatase (TRAP) staining was used. Multinucleated TRAP positive staining is a characteristic marker of osteoclasts, which is the basis for the design of this trial. RAW 264. 7 cells were used as test materials, 1 10 ⁴ cells/well were seeded in 24-well plates, and cultured for 24 hours, respectively, with 50 ng/ml RAML and different concentrations (10 ng to 500 ng/ml), cells were changed every 3 days. 1 time. Day 6 anti-tartaric acid phosphatase (TRAP) staining (Sigma-Aldrich, 387-A, USA), cells were fixed in citrate/acetone solution for 1 minute, and incubation solution with naphthol AS BI phosphate as substrate Incubate for 1 hour at 37 °C, wash 3 times with distilled water, counterstain for 2 minutes with hematoxylin. After drying, the xylene is transparent, sealed with DPX (polystyrene + butyrate + xylene), and observed under light microscope. TRAP-positive cells were osteoclasts, and the number of TRAP-positive multinucleated osteoclasts (3 nuclei) was counted by microscopy.

The test results are shown in Figure 3. The antibody concentrations were: 0, 0. 001, 0. 01, 0. 05, 0. 1, 1. 0, 10. Ong/ml o As can be seen from the data in the figure, the anti-human RANKL antibody of the present invention has good The ability of neutralizing RANKL to stimulate osteoclast formation.

2. Affinity identification

Affinity measurements were performed using Scatchard analysis (Munson et al, 1980, Anal. BioChem, 107: 220) and the results showed that the affinity of 2G8 reached 3. 2X10E-9.

The above results indicate that the affinity of 2G8 has reached 0. InM, which has reached the internationally recognized requirement for the affinity of the therapeutic antibody to reach the InM level. 3. DNA sequencing of anti-human RANKL monoclonal antibody gene

According to the pedigree, the variable region of the above 2G8 anti-human RANKL antibody was subjected to DNA sequencing, and the sequencing work was entrusted to Shanghai Keikang Co., Ltd. The results are as follows:

2G8 light chain variable region gene sequence (5'→3', 324bp)

gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgtagggccag tcagagtgttcgcggcaggtacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtg cat ccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccat cage agactggagcctgaagattttgcagtgtgggactgtcagcagtatggtagttcacctcggacgttcggccaagggac caaggtggaaatcaa (SEQ ID NO: 1)

Presumed amino acid sequence of the 2G8 light chain variable region (108 amino acids)

RLEPEDFAVYYCQQRLNWPLTFGGGTKVEIK (SEQ ID NO : 2)

2G8 heavy chain variable region gene sequence (5'→3', 366bp)

gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctgg attcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaggtatta ctgggagtggtggtagtacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaac acgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattact tgcgaaagatccagggactac ggt attat agttggttcgacccctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 3) deduced amino acid sequence of a heavy chain variable region of 2G8 (122)

TLYLQMNSARLEDTSVYYCAKDPGTTVIMSWFDPWGQGTLVTVSS (SEQ ID NO: 4) Example 4: Cloning and expression of anti-human RANKL-Fab and PEGylation

(1) Fab expression and miniprep l (^g The above DNA fragments SEQ ID NO: 1 and SEQ ID NO: 3 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 encephalitis virus neutral ization. Vaccine. 25 ; 23 ( 2 ) : 163-71 ), electroporation of 5 ml of E. coli DH5a (from New England Biolabs or Takara) After the cells, the products were screened and identified on IPTG/X-gal plates. 20 plaques were inoculated into liquid LB medium containing ampicillin for amplification. After IPTG induction, ELISA was used to prepare small amounts of Fab antibody. , called 2G8Fab.

(2) PEGylation of Fab The 2G8 Fab obtained above is PEGylated, and the PEGylation and purification method of Fab is referred to AP Champman et al. [Reference: Therapeutic antibody fragments with prolonged in vivo half l ife. Nature Biotech. V17 1999 August, Page The method of 780-783] was carried out to obtain PEGylated anti-human RANKL 2G8 Fab-PEG.

Example 5: Study on the antagonism of RAML by 2G8Fab-PEG. The purified antibody of the above Example 4 was tested for its neutralizing ability to RANKL by the following method.

The prepared Fab was antagonized by the RANKL neutralization ability test as described in Example 3, and it was found that 2G8Fab has a good neutralization effect (see Fig. 3 for the results).

Claims

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An anti-human RANKL antibody, characterized in that the light chain variable region amino acid sequence is SEQ ID NO: 2, and the heavy chain variable region amino acid sequence is SEQ ID NO: 4.

A polynucleotide characterized by encoding the light chain variable region of the anti-human RANKL 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 variable region of the anti-human RANKL antibody of claim 1.

The polynucleotide according to claim 4, wherein the sequence of the polynucleotide is SEQ ID NO: 3.

A polynucleotide, which encodes the full length of the anti-human RANKL antibody of claim 1.

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

8. A Fab antibody against an anti-human RANKL antibody according to 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 preventing or treating osteoporosis and/or bone damage associated with RANKL.