WO2011116527A1 - Anti human rankl monoclonal antibodies and uses thereof - Google Patents

Abstract

The invention provides antibodies that specially bind to human RANKL and uses of said antibodies for the treatment and/or prevention of RANKL-related osteoporosis and/or bone lesions.

Description

Anti human RANKL monoclonal antibodies developed by PAE Technology and their use

Field of the invention

The present invention relates to recombinant antibodies that bind human Receptor Activator for Nuclear Factor KB Ligand (RANKL). Their impacts on osteoclast formation, and further their potentiality for treatment of bone diseases, such as osteoporosis, bone erosions caused by autoimmune diseases, cancers and hormone treatment are described.

Background of the invention

Bone formation and remodeling is a very complex biological procedure that involves RANK, RANKL and OPG(osteoprotegerin).

Osteoclasts and osteoblasts dictate skeletal mass, structure, and strength via their respective roles in resorbing and forming bone. Bone remodeling is a spatially coordinated lifelong process whereby old bone is removed by osteoclasts and replaced by bone-forming osteoblasts. The refilling of resorption cavities is incomplete in many pathologic states, which leads to a net loss of bone mass with each remodeling cycle. Postmenopausal osteoporosis and other conditions are associated with an increased rate of bone remodeling, which leads to accelerated bone loss and increased risk of fracture.

RANK, also known as TNF-related activation-induced cytokine (TRANCE), osteoprote- gerin ligand (OPGL), and ODF (osteoclast differentiation factor), is a functional acceptor that mainly expresses on the cell surface of preosteoclasts and osteoclasts. It has been proved by many results that RANK is a key factor in bone resorption, and be able to accelerate osteoclast cell differentiation to play bone resorption. RANK and RANKL play pivotal roles in regulating various biological processes such as bone homeostasis, immune function, vascular disease and mammary gland development [3, 5-8].

RANKL overproduction is implicated in a variety of degenerative bone diseases, such as Rheumatoid arthritis (RA) and psoriatic arthritis. RANKL also has a function in the immune system, where it is expressed by T helper cells and is thought to be involved in dendritic cell maturation. For details see: (1) Buckley KA, Fraser WD, 2003, Receptor activator for nuclear factor kappa B ligand and osteoprotegerin: regulators of bone physiology and immune responses/potential therapeutic agents and biochemical markers. Ann. Clin. Biochem. 39 (Pt 6): 551-6. (2) Whyte MP, Mumm S, 2005, Heritable disorders of the RANKL/OPG/RANK signaling pathway. J. of musculoskeletal & neuronal interactions 4 (3): 254-67.

OPQ also known as osteoclastogenesis inhibitory factor (OCIF), is a cytokine, which can inhibit the production of osteoclasts. It is a member of TNF receptor superfamily. OPG inhibits the differentiation of osteoclast precursors into osteoclasts and also regulates the resorption of osteoclasts in vitro and in vivo. OPG is a RANK homolog, and works by binding to RANKL on osteoblast/stromal cells, thus blocking the RANKL-RANK ligand interaction between osteoblast/stromal cells and osteoclast precursors. This has the effect of inhibiting the differentiation of the osteoclast precursor into a mature osteoclast. Recombinant human OPG specifically acts on bone, increasing bone mineral density and bone volume. Space shuttle flight STS-108 in 2001 tested the effects of OPG on mice in microgravity, finding that it did prevent increase in resorption and maintained mineralization. OPG has been used experimentally to decrease bone resorption in women with postmenopausal osteoporosis and in patients with lytic bone metastases. For details see: Khosla S, 2001, Minireview: The OPG/RANKL/RANK System. Endocrinology, 142:5050-5055. OPG can directly binds to RANK and competitively inhibits the binding process between RANKL and RANK and inhibit osteoclast differentiation and function. On the other hand, OPG can form trimer with RANKL/RANK complex and directly inhibit the function of RANKL/RANK [14]. Abnormal bone resorption and osteosclerosis have been found in RANKL or RANK deficient mouse. Osteoporosis was involved in OPG-deficient mouse while bone density will be improved after recombinant OPG injected. These results support that RANKL/RANK/OPG forms a key modulation system for osteoclast differentiation and function [1, 2, 5, 6].

RANKL blockades has been proved being able to prevent and/or improve bone loss and erosions caused by osteoporosis, chronic inflammatory disorders, and malignant tumors in animal models, and may emerge as a therapy in humans based on studies in postmenopausal osteoporosis, myeloma, bone disease, and osteolytic metastases [Bekker PJ, et al, 2005, J Bone and Mineral Res., 202275-2282, and 1, 3]. This factor is expressed osteoblast/stromal cells and activated lymphocyte. RANKL inhibitors also prevent focal bone erosions that occur in animal models of RA and bone metastasis. For details, see: Kearns AE, et al, 2008, Receptor activator of nuclear factor kappa B ligand and osteoprotegerin regulation of bone remodeling in health and disease. Endocr Rev. 29(2): 155-92.

Based on elucidation of OPG/RANK/RANKL function and their relationship, mechanism of osteoporosis and bone loss and erosion in patients with osteoporosis, bone metastasis, myeloma and RA has been substantially understood. All these understandings bring us a possibility to design and/or develop new therapies for the diseases [1, 3, 6]. AMGEN developed Denosumab, a new generation drug based on monoclonal antibody technology targeting RANKL to treat bone erosions caused by several diseases (see for details: US Patent Application No. 20040033535).

Osteopenia is a condition where bone mineral density is lower than normal. It may arise from a decrease in the rate of bone synthesis or an increase in the rate of bone destruction or both, and is considered by many doctors to be a precursor to osteoporosis, also referred to as postmenopausal and senile osteoporosis. However, not every person diagnosed with osteopenia will develop osteoporosis. More specifically, osteopenia is defined as a bone mineral density T-score between -1.0 and -2.5 (For details see: 1. Wikipedia, the free encyclopedia online; 2. WHO Scientific Group on the Prevention and Management of Osteoporosis (2000: Geneva) 3. Prevention and management of osteoporosis : report of a WHO scientific group" (pdf) http://whqlibdoc.who.int/trs/WHO_TRS_921.pdf. Retrieved 2007-05-31).

Osteoporosis occurs when body fails to form enough new bone, when too much old bone is reabsorbed by the body, or both. Calcium and phosphate are two minerals that are essential for normal bone formation. Throughout youth, human body uses these minerals to produce bones. If a body does not get enough calcium, or if your body does not absorb enough calcium from the diet, bone production and bone tissues may suffer. As aging, calcium and phosphate may be reabsorbed back into the body from the bones, which makes the bone tissue weaker. This can result in brittle, fragile bones that are more prone to fractures, even without injury.

The leading causes of osteoporosis are a drop in estrogen in women at the time of menopause and a drop in testosterone in men. Women over age 50 and men over age 70 have a higher risk for osteoporosis.

Many factors can cause bone loss and erosion.

1. aging and woman postmenopausal.

2. inflammatory diseases, such as RA, may cause severe bone erosion in certain instances.

3. bone metastasis and myeloma, they can cause bone erosion by the following ways: ( 1 ) erosion by resorption resulting in osteolytic lesions and leaving tini holes in bone. (2) trigger of unusual bone growth in abnormal places and leaving osteosclerotic lesions.

4. some hormone therapy, such as taking corticosteroid medications (prednisone, methylprednisolone) every day for more than 3 months, or taking some anti-seizure drugs.

5. Hyperparathyroidism.

6. eating disorders, not enough calcium consumption.

7. being confined to a bed.

Several factors have been identified which may contribute to postmenopausal and senile osteoporosis. They include alteration in hormone levels with aging and inadequate calcium consumption attributed to decreased intestinal absorption of calcium and other minerals. Certain treatments have included hormone therapy or dietary supplements in an attempt to retard the process. More recently, anti-resorptive agents such as bisphosphonates and selective estrogen receptor modifiers (SERMs) have emerged for the prevention and treatment of reduced bone mass. Thus, it may be useful to combine those treatments with molecules that can regulate the activity of RANKL in treating certain osteopenic disorders.

Monoclonal Antibody Therapy:

Monoclonal antibody therapy is the use of monoclonal antibodies (or mAb) to specifically bind to target cells. This may then stimulate the patient's immune system to attack those cells. It is possible to create a mAb specific to almost any extracellular/cell surface target, and thus there is a large amount of research and development currently being undergone to create mAb for numerous serious diseases (such as RA, multiple sclerosis and different types of cancers). There are a number of ways that mAbs can be used for therapy. For example: mAb therapy can be used to destroy malignant tumor cells and prevent tumor growth by blocking specific cell receptors. Variations also exist within this treatment, e.g. radioimmunotherapy, where a radioactive dose localizes on target cell line, delivering lethal chemical doses to the target.

In last decade, monoclonal antibody therapy gains great achievements and have shown very high potentiality and prospect in many serious diseases including but not limited to cancers, cardiovascular diseases, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infection, and will show very prospective future. This category of products is expending at a dramatic speed because of their unbeatable efficacy , safety and huge market demand [9, 10, 11, 12].

Up to now, the prevailing procedure to obtain mAb is hybridoma involving rodent (e.g. mouse and rabbits), Primates (e.g. macaque, ape and monkey). One problem in medical applications is that the standard procedure of producing mAb yields human anti-mouse antibodies (HAMA). Although murine antibodies are very similar to human ones there are differences. The human immune system hence recognizes mouse antibodies as foreign, rapidly removing them from circulation and causing systemic inflammatory effects. Such responses are recognized as producing HACA (Human Anti-Chimeric) antibody antibodies or HAMA.

A solution to this problem would be to generate human antibodies directly from humans or human cells. Abraham Karpas in the University of Cambridge developed human-human hybridoma technology with his human myeloma cell line Karpas-707H that can be used in research and for the production of human mAb. The cell line has the potential to immortalize most of the antibodies the human body produces to fight infections, cancer, AIDS and other pathological conditions (Karpas A, et al, 2005, Studies of four new human myeloma cell lines. Leuk Lymphoma 46(1): 101-12). However, this is not easy primarily because it is generally not seen as ethical to challenge humans with antigen in order to produce antibody; the ethics of doing the same to non-humans is a matter of debate. Furthermore, it is not easy to generate human antibodies against human tissues or human proteins. Another important and obvious problem is that a bottleneck when human is involved in development of novel mAb even though human immune system can produce very large population of antibody-producing cells: the abundance of target-specific precursor B cell is extremely low in human, at the same time the positive precursor cell rates in human are very low compared with the demand for human hybridoma construction. And hence, precursor B cell isolation, purification and enrichment will be one of the key technologies for human hybridoma.

Various approaches using recombinant DNA technology to overcome this problem have been tried since the late 1980s. In one approach, one takes the DNA that encodes the binding portion of monoclonal mouse antibodies and merges it with human antibody-producing DNA. One then uses mammalian cell cultures to express this DNA and produce these half-mouse and half-human antibodies. (Bacteria cannot be used for this purpose, since they cannot produce this kind of glycoprotein).

In the past scientists could produce only mouse mAb, which cannot be used for immunotherapy without being humanized as they provoke HAMA.

Genetically engineered mice, so called transgenic mice, can be modified to produce human antibodies[16], and this has been exploited by a number of commercial organizations:

1. Medarex - who market their UltiMab platform[17]

2. Abgenix - who marketed their Xeno mouse technology. Abgenix were acquired in April 2006 by Amgen[18].

3. Regeneron's Veloclmmune technology[ 19] .

In August 2006 the Pharmaceutical Research and Manufacturers of America reported that U.S. companies had 160 different mAb in clinical trials or awaiting approval by the Food and Drug Administration[20] .

Humanized animal is powerful and repeatable as shown by many successful cases in the last decade. Ever since the discovery that mAb could be generated in-vitro, scientists have targeted the creation of 'fully' human antibodies to avoid some of the side effects of humanized and chimeric antibodies. Two successful approaches were identified - phage display-generated antibodies and mice genetically engineered to produce more human-like antibodies.

One of the most successful commercial organizations behind therapeutic mAb was Cambridge Antibody Technology (CAT). Scientists at CAT demonstrated that phage display could be used such that variable antibody domains could be expressed on filamentous phage antibodies. This was reported in a key Nature publication[10]. Other significant publications include references 22 and 23.

CAT developed their display technologies further into several, patented antibody discovery/functional genomics tools which were named Proximol [12] and ProAb. ProAb was announced in December 1997[13] and involved high throughput screening of antibody libraries against diseased and non-diseased tissue, whilst Proximol used a free radical enzymatic reaction to label molecules in proximity to a given protein[14][15].

Antibody library and phage display technology have been powerful tools to develop novel mAb with high quality avoid hybridoma procedure. In the last two decades, these technologies gained great progress for development of therapeutic mAbs, for instance, the directed molecular evolution technologies disclosed in US patent 007175996 and others. These technologies are becoming indispensable tools in antibody maturation and so forth [13, 14, 15].

RANKL antagonists and their application in clinic

As mentioned above, RANKL is a key factor causing osteoporosis, and there are a huge amount of research results showed that it is a critical factor of bone erosions accompanying Ankylosing Spondylitis, Psoriasis, Rhumatoid arthritis, and of osteoporosis that is very popular in aged people and postmenopausal women allover the world[16, 17, 18, 19, 20].

Organizations developing drugs and therapies preventing or treating osteoporosis and bone erosions is blooming whereas these diseases has a very large population and very high disability rate [21]. Up to now several RANKL antagonists such as ligand-Fc fusion proteins and anti human RANKL mAb entered preclinical or clinical phases. Some of them, for instance, recombinant OPG and recombinant therapeutic mAb, gained very prospective results. This invention herein discloses a procedure to develop therapeutic mAb. This procedure involves (1) panning a fully-human Naive combinatorial antibody library, (2)improve affinity of the mAb obtained from the library mentioned above by programmed artificial molecular evolution (PAE) technology, and (3) identify mAbs with high technological parameters, mainly high affinity.

SUMMARY OF THE INVENTION

In certain embodiments, the invention provides for a light chain, comprising an amino acid sequence as set forth in SEQ ID NO: 2 or a fragment thereof.

In certain embodiments, the invention provides for a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 4 or a fragment thereof.

In certain embodiments, the invention provides for a light chain, comprising a nucleotide sequence as set forth in SEQ ID NO: 1 or a fragment thereof.

In certain embodiments, the invention provides for a heavy chain comprising a nucleotide sequence as set forth in SEQ ID NO: 3 or a fragment thereof.

In certain embodiments, the invention provides for a full-length antibody light chain specific to human RANKL, comprising a nucleotide sequence as set forth in SEQ ID NO: 5 or a fragment thereof.

In certain embodiments, the invention provides for a full-length antibody heavy chain specific to human RANKL, comprising a nucleotide sequence as set forth in SEQ ID NO: 6 or a fragment thereof.

In certain embodiments, the invention provides for a Fab fragment of above antibody specific to human RANKL. Preferably this Fab is PEGylated format.

In certain embodiments, the invention provides methods for the full-length or Fab fragment of above antibodies to treat osteoporosis in postmenopause women or aged people, or bone erosions caused by inflammatory diseases, cancer bone metastasis, hormone treatment or others.

Advantages of the invention:

There are several kinds of drugs such as Bisphosphonates and Chinese medicines for osteoporosis and bone erosions with good efficacy even though they are of the following problems.

Bisphosphonates (also called diphosphonates) are a class of drugs that prevent bone loss, used to treat osteoporosis and similar diseases. Bone has constant turnover, and is kept in balance (homeostasis) by osteoblasts creating bone and osteoclasts digesting bone. Bisphosphonates inhibit the digestion of bone by osteoclasts. Osteoclasts also have constant turnover and normally destroy themselves by apoptosis, a form of cell suicide. Bisphosphonates encourage osteoclasts to undergo apoptosis. The uses of bisphosphonates include the prevention and treatment of osteoporosis, osteitis deformans ("Paget's disease of bone"), bone metastasis (with or without hypercalcaemia), multiple myeloma, primary hyperparathyroidism, osteogenesis imperfecta and other conditions that feature bone fragility.

Bisphosphonates have the following side effects (For details, see: http:// en.wikipedia.org/wiki/Bisphosphonate #Side-effects): ( 1 ) Oral bisphosphonates can cause upset stomach and inflammation and erosions of the esophagus, which is the main problem of oral N-containing preparations. This can be prevented by remaining seated upright for 30 to 60 minutes after taking the medication.

(2) Intravenous bisphosphonates can give fever and flu-like symptoms after the first infusion, which is thought to occur because of their potential to activate human γδ T cells. These symptoms do not recur with subsequent infusions.

( 3 ) There is a slightly increased risk for electrolyte disturbances, but not enough to warrant regular monitoring.

(4) In chronic renal failure, the drugs are excreted much more slowly, and dose adjustment is required.

( 5 ) Bisphosphonates have been associated with osteonecrosis of the jaw; with the mandible twice as frequently affected as the maxilla and most cases occurring following high-dose intravenous administration used for some cancer patients. Some 60% of cases are preceded by a dental surgical procedure (that involve the bone), and it has been suggested that bisphosphonate treatment should be postponed until after any dental work to eliminate potential sites of infection (the use of antibiotics may otherwise be indicated prior to any surgery).

( 6) A number of cases of severe bone, joint, or musculoskeletal pain have been reported, prompting labeling changes.

( 7) Recent studies have reported bisphosphonate use (specifically zoledronate and alendronate) as a risk factor for atrial fibrillation in women. The inflammatory response to bisphosphonates or fluctuations in calcium blood levels have been suggested as possible mechanisms. One study estimated that 3% of atrial fibrillation cases might have been due to alendronate use. Until now however, the benefits of bisphosphonates generally outweigh this possible risk, although care needs to be taken in certain populations at high risk of serious adverse effects from atrial fibrillation (such as patients with heart failure, coronary artery disease or diabetes). FDA has not yet confirmed a causal relationship between bisphosphonates and atrial fibrillation.

( 8 ) Matrix metalloproteinase 2 may be a candidate gene for bisphosphonate- associated osteonecrosis of the jaws, since it is the only gene known to be associated with bone abnormalities and atrial fibrillation, both of which are side-effects of bisphosphonates.

( 9) There are concerns that long-term bisphosphonate use can result in severe or over-suppression of bone turnover especially at the femur sub-trochanteric region. It is thought that micro-cracks in the bone are unable to heal and eventually unite and propagate, resulting in atypical fractures. Such fractures tend to heal poorly and often require some form of bone stimulation, for example bone grafting as a secondary procedure. This complication is not common, and the benefit of overall fracture reduction still holds.

The product of this invention has different therapeutic mechanisms compared with the current first-line drugs such as Fosamax, a bisphosphonate from MERK, and has better affectivity and safety while it needs only twice a year compared to more than once a week.

This invention isolated a fully-human anti human RANKL from a human antibody library and further improved by PAE technology and led potentially to a novel therapeutic monoclonal antibody with high affinity designated PAE30A. The results showed that PAE30A has high ability to neutralize human RANKL and inhibits the differentiation, activity, and survival of osteoclasts both in vitro and in vivo. Firstly, this product is a fully-human antibody that has significantly decreased immunogenicity so that the adverse side effects decreased greatly compared with chimeric or humanized mAb.

One problem in medical applications is that the standard procedure of producing mAbs yields mouse antibodies. Although murine antibodies are very similar to human ones there are differences between them. The human immune system hence recognizes mouse antibodies as foreign, rapidly removing them from circulation and causing systemic inflammatory effects. Such responses are recognized as producing HAC A (Human Anti-Chimeric) antibodies or HAMA (Human Anti-Mouse) antibodies.

Secondly, this invention utilizes the Fab fragment as one alternative format of this product that will decrease the product costs without loss of the above advantages. Otherwise, this format will have not CDC and ADCC effects that will possibly bring adverse side-effects.

And thirdly, the Fab format adopts Surface Modification Technology by PEGylation so that its life in the blood circulation was dramatically extended and the product become sustained-released. PEGylation is the process of covalent attachment of Polyethylene glycol polymer chains to another molecule, normally a drug or therapeutic protein. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule. The covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity), increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.

References

1. Schwaber J, EP Cohen, 1973, Humanxmouse somatic cell hybrid clone secreting immunoglobulins of both parental types. Nature 244 (5416): 444-7.

2. Cambrosio A,P Keating, 1992, Between fact and technique: The beginnings of hybridoma technology. J History of Biology 25 (2): 175.

3. KOhler QC Milstein, 1975, Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495.

4. Riechmann L, M Clark, H Waldmann, G Winter, 1988, Reshaping human antibodies for therapy.

Nature, 332:323-7. PMID 3127726.

5. Siegel DL, 2002, Recombinant monoclonal antibody technology. Transfusion clinique et biologique: journal de la Societe francaise de transfusion sanguine 9 (1): 15-22. PMID 11889896. 6. Schmitz U, A Versmold, P Kaufmann,HG Frank, 2000, Phage display: a molecular tool for the generation of antibodies-a review. Placenta 21 Suppl A: SI 06-12.

7. Modified from Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1 : 118-129

8. Takimoto CH, E Calvo,2008,Principles of Oncologic Pharmacotherapy" in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ Cancer Management: A Multidisciplinary Approach. 11 ed. 2008.

9. McCafferty, J Griffiths, A, Winter, Q Chiswell, D, 1990, Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552-554.

10. http : //bfgp . oxfordj ournal s . org/ cgi/ content/ ab stract/ 1/2/189

11. http://www.ncbi.nlm.nih.gov/pubmed/11968489

12. http://www.ukbusinesspark.co.uk/cay92125.htm

13. http ://www.sciencedirect.com/science?_ob= Article

0&_rdoc=l&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=960781456 &_rerunOrigin=google&_acct=C000050221 &_ver sion= 1 &_url Version=0&_userid= 10&md5= eacedcd4604792172fc 18143 a590a454

14. http://www.chidb.com/newsarticles/issue3_l .ASP

15. http://www.ncbi.nlm.nih.gov/pubmed/7494109

16. http://www.medarex.com/Development/UltiMAb.htm

17. http://wwwext.amgen.com/media/media_pr_detail.jsp?year=2006&releaseID=837754

18. http ://www.regeneron. com/velocimmune. html

19. PhRMA Reports Identifies More than 400 Biotech Drugs in Development. Pharmaceutical Technology, August 24, 2006. Retrieved 2006-09-04.

20. Edward M Schwarz, TR Christopher, 2007, Clinical development of anti-RANKL therapy, Arthritis Research & Therapy , 9 Suppl 1.

21. Marks JD, HR Hoogenboom, TP Bonnert, J McCafferty, AD Griffiths, G Winter, 1991, By-passing immunization: human antibodies from V-gene libraries displayed on phage. J Mol Biol, 222:581-597.

22. Carmen and L. Jermutus, 2002, Concepts in antibody phage display". Briefings in Functional Genomics and Proteomics 1(2): 189-203.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Mammalian expression vector schematic diagram. Antibiotic gene (AMP) and replication origin ( O ) are derived from pUC57, eukaryotic promoter (Pcmv) is derived from human cytomegalovirus, SV40 Ori and SV40 PolyA signal are derived from SV40, antibiotic gene (Neo) derived from pCDNA3.1(Invitrogen).

Figure 2: HPLC diagram showing the quality of the target monoclonal antibody after purification. As shown in the diagram, the produced protein has only one band to a degree of purity approximating 98% by integral. Figure 3 : Inhibition of the present antibodies on osteoclast differentiation. PAE36, a mAb with Fc fragment identical to IgG2, is the negative control, and AMG162 the positive. The mAb concentrations: 0.00, 1.00, 5.00, 10.00, 20.00, 40.00, 80.00, 160.00 ng/ml. The data showed that both 3K7F5/Full ( designated as PAE30Full in other places ) and 3K7F5/Fab ( designated as PAE30Fab) are able to neutralize the stimulation effect of RANKL on osteoclast formation, and more importantly, this effect is closely dose-dependent. On the other hand, this data showed clearly that 3K7F5/Full and 3K7F5/Fab are more effective on the inhibition effect than its positive control.

EXAMPLES

The following examples, including the experiments conducted and results achieved are provided for illustrative purposes only and are not to be construed as limiting the present invention.

The basic technologic strategy of this invention includes: (1) isolating variable region specific to human RANKL with high affinity from a fully-human antibody library, and (2) further improve its affinity by PAE technology, before (3) evaluating their therapeutic values after serial experiments in vitro and in vivo for treatment of osteoporosis and bone erosions.

Example 1: Panning for Variable-fragments specific to human RANKL

( 1 ) construction of combinatorial fully-human Naive anti body library

An ultra large antibody library was constructed with blood samples of over 3000 individuals from different provinces and different nationalities, based on the procedure disclosed in the following publications. Phage lysate from the constructed phage-displayed library diluted to 10E10 in YT Medium containing 7% DMSO was stored at -80°C in 1ml aliquots for later use.

1. Hoogenboom FIR, and G Winter, 1992, By-passing immunisation: human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J Mol Biol,227(2): 381-388.

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

3. Nissim, A,HR Hoogenboom, IM Tomlinson, G Flynn, C Midgley, D Lane, G Winter, 1994, Antibody fragments from a' single pot' phage display library as immunochemical reagents. EMBO J, 13 :692-698.

4. Marks JD, HR Hoogenboom, TP Bonnert, J McCafferty, AD Griffiths, G Winter, 1991, By-passing immunization: human antibodies from V-gene libraries displayed on phage. J Mol Biol, 222:581-597.

5. Haidaris CQ J Malone, LA Sherrill, JM Bliss, AA Gaspari, RA Insel, MA Sullivan, 2001, Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens. J Immunol Methods. 257 ( 1-2) : 185-202.

(2) panning against human RANKL

1. Thaw one tube of E. coli TGI strain and transfer to a 50ml flask, add fresh LB to 15ml, and culture for 16 hours at 37°C, 225RPM.

2. Thaw one tube of the above mentioned phage lysate in an incubator at 40C as quickly as possible, add to the above TGI culture and and culture for 16 hours at 37°C, 225RPM.

3. spin at 12000rpm for lOminutes, and transfer the supernatant to a sterilized 50ml centrifugal tube, store at 4C for later use. Its titer should be at 2^χ 10E11 or more.

4. coat a 25-ml cell culture flask with recombinant human sRANKL ( from Orbigen) for at least two hours, and add at least 3 >< 10E10 phage particles.

5. incubate at 37°C for 1 hour.

6. decant the supernatant and wash the tube 10 times with 1 xPBS containing .

7. add 1ml freshly prepared TGI cells at log phase into the flask, incubate at 37°C for 16 hours.

8. repeat the above steps four times from step 3 to 7.

9. dilute above cell to 100000 cells/ml before spreading on 1.5 %agar plate containing 0.1% AMP until single colonies with phage plaque can be identified.

10. isolate colonies with phage plaque and culture in 10 deep 96-well plates.

11. spin the plates at 5000RPM for 20 tminutes before transfer the supernatant to new 96-well plates, seal the plates and store at before use.

12. coat ten 96-well plates with recombinant human RANKL at 10μg/m, ΙΟμΙ/well before add Ι μΐ phage supernatant from the above step, incubate at 37°C for lhour before wash 20 times with 1XPBS containing l%Tween-20.

13. add Ι μΐ HRP-labeled goat anti-M13 monoclonal antibody and incubate at 37°C for 30minutes before wahs 10 times with l xPBS containing l%Tween-20.

14. add 200ul PBS containing 0.025%DAB and Ι μΐ 1% H202, incubate for 20minutes at 37°C, then read the absorption data at 595nm.

15. identify the strongest wells with color reaction data, and the corresponding clones are positive clones with relatively high affinity. From this step, 12 clones with relatively higher readings were identified from 876 positive clones. Positive control AMG162 should be included if needed.

16. small scale protein preparations were made for the above 12clones for measuring their affinity.

It's confirmed by three repeated tests that 6E2 and 3K7 were the highest among them. Further experiments with Scatchard analysis (Munson et al, 1980, Anal. BioChem, 107:220) revealed that their affinities were as high as 4.27>< 10E-9 and 3.56>< 10E-9 nM respectively, and the later one is substantially better.

(3) Molecular Evolution by PAE technology

The above results show that 3K7 were at sub-nanomolar level. Programmed Artificial molecular Evolution (PAE) technology, as described in PCT/CN2009/074839 and CN200910198282.X in detail, was used to improve 3K7 affinity to meet the requirements for therapy. For this purpose, a phage-displayed sub-library with the mutated CDR2 and CDR3 in both heavy and light chains of 3K7 was panned against human sRANKL with AMG162 as positive control. After four rounds of mutation and panning, fifteen clones with higher affinity compared with the positive control were obtained. As shown by affinity analysis, the affinities of the tested clones 3K7B8 and 3K7F5 are as low as 0.42x and 0.17x lO^"12nM, much more better than its positive control that showed 5.23 x lO^"12 in parallel tests.

The clone 3K7F5 was used for the following experiments after sequenced as described in Example 6. The sequences are shown as SEQ ID NO. 1 to SEQ ID NO 4. SEQ ID NO. l is the DNA sequence and SEQ ID NO. 2 the deduced amino acid sequence for the light chain, and SEQ ID NO.3 is the DNA sequence and SEQ ID NO.4 the deduced amino acid sequence for heavy chain of the variable region of the clone 3K7F5.

( 4) Cloning the target genes into mammalian expression vector

1. prepare plasmid DNA with PROMEGAs Plasmid DNA Preparation Kit from the bacterial strain containing the above 3K7F5 plasmid from 100ml culture in LB.

2. digest the above plasmid with proper restriction enzymes and separate on 1.5 % agarose gel to isolate the 360bp fragments for heavy chain and 320bp fragments for light chain. Then purify the fragments from the gel with DNA Recovery Kit from Promega or equivalents.

3. combine the above obtained variable fragments with their corresponding constant regions coding sequences of light chain or heavy chain by overlapping PCR method to form full length genes for both heavy and light chains. The overlapping method is as described in: (1) Young L and Dong Q, 2004, Two-step total gene synthesis method, NAR,32(7) e59 DOI: 10.1093/nar/gnh058; (2) Roman Rydzanicz, X. Sharon Zhao and Philip E. Johnson, 2005, Assembly PCR oligo maker: a tool for designing oligo deoxynucleotides for constructing long DNA molecules for RNA production, NAR, V33, W521-W525doi: 10.1093/nar/gki380.

The constant DNA sequences are shown in capital letter in SEQ ID NO.5 and SEQ ID NO.6, respectively. These DNA sequences are pre-cloned into Smal site of pBluescript II SK, designated as pLightCon and pHeavyCon, respectively. The sequences were verified by sequencing, and used as template in the overlapping PCR reactions to form full length light chain and heavy chain with the above obtained variable fragments.

The overlapping regions between the constant and variable fragments should be as long as about 20±l~6bps with Tm at about 60±1 ^°C PCR cycling was set as: 2min at 95 ^°C to pre-denature before 20 cycles of lOsec at 95 ^°C-35sec at 56^°C-45sec at 72^°C, then post extending for 5mins at 72 ^°C .

The full length sequences after added to their constant regions for heavy and light chain are shown in SEQ ID NO. 5 and Seq ID No. 6.

4. insert the above full-length heavy and light chains into the corresponding sites of the mammalian expression vector phCMV-II ( as shown in Fig. l, other mammalian expression vectors, such as pcDNA3.1 from Invitrogen, pCI-Neo from Promega, can be alternatives). Both full light and heavy chains, having Nhel and NotI sites at 5'- and 3' respectively, were digested by Nhel/Notl before ligated to predigested phCMVII with the same restriction enzyme combination. Then, isolate single colonies and verify by sequencing.

5. transform E. coli strain DH5a and verify by DNA sequencing. The resulted correct plasmid was designated as phCMV-II/3K7F5. This clone was used in the following experiments.

Example 2: preparation of recombinant mAb protein

1. Preparation of endotoxin-free plasmid DNA. Inoculate 100ml fresh LB medium with the cell harboring phCMV-II/3K7F5, and prepare plasmid DNA with Ultrapure Plasmid DNA Purification Kit from Qiagen. Other commercially-available alternatives can be used.

2. Transfection and culture of CHO cells. For transfecting mammalian cells with the above prepared plasmid DNA, Lipofamine 2000 from Invitrogen is used as transfection reagent based on the protocol provided by the provider. Alternatives such as PEI25000 can be used, too.

3. Transfection conditions. (1) To 5ml freshly-prepared Ex302 medium (Sigma Aldrich) , add CHO DHFR ( -) cells ( ATCC No. CRL-9096) to final density of 2^χ 10Ε5 cells/ml. Collect cells by centrifugation at 450xg for lOminutes after cultured at 37° for 48 hours. (2) Add 1ml fresh DMEM medium to the tube and resuspend the cells by tapping the tube bottom. Then, transfect the cells with above prepared plasmid DNA. (3)Sampling should be made after 48 hours and expression was monitored by ELISA with 50ul supernatant. If the expression is roughly good, go to next step. (4) add 45ml fresh EX302 medium and add G418 to the final concentration of 50ug/ml, culture at 37° for 96 hours.

4. mAb purification. Centrifuge the culture at 2000RPM for lOminutes and collect the supernatant. Then pass the supernatant through HiTrap Protein A HP before Capto S and Capto Q columns. The eluent purity was verified by SDA-PAGE electrophoresis. The purity should be higher than 95 % .

5. Polish of the eluent. Gel filtration chromatography on HP Superdex 200, 10/300 column was used as a final "polishing" step. After this, the purity can be reach >98% (Fig.2 ). The final polished eluent with high purity can be stored at -80C for later use.

Example 3: expression and recombinant protein preparation of Fab format

(1) constructing expression vectors

10μ§ DNA fragments containing the above SEQ ID NO: l and SEQ ID NO: 3 were cloned into pCOM3H (Wu SC, Lin YJ, Chou JW, Lin CW. 2004, Construction and characterization of a Fab recombinant protein for Japanese encephalitis virus neutralization. Vaccine. 25;23 (2) : 163-71 ) . Light with Nhel and NotI sites at 5'- and 3' -end, was digested by Sacl/Xbal before ligated to predigested pCOM3H with the same restriction enzyme combination. Then, heavy chain with Xhol and Spel was digested with the same restriction combination and ligated to the Xhol/Spel site. Transform competent E. coli strain DH5a cells ( from NEB ) and identify and isolate single colonies on a agar plate containing IPTG/X-gal and ampicillin before verification by restriction digestion. A correct clone was inoculated into 500ml LB medium with Amp for 6 hours before induction by ImM IPTG to produce recombinant Fab. Collect the supernatant after another 2hours induction. (2) purifying Fab and PEGylation

Purification was carried out through KappaSelect ( GE) /ion-exchange/molecular sieve according to supplier's manual, and the obtained protein was PEGylated and purified according to AP Champman et al(1999), [Ref: Therapeutic antibody fragments with prolonged in vivo half life. Nature Biotech. 17:780-783 ]. The purified PEGylated anti human RANKL mAb, 3K7F5Fab-PEQ was stored at -80 ^°C for later use.

Example 4: In vitro Studies on neutralization and affinity of the mAbs

1. Neutralization ability of recombinant mAb 3K7F5 and its Fab format

( 1 ) in vitro inhibition of osteoclast formation

RAW264.7 (ATCC No. TIB-71, Manassas, Va.) is a murine macrophage cell line and can be induced by RANKL to differentiate into osteoclast-like cells. Simonet et al. ( 1997, Cell 89:309) and Lacey et al. ( 1998, Cell 93 : 165 ) described in detail the experimental methods.

TRAP test: Osteoclasts are multinucleate and TRAP positive cells. RAW 264.7 is a universally-used cell line for testing osteoporosis treatments. In this study, 1 >< 10E4 RAW cells/well were inoculated into 24-well plates and culture for 24h before add 50 ng/ml RANKL and a serial dose of the present monoclonal antibody from 10 to 500ng/ml) . Change the medium once by a 3d interval. At the sixth day, Trap treatment was conducted with the above mentioned TRAP test method. After the color reaction, cells were fixed with Citrate /acetone for lmin, then AS-BI as substrate treat for lhour at 37°, and stained with Mayer's hematoxylin (Sigma Chemical Co., St. Louis, Mo.) for two minutes before dryness, clearance with dimethylbenzene and envelope with neutral resin for visualization. The number of Trap-positive cells (osteoclasts with multinucleate, >3 nucleoli/cell ) were counted by microscope observation. To test the function of the present antibody, varying doses of purified protein were added to the culture while AMG162 was used as a positive and Rituxan* negative control for inhibition of osteoclast formation. Media of each culture was replaced with fresh media every 3 days.

It was demonstrated as shown in Fig.3 that the present monoclonal antibody specific to human RANKL is able to neutralize RANKL, and stimulate osteoclast formation.

Importantly, this result showed that addition of the present antibody inhibited, in a dose-dependent manner, osteoclast differentiation of the RAW cell, an osteoclast-like precursor, stimulated. This demonstrates the anti- osteoclastogenic properties of the present antibody.

2. Affinity analysis

Scatchard method (Munson et al, 1980, Anal. BioChem, 107:220 )was used to analyze the affinity of 3K7F5/Full and 3K7F5/Fab, the results showed that their affinities are as high as 8.7><and 6.5>< 10^"12 M compared with AMG162 at approximately 0.37^χ 10 ^"12 M as shown in the parallel analysis results. This means that both 3K7F5/Full and 3K7F5/Fab have affinity much higher than the widely accepted 0. lnM standard for therapeutic use and the positive control.

Example 5: In vivo experiments in model animal rats

Experimental Design: Seventy-two three-month old female Sprague-Dawley rats weighted 120 to 180 grams were ovariectomized, and randomly divided into 12 groups, 6 animals in each group according to their weight. PAE30Full and PAE30Fab (Test Groups) were tested for their inhibition ability on bone loss in the model system. AMG162 (an anti human RANKL IgG2 mAb) was as positive control, PAE36 as negative control. PAE36 is an IgG2 mAb related to human CD20 with no binding with RANKL).

For all groups, animals received mAbs by injection of a single dose at 0.8, 1.6 or 2.4mg/kg in the third day after surgery. The Treatment Groups received PAE30Full or PAE30Fab. Positive and Negative Control Groups received PAE36 and AMG162 respectively in the same method. The animals were killed in eighty-fourth day, and their right-side femurs were taken to be weighted after dried at 105 ^°C to constant weight. Then, bone density and Bone Calcium Content were tested.

Femoral Bone Density: Femoral bone density (g/cm2) was measured by dual-energy X-ray absorptiometry (DEXA; Piximus Mouse Densitometer, GE Medical Systems). BMD, calculated by dividing bone mineral content (g) by the projected bone area (cm2 ), was assessed for the total femur as well as for three equivalent femoral sub regions: the proximal third, central third, and distal third of each femur.

Femoral Bone Calcium content: Femoral bone calcium content (mg/g) was measured by TRACE 1200 atomic absorption spectrometer (Aurora Biomed Co.).

Statistic Analysis: Unless otherwise noted, all values are expressed as mean ± standard deviation. Bone densities were compared between Treatment and Positive or Negative Control groups using student's paired t-test (SPSS for Windows 11.0.1, SPSS Inc., Chicago, IL). For comparisons of all other variables among treatment groups, significant main effects were tested by multivariate analysis (generalized linear model). Where main effects were significant (p<0.05), treatment groups were compared using Tukey post-hoc comparison (p<0.05). To test whether or not either rat body mass or uterine mass had a confounding effect on bone properties, all data were also compared by multivariate analysis with rat body mass and uterine mass as covariates.

Results

The results were shown in the following table.

Table. Impact of mAbs on bone density and bone calcium content in ovariectomized rats ( ±S, n=6)

* compared with negative control; +compared with positive control; * or + means p<0.05, and ** or ++ means p<0.0\ .

It was shown that compared with negative control, 3K7F5/Full, 3K7F5/Fab and positive control can increase significantly the bone densities and bone calcium content (p<0.01); and compared with positive control, both 3K7F5/Full and 3K7F5/Fab have higher potential in increasing bone densities and bone calcium content (p<0.01). Otherwise no statistic difference was found between 3K7F5/Full and 3K7F5/Fab.

Statistic analysis indicated that (1) ovariectomization can significantly decrease bone density and calcium content, and concludes that ovariectomization is an effective and reasonable method to set up model animals; (2) compared with positive control, both 3K7F5/Full, and 3K7F5/Fab can increase more significantly bone density and calcium; (3) all three anti RANKL mAbs, positive control, 3K7F5/Full and 3K7F5/Fab, work in a clear dose-dependent manner.

Above in vitro and in vivo experiments show that both 3K7F5/Full and 3K7F5/Fab are able to increase bone density and calcium in the model rats with good dose-dependency, and all these are reflections of their potentiality in further development.

Example 6: Sequencing of anti human RANKL mAb 3K7F5

The variable region of the above anti human RANKL mAb was sequenced with BigDye sequencing kit (PE). The SEQ ID No. 1 to 4 are the integrated sequencing nucleotide sequences of variable fragments of both light and heavy chains, and deduced amino acid sequences.

In the deduced amino acid sequences, the antigen binding fragments composed mainly of the CDRs designated by Kabat method are shown in the following:

CDRs in the deduced light chain amino acid sequence ( 108 amino acid residues)

EIVLTOSPGTLSLSPGERATLSCRVSOSARGRYFGWYOOKPGOAPRLLIYGGSSRPTGIPDRF

S GS GS GTDFTLTL SRLEPEDFAVF YC 00 YL S SPKTFGO GTKVEIK (SEQ ID NO:2)

CDRs in the deduced heavy chain amino acid sequence (122 amino acid residues) EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYVMSWLROAPGKGLEWVSGLTGSVGSTYY LDSAKGRFTISRDNSKNTLYLOMNSVRIEDTPLYYCVKDPGTTVIMSWFDPWGOGTLVTVS

S (SEQ ID NO:4)

The underlined sequences are CDRs of the heavy and light chains.

Claims

Claims

An anti-RANKL monoclonal antibody or antibody fragment thereof comprising a light chain variable region comprising CRDs with SEQ ID NO: 7-9 as antigen-binding sites, and further comprising a heavy chain variable region comprising CDRs with SEQ ID NO: 10-12 as antigen-binding sites.

The anti-RANKL monoclonal antibody or antibody fragment thereof of claim 1, wherein the antibody fragment is a scFv, a Fab Fab 'or antibody fragment or a full length antibody containing the said CDRs or a part that is of similarity over 85% with them.

The anti-RANKL monoclonal antibody or antibody fragment thereof of claim 1, wherein the said light chain comprises an amino acid sequence as set forth in SEQ ID NO: 2 or a fragment thereof, and the said heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 4 or a fragment thereof.

The anti RANKL monoclonal antibody or antibody fragment thereof of claim 1, wherein the said monoclonal antibody or the antigen binding fragment thereof is PEGylated or not PEGylated, to attach polymers.

A method for the treatment and/or prevention of RANKL-related osteoporosis and/or bone lesions in a subject comprising: administering to the subject an effective amount of the anti-RANKL antibody or the antibody fragment as described in claim 1.