WO2015030701A1 - Fully human antibodies against human rankl - Google Patents

Fully human antibodies against human rankl Download PDF

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Publication number
WO2015030701A1
WO2015030701A1 PCT/US2013/056539 US2013056539W WO2015030701A1 WO 2015030701 A1 WO2015030701 A1 WO 2015030701A1 US 2013056539 W US2013056539 W US 2013056539W WO 2015030701 A1 WO2015030701 A1 WO 2015030701A1
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WIPO (PCT)
Prior art keywords
antibody
seq
heavy chain
variable region
sequence
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PCT/US2013/056539
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French (fr)
Inventor
Ilya ALEXANDROV
Tajib Mirzabekov
Roman MIKHAILOV
Khikmet SADYKOV
Anton CHESTUKHIN
Alexey REPIK
Vasily IGNATIEV
Yan Lavrovsky
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R-Pharm Overseas, Inc.
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Priority to PCT/US2013/056539 priority Critical patent/WO2015030701A1/en
Publication of WO2015030701A1 publication Critical patent/WO2015030701A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to the field of biological pharmaceuticals as well as their in conditions associated with autoimmune and inflammatory diseases and oncology. More specifically, the invention relates to antibodies that bind to human receptor RANKL.
  • RANKL Receptor activator of nuclear factor kappa-B ligand
  • TNF tumor necrosis factor
  • OPG osteoprotegerin
  • RANKL is expressed by T helper cells and has a function in the immune system, been involved in dendritic cell maturation.
  • RANKL has been shown to be a dendritic cell survival factor and is involved in the regulation of T-cell dependent immune response. T cell activation was reported to induce expression of this gene and lead to an increase of osteoclastogenesis and bone loss.
  • RANKL activates antiapoptotic kinase AKT/PKB through a signaling complex that involve SRC kinase and tumor necrosis factor receptor-associated factor 6 (TRAF6), evidencing that this protein plays a role in the regulation of cell apoptosys.
  • RANKL is also referred to as tumor necrosis factor ligand superfamily member 11 (TNFSF11), TNF -related activation-induced cytokine (TRANCE), osteoprotegerin ligand (OPGL), and osteoclast differentiation factor (ODF).
  • TNFSF11 tumor necrosis factor ligand superfamily member 11
  • TRANCE TNF -related activation-induced cytokine
  • OPGL osteoprotegerin ligand
  • ODF osteoclast differentiation factor
  • the protein is also known as CD254.
  • RANKL is encoded by the TNFSF11 gene.
  • RANKL protein is highly critical for adequate body bone metabolism.
  • the protein is a cell surface-bound molecule and highly expressed on osteoblasts, where it serves to activate oteoclasts, the cells involved in bone resorption. Osteoclastic activity is triggered via the osteoblasts' surface- bound RANKL activating the osteoclasts' surface-bound receptor activator of nuclear factor kappa- B (RANK).
  • RANK nuclear factor kappa- B
  • Overproduction of RANKL is implicated in a variety of degenerative bone diseases, such as rheumatoid arthritis and psoriatic arthritis.
  • Denosumab is a high affinity fully human monoclonal antibody that binds to human RANKL and inhibits its interactions with RANK, thus having a similar to OPG mode of action.
  • Denosumab under the trade name Prolia was approved by U.S. Food and Drug Administration (FDA) for prevention and treatment of osteoporosis in postmenopausal women.
  • Denosumab under the trade name Xgeva was approved by U.S. Food and Drug Administration (FDA) for the prevention of skeletal-related events in patients with bone metastases from solid tumors. Further clinical trials of denosumab for other bone remodeling related conditions are currently under way, i.e. for bone metastases from other forms of cancer.
  • TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J. Biol. Chem. 272 (40): 25190-4.
  • RANKL/RANK/OPG system is associated with bone metastasis in human non-small cell lung cancer.
  • Lamothe B Lai Y, Xie M, Schneider MD, Darnay BG.
  • TAK1 is essential for osteoclast differentiation and is an important modulator of cell death by apoptosis and necroptosis. Mol Cell Biol. 2013 Feb;33(3):582-95.
  • Schmiedel BJ Scheible CA, Nuebling T, Kopp HG, Wirths S, Azuma M, Schneider P, Jung G, Grosse-Hovest L, Salih HR. RANKL expression, function, and therapeutic targeting in multiple myeloma and chronic lymphocytic leukemia. Cancer Res. 2013 Jan 15;73(2):683-94. Cowan RW, Singh G. Giant cell tumor of bone: a basic science perspective.Bone. 2013 Jan;52(l):238-46.
  • Roodman GD Genes associate with abnormal bone cell activity in bone metastasis. Cancer Metastasis Rev. 2012 Dec;31(3-4):569-78. Brown HK, Ottewell PD, Evans CA, Holen I. Location matters: osteoblast and osteoclast distribution is modified by the presence and proximity to breast cancer cells in vivo. Clin Exp Metastasis. 2012 Dec;29(8):927-38. Lacey DL, Boyle WJ, Simonet WS, gleichuik PJ, Dougall WC, Sullivan JK, San Martin J, Dansey R. Bench to bedside: elucidation of the OPG-RANK-RANKL pathway and the development of denosumab.Nat Rev Drug Discov. 2012 May;l 1(5):401-19.
  • Kishimoto K, Sasaki A Bone destruction by invading oral squamous carcinoma cells mediated by the transforming growth factor-beta signalling pathway. Anticancer Res. 2010
  • Nannuru KC Futakuchi M, Varney ML, Vincent TM, Marcusson EG, Singh RK.
  • Matrix metalloproteinase (MMP)- 13 regulates mammary tumor-induced osteolysis by activating
  • Osteoclast-derived matrix metalloproteinase-7 but not matrix metalloproteinase-9, contributes to tumor-induced osteolysis. Cancer Res. 2009 Aug 15;69(16):6747-6755.
  • the invention is based, in part, upon the discovery of antibodies that specifically bind to human RANKL blocking the RANK/RANKL interactions.
  • Series of antibodies were isolated from antibody libraries that contained fully human antibody frames.
  • the CDRs (Complementarity Determining Regions) of RANKL -specific antibodies differ from each other, consistent with the design of the antibody libraries used.
  • CDRl and CDR2 of the antibody heavy chain were randomized to provide limited number of variation and CDR3 of the heavy chain was randomized to include all possible amino acid combinations.
  • CDRl and CDR2 were invariant and CDR3 was randomized completely. Since the frameworks for both heavy and light chain of antibodies were corresponding to human sequences, the antibodies described herein could be administered to humans without additional modifications such as a "humanization" process.
  • the antibodies could also be used as diagnostics and for research purposes.
  • the monoclonal antibodies disclosed herein are applicable for RANKL /RANK signaling cascade targeting in drug development for osteoporosis, bone metastasis and others.
  • the invention provides for an isolated antibody (RLl), or an antigen binding fragment of the antibody, that binds human RANKL receptor.
  • the antibody comprises an immunoglobulin light chain of SEQ. ID NO. 154, and an immunoglobulin heavy chain of SEQ. ID NO. 139.
  • the antibody can be a monoclonal antibody.
  • the invention provides for an isolated antibody (RL2), or an antigen binding fragment of the antibody, that binds human RANKL receptor.
  • the antibody comprises an immunoglobulin light chain of SEQ. ID NO. 155, and an immunoglobulin heavy chain of SEQ. ID NO. 140.
  • the antibody can be a monoclonal antibody.
  • the invention provides for an isolated antibody (RL4), or an antigen binding fragment of the antibody, that binds human RANKL receptor.
  • the antibody comprises an immunoglobulin light chain of SEQ. ID NO. 157, and an immunoglobulin heavy chain of SEQ. ID NO. 142.
  • the antibody can be a monoclonal antibody.
  • the invention provides for an isolated antibody (RL7), or an antigen binding fragment of the antibody, that binds human RANKL receptor.
  • the antibody comprises an immunoglobulin light chain of SEQ. ID NO. 158, and an immunoglobulin heavy chain of SEQ. ID NO. 143.
  • the antibody can be a monoclonal antibody.
  • the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL.
  • the antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 108.
  • the antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 31, a CDRH2 comprising the sequence of SEQ. ID NO. 32, and a CDRH3 comprising the sequence of SEQ. ID NO. 33.
  • the CDR sequences of the antibody can be interposed between human or humanized framework sequences.
  • the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL.
  • the antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 109.
  • the antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 34, a CDRH2 comprising the sequence of SEQ. ID NO. 35, and a CDRH3 comprising the sequence of SEQ. ID NO. 36.
  • the CDR sequences of the antibody can be interposed between human or humanized framework sequences.
  • the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL.
  • the antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 111.
  • the antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 40, a CDRH2 comprising the sequence of SEQ. ID NO. 41, and a CDRH3 comprising the sequence of SEQ. ID NO. 42.
  • the CDR sequences of the antibody can be interposed between human or humanized framework sequences.
  • the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL.
  • the antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 112.
  • the antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 43, a CDRH2 comprising the sequence of SEQ. ID NO. 44, and a CDRH3 comprising the sequence of SEQ. ID NO. 45.
  • the CDR sequences of the antibody can be interposed between human or humanized framework sequences.
  • the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL.
  • the antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ. ID NO. 76, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ. ID NO. 01.
  • the antibody can be a monoclonal antibody.
  • the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL.
  • the antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ. ID NO. 78, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ. ID NO. 03.
  • the antibody can be a monoclonal antibody.
  • the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL.
  • the antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ. ID NO. 82, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ. ID NO. 07.
  • the antibody can be a monoclonal antibody.
  • the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL.
  • the antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ. ID NO. 84, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ. ID NO. 09.
  • the antibody can be a monoclonal antibody.
  • Figure 1 schematically shows representation of a typical antibody; gray areas on the scheme depict Constant Regions of both antibody light and heavy chains, black areas depict Variable Regions of the antibody light chain, white areas depict Variable Regions of the antibody heavy chain. CHI, CH2 and CH3 - constant regions 1, 2 and 3of the antibody heavy chain respectively. VH - Variable Regions of the antibody heavy chain; VL - Variable Regions of the antibody light chain; CL - Constant Region of the antibody light chain. Links between heavy and light chains and between two heavy chains depict intermolecular disulfide bridges;
  • Figure 2 schematically shows the amino acid sequences defining a complete immunoglobulin Heavy Chain Variable Region of the antibodies of the present teachings; the amino acid sequences are aligned relative to each other where the regions defining CDR1, CDR2 and CDR3, respectively, are identified in boxes; the unboxed sequences represent immunoglobulin framework;
  • Figure 3 schematically shows aligned amino acid sequences of CDR1, CDR2 and CDR3 for each immunoglobulin Heavy Chain Variable Region shown in Figure 2;
  • Figure 4 schematically shows the amino acid sequences defining a complete immunoglobulin Light Chain (Kappa) Variable Region of the the antibodies of the present teachings; the amino acid sequences are aligned relative to each other where the regions defining CDR1 , CDR2 and CDR3 respectively are identified in boxes; the unboxed sequences represent immunoglobulin framework; and
  • FIG. 5 schematically shows amino acid sequences of CDR1, CDR2 and CDR3 for each immunoglobulin Light Chain (Kappa) Variable Region shown in Figure 4.
  • the invention is based, in part, upon the discovery of antibodies that specifically bind to human RANKL (UniProtKB/Swiss-Prot: 014788) and block interactions with its cognate receptor RANK (UniProtKB/Swiss-Prot: Q9Y6Q6).
  • the antibodies could be used for a variety of diagnostic and therapeutic applications and as research tools.
  • the antibodies were selected for their ability to bind to human RANKL with high affinity, specificity and selectivity. Additionally, the functional properties of the antibodies were tested in a competition assay with commercial antibody - Prolia.
  • the antibodies described herein are engineered on the basis of human sequences, all of them could be administered to humans directly. Depending on a particular application, the described antibodies could be used as targeting moieties for various payloads such as
  • radionuclides drugs, toxins and other effector molecules. Certain features and aspect of the application of the invention are described in more details below.
  • the invention provides for an isolated antibody that specifically binds to human RANKL.
  • the antibody is comprised of (1) an immunoglobulin light chain variable region comprised of three CDRs and (2) an immunoglobulin heavy chain variable region comprised of three other CDRs.
  • the CDRs are embedded into the immunoglobulin framework generated by less variable FR domains.
  • the CDRs of the immunoglobulin light and heavy chain brought together in immunoglobulin molecule define a unique binding site that specifically binds to a native conformation of RANKL.
  • the terms "binds specifically” or “specifically binds” are interchangeable and mean that binding affinities (EC50 values) of the antibodies described herein are below 50 nM (5* 10 ⁇ 8 M).
  • the antibodies can comprise both immunoglobulin heavy and light chain sequences of fragments thereof, such as Fab or Fab 2 fragments. It is understood that specific binding and functional properties can be displayed by a full-length intact immunoglobulin or antigen binding fragment thereof or biosynthetic antibody site.
  • each of the antibody molecules can be an intact antibody, for example, a monoclonal antibody.
  • the antigen binding could be displayed by an antigen binding fragment of an antibody or can be a biosynthetic antibody binding site.
  • Antibody fragments include Fab, Fab 2 or Fv fragments. Techniques for making such antibody fragments are known to those skilled in the art.
  • a number of biosynthetic antibody binding sites are known in the art and include single Fv or sFv molecules, for example as described in US Patent # 5,476,786.
  • Other biosynthetic antibody binding sites include bi-specific or bi-functional antibodies that bind to at least two different target molecules.
  • a bi-specific antibody can bind to human RANKL and to another antigen of interest. Methods for making bi-specific antibodies are known in art and include fusing hybridomas or linking Fab fragment together.
  • Antibodies described in this invention can be produced in different ways utilizing previously developed approaches.
  • DNA encoding variable regions of light and heavy chains can be synthesized chemically using commercially available services and sequence information provided in this invention.
  • the DNA encoding variable regions of heavy and light chains can be amplified by Polymerase Chain Reaction (PCR) using the original clones of Fab fragments of the antibodies described herein, as templates.
  • Synthetic or PCR-amplified DNA fragments can be genetically fused with appropriate nucleotide sequences to generate full-size antibodies or fragments thereof.
  • Antibody expression constructs can be generated by including immunoglobulin constant region coding sequences, sequences providing expression control and other standard elements of expression systems. Generation of specific gene expression constructs is within ordinary skill in the art.
  • the DNA sequences encoding antibodies of interest can be genetically inserted into expression vectors that can be introduced into host cells using standard transfection of transformation procedures known in the art.
  • expression approaches include bacterial expression (E. coli) or mammalian expression (Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney (COS) cells, Human Embryo Kidney (HEK- 293) cells and myeloma cells that do not produce endogenous immunoglobulins.
  • Transfected or transformed host cells can be propagated under conditions providing expression of genes of interest, such as immunoglobulin light and heavy chains and fragments thereof.
  • the expressed proteins can be harvested using common techniques known in the art.
  • E. co/z ' -based expression system is particularly suitable for production of Fab, Fab 2 or sFv antibody fragments.
  • the engineered antibody gene is cloned into a vector suitable for bacterial expression downstream from a commonly used bacterial promoters, e.g. T5 of Lac. Genetic fusion of a signal sequence providing targeting on the expressed protein into the periplasm may enable production and accumulation of soluble forms of antibody fragments into the periplasm of bacterial cells. Extraction of proteins of interest and, specifically, of antibody fragments from the periplasm of bacteria is a well-established array of standard methods known in the art.
  • DNA coding sequences must be inserted into appropriate expression vectors containing adequate eukaryotic promoter, signal peptide for secretion from the cells and other genetic elements known in the arts.
  • Mammalian expression systems are particularly suitable for production of full-size
  • immunoglobulins One of the approaches for antibody production is transient co-expression of heavy (variable + constant) and light (variable + constant) chains of immunoglobulin genetically introduced into two separate expression vectors. Another approach for antibody production is expression on both heavy and light chains from a single bi-cistronic vector. Alternatively, stable cell lines constitutively expressing both heavy and light chains can be generated using single vector approach or utilizing two-vector systems.
  • epitope or purification tags or peptide and protein toxins can be genetically fused to the expression constructs encoding heavy chain, light chain or combination of both immunoglobulin chains.
  • the antibodies disclosed herein can be modified to improve performance which largely depends on the intended use.
  • the antibody can be genetically modified to reduce its immunogenicity in the intended recipient.
  • the antibody can be genetically fused or coupled to another peptide or protein, such as epitope tag, purification tag, a growth factor, cytokine or natural or modified toxin. These modifications can be readily achieved by utilizing genetic manipulations known in the art.
  • the RANKL -specific antibodies described herein can be used as therapeutic agents, and diagnostic agents or as reagents for basic and applied research and development.
  • the antibodies in the invention specifically bind to human RANKL and block interactions with its cognate receptor RANK, they can be utilized in a variety of therapeutic applications. It is contemplated that the antibodies of the invention can be used for the treatment of a variety of disorders in which RANKL mediated signaling is involved. This includes osteoporosis, bone metastasis and alike, as well as various types of cancers.
  • the antibodies are typically modified directly or indirectly with a detection moiety.
  • the detection moiety is a functional addition to the antibody that can be detected either directly or indirectly or is capable of generating a detectable signal.
  • the detectable moiety can be a
  • radionuclide 125 Iodine, 32 Phosphorus, 14 Carbon or others
  • fluorescent or chemiluminescent compound such as fluorescein, rhodamine or luciferine.
  • Enzyme moieties include alkaline phosphatase, horse radish peroxidase, beta-galactozidase and others. Methods for conjugation of the detection moieties largely depend on the nature of the moiety and routinely can be reproduced by those experienced in the art.
  • the antibodies of the invention can be used in a broad range of immunological techniques know in the art. Examples of such techniques include sandwich immunoassays (ELISA), competitive immunoassays, cell surface staining procedures combined with FACS analysis, immunocyto- and immunohistochemical procedures. Protocols and method all of these procedures and assays are well-established and can be routinely carried out by those skilled in the art.
  • sandwich immunoassays ELISA
  • competitive immunoassays cell surface staining procedures combined with FACS analysis
  • immunocyto- and immunohistochemical procedures Protocols and method all of these procedures and assays are well-established and can be routinely carried out by those skilled in the art.
  • Example 1 Generation of Magnetic Proteoliposomes as antigen presenting platforms.
  • Proteoliposomes containing an integral membrane protein having one or more transmembrane domains US Patent 6,761,902; has been used for isolation of antibodies against RANKL.
  • a stable cell line was prepared, utilizing established protocols, overexpressing human recombinant RANKL.
  • the condition for extraction of human RANKL were tested and optimized prior to the initiation of the antibody selection procedure.
  • An extensive matrix of combinations of various detergents, salts and buffer components was analyzed to identify conditions providing a balance between effective extraction of the RANKL and retaining of its function and native conformation.
  • the RANKL functionality was tested by assaying binding of its receptor, RANK, to the RANKL immobilized on the surface of the MPLs. Stability of the MPL particles (as judged by RANK and Prolia (Denosumab)) binding was also tested to ensure that the native conformation of the RANKL is retained for the duration of the antibody selection protocol.
  • Example 2 Antibody libraries and selection of anti-RANKL antibodies.
  • RANKL-specific antibodies were carried out from antibody libraries encoding a series of fully human Fab antibody fragments consisting of 10 10 - 10 11 independent antibody clones. Randomization of all three CDRs (CDR1, CDR2 and CDR3) was carried out for the heavy chain of Fab fragments. Randomization of CDR3 was carried out for the light chain of Fab fragments whereas CDR1, CDR2 were kept invariant.
  • a phage display library represents a collection of individual phage particles that express only a certain type of a genetic fusion of an individual Fab antibody fragment with a surface protein intrinsic for this particular type of phage. A fraction of the phage display library usually containing 10 12 -10 13 phage particles is used as a primary source of the antibody variety.
  • a fraction of the phage display library (10 12 -10 13 phage particles) was incubated with MPL preparations containing functional RANKL in its native conformation.
  • the phage particles that did not bind to the RANKL-MPLs were removed by a series of subsequent washes under conditions providing retention of the native conformation of RANKL.
  • the pool of phage particles that was bound to RANKL-MPLs was removed by acidic elution.
  • the deconvoluted phage output (usually 10 6 -10 8 phage particles) was harvested and further amplified by propagation in E. coli.
  • the pool of the amplified phage is then used further for the second selection round as described for the first round above.
  • a minimum of 2 and maximum of 4 selection rounds are carried out for a standard selection procedure.
  • This example describes a procedure for screening antibodies specific against native conformations of RANKL receptor.
  • the screening procedure is based on the usage of live cells expressing human RANKL receptor on their surface. Generation of stable cell lines expressing RANKL is described in Example 6 below. R1610 cells expressing RANKL were used for screening.
  • Phage outputs from 3 rd or 4 th selection rounds represented pools of page particles that were used to infect E. coli to produce phagemid DNA.
  • the phagemid DNA was digested with Nhel and BstEII to excise the entire Fab fragment and further introduced (ligated) into pQE3-Kan expression vector (Quiagen) digested with the same restriction enzymes, Nhel-BstEII.
  • the resulting genetic construct was suitable for expression of Fab antibody fragment in bacteria under control of T5 promoter.
  • the cells expressing human RANKL were mixed with individual Fab preparations and allowed to interact for 30 min at 4°C. Unbound material was removed by pelleting the cells and by aspirating the supernatant. Binding the Fab antibody fragments to the cells was analyzed by secondary antibodies conjugated to phycoerythrin. The antibody binding was quantified by FACS analysis using 96-well plate compatible Guava flow cytometer. Clones that gave increase of the signal >5 fold over the background were scored as positive and kept for further analysis.
  • the antibody clones that bound to RANKL were re -tested for their ability to interact with parental cells that did not express RANKL in order to identify specific RANKL binders.
  • the test was performed as described above and the final candidates from the screening had the following properties: they bind to the RANKL-expressing cells but not the RANKL-negative parental cells.
  • Example 3 The candidate Fab antibody clones identified in Example 3 were subjected to a sequencing analysis to identify independent clones. Bacterial cultures were submitted to Beckman Genomics automated sequencing facility.
  • the experimental sequencing data were analyzed by appropriate software, specifically the nucleic acid sequences were aligned to identify identical clones and to determine the differences in the deduced protein sequences.
  • variable regions can be identified using IMGT ACQUEST webserver-based software at http://www.imgt.org/] MGT vqxtest/share/textes/.
  • the sequencing analysis enabled detection of clones with identical sequences and only clones with unique and distinct DNA and protein sequences were kept for further evaluation.
  • variable region of the sequences shown below are combined with the corresponding constant region sequences.
  • Signal peptides are not presented in the sequence; each sequence starts from the actual beginning of the antibody molecule according to Kabat numbering system.
  • RLl Variable Region, Heavy Chain (Seq. ID NO. 02)
  • AAATCTTGTG ACAAAACTCA CACATGCCCA CCGTGCCCAG CACCTGAACT CCTGGGGGGA
  • Heavy Chain of the antibody RL4 full amino acid sequence (Seq. ID NO. 142) 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFN RTDMSWVRQA PGKGLEWVSG INSYGGATNY
  • VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
  • DKSRWQQGNV 421 FSCSVMHEAL HNHYTQKSLS LSPGK
  • Light Chain Kappa of the antibody RL2 full amino acid sequence (Seq. ID NO. 155) 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
  • Light Chain Kappa of the antibody RL8, full amino acid sequence (Seq. ID NO. 159) 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
  • EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QASYYSPVTF GQGTKVEIKR TVAAPSVFIF 121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC
  • Light Chain Kappa of the antibody RL11, full amino acid sequence (Seq. ID NO. 162) 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
  • EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QSYDSYPFTF GQGTKVEIKR TVAAPSVFIF 121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC
  • Light Chain Kappa of the antibody RL21, full amino acid sequence (Seq. ID NO. 167) 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
  • Table 1 provides a correspondence between sequences discussed in these Examples with Sequence Listing (Seq. ID NO).
  • Heavy Chain of the antibody RL7 full amino acid sequence 144 Heavy Chain of the antibody RL8, full amino acid sequence
  • Example 5 Production of recombinant human RANKL.
  • This Example describes design and generation of expression constructs for inducible expression of human RANKL receptor.
  • this example describes epitope and purification tags genetically fused to the nucleic acid sequences encoding RANKL and its orthologs.
  • Human RANKL (NCBI Reference Sequence: NM_003701.3) was amplified by PCR using Clone ID HsCD00436583 (DNASU Plasmid Repository) as a template.
  • Clone ID HsCD00436583 DNASU Plasmid Repository
  • the endogenous native Stop codon of RANKL was removed using IA-014 Reverse primer to provide in- frame genetic fusion with Strep-tag (ht : 7www.iba-go.de/prottoots/ rot streptag.htmi.
  • IA-013 direct primer (Seq. ID NO. 124)
  • Example 6 Generation of stable cell lines expressing recombinant RANKL.
  • the expression constructs encoding human RANKL receptor described in Example 5 were used for generation of stable cell lines.
  • Commercially available cell lines, R1610, Cf2Th and HEK-293 (ATCC) were used.
  • the expression constructs were verified for the expression of the protein of interest in a transient transfection experiment and then the cells were propagated on a medium containing Zeocin to select for stable cell lines harboring the gene of interest.
  • Expression of the RANKL was verified by Western blot and by FACS analysis to ensure that the expressed protein was translocated to the plasma membrane. For both techniques, commercially available antibodies were used. All steps of the cell line generation were carried out according to the manufacturers' protocols.
  • Example 7 Conversion of the Fab antibody fragments into immunoglobulins and their production.
  • This Example provides description an approach for subcloning of Fab fragments into mammalian expression vectors for production of fully functional immunoglobulins.
  • a protein production approach is also provided herein.
  • the candidate Fab antibody Heavy Chain variable region fragments described in the foregoing Examples 2-4 were converted into full size immunoglobulins of IgGl framework.
  • Variable region of the heavy chain was fused to the constant region of human IgGl isotype using expression vector pTT-5 ( RC Biotechnology Research Institute, National Research Council of Canada) modified by introducing the constant region of human IgGl from pFUSE-CHIg-hGl expression vector (Invivogen) resulting in pTT-IgGl-HC vector.
  • the signal peptide from immunoglobulin kappa light chain variable region (Mus musculus, gb
  • Immunoglobulin 20-amino acid signal peptide sequence where starting methionine is underlined (Seq. ID NO. 130)
  • variable regions of the heavy chain were amplified by PCR to introduce into the following cloning sites: 5 '-end cloning restriction site is Sail, 3 '-end restriction site is Nhel.
  • A-371 Reverse PCR primer for amplification of Heavy Chain Variable Region (Nhel restriction site is underlined) (SEQ. ID NO 132) 5 ' - TTGTGCTAGCACTCGAGACGGTGACCAAGGTTCCCTGGCC - 3
  • the candidate Fab antibody Light Chain variable region fragments described in the foregoing Examples 2-4 were converted into full size immunoglobulins of IgGl framework.
  • Variable Region of light chain was fused to the constant region of human light chain kappa using expression vector pTT-5 (NRC Biotechnology Research Institute, National Research Council of Canada) modified by introduction of the constant region of human light chain fragmen from pFUSE2- CLIg-hk expression vector (Invivogen) resulting in pTT-LC-Kappa.
  • the signal peptide from immunoglobulin kappa light chain variable region (Mus musculus, gb
  • variable regions of the light chain were amplified by PCR to introduce for the following cloning sites: 5'-end cloning restriction site is Sail, 3'-end restriction site is BsiWI.
  • the resulting PCR fragment was digested with Sall-BsiWI restriction enzymes and then introduced into pTT-LC-Kappa vector digested with the same enzymes.
  • A-340 direct PCR primer for amplification of Light Chain Kappa Variable Region (Sail restriction site is underlined) (Seq. ID NO. 133)
  • the antibodies in a format of human IgGl framework were produced using a protocol for transfection of CHO-3E7 cells using LPEI MAX in shake flask cultures.
  • CHO-3E7 cells provided by NRC Biotechnology Research Institute, National Research Council of Canada, were diluted to 0.8 xlO 6 cells/ml 24 h before transfection.
  • On the day of transfection cell density was adjusted to 2.0 to 2.2xl0 6 cells/ml using complete FreeStyleTM CHO medium and cell viability was greater than 97%.
  • PEI Polyethylenimine
  • Polyethylenimine "MAX" linear MW 25 kDa (40 kDa nominal), 3 mg/ml stock solution in water, pH 7.0 (Polysciences Inc. cat# 24765-2) was mixed with purified and quantified plasmid DNA of interest.
  • A260/A280 ratio (use 50 mM Tris-HCl pH 8.0 to dilute the plasmid DNA) was between 1.85 and 1.95.
  • the cells were used in exponential growth phase, 2-2.2xl0 6 cells/ml in CHO FreeStyle medium.
  • DNA preparations (0.75mg/L) encoding for Heavy or Light chains of immunoglobulin were mixed with PEI in CHO FreeStyle medium at 1 :5 (w:w) ratio, the mixture was then incubated 8-10 min, add then added to culture. Volume of the transfection mixture was 1/10 of the final volume of the production culture.
  • immunoglobulins were from 20 to 100 mg/L.
  • the immunoglobulin production was monitored by commercially available ELISA kit (Bethyl Laboratories).
  • the Protein A Plus Agarose resin was harvested and placed into 1 -ml columns (Pierce) and then washed with 10 volumes of lx PBS, then with 10 volumes of 25mM Tris-HCl, 0.12M Glycine, 1.5M NaCl (pH 8.5), then with 10 volumes of TBS-Tween-20, then with 10 volumes of 20mM Sodium Citrate Buffer, 1M NaCl (pH 5.5) and the final wash with 10 volumes of 150mM Sodium Chloride without any buffer.
  • the elution of bound immunoglobulins was carried out with Elution Buffer (0.1 M Glycine pH 3.0, 10% Sucrose, 150mM NaCl) that was added at ratio of 1 : 1 to the volume of Protein A Plus Agarose resin.
  • Elution Buffer 0.1 M Glycine pH 3.0, 10% Sucrose, 150mM NaCl
  • the elution buffer was incubated with the Protein A Plus Agarose resin for 3 min, removed and then another portion of fresh Elution Buffer was added to the Protein A Plus Agarose resin.
  • the eluted material was immediately neutralized by 0.5 M Sodium Citrate Buffer, pH 6.0, at the 1/10 volume ratio to the eluted volume.
  • the concentration of the resulting immunoglobulin preparations was determined by measure optical density of the solution at 280nm in UV-transparent cuvettes where a mixture of 0.1 ml of 0.5 M Sodium Citrate, pH 6, with 1ml of the Elution Buffer was used as a Reference Buffer.
  • concentration of IgG the following formula that provides IgG concentration in mg/ml, was used:
  • [IgG] OD 2W * DilutionFactor ⁇ w ⁇ Qre j ⁇ ⁇ s ⁇ s ⁇ an ⁇ ar( j extinction coefficient for an IgG.
  • This example describes the method for determination of the affinities of the antibodies against human RANKL receptor and provides means of comparison of properties of different antibody clones.
  • Zometa is a registered trademark of Novartis AG Corporation, Switzerland.
  • Prolia and Xgeva are registered trademarks of Amgen Inc., a Delaware Corporation.

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Abstract

Therapeutic antibodies are described that can be used for treating or prevention of diseases associated with modulation of activity of human RANKL. In certain aspects, the disclosed invention is based upon discovering antibodies that are capable of binding to human RANKL. The disclosed antibodies are capable of blocking the interactions between RANKL and its receptor RANK. The disclosed antibodies could be administered to humans without additional modifications such as a "humanization" process. The antibodies could also be used as diagnostics and for research purposes. The described monoclonal antibodies are applicable for RANKL /RANK signaling cascade targeting in drug development for cancer and immunological disorders.

Description

Fully Human Antibodies Against Human RANKL.
FIELD
Generally, the invention relates to the field of biological pharmaceuticals as well as their in conditions associated with autoimmune and inflammatory diseases and oncology. More specifically, the invention relates to antibodies that bind to human receptor RANKL.
BACKGROUND The approaches described in this section could be pursued, and are not necessarily approaches that have previously been conceived or pursued. Therefore, unless otherwise indicated, it should not be assumed that any of the approaches described in this section qualify as prior art, merely by virtue of their inclusion into this section.
RANKL (Receptor activator of nuclear factor kappa-B ligand) is a member of the tumor necrosis factor (TNF) cytokine family. It is a ligand for osteoprotegerin (OPG) and plays a principal function in bone formation, osteoclast differentiation and activation. RANKL is expressed by T helper cells and has a function in the immune system, been involved in dendritic cell maturation. RANKL has been shown to be a dendritic cell survival factor and is involved in the regulation of T-cell dependent immune response. T cell activation was reported to induce expression of this gene and lead to an increase of osteoclastogenesis and bone loss. RANKL activates antiapoptotic kinase AKT/PKB through a signaling complex that involve SRC kinase and tumor necrosis factor receptor-associated factor 6 (TRAF6), evidencing that this protein plays a role in the regulation of cell apoptosys. RANKL is also referred to as tumor necrosis factor ligand superfamily member 11 (TNFSF11), TNF -related activation-induced cytokine (TRANCE), osteoprotegerin ligand (OPGL), and osteoclast differentiation factor (ODF). The protein is also known as CD254. In humans RANKL is encoded by the TNFSF11 gene.
RANKL protein is highly critical for adequate body bone metabolism. The protein is a cell surface-bound molecule and highly expressed on osteoblasts, where it serves to activate oteoclasts, the cells involved in bone resorption. Osteoclastic activity is triggered via the osteoblasts' surface- bound RANKL activating the osteoclasts' surface-bound receptor activator of nuclear factor kappa- B (RANK). Overproduction of RANKL is implicated in a variety of degenerative bone diseases, such as rheumatoid arthritis and psoriatic arthritis.
Denosumab is a high affinity fully human monoclonal antibody that binds to human RANKL and inhibits its interactions with RANK, thus having a similar to OPG mode of action. Denosumab under the trade name Prolia was approved by U.S. Food and Drug Administration (FDA) for prevention and treatment of osteoporosis in postmenopausal women. Denosumab under the trade name Xgeva was approved by U.S. Food and Drug Administration (FDA) for the prevention of skeletal-related events in patients with bone metastases from solid tumors. Further clinical trials of denosumab for other bone remodeling related conditions are currently under way, i.e. for bone metastases from other forms of cancer.
Further discussion of RANKL and its therapeutic utility can be found in the references provided below:
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development of progestin-driven mammary cancer". 2010. Nature 468 (7320): 98-102.
Gonzalez-Suarez E, Jacob AP, Jones J, Miller R, Roudier-Meyer MP, Erwert R, Pinkas J, Branstetter D, Dougall WC. RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. 2010. Nature 468 (7320): 103-7. Luan X, Lu Q., Zhang S, Wang, Q., Yuan, H. Zhao, W., Wang, J. Wang, X. Crystal structure of human RANKL complexed with its decoy receptor osteoprotegerin." J. Immunol. 2012, 189: 245-252. Cummings SR at al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med. 2009 Aug 20;361(8):756-65.
Monteiro AC, Leal AC, Goncalves-Silva T, Mercadante AC, Kestelman F, Chaves SB,Azevedo RB, Monteiro JP, Bonomo A. T cells induce pre-metastatic osteolytic disease and help bone metastases establishment in a mouse model of metastatic breast cancer. PLoS One. 2013 Jul 18;8(7):e68171.
Xiao Y, Song JY, de Vries TJ, Fatmawati C, Parreira DB, Langenbach GE, Babala N, Nolte MA, Everts V, Borst J. Osteoclast precursors in murine bone marrow express CD27 and are impeded in osteoclast development by CD70 on activated immune cells. Proc Natl Acad Sci U S A. 2013 Jul 23;110(30):12385-90.
Lacroix AZ, Jackson RD, Aragaki A, Kooperberg C, Cauley JA, Chen Z, Leboff MS, Duggan D, Wactawski-Wende J. OPG and sRANKL serum levels and incident hip fracture in postmenopausal Caucasian women in the Women's Health Initiative Observational Study. Bone. 2013 Jun 2;56(2):474-481.
Casimiro S, Mohammad KS, Pires R, Tato-Costa J, Alho I, Teixeira R, Carvalho A, Ribeiro S, Lipton A, Guise TA, Costa L. RANKL/RANK/MMP- 1 molecular triad contributes to the metastatic phenotype of breast and prostate cancer cells in vitro.
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RANKL/RANK/OPG system is associated with bone metastasis in human non-small cell lung cancer. PLoS One. 2013;8(3):e58361).
Sato K, Lee JW, Sakamoto , Iimura T, Kayamori K, Yasuda H, Shindoh M, Ito M, Omura K, Yamaguchi A. RANKL synthesized by both stromal cells and cancer cells plays a crucial role in osteoclastic bone resorption induced by oral cancer. Am J Pathol. 2013 May; 182(5): 1890-9.
Ney JT, Juhasz-Boess I, Gruenhage F, Graeber S, Bohle RM, Pfreundschuh M, Solomayer EF, Assmann G. Genetic polymorphism of the OPG gene associated with breast cancer. BMC Cancer. 2013 Jan 31;13:40.).
Rajpar S, Fizazi K. Bone targeted therapies in metastatic castration-resistant prostate cancer. Cancer J. 2013 Jan-Feb;19(l):66-70. Owen S, Ye L, Sanders AJ, Mason MD, Jiang WG. Expression profile of receptor activator of nuclear-κΒ (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) in breast cancer .Anticancer Res. 2013 Jan;33(l):199-206.
Schmiedel BJ, Nuebling T, Steinbacher J, Malinovska A, Wende CM, Azuma M, Schneider P, Grosse-Hovest L, Salih HR. Receptor activator for NF-κΒ ligand in acute myeloid leukemia: expression, function, and modulation of NK cell immunosurveillance.J Immunol. 2013 Jan 15;190(2):821-31.
Logan JG, Sophocleous A, Marino S, Muir M, Brunton VG, Idris AI. Selective tyrosine kinase inhibition of insulin-like growth factor- 1 receptor inhibits human and mouse breast cancer- induced bone cell activity, bone remodeling, and osteolysis.J Bone Miner Res. 2013
May;28(5): 1229-42.
Jin Z, Wei W, Dechow PC, Wan Y. HDAC7 inhibits osteoclastogenesis by reversing RANKL- triggered β-catenin switch. Mol Endocrinol. 2013 Feb;27(2):325-35.
Lamothe B, Lai Y, Xie M, Schneider MD, Darnay BG. TAK1 is essential for osteoclast differentiation and is an important modulator of cell death by apoptosis and necroptosis. Mol Cell Biol. 2013 Feb;33(3):582-95.
Schmiedel BJ, Scheible CA, Nuebling T, Kopp HG, Wirths S, Azuma M, Schneider P, Jung G, Grosse-Hovest L, Salih HR. RANKL expression, function, and therapeutic targeting in multiple myeloma and chronic lymphocytic leukemia. Cancer Res. 2013 Jan 15;73(2):683-94. Cowan RW, Singh G. Giant cell tumor of bone: a basic science perspective.Bone. 2013 Jan;52(l):238-46.
Sottnik JL, Keller ET. Understanding and targeting osteoclastic activity in prostate cancer bone metastases.Curr Mol Med. 2013 May;13(4):626-39.)
Yuasa T, Yamamoto S, Urakami S, Fukui I, Yonese J. Denosumab: a new option in the treatment of bone metastases from urological cancers. Onco Targets Ther. 2012;5:221-9.
Branstetter DG, Nelson SD, Manivel JC, Blay JY, Chawla S, Thomas DM, Jun S, Jacobs I. Denosumab induces tumor reduction and bone formation in patients with giant-cell tumor of bone.Clin Cancer Res. 2012 Aug 15;18(16):4415-24
Roodman GD. Genes associate with abnormal bone cell activity in bone metastasis. Cancer Metastasis Rev. 2012 Dec;31(3-4):569-78. Brown HK, Ottewell PD, Evans CA, Holen I. Location matters: osteoblast and osteoclast distribution is modified by the presence and proximity to breast cancer cells in vivo. Clin Exp Metastasis. 2012 Dec;29(8):927-38. Lacey DL, Boyle WJ, Simonet WS, Kostenuik PJ, Dougall WC, Sullivan JK, San Martin J, Dansey R. Bench to bedside: elucidation of the OPG-RANK-RANKL pathway and the development of denosumab.Nat Rev Drug Discov. 2012 May;l 1(5):401-19.
Zhang L, Teng Y, Zhang Y, Liu J, Xu L, Qu J, Hou K, Yang X, Liu Y, Qu X. C-Src-mediated RANKL-induced breast cancer cell migration by activation of the ERK and Akt pathway. Oncol Lett. 2012: 395-400.
Tai YT, Chang BY, Kong SY, Fulciniti M, Yang G, Calle Y, Hu Y, Lin J, Zhao JJ, Cagnetta A, Cea M, Sellitto MA, Zhong MY, Wang Q, Acharya C, Carrasco DR, Buggy JJ, Elias L, Treon SP, Matsui W, Richardson P, Munshi NC, Anderson KC. Bruton tyrosine kinase inhibition is a novel therapeutic strategy targeting tumor nin the bone marrow microenvironment in multiple myeloma. Blood. 2012 Aug 30;120(9):1877-87.
Lee JH, Kim FIN, Kim KO, Jin WJ, Lee S, Kim HH, Ha H, Lee ZH. CXCL10 promotes osteolytic bone metastasis by enhancing cancer outgrowth and osteoclastogenesis. Cancer Res. 2012 Jul l;72(13):3175-86.
Lacey DL, Boyle WJ, Simonet WS, Kostenuik PJ, Dougall WC, Sullivan JK, San Martin J, Dansey R. Bench to bedside: elucidation of the OPG-RANK-RANKL pathway and the development of denosumab. Nat Rev Drug Discov. 2012 May;l 1(5):401-1 .
Palafox M, Ferrer I, Pellegrini P, Vila S, Hernandez-Ortega S, Urruticoechea A, Climent F, Soler MT, Munoz P, Vinals F, Tometsko M, Branstetter D, Dougall WC, Gonzalez-Suarez E. RANK induces epithelial-mesenchymal transition and sternness in human mammary epithelial cells and promotes tumorigenesis and metastasis. Cancer Res. 2012 Jun 1;72(11):2879-88. Yang Y, Ren Y, Ramani VC, Nan L, Suva LJ, Sanderson RDHeparanase enhances local and systemic osteolysis in multiple myeloma by upregulating the expression and secretion of RANKL. Cancer Res. 2010 Nov l;70(21):8329-38. Gonzalez-Suarez E, Jacob AP, Jones J, Miller R, Roudier-Meyer MP, Erwert R, Pinkas J, Branstetter D, Dougall WC. RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Nature. 2010 Nov 4;468(7320):103-7.
Schramek D, Leibbrandt A, Sigl V, Kenner L, Pospisilik JA, Lee HJ, Hanada R, Joshi PA, Aliprantis A, Glimcher L, Pasparakis M, Khokha R, Ormandy CJ, Widschwendter M, Schett G, Penninger JM. Osteoclast differentiation factor RANKL controls development of progestin- driven mammary cancer. Nature. 2010 Nov 4;468(7320):98-102.
Onishi T, Hayashi N, Theriault RL, Hortobagyi GN, Ueno NT .Future directions of bone- targeted therapy for metastatic breast cancer. Nat Rev Clin Oncol. 2010 Nov;7(l 1):641-51.
Goda T, Shimo T, Yoshihama Y, Hassan NM, Ibaragi S, Kurio N, Okui T, Honami T,
Kishimoto K, Sasaki A. Bone destruction by invading oral squamous carcinoma cells mediated by the transforming growth factor-beta signalling pathway. Anticancer Res. 2010
Jul;30(7):2615-23.
Rizzoli R, Yasothan U, Kirkpatrick P. Denosumab. Nat Rev Drug Discov. 2010 Aug;9(8):591- 592. Rucci N, Millimaggi D, Mari M, Del Fattore A, Bologna M, Teti A, Angelucci A, Dolo V. Receptor activator of NF-kappaB ligand enhances breast cancer-induced osteolytic lesions through upregulation of extracellular matrix metalloproteinase inducer/CD147. Cancer Res. 2010 Aug l;70(15):6150-60. Sabbota AL, Kim HR, Zhe X, Fridman R, Bonfil RD, Cher ML. Shedding of RANKL by tumor-associated MT 1 -MMP activates Src-dependent prostate cancer cell migration. Cancer Res. 2010 Jul l;70(13):5558-66. Joshi PA, Jackson HW, Beristain AG, Di Grappa MA, Mote PA, Clarke CL, Stingl J,
Waterhouse PD, Khokha R. Progesterone induces adult mammary stem cell expansion. Nature. 2010 Jun 10;465(7299):803-807.
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MMP9 and transforming growth factor-beta signaling at the tumor-bone interface. Cancer Res. 2010 May l;70(9):3494-504.
Akiyama T, Choong PF, Dass CR. RANK-Fc inhibits malignancy via inhibiting ERK activation and evoking caspase-3 -mediated anoikis in human osteosarcoma cells. Clin Exp Metastasis. 2010 Apr;27(4):207-215.
Asselin-Labat ML, Vaillant F, Sheridan JM, Pal B, Wu D, Simpson ER, Yasuda H, Smyth GK, Martin TJ, Lindeman GJ, Visvader JE. Control of mammary stem cell function by steroid hormone signalling.Nature. 2010 Jun 10;465(7299):798-802.
Canon J, Bryant R, Roudier M, Osgood T, Jones J, Miller R, Coxon A, Radinsky R, Dougall WC. Inhibition of RANKL increases the anti-tumor effect of the EGFR inhibitor panitumumab in a murine model of bone metastasis.Bone. 2010 Jun;46(6): 1613-1619.
Beleut M, Rajaram RD, Caikovski M, Ayyanan A, Germano D, Choi Y, Schneider P, Brisken C. Two distinct mechanisms underlie progesterone-induced proliferation in the mammary gland. Proc atl Acad Sci U S A. 2010 Feb 16;107(7):2989-2994. Vanhara P, Lincova E, Kozubik A, Jurdic P, Soucek K, Smarda J. Growth/differentiation factor- 15 inhibits differentiation into osteoclasts— a novel factor involved in control of osteoclast differentiation.Differentiation. 2009 Nov;78(4):213-22. Thiolloy S, Halpern J, Holt GE, Schwartz HS, Mundy GR, Matrisian LM, Lynch CC.
Osteoclast-derived matrix metalloproteinase-7, but not matrix metalloproteinase-9, contributes to tumor-induced osteolysis. Cancer Res. 2009 Aug 15;69(16):6747-6755.
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SUMMARY The invention is based, in part, upon the discovery of antibodies that specifically bind to human RANKL blocking the RANK/RANKL interactions. Series of antibodies were isolated from antibody libraries that contained fully human antibody frames. The CDRs (Complementarity Determining Regions) of RANKL -specific antibodies differ from each other, consistent with the design of the antibody libraries used. In these libraries, CDRl and CDR2 of the antibody heavy chain were randomized to provide limited number of variation and CDR3 of the heavy chain was randomized to include all possible amino acid combinations. For the light chain, CDRl and CDR2 were invariant and CDR3 was randomized completely. Since the frameworks for both heavy and light chain of antibodies were corresponding to human sequences, the antibodies described herein could be administered to humans without additional modifications such as a "humanization" process. The antibodies could also be used as diagnostics and for research purposes.
The monoclonal antibodies disclosed herein are applicable for RANKL /RANK signaling cascade targeting in drug development for osteoporosis, bone metastasis and others.
In some aspects the invention provides for an isolated antibody (RLl), or an antigen binding fragment of the antibody, that binds human RANKL receptor. The antibody comprises an immunoglobulin light chain of SEQ. ID NO. 154, and an immunoglobulin heavy chain of SEQ. ID NO. 139. The antibody can be a monoclonal antibody. In some aspects the invention provides for an isolated antibody (RL2), or an antigen binding fragment of the antibody, that binds human RANKL receptor. The antibody comprises an immunoglobulin light chain of SEQ. ID NO. 155, and an immunoglobulin heavy chain of SEQ. ID NO. 140. The antibody can be a monoclonal antibody.
In some aspects the invention provides for an isolated antibody (RL4), or an antigen binding fragment of the antibody, that binds human RANKL receptor. The antibody comprises an immunoglobulin light chain of SEQ. ID NO. 157, and an immunoglobulin heavy chain of SEQ. ID NO. 142. The antibody can be a monoclonal antibody.
In some aspects the invention provides for an isolated antibody (RL7), or an antigen binding fragment of the antibody, that binds human RANKL receptor. The antibody comprises an immunoglobulin light chain of SEQ. ID NO. 158, and an immunoglobulin heavy chain of SEQ. ID NO. 143. The antibody can be a monoclonal antibody.
In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL. The antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 108. The antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 31, a CDRH2 comprising the sequence of SEQ. ID NO. 32, and a CDRH3 comprising the sequence of SEQ. ID NO. 33. The CDR sequences of the antibody can be interposed between human or humanized framework sequences.
In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL. The antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 109. The antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 34, a CDRH2 comprising the sequence of SEQ. ID NO. 35, and a CDRH3 comprising the sequence of SEQ. ID NO. 36. The CDR sequences of the antibody can be interposed between human or humanized framework sequences. In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL. The antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 111. The antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 40, a CDRH2 comprising the sequence of SEQ. ID NO. 41, and a CDRH3 comprising the sequence of SEQ. ID NO. 42. The CDR sequences of the antibody can be interposed between human or humanized framework sequences.
In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL. The antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 112. The antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 43, a CDRH2 comprising the sequence of SEQ. ID NO. 44, and a CDRH3 comprising the sequence of SEQ. ID NO. 45. The CDR sequences of the antibody can be interposed between human or humanized framework sequences.
In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL. The antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ. ID NO. 76, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ. ID NO. 01. The antibody can be a monoclonal antibody.
In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL. The antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ. ID NO. 78, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ. ID NO. 03. The antibody can be a monoclonal antibody.
In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL. The antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ. ID NO. 82, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ. ID NO. 07. The antibody can be a monoclonal antibody.
In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human RANKL. The antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ. ID NO. 84, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ. ID NO. 09. The antibody can be a monoclonal antibody.
These and other aspects and advantages of the invention described herein will become apparent upon consideration of the Figures and detailed description of antibody properties below.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings and descriptions provide complete understanding of the invention: Figure 1 schematically shows representation of a typical antibody; gray areas on the scheme depict Constant Regions of both antibody light and heavy chains, black areas depict Variable Regions of the antibody light chain, white areas depict Variable Regions of the antibody heavy chain. CHI, CH2 and CH3 - constant regions 1, 2 and 3of the antibody heavy chain respectively. VH - Variable Regions of the antibody heavy chain; VL - Variable Regions of the antibody light chain; CL - Constant Region of the antibody light chain. Links between heavy and light chains and between two heavy chains depict intermolecular disulfide bridges;
Figure 2 schematically shows the amino acid sequences defining a complete immunoglobulin Heavy Chain Variable Region of the antibodies of the present teachings; the amino acid sequences are aligned relative to each other where the regions defining CDR1, CDR2 and CDR3, respectively, are identified in boxes; the unboxed sequences represent immunoglobulin framework;
Figure 3 schematically shows aligned amino acid sequences of CDR1, CDR2 and CDR3 for each immunoglobulin Heavy Chain Variable Region shown in Figure 2;
Figure 4 schematically shows the amino acid sequences defining a complete immunoglobulin Light Chain (Kappa) Variable Region of the the antibodies of the present teachings; the amino acid sequences are aligned relative to each other where the regions defining CDR1 , CDR2 and CDR3 respectively are identified in boxes; the unboxed sequences represent immunoglobulin framework; and
Figure 5 schematically shows amino acid sequences of CDR1, CDR2 and CDR3 for each immunoglobulin Light Chain (Kappa) Variable Region shown in Figure 4.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based, in part, upon the discovery of antibodies that specifically bind to human RANKL (UniProtKB/Swiss-Prot: 014788) and block interactions with its cognate receptor RANK (UniProtKB/Swiss-Prot: Q9Y6Q6). The antibodies could be used for a variety of diagnostic and therapeutic applications and as research tools. The antibodies were selected for their ability to bind to human RANKL with high affinity, specificity and selectivity. Additionally, the functional properties of the antibodies were tested in a competition assay with commercial antibody - Prolia.
Since the antibodies described herein are engineered on the basis of human sequences, all of them could be administered to humans directly. Depending on a particular application, the described antibodies could be used as targeting moieties for various payloads such as
radionuclides, drugs, toxins and other effector molecules. Certain features and aspect of the application of the invention are described in more details below.
I - ANTIBODIES AGAINST HUMAN RANKL
In one aspect, the invention provides for an isolated antibody that specifically binds to human RANKL. The antibody is comprised of (1) an immunoglobulin light chain variable region comprised of three CDRs and (2) an immunoglobulin heavy chain variable region comprised of three other CDRs. The CDRs are embedded into the immunoglobulin framework generated by less variable FR domains. The CDRs of the immunoglobulin light and heavy chain brought together in immunoglobulin molecule define a unique binding site that specifically binds to a native conformation of RANKL. The terms "binds specifically" or "specifically binds" are interchangeable and mean that binding affinities (EC50 values) of the antibodies described herein are below 50 nM (5* 10~8 M).
It is understood that the antibodies can comprise both immunoglobulin heavy and light chain sequences of fragments thereof, such as Fab or Fab2 fragments. It is understood that specific binding and functional properties can be displayed by a full-length intact immunoglobulin or antigen binding fragment thereof or biosynthetic antibody site.
It is understood that each of the antibody molecules can be an intact antibody, for example, a monoclonal antibody. Alternatively, the antigen binding could be displayed by an antigen binding fragment of an antibody or can be a biosynthetic antibody binding site. Antibody fragments include Fab, Fab2 or Fv fragments. Techniques for making such antibody fragments are known to those skilled in the art. A number of biosynthetic antibody binding sites are known in the art and include single Fv or sFv molecules, for example as described in US Patent # 5,476,786. Other biosynthetic antibody binding sites include bi-specific or bi-functional antibodies that bind to at least two different target molecules. For example, a bi-specific antibody can bind to human RANKL and to another antigen of interest. Methods for making bi-specific antibodies are known in art and include fusing hybridomas or linking Fab fragment together.
II - PRODUCTION OF RANKL ANTIBODIES
Antibodies described in this invention can be produced in different ways utilizing previously developed approaches. For example, DNA encoding variable regions of light and heavy chains can be synthesized chemically using commercially available services and sequence information provided in this invention. Alternatively, the DNA encoding variable regions of heavy and light chains can be amplified by Polymerase Chain Reaction (PCR) using the original clones of Fab fragments of the antibodies described herein, as templates. Synthetic or PCR-amplified DNA fragments can be genetically fused with appropriate nucleotide sequences to generate full-size antibodies or fragments thereof. Antibody expression constructs can be generated by including immunoglobulin constant region coding sequences, sequences providing expression control and other standard elements of expression systems. Generation of specific gene expression constructs is within ordinary skill in the art. The DNA sequences encoding antibodies of interest can be genetically inserted into expression vectors that can be introduced into host cells using standard transfection of transformation procedures known in the art. Examples of expression approaches include bacterial expression (E. coli) or mammalian expression (Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney (COS) cells, Human Embryo Kidney (HEK- 293) cells and myeloma cells that do not produce endogenous immunoglobulins. Transfected or transformed host cells can be propagated under conditions providing expression of genes of interest, such as immunoglobulin light and heavy chains and fragments thereof. The expressed proteins can be harvested using common techniques known in the art.
The particular conditions for production of antibodies or fragments thereof vary depending on the expression system utilized. For example, E. co/z'-based expression system is particularly suitable for production of Fab, Fab2 or sFv antibody fragments. The engineered antibody gene is cloned into a vector suitable for bacterial expression downstream from a commonly used bacterial promoters, e.g. T5 of Lac. Genetic fusion of a signal sequence providing targeting on the expressed protein into the periplasm may enable production and accumulation of soluble forms of antibody fragments into the periplasm of bacterial cells. Extraction of proteins of interest and, specifically, of antibody fragments from the periplasm of bacteria is a well-established array of standard methods known in the art.
If the antibodies are produced in mammalian expression systems, DNA coding sequences must be inserted into appropriate expression vectors containing adequate eukaryotic promoter, signal peptide for secretion from the cells and other genetic elements known in the arts.
Mammalian expression systems are particularly suitable for production of full-size
immunoglobulins. One of the approaches for antibody production is transient co-expression of heavy (variable + constant) and light (variable + constant) chains of immunoglobulin genetically introduced into two separate expression vectors. Another approach for antibody production is expression on both heavy and light chains from a single bi-cistronic vector. Alternatively, stable cell lines constitutively expressing both heavy and light chains can be generated using single vector approach or utilizing two-vector systems.
Alternative approaches for antibody production include expression in yeast (P. Pastoris or similar strains) or plant cells. Each expression system requires generation of host-specific genetic constructs and generation of expression constructs is very similar to described above bacterial and mammalian expression systems.
It is understood that regardless of the expression system utilized for production of antibodies of fragments thereof, the protein sequence of each antibody remains the same and directly corresponds to the sequence of this invention. It is also understood that DNA sequence may be altered, for example, by the process known as codon optimization that provides higher protein production, depending on host-specific codon usage.
It is understood that during the process of generation of expression constructs for antibodies and fragments thereof, various genetic modifications can be introduced. For example, epitope or purification tags or peptide and protein toxins can be genetically fused to the expression constructs encoding heavy chain, light chain or combination of both immunoglobulin chains.
Ill - MODIFICATIONS OF RANKL ANTIBODIES It is understood that the antibodies disclosed herein can be modified to improve performance which largely depends on the intended use. For example, if used as therapeutic agent, the antibody can be genetically modified to reduce its immunogenicity in the intended recipient. Additionally, or as an alternative, the antibody can be genetically fused or coupled to another peptide or protein, such as epitope tag, purification tag, a growth factor, cytokine or natural or modified toxin. These modifications can be readily achieved by utilizing genetic manipulations known in the art.
IV - USE OF RANKL ANTIBODIES
The RANKL -specific antibodies described herein can be used as therapeutic agents, and diagnostic agents or as reagents for basic and applied research and development.
(1) Therapeutic Applications
Because the antibodies in the invention specifically bind to human RANKL and block interactions with its cognate receptor RANK, they can be utilized in a variety of therapeutic applications. It is contemplated that the antibodies of the invention can be used for the treatment of a variety of disorders in which RANKL mediated signaling is involved. This includes osteoporosis, bone metastasis and alike, as well as various types of cancers.
(2) Diagnostic Applications
Whenever the antibodies are used for diagnostic purposes, either in vivo or in vitro, the antibodies are typically modified directly or indirectly with a detection moiety. The detection moiety is a functional addition to the antibody that can be detected either directly or indirectly or is capable of generating a detectable signal. For example, the detectable moiety can be a
radionuclide ( 125 Iodine, 32 Phosphorus, 14 Carbon or others), fluorescent or chemiluminescent compound, such as fluorescein, rhodamine or luciferine. Enzyme moieties include alkaline phosphatase, horse radish peroxidase, beta-galactozidase and others. Methods for conjugation of the detection moieties largely depend on the nature of the moiety and routinely can be reproduced by those experienced in the art.
The antibodies of the invention can be used in a broad range of immunological techniques know in the art. Examples of such techniques include sandwich immunoassays (ELISA), competitive immunoassays, cell surface staining procedures combined with FACS analysis, immunocyto- and immunohistochemical procedures. Protocols and method all of these procedures and assays are well-established and can be routinely carried out by those skilled in the art.
EXAMPLES
The following examples illustrate selection, identification and characterization of a number of fully human recombinant antibodies against human RANKL receptor.
Example 1 - Generation of Magnetic Proteoliposomes as antigen presenting platforms.
Isolation of antibodies that recognize native conformations of RANKL is absolutely critical for the development of such antibodies as therapeutics. In the human body and tissues RANKL is present in its native form, therefore only the antibodies that bind to the native receptor have practical utility. Preparation of antigen-presenting platforms that provide oriented and functional receptor in its native conformation is far from being trivial. Many methods of antibody generation, for example generation of antibodies against peptide epitopes or receptor fragments, usually are not successful for complex targets such as RANKL. Previously patented technology that relies on the generation of magnetic proteoliposomes (MPLs) (Sodroski, J.G. and T. Mirzabekov,
Proteoliposomes containing an integral membrane protein having one or more transmembrane domains; US Patent 6,761,902;) has been used for isolation of antibodies against RANKL.
The main advantage of the core technology that relies on usage of MPLs is the ability to present highly purified and concentrated antigen (human RANKL in this invention) properly oriented and, most importantly, in its native conformation and functional state. Although the technology disclosed in US Patent 6,761 ,902 can be applied to a variety of complex membrane proteins, each target requires extensive optimization of protein extraction and MPL formation conditions.
Prior to preparing MPL particles, a stable cell line was prepared, utilizing established protocols, overexpressing human recombinant RANKL. The condition for extraction of human RANKL were tested and optimized prior to the initiation of the antibody selection procedure. An extensive matrix of combinations of various detergents, salts and buffer components was analyzed to identify conditions providing a balance between effective extraction of the RANKL and retaining of its function and native conformation. The RANKL functionality was tested by assaying binding of its receptor, RANK, to the RANKL immobilized on the surface of the MPLs. Stability of the MPL particles (as judged by RANK and Prolia (Denosumab)) binding was also tested to ensure that the native conformation of the RANKL is retained for the duration of the antibody selection protocol.
Example 2 - Antibody libraries and selection of anti-RANKL antibodies.
Selection of human RANKL-specific antibodies was carried out from antibody libraries encoding a series of fully human Fab antibody fragments consisting of 1010 - 1011 independent antibody clones. Randomization of all three CDRs (CDR1, CDR2 and CDR3) was carried out for the heavy chain of Fab fragments. Randomization of CDR3 was carried out for the light chain of Fab fragments whereas CDR1, CDR2 were kept invariant. A phage display library represents a collection of individual phage particles that express only a certain type of a genetic fusion of an individual Fab antibody fragment with a surface protein intrinsic for this particular type of phage. A fraction of the phage display library usually containing 1012-1013 phage particles is used as a primary source of the antibody variety.
A fraction of the phage display library (1012-1013 phage particles) was incubated with MPL preparations containing functional RANKL in its native conformation. The phage particles that did not bind to the RANKL-MPLs were removed by a series of subsequent washes under conditions providing retention of the native conformation of RANKL. The pool of phage particles that was bound to RANKL-MPLs was removed by acidic elution. The deconvoluted phage output (usually 106-108 phage particles) was harvested and further amplified by propagation in E. coli. The pool of the amplified phage is then used further for the second selection round as described for the first round above. A minimum of 2 and maximum of 4 selection rounds are carried out for a standard selection procedure.
Example 3 - Screening of anti-RANKL antibodies.
This example describes a procedure for screening antibodies specific against native conformations of RANKL receptor.
The screening procedure is based on the usage of live cells expressing human RANKL receptor on their surface. Generation of stable cell lines expressing RANKL is described in Example 6 below. R1610 cells expressing RANKL were used for screening.
Phage outputs from 3rd or 4th selection rounds represented pools of page particles that were used to infect E. coli to produce phagemid DNA. The phagemid DNA was digested with Nhel and BstEII to excise the entire Fab fragment and further introduced (ligated) into pQE3-Kan expression vector (Quiagen) digested with the same restriction enzymes, Nhel-BstEII. The resulting genetic construct was suitable for expression of Fab antibody fragment in bacteria under control of T5 promoter.
Pool of pQE3-Kan vector with inserted Fab fragments was used for transformation of E. coli. The individual colonies resulting from the transformation were picked up and propagated in 96- well plates. Expression of Fab fragments was induced by IPTG and the expressed protein was harvested using standard biochemical approaches know to those in the art. Each well of 96-well plate containing single Fab clone was harvested separately and used further for binding property testing. A minimum of 500 and maximum of 50,000 individual clones were usually analyzed in the screening procedure.
The cells expressing human RANKL were mixed with individual Fab preparations and allowed to interact for 30 min at 4°C. Unbound material was removed by pelleting the cells and by aspirating the supernatant. Binding the Fab antibody fragments to the cells was analyzed by secondary antibodies conjugated to phycoerythrin. The antibody binding was quantified by FACS analysis using 96-well plate compatible Guava flow cytometer. Clones that gave increase of the signal >5 fold over the background were scored as positive and kept for further analysis.
The antibody clones that bound to RANKL were re -tested for their ability to interact with parental cells that did not express RANKL in order to identify specific RANKL binders. The test was performed as described above and the final candidates from the screening had the following properties: they bind to the RANKL-expressing cells but not the RANKL-negative parental cells.
Example 4 - Sequencing of anti-RANKL antibodies.
The candidate Fab antibody clones identified in Example 3 were subjected to a sequencing analysis to identify independent clones. Bacterial cultures were submitted to Beckman Genomics automated sequencing facility.
The experimental sequencing data were analyzed by appropriate software, specifically the nucleic acid sequences were aligned to identify identical clones and to determine the differences in the deduced protein sequences. For example, variable regions can be identified using IMGT ACQUEST webserver-based software at http://www.imgt.org/] MGT vqxtest/share/textes/. The sequencing analysis enabled detection of clones with identical sequences and only clones with unique and distinct DNA and protein sequences were kept for further evaluation.
In order to create a complete heavy chain and light chain immunoglobulin sequences, the variable region of the sequences shown below are combined with the corresponding constant region sequences. Signal peptides are not presented in the sequence; each sequence starts from the actual beginning of the antibody molecule according to Kabat numbering system. RLl Variable Region, Heavy Chain (Seq. ID NO. 02)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGTGCGG CCAGTGGCTT TACCTTCAAT AACACTTATA TGAGCTGGGT GCGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCGGT ATTTATCCGA AGTCTGGCAC TACCGACTAT 181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC 241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGATTCCGC 301 GTCCTGTTCG ACTACTGGGG CCAGGGAACC TTGGTCACCG TCTCGAGT RL2 Variable Region, Heavy Chain (Seq. ID NO . 04)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGTGCGG CCAGTGGCTT TACCTTCACC GGCAGCACCA TGAACTGGGT ACGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCCGC ATTGGCCCGG AAGGCGGCGG CACCGATTAT 181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC 241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGAGGCATT 301 ATTATTTTCG ACTACTGGGG CCAGGGAACC TTGGTCACCG TCTCGAGT RL3 Variable Region, Heavy Chain (Seq. ID NO. 06)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGCGAGG CCAGTGGCTT TACCTTCAGT AGTAATTCTA TGAGCTGGGT GCGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCTCT ATTACGCCGT CTAATAGCGC GACCTACTAT 181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC 241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGAAGGGCC 301 TTCTGGTTCG ACTACTGGGG CCAGGGAACC TTGGTCACCG TCTCGAGT
RL4 Variable Region, Heavy Chain (Seq. ID NO. 08)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGTGCGG CCAGTGGCTT TACCTTCAAC CGCACCGATA TGAGCTGGGT ACGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCGGC ATTAACAGCT ATGGCGGCGC GACCAATTAT 181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC 241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGACGCGGC 301 CTGTGG RL7 Variable Region, Heavy Chain (Seq. ID NO. 10)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGTGCGG CCAGTGGCTT TACCTTCACT AGATCCGCTA TGAGCTGGGT GCGTCAGGCT
121 CCGGGCAAAG GTCTGGAATG GGTTAGCTCG ATTTCGCCGA ATAGCTGGAC CAACTATGCG 181 GATAGCGTGA AAGGCCGTTT TACCATTTCT CGCGACAACA GCAAGAACAC GCTGTACCTG 241 CAGATGAACT CACTGCGTGC CGAAGATACG GCCGTGTATT ACTGTGCGAG ACGGGGGCTC 301 GTGTTCGACT ACTGGGGCCA GGGAACCTTG GTCACCGTCT CGAGT
RL8 Variable Region, Heavy Chain (Seq. ID NO. 12)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT TTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGTGCGG CCAGTGGCTT TACCTTCAGT AGTTATAGGA TGAACTGGGT GCGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCGTT ATTTCTCCGG GTGGTGGCTG GACCAACTAT 181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC 241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGAACGAGT 301 ACTTCGTCGG ATTATTACTA TTACGGCTTG GACTACTGGG GCCAGGGAAC CTTGGTCACC 361 GTCTCGAGT
RL9 Variable Region, Heavy Chain (Seq. ID NO. 14)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGCGCGG CCAGTGGCTT TACCTTCACC CGCTATAGCA TGAGCTGGGT GCGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCAGC ATTCGCAGCG ATAACAGCAC CACCGATTAT 181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC 241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGACATGCG 301 CTGATTTTCG ACTACTGGGG CCAGGGAACC TTGGTCACCG TCTCGAGT
RL10 Variable Region, Heavy Chain (Seq. ID NO. 16)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGTGCGG CCAGTGGCTT TACCTTCAGT GGTTCTGCGA TGAGCTGGGT ACGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCTTG ATTAGGCCGA GTCGTGGCGT TACCAATTAT
181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC 241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGACGGGGC 301 TTCTGGTTCG ACTACTGGGG CCAGGGAACC TTGGTCACCG TCTCGAGT RLl 1 Variable Region, Heavy Chain (Seq. ID NO. 18)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGCGCGG CCAGTGGCTT TACCTTCAGC GGCAGCGGCA TGAGCTGGGT GCGTCAGGCT
121 CCGGGCAAAG GTCTGGAATG GGTTAGCAGC ATTTGGCCGG AAGGCGGCTA TACCTATTAT
181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC
241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGATTTGTG
301 TTTCTGTTTG ATTATTGGGG CCAGGGAACC TTGGTCACCG TCTCGAGT
RL12 Variable Region, Heavy Chain (Seq. ID NO. 20)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG
61 AGTTGTGCGG CCAGTGGCTT TACCTTCAAT GACAATTATA TGAGCTGGGT GCGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCAGT ATTCGTTCGG GGGGGGGCGT GACCCGGTAT
181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC
241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGACATCAC
301 TACTCTGTTC AGGGTGGCTT GGACTACTGG GGCCAGGGAA CCTTGGTCAC CGTCTCGAGT RL 13 Variable Region, Heavy Chain (Seq. ID NO. 22)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG
61 AGTTGCGCGG CCAGTGGCTT TACCTTCAGT AACAATGCTA TGAGCTGGGT GCGTCAGGCT
121 CCGGGCAAAG GTCTGGAATG GGTTAGCGCG ATTAGCGGTA GTGGCGGTAG CACGTACTAT 181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC
241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGACACTTC
301 TTGTTGTTCG ACTACTGGGG CCAGGGAACC TTGGTCACCG TCTCGAGT
RLl 8 Variable Region, Heavy Chain (Seq. ID NO. 24)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG
61 AGTTGCGCGG CCAGTGGCTT TACCTTCAGT GATTATTATA TGAGCTGGGT GCGTCAGGCT
121 CCGGGCAAAG GTCTGGAATG GGTTAGCAGT ATTGCTTCGT CTGATGGCTA TACCAACTAT
181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC 241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGATCTTAC
301 GCTTACCAGT ACCGTGGCTT GGACTACTGG GGCCAGGGAA CCTTGGTCAC CGTCTCGAGT RLl 9 Variable Region, Heavy Chain (Seq. ID NO. 26)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG
61 AGTTGCGCGG CCAGTGGCTT TACCGTCACT AGGTCTGTGA TCTGGTGGGT GCGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCGGG ATTGGGCCGG GGGGGGGCTG GACCCGGTAT
181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC
241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGAAGCATC
301 GGGCGCCGGG TGTTCGACTA CTGGGGCCAG GGAACCTTGG TCACCGTCTC GAGT RL21 Variable Region, Heavy Chain (Seq. ID NO. 28)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGCGCGG CCAGTGGCTT TACCTTCAGT GATTATTATA TGAGCTGGGT GCGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCGGG ATTGGGCCGG GGGGGGGCTG GACCCGGTAT 181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC 241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGAAGCATC 301 GGGCGCCGGG TGTTCGACTA CTGGGGCCAG GGAACCTTGG TCACCGTCTC GAGT
RL22 Variable Region, Heavy Chain (Seq. ID NO. 30)
1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG 61 AGTTGCGCGG CCAGTGGCTT TACCTTCAGT GATTATTATA TGAGCTGGGT GCGTCAGGCT 121 CCGGGCAAAG GTCTGGAATG GGTTAGCAGT ATTGCTTCGT CTGATGGCTA TACCAACTAT 181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC 241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGATCTTAC 301 GCTTACCAGT ACCGTGGCTT GGACTACTGG GGCCAGGGAA CCTTGGTCAC CGTCTCGAGT
RLl Variable Region, Light Chain, Kappa (Seq. ID NO. 77)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT 61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA 121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA 181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA 241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAATCTTCTT ATTATGCTCC TGTCACGTTC
301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A RL2 Variable Region, Light Chain, Kappa (Seq. ID NO. 79)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT
61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA 121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA
181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA
241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGCTTCTT ATTATTCTCC TTTCACGTTC
301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A
RL3 Variable Region, Light Chain, Kappa (Seq. ID NO. 81)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT
61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA 121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA
181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA
241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGCTTCTT ATTATTCTCC TGTCACGTTC
301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A RL4 Variable Region, Light Chain, Kappa (Seq. ID NO. 83)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT
61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA
121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA 181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA
241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGCTTCTT ATTATTCTCC TGTCACGTTC
301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A
RL7 Variable Region, Light Chain, Kappa (Seq. ID NO. 85)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT
61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA
121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA
181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA 241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGCTTCTT ATTATTCTCC TGTCACGTTC
301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A RL8 Variable Region, Light Chain, Kappa (Seq. ID NO. 87)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT 61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA
121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA
181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA
241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAATCTTCTT ATTCTCCTCT CACGTTCGGC
301 CAAGGGACCA AGGTGGAAAT CAAACGTA
RL9 Variable Region, Light Chain, Kappa (Seq. ID NO. 89)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT
61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA 121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA
181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA
241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAATCTTCTT ATTATTCTCC TGGCACGTTC
301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A RL10 Variable Region, Light Chain, Kappa (Seq. ID NO. 91)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT
61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA
121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA 181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA
241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGCTTCTT ATTATTCTCC TGTCACGTTC
301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A
RLl 1 Variable Region, Light Chain, Kappa (Seq. ID NO. 93)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT
61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA
121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA
181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA 241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGCTTCTT ATTATTCTCC TGTCACGTTC
301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A RL12 Variable Region, Light Chain, Kappa (Seq. ID NO. 95)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT 61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA
121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA
181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA
241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAATATTATA GCAGCCCGAT TACCGGCCAA 301 GGGACCAAGG TGGAAATCAA ACGTA
RL13 Variable Region, Light Chain, Kappa (Seq. ID NO. 97)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT 61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA 121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA 181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA 241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGCTTCTT ATTATTCTCC TTTCACGTTC 301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A
RL 18 Variable Region, Light Chain, Kappa (Seq. ID NO. 99)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT
61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA
121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA
181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA 241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAATCTTATG ATTCTTATCC TTTCACGTTC
301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A
RL19 Variable Region, Light Chain, Kappa (Seq. ID NO. 101) 1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT
61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA 121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA 181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA 241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAATCTTATG ATTCTTATCC TTTCACGTTC 301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A RL21 Variable Region, Light Chain, Kappa (Seq. ID NO. 103)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT 61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA 121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA 181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA 241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAATCTGATT CTTATCCTAT CACGTTCGGC 301 CAAGGGACCA AGGTGGAAAT CAAACGTA RL22 Variable Region, Light Chain, Kappa (Seq. ID NO. 105)
1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT 61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA 121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA 181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA 241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGCTAGGA AGAGTCCTAT CACGTTCGGC 301 CAAGGGACCA AGGTGGAAAT CAAACGTA
Reference Human IgGl Heavy Chain Constant Region, nucleotide sequence (Seq. ID NO. 135)
1 GCT GCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT CCTCCAAGAG CACCTCTGGG
61 GGCACAGCGG CCCTGGGCTG CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG
121 TGGAACTCAG GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA
181 GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC
241 TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAA AGTTGAGCCC
301 AAATCTTGTG ACAAAACTCA CACATGCCCA CCGTGCCCAG CACCTGAACT CCTGGGGGGA
361 CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT
421 G GGTCAC T GCGTGGTGGT GGACGTGAGC CACGAAG CC CTGAGGTCAA GTTCAACTGG
481 TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA GCAGTACAAC
541 AGCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC AGGACTGGCT GAATGGCAAG
601 GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC CCATCGAGAA AACCATCTCC
661 AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC TGCCCCCATC CCGGGAGGAG
721 ATGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTATCC CAGCGACATC
781 GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC GCCTCCCGTG
841 CTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA CCGTGGACAA GAGCAGGTGG
901 CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG
961 CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA TGA Reference Human IgGl Heavy Chain Constant Region, amino acid sequence (Seq. ID NO. 136)
1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 61 GLYSLSSWT VPSSSLGTQT YI CNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG
12 1 PSVFLFPPKP KDTLMISRTP EVTCWVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN
181 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPI EKTI S KAKGQPREPQ VYTLPPSREE
24 1 MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 3 01 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
Reference Human Light Chain Kappa Constant Region, nucleotide sequence (Seq. ID NO. 137)
1 CCGGTCACCA TGGAAAT CAA ACGTACGGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA 61 TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT 12 1 C C C G G AGG C CAAAGT AC A GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG 181 GAGAGTGTCA C AG AG C AGG A C AG C AAGG AC AGCACCTACA GCCTCAGCAG CACCCTGACG 24 1 CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC 3 01 CTGAGCTCGC CCGTCACAAA GAGCTTCAAC AGGGGAGAGT GTTAG Reference Human Light Chain Kappa Constant Region, amino acid sequence (Seq. ID NO. 138)
1 PVTMEIKRTV AAPSVFI FPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ 61 ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Heavy Chain of the antibody RL1, full amino acid sequence (Seq. ID NO. 139)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFN NTYMSWVRQA PGKGLEWVSG I YPKSGTTDY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARFR VLFDYWGQGT LVTVSSASTK
12 1 GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS 181 LSSWTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF
24 1 LFPPKPKDTL MI SRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR
3 01 WSVLTVLHQ DWLNGKEYKC KVSNKALPAP I EKTI SKAKG QPREPQVYTL PPSREEMTKN
361 QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN
42 1 VFSCSVMHEA LHNHYTQKSL SLSPGK Heavy Chain of the antibody RL2, full amino acid sequence (Seq. ID NO. 140)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFT GSTMNWVRQA PGKGLEWVSR IGPEGGGTDY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGI I IFDYWGQGT LVTVSSASTK 121 GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS
181 LSSWTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF
241 LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR
301 WSVLTVLHQ DWLNGKEYKC KVSNKALPAP lEKTISKAKG QPREPQVYTL PPSREEMTKN
361 QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN 421 VFSCSVMHEA LHNHYTQKSL SLSPGK
Heavy Chain of the antibody RL3, full amino acid sequence (Seq. ID NO. 141)
1 EVQLLESGGG LVQPGGSLRL SCEASGFTFS SNSMSWVRQA PGKGLEWVSS ITPSNSATYY 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARRA FWFDYWGQGT LVTVSSASTK
121 GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS
181 LSSWTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF
241 LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR
301 WSVLTVLHQ DWLNGKEYKC KVSNKALPAP lEKTISKAKG QPREPQVYTL PPSREEMTKN 361 QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN 421 VFSCSVMHEA LHNHYTQKSL SLSPGK
Heavy Chain of the antibody RL4, full amino acid sequence (Seq. ID NO. 142) 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFN RTDMSWVRQA PGKGLEWVSG INSYGGATNY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARRG LWFDYWGQGT LVTVSSASTK
121 GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS
181 LSSWTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF
241 LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR 301 WSVLTVLHQ DWLNGKEYKC KVSNKALPAP lEKTISKAKG QPREPQVYTL PPSREEMTKN
361 QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN 421 VFSCSVMHEA LHNHYTQKSL SLSPGK Heavy Chain of the antibody RL7, full amino acid sequence (Seq. ID NO. 143)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFT RSAMSWVRQA PGKGLEWVSS ISPNSWTNYA
61 DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCARRGL VFDYWGQGTL VTVSSASTKG 121 PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL
181 SSWTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAP ELLGGPSVFL
241 FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV
301 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ
361 VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 421 FSCSVMHEAL HNHYTQKSLS LSPGK
Heavy Chain of the antibody RL8, full amino acid sequence (Seq. ID NO. 144)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYRMNWVRQA PGKGLEWVSV ISPGGGWTNY 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARTS TSSDYYYYGL DYWGQGTLVT
121 VSSASTKGPS VFPLAPLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKTHT
181 CPPCPALFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ
241 YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR
301 EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS 361 RWQQGNLHNH YTQKSLSLSP GK
Heavy Chain of the antibody RL9, full amino acid sequence (Seq. ID NO. 145)
1 EVQLLESGGG LVQPGGSLRL SCEASGFTFS SNSMSWVRQA PGKGLEWVSS ITPSNSATYY 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARRA FWFDYWGQGT LVTVSSASTK
121 GPSVFPLAPL SSWTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAL
181 FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV
241 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ
301 VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNL 361 HNHYTQKSLS LSPGK Heavy Chain of the antibody RL10, full amino acid sequence (Seq. ID NO. 146)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS GSAMSWVRQA PGKGLEWVSL IRPSRGVTNY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARRG FWFDYWGQGT LVTVSSASTK 121 GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS
181 LSSWTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF
241 LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR
301 WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN
361 QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN 421 VFSCSVMHEA LHNHYTQKSL SLSPGK
Heavy Chain of the antibody RL11, full amino acid sequence (Seq. ID NO. 147)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS GSGMSWVRQA PGKGLEWVSS IWPEGGYTYY 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARFV FLFDYWGQGT LVTVSSASTK
121 GPSVFPLAPL SSWTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAL
181 FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV
241 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ
301 VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNL 361 HNHYTQKSLS LSPGK
Heavy Chain of the antibody RL12, full amino acid sequence (Seq. ID NO. 148)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFN DNYMSWVRQA PGKGLEWVSS IRSGGGVTRY 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARHH YSVQGGLDYW GQGTLVTVSS
121 ASTKGPSVFP LAPLSSWTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP
181 CPALFPPKPK DTLMISRTPE VTCWVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
241 TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM
301 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ 361 QGNLHNHYTQ KSLSLSPGK
Heavy Chain of the antibody RL13, full amino acid sequence (Seq. ID NO. 149)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS NNAMSWVRQA PGKGLEWVSA ISGSGGSTYY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARHF LLFDYWGQGT LVTVSSASTK 121 GPSVFPLAPL SSWTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAL
181 FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV
241 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ
301 VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNL
361 HNHYTQKSLS LSPGK
Heavy Chain of the antibody RL18, full amino acid sequence (Seq. ID NO. 150)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVSS IASSDGYTNY 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARSY AYQYRGLDYW GQGTLVTVSS
121 ASTKGPSVFP LAPLSSWTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP
181 CPALFPPKPK DTLMISRTPE VTCVWDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
241 TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM
301 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ 361 QGNLHNHYTQ KSLSLSPGK
Heavy Chain of the antibody RL19, full amino acid sequence (Seq. ID NO. 151)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTVT RSVIWWVRQA PGKGLEWVSG IGPGGGWTRY 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARSI GRRVFDYWGQ GTLVTVSSAS
121 TKGPSVFPLA PLSSWTVPS SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP
181 ALFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
241 RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK
301 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG 361 NLHNHYTQKS LSLSPGK Heavy Chain of the antibody RL21, full amino acid sequence (Seq. ID NO. 152)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVSG IGPGGGWTRY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARSI GRRVFDY GQ GTLVTVSSAS 121 TKGPSVFPLA PLSSWTVPS SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP
181 ALFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
241 RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK
301 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG 361 NLHNHYTQKS LSLSPGK
Heavy Chain of the antibody RL22, full amino acid sequence (Seq. ID NO. 153)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVSS IASSDGYTNY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARSY AYQYRGLDYW GQGTLVTVSS 121 ASTKGPSVFP LAPLSSWTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP
181 CPALFPPKPK DTLMISRTPE VTCVWDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
241 TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM
301 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ 361 QGNLHNHYTQ KSLSLSPGK
Light Chain Kappa of the antibody RL1, full amino acid sequence (Seq. ID NO. 154)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QSSYYAPVTF GQGTKVEIKR TVAAPSVFIF 121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC
Light Chain Kappa of the antibody RL2, full amino acid sequence (Seq. ID NO. 155) 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QASYYSPFTF GQGTKVEIKR TVAAPSVFIF
121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC Light Chain Kappa of the antibody RL3, full amino acid sequence (Seq. ID NO. 156)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QASYYSPVTF GQGTKVEIKR TVAAPSVFIF 121 TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK 181 HKVYACEVTH QGLSSPVTKS FNRGEC
Light Chain Kappa of the antibody RL4, full amino acid sequence (Seq. ID NO. 157)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QASYYSPVTF GQGTKVEIKR TVAAPSVFIF
121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST
181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC
Light Chain Kappa of the antibody RL7, full amino acid sequence (Seq. ID NO. 158)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QASYYSPVTF GQGTKVEIKR TVAAPSVFIF 121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST
181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC
Light Chain Kappa of the antibody RL8, full amino acid sequence (Seq. ID NO. 159) 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QSSYSPLTFG QGTKVEIKRT VAAPSVFIFT
121 ASWCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH 181 KVYACEVTHQ GLSSPVTKSF NRGEC Light Chain Kappa of the antibody RL9, full amino acid sequence (Seq. ID NO. 160)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QSSYYSPGTF GQGTKVEIKR TVAAPSVFIF
121 TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK 181 HKVYACEVTH QGLSSPVTKS FNRGEC Light Chain Kappa of the antibody RL10, full amino acid sequence (Seq. ID NO. 161)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QASYYSPVTF GQGTKVEIKR TVAAPSVFIF 121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC
Light Chain Kappa of the antibody RL11, full amino acid sequence (Seq. ID NO. 162) 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QASYYSPVTF GQGTKVEIKR TVAAPSVFIF
121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST
181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC Light Chain Kappa of the antibody RL12, full amino acid sequence (Seq. ID NO. 163)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYYSSPITGQ GTKVEIKRTV AAPSVFIFTA
121 SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK 181 VYACEVTHQG LSSPVTKSFN RGEC
Light Chain Kappa of the antibody RL13, full amino acid sequence (Seq. ID NO. 164)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QASYYSPFTF GQGTKVEIKR TVAAPSVFIF
121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST
181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC
Light Chain Kappa of the antibody RL18, full amino acid sequence (Seq. ID NO. 165)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QSYDSYPFTF GQGTKVEIKR TVAAPSVFIF
121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST
181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC Light Chain Kappa of the antibody RL19, full amino acid sequence (Seq. ID NO. 166)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QSYDSYPFTF GQGTKVEIKR TVAAPSVFIF 121 PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 181 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC
Light Chain Kappa of the antibody RL21, full amino acid sequence (Seq. ID NO. 167) 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QSDSYPITFG QGTKVEIKRT VAAPSVFIFP
121 PSDEQLKSGT ASWCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL
181 TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC Light Chain Kappa of the antibody RL22, full amino acid sequence (Seq. ID NO. 168)
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QARKSPITFG QGTKVEIKRT VAAPSVFIFP
121 PSDEQLKSGT ASWCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL 181 TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC
The sequences of Fab antibody fragments defining Variable Regions of immunoglobulin heavy chain selected from the phage display libraries as described in Example 2, screened as set forth in Example 3 and sequences of which identified in the Example 4 are shown in Figure 2. The sequences are aligned to each other due to homology of the antibody framework where CDRl, CDR2 and CDR3 are identified by boxes. Figure 3 shown an alignment of each CDR separately where dashes (-) are inserted to shorter CDRs for the alignment purpose.
The sequences of Fab antibody fragments defining Variable Regions of immunoglobulin light chain (kappa) selected from the phage display libraries as described in Example 2, screened as set forth in Example 3 and sequences of which identified in the Example 4 are shown in Figure 4. The sequences are aligned to each other due to homology of the antibody framework where CDRl, CDR2 and CDR3 are identified by boxes. Figure 5 shown an alignment of each CDR separately where dashes (-) are inserted to shorter CDRs for the alignment purpose.
Table 1 provides a correspondence between sequences discussed in these Examples with Sequence Listing (Seq. ID NO).
TABLE 1
Seq. ID
Protein or Nucleic acid Description
NO
01 Heavy Chain Variable region of RLl - Protein
02 Heavy Chain Variable region of RLl - Nucleic acid
03 Heavy Chain Variable region of RL2 - Protein
04 Heavy Chain Variable region of RL2 - Nucleic acid
05 Heavy Chain Variable region of RL3 - Protein
06 Heavy Chain Variable region of RL3 - Nucleic acid
07 Heavy Chain Variable region of RL4 - Protein
08 Heavy Chain Variable region of RL4 - Nucleic acid
09 Heavy Chain Variable region of RL7 - Protein
10 Heavy Chain Variable region of RL7 - Nucleic acid
11 Heavy Chain Variable region of RL8 - Protein
12 Heavy Chain Variable region of RL8 - Nucleic acid
13 Heavy Chain Variable region of RL9 - Protein
14 Heavy Chain Variable region of RL9 - Nucleic acid
15 Heavy Chain Variable region of RLl 0 - Protein
16 Heavy Chain Variable region of RLl 0 - Nucleic acid
17 Heavy Chain Variable region of RLl 1 - Protein
18 Heavy Chain Variable region of RLl 1 - Nucleic acid
19 Heavy Chain Variable region of RLl 2 - Protein
20 Heavy Chain Variable region of RL12 - Nucleic acid
21 Heavy Chain Variable region of RLl 3 - Protein Heavy Chain Variable region of RL13 - Nucleic acid
Heavy Chain Variable region of RL18 - Protein
Heavy Chain Variable region of RL18 - Nucleic acid
Heavy Chain Variable region of RL19 Protein
Heavy Chain Variable region of RL19 - Nucleic acid
Heavy Chain Variable region of RL21 - Protein
Heavy Chain Variable region of RL21 - Nucleic acid
Heavy Chain Variable region of RL22 - Protein
Heavy Chain Variable region of RL22 - Nucleic acid
Heavy Chain CDRl of RLl - Protein
Heavy Chain CDR2 of RLl - Protein
Heavy Chain CDR3 of RLl - Protein
Heavy Chain CDRl of RL2- Protein
Heavy Chain CDR2 of RL2- Protein
Heavy Chain CDR3 of RL2- Protein
Heavy Chain CDRl of RL3- Protein
Heavy Chain CDR2 of RL3- Protein
Heavy Chain CDR3 of RL3- Protein
Heavy Chain CDRl of RL4 - Protein
Heavy Chain CDR2 of RL4 - Protein
Heavy Chain CDR3 of RL4 - Protein
Heavy Chain CDRl of RL7 - Protein
Heavy Chain CDR2 of RL7 - Protein
Heavy Chain CDR3 of RL7 - Protein
Heavy Chain CDRl of RL8 - Protein
Heavy Chain CDR2 of RL8 - Protein
Heavy Chain CDR3 of RL8 - Protein
Heavy Chain CDRl of RL9- Protein
Heavy Chain CDR2 of RL9- Protein
Heavy Chain CDR3 of RL9- Protein
Heavy Chain CDRl of RLIO- Protein Heavy Chain CDR2 of RLIO Protein
Heavy Chain CDR3 of RL10- Protein
Heavy Chain CDR1 of RL11 - - Protein
Heavy Chain CDR2 of RL11 - Protein
Heavy Chain CDR3 of RL11 - - Protein
Heavy Chain CDR1 of RL12 - - Protein
Heavy Chain CDR2 of RL12 - Protein
Heavy Chain CDR3 of RL12 - - Protein
Heavy Chain CDR1 of RL13 - - Protein
Heavy Chain CDR2 of RL13 - - Protein
Heavy Chain CDR3 of RL13 - - Protein
Heavy Chain CDR1 of RL18 - - Protein
Heavy Chain CDR2 of RL18 - - Protein
Heavy Chain CDR3 of RL18 - - Protein
Heavy Chain CDR1 of RL19 - - Protein
Heavy Chain CDR2 of RL19 - - Protein
Heavy Chain CDR3 of RL19 - - Protein
Heavy Chain CDR1 of RL21 - - Protein
Heavy Chain CDR2 of RL21 - - Protein
Heavy Chain CDR3 of RL21 - - Protein
Heavy Chain CDR1 of RL22 - - Protein
Heavy Chain CDR2 of RL22 - - Protein
Heavy Chain CDR3 of RL22 - - Protein
Light Chain (Kappa) Variable region of RL1 - Protein
Light Chain (Kappa) Variable region of RL1 - Nucleic acid
Light Chain (Kappa) Variable region of RL2- - Protein
Light Chain (Kappa) Variable region of RL2- - Nucleic acid
Light Chain (Kappa) Variable region of RL3- - Protein
Light Chain (Kappa) Variable region of RL3- - Nucleic acid
Light Chain (Kappa) Variable region of RL4 - Protein
Light Chain (Kappa) Variable region of RL4 - Nucleic acid Light Chain (Kappa) Variable region of RL7 - Protein
Light Chain (Kappa) Variable region of RL7 - Nucleic acid
Light Chain (Kappa) Variable region of RL8 - Protein
Light Chain (Kappa) Variable region of RL8 - Nucleic acid
Light Chain (Kappa) Variable region of RL9- Protein
Light Chain (Kappa) Variable region of RL9- Nucleic acid
Light Chain (Kappa) Variable region of RL10 Protein
Light Chain (Kappa) Variable region of RLl 0- - Nucleic acid
Light Chain (Kappa) Variable region of RLl 1 - Protein
Light Chain (Kappa) Variable region of RLl 1 - Nucleic acid
Light Chain (Kappa) Variable region of RLl 2 - Protein
Light Chain (Kappa) Variable region of RLl 2 - Nucleic acid
Light Chain (Kappa) Variable region of RLl 3 - Protein
Light Chain (Kappa) Variable region of RLl 3 - Nucleic acid
Light Chain (Kappa) Variable region of RLl 8- - Protein
Light Chain (Kappa) Variable region of RLl 8- - Nucleic acid
Light Chain (Kappa) Variable region of RLl 9- - Protein
Light Chain (Kappa) Variable region of RLl 9- - Nucleic acid
Light Chain (Kappa) Variable region of RL21 - Protein
Light Chain (Kappa) Variable region of RL21- - Nucleic acid
Light Chain Kappa^ Variable region of RL22 - Protein
Light Chain (Kappa) Variable region of RL22 - Nucleic acid
Light Chain (Kappa) CDRl of RLl; RL2; RL3; RL4; RL7; RL8; RL9; RL10; RL11; RL12 , RL13; RL18; RL19; RL21; RL22 - Protein
Light Chain (Kappa) CDR2 of RLl; RL2; RL3; RL4; RL7; RL8; RL9; RL10; RL11; RL12 , RL13; RL18; RL19; RL21; RL22 - Protein
Light Chain Kappa^ CDR3 of RLl - Protein
Light Chain (Kappa) CDR3 of RL2- Protein
Light Chain (Kappa) CDR3 of RL3- Protein
Light Chain Kappa^ CDR3 ofRL4 - Protein
Light Chain (Kappa) CDR3 ofRL7 - Protein Light Chain (Kappa) CDR3 of RL8 Protein
Light Chain (Kappa) CDR3 of RL9 - Protein
Light Chain (Kappa) CDR3 of RL 10 - Protein
Light Chain (Kappa) CDR3 of RL 1 1 Protein
Light Chain (Kappa) CDR3 of RL 12 - Protein
Light Chain (Kappa) CDR3 of RL 13 - Protein
Light Chain (Kappa) CDR3 of RL 18- Protein
Light Chain (Kappa) CDR3 of RL 19 - Protein
Light Chain (Kappa) CDR3 of RL21 - Protein
Light Chain (Kappa) CDR3 of RL22 - Protein
Epitope tag that was cloned into pcDNA3.1 -Zeo-(+)
IA-003 direct primer
IA-004 reverse primer
AC-001 Direct 'structural' primer
AC-002 Reverse 'structural' primer
AC-003 direct primer
AC -004 reverse primer
Immunoglobulin 20-amino acid signal peptide sequence
A-370 Direct PCR primer for Variable Region of Heavy Chain IgG 1 A-371 Reverse PCR primer for Variable Region of Heavy Chain IgGl A-340 Direct PCR primer for Variable Region of Light Chain Kappa A-341 Reverse PCR primer for Variable Region of Light Chain Kappa Reference Human IgGl Heavy Chain Constant Region, nucleotide sequence Reference Human IgGl Heavy Chain Constant Region, amino acid sequence Reference Human Light Chain Kappa Constant Region, nucleotide sequence Reference Human Light Chain Kappa Constant Region, amino acid sequence Heavy Chain of the antibody RLl, full amino acid sequence
Heavy Chain of the antibody RL2, full amino acid sequence
Heavy Chain of the antibody RL3, full amino acid sequence
Heavy Chain of the antibody RL4, full amino acid sequence
Heavy Chain of the antibody RL7, full amino acid sequence 144 Heavy Chain of the antibody RL8, full amino acid sequence
145 Heavy Chain of the antibody RL9, full amino acid sequence
146 Heavy Chain of the antibody RL10, full amino acid sequence
147 Heavy Chain of the antibody RL 11 , full amino acid sequence
148 Heavy Chain of the antibody RL12, full amino acid sequence
149 Heavy Chain of the antibody RL13, full amino acid sequence
150 Heavy Chain of the antibody RL18, full amino acid sequence
151 Heavy Chain of the antibody RL19, full amino acid sequence
152 Heavy Chain of the antibody RL21, full amino acid sequence
153 Heavy Chain of the antibody RL22, full amino acid sequence
154 Li. iht Chain Kappa of the antibody RL 1 , full amino acid sequence
155 Li. iht Chain Kappa of the antibody RL2, full amino acid sequence
156 L¾ iht Chain Kappa of the antibody RL3, full amino acid sequence
157 Li. iht Chain Kappa of the antibody RL4, full amino acid sequence
158 Li. iht Chain Kappa of the antibody RL7 full amino acid sequence
159 Li. iht Chain Kappa of the antibody RL8, full amino acid sequence
160 Li. iht Chain Kappa of the antibody RL9, full amino acid sequence
161 Li. >ht Chain Kappa of the antibody RL 10, full amino acid sequence
162 Li. ^ht Chain Kappa of the antibody RL 11 , full amino acid sequence
163 Li. iht Chain Kappa of the antibody RL 12, full amino acid sequence
164 Li. ht Chain Kappa of the antibody RL13 full amino acid sequence
165 Li. iht Chain Kappa of the antibody RL 18, full amino acid sequence
166 Li. iht Chain Kappa of the antibody RL 19, full amino acid sequence
167 Li. ht Chain Kappa of the antibody RL21 full amino acid sequence
168 Li. iht Chain Kappa of the antibody RL22 full amino acid sequence
Example 5 - Production of recombinant human RANKL. This Example describes design and generation of expression constructs for inducible expression of human RANKL receptor. In particular, this example describes epitope and purification tags genetically fused to the nucleic acid sequences encoding RANKL and its orthologs.
Human RANKL (NCBI Reference Sequence: NM_003701.3) was amplified by PCR using Clone ID HsCD00436583 (DNASU Plasmid Repository) as a template. To the coding sequence of RANKL, Nhel restriction site for cloning purpose and Kozak sequence was genetically fused upstream of the starting native ATG codon using IA-013 Direct primer. The endogenous native Stop codon of RANKL was removed using IA-014 Reverse primer to provide in- frame genetic fusion with Strep-tag (ht : 7www.iba-go.de/prottoots/ rot streptag.htmi. a 8-amino acid peptide tag) and FLAG epitope tag for detection purposes, and Aflll restriction site was introduced for cloning purpose . The PCR product obtained from the reaction using IA-013 and IA-014 primers and human RANKL as the template, was Nhel-Aflll digested and cloned into pcDNA3.1-FlagStr vector (Invitrogen). Cloning of pcDNA3.1-FlagStr vector was performed by PCR assembly of the epitope tag: -Link-FLAG-SBP-* (where SBP stands for Streptavidin Binding Peptide) into the cloning site between Aflll and Apal cloning clonig sites. Combining of AC-001, AC-002
'structural' primers and AC-003 and AC -004 direct and reverse PCR primers was required for assembly of the tag.
Following is the epitope tag that was cloned into pcDNA3.1-Zeo-(+) using 5 '-end Aflll and 3'-end Apal restriction sites (Seq. ID NO. 123): ATCCTTAAGGGCAGCGGGTCCTCTGGAGGGGGAGACTATAAGGATGACGATGACA
AGagtatggatgagaaaaccacaggttggcgcggcgggcatgtcgttgaaggac tggccggtgagctggaacaactcagggctagattggagcaccaccctcagggcca gcgggaaccttagGGCCCATA where Aflll cloning site (CTTAAG) and Apal cloning site (GGGCCC) are shown
underlined, GS-linker in shown in italics, and SBP is shown in small cap letters.
IA-013 direct primer (Seq. ID NO. 124)
5 ' - ATGCGCCGCGCCAGCAGAGACTAC - 3 ' IA-014 reverse primer (Seq. ID NO. 125)
5 ' - ATCTATATCTCGAACTTTAAAAGCCCCA - 3 '
AC-001 direct 'structural' primer (Seq. ID NO. 126)
5 ' - AGCGGGTCCTCTGGAGGGGGAGACTATAAGGATGACGATGACAAGAGTA
TGGATGAGAAAACGACAGGTTGGCGCGGCGGGCATGTCGTT - 3 '
AC-002 reverse 'structural' primer (Seq. ID NO. 127)
5 ' - GTTCCCGCTGGCCCTGAGGGTGGTGCTCCAATCTAGCCCTGAGTTGTTCC
AGCTCACCGGCCAGTCCTTCAACGACATGCCCGCCGCGCC - 3 '
AC-003 direct PCR primer (Seq. ID NO. 128)
5 ' - ATCCTTAAGGGCAGCGGGTCCTCTGGAGGGGGAG - 3 '
AC-004 reverse PCR primer (Seq. ID NO. 129)
5 ' - TATGGGCCCTAAGGTTCCCGCTGGCCCTGAGGG - 3 '
Example 6 - Generation of stable cell lines expressing recombinant RANKL. The expression constructs encoding human RANKL receptor described in Example 5 were used for generation of stable cell lines. Commercially available cell lines, R1610, Cf2Th and HEK-293 (ATCC) were used. The expression constructs were verified for the expression of the protein of interest in a transient transfection experiment and then the cells were propagated on a medium containing Zeocin to select for stable cell lines harboring the gene of interest. Expression of the RANKL was verified by Western blot and by FACS analysis to ensure that the expressed protein was translocated to the plasma membrane. For both techniques, commercially available antibodies were used. All steps of the cell line generation were carried out according to the manufacturers' protocols. Example 7 - Conversion of the Fab antibody fragments into immunoglobulins and their production.
This Example provides description an approach for subcloning of Fab fragments into mammalian expression vectors for production of fully functional immunoglobulins. A protein production approach is also provided herein.
The candidate Fab antibody Heavy Chain variable region fragments described in the foregoing Examples 2-4 were converted into full size immunoglobulins of IgGl framework.
Variable region of the heavy chain was fused to the constant region of human IgGl isotype using expression vector pTT-5 ( RC Biotechnology Research Institute, National Research Council of Canada) modified by introducing the constant region of human IgGl from pFUSE-CHIg-hGl expression vector (Invivogen) resulting in pTT-IgGl-HC vector. The signal peptide from immunoglobulin kappa light chain variable region (Mus musculus, gb|AAG35718.1|AF207705_l) was introduced into the construct upstream of the antibody variable sequences disclosed herein. Immunoglobulin 20-amino acid signal peptide sequence where starting methionine is underlined (Seq. ID NO. 130)
METDTILLWVLLLWVPGSTG The variable regions of the heavy chain were amplified by PCR to introduce into the following cloning sites: 5 '-end cloning restriction site is Sail, 3 '-end restriction site is Nhel. A set of two primers, A-370 direct primer and A-371 reverse primer, was used for the PCR
amplification. The resulting PCR fragment was digested with Sall-Nhel restriction enzymes and then introduced into pTT-IgGl-HC vector digested with the same enzymes. A-370 direct PCR primer for amplification of Heavy Chain Variable Region (Sail restriction site is underlined) (Seq. ID NO. 131)
5 ' - TGTGTCGACCGGAGAAGTTCAACTGCTGGAGTCCGGTGGTGGTCTGG
TACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCG - 3 '
A-371 Reverse PCR primer for amplification of Heavy Chain Variable Region (Nhel restriction site is underlined) (SEQ. ID NO 132) 5 ' - TTGTGCTAGCACTCGAGACGGTGACCAAGGTTCCCTGGCC - 3
The candidate Fab antibody Light Chain variable region fragments described in the foregoing Examples 2-4 were converted into full size immunoglobulins of IgGl framework. Variable Region of light chain was fused to the constant region of human light chain kappa using expression vector pTT-5 (NRC Biotechnology Research Institute, National Research Council of Canada) modified by introduction of the constant region of human light chain fragmen from pFUSE2- CLIg-hk expression vector (Invivogen) resulting in pTT-LC-Kappa. The signal peptide from immunoglobulin kappa light chain variable region (Mus musculus, gb|AAG35718.1|AF207705 1) was introduced into the construct upstream of the antibody variable sequences reported in this invention.
The variable regions of the light chain were amplified by PCR to introduce for the following cloning sites: 5'-end cloning restriction site is Sail, 3'-end restriction site is BsiWI. The set of two primers, A-340 direct primer and A-341 reverse primer, was used for the PCR amplification. The resulting PCR fragment was digested with Sall-BsiWI restriction enzymes and then introduced into pTT-LC-Kappa vector digested with the same enzymes.
A-340 direct PCR primer for amplification of Light Chain Kappa Variable Region (Sail restriction site is underlined) (Seq. ID NO. 133)
5 ' - TGTGTCGACCGGGGAAATTGTGTTGACGCAGTCTCCG - 3 A-341 reverse PCR primer for amplification of Light Chain Kappa Variable Region (BsiWI restriction site is underlined) (Seq. ID NO. 134)
5 ' - ATGGTGCAGCCACCGTACGTTTGATTTCCACC - 3 '
The antibodies in a format of human IgGl framework were produced using a protocol for transfection of CHO-3E7 cells using LPEI MAX in shake flask cultures. CHO-3E7 cells provided by NRC Biotechnology Research Institute, National Research Council of Canada, were diluted to 0.8 xlO6 cells/ml 24 h before transfection. On the day of transfection cell density was adjusted to 2.0 to 2.2xl06 cells/ml using complete FreeStyle™ CHO medium and cell viability was greater than 97%.
The working solution of Polyethylenimine (PEI) was prepared as follows. To the 450 ml Milli-Q water, 1500 mg PEI "MAX" was added and stirred until complete dissolution and a final concentration of 3 mg/ml. The initial pH of the solution was around 2.2 and then it was adjusted to pH 7.0 by NaOH. The final pH adjustments were made using HC1 and/or NaOH. The final volume of the solution was adjusted to 500 ml, filter-sterilize using a 0.22 μιτι membrane and stored at -20°C.
Polyethylenimine "MAX" linear, MW 25 kDa (40 kDa nominal), 3 mg/ml stock solution in water, pH 7.0 (Polysciences Inc. cat# 24765-2) was mixed with purified and quantified plasmid DNA of interest. A260/A280 ratio (use 50 mM Tris-HCl pH 8.0 to dilute the plasmid DNA) was between 1.85 and 1.95. The cells were used in exponential growth phase, 2-2.2xl06 cells/ml in CHO FreeStyle medium. DNA preparations (0.75mg/L) encoding for Heavy or Light chains of immunoglobulin were mixed with PEI in CHO FreeStyle medium at 1 :5 (w:w) ratio, the mixture was then incubated 8-10 min, add then added to culture. Volume of the transfection mixture was 1/10 of the final volume of the production culture.
Routinely, expression level for various immunoglobulins was from 20 to 100 mg/L. The immunoglobulin production was monitored by commercially available ELISA kit (Bethyl Laboratories).
Purification of immunoglobulin preparations was carried out as follows. Tris-Gly cine-Native Buffer, lOx, pH 8.5 (Boston Bioproducts; Cat# BP- 160) was added to the supernatant (1/10 volume/volume) containing IgG at a final concentration of 20-100 mg/L and gently mixed. The supernatant volume was evenly distributed into 50-ml centrifuge tubes, 50ml per tube. Protein A Plus Agarose (Pierce, Cat. # 22812), a wet pellet, was used at 1/100 ratio to the total volume of the supernatant material. The appropriate amount of Protein A Plus Agarose was incubated with the IgG-containing supernatants at an orbital shaker for overnight. After the incubation, the Protein A Plus Agarose resin was harvested and placed into 1 -ml columns (Pierce) and then washed with 10 volumes of lx PBS, then with 10 volumes of 25mM Tris-HCl, 0.12M Glycine, 1.5M NaCl (pH 8.5), then with 10 volumes of TBS-Tween-20, then with 10 volumes of 20mM Sodium Citrate Buffer, 1M NaCl (pH 5.5) and the final wash with 10 volumes of 150mM Sodium Chloride without any buffer. The elution of bound immunoglobulins was carried out with Elution Buffer (0.1 M Glycine pH 3.0, 10% Sucrose, 150mM NaCl) that was added at ratio of 1 : 1 to the volume of Protein A Plus Agarose resin. The elution buffer was incubated with the Protein A Plus Agarose resin for 3 min, removed and then another portion of fresh Elution Buffer was added to the Protein A Plus Agarose resin. The eluted material was immediately neutralized by 0.5 M Sodium Citrate Buffer, pH 6.0, at the 1/10 volume ratio to the eluted volume. The concentration of the resulting immunoglobulin preparations was determined by measure optical density of the solution at 280nm in UV-transparent cuvettes where a mixture of 0.1 ml of 0.5 M Sodium Citrate, pH 6, with 1ml of the Elution Buffer was used as a Reference Buffer. To calculate the
concentration of IgG, the following formula that provides IgG concentration in mg/ml, was used:
[IgG] = OD2W * DilutionFactor ^ w^Qre j ^ ^s ^ s^an^ar(j extinction coefficient for an IgG.
Example 8 - Determination of affinities (EC50 values) of RANKL antibodies
This example describes the method for determination of the affinities of the antibodies against human RANKL receptor and provides means of comparison of properties of different antibody clones.
Determination of EC50 values for the antibodies against RANKL was performed using cells overexpressing human RANKL. The antibody at various concentrations (from 0.02 nM to 500 nM) was allowed to interact with the indicated cell lines and then the binding of the antibody to the cells was revealed by fluorescently labeled secondary antibodies. The stained cells were analyzed by FACS where Mean Fluorescence Intensity (MFI) was measured. The obtained data were analyzed by GraphPad Prism 5.0 software and EC50 values were calculated using agonistic 4- parameter curve fit algorithm
TABLE 2
Figure imgf000052_0001
All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
"Zometa" is a registered trademark of Novartis AG Corporation, Switzerland. "Prolia" and "Xgeva" are registered trademarks of Amgen Inc., a Delaware Corporation.
While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variation

Claims

claimed is:
An isolated antibody that binds human RANKL, comprising
an immunoglobulin light chain of SEQ. ID NO. 154;
and an immunoglobulin heavy chain of SEQ ID NO. 139;
or an antigen binding fragment of the antibody.
An isolated antibody that binds human RANKL, comprising
an immunoglobulin light chain of SEQ. ID NO. 155;
and an immunoglobulin heavy chain of SEQ. ID NO . 140;
or an antigen binding fragment of the antibody.
An isolated antibody that binds human RANKL, comprising
an immunoglobulin light chain of SEQ. ID NO. 157;
and an immunoglobulin heavy chain of SEQ. ID NO . 142;
or an antigen binding fragment of the antibody.
An isolated antibody that binds human RANKL, comprising
an immunoglobulin light chain of SEQ. ID NO. 158;
and an immunoglobulin heavy chain of SEQ. ID NO . 143;
or an antigen binding fragment of the antibody.
The antibody of claim 1 , wherein the antibody is a monoclonal antibody.
The antibody of claim 2, wherein the antibody is a monoclonal antibody.
The antibody of claim 3 , wherein the antibody is a monoclonal antibody
The antibody of claim 4, wherein the antibody is a monoclonal antibody
8. An isolated antibody that binds human RANKL, comprising
(i) an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 108; and
(ii) an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 31, a CDRH2 comprising the sequence of SEQ. ID NO. 32, and a CDRH3 comprising the sequence of SEQ. ID NO. 33; or an antigen binding fragment of the antibody.
9. An isolated antibody that binds human RANKL, comprising
(i) an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 109; and
(ii) an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 34, a CDRH2 comprising the sequence of SEQ. ID NO. 35, and a CDRH3 comprising the sequence of SEQ. ID NO. 36; or an antigen binding fragment of the antibody.
An isolated antibody that binds human RANKL, comprising
(i) an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. I l l; and
(ii) an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 40, a CDRH2 comprising the sequence of SEQ. ID NO. 41, and a CDRH3 comprising the sequence of SEQ. ID NO. 42; or an antigen binding fragment of the antibody.
11. An isolated antibody that binds human RANKL, comprising
(i) an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ. ID NO. 106, a CDRL2 comprising the sequence of SEQ. ID NO. 107, and a CDRL3 comprising the sequence of SEQ. ID NO. 112; and (ii) an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ. ID NO. 43, a CDRH2 comprising the sequence of SEQ. ID NO. 44, and a CDRH3 comprising the sequence of SEQ. ID N0.45; or an antigen binding fragment of the antibody.
12. The antibody of claim 8, wherein the CDR sequences are interposed between human or humanized framework sequences.
13. The antibody of claim 9, wherein the CDR sequences are interposed between human or humanized framework sequences.
14. The antibody of claim 10, wherein the CDR sequences are interposed between human or humanized framework sequences.
15. The antibody of claim 11 , wherein the CDR sequences are interposed between human or humanized framework sequences.
16. An isolated antibody that binds human RANKL, comprising
an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 76, and
an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 01; or
an antigen binding fragment of the antibody.
17. An isolated antibody that binds human RANKL, comprising
an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 78, and
an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 03; or
an antigen binding fragment of the antibody.
18. An isolated antibody that binds human RANKL, comprising
an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 82, and
an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 07; or
an antigen binding fragment of the antibody.
19. An isolated antibody that binds human RANKL, comprising
an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 84, and
an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 09; or
an antigen binding fragment of the antibody.
20. The antibody of claim 16, wherein the antibody is a monoclonal antibody.
21. The antibody of claim 17, wherein the antibody is a monoclonal antibody.
22. The antibody of claim 18, wherein the antibody is a monoclonal antibody.
23. The antibody of claim 19, wherein the antibody is a monoclonal antibody.
PCT/US2013/056539 2013-08-24 2013-08-24 Fully human antibodies against human rankl WO2015030701A1 (en)

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WO2018080914A1 (en) * 2016-10-28 2018-05-03 Eli Lilly And Company Anti-rankl antibodies and uses thereof
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