Patent Application
20100239588
The present invention relates to immunology field. In particular, the present invention discloses a functional protein epitope, monoclonal antibodies specially binding to the same, and the use of these antibodies.
Autoimmune refers to the phenomenon that the organism immune system produces antibodies and sensitized lymphocytes against its own tissue or organ, and induces immune response. When autoimmune causes functional dysfunctions of the tissue or organ and results in clinical symptoms, it is termed as autoimmune disease (AID). More than 30 autoimmune diseases are currently known, most of which are primary diseases and only a few of which are secondary diseases. Primary autoimmune disease, which is caused by unknown causes, is closely related to genetic factors and can be divided into organ specific and non-organ specific. The target antigens and the lesions of organ-specific AID are often restricted to a particular organ, while the target antigens and the lesions of non-organ specific AID often appear as systemic or systematic. In recent years, autoimmune and autoimmune diseases are of more and more attention. About several hundreds millions of people are suffering from the pain caused by the disease throughout the world. The disease has a longer duration, can cause disability, and is even deadly when serious. Due to the universality and seriousness of this problem, a global research wave of autoimmune disease is set off.
The pathogenesis of autoimmune diseases is very complex, and the exact pathogenesis is currently not fully understood. For the time being, it is believed that, individuals with certain genetic characteristics (determined by AID-related gene polymorphisms) may suffer from autoimmune disease for some internal and external pathogenic factors stimuli (autoantigen release, allosteric and ultra-antigen molecular simulation, etc.) or gene mutation, modification of autoreactive immune cells, which result in antigen-presenting cell (APC) or dendritic cell abnormal activation, immune regulatory network imbalance (TH1/TH2 balance destruction) and changes in autoreactive immune cells polyclonal activation or apoptosis inhibition. The onsets of autoimmune diseases related to many factors, including genetic, infection, immune dysfunction and so on. Although the pathogenesis is still not clear, but a large number of studies have indicated that the levels of a variety of inflammatory factors, such as TNF-a, IL-13 and IL-6 and so on in autoimmune disease patients are much higher than the health individual. Furthermore, anti-TNF-a monoclonal antibody and IL-1 receptor antagonist have showed significant therapeutic effect, which greatly improved the patient's condition, especially for some autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, etc. (“The therapeutic effects of an engineered human anti-tumour necrosis factor alpha antibody (CDP571) in rheumatoid arthritis”. 1995, Br, J. Rheumatol. 34: 334-342; “Infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial”. ATTRACT Study Group. Lancet. 1999. 354: 1932-1939; “Infliximab and methotrexate in the treatment of rheumatoid arthritis”. N. Engl. J. Med. 2000. 343: 1594-1602). Although these biological treatments have produced significant therapeutical effects, they still can not completely alleviate and cure these diseases. In recent years, a large number of studies indicated that chemokines also play an important role in autoimmune diseases, they can accurately regulate and control the migration of leukocytes to the inflammatory tissue, and reduce the apoptotic activity, extend the life of B-cell and T-cell, so as to attack the target tissue.
Osteopontin (OPN), also known as early T-lymphocyte-activation factor I (Eta-1) or secreted phosphoprotein I, contains Arg-Gly-Asp (RGD, arginine-glycine-aspartic acid) tripeptide sequence, has a molecular weight of about 44-66 kD depending on the dephosphorylation and glycosylation foil ns, and is an important multifunctional extracellular matrix protein (“Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity”. Science. 2000. 287: 860-864). OPN is expressed in bone tissue cells, macrophages, activated T cells and epithelial cells, etc., and plays an important role in mediating cell chemotaxis, adhesion, proliferation and migration, tumor metastasis, bone mineralization and reconstruction, immune regulation, signal transduction, as well as immunity to infectious diseases, etc. (“Osteoprotegerin Ligand Is a Cytokine that Regulates Osteoclast Differentiation and Activation”. Cell, 1998. 93: 65-176, “Osteopontin: role in cell signaling and cancer progression”. TRENDS in Cell Biology 2006, 16: 79-87.). OPN binds to cell surface receptors mainly through RGD pathway and non-RGD pathway so as to achieve its function (“Inhibition of Arg-Gly-Asp (ROD)-mediated Cell Adhesion to osteopontin by a Monoclonal Antibody against osteopontin”. J. Bio. Chem. 1994. 269: 23280-23285), wherein in the ROD pathway, OPN mainly binds to cell surface integrin receptors (avβ3, avβ1, avβ5), and in non-ROD pathway, it mainly binds to some variants of CD44 (V6, V7), thereby mediates a variety of cell physiological functions (“Intracellular Osteopontin Is an Integral Component of the CD44-ERM Complex Involved in Cell Migration”. Journal of Cellular Physiology. 2000. 184: 118-130). In general, OPN secreted by cells exists in the body' in many' forms after a series of post-expression modifications such as phosphorylation, glycosylation, and cleavage by thrombin, and every form may: have its unique function which is not fully explained based on the current study' and needs further research and discussion.
A large number of studies indicate that in autoimmune diseases (rheumatoid arthritis, multiple sclerosis, autoimmune hepatitis, Crohn's disease, etc.), the tissue and plasma OPN levels were significantly up-regulated (“Expression of osteopontin at sites of bone erosion in a murine experimental arthritis model of collagen-induced arthritis: possible involvement of osteopontin in bone destruction in arthritis”. Arthritis Rheum, 2002. 46: 1094-1101; “Role of osteopontin in amplification and perpetuation of rheumatoid synovitis”. J. Clin. Invest. 2005, 115: 1060-1067. “Osteopontin/Eta-1 upregulated in Crohn's disease regulates the Th1 immune response”. Gut. 2005. 54: 1254-1262. “Osteopontin as a Mediator of NKT Cell Function in T Cell-Mediated Liver Diseases”. Immunity, 2004, 21: 539-550), OPN recruits inflammatory cells in the target organ by binding to CD44 and other integrin receptors on the surface of lymphocytes and macrophages, and further promotes the activation of various inflammatory cells, which secretes a large number of pro-inflammatory cytokines (TNF-α, IL-13 and IL-6) (“Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis”, J. Clin. Invest. 2003. 112: 181-188). In addition, in rheumatoid arthritis, OPN can mediate bone destruction and absorption through binding to avβ3 and avβ5 receptors on the surface of osteoclast, thereby increase the onset of RA (“Osteopontin”. Crit Rev Oral Biol Med. 2000; 11(3): 279-303). Studies have shown that, in OPN gene deletion mice, the incidence and severity of arthritis decreased significantly' (“Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody induced arthritis in mice”. PNAS. 2002. 99(7): 4556-4561), the incidence of ConA-induced autoimmune hepatitis and hepatic injury level also decreased significantly (“Osteopontin as a Mediator of NKT Cell Function in T Cell-Mediated Liver Diseases”. Immunity, 2004. 21: 539-550), and a protective effect on DSS-induced Crohn's disease in mice was also observed (“Osteopontin deficiency' protects mice from Dextran sodium sulfate-induced colitis”. Inflamm Bowel Dis. 2006. 12(8): 790-6).
In the above models, the anti-OPN polyclonal antibody can significantly lower the incidence in rheumatoid arthritis model mice, reduce the extent of joint swelling and deformity (“Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis”. J. Clin. Invest. 2003. 112: 181-188), and can also significantly reduce the liver inflammation (“Osteopontin as a Mediator of NKT Cell Function in T Cell-Mediated Liver Diseases”, Immunity, 2004. 21: 539-550). Thus, OPN is a major pathogenic factor of autoimmune diseases and will be an effective drug target for the treatment of autoimmune diseases.
The purpose of this invention is to provide a monoclonal antibody against the therapeutic target, osteopontin, for the treatment of autoimmune diseases.
The first aspect of the present invention provides a functional osteopontin epitope, wherein the functional epitope is WLXPDP, and wherein X is any amino acid.
In a preferred embodiment, the preferred X in the functional epitope is: N, M or L.
The second aspect of the present invention provides an anti-osteopontin monoclonal antibody which specifically binds to the above functional epitope.
In one embodiment, the amino acid sequence of the CDR in the VII of the monoclonal antibody is selected from the group consisting of: IYAMD, RIRSQSNNYTTYYADSVKD and QMGDY (See, for example, H23C3VHb in
In another embodiment, the amino acid sequence of the VH in the monoclonal antibody is as set forth in SEQ ID NO: 4 or SEQ ID NO: 8, and the amino acid sequence of the VL is as set forth in SEQ ID NO: 6 or SEQ ID NO: 10.
In another embodiment, the constant region of the monoclonal antibody is a mouse constant region or a human constant region.
In a preferred embodiment, the constant region is a human constant region. In another preferred embodiment, the amino acid sequence of the heavy chain constant region in the constant region is as set forth in SEQ ID NO: 13. In another preferred embodiment, the amino acid sequence of the light chain constant region in the constant region is as set forth in SEQ ID NO: 15. In another preferred embodiment, the monoclonal antibody is obtained by hybridoma method or recombinant DNA method, or is isolated from phage antibody library. In another preferred embodiment, the monoclonal antibody is a chimeric antibody or a humanized antibody.
The third aspect of the present invention provides a DNA molecule, which encodes the monoclonal antibody of this invention as mentioned above.
In another preferred embodiment, the VH encoding nucleotide sequence in the DNA molecule is as set forth in SEQ ID NO: 3 or SEQ ID NO: 7; and the VL encoding nucleotide sequence is as set forth in SEQ ID NO: 5 or SEQ ID NO: 9.
The fourth aspect of the present invention provides a vector, which contains the DNA molecule of the present invention as mentioned above.
The fifth aspect of the present invention provides a host cell, which contains the vector of the present invention, or has the DNA molecule of the present invention integrated in its genome.
In a preferred embodiment, the host cell is a prokaryotic cell, preferably a bacterial cell; a lower eukaryotic cell, preferably a yeast cell; or a higher eukaryotic cell, preferably a mammalian cell.
The sixth aspect of the present invention provides an immunoconjugate, which contains: (a) the monoclonal antibody of the present invention; and (b) a conjugate moiety selected from the group consisting of drugs, toxins, cytokines, radionuclides, and enzymes.
The seventh aspect of the present invention provides a use of the anti-osteopontin monoclonal antibody or immunoconjugate of the present application in the manufacture of a medicament for treating autoimmune diseases.
In another preferred embodiment, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, autoimmune hepatitis, ankylosing spondylitis, Crohn's disease, rheumatoid spondylitis, osteoarthritis, gouty arthritis, autoimmune diabetes, autoimmune uveitis, nephrotic syndrome, ulcerative colitis, psoriatic arthritis, undifferentiated spondyloarthropathy, chronic liver failure, and other rheumatoid arthritis-related conditions including weight gain, joint distortion, joint swelling, joint deformity, joint bending stiffness, severe movement disorders, and combinations thereof.
The eighth aspect of the present invention provides a pharmaceutical composition comprising the anti-osteopontin monoclonal antibody or immunoconjugate of the invention and the pharmaceutically acceptable carriers.
In a preferred embodiment, the pharmaceutical composition contains 0.00001-99.9%, preferably 0.0001-90 wt %, more preferably 0.001-75 wt %, even more preferably 0.01-50 wt % of the monoclonal antibody or immunoconjugate. In another preferred embodiment, the pharmaceutical composition further comprises other active agents selected from the group consisting of anti-inflammatory cytokines including IL-4, IL-6, IL-10 and/or IL-4 antagonist; ENBREL®; phosphodiesterase IV inhibitors; anti-inflammatory inhibitors and cytokine inhibitors such as leflunomide; non-steroidal inflammatory drugs such as naproxen, cortisol, or cyclosporine.
The ninth aspect of the present invention provides a kit for detection of osteopontin, which contains the anti-osteopontin monoclonal antibody or immunoconjugate of the invention.
The tenth aspect of the present invention provides a method for the detection of the presence or the content of osteopontin in a biological sample, which includes the following steps: (i) contacting the sample with the anti-osteopontin monoclonal antibody or immunoconjugate of the invention; (ii) detecting the formation of antigen—antibody complex, wherein the formation of antigen—antibody complex suggests the presence of osteopontin in the sample, or the quantitative determined content of the antigen—antibody complex reflects the content of osteopontin in the sample.
In a preferred embodiment, the sample can be optionally pre-treated, preferably pretreated by extraction, purification, or enrichment.
Other aspects of the present invention will be obvious to one skilled in the art from the disclosure herein.
The inventors obtained the monoclonal antibody against specific functional epitope of osteopontin after long and thorough studies, further produced chimeric monoclonal antibody and humanized antibody, and determined the encoding sequences thereof. By studying, the inventors also demonstrated that the monoclonal antibody of the present invention can relieve or alleviate the symptoms of for example, arthritis, autoimmune hepatitis, Crohn's disease and other autoimmune diseases by inhibiting osteopontin, protect related organs, and thus can be used for the treatment of autoimmune diseases. On the basis of these findings, the present invention was accomplished.
In particular, the applicant of the present invention cloned human and mouse OPN genes using molecular biology technology, expressed human and mouse OPN proteins in eukaryotic cells, and prepared anti-human OPN monoclonal antibody (for example, 23C3 of the present invention) by cell fusion—hybridoma method, as well as cloned and sequenced the obtained monoclonal antibody gene.
The invention further disclose a method for the preparation of a humanized anti-osteopontin antibody, including the steps of computer-aided designing of humanized antibody h23C3 amino acid sequence; whole gene synthesizing h23C3 heavy chain and VL genes and splicing the same with human antibody heavy and light chain constant regions by gene recombination; cloning the obtained recombinant into an eukaryotic expression vector to construct humanized antibody light and heavy chain expression vectors respectively; co-transfecting a host cell (eg. CHO) with said light and heavy chain expression vectors by liposome transfection method; screening and culturing the vectors; and obtaining the antibody by purification. Chimeric monoclonal antibody c23C3 can also be obtained according to this method.
The applicant of the present invention has also established a collagen-induced arthritis DBA/1J mouse model, ConA-induced autoimmune hepatitis model, and DSS-induced Crohn's disease mouse model. Experiments were carried out in the three autoimmune disease models with the above monoclonal antibodies to determine the protective effects.
The experimental results of arthritis model showed that, monoclonal antibody of the present invention could significantly reduce the clinical scores of arthritis, delay onset time, inhibit the function of osteoclasts, reduce the bone destruction and absorption caused by arthritis, as well as protect bone resorption, effectively block lymphocyte migration, and so as to reduce or alleviate arthritis symptoms.
The experimental results of autoimmune hepatitis model showed that the monoclonal antibodies could neutralize OPN protein in liver tissue, regulate OPN mRNA level, reduce the level of pro-inflammatory cytokines, down-regulate NF-κB level in Th1-type immune response, so as to achieve the purpose of protecting liver and reducing mortality.
The result of experiments using Crohn's disease model showed that the monoclonal antibodies could neutralize OPN protein in liver tissue, regulate OPN mRNA level, decrease MPO activity, reduce lymphocyte and neutrophil infiltration, reduce the level of pro-inflammatory cytokines, thereby' reducing inflammation and ulceration, protecting intestinal and/or colon tissue, alleviating the symptoms of Crohn's disease, so as to achieve the prevention and/or therapeutic purposes.
The applicant of the present invention has also identified the OPN functional epitope interacting with 23C3 mAb by phage display technology, wherein the functional epitope is WLXPDP, and X can be any amino acids, more preferably X in the functional epitope is N, M or L. Therefore, the target of OPN molecular was further disclosed. More specifically, the applicant of the present invention has inferred different OPN functional epitopes by monoclonal antibody epitope panning, phage ELISA and Western blot, sequencing and sequence analysis, and further synthesized a series of short peptides by analyzing phage and antibody binding ability and choosing the clone with the strongest binding ability. The functional epitopes were identified by short peptide-specific antibody binding assay.
Monoclonal Antibody of the Invention and the Preparation Thereof.
As used herein, the term “monoclonal antibody (mAb)” refers to antibodies from a substantially homogeneous population, that is, the individual antibodies in the population are substantially the same, except for some naturally occurring mutations. A monoclonal antibody is highly specific to a unique antigenic site. Moreover, unlike conventional polyclonal antibody formulations (which are usually different antibodies specific to different epitopes), each monoclonal antibody is specific to a unique epitope on an antigen. In addition to the specificity, the advantages of monoclonal antibodies also include that, they can be synthesized by a hybridoma culture and will not be contaminated by other immunoglobulins. The modifier “monoclonal” indicates the characteristics of the antibody, which is from a substantially homogeneous antibody population, and should not be interpreted as being limited to antibodies produced by any special method.
As used herein, the term “antibody” or “immunoglobulin” refers to a heterotetramerized glycoprotein of about 150,000 Dalton and having the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain through a covalent disulfide bond, and different immunoglobulin isotypes have different numbers of disulfide bonds between the two heavy chains. Each heavy chain and each light chain also has regularly spaced interchain disulfide bonds. One end of each heavy chain is a variable region (VH), followed by multiple constant regions. Each light chain has a variable region (VL) at one end, and a constant region at the other end. The light chain constant region is located opposite to the first heavy chain constant region, and the light chain variable region is opposite to the heavy chain variable region. Specific amino acid residues form an interface between the light chain and the heavy chain variable regions.
As used herein, the term “variable” refers to some parts of the antibody variable regions, which have difference sequences and contribute to the binding specificity between specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable regions of antibody. It is concentrated in three fragments in the light chain and heavy chain variable regions, which are known as complementarily determining region (CDR) or hypervariable region. The relatively conservative part of the variable regions is known as the framework region (FR). Each of the naturally occurred heavy chain and light chain variable regions independently contains four FRs, which are generally in folding configuration, connected with three CDRs forming connection loops, and in some cases can form part of the β-folding structure. The CDRs in each chain are closely positioned through FR and form the antigen binding site of the antibody together with the CDRs of another chain (see Kabat, etc., NIH Publ. No, 91-3242, Volume I, p 647-669, (1991)). The constant regions are not directly involved in the binding of antibody and antigen, but show different effects, such as involved in antibody-dependent cytotoxicity of an antibody.
The “light chains” of a vertebrate antibody (immuneglobulin) may be classified as one of two significantly different classes (known as K and X) based on the amino acid sequences of its constant region. In accordance with the amino acid sequences of CH, immunoglobulins can be divided into different types. There are five types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subtypes (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant region corresponding to different types of immunoglobulin, are known as α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known in the art.
For example, in the present invention, the amino acid sequence of the VH in the monoclonal antibody is preferably as set forth in SEQ ID NO: 4 or SEQ ID NO: 8, the amino acid sequence of the VL is preferably as set forth in SEQ ID NO: 6 or SEQ ID NO: 10, while the constant region can be from mouse or human, such as IgG from mouse.
The monoclonal antibody obtained by the present molecular biology techniques also comprise the chimeric monoclonal antibody c23C3 and humanized antibody h23C3 which specific bind to the above osteopontin functional epitope. In c23C3, the amino acid sequence of the VH is as set forth in SEQ ID NO: 4, the amino acid sequence of the VL is as set forth in SEQ ID NO: 6, and the constant region is from human antibody. In h23C3, the amino acid sequence of the VII is as set forth in SEQ ID NO: 8, the amino acid sequence of the VL is as set forth in SEQ ID NO: 10, and the constant region is from human antibody.
In one embodiment of the present invention, the CDR1-3 amino acid sequences of the VH are independently selected from the group consisting of IYAMD, RIRSQSNNYTTYYADSVKD and QMGDY (see H23C3VHb in
The monoclonal antibodies can be prepared using a variety of well known methods in the art. For example, the monoclonal antibodies can be prepared by hybridoma method (first proposed by Kohler et al., Nature, 256:495 (1975)), or recombinant DNA method (U.S. Pat. No. 4,816,567). The monoclonal antibodies can also be isolated from phage antibody library according to the techniques as disclosed for example, in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).
The monoclonal antibody of the present invention can also be a chimeric antibody or a humanized antibody, such as the chimeric monoclonal antibody c23C3 and the humanized antibody h23C3 obtained by known molecular biology techniques. In c23C3, the amino acid sequence of the VH is as set forth in SEQ ID NO: 4, the amino acid sequence of VL is as set forth in SEQ ID NO: 6, and the constant region is from human antibody. In h23C3, the amino acid sequence of the VH is as set forth in SEQ ID NO: 8, the amino acid sequence of the VL is as set forth in SEQ ID NO: 10, and the constant region is from human antibody.
The invention also includes the monoclonal antibody having corresponding amino acid sequence of the anti-hOPN monoclonal antibody, the monoclonal antibody having the variable region chain(s) of the anti-hOPN monoclonal antibody, and other proteins or protein conjugates as well as products of fusion expression having these chains. Specifically, the present invention includes any proteins or protein conjugates as well as any product of fusion expression (i.e. immunoconjugate and fusion expression products) having hypervariable region (complementary determining region, CDR) in the light chain and the heavy chain thereof, as long as the hypervariable regions in the light chain and the heavy chain are identical or have at least 90% homology, preferably at least 95% homology, to the variable regions in the light chain and the heavy chain of the present invention. As known to the skilled in the art, the immunoconjugate and the products of fusion expression include the conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the anti-hOPN monoclonal antibody or the fragments thereof. The invention also includes cell surface markers or antigens binding to the anti-hOPN monoclonal antibody or the fragments thereof.
The invention includes not only the complete monoclonal antibody, but also immuno-active antibody fragments, such as Fab or (Fab)2 fragments; a heavy chain of the antibody; and a light chain of the antibody.
Molecules Encoding Anti-hOPN Monoclonal Antibody or its Fragments, Expression Vectors and Host Cells Containing the Molecules
The present invention also provides DNA molecules that encode the anti-hOPN monoclonal antibody or the fragments thereof. The sequences of these DNA molecules can be obtained by conventional techniques, such as PCR amplification or genome library screen. In addition, a sequence encoding the light chain can be fused with a seqence encoding the heavy chain to form a single-chain antibody.
For example, the DNA molecule of the present invention may contain VH encoding nucleotide sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 7; and VL encoding nucleotide sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 9. For example, the encoding nucleotide sequence of c23C3 VH is shown in SEQ ID NO: 3, and the encoding nucleotide sequence of c23C3 VL is shown in SEQ ID NO: 5; the encoding nucleotide sequence of h23C3 VH is shown in SEQ ID NO: 7, and the encoding nucleotide sequence of h23C3 VL is shown in SEQ ID NO: 9.
Once the relevant sequence is obtained, we can use recombination methods to get a large amount of relevant sequences. Usually, the obtained relevant sequence is cloned into vectors to be transferred to host cells, and then isolated from the proliferated host cell by conventional method.
In addition, the relevant sequence can be synthesized by artificial synthesis methods, especially for short ones. Typically, multiple short fragments are first synthesized, which are then connected into a long sequence.
At present, the DNA sequence encoding the anti-hOPN monoclonal antibody (or the fragments or derivatives thereof) of the present invention can be obtained completely through chemical synthesis. Then the DNA sequence can be introduced into a variety of DNA molecules (or vectors) and cells known in the art. In addition, mutations can be introduced into the sequence of proteins according to the present invention by chemical synthesis.
The present invention also involves vectors containing the above DNA sequence and a suitable promoter or other regulatory sequence(s). These vectors can be used to transform suitable host cells to express the protein. The host cell can be prokaryotic cells, such as bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
The invention also provides a hybridoma cell line that can produce the anti-hOPN monoclonal antibodies of the present invention. Preferably, the present invention provides a hybridoma cell line that can produce the anti-hOPN monoclonal antibodies having high efficiency.
Upon obtaining the hybridoma which can produce the anti-hOPN monoclonal antibody of the present invention, the skilled in the art can easily determine the structure of the antibody of the invention (such as the VH and the VL of the antibody), and then prepare the monoclonal antibody of the invention (for example) in the ways as described below.
An expression vector which contains the nucleotide sequence of the monoclonal antibody of the invention and expression-regulatory sequences operably linked to said sequence is first constructed.
As used herein, the term “expression-regulatory sequence” usually refers to a sequence contributing to the regulation of the expression of nucleotide sequences. The expression-regulatory sequences include promoters and termination signals operably linked to target nucleotide sequences. Usually, expression-regulatory sequences also include the sequences which are need for the proper translation of the nucleotide sequences. The term “Operably linked” refers to certain parts of a linear DNA sequence which can affect the activity of other parts of the same linear DNA sequence. For example, if a promoter or enhancer increases the transcription of an encoding sequence, then it is operably linked to the encoding sequence.
The DNA sequences encoding the present monoclonal antibody can be produced by well-known techniques in the art. For example, the VH and VL encoding nucleotide sequences of the monoclonal antibody can be artificially synthesized or amplified by PCR based on the herein disclosed sequences. Then, various methods known in the art can be used to introduce these nucleotide sequences into appropriate expression vectors by selecting appropriate enzyme cleavage sites so as to make them separately located ahead of the CH encoding sequence and CL encoding sequence carried by the expression vector, and in the same reading frame of the CH and CL encoding sequences. The expression vectors used in the present invention are well-known and commercially available, such as pPICZa and pPIC9K. The expression vectors containing the above nucleotide sequences of the present invention can be, for example, pcDNA3.1/ZEO (+) and pcDNA3.1 (+).
The above obtained expression vector is used to transform suitable host cells. The term “host cell” generally includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include E. coli, Bacillus subtilis and so on. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. In the invention, mammalian cell is preferred. Mammalian cell line is usually used as host cell to express eukaryotic cell-derived peptides. The proliferation of mammalian cells in culture is in well known in the art. See “Tissue Culture”, Academic Press, Kruse and Patterson Ed. (1973), the disclosures of which are hereby incorporated by reference. Preferred mammalian cells are commercially available immortalized cell lines. These immortalized cell lines include but not limited to the Chinese hamster ovary (CHO) cells, the Vero cell, the Hela cells, the baby hamster kidney (BHK) cells, the monkey kidney cells (COS), the human hepatocellular carcinoma cells (such as Hep G2) and many other cell lines. They provide post-translation modification to the proteins, including correct folding, correct formation of disulfide bond and correct glycosylation sites.
There are many methods for transforming host cell with expression vector, and the transformation procedure used depends on the host to be transformed. The methods for introducing heterologous polynucleotide into mammalian cells are well known in the art, which includes dextran-mediated transfection, calcium phosphate precipitation, Polybrene (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide)-mediated transfection, protoplast fusion, electroporation, and liposome-mediated transfection, as well as direct DNA microinjection into the nucleus. In the present invention, the preferred method is electroporation, or liposome-mediated transfection. For example, the Invitrogen liposome transformation kit can be used to transfect host cells such as CHO cells and so on. In the present invention, the preferred host cell is COS or CHO cell.
The transformed host cell is then cultured under conditions suitable for the expression of the monoclonal antibody of the present invention. Then the humanized anti-hOPN monoclonal antibody of the invention can be obtained using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion-exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography and other well known conventional separation and purification methods.
Identification, Expression and Purification of Monoclonal Antibody
The resultant monoclonal antibody can be identified by conventional procedures. For example, the binding specificity of the monoclonal antibody can be determined by' immunoprecipitation or in vitro binding assay (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of the monoclonal antibody can be determined by, for example, the Scatchard analysis of Munson, et al., Anal. Biochem., 107:220 (1980).
The anti-hOPN monoclonal antibody of the present invention can be expressed in the cell, or expressed on the surface of the cell, or can be secreted to the outside. If necessary, the recombinant proteins can be separated and purified using a variety of separation methods based on their physical, chemical, and other features. These methods are well known in the art. Examples of these methods include, but not limited to, conventional renaturation treatment, treatment with the protein precipitating agent (salting-out method), centrifugation, osmosis, ultrasound treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and their combinations.
Pharmaceutical Composition
The present invention also provides a pharmaceutical composition for the treatment of autoimmune disease, which comprises pharmaceutically effective amount of the monoclonal antibody of the invention or immunoconjugate thereof, as well as pharmaceutically acceptable carriers.
As used herein, the term “pharmaceutically acceptable” refers to the property of a molecular or combination comprising the same that when administering to animals or human, the molecular itself and combinations thereof will not cause disadvantaged, allergic or other adverse reactions. As used herein, the term “pharmaceutically acceptable carrier” should be compatible with the active components of the present invention, that is, can be combined with the active components and usually will not significantly reduce the effect of the pharmaceutical composition. These carriers are well known in the art. A thorough discussion of the pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).
Such carriers include, but not limited to: saline, buffers, glucose, water, glycerin, ethanol, adjuvants, and combinations thereof. In addition, these carriers may also comprise auxiliary substances, such as wetting agents or emulsifying agent, pH buffering agents and so on.
The composition of the invention can be administered via conventional manners such as via oral, intravenous injection, intramuscular injection or subcutaneous injection, preferably via oral or intravenous administration is preferred.
Typically, the pharmaceutical composition of the present invention comprises 0.00001-99.9 wt %; preferably 0.0001-90 wt %, more preferably 0.001-75 wt %, even more preferably 0.01-50 wt % of the monoclonal antibody of the present invention or immunoconjugate thereof, based on the total weight of the composition. The rest of the composition is consisted of pharmaceutically acceptable carrier(s) and other additive(s).
The pharmaceutical compositions of the invention can be formulated into a variety of dosage forms according to particular needs, and can be administered in an effective amount by a physician according to the factors, such as the type, age, body weight and general conditions of the patient, and routes of administration. The route of administration can be, for example, infusion and other therapeutical ways.
When applying the pharmaceutical composition, a safe and effective amount of anti-hOPN monoclonal antibody or immunoconjugate is administered to mammals, wherein the safe and effective amount is usually about 0.1 μg-5 mg/kg body weight, and in most cases, not more than about 5 mg/kg body weight, preferably in the range of about 1-10 μg/kg body weight to about 1 mg/kg body weight. Of course, when determining the particular dosage, the factors such as administration routes and the health status of the patient should also be taken into consideration, which are within the skills of the practiced physicians.
The monoclonal antibody or immunoconjugate of the invention can be used for the treatment of autoimmune diseases, in which osteopontin acts as an important pathogenic factor, by targeting osteopontin. Such autoimmune diseases including, but not limited to: rheumatoid arthritis, multiple sclerosis, autoimmune hepatitis, ankylosing spondylitis, Crohn's disease, rheumatoid spondylitis, osteoarthritis, gouty arthritis, autoimmune diabetes, autoimmune uveitis, nephrotic syndrome, ulcerative colitis, psoriatic arthritis, undifferentiated spondyloarthropathy, chronic liver failure, and other conditions relating to rheumatoid arthritis including weight gain, joint distortion, joint swelling, joint deformity, joint bending stiffness, severe movement disorders, and combinations thereof.
The pharmaceutical composition of the invention can also comprise the substances having therapeutic or ameliorative activity on autoimmune disease, or can be used in combination with other active agents, to obtain improved therapeutic effects. The other substances having therapeutic or ameliorative activity on autoimmune disease include, but not limited to: anti-inflammatory cytokines including IL-4, IL-6, IL-10 and/or IL-4 antagonist, ENBREL®, phosphodiesterase IV inhibitors, anti-inflammatory inhibitors and cytokine inhibitors such as leflunomide, non-steroidal inflammatory drugs such as naproxen, cortisol, or cyclosporine.
When two or more drugs are administrated in combination, the effect thereof is usually better than the effect produced by separately administrating the individual drugs. Preferably, the drugs or other agents administrated in combination with the monoclonal antibody of the present invention do not interfere with the therapeutic activity of said monoclonal antibody.
Kit for the Detection of hOPN
The present invention also provides a kit for the detection of hOPN, which contains anti-hOPN monoclonal antibody as well as the active fragment or immunoconjugate thereof. The kit of the present invention can be used to detect the presence or content of osteopontin in the biological sample. The method for detection comprises the following steps: (i) contacting the biological sample with the anti-osteopontin monoclonal antibody or immunoconjugate of the invention; (ii) detecting the formation of antigen—antibody complex, wherein the formation of antigen—antibody complex suggests the presence of osteopontin in the sample, or quantitatively determining the content of the formed antigen—antibody complex so as to reflect the content of osteopontin in the sample. The sample can be pre-treated or not, for example by extraction, purification; or enrichment.
The kit comprises container and the monoclonal antibody of the present invention contained in the container, or plate(s) used for detection containing said monoclonal antibody, and instruction. The kit can also comprise other reagent(s) needed for detection, such as buffer, indicators and so on. The skilled in the art can adjust the contents in the kit according to particular need.
The invention will be further described in the following examples and experiments, which do not limit the scope of the invention. Examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, to insert protein encoding genes into such vectors and plasmids, to introduce plasmids into host cells, or the classic cell fusion method and monoclonal antibody screening and purification methods. Such methods are well known in the art and have been described in many publications, including Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) “Molecular Cloning: A Laboratory Manual”, Cold spring Harbor Laboratory Press. All percentages and parts are based on weight, unless otherwise indicated. All the materials and adjuvants in the present examples or experiments are commercially available, unless specially indicated.
The following primers were synthesized based on human OPN information and sequence provided in GENEBANK: OPN sense primer (primer 1): GGG AAGCTT ACCATGAGAATTGCAGTGATTTG (Hind III) (SEQ ID NO: 16); and OPN antisense primer (primer 2): GCC GGTACC ATTGACCTCAGAAGATGCAC (Kpn I) (SEQ ID NO: 17). Human liver cancer cell line LM3 (purchased from Shanghai Zhongshan Hospital) was cultured and proliferated before extracting RNA by RT-PCR (PROMEGA) using TRISOL kit (INVITROGEN). The PCR condition was: 94° C. for 5 mins; 30 cycles of 94° C. for 45 sec, 58° C. for 30 sec, and 72° C. for 45 sec; and 72° C. for 10 mins. The resultant DNA fragment having 963 bp was recovered by gel extraction kit (Shanghai Sangon) and cleavaged with restriction enzymes Hind III and Kpn I. The cleavaged fragment was recovered by gel electrophoresis and was linked to the Hind III and Kpn I double-digested plasmid vector. The vector was then transformed into E. coli DH10B, and the positive clones with inserts were screened. The DNA sequence was confirmed by sequencing. The nucleotide sequence of OPN gene was as set forth in SEQ ID NO: 1.
The following primers were synthesized based on mouse OPN information and sequence provided in GENEBANK: mouse OPN sense primer (primer 3): ATAAGCTTGGATGACGACGACAAGATGAGAATTGCAGTGATT (Hind III) (SEQ ID NO: 18) and mouse OPN antisense primer (primer 4): ATCTCGAGTTAATTGACCTCAGAAGA (Kpn I) (SEQ ID NO: 19). The mouse spleen T lymphocyte was separated and activated with ConA for 30 hours. Then, RNA was extracted by RT-PCR (PROMEGA) using TRISOL kit (INVITROGEN). The PCR condition was: 94° C. for 5 mins; 30 cycles of 94° C. for 45 sec, 55° C. for 30 sec, 72° C. for 45 sec; and 72° C. for 10 mins. The target DNA fragment was obtained, revocered by gel extraction kit (Shanghai Sangon), and then cleavaged with restriction enzymes Hind III and Kpn I. The cleavaged fragment was recovered by gel electrophoresis and was linked to the Hind III and Kpn I double-digested plasmid vector. The vector was transformed into E. coli DH10B, and the positive clones with inserts were screened. The DNA sequence was confirmed by sequencing. The nucleotide sequence of mouse OPN gene was as set forth in SEQ ID NO: 2.
The correct human and mouse OPN gene fragments obtained in example 1 and example 2 were recovered after corresponding restriction enzyme digestion, and introduced into pPICZa plasmid. Saccharomyces Pichia cell was transfected with the plasmid, and individual clone was picked out and induced to express human and mouse OPN proteins, The expression supernatant of yeast cell was collected and purified with anion exchange and molecular sieve. The result of SDS-PAGE proved the obtained proteins to be pure human and mouse OPN proteins. Then the purified proteins were subjected to SDS-PAGE electrophoresis, and the result was shown in
One hundred of human OPN and an equal volume of Freund's adjuvant were emulsified, and the resultant was administrated to BALB/C mouse (i.p.). The mice were boosted every two weeks with the same dose. After three immunizations, the mice with relatively high serum anti-OPN antibody titer were selected. The spleen lymphocytes of the selected mouse were separated and fused with NS-1 cells using classic PEG method. 96-well plate coated with 10 μg/ml OPN was used for repeated screening by ELISA method to obtain hybridoma cell line 23C3 which was capable of stably expressing anti-human OPN antibody. The monoclonal cell line 23C3 was amplified, and administrated to BALB/C mice in 5×106 cells/mouse (i.p.). Mouse ascites were collected after 10 days, and anti-human OPN monoclonal antibody was purified by affinity chromatography using protein A column. The result of western blot showed that mouse anti-human OPN monoclonal antibody 23C3 not only specifically bound with human OPN protein, but also cross acted with mouse OPN protein. The result was shown in
23C3 (5×106-1×107) hybridoma cell were collected, and total RNA were extracted using TRIzol (Invitrogen, Catalog No. 15596-026). The following primers were designed according to mouse antibody constant region sequences:
By using Invitrogen 5′RACE kit (Catalog No. 18374-058), the first-strand cDNA was synthesized using the primers HGSP1 and LGSP1 respectively; Poly C was added to 3′ terminal of the first-strand cDNA using TdT and dCTP; the PCR products of the VH and VL were obtained using HGSP2, HGSP3, LGSP2 and LGSP3 as 5 primer in nested-PCR; The PCR products were introduced into pGEM-T vector. Clones were picked out to extract plasmid, and the positive clones were identificated by using restriction enzymes and then subject to sequencing. The nucleotide and amino acid sequences of the VH in 23C3 were as set forth in SEQ ID NO: 3 and SEC) ID NO: 4 respectively, and the nucleotide and amino acid sequences of the VL in 23C3 were as set forth in SEQ ID NO: 5 and SEQ ID NO: 6 respectively.
Healthy human lymphocytes were isolated using lymphocyte separation medium (DingGuo Biotechnology Development Co.). Total RNA was then extracted using Trizol reagent (Invitrogen). Primers were designed according to the sequences reported in Cell, 1980, 22: 197-207 and Nucleic Acids Research, 1982, 10: 4071-4079, and antibody heavy chain and light chain constant region genes were amplified by RT-PCR. PCR products were purified and collected by agarose gel electrophoresis and cloned into pGEM-T vector, verified by sequencing to obtain correct clone. The nucleotide sequence and amino acid sequence of the heavy chain constant region (CH) were as set forth in SEQ ID NO: 12 and SEQ ID NO: 13, respectively. The nucleotide sequence and amino acid sequence of the light chain constant region (CL) were as set forth in SEQ ID NO: 14 and SEQ ID NO: 15. The correct clones of this example were known as pGEM-T/CH and pGEM-T/CL.
The chimeric antibody heavy chain gene was synthesized by overlap PCR using the VH genes 23C3VH of 23C3 and pGEM-T/CH vector as template. The reaction condition was: 95° C. for 15 mins; 30 cycles of 94° C. for 50 sec, 58° C. for 50 sec, and 72° C. for 50 sec; 72° C. for 10 mins. The chimeric heavy chain gene had a Hind III restriction enzyme site and a signal peptide gene sequence at 5 end, and a translation stop codon TAA and an EcoRI restriction enzyme site at 3′ end. The sequence of the signal peptide gene was ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAA TATCCAGAGGA (SEQ ID NO: 52). PCR amplified product was separated by agarose gel electrophoresis. The target band was recovered and cloned into pGEMT vector. Positive clone was screened and sequenced. The correct clones identified by sequencing were digested with Hind III and EcoR I. Chimeric antibody heavy chain fragment c23C3VHCH was purified and recovered via agarose gel electrophoresis, linked to Hind III and EcoR I double-digested plasmid pcDNA3.1 (+) (Invitrogen, USA), and constructed into chimeric heavy chain eukaryotic expression vector pcDNA3.1 (+) (c23C3VHCH).
The light chain gene of the chimeric antibody was synthesized by overlap PCR using the VL gene 23C3VL of 23C3 and pGEM-T/CL vector as template. The reaction condition was: 95° C. for 15 mins; 30 cycles of 94° C. for 50 sec, 58° C. for 50 sec, and 72′C for 50 sec; 72° C. for 10 mins. The obtained PCR product had a Hind III restriction enzyme site and a signal peptide gene sequence at 5′ end, and a translation stop codon TAA and an EcoR I restriction enzyme site at 3′ end. The signal peptide gene sequence was ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAA TATCCAGAGGA (SEQ ID NO: 52). The PCT products were recovered by agarose gel electrophoresis and cloned into pGEM-T vector (Promega). Positive clones were screened and sequenced. The clones with correct sequence were picked out and digested with Hind III and EcoR I. The light chain fragments c23C3VLCL of the chimeric antibody were purified and recovered by agarose gel electrophoresis, linked to Hind III and EcoR I double-digested plasmid pcDNA3.1/ZEO(+) (Invitrogen, USA), and constructed into chimeric light chain eukaryotic expression vector pcDNA3.1/ZEO(+) (c23C3VLCL).
The successfully constructed chimeric heavy chain and light chain expression vectors were used to co-transfect COS-1 cell (ATCC CRL 1650) by liposomal transfection method. The supernatant was collected for analysis after 72 hours. The content of chimeric antibody c23C3 in supernatant was determined by ELISA. ELISA plate was coated with goat anti-human IgG (Fc), and blocked with 2% BSA-PBS at 37° C. for 2 hours. The supernatant to be tested and the standard (Human myeloma IgG1, k) were added into the plate and incubated at 37° C. for 2 hours. HRP-goat anti-human κ was then added to perform binding reaction at 37° C. by incubated for 1 hour. TMB was added and incubated at 37° C. for 5 minutes before adding H2SO4 to terminate the reaction. OD450 was measured.
Insight II package (Accelrys) was used to simulate the three-dimensional structure for 23C3 murine monoclonal antibody variable region. First, the template proteins for the HL and VL proteins of 23C3 were searched in Protein Data Bank (PDB) using BLAST program, Antibody 1 cbv and 1F6L with highest homology were selected as the modeling templates for 23C3, and the three-dimensional structure of 23C3 was constructed using Insight II program (
BLAST program was used to search the most similar human origin templates for the VL and the VH of 23C3 respectively in Genebank database. The human origin antibody which has the highest homologous to the VH of 23C3 was human antibody AAT51715.1 (gb|AAT51715.1|), with a similarity of 72%, and the human origin antibody which has the highest homology to the VL of 23C3 was BAC01726.1 (db|BAC01726.1|), with a similarity of 72%. Therefore, AAT51715.1 and BAC01726.1 were used as the templates for humanizing 23C3 heavy chain and light chain.
The CDRs in the heavy chain and light chain of 23C3 were transplanted directly into human origin template AAT51715.1 and BAC01726.1 respectively to construct a CDR graft antibody, which had heavy chain h23C3Ha and light chain h23C3La. The sequences of h23C3Ha and variable region sequences were shown in
Humanized antibody light chain gene was synthesized by overlap PCR by using h23C3VLa gene and pGEM-T/CL vector as templates. The reaction condition was: 95° C. for 15 mins; 30 cycles of 94° C. for 50 sec, 58° C. for 50 sec, 72° C. for 50 sec; 72° C. for 10 mins. The obtained PCR product, h23C3VlaCL, had a Hind III restriction enzyme site and a signal peptide gene sequence at 5′ end, and a translation stop codon TAA and an EcoRI restriction enzyme site at 3′ end. The sequence of signal peptide gene was ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAA TATCCAGAGGA (SEQ ID NO: 52), The clones with correct sequence were picked out and digested with Hind III and EcoR I. The light chain fragment the humanized antibody agarose gel electrophoresis, and linked to Hind III and EcoR I double-digested plasmid pcDNA3.1ZEO(+) (Invitrogen, USA) to construct a humanized light chain eukaryotic expression vector pcDNA3.1/ZEO(+) (h23C3VLaCL).
The successfully constructed heavy chain and light chain expression vectors were used to co-transfect COS-1 cell (ATCC CRL 1650) by liposome transfection method. The supernatant was collected for analysis after 72 hours. The content of humanized antibody (H23C3Ha/H23C3La) in the supernatant was determined by ELISA. ELISA plate were coated with goat anti-human IgG (Fc), and blocked with 2% BSA-PBS at 37° C. for 2 hours. The supernatant to be tested and the standard (Human myeloma IgG 1, κ) were added into the wells, and incubated at 37° C. for 2 hours. HRP-goat anti-human κ was then added for the binding reaction (7° C. incubated for 1 hour). TMB was then added and incubated at 37° C. for 5 minutes before adding H2SO4 to terminate the reaction. OD450 was measured.
ELISA plate was coated with 2 ug/ml OPN. The binding activity of transfected humanized antibody to OPN was detected by ELISA. The result showed that the activity of humanized antibody composed of H23C3Ha and H23C3La (H23C3a/H23C3La) was almost lost when comparing with C23C3 chimeric antibody. Therefore, in order to obtain humanized antibody with high-affinity, murine FR residues which might affect h23C3 antibody binding activity should be subject to analysis and reverse mutation. By analyzing the three-dimensional structure of modeled 23C3 monoclonal antibody variable region (
The binding activity of H23C3 to OPN was similar to that of c23C3 chimeric antibody, and the humanized antibody (h23C3Hb/h23C3Lb) was named as h23C3.
The successfully constructed c23C3 or h23C3 heavy chain and light chain plasmids were used to co-transfect CHO-K1 cell (ATCC CRL-9618) by liposome transfection method. After 24 hours, the media was changed to a selective media comprising 600 μg/ml G418 and 250 μg/ml Zeocin for the selection of resistant clones. The supernatant of the cell culture was collected and was screened for high expression clones by ELISA assay. The selected high expression clones were expanded in serum-free culture medium. Chimeric antibody c23C3 or humanized antibody h23C3 were isolated and purified by protein A affinity column (GE), and were finally quantified by UV absorption.
SDS-PAGE (10%) electrophoresis was carried out (5 ug hOPN/lane). hOPN was transferred to nitrocellulose membrane by 360 mA constant current for 1 hour. The membrane was blocked with 10% skimmed milk at 4′C overnight or incubated at room temperature for two hours; washed with 1×TBST (Tween-20, 0.1%) for 3 times (10 minutes per time); reacted with bug/ml c23C3 and h23C3 respectively at room temperature for 1 hour, washed with 1×TBST (Tween-20, 0.1%) for five times, 10 minutes per time; and then reacted with 1:1000 diluted HRP labeled mouse anti-human IgG (Beijing Zhongshan Company) at room temperature for 1 hour, treated with ECL kit (Tiangen Company) for 1-2 minutes, pressed and exposed with medical blue-sensitive X-ray film. The result was shown in
Experiment I. Effect of 23C3 Monoclonal Antibody on CIA Arthritis Model Mice
Experiment I-1. Effect of 23C3 mAb Treatment on Clinical Scores for Arthritis, Onset Time and Incidence Rate in CIA Arthritis Model Mice
One hundred μg of chicken type II collagen (Condrex) and equal volume of Freund's complete adjuvant were emulsified, injected subcutaneously into DBA/1J mice (32 mice, male, 8-10 weeks old) at the tail for primary immunization. The mice were boosted after 21 days, i.e. 100 μg of chicken type II collagen and equal volume of Freund's complete adjuvant were emulsified and injected subcutaneously at the tail for boosting. The mice which had been immunized for two times, were divided into four groups and administrated with control antibody (mouse IgG, 200 μg/mouse, Sigma), and 23C3 monoclonal antibody (200 μg/mouse, 100 μg/mouse, and 50 μg/mouse) respectively, All the treatment groups were administered by intraperitoneal injection, and administered every other day for six times from the day of the second immunization. The clinical scores for arthritis, onset time and incidence rate were observed from the day of the second immunization and for 30 days, The arthritis severity in mice was scored with reference to the literature (“A Role for the Lymphotoxin/LIGHT Axis in the Pathogenesis of Murine Collagen-Induced Arthritis”. Roy A. Fava, Evangelia Notidis, Jane Hunt, Veronika Szanya, Nora Ratcliffe, A pinya Ngarn-ek, Antonin R. de Fougerolles, Andrew Sprague, and Jeffrey L. Browning. In particular, each leg was scored as 0-4 according to the severity of inflammation, wherein 0—no swelling; 1—swelling in one finger or toe, or slight edema; 2—swelling in more than one fingers or toes, or moderate edema; 3—swelling in most of the fingers or toes, or severe edema; 4—swelling in all of the fingers or toes, or deformity. The clinical scores for arthritis in mice was the sum of the inflammation scores of fore limbs and hind limbs.
The experimental results were shown in
Experiment I-2. The Protective Effect of 23C3 mAb Treatment on Joint Bone Resorption in CIA Mouse
The CIA mice in the group receiving 200 μg 23C3 monoclonal antibody treatment and the control group (as indicated in Experiment I-1) were subject to X-ray detection at the 30th day after the second immunization to observe the bone destruction of the sick joints. The result was shown in
Experiment I-3. The In Vitro Inhibitory Effect of 23C3 mAb on Lymphocyte Migration
The spleens of the CIA mice (experiment I-1) were aseptically separated, passed 100 mesh steel screen, washed with PBS for two times, and treated with lymphocyte separating medium to separate spleen lymphocytes, and the resultant was centrifuged at 2000 rpm for 20 minutes. The lymphocyte layer was pipetted and washed with PBS for 2 times. The lymphocytes were counted, and adjusted to a concentration of 5×106 cells/ml. 24-well tissue culture plate having a 5 μm filter membrane at the upper layer was pre-coated with 10 ug OPN at the lower layer. 5×105 lymphocytes as well as the following antibodies were added into the upper layer of each well: 23C3 (anti-OPN monoclonal antibody), Her-2 antibody (irrelevant antibody control), and anti-OPN polyclonal antibody (CHEMICON). The plate was incubated at 37° C., 5% CO2 for 2 hours, and then washed with PBS twice. The number of cells in the lower layer was calculated by taking the average of the counted numbers in five high power fields and then multiplied by 50.
The experimental result was shown in
Experiment I-4. OPN Level Changes in the Plasma and Joint Tissue of CIA Mice in Different Treatment Groups
The DBA/1J mice plasma of different time points (0, 7, 14 or 28 days) in different treatment groups (experiment I-1) were collected, and detected for the OPN protein concentration using mouse OPN ELISA kit (R & D Company). Specifically, venous blood from mice outer canthus was collected, and centrifuged immediately at 4000 rpm for 10 minutes to separate the plasma. The 1:200 dilution was added into the 96 well plate of ELISA detection kit. The plate was incubated at room temperature for 1 hour, and washed with PBST for five times. Biotin-labeled anti-OPN primary antibody was then added into the plate before incubating the plate at room temperature for 1 hour. After the plate was washed for five times, avidin labeled secondary antibody was added into the plate. The plate was incubated for 1 hour at room temperature, washed for six times, and developed with TMB substrate. OD450 absorbance was detected. The plasma OPN concentration was calculated according to the standard curve.
The result was shown in
At day 14, the joint tissues of CIA mice in different treatment groups of experiment I-1 were collected. The skin and muscle tissues were removed. Total RNA was extracted with RNeasy Mini Kit (QIAGEN) and subjected to reverse transcription with Sensiscript RT Kit (QIAGEN) for cDNA. OPN primers were designed according to the requirements of real-time PCR (as shown in Table 1), OPN mRNA level was detected by real-time PCR using SYBR Green Master Mix (Applied Biosystems). The PCR procedure was: 50° C. for 2 mins; 95° C. for 10 mins; 40 s of 95° C. for 15 sec and 60° C. for 60 sec. The internal standard was GAPDH gene. The experimental data was analyzed using ABI PRISM 7900 sequencing system (Applied Biosystems).
The experimental results were shown in
Experiment I-5. The mRNA Levels of OPN Receptors in the Joint Tissue of CIA Mice in Different Treatment Groups
At day 14, the joint tissues of CIA mice in different treatment groups of experiment I-1 were collected. The skin and muscle tissues were removed. Total RNA was extracted with RNeasy Mini Kit (QIAGEN) and subjected to reverse transcription with Sensiscript RT Kit (QIAGEN) for cDNA. Primers for OPN receptors were designed according to the requirements of real-time PCR (as shown in Table 1). OPN receptor mRNA level was detected by real-time PCR using SYBR Green Master Mix (Applied Biosystems). The PCR procedure was the same as that used in experiment 4. The experimental data was analyzed using ABI PRISM 7900 sequencing system (Applied Biosystems).
The experimental results were shown in
Experiment I-6 Cytokines mRNA Levels in CIA Mice Joint Issue of Different Treatment Groups
At day 14, the joint tissues of CIA mice in different treatment groups of experiment I-1 were collected. The skin and muscle tissues were removed. Total RNA was extracted with RNeasy Mini Kit (QIAGEN) and subjected to reverse transcription with Sensiscript RT Kit (QIAGEN) for cDNA. Cytokine primers were designed according to the requirements of real-time PCR (as shown in Table 1). The tissue mRNA levels of cytokines were detected by real-time PCR using SYBR Green Master Mix (Applied Biosystems). The PCR procedure was the same as that used in experiment 4. The experimental data was analyzed using ABI PRISM 7900 sequencing system (Applied Biosystems).
The experimental result was shown in
Experiment I-7. In Vitro Lymphocyte Recall Response Changes in Different Treatment Groups
The spleens and lymph nodes of CIA mice in different treatment groups of Experiment I-1 were aseptically separated, passed 100 mesh steel screen, washed with PBS for two times, and treated with lymphocyte separating medium to separate spleen lymphocyte. The resultant was centrifuged at 2000 rpm for 20 minutes. The lymphocyte layer was pipetted and washed with PBS for 2 times. The cells were counted, and the lymphocyte concentration was adjusted to 5×106 cells/ml. 5×105 lymphocytes were added to each well and were stimulated with different concentrations of chicken type II collagen. The plate was incubated at 37° C., 5% CO2 for 72 hours. The supernatant collected at hour 56 was used for the detection of cytokine IFN-γ and IL-10. At the same time, 100 ul culture medium containing 3H (0.5 μCi) was added into each well, and the plate was cultured for another 16 hours, Cells were then collected with cell collector and detected with γ liquid scintillation counter.
The experimental result was shown in
Experiment I-8. The Serum Autoantibodies Levels in CIA Mice of Different Treatment Groups
The DBA/1J mice serums of different time points (0, 7, 14 or 28 days) from different treatment groups (experiment I-1) were collected, and detected for the serum titer of anti-type II collagen autoantibodies IgG1, IgG2a and total Ig using ELISA method. Specifically, venous blood from mice outer canthus was collected, allowed to stand for 2 hours at room temperature, and centrifuged at 4000 rpm for 10 minutes to separate the serum. 96-well plate was coated with 100 ng/well chicken type II collagen, 4° C. overnight, washed with PBS, blocked with 10% skimmed milk for 2 hours, and then washed with PBST for three times. The serum samples were serial diluted beginning with 1:2000, and diluted samples were added to the 96-well plate, incubated at room temperature for 1 hour. After the plate was washed with PBST for five times, HRP labeled rabbit anti-mouse IgG1, IgG2a and total Ig antibody were added into the plate. The plate was incubated for 2 hours at 37° C., washed for five times, and developed with substrate o-phenylenediamine. OD450 absorbance was detected. The autoantibody titer of autoantibody in serum sample was calculated according to the standard curve.
The result was shown in
Experiment I-9. RANKL, RANK and OPG mRNA Levels in the Joint Tissue of CIA Mice in Different Treatment Groups
At day 28, the joint tissues of mice in different treatment groups (experiment I-1) were collected. The skin and muscle tissues were removed, Total RNA was extracted with RNeasy Mini Kit (QIAGEN) and subjected to reverse transcription with Sensiscript RT Kit (QIAGEN) for cDNA. Primers for RANKL, RANK and OPG were designed according to the requirements of real-time PCR (as shown in Table 1). The tissue mRNA levels of RANKL, RANK and OPG were detected by real-time PCR using SYBR Green Master Mix (Applied Biosystems). The PCR procedure was the same as that used for human and mouse eukaryotic expression and purification step as indicated in the examples. The experimental data was analyzed using ABI PRISM 7900 sequencing system (Applied Biosystems).
The experimental result was shown in
Experiment I-10. The Urine D-pyr Level in CIA Mice of Different Treatment Groups
The urine of DBA/1J mice (experiment I-1) in different treatment groups was collected 30 days after the secondary immunization, and detected for D-pyr level using mice D-pyr ELISA kit (R & D). The result was shown in
Experiment II. Effect of 23C3 Monoclonal Antibody on Hepatitis Model Mice
Experiment II-1. The effect of 23C3 Monoclonal Antibody on the Mortality of Hepatitis Model Mice
C57BL/6 (male, 8-10 weeks old) mice (purchased from Experimental Animal Center of the Second Military Medical University) were administrated with irrelevant control antibody Ig (Sigma) or 23C3 monoclonal antibody (200 μg/mouse, and 10 mice for each group), via intraperitoneal injection. After 2 hours, 200 μl ConA (30 mg/ml) was injected to the tail veins of the mice to induce acute severe hepatitis. The effect of 23C3 monoclonal antibody on mortality in hepatitis mice was continually observed for 24 hours.
The experimental result was shown in
Experiment II-2. The Liver Protective Effect of 23C3 Monoclonal Antibody on Hepatitis Model Mice
C57BL/16 (male, 8-10 weeks old) mice were administrated with irrelevant control antibody Ig (Sigma) or 23C3 monoclonal antibody (200 μg/mouse, and 10 mice for each group), via intraperitoneal injection. After 2 hours, 200 μl ConA (30 mg/ml) was injected to the tail vein of to the mice to induce acute severe hepatitis. 0, 3, 6, 12, 24 and 48 hours after injection of ConA, the mice serum of each treatment group was collected and detected for ALT and AST levels. The mouse livers of each group were taken at 12 hour, stained by HE and TUNEL for the observations of liver necrosis, inflammatory infiltration and apoptosis.
The experimental result was shown in
Experiment II-3. The OPN Level in the Plasma and Liver Tissue of Hepatitis Model Mice in Different Treatment Groups
The C57BL/6 mice plasma of different time points (0, 3, 12 or 24 hours) in different treatment groups (experiment II-2) was collected, and detected for OPN protein concentration using mouse OPN ELISA kit. Specifically, venous blood from mice outer canthus was collected, and centrifuged immediately at 4000 rpm for 10 minutes to separate the plasma. The 1:200 dilution was added into 96 well plate of ELISA detection kit, incubated at room temperature for 1 hour, and washed with PBST for five times. Biotin labeled anti-OPN primary antibody was then added into the plate, and the plate was incubated at room temperature for 1 hour and washed for five times. Then, avidin labeled secondary antibody was added into the plate. The plate was incubated at room temperature for 1 hour, washed for six times, and developed with TMB substrate. OD450 absorbance was detected. The plasma OPN concentration was calculated according to the standard curve.
The result was shown in
At hour 6, the liver tissue of different treatment groups (experiment II-2) was collected. The total RNA was extracted from the tissue with RNeasy Mini Kit (QIAGEN) and subjected to reverse transcription using Sensiscript RT Kit (QIAGEN) for cDNA. OPN primers were designed according to the requirements of real-time PCR (as shown in Table 1), OPN mRNA level was detected by real-time PCR using SYBR Green Master Mix (Applied Biosystems). The PCR procedure was: 50° C. for 2 mins; 95° C. for 10 mins; 40 s of 95° C. for 15 sec and 60° C. for 60 sec. The internal standard was GAPDH gene. The experimental data was analyzed using ABI PRISM 7900 sequencing system (Applied Biosystems).
The experimental result was shown in
Experiment II-4. The Cytokines Levels in the Plasma and Liver Tissue of Hepatitis Model Mice in Different Treatment Groups
The C57BL/6 mice plasma of different time points (0, 3, 6, 12 or 24 hours) in different treatment groups (experiment II-2) were selected, the TNF-α and IFN-γ protein concentrations were detected using mouse TNF-α, IFN-γ ELISA kit (R&D). Specifically, venous blood from mice outer canthus was collected, allowed to stand at room temperature for 2 hours and separated for the serum.
The 1:100 dilution was added into 96 well plate of ELISA detection kit, incubated at room temperature for 1 hour and washed with PBST for five times. Biotin labeled anti-TNF-α or IFN-γ primary antibody was then added into the plate, and the plate was incubated at room temperature for 1 hour and washed for five times. Avidin labeled secondary antibody was then added into the plate. The plate was incubated at room temperature for 1 hour, washed for six times, and developed with TMB substrate. OD450 absorbance was detected. The plasma TNF-α and IFN-γ concentrations were calculated according to the standard curve.
The result was shown in
At hour 6, the liver tissues of different treatment groups were collected. The total RNA in the tissue was extracted with RNeasy Mini Kit (QIAGEN) and subjected to reverse transcription using Sensiscript RT Kit (QIAGEN) for cDNA. Primers for cytokines were designed according to the requirements of real-time PCR (as shown in Table 1). Cytokine mRNA level was detected by real-time PCR using SYBR Green Master Mix (Applied Biosystems). The PCR procedure was the same as that used in example 3. The experimental data was analyzed using ABI PRISM 7900 sequencing system (Applied Biosystems).
The experimental result was shown in
Experiment III. The Effect of 23C3 mAb on Crohn's Disease Model Mice
Experiment III-1. The Effect of 23C3 Monoclonal Antibody on Body Weight, Disease Activity Index (DAI) and Spleen INDEX in Crohn's Disease Model Mice
Dextran sulfate sodium (DSS) was used to induce experimental colitis in 30 mice. That is, 30 g/L DSS was given to the mice instead of drinking water for 7 days. The above mice were randomly divided into 3 groups: control. Ig control treatment group and 23C3 monoclonal antibody treatment group. Intraperitoneal administration began at the modeling day and lasted for 3 consecutive days, 200 ug/mouse/time. The body weights of the experimental mice were recorded every day. The disease activity index was scored every day according to stool character, occult blood test and the change of body weight (scoring criteria was shown in table 2);
At day 7, the mouse spleens were taken and weighed to calculate the spleen index (spleen weight/body weight).
The experimental results were shown in
Experiment III-2. The Histopathologic Changes of Colon in Crohn's Disease Model Mice of Different Treatment Groups
At day 7, the colon tissue with pathologic changes of different treatment groups (experiment III-1) were taken, washed with PBS, fixed in 10% formalin, paraffin-embedded, sliced continuously into 2 μm sections, and stained with HE. The histological changes in different treatment groups were observed using light microscope. The specific scoring criteria were shown in Table 3.
The result was shown in
Experiment III-3. Effect of 23C3 Monoclonal Antibody on Intestinal Tissue Myeloperoxidase (MPO) Activity in Crohn's Disease Model Mice
Intestinal tissue myeloperoxidase (MPO) activity was detected using commercial reagent for detection. At day 0, 3 and 7, the colon tissues of each treatment group (experiment III-1) were taken, weighted the same weight, shredded, and homogenized for 30 sec in ice bath (in 0.5% HTAB containing buffer). The homogenate was sonicated for 10 sec, repeated freeze-thaw for three times, centrifuged at 15000 g for 15 mins. 0.1 ml homogenate supernatant was added to 2.9 ml of 0.167 mg/ml O-dianisidine hydrochloride, and then 0.0005% H2O2 was added. OD460 was detected. The MPO activity was calculated as follow, one MPO activity unit equaled to 1 mol peroxidase degradation in 1 minute.
The result was shown in
Experiment III-4. The Intestinal Tissue TNF-α, IL-1β and IL-6 mRNA Levels in Crohn's Disease Model Mice of Different Treatment Groups
At day 3 and 7, the mice intestinal tissues of different treatment groups (experiment III-1) were taken. Total RNA was extracted using RNeasy Mini Kit (QIAGEN) and subjected to reverse transcription with Sensiscript RT Kit (QIAGEN) for cDNA. Primers for various cytokines were designed according to the requirements of real-time PCR (as shown in Table 1). Cytokine mRNA levels were detected by real-time PCR using SYBR Green Master Mix (Applied Biosystems). PCR procedure was the same as that used in Example 3. The experimental data was analyzed using ABI PRISM 7900 sequencing system (Applied Biosystems).
The experimental result was shown in
Example III-5. The Intestinal Tissue and Plasma OPN Levels in Crohn's Disease Model Mice of Different Treatment Groups
The C57BL/6 mice plasma of different treatment groups (experiment III-1) were collected at different time points (day 0, 3 and 7), and detected for OPN protein concentration using mouse OPN ELISA kit. Specifically, venous blood from mice outer canthus was collected, and centrifuged immediately at 4000 rpm for 10 minutes to separate the plasma. The 1:200 dilution was added into 96 well plate of ELISA detection kit, incubated at room temperature for 1 hour, and washed with PBST for five times. Biotin labeled anti-OPN primary antibody was then added into the plate, and the plate was incubated at room temperature for 1 hour. After the plate was washed for five times, avidin labeled secondary antibody was added. Then, the plate was incubated at room temperature for 1 hour, washed for six times, and developed with TMB substrate. OD450 absorbance was detected. The plasma OPN concentration was calculated according to the standard curve.
The result was shown in the left panel of
At day 7, the colon tissues of different treatment groups (experiment III-1) were taken. The total RNA was extracted with RNeasy Mini Kit (QIAGEN) and subjected to reverse transcription using Sensiscript RT Kit (QIAGEN) for cDNA. OPN primers were designed according to the requirements of real-time PCR (as shown in Table 1). OPN mRNA level was detected by real-time PCR using SYBR Green Master Mix (Applied Biosystems). The PCR procedure was: 50° C. for 2 mins; 95° C. for 10 mins; 40 cycles of 95° C. for 15 sec and 60° C. for 60 sec. The internal standard was GAPDH gene. The experimental data was analyzed using ARI PRISM 7900 sequencing system (Applied Biosystems).
The experimental result was shown in the right panel of
Experiment IV. Effect of c23C3 and h23C3 on In Vitro Lymphocyte Migration, Immune Response and Survival
Experiment IV-1. The Inhibitory Effect of c23C3 and h23C3 Antibodies on Lymphocyte Migration
CIA mice spleens were aseptically separated, passed 100 mesh steel screen, washed with PBS for two times, treated with lymphocyte separating medium to separate spleen lymphocyte, and centrifuged at 2000 rpm for 20 minutes. The lymphocyte layer was pipetted and washed with PBS for 2 times. The lymphocytes were counted, and adjusted to a concentration of 5×106 cells/ml. 24-well tissue culture plate having a 5 μm filter membrane at the upper layer was pre-coated with 10 μg OPN at the lower layer. 5×105 lymphocyte as well as the following antibodies were added into each well in the upper layer: 23C3, Her-2 antibody (irrelevant antibody control), c23C3 and h23C3. The plate was incubated at 37° C., 5% CO2 for 2 hours, washed with PBS twice. The number of cell in the lower layer was calculated by taking the average of counted numbers in five high power fields and then multiplied by 50.
The experimental result was shown in
Experiment IV-2. The Inhibitory Effect of c23C3 and h23C3 Antibodies on Lymphocyte Immune Response
CIA mice spleens were aseptically separated, passed 100 mesh steel screen, washed with PBS for two times, treated with lymphocyte separating medium to separate spleen lymphocyte, and centrifuged at 2000 rpm for 20 minutes. The lymphocyte layer was pipetted and washed with PBS for 2 times. The lymphocytes were counted, and adjusted to a concentration of 5×106 cells/ml. 5×105 lymphocytes as well as 30 μg/ml of chicken type II collagen and different concentrations of 23C3, control Ig, c23C3 and h23C3 were added into each well. The plate was incubated at 37° C., 5% CO2 for 72 hours. 100 ul culture medium containing 3H (0.5 μCi) was added into each well, and the plate was cultured for another 16 hours. Cells were then collected with cell collector and detected with γ liquid scintillation counter.
The experimental result was shown in
Experiment IV-3. Effect of c23C3 and h23C3 Antibodies on the Survival of Activated Lymphocyte
CIA mice spleens were aseptically separated, passed 100 mesh steel screen, washed with PBS for two times, treated with lymphocyte separating medium to separate spleen lymphocytes, and centrifuged at 2000 rpm for 20 minutes. The lymphocyte layer was pipetted and washed with PBS for 2 times. The lymphocytes were counted, and adjusted to a concentration of 5×106 cells/ml, 5×105 lymphocyte as well as 100 μg/ml of chicken type II collagen and different concentrations of 23C3, control Ig, c23C3 and h23C3 were added into each well. The plate was incubated at 37° C., 5% CO2 for 72 hours. The cells were collected, to which Annexin V-FITC was added, Cell apoptosis was detected through flow cytometry.
The result was shown in
Experiment V. Identification of the Epitope of the Anti-hOPN (23C3) Monoclonal Antibody
Experiment V-1. Panning of the Epitope of Anti-hOPN (23C3) Monoclonal Antibody
The experiment was performed using (Phage) random peptide library panning method, and the whole process was carried out in a 96-well plate. The plate was coated with 100 ug/ml 23C3 monoclonal antibody (100 μl/well) at 4° C. overnight, blocked with 10% skimmed milk (diluted with TBST) overnight, and washed with 1×TBST (Tween-20, 0.1%) for six times. To the plate were added the phage random peptide library (purchased from NEB, Ph.D.-12™ Phage Display Peptide Library Kit) 4×1010 pfu and 100 μl normal mouse serum, and gently agitated at room temperature for 1 hour. The plate was washed with 1×TBST (Tween-20, 0.1%), 1×PBS (pH5.0), and 1×PBS (pH3.5) (each for five times), and then eluted with glycine-Cl (containing 1 mg/ml BSA, pH 2.2) with gently agitated at room temperature for 15 minutes before neutralizing with 15 ul Tris-Cl (pH 9.1). 10 μl resultant was taken out for titer, and the rest was used for amplification. Amplification products were precipitated by PEG/NaCl, and the titer was measured, on which the second round of panning was performed. The third round of panning was performed with the same process. Each time the same number of phages (4×1010 pfu) was added, and the number of the output phage after three rounds of panning was 2.76×103 pfu, 3.45×105 pfu and 3.08×106 pfu, respectively. The panning efficiency increased at a 125-fold and 1116-fold relative to that of the first round, respectively. The result suggests that the panning enrichment effect is significant (as shown in
Experiment V-2. Phage Clone ELISA and Western Blot Assay
ELISA was carried on a 96-well plate. Each well was coated with 50 μl of 100 μg/ml 23C3 monoclonal antibody at 4° C. overnight, blocked with 10% skimmed milk (TBST diluted) at 37° C. for 2 hours, washed with 1×TBST (Tween-20, 0.1%) for five times. The supernatant of each monoclonal phage amplification product was diluted with 1×TBS to 5×108 pfu/50 μl. The control antibody was mouse monoclonal antibody SM5-1 (prepared by the Cancer Institute of the Second Military Medical University, and according to the procedure disclosed in “Arch Dermatol Res. December 2000; 292 (12): 583-9, the same method was used hereinafter). The negative control phage was 4E4 (SM5-1 positive clone, ibid. The same negative control phage was used hereinafter). Binding was carried out at room temperature for 1 hour. The resultant was washed with 1×TBST (Tween-20, 0.1%) for five times, and then 200 μl of 1:5000 diluted HRP labeled anti-M13 antibody (Pharmacia # 27-9411-01) was added into each well. The plate was shaken at room temperature for 1 hour and washed with 1×TBST (Tween-20, 0.1%) for five times. Fresh substrate prepared by mixing reagent A and reagent B of ELISA detection kit (Jinmei, A:B=1:1, 50 ul/well) was added into each well, and the plate was incubated at room temperature for 1-5 minutes before adding 2NH2SO4 to terminate the reaction. The reaction of each clone to 23C3 and control antibody SM5-1 was detected in triplicate. OD450 data showed that the response of the positive clones to the antibody was specific (as shown in
Western blot process was as follows: the supernatant of the amplified phage monoclonal was purified by 20% PEG/NaCl precipitation, then subject to 10% SDS-PAGE electrophoresis (1×1010 pfu/lane) under 360 mA constant current for 1 hour to transfer to a nitrocellulose membrane. The membrane was blocked with 10% skim milk at 4′C overnight or at room temperature for two hours. After being washed with 1×TBST (Tween-20, 0.1%) for three times (10 minutes/time), the membrane was reacted with 10 μg/ml primary antibody at room temperature for 1 hour, and washed with 1×TBST (Tween-20, 0.1%) for five times (10 minutes/time). The membrane was then reacted with 1:1000 diluted HRP labeled rabbit anti-mouse IgG (Beijing Zhongshan Company) at room temperature for 1 hour, treated with ECL kit (Tiangen Company) for 1-2 minutes, and then pressed and exposed with medical blue-sensitive X-ray film. The result was shown in
Experiment V-3. Sequencing and Sequence Analysis
Single-stranded DNA Extraction Kit (Shanghai Jierui Company) was used to prepare the template. The sequencing was performed using −96 primer, and the sequence was read with Chromas. Among 25 positive clones were panned out 15 independent sequences (analyzed by AlignX). The result was shown in
Experiment V-4. The Binding Ability of Phage Clone to Antibody
Each clone obtained in experiment V-3 was added at 5×107 pfu into an antibody coated 96 well plate, and was panned with the same conditions. (Control antibody: SM5-1; irrelevant control phage: 4E4). The titers of the phages were detected after elution (referring to Blood 2006-04-014639).
The results showed that: the epitope in hOPN sequence was W43LNP46DP48, wherein W43, P46, and P48 played an important role in mediating 23C3-hOPN binding. Moreover, the clones with the highest binding ability were: 5F12: DVYWLMPDPPTS synthetic short peptide, and 5D2:GTSTEVWLAPDP synthetic short peptide. The result was shown in
Experiment V-5. The Specific Binding Assay of Synthetic Short Peptides and Antibody
Dot-blot assay was as follows: three dots of 3×10 ng KLH-4B5 (irrelevant control) and KLH-5F12 respectively, were pointed. Each short peptide obtained in experiment V-4 was used to hybrid with three different antibodies. After being blocked and washed, the membrane was reacted with primary antibody at room temperature for 1 hour. Then, the membrane was washed again, and reacted with HRP-labeled anti-mouse secondary antibody at room temperature for 1 hour. After being washed, the membrane was pressed and exposed. Irrelevant short peptide did not react with 23C3, while KLH-5F12 specifically reacted with the corresponding antibody. The result was shown in
Blocking assay of short peptide on phage-antibody binding was as follows: 96 well plate was coated with 100 ug/ml antibody (50 μl/well) at 4° C. overnight, blocked with 10% skimmed milk, and washed with 1×PBST for three times until use. 100 μg/ml short peptide was mixed with phage until homogenous, and the homogenous solution was then added to the antibody-coated plate. The plate was gently shook at room temperature for 1 hour before washed with 1×TBST (Tween-20, 0.1%), 1×PBS (pH5.0), and 1×PBS (pH3.5) (each for 5 times). After being eluted with glycine-Cl (containing 1 mg/ml BSA, pH 2.2) for 15 minutes, the plate was neutralized with Tris-Ci (pH 9.1). Then, the titer was measured. Each peptide was divided into three groups: (1) phage; (2) plage+peptide; and (3) phage irrelevant peptide. The blocking efficiency of the short peptide 5F12 having such a concentration was 71%. The result was shown in
The results showed that, the sequence of the epitope of 23C3 antibody was WLNPDP, which was located in the fourth exon of OPN. The location and sequence of the epitope in OPN protein were shown in
All documents mentioned in this specification are herein incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Further, it is understood that, after reading this specification, those skilled in the art can make variations and modifications to the present invention, and the scope of protection of the invention is determined in particular by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 200710039878.6 | Apr 2007 | CN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN08/70582 | 3/25/2008 | WO | 00 | 11/16/2009 |
