HK1085932B - Degraded agonist antibody - Google Patents
Degraded agonist antibody Download PDFInfo
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- HK1085932B HK1085932B HK06106076.8A HK06106076A HK1085932B HK 1085932 B HK1085932 B HK 1085932B HK 06106076 A HK06106076 A HK 06106076A HK 1085932 B HK1085932 B HK 1085932B
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Description
The present application is a divisional application of the chinese patent application "agonist antibody with reduced molecular weight" with application number 01817431.0, applied on 10/22/2001.
Technical Field
The present invention relates to altered antibodies comprising two or more H chain V regions and two or more L chain V regions of a monoclonal antibody that exhibits agonist activity by cross-linking cell surface molecules or intracellular molecules. The altered antibodies have agonist activity to transduce signals into cells by cross-linking cell surface molecules, and are useful as drugs for a variety of purposes.
Background
JP-A9-295999 discloses the production of a specific monoclonal antibody using a spleen stromal cell line as a sensitizing antigen, the object of which is to develop a specific antibody recognizing the above spleen stromal cells, and the production of a novel monoclonal antibody recognizing a mouse integrin-associated protein (mouse IAP) as an antigen. JP-A.9-295999 also discloses that these monoclonal antibodies are capable of inducing apoptosis in bone marrow cells.
WO99/12973 discloses monoclonal antibodies whose antigen is a human integrin-associated protein (herein incorporated by reference human IAP; the amino acid and nucleotide sequences of which are described in J.cell biol., 123, 485-496, 1993; see also Journal of cell Science, 108, 3419-3425, 1995) which are capable of inducing apoptosis in human nucleated blood cells (myeloid and lymphoid cells) bearing said human IAP. These monoclonal antibodies are referred to as antibody MABL-1 and antibody MABL-2, and the hybridomas that produce these antibodies are also referred to as MABL-1(FERM BP-6100) and MABL-2(FERM BP-6101), respectively.
Japanese patent application 11-63557 describes the preparation of single-chain Fv with single-chain Fv region of a monoclonal antibody with human IAP as antigen. The single chain Fv is capable of inducing apoptosis in nucleated blood cells having human IAP.
Monoclonal antibodies recognizing IAP as an antigen induce apoptosis of nucleated blood cells having human IAP, but also causeIn vitroHemagglutination reaction. It is suggested that administration of large amounts of monoclonal antibodies recognizing IAP as an antigen may produce side effects such as hemagglutination.
The present inventors have conducted intensive studies using a monoclonal antibody against human IAP as a therapeutic agent for hematological diseases, and have obtained a single-chain Fv having a single-chain Fv region capable of inducing apoptosis of nucleated blood cells having human IAP.
On the other hand, altered antibodies, particularly those with reduced molecular size, for example, by reducing molecular size, single chain Fv's that increase permeability into tissues and tumors are developed and produced by recombinant methods. More recently, dimers, particularly bispecific dimers, of single chain Fv's have been used to crosslink cells. A typical example of such a dimer is the heterodimer of a single-chain Fv which recognizes cancer cell antigens as well as antigens of host cells such as NK cells and neutrophils (Kipriyanov et al, int. J. cancer, 77, 9763-9772, 1998). They are prepared as altered antibodies using single chain Fv construction techniques to more effectively treat cancer by inducing intercellular cross-linking. Intercellular cross-linking has been suggested to be induced by antibodies and fragments thereof (e.g., Fab fragments), bispecific altered antibodies, or even monospecific single chain Fv dimers.
As antibodies capable of transducing signals by cross-linking cell surface molecules, there are known an antibody to EPO receptor involved in cell differentiation and proliferation (JP-A2000-95800), an antibody to MuSK receptor (Xie et al, Nature Biotech.15, 768 771, 1997), and others. However, no altered antibodies have been reported that reduce molecular size.
Note that single chain Fv monomers from MABL-1 and MABL-2 antibodies do not induce apoptosis, whereas single chain Fv dimers induce apoptosis with IAPs, which the inventors found cross-links (dimerizes) IAP receptors on the cell surface, thereby transducing signals into the cell, resulting in induction of apoptosis. This suggests that monospecific single chain Fv dimers cross-link cell surface molecules (e.g., receptors) and transduce signals like ligands, thereby acting as agonists.
Cell-cell cross-linking was investigated and found that the single-chain Fv dimer did not cause hemagglutination, whereas the monoclonal antibody did. The same results were observed for single chain diabodies (single chain polypeptides comprising two H chain V regions and two L chain V regions). This suggests that monoclonal antibodies may form intercellular crosslinks, while altered antibodies, such as single-chain Fv dimers and single-chain diabodies, crosslink cell surface molecules but do not form intercellular crosslinks.
Based on these observations, the inventors have recently discovered that altered antibodies, such as single chain Fv dimers and single chain diabodies, cross-link cell surface molecules or intercellular molecules of the same cell, in addition to known intercellular cross-links, are suitable as ligands for the molecule (particularly as ligands that mimic the action of the native ligand).
The present inventors have completed the present invention by further finding that antibody molecules (complete IgG) can be changed to single-chain Fv dimers, single-chain diabodies, etc., which are cross-linked cell surface molecules, thereby reducing side effects caused by intercellular cross-linking and providing a new drug which induces only the desired effects of cells. The altered antibodies of the invention have a very high activity compared to natural ligands, such as TPO, EPO or G-CSF, or to complete antibodies (IgG) having the same V region as the altered antibody. Its permeability into tissues is increased due to the reduced molecular size and the absence of the constant region compared to antibody molecules.
Disclosure of Invention
It is an object of the present invention to provide an agonist-altered antibody of low molecular size, which comprises two or more H chain V regions and two or more L chain V regions of a monoclonal antibody and has an agonist effect by crosslinking a cell surface molecule or an intracellular molecule.
Thus, the present invention relates to altered antibodies comprising two or more H chain V regions and two or more L chain V regions, preferably 2-6 each, particularly preferably 2-4 each, most preferably two each, and which exhibit agonist activity by cross-linking cell surface or intracellular molecules.
The term "altered antibody" as used herein refers to any substance comprising two or more H chain V regions and two or more L chain V regions, wherein the V regions are linked by a covalent or non-covalent bond, either directly or through a linker. For example, polypeptides and compounds prepared by linking each V region of an antibody via a peptide linker or a chemical cross-linking agent, or the like. The two or more H chain V regions and the two or more L chain V regions used in the present invention may be derived from the same antibody or different antibodies.
Preferred examples of the altered antibody of the present invention are multimers, such as dimers, trimers or tetramers, of single chain Fv comprising an H chain V region and an L chain V region, or single chain polypeptides comprising two or more H chain V regions and two or more L chain V regions. If the altered antibody of the present invention is a multimer, e.g., a dimer, trimer or tetramer, of a single-chain Fv comprising an H chain V region and an L chain V region, it is preferred that the H chain V region and the L chain V region on the same chain do not bind to form an antigen-binding site.
More preferred examples are dimers of single chain Fv comprising H chain V regions and L chain V regions, or single chain polypeptides comprising two H chain V regions and two L chain V regions. The H chain V region and the L chain V region in the altered antibody are preferably linked by a linker.
"agonist effect" in the present specification refers to a biological effect occurring in a cell which is transmitted a signal by crosslinking a cell surface molecule or an intracellular molecule, such as induction of apoptosis, induction of cell proliferation, induction of cell differentiation, induction of cell division or cell cycle regulation.
The ED50 of the agonist effect of the invention is determined by known methods of measuring agonist effect. For example, detecting agonist-specific cell death or cell proliferation, detecting expression of cell differentiation-specific proteins (e.g., specific antigens), or measuring cell cycle-specific kinase activity. ED50 is the dose required to reach 50% of the maximum activity (which is set as 100% in the dose-response curve).
Preferred altered antibodies of the invention have an agonist effect (ED50) that is equal to or better than an antibody having the same antigen binding region as the altered antibody, i.e., a complete antibody, such as an IgG (hereinafter "parent antibody"), having the same H chain V region and L chain V region pair as the H chain V region and L chain V region pair forming the antigen binding region of the altered antibody. More preferably an altered antibody which has an agonist effect (ED50) which is more than two times, even more preferably more than 5 times, most preferably more than 10 times higher than the parent antibody. The invention encompasses altered antibodies with agonist action comprising H chain V regions and L chain V regions that form the same antigen binding region as the parent antibody (which can bind to a target cell surface molecule or intracellular molecule, but which has no agonist action on the molecule).
The compound of the present invention comprising two or more H chain V regions and two or more L chain V regions may be any compound comprising two or more H chain V regions and two or more L chain V regions of an antibody, which exhibits an agonist action (ED50) equal to or better than that of a natural ligand which binds to a cell surface molecule or an intracellular molecule. Preferred are compounds with an agonist effect (ED50) that is more than 2-fold, more preferably more than 5-fold, most preferably more than 10-fold, greater than the natural ligand.
As used herein, the term "compound" includes not only the altered antibodies of the invention, but also any compound comprising two or more, preferably 2-6, more preferably 2-4, most preferably 2 antigen binding regions, such as a complete antibody or F (ab')2。
The altered antibodies or compounds of the invention comprising two or more H chain V regions and two or more L chain V regions of the antibody preferably do not have substantial intercellular adhesion. When the H chain V region and L chain V region of the altered antibody of the invention are derived from the same antibody, they preferably have an intercellular adhesion (ED50) of no more than 1/10 compared to the original antibody.
The ED50 for cell-cell adhesion according to the invention is determined using known methods for measuring agonist action, for example by measuring agglutination of cells expressing the cell surface molecule, for example a hemagglutination test.
The present invention relates to DNA encoding altered antibodies.
The present invention relates to animal cells or microorganisms that produce altered antibodies.
The present invention relates to the use of altered antibodies as agonists.
The present invention relates to methods of inducing cell agonist effects (e.g., inducing apoptosis, inducing cell proliferation, inducing cell differentiation, inducing cell division, or cell cycle regulation) by transducing signals into cells by cross-linking cell surface molecules or intracellular molecules using altered antibodies.
The present invention relates to medicaments comprising altered antibodies.
The present invention relates to the use of altered antibodies as medicaments.
The present invention relates to a method for screening or measuring an altered antibody comprising two or more H chain V regions and two or more L chain V regions of an antibody and exhibiting an agonist effect by crosslinking a cell surface molecule or an intracellular molecule, which comprises 1) preparing an altered antibody comprising two or more H chain V regions and two or more L chain V regions of an antibody and specifically binding to said molecule, 2) contacting a cell expressing said molecule with the altered antibody and 3) measuring the agonist effect occurring in the cell caused by crosslinking said molecule. Assay methods are used for quality control in the preparation of the altered antibodies of the invention for pharmaceutical and other purposes.
The above-mentioned single-chain Fv dimers include non-covalent-bond dimers, covalent-bond dimers through a crosslinking group, and dimers through a crosslinking agent (antibodies, antibody fragments, or bivalent-altered antibodies). Conventional crosslinking groups for crosslinking peptides can be used as the crosslinking group for forming a dimer. For example by disulfide crosslinking of cysteine residues,other crosslinking groups, e.g. C4-C10Alkylene (e.g. tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, etc.) or C4-C10Alkenylene (cis/trans-3-butenylene, cis/trans-2-pentenylene, cis/trans-3-hexenylene, etc.).
Furthermore, examples of the crosslinking agent capable of binding to a single-chain Fv include an antibody or a fragment thereof, which can optionally introduce an amino acid sequence of an Fv, for example, a FLAG sequence, or a modified antibody derived from an antibody, for example, a single-chain Fv.
The invention also relates to methods of inducing an effect on a cell agonist by administering a first ligand and a second ligand that bind a cell surface molecule or an intracellular molecule, and administering a substance that binds the first and second ligands and crosslinks the first and second ligands. The first and second ligands may be any substance that comprises a binding site for the molecule and is capable of inducing an agonist effect via cross-linking. Preferred examples are antibodies of varying valency, e.g., the same or different single chain Fv monomers, antibody fragments, and the like. The substance for crosslinking the above ligands may be any substance which induces an effect on a cell agonist by crosslinking the first ligand and the second ligand. Preferred examples are antibodies, antibody fragments, (Fab)2Or a bivalent altered antibody. An example of a diabody is (Fab)2Single chain Fv dimers comprising one H chain V region and one L chain V region, and single chain polypeptides comprising two H chain V regions and two L chain V regions. The method is effective for exploring a receptor which transduces a signal into a cell by crosslinking, desirably a DDS for delivering a drug into a target cell, and also as a drug delivery system which suppresses side effects and makes the drug effective at a desired time and for a desired period of time.
The altered antibody of the present invention may be any substance comprising the L chain V region and H chain V region of an antibody (e.g., antibody MABL-1, antibody MABL-2, antibody 12B5, antibody 12E10, etc.), and any substance that specifically recognizes a cell surface molecule or an intracellular molecule (e.g., a protein (receptor or protein involved in signal transduction), or a sugar chain of the above protein or cell membrane protein) and cross-links the cell surface molecule, thereby transducing a signal into a cell. Including altered antibodies in which a portion of the amino acid sequence of the V region has been altered.
The altered antibody may be monospecific or multispecific (e.g., bispecific) depending on the characteristics of the cell surface molecule or intracellular molecule to be bound, e.g., molecular structure or mechanism of action. Monospecific altered antibodies are preferred if they bind to receptor molecules (e.g., erythropoietin receptor, thrombopoietin receptor, G-CSF receptor, SCF receptor, EGF receptor, IAP (CD47), etc.) that homodimerize and transduce a signal into the cell. If it binds to a receptor molecule (e.g., IL-6 receptor, LIF receptor, IL-11 receptor) that heterodimerizes and transduces signals into cells, bispecific altered antibodies are preferred. Trispecifically altered antibodies are preferred if they bind to receptor molecules (e.g., IL-2 receptor, CNTF receptor, OSM receptor) that heterotrimerize and transduce signals into the cell. Methods for the preparation of bispecific single chain Fv dimers are described in WO9413804 et al.
The present invention also relates to an altered antibody in which the H chain V region and/or the L chain V region are derived from a human antibody H chain V region and/or derived from a human antibody L chain V region. The H chain V region and/or L chain V region derived from a human antibody can be obtained by screening a human monoclonal antibody library as described in WO 99/10494. Also included are H chain V regions and L chain V regions derived from human monoclonal antibodies.
The present invention further relates to an altered antibody in which the H chain V region and/or L chain V region is a humanized H chain V region and/or a humanized L chain V region. Specifically, the humanized altered antibody is composed of a humanized L chain V region comprising a Framework Region (FR) derived from the L chain V region of a human monoclonal antibody and a complementarity determining region (hereinafter "CDR") derived from the L chain V region of a non-human mammal (e.g., mouse, rat, bovine, ovine, simian) monoclonal antibody and/or a humanized H chain V region comprising a FR derived from the H chain V region of a human monoclonal antibody and a CDR derived from the H chain V region of a non-human mammal (e.g., mouse, rat, bovine, ovine, simian) monoclonal antibody. In this case, the amino acid sequences of the CDR and FR may be partially changed, for example, deleted, substituted or added.
The H chain V region and/or the L chain V region of the altered antibody of the present invention may be those derived from a monoclonal antibody of an animal other than human (e.g., mouse, rat, bovine, sheep, ape, chicken, etc.). In this case, the amino acid sequences of the CDR and FR may be partially changed, for example, deleted, substituted or added.
The present invention also relates to DNA encoding the above-described various altered antibodies, and genetic engineering techniques for preparing recombinant vectors comprising the DNA.
The invention also relates to host cells transformed with the recombinant vectors. Examples of the host cell are animal cells such as human cells, mouse cells and the like, and microorganisms such as Escherichia coli (E.coli) ((E.coli))E. coli)、Bacillus subtilis (Bacillus subtilis)Yeast, etc.
The present invention relates to methods for producing an altered antibody comprising culturing the above-described host and extracting the altered antibody from the culture thereof.
The present invention further relates to a method for producing a single-chain Fv dimer comprising culturing a host animal cell producing the single-chain Fv in a serum-free medium to secrete the single-chain Fv into the medium, and isolating the single-chain Fv dimer formed in the medium.
The invention also relates to the use of the altered antibodies as agonists. That is, it relates to a signal transduction agonist comprising the altered antibody obtained as described above as an active ingredient. Since the altered antibodies used in the present invention cross-link a cell surface molecule or an intracellular molecule and induce signal transduction, the molecule can be any molecule that oligomerizes (e.g., dimerizes) via a binding ligand, thereby transducing a signal into a cell.
Such cell surface molecules include hormone receptors and cytokine receptors. Hormone receptors include, for example, estrogen receptors. Cytokine receptors and the like include hematopoietic factor receptors, lymphokine receptors, growth factor receptors, differentiation control factor receptors and the like. Examples of cytokine receptors are the Erythropoietin (EPO) receptor, the Thrombopoietin (TPO) receptor, the granulocyte colony-stimulating factor (G-CSF) receptor, the macrophage colony-stimulating factor (M-CSF) receptor, the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor, the Tumor Necrosis Factor (TNF) receptor, the interleukin-1 (IL-1) receptor, the interleukin-2 (IL-2) receptor, the interleukin-3 (IL-3) receptor, the interleukin-4 (IL-4) receptor, the interleukin-5 (IL-5) receptor, the interleukin-6 (IL-6) receptor, the interleukin-7 (IL-7) receptor, the interleukin-9 (IL-9) receptor, the like, Interleukin-10 (IL-10) receptor, interleukin-11 (IL-11) receptor, interleukin-12 (IL-12) receptor, interleukin-13 (IL-13) receptor, interleukin-15 (IL-15) receptor, interferon-alpha (IFN-alpha) receptor, interferon-beta (IFN-beta) receptor, interferon-gamma (IFN-gamma) receptor, Growth Hormone (GH) receptor, insulin receptor, blood stem cell proliferation factor (SCF) receptor, Vascular Endothelial Growth Factor (VEGF) receptor, epithelial cell growth factor (EGF) receptor, Nerve Growth Factor (NGF) receptor, Fibroblast Growth Factor (FGF) receptor, platelet-derived growth factor (PDGF) receptor, transforming growth factor-beta (TGF-beta) receptor, insulin receptor, interferon-alpha (IFN-alpha) receptor, interferon-beta (IFN-beta) receptor, interferon-gamma (IFN-gamma-, Leukocyte migration inhibitory factor (LIF) receptors, ciliary neurotrophic factor (CNTF) receptors, oncostatin M (OSM) receptors, Notch family receptors, and the like.
Intracellular surface molecules include TAK1, TAB1, and the like. TAK1 and TAB1 act on the TGF- β signal transduction pathway, activating MAP kinase by forming heterodimers, transducing a range of signals. Many cancer cells have mutations in their TGF-beta receptors that inhibit cancer growth and are therefore unable to transduce TGF-beta signals. Antibodies that can cross-link TAK1 and TAB1 to transduce an alteration in signal are capable of inducing TGF- β signaling by the agonist effect of binding TAK1/TAB 1. Such altered antibodies of the invention are capable of inhibiting the growth of TGF- β resistant cancer cells, and provide novel methods for treating cancer. Other examples of intracellular molecules are the transcription factor E2F homodimer and the E2F/DP1 heterodimer, which have a cell-proliferating effect. The altered antibodies of the invention are also capable of inducing agonist effects on these molecules and can therefore be used to treat a variety of cell proliferation-related diseases. The altered antibodies of the invention are also capable of inducing agonist effects by cross-linking intracellular factors involved in apoptosis-inducing related signal transduction, and thus are capable of inducing apoptotic cell death in cancer cells or cells associated with autoimmune diseases.
To achieve The interaction of The altered antibodies of The invention with intracellular molecules, peptides with cell membrane permeability (e.g., Pegelin, penetretin) can be used to transport The altered antibodies into cells (Martine Mazel et al, Doxorubicin-peptide conjugate lipid multiple Drugs resistance. anti-Cancer Drugs 2001, 12, Dcs Cross D. et al, The third helix of The anti-parasitic peptide complexes through biological membranes, J.biol.chem.1994, 269, 10444-.
Therefore, a pharmaceutical preparation comprising an agonist-modified antibody as an active ingredient is useful as a prophylactic and therapeutic drug for various diseases such as cancer, inflammation, hormonal disorders, blood diseases, and autoimmune diseases, and the like.
The oligomers that can be formed from the receptor protein can be homo-oligomers or hetero-oligomers, as well as any oligomers, such as dimers, trimers, and tetramers. For example, it is known that erythropoietin receptor, thrombopoietin receptor, G-CSF receptor, SCF receptor, EGF receptor and the like form homodimers, IL-6 receptor, LIF receptor and IL-11 receptor form heterodimers, and IL-2 receptor, CNTF receptor, OSM receptor form heterotrimers.
The altered antibodies of the invention comprise 2 or more H chain V regions and 2 or more L chain V regions derived from a monoclonal antibody. The structure of the altered antibody may be a dimer of a single chain Fv comprising 1H chain V region and 1L chain V region, or a polypeptide comprising 2H chain V regions and 2L chain V regions. In the altered antibody of the present invention, the V regions of the H chain and L chain are preferably linked by a peptide linker consisting of one or more amino acids. The resulting altered antibodies comprise the variable regions of the antibody to bind antigen with the same specificity as the original monoclonal antibody.
H chain V region
In the present invention, the H chain V region derived from an antibody recognizes a cell surface molecule or an intracellular molecule such as a protein (receptor or signal transduction-related protein) or a sugar chain on a protein or a cell membrane, and an oligomer, which is dimerized, for example, by crosslinking the molecule, thereby transducing a signal into a cell. The H chain V region of the present invention includes H chain V regions derived from mammals (e.g., humans, mice, rats, cows, sheep, apes, etc.), and H chain V regions having partially changed amino acid sequences of the H chain V regions. More preferred are humanized H chain V regions comprising the FRs of the H chain V region of the human monoclonal antibody, and the CDRs of the H chain V region of the mouse monoclonal antibody. Also preferred is an H chain V region having an amino acid sequence derived from human, which can be prepared by recombinant techniques. The H chain V region of the present invention may be a fragment of the above H chain V region that retains the antigen binding ability.
L chain V region
In the present invention, the L chain V region recognizes a cell surface molecule or an intracellular molecule such as a protein (receptor or signal transduction-related protein) or a sugar chain on a protein or a cell membrane, and oligomers which dimerize, for example, by crosslinking the molecule, thereby transducing a signal into a cell. The L chain V region of the present invention includes L chain V regions derived from mammals (e.g., human, mouse, rat, bovine, ovine, simian, etc.), and L chain V regions having partially changed amino acid sequences of the L chain V regions. More preferred are humanized L chain V regions comprising the FRs of the L chain V region of the human monoclonal antibody and the CDRs of the L chain V region of the mouse monoclonal antibody. It is also preferable to have an L chain V region derived from an amino acid sequence of a human antibody, which can be prepared by using a recombinant technique. The L chain V region of the present invention may be a fragment of the L chain V region that retains the antigen binding ability.
Complementarity Determining Region (CDR)
Each V region of the L chain and H chain forms an antigen binding site. The variable regions of the L and H chains consist of 4 common framework regions that are fairly conserved linked to 3 highly variable regions or Complementarity Determining Regions (CDRs) (Kabat, e.a. et al, "Sequences of proteins of immunological Interest", US dept.
The major part of the 4 Framework Regions (FR) form a β -sheet structure, so that 3 CDRs form a loop. In some cases, the CDRs may form part of the sheet structure. The 3 CDRs are spatially located close to each other by the FRs, together with the 3 CDRs forming an antigen binding junction.
These CDRs are identified by comparing the amino acid sequence of the V region of the antibody obtained with the known amino acid Sequences of the V regions of known antibodies according to the rules of thumb in Kabat, E.A. et al, "Sequences of proteins of immunological interest".
Single chain Fv
Single-chain Fv is a polypeptide monomer comprising an H chain V region and an L chain V region linked to each other and derived from a monoclonal antibody. The resulting single-chain Fv comprises the variable region of the parent monoclonal antibody and retains its complementarity determining regions, and thus binds to an antigen with the same specificity as the parent monoclonal antibody (JP-appl.11-63557). A portion of the variable region and/or CDR of the single-chain Fv of the present invention, or a portion of the amino acid sequence thereof, may be partially altered, e.g., deleted, substituted or added. The H chain V region and the L chain V region, which have been described above as constituting the single-chain Fv of the present invention, may be directly linked or linked via a linker, preferably a peptide linker. The structure of single-chain Fv may be [ H chain V region ] - [ L chain V region ], or [ L chain V region ] - [ H chain V region ]. In the present invention, it is possible to form single-chain Fv's as dimers, trimers or tetramers from which the altered antibodies of the present invention are formed.
Single chain altered antibodies
The present invention comprises single chain altered antibodies comprising two or more H chain V regions and two or more L chain V regions (preferably 2-4 each, and particularly preferably 2 each), comprising two or more H chain V regions and L chain V regions as described above. Each region of the peptide should be arranged so that the altered single chain antibody forms a specific spatial structure, precisely mimicking the spatial structure formed by a single chain Fv dimer. For example, the V regions are arranged in the following order: [ H chain V region ] - [ L chain V region ] - [ H chain V region ] - [ L chain V region ]; or [ L chain V region ] - [ H chain V region ] - [ L chain V region ] - [ H chain V region ], wherein these regions are connected by a peptide linker, respectively.
Joint
In the present invention, the linker connecting the H chain V region and the L chain V region may be any peptide linker that can be introduced by genetic engineering methods, or any linker that is chemically synthesized. For example, linkers disclosed in the literature, such as Protein engineering, 9(3), 299-. These linkers may be the same or different in the same molecule. If a peptide linker is desired, the following are cited as examples of linkers:
Ser
Gly-Ser
Gly-Gly-Ser
Ser-Gly-Gly
Gly-Gly-Gly-Ser
Ser-Gly-Gly-Gly
Gly-Gly-Gly-Gly-Ser
Ser-Gly-Gly-Gly-Gly
Gly-Gly-Gly-Gly-Gly-Ser
Ser-Gly-Gly-Gly-Gly-Gly
Gly-Gly-Gly-Gly-Gly-Gly-Ser
Ser-Gly-Gly-Gly-Gly-Gly-Gly
(Gly-Gly-Gly-Gly-Ser)nand
(Ser-Gly-Gly-Gly-Gly)n
wherein n is an integer of not less than 1. The preferred peptide linker length varies depending on the receptor as antigen, and for single chain Fv, the range of 1-20 amino acids is generally preferred. For single chain altered antibodies comprising two or more H chain V regions and two or more L chain V regions, the peptide linker to which they are linked to form the same antigen binding site comprising [ H chain V region ] - [ L chain V region ] (or [ L chain V region ] - [ H chain V region ]) is 1-30 amino acids in length, preferably 1-20 amino acids, more preferably 3-18 amino acids. The peptide linker to which the same antigen-binding site comprising [ H chain V region ] - [ L chain V region ] (or [ L chain V region ] - [ H chain V region ]) is linked is 1 to 40 amino acids, preferably 3 to 30 amino acids, and more preferably 5 to 20 amino acids in length. In the description of the DNA constructs encoding the altered antibodies of the invention, methods of introducing these linkers will be described.
According to the present invention, the chemically synthesized linker, i.e., the chemical cross-linking agent, may be any linker conventionally used for linking peptides. Examples of linkers include N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl suberate) (BS)3) Dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethyleneglycol bis (succinimidyl succinate) (EGS), ethyleneglycol bis (sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (sulfo-DST), bis [2- (succinimidyoxycarbonyloxy) ethyl]Sulfone (BSOCOES), bis [2- (sulfosuccinimidyloxycarbonyloxy) ethyl]Sulfone (sulfo-BSOCOES), and the like. These are all commercially available. Chemically synthesized linkers of the same length as the peptide linker are preferred.
To form dimers of single chain Fv's, it is preferred that the linker is suitable to dimerize in solution, e.g., in culture, the single chain Fv produced by the host cell by more than 20%, preferably by more than 50%, more preferably by more than 80%, and most preferably by more than 90%. In particular, it is preferred that the linker consists of 2-12 amino acids, preferably 3-10 amino acids, or other corresponding linkers.
Preparation of altered antibodies
By linking known or novel monoclonal antibodies derived from molecules that specifically bind to cell surface using the above linkersAltered antibodies can be made from the H chain V region and L chain V region of the body. Examples of single chain Fv's of interest are the MABL1-scFv and the MABL2-scFv, which comprise the H chain V region and the L chain V region derived from antibody MABL-1 and antibody MABL-2, respectively. As examples of the related single-chain polypeptides comprising two H chain V regions and two L chain V regions, MABL1-sc (fv) comprising the H chain V region and the L chain V region derived from the above-mentioned antibody2And MABL2-sc (fv)2。
To prepare the polypeptide, a signal peptide may be attached to the N-terminus of the polypeptide if it is desired that the polypeptide be a secretory peptide. For efficient purification of the polypeptide, amino acid sequences well known for polypeptide purification, such as FLAG sequences, may be ligated. In this case, a dimer may be formed using an anti-FLAG antibody.
To prepare the altered antibodies of the invention, DNA must be obtained, i.e., DNA encoding a single chain Fv or DNA encoding a reconstituted single chain polypeptide. These DNAs, particularly MABL1-scFv, MABL2-scFv, MABL1-sc (fv)2And/or MABL2-sc (fv)2The DNA of (4), which is obtainable from a DNA encoding the H chain V region and the L chain V region derived from said Fv. They can also be obtained by a PCR method which amplifies a DNA portion containing the desired amino acid sequence using these DNAs as templates and primer pairs corresponding to both ends thereof.
When it is desired to have a partially altered amino acid sequence per V region, a V region in which one or some amino acids are altered, i.e., deleted, substituted or added, can be obtained by methods known in the art using PCR. To prepare an altered antibody with sufficient activity for a particular antigen, the partial amino acid sequence in the V region is preferably altered using PCR methods known in the art.
To determine the primers for PCR amplification, the types of H chain and L chain of the antibody of interest must be determined. However, the antibodies MABL-1 and MABL-2 have been reported that the antibody MABL-1 is a kappa type L chain and a gamma 1 type H chain, and the antibody MABL-2 is a kappa type L chain and a gamma 2a type H chain (JP-APPl.11-63557). For PCR amplification of DNA encoding the H chain and L chain of antibody MABL-1 and/or antibody MABL-2, primers described in Jones, S.T. et al, Bio/Technology, 9, 88-89, 1991 can be used.
For PCR amplification of the L chain V regions of antibody MABL-1 and antibody MABL-2, oligonucleotide primers for the 5 'and 3' ends were determined as described above. The 5 '-end and 3' -end oligonucleotide primers for amplifying the H chain V regions of antibody MABL-1 and antibody MABL-2 were determined in the same manner.
In an embodiment of the invention, a 5 'primer is used comprising a "GANTC" sequence providing a restriction enzyme Hinf I recognition site near its 5' end and a 3 'primer comprising a nucleotide sequence "CCCGGG" providing an XmaI recognition site near its 5' end. Other restriction enzyme recognition sites may be used in place of these sites as long as they are useful for subcloning the DNA fragment of interest into a cloning vector.
Specifically designed PCR primers are used to provide appropriate nucleotide sequences at the 5 'and 3' ends of the V region encoding cdnas for antibodies MABL-1 and MABL-2, thereby allowing the cdnas to be easily inserted into expression vectors and to function properly in expression vectors (e.g., the present invention is designed to increase translation efficiency by inserting Kozak sequences). The V regions of antibodies MABL-1 and MABL-2 obtained by PCR amplification using these primers were inserted into a HEF expression vector containing the human C region of interest (see WO 92/19759). The cloned DNA may be sequenced by any conventional method, for example, by using an automated DNA sequencer (Applied Biosystems).
A linker, e.g., a peptide linker, can be introduced into the altered antibodies of the invention as follows. Primers having partial complementary sequences of the above H chain V region and L chain V region primers, and encoding the N-terminus or C-terminus of the linker were designed. Then, PCR was performed using these primers to prepare a DNA encoding a peptide linker having the desired amino acid sequence and length. The DNA encoding the altered antibody of the present invention having the peptide linker of interest is prepared by using the DNA encoding the H chain V region and the L chain V region linked by the obtained DNA. Once the DNA encoding an altered antibody is prepared, the DNA encoding the altered antibody with or without the desired peptide linker can be easily prepared by designing various primers for the linker and performing PCR using the primers and the above DNA as templates.
Each V region of the altered antibodies of the invention can be humanized using conventional techniques (e.g., Sato k. et al, Cancer res., 53, 1-6 (1993)). Once a DNA encoding each humanized Fv has been prepared, a humanized single-chain Fv, a fragment of a humanized single-chain Fv, a humanized monoclonal antibody, and a fragment of a humanized monoclonal antibody can be easily prepared according to a conventional method. Preferably, partial changes in the amino acid sequence of the V region thereof may be made, if necessary.
Furthermore, DNAs derived from other mammals, for example, DNAs encoding each V region of human antibodies, can be prepared in the same manner as used for preparing DNAs encoding mouse-derived H chain V region and L chain V region, using the above-mentioned conventional methods. The obtained DNA can be used for preparing other mammals, particularly H chain V region and L chain V region derived from human antibody, single chain Fv derived from human and fragments thereof, and human monoclonal antibody and fragments thereof.
If the altered antibody of the invention is a bispecific altered antibody, it may be prepared by known methods (e.g. as described in WO 9413804).
As described above, if the desired coding DNA encoding the V region of the altered antibody and the V region of the humanized altered antibody is prepared, an expression vector comprising them and a host transformed with the vector can be obtained according to a conventional method. In addition, the host can be cultured according to a conventional method to prepare a reconstituted single-chain Fv, a reconstituted humanized single-chain Fv, a humanized monoclonal antibody, and a fragment thereof. It can be isolated from the cells or culture medium and purified as a homogeneous substance. For this purpose, any conventional method for protein isolation and purification may be used in combination, such as, for example, chromatography, ultrafiltration, salting out and dialysis, if necessary, but not limited thereto.
When the reconstituted single-chain Fv of the present invention is prepared by culturing animal cells, such as COS7 cells or CHO cells, preferably CHO cells, in a serum-free medium, the dimer of the single-chain Fv formed in the medium can be stably recovered and purified in high yield. The thus purified dimer can be stably preserved for a long period of time. The serum-free medium used in the present invention may be any medium conventionally used for the preparation of recombinant proteins, but is not limited thereto.
For the preparation of the altered antibodies of the invention, any expression system may be used, e.g., eukaryotic cells such as animal cells, e.g., established mammalian cell lines, filamentous fungi and yeasts, and prokaryotic cells such as bacterial cells, e.g.E.coli. Preferably, the altered antibodies of the invention are expressed in mammalian cells, such as COS7 cells or CHO cells.
In these cases, conventional promoters useful for mammalian cell expression may be used. Preferably, the immediate early promoter of Human Cytomegalovirus (HCMV) is used. Expression vectors containing the HCMV promoter include HCMV-VH-HC γ 1, HCMV-VL-HCK, etc., derived from pSV2neo (WO 92/19759).
In addition, other promoters useful for mammalian cell-based expression that can be used in the present invention include viral promoters derived from retroviruses, polyoma viruses, adenoviruses, and simian virus 40(SV40), as well as promoters derived from mammals, such as human polypeptide chain elongation factor 1 α (HEF-1 α). The SV40 promoter (Nature 277, 108-114(1979)) can be readily used according to the method of Mullingan R.C. et al, and the HEF-1. alpha. promoter (Nucleic Acids Research, 18, 5322(1990)) can also be used according to the method of Mizushima S. et al.
Origins of replication (ori) useful in the present invention include origins derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV), and the like. The expression vector may comprise the phosphotransferase APH (3') II or I (neo) gene, the Thymidine Kinase (TK) gene, the,E.coliXanthine-guanine phosphoribosyl transferase (Ecogpt) gene or dihydrofolate reductase (DHFR) gene as selectable markers.
The altered antibodies prepared as described above can be evaluated for antigen binding activity using conventional methods such as Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or surface plasmon resonance (surface plasmon resonance). The activity of the original antibody can also be evaluated using its binding-inhibiting ability as an index, for example, as to whether or not the binding of the monoclonal antibody to an antigen is inhibited in a concentration-dependent manner.
In more detail, animal cells, such as COS7 cells or CHO cells, transformed with an expression vector comprising DNA encoding the altered antibody of the present invention are cultured. Cultured cells and/or culture medium supernatant, or altered antibodies purified therefrom, are used to determine binding to an antigen. The supernatant of the medium, which was used to culture cells transformed with the expression vector alone, was used as a control. For antigens, such as antibody MABL-1 and antibody MABL-2, test samples or control supernatants of the altered antibodies of the invention are added to a mouse leukemia L1210 cell line expressing human IAP, and then subjected to, for example, flow cytometry assays to evaluate antigen binding activity.
By using the following methodIn vitroEvaluation of signal transduction effects (apoptosis-inducing effects for antibody MABL-1 and antibody MABL-2): a test sample of the altered antibody is added to cells expressing the antibody, or cells into which the antibody gene has been transferred, and evaluated by signal transduction for changes such as whether cell death is induced in a human IAP-antigen specific manner using conventional methods.
By using the following methodIn vivoEvaluating an apoptosis-inducing effect, e.g., in the case of an altered antibody recognizing human IAP (e.g., the altered antibody is derived from antibody MABL-1 and antibody MABL-2): a mouse model of human myeloma was prepared. Intravenous administration of monoclonal antibodies or altered antibodies of the invention to mice induces apoptosis of nucleated blood cells having IAP. Control mice were given PBS only. Based on the change of the human IgG content in the mouse serum and the survival time thereof, the apoptosis induction effect of the mouse is evaluated by the anti-tumor effect.
By preparing an altered antibody comprising two or more H chain V regions and two or more L chain V regions and specifically binding to a target cell surface molecule or intracellular molecule as described aboveIn vivoOrIn vitroEvaluation screening of the altered antibody can result in an altered antibody of the invention.
The altered antibodies of the invention comprising two or more H chain V regions and two or more L chain V regions, preferably 2-4 each, more preferably 2 each, may be dimeric in a single chain Fv comprising one H chain V region and one L chain V region, or a single chain polypeptide in which two or more H chain V regions and two or more L chain V regions are linked. It is believed that due to such a configuration, the peptide mimics the three-dimensional structure of the natural ligand, thereby maintaining superior antigen binding properties and agonist activity.
The altered antibodies of the invention have a significantly reduced molecular size compared to the antibody molecule (complete IgG) and thus have high permeability to tissues or tumors, with higher activity than the original agonist monoclonal antibody. Thus, appropriate selection of the parent antibody may be possible to transduce various signals into the cell and induce various effects of the cell, such as induction of apoptosis, induction of cell proliferation, induction of cell differentiation, induction of cell division or cell cycle regulation. Pharmaceutical preparations containing them are useful for the treatment of diseases treatable by induction of signal transduction, such as cancer, inflammation, hormonal disorders, autoimmune diseases and hematological disorders, such as leukemia, malignant lymphoma, aplastic anemia, myelodysplastic syndrome and polycythemia vera. It is also contemplated that the antibodies of the invention may be used as contrast agents after RI labeling. The effect can be enhanced by the attachment of an RI compound or toxin.
Best mode for working the invention
The present invention will be specifically illustrated with reference to the following examples, but does not limit the scope of the present invention.
To illustrate the method of preparing the altered antibodies of the invention, examples of single chain Fv preparations are shown below. Examples of making altered antibodies use mouse anti-human IAP antibodies, MABL-1 and MABL-2. On 11/9/1997, hybridomas producing these two antibodies, MABL-1 and MABL-2, were deposited internationally as FERM BP-6100 and FERM BP-6101 at the Institute of Biotechnology and human research, National Institute of Bioscience and human Technology, International Trade and Industry (National Institute of Bioscience and dhuman Technology, Agency of Industrial Science and Technology, Minister of International Trade and Industry) (1-3Higasi 1-Chome, Tsukuba-shi, Ibaraki-Ken, Japan), respectively.
Examples
Example 1 (cloning of DNA encoding V region of mouse anti-human IAP monoclonal antibody)
The encoding DNA for the variable regions of the mouse anti-human IAP monoclonal antibodies MABL-1 and MABL-2 was cloned as follows.
1.1 preparation of messenger RNA (mRNA)
mRNA of hybridomas MABL-1 and MABL-2 was obtained using mRNA Purification kit (Pharmacia Biotech).
1.2 Synthesis of double-stranded cDNA
Double-stranded cDNA was synthesized from approximately 1. mu.g mRNA using the Marathon cDNA Amplification kit (CLONTECH), and ligated with a linker.
1.3PCR amplification of coding groups for antibody variable regions
PCR was performed using Thermal Cycler (PERKIN ELMER).
(1) Amplification of coding gene of MABL-1L chain V region
The primers used for the PCR method were Adapter primer-1 (CLONTECH) shown in SEQ ID No.1, which hybridizes with a partial sequence of the linker, and MKC (murine Kappa constant) primer (Bio/Technology, 9, 88-89, 1991) shown in SEQ ID No.2, which hybridizes with a mouse Kappa type L chain V region.
50 μ l of PCR solution contained 5 μ l of 10 XPCR buffer II, 2mM MgCl20.16mM dNTPs (dATP, dGTP, dCTP and dTTP), 2.5 units of DNA polymerase AmpliTaqgold (PERKIN ELMER), 0.2. mu.M of the linker primer of SEQ ID No.1, 0.2. mu.M of the primer of SEQ ID No.2MKC and 0.1. mu.g of double-stranded cDNA derived from MABL-1. Solutions ofPreheating at an initial temperature of 94 ℃ for 9 minutes, and then heating at 94 ℃ for 1 minute, 60 ℃ for 1 minute, and 72 ℃ for 1 minute and 20 seconds in sequence. This temperature cycle was repeated 35 times and the reaction mixture was then heated for an additional 10 minutes at 72 ℃.
(2) cDNA encoding for amplification of MABL-1H chain V region
The Adapter primer-1 shown in SEQ ID No.1 and the MHC- γ 1 (mouse heavy chain constant) primer shown in SEQ ID No.3 (Bio/Technology, 9, 88-89, 1991) were used as PCR primers.
cDNA amplification was performed according to the amplification method of L chain V region gene described in example 1.3- (1), except that 0.2. mu.M MHC-. gamma.1 primer was used instead of 0.2. mu.M MKC primer.
(3) cDNA encoding amplified MABL-2L chain V region
Adap primer-1 of SEQ ID No.1 and MKC primer of SEQ ID No.2 were used as PCR primers.
cDNA amplification was carried out in accordance with the amplification method of the V region gene of MABL-1L strand described in example 1.3- (1), except that 0.1. mu.g of the double-stranded cDNA derived from MABL-2 was used instead of 0.1. mu.g of the double-stranded cDNA of MABL-1.
(4) cDNA encoding for amplification of MABL-2H chain V region
Adapter primer-1 of SEQ ID No.1 and MHC-. gamma.2a primer shown in SEQ ID No.4 (Bio/Technology, 9, 88-89, 1991) were used as PCR primers.
cDNA amplification was performed according to the L chain V region gene amplification method described in example 1.3- (3), except that 0.2. mu.M MHC-. gamma.2a primer was used instead of 0.2. mu.M MKC primer.
1.4 purification of PCR products
The above PCR-amplified DNA fragment was purified using QIAquick PCR purification kit (QIAGEN) and dissolved in 10mM Tris-HCl (pH8.0) containing 1mM EDTA.
1.5 ligation and transformation
About 140ng of the DNA fragment containing the gene encoding the V region derived from the Kappa-type mouse MABL-1L chain prepared above was ligated with 50ng of pGEM-T Easy vector (Promega) at 15 ℃ for 3 hours in a reaction buffer containing 30mM Tris-HCl (pH7.8), 10mM MgCl210mM dithiothreitol, 1mM ATP, and 3 units of T4DNA ligase (Promega).
Then 1. mu.l of the reaction mixture was added to 50. mu.lE.ColiIn DH 5. alpha. competent cells (Toyobo Inc.), the cells were stored on ice for 30 min, incubated at 42 ℃ for 1 min and stored on ice for a further 2 min. Add 100. mu.l SOC medium (GIBCO BRL). Will be provided withE.coliCells were inoculated in LB agar medium (Molecular Cloning: A laboratory Manual, Sambrook et al, Cold spring Harbor laboratory Press, 1989) containing 100. mu.g/ml ampicillin (SIGMA), and cultured overnight at 37 ℃ to obtainE.coliTransformants.
The transformant was cultured overnight at 37 ℃ in 3ml of LB medium containing 50. mu.g/ml ampicillin, and plasmid DNA was prepared from the culture using QIAprep Spin Minprep kit (QIAGEN).
The resulting plasmid containing the gene encoding the mouse Kappa-type L chain V region derived from hybridoma MABL-1 was designated pGEM-M1L.
A plasmid containing the mouse H chain V region-encoding gene derived from hybridoma MABL-1, designated pGEM-M1H, was prepared from the purified DNA fragment in the same manner as described above.
A plasmid containing the mouse Kappa-type L chain V region-encoding gene derived from hybridoma MABL-2, designated pGEM-M2L, was prepared from the purified DNA fragment.
From the purified DNA fragment, a plasmid was prepared which contained the mouse H chain V region-encoding gene derived from hybridoma MABL-2 and was designated pGEM-M2H.
Example 2(DNA sequencing)
The nucleotide sequence of the cDNA coding region in the above plasmid was determined using Auto DNA sequence (Applied Biosystem) and ABI PRISMDYe Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystem) according to the manufacturer's protocol.
The plasmid pGEM-M1L contains an L chain V region coding gene of a mouse antibody MABL-1, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 5.
The plasmid pGEM-M1H contains the H chain V region coding gene of the mouse antibody MABL-1, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 6.
The plasmid pGEM-M2L contains an L chain V region coding gene of a mouse antibody MABL-2, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 7.
The plasmid pGEM-M2H contains the H chain V region coding gene of the mouse antibody MABL-2, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 8.
Example 3 (determination of CDR)
The L chain is generally structurally similar to the V region of the H chain, with every four framework regions connected by three hypervariable regions, the Complementarity Determining Regions (CDRs). The amino acid sequence of the framework is relatively well conserved, while the amino acid sequence of the CDRs is extremely highly variable (Kabat E.A. et al, "Sequences of proteins of Immunological Interest", US Dept. health and HumanServices, 1983).
Based on these facts, the amino acid sequence of the variable region of a mouse anti-human IAP monoclonal antibody was applied to the amino acid sequence database of an antibody established by Kabat et al to search for homology.
Table 1 shows the CDR regions determined by homology.
TABLE 1
| Plasmids | SEQ ID No. | CDR(1) | CDR(2) | CDR(3) |
| pGEM-M1LpGEM-M1HpGEM-M2LpGEM-M2H | 5678 | 43-5850-5443-5850-54 | 74-8069-8574-8069-85 | 113-121118-125113-121118-125 |
Example 4 (identification of expression of cloned cDNA, preparation of chimeric MABL-1 antibody and chimeric MABL-2 antibody)
4.1 preparation of vectors expressing chimeric MABL-1 antibodies
cDNA clones pGEM-M1L and pGEM-M1H encoding the L chain and H chain V regions, respectively, of the mouse antibody MABL-1 were modified by PCR, and HEF expression vectors (WO92/19759) were introduced to prepare vectors for expressing the chimeric MABL-1 antibody.
The forward primer MLS for the L chain V region (SEQ ID No.9) and the forward primer MHS for the H chain V region (SEQ ID No.10) were designed to hybridize to the coding DNA at the beginning of each V region leader and to contain a Kozak consensus sequence (J.mol.biol., 196, 947-950, 1987) and a HindIII restriction enzyme site. An L chain V region reverse primer MLAS (SEQ ID No.11) and an H chain V region reverse primer MHAS (SEQ ID No.12) were designed to hybridize to the J region terminal-encoding DNA and to contain a splice donor sequence and a BamHI restriction enzyme site.
Will contain 10. mu.l of 10 XPCR buffer II, 2mM MgCl2100. mu.l of PCR solution of 0.16mM dNTPs (dATP, dGTP, dCTP and dTTP), 5 units of DNA polymerase AmpliTaq Gold, 0.4uM of each primer and 8ng of template DNA (pGEM-M1L or pGEM-M1H) was preheated at an initial temperature of 94 ℃ for 9 minutes, and then sequentially heated at 94 ℃ for 1 minute, 60 ℃ for 1 minute and 72 ℃ for 1 minute and 20 seconds. This temperature cycle was repeated 35 times and the reaction mixture was heated for an additional 10 minutes at 72 ℃.
The PCR product was purified using QIAquick PCR Purification kit (QIAGEN), and then digested with HindIII and BamHI. Cloning the product of the L chain V region into an HEF expression vector HEF-K, and cloning the product of the H chain V region into an HEF expression vector HEF-gamma. After DNA sequencing, plasmids containing the correct sequence DNA fragments were designated HEF-M1L and HEF-M1H, respectively.
4.2 preparation of vectors expressing chimeric MABL-2 antibodies
The cDNA was altered and cloned in the same manner as described in example 4.1, except that pGEM-M2L and pGEM-M2H were used as template DNA instead of pGEM-M1L and pGEM-M1H. After DNA sequencing, the plasmids containing the DNA fragments with the correct sequence were named HEF-M2L and HEF-M2H, respectively.
4.3 transfection of COS7 cells
The above expression vectors were tested in COS7 cells and transient expression of chimeric MABL-1 and MABL-2 antibodies was observed.
(1)Gene of transfection chimeric MABL-1 antibody
The HEF-M1L and HEF-M1H vectors were co-transformed into COS7 cells by electroporation using a Gene Pulser apparatus (BioRad). Each DNA (10. mu.g) and 0.8ml of 1X 10 DNA were added to the cuvette7Cells/ml PBS. The mixture was pulsed with 1.5KV 25 μ F capacitance.
After 10 min of room temperature recovery, the electroporated cells were transferred to DMFM medium containing 10% gamma-globulin free fetal bovine serum (GIBCO BRL). After 72 hours of culture, the supernatant was collected, centrifuged to remove cell debris and recovered.
(2)Transfection of Gene encoding chimeric MABL-2 antibody
COS7 cells were co-transfected with the gene encoding the chimeric MABL-2 antibody using the same method as described in example 4.3- (1), except that HEF-M2L and HEF-M2H vectors, instead of HEF-M1L and HEF-M1H vectors, were used. The supernatant was recovered in the same manner.
4.4 flow cytometry
The antigen binding was measured by flow cytometry analysis using the above COS7 cell culture supernatant. 4X 10 of the culture supernatant of COS7 cells expressing the chimeric MABL-1 antibody or COS7 cells expressing the chimeric MABL-2 antibody or human IgG antibody (SIGMA) as a control was added5Human IAP expressing mouse leukemia cell line L1210 cells were ice-washed. After washing, FITC-labeled anti-human IgG antibody (Cappel) was added thereto. After incubation and washing, the fluorescence intensity was measured with a FACScan device (BECTON DICKINSON).
Since the chimeric MABL-1 and MABL-2 antibodies specifically bound to L1210 cells expressing human IAP, it was confirmed that these chimeric antibodies had the correct V-region structures of the mouse monoclonal antibodies MABL-1 and MABL-2, respectively (fig. 1-3).
Example 5 (preparation of reconstructed Single chain fv (scFv) of antibody MABL-1 and antibody MABL-2)
5.1 preparation of reconstructed Single chain Fv of antibody MABL-1
A reconstituted single chain Fv of the antibody MABL-1 was prepared as follows. The H chain V region and the L chain V region of the antibody MABL-1, and a linker were amplified separately by PCR and ligated together to prepare a reconstituted single chain Fv of the antibody MABL-1. The preparation method is shown in figure 4. Single-chain Fv of antibody MABL-1 was prepared using six primers (A-F). Primers A, C and E have sense sequences and primers B, D and F have antisense sequences.
The forward primer VHS (primer A, SEQ ID No.13) for the H chain V region was designed to hybridize with the DNA encoding the N-terminus of the H chain V region and contains a NcoI restriction enzyme recognition site. The reverse primer VHAS (primer B, SEQ ID No.14) of the H chain V region was designed to hybridize to the C-terminal coding DNA of the H chain V region and overlap the linker.
The forward primer LS (primer C, SEQ ID No.15) of the linker was designed to hybridize to the DNA encoding the N-terminus of the linker and overlap with the DNA encoding the C-terminus of the H chain V region. The reverse primer LAS (primer D, SEQ ID No.16) of the linker is designed to hybridize to the C-terminal coding DNA of the linker and overlap with the N-terminal coding DNA of the L chain V region.
The forward primer VLS (primer E, SEQ ID No.17) for the L chain V region was designed to hybridize to the linker C-terminal coding DNA and overlap with the L chain V region N-terminal coding DNA. The reverse primer VLAS-FLAG (primer F, SEQ ID No.18) of the L chain V region was designed to hybridize with the DNA coding for the C-terminus of the L chain V region and has a FLAG peptide coding sequence (Hopp T.P. et al, Bio/Technology, 6, 1204-.
In the first PCR step, three reactions A-B, C-D, E-F were performed and their PCR products were purified. The three PCR products obtained from the first PCR step were assembled according to their complementarity. Primers A and F were then added to amplify the full-length DNA encoding the reconstituted single-chain Fv of antibody MABL-1 (second PCR). Plasmids used as templates in the first PCR step respectively include the plasmid pGEM-M1H encoding the V region of the H chain of the antibody MABL-1 (see example 2), the plasmid pSC-DP1 comprising a DNA sequence encoding a linker region comprising Gly GlyGly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID No.19) (Huston J.S. et al, Proc. Natl Acad. Sci. USA, 85, 5879-5883, 1988), and the plasmid pGEM-M1L encoding the V region of the L chain of the antibody MABL-1 (see example 2).
50 μ l of the first PCR reaction contained 5 μ l of 10 XPCR buffer II, 2mM MgCl20.16mM dNTPs, 2.5 units of DNA polymerase AmpliTaq Gold (PERKIN ELMER), 0.4. mu.M of each primer and 5ng of each template DNA. The PCR solution was preheated at an initial temperature of 94 ℃ for 9 minutes, and then heated at 94 ℃ for 1 minute, 65 ℃ for 1 minute, and 72 ℃ for 1 minute and 20 seconds in this order. This temperature cycle was repeated 35 times, then 72 deg.CThe reaction mixture was heated for an additional 7 minutes.
The PCR products A-B (371bp), C-D (63bp) and E-F (384bp) were purified using QIApuick PCR Purification kit (QIAGEN) and assembled in the second PCR step. In the second PCR, 120ng of the first PCR product A-B, 20ng of the PCR product C-D and 120ng of the PCR product E-F, 10. mu.l of 10 XPCR buffer II, 2mM MgCl20.16mM dNTPs, 5 units of DNA polymerase AmpliTaq Gold (PERKINELMER), and the initial temperature of 94 ℃ preheating 8 minutes, and then 94 ℃ heating 2 minutes, 65 ℃ 2 minutes, 72 ℃ 2 minutes. This temperature cycle was repeated 2 times, and then 0.4. mu.M of each of primers A and F was added to the reaction. The mixture was preheated at an initial temperature of 94 ℃ for 1 minute, and then heated at 94 ℃ for 1 minute, 65 ℃ for 1 minute, and 72 ℃ for 1 minute and 20 seconds in this order. This temperature cycle was repeated 35 times and the reaction mixture was then heated for an additional 7 minutes at 72 ℃.
The 843bp DNA fragment prepared by the second PCR was purified and digested with NcoI and EcoRI. The resulting DNA fragment was cloned into the pSCFVT7 vector. The expression vector pSCFVT7 containsE.coliThe pelB signal sequence of the periplasmic expression system (Lei S.P. et al, J.bacteriology, 169, 4379-4383, 1987). After DNA sequencing, the plasmid encoding the DNA fragment containing the reconstructed single chain Fv correct amino acid sequence of the antibody MABL-1 was designated "pscM 1" (see fig. 5). The plasmid pscM1 contains the nucleotide and amino acid sequence of the antibody MABL-1 reconstituted single chain Fv as shown in SEQ ID No. 20.
The pscM1 vector was altered by PCR to prepare a vector for reconstitution of single chain Fv by expressing the antibody MABL-1 in mammalian cells. The resulting DNA fragment was introduced into the pCHO1 expression vector. The expression vector pCHO1 was constructed by digesting DHFR-. DELTA.E-rvH-PM 1-f (WO92/19759) with EcoRI and SmaI, removing the antibody gene, and ligating an EcoRI-NotI-BamHI adaptor (Takara ShuZo).
A Sal-VHS primer shown in SEQ ID No.21 was designed as a forward primer for PCR, which hybridizes to the DNA coding for the N-terminus of the V region of the H chain and contains a Sal I restriction enzyme recognition site. The FRH1anti primer shown in SEQ ID No.22 was designed as a PCR reverse primer that hybridizes to the coding DNA at the end of the first framework sequence.
Will contain 10. mu.l of 10 XPCR buffer II, 2mM MgCl20.16mM dNTPs, 5 units of DNA polymerase AmpliTaq Gold, 0.4. mu.l of each primer and 100. mu.l of a PCR solution of 8ng of template DNA (pscM1) were preheated at an initial temperature of 95 ℃ for 9 minutes, and then heated at 95 ℃ for 1 minute, 60 ℃ for 1 minute and 72 ℃ for 1 minute and 20 seconds in this order. This temperature cycle was repeated 35 times and the reaction mixture was then heated for an additional 7 minutes at 72 ℃.
The PCR product was purified using QIAquick PCR purification kit (QIAGEN), and digested with SalI and MboII to obtain a DNA fragment encoding the N-terminus of the reconstituted single-chain Fv of antibody MABL-1. The pscM1 vector was digested with MboII and EcoRI to obtain a reconstructed single-chain Fv C-terminal encoding DNA fragment of antibody MABL-1. The SalI-MboII DNA fragment and the MboII-EcoRI DNA fragment were cloned into the pCH01-IgS vector. After DNA sequencing, the plasmid containing the DNA sequence of interest was designated "pCHOM 1" (see FIG. 6). The expression vector pCHO1-Igs contains the mouse IgG1 signal sequence suitable for the secretory expression system of mammalian cells (Nature, 322, 323-327, 1988). The nucleotide sequence and the amino acid sequence of the antibody MABL-1 reconstructed single-chain Fv contained in the plasmid pCHOM1 are shown in SEQ ID No. 23.
5.2 preparation of reconstructed Single chain Fv of antibody MABL-2
A reconstituted single chain Fv of the antibody MABL-2 was prepared as described in example 5.1 above. The plasmid pGEM-M2H encoding the H chain V region of MABL-2 (see example 2) but not pGEM-M1H, and the plasmid pGEM-M2L encoding the L chain V region of MABL-2 (see example 2) but not pGEM-M1L were used in the first PCR step to obtain a DNA fragment encoding the amino acid sequence of interest of the plasmid pscM2, which contains the single chain Fv of antibody MABL-2. The plasmid pscM2 contains a reconstituted single chain Fv of the antibody MABL-2, the nucleotide and amino acid sequence of which is shown in SEQ ID No. 24.
The vector pCHOM2, which contains a DNA fragment encoding the correct amino acid sequence of the reconstituted single chain Fv of the antibody MABL-2, was prepared for expression in mammalian cells by PCR method with the pscM2 vector altered. The nucleotide sequence and amino acid sequence of the reconstructed single-chain Fv of the antibody MABL-2 contained in plasmid pCHOM2 are shown in SEQ ID No. 25.
5.3 transfection of COS7 cells
The pCHOM2 vector was tested in COS7 cells and transient expression of the reconstituted single chain Fv of antibody MABL-2 was observed.
Using a Gene Pulser apparatus (BioRad), COS7 cells were transformed with pCHOM2 by electroporation. To a cuvette, DNA (10. mu.g) and 0.8ml of 1X 10 DNA were added7Cells/ml PBS. The mixture was pulsed with a 1.5KV 25 μ F capacitance.
After 10 min of room temperature recovery, the electroporated cells were transferred to IMDM medium containing 10% fetal bovine serum (GIBCO BRL). After 72 hours of culture, the supernatant was collected, centrifuged to remove cell debris and recovered.
5.4 detection of reconstituted Single chain Fv of antibody MABL-2 in COS7 cell culture supernatant
The presence of single-chain Fv of antibody MABL-2 was confirmed by Western blotting in the culture supernatant of COS7 cells transfected with pCHOM2 vector.
Culture supernatants of COS7 cells transfected with pCHOM2 vector and COS7 cells transfected with pCHO1 as a control were subjected to SDS electrophoresis and transferred to REINFORCED NC membrane (Schleicher & Schuell). The membranes were blocked with 5% skim milk (Morinaga Nyu-gyo), washed with 0.05% Tween20-PBS, and mixed with anti-FLAG antibody (SIGMA). The membranes were incubated at room temperature, washed and mixed with alkaline phosphatase-conjugated mouse IgG antibody (Zymed). After incubation and washing at room temperature, the substrate solution (Kirkegaard Perry Laboratories) was added for color development (FIG. 7).
The FLAG peptide-specific protein was detected only in the culture supernatant of COS7 cells introduced with pCHOM2 vector, thereby confirming the secretion of the reconstituted single-chain Fv of antibody MABL-2 into the culture supernatant.
5.5 flow cytometry
Antigen binding was determined by flow cytometry using the COS7 cell culture supernatant described above. COS7 cell culture supernatant of reconstituted single chain Fv expressing antibody MABL-2, or COS7 cell culture supernatant transformed with pCHO1 vector as control, was added with 2X 105The mouse leukemia cell line L1210 expressing human integrin-associated protein (IAP) or the L1210 cell line transformed with pCOS1 as a control. After ice-bath and washing, mouse anti-FLAG antibody (SIGMA) was added. The cells were then incubated and washed. After that, FITC-labeled anti-mouse IgG antibody (BECTONDICKINSON) was added thereto, and the cells were incubated and washed again. The fluorescence intensity was subsequently measured with a FACScan device (BECTON DICKINSON).
Since the single-chain Fv of the antibody MABL-2 specifically binds to L1210 cells expressing human IAP, it was determined that the reconstituted single-chain Fv of the antibody MABL-2 has affinity for human integrin-associated protein (IAP) (see fig. 8-11).
5.6 competitive ELISA
The binding activity of the reconstituted single-chain Fv of the antibody MABL-2 was determined on the basis of the inhibitory activity against the binding of a mouse monoclonal antibody to an antigen.
The anti-FLAG antibody was adjusted to 1. mu.g/ml, added to each well of a 96-well plate, and incubated at 37 ℃ for 2 hours. After washing, blocking with 1% BSA-PBS was performed. After incubation and washing at room temperature, COS7 cell culture supernatant into which the secreted human IAP antigen gene (SEQ ID No.26) was introduced was diluted to two volumes with PBS and added to each well. After incubation and washing at room temperature, 50. mu.l of a mixture of biotinylated MABL-2 antibody adjusted to 100ng/ml and 50. mu.l of serially diluted COS7 cell culture supernatants expressing the reconstituted single chain Fv of antibody MABL-2 were added to each well. After incubation and washing at room temperature, alkaline phosphatase-conjugated streptavidin (Zymed) was added to each well. After incubation and washing at room temperature, substrate Solution (SIGMA) was added and the absorbance of the reaction mixture at 405nm was measured in each well.
The results showed that the reconstituted single chain Fv (MABL2-scFv) concentration-dependent significant inhibition of the binding of the mouse antibody MABL-2 to human IAP antigen, compared to the control COS7 cell culture supernatant introduced with pCHO1 (fig. 12). Thus, it was suggested that the structure of each V region derived from the mouse monoclonal antibody MABL-2 was correct in the reconstituted single-chain Fv of the antibody MABL-2.
5.7 in vitro apoptosis inducing Effect
Apoptosis-inducing effect of reconstituted single-chain Fv of antibody MABL-2 was examined by Annexin-V staining (Boehringer Mannheim) using L1210 cells transfected with human IAP gene, L1210 cells transfected with pCOS1 vector as a control, and CCRF-CEM cells.
At 1X 105To each of the above cells, COS7 cell culture supernatant expressing reconstituted single chain Fv of antibody MABL-2, or COS7 cell culture supernatant transfected with pCHO1 vector as a control was added at a final concentration of 50%, and the mixture was cultured for 24 hours. Then, Annexin-V staining was performed, and the fluorescence intensity was measured by a FACScan apparatus (BECTON DICKINSON).
The results of Annexin-V staining are shown in FIGS. 13-18, respectively. The lower left-hand spot represents live cells, the lower right-hand spot represents early apoptotic cells, and the upper right-hand spot represents late apoptotic cells. The results show that the reconstituted single-chain Fv of the antibody MABL-2 (MABL2-scFv) significantly induced L1210 cell death specific for human IAP antigen (fig. 13-16), and compared to the control, the reconstituted single-chain Fv also significantly induced CCRF-CEM cell death (fig. 17-18).
5.8 expression of MABL-2-derived Single-chain Fv in CHO cells
CHO cells were transfected with pCHOM2 vector to establish CHO cell lines that constantly express single chain Fv (polypeptide) derived from antibody MABL-2.
The pCHOM2 vector was transformed into CHO cells by electroporation using the Gene Dulser apparatus (BioRad). The cuvette was filled with DNA (10. mu.g) and 0.7ml of CHO-containing cells (1X 10)7Cells/ml) in PBS. The mixture was pulsed with a 1.5KV 25 μ F capacitance. Recovery at Room temperature 1After 0 min, the electroporated cells were transferred to nucleic acid-free α -MEM medium (GIBCO BRL) containing 10% fetal bovine serum for culture. The expression of the target protein in the obtained clones was confirmed by SDS-PAGE, and the clones with a high expression level were selected as a production cell line for single-chain Fv derived from the antibody MABL-2. The cell line was cultured in 10nM methotrexate (SIGMA) in CHO-S-SFM II (GIBCO BRL) serum-free medium. Then, the culture supernatant was collected, centrifuged to remove cell debris, and recovered.
5.9 purification of MABL-2-derived Single chain Fv produced in CHO cells
The culture supernatants of the single-chain Fv-expressing CHO cell lines obtained in example 5.8 were concentrated up to 20-fold using an artificial dialysis column (PAN130SF, ASAHI MEDICALS). The concentrate was stored at-20 ℃ and thawed upon purification.
Single-chain Fv was purified from CHO cell culture supernatant using three chromatographic methods, Blue-sepharose, hydroxyapatite and gel filtration.
(1)Blue-sepharose column chromatography
The concentrated supernatant was diluted 10-fold with 20mM acetate buffer (pH6.0), and insoluble matter was removed by centrifugation (10000 Xrpm, 30 minutes). The Blue-sepharose column (20ml) was equilibrated with the same buffer and the supernatant was applied. After washing the column with the same buffer, the adsorbed proteins in the column were eluted with a stepwise gradient of 0.1, 0.2, 0.3, 0.5 up to 1.0M NaCl in the same buffer. The flow through fractions and each eluted fraction were analyzed by SDS-PAGE. Fractions confirmed to contain single chain Fv (0.1-0.3M NaCl eluate) were mixed together and concentrated up to about 20-fold using Centri Prep-10 (AMICON).
(2)Hydroxyapatite
(1) The concentrate obtained in (1) was diluted 10-fold with 10mM phosphate buffer (pH7.0) and loaded on a hydroxyapatite column (20ml, BIORAD). The column was washed with 60ml of 10mM phosphate buffer (pH 7.0). The adsorbed proteins in the column were then eluted with a linear gradient of up to 200mM sodium phosphate buffer (see FIG. 19). Each fraction was analyzed by SDS-PAGE to confirm the presence of single-chain Fv in fraction A and fraction B.
(3)Gel filtration
Fractions A and B from (2) were each concentrated with CentriPrep-10 and applied to a TSKgelG3000SWG column (21.5X 600mM) equilibrated with 20mM acetate buffer (pH6.0) containing 0.15M NaCl. The chromatogram is shown in FIG. 20. The fractions were analyzed by SDS-PAGE, confirming that the two main peaks (AI and BI) are the desired single chain Fv. In the gel filtration analysis, fraction A eluted at an apparent molecular weight of 36kDa and fraction B eluted at 76 kDa. The purified single chain Fv (AI, BI) was analyzed on a 15% SDS polyacrylamide gel. Samples were treated in the presence or absence of reducing agents and electrophoresed according to the Laemmli's method. The protein was then stained with Coomassie Brilliant blue. As shown in FIG. 21, AI and BI exhibited a single strand at an apparent molecular weight of 35kDa, regardless of the presence or absence of reducing agent. From the above, it can be inferred that AI is a monomer of a single-chain Fv and BI is a non-covalently bound dimer of a single-chain Fv. Analysis of component AI and BI by gel filtration using a tskgel g3000SW column (7.5 × 60mm) showed that the monomeric peak was detected only in component AI and the dimeric peak was detected only in component BI (figure 22). The dimer component (component BI) comprises four percent of the total single chain Fv. More than 90% of the dimer in the dimer component was stable for more than 1 month at 4 ℃.
5.10 construction of vector for expressing Single chain Fv derived from antibody MABL-2 in E.coli cells
The vector of pscM2 was modified by PCR to prepareE.coliA vector for efficiently expressing a single-chain Fv of the antibody MABL-2 in a cell. The resulting DNA fragment was introduced into the pSCFVT7 expression vector.
The Nde-VHSm02 primer shown in SEQ ID No.27 was designed to hybridize with DNA encoding the N-terminus of the V region of the H chain as the PCR forward primer and to contain the initiation codon and the NdeI restriction enzyme recognition site. The VLAS primer shown in SEQ ID No.28 was designed as a PCR reverse primer which hybridizes to the DNA coding for the C-terminus of the V region of the L chain and contains two stop codons and an EcoRI restriction enzyme recognition site. Is at the same timeE.coliTherein is provided withEfficiently expressed, 5 point mutations were contained in the portion of the forward primer Nde-VHSm02 hybridizable with the DNA encoding the N-terminus of the H chain V region.
Will contain 10. mu.l of 10 XPCR buffer #1, 1mM MgCl20.2mM dNTPs, 5 units KOD DNA polymerase (all from TOYOBO), 1. mu.M of each primer and 100ng of template DNA (pscM2) in 100. mu.l of PCR solution were heated at 98 ℃ for 15 seconds, 65 ℃ for 2 seconds and 74 ℃ for 30 seconds in this order. This temperature cycle was repeated 25 times.
The PCR product was purified using QIAquick PCR purification kit (QIAGEN), digested with NdeI and EcoRI, and the resulting DNA fragment was then cloned into the pSCFVT7 vector from which the pel B signal sequence had been removed by digestion with NdeI and EcoRI. After DNA sequencing, the resulting plasmid containing the DNA fragment carrying the DNA sequence of interest was designated "pscM 2DEm 02" (see FIG. 23). The nucleotide sequence and amino acid sequence of the single chain Fv derived from the antibody MABL-2 contained in the plasmid pscM2DEm02 are shown in SEQ id No. 29.
5.11 expression of Single chain Fv derived from antibody MABL-2 in E.coli cells
Transformation with the pscM2DEm02 vectorE.coliBL21(DE3) pLysS (STRATAGENE) for expression of single chain Fv derived from antibody MABL-2E.coliAnd (3) strain. The expression of the target protein in the obtained clones was examined by SDS-PAGE, and the clones with a high expression level were selected as strains producing single-chain Fv derived from the antibody MABL-2.
5.2 purification of Single chain Fv derived from antibody MABL-2 produced in E.coli
Obtained by transformationE.coliThe single clones were cultured in 3ml of LB medium at 28 ℃ for 7 hours and then in 70ml of LB medium at 28 ℃ overnight. The preculture was transferred to 7L of LB medium, stirred at 300 rpm and cultivated at 28 ℃ in a Jar fermenter. When the absorbance of the medium reached o.d. ═ 1.5, the bacteria were induced with 1mM IPTG, followed by culture for 3 hours.
The bacterial pellet was recovered by centrifugation (10000 Xg, 10 min) of the medium. To the bacteria, 50mM Tris-HCl buffer (pH8.0) containing 5mM EDTA, 0.1M NaCl and 1% Triton x-100 was added, and the bacteria were disrupted by ultrasonication (output: 4, duty cycle: 70%, 1 minute X10 times). The suspension of disrupted bacteria was centrifuged (12000g, 10 min) to pellet the inclusion bodies. The separated inclusion bodies were mixed with 50mM Tris-HCl buffer (pH8.0) containing 5mM EDTA, 0.1M NaCl and 4% Tritonx-100, sonicated again (output: 4, duty cycle: 50%, 30 seconds. times.2 times), centrifuged (12000g, 10 minutes) to separate the objective protein precipitate, and the protein contained in the supernatant was removed.
Inclusion bodies containing the protein of interest were lysed in 50mM Tris-HCl buffer (pH8.0) containing 6M urea, 5mM EDTA and 0.1M NaCl and loaded onto a Sephacryl S-300 gel filtration column (5X 90cm, Amersham Pharmacia) equilibrated with 50mM Tris-HCl buffer (pH8.0) containing 4M urea, 5mM EDTA, 0.1M NaCl and 10mM mercaptoethanol at a flow rate of 5 ml/min to remove the relevant high molecular weight single chain Fv. Analyzing the resulting fraction by SDS-PAGE, diluting the high purity protein fraction to O.D. with the buffer used for gel filtration2800.25. The fraction was then dialyzed 3 times against 50mM Tris-HCl buffer (pH8.0) containing 5mM EDTA, 0.1M NaCl, 0.5 MAGg, 2mM reduced glutathione and 0.2mM oxidized glutathione to refold the protein. The fraction was further dialyzed against 20mM acetate buffer (pH6.0) containing 0.15M NaCl three times to change the buffer.
The dialyzed product was applied to a Superdex200 pg gel filtration column (2.6X 60cm. Amersham Pharmacia) which had been equilibrated with 20mM acetate buffer (pH6.0) containing 0.15M NaCl to remove a small amount of high molecular weight protein crosslinked by intermolecular S-S bonds. As shown in fig. 24, two peaks, a main peak and a secondary peak, eluted after a broad peak (expected to be a high molecular weight aggregate). SDS-PAGE analysis (see FIG. 21) and gel filtration analysis of the elution positions of the two peaks suggested that the main peak was single-chain Fv monomer and the secondary peak was non-covalently bound single-chain Fv dimer. The non-covalently bound dimer accounts for 4% of the total single chain Fv.
5.13 in vitro apoptosis of Single chain Fv derived from antibody MABL-2Inducing Activity
CHO cells and hIAP were detected by two Annexin-V staining (Boehringer Mannheim) methods using L1210 cells into which human IAP genes were introduced (hIAP/L1210)E.coliApoptosis induction of the resulting single chain Fv derived from the antibody MABL-2 (MABL 2-scFv).
In the first method, the direction is 5X 104To each hIAP/L1210 cell, a sample antibody was added at a final concentration of 3. mu.g/ml, and cultured for 24 hours. Analysis of sample antibodies, monomer and dimer of MABL-2 Single chain Fv from CHO cells obtained in example 5.9, example 5.12E.coliThe resulting MABL-2 single-chain Fv monomers and dimers, and the control mouse IgG antibody. After incubation, Annexin-V staining was performed, and the fluorescence intensity was measured using a FACScan apparatus (BECTON DICKINSON).
In the second method, the direction is 5X 104To each hIAP/L1210 cell, the sample antibody was added at a final concentration of 3. mu.g/ml, cultured for 2 hours, and mixed with anti-FLAG antibody (SIGMA) at a final concentration of 15. mu.g/ml, and cultured for another 22 hours. Sample antibodies, which are monomers of MABL-2 single-chain Fv obtained from CHO cells of example 5.9, and control mouse IgG antibodies were analyzed. After the incubation, Annexin-V staining was performed, and the fluorescence intensity was measured using a FACScan apparatus.
The results of Annexin-V staining analysis are shown in FIGS. 25-31. The results showed that CHO cells andE.colithe resulting dimers of the MABL-2 single-chain Fv polypeptide significantly induced cell death (fig. 26, 27) compared to the control (fig. 25), while targeting CHO cells andE.colino induction of apoptosis was observed with the monomer of the resulting MABL-2 single-chain Fv polypeptide (fig. 28, 29). When anti-FLAG antibodies were used together, the single chain Fv polypeptide monomer derived from MABL-2 antibody produced in CHO cells induced significant cell death compared to the control (fig. 30) (fig. 31).
5.14 antitumor Effect of monomers and dimers of scFv/CHO Polypeptides against human myeloma model mice
Should be taken
(1) Quantitative measurement of human IgG in mouse serum
Human IgG (M protein) produced by human myeloma cells contained in mouse serum was measured by the following ELISA method. Goat anti-human IgG antibody (BIOSOURCE, Lot #7902) was diluted to 1. mu.g/ml with 0.1% bicarbonate buffer (pH9.6), and 100. mu.l was added to each well of a 96-well plate (Nunc), and incubated overnight at 4 ℃ to immobilize the antibody. After blocking, 100 μ l of stepwise diluted mouse serum or human IgG as standard (CAPPEL, Lot #00915) was added to each well and incubated for 2 hours at room temperature. After washing, 10. mu.l of 5000-fold diluted alkaline phosphatase-labeled anti-human IgG antibody (BIOSOURCE, Lot #6202) was added and incubated at room temperature for 1 hour. After washing, the substrate solution was added. After incubation, absorbance at 405nm was measured using a microplateder Model 3550 (BioRad). The concentration of human IgG in mouse serum was calculated based on a calibration curve obtained from the absorbance values of human IgG standards.
(2)Preparation of antibody for administration
On the day of administration, the scFv/CHO polypeptide monomers and dimers were diluted to 0.4mg/ml or 0.25mg/ml with sterile filtered PBS (-) respectively to prepare samples for administration.
(3)Preparation of mouse model of human myeloma
A mouse model of human myeloma was prepared as follows. KPMM2 cells (JP-appl.7-236475) passaged in SCID mice (Japan Clare) were suspended in RPMI1640 medium containing 10% fetal bovine serum (GIBCO-BRL) and adjusted to 3X 107Cells/ml. 200. mu.l of KPMM2 cell suspension (6X 10) were placed through the tail vein6Cells/mouse) were transplanted into SCID mice (male, 6 weeks old) that had been injected subcutaneously with asialo GM1 antibody (WAKO JUNYAKU, 1 vial in 5ml) the day before transplantation.
(4)Administration of antibodies
The antibody sample prepared in (2), monomer (250. mu.l) and dimer (400. mu.l) were administered to the human myeloma cell model mouse prepared in (3) via the tail vein. Administration was started 3 days after transplantation of KPMM2 cells, 2 times daily for 3 days. As a control, 200gl sterile-filtered PBS (-) was also administered through the tail vein 2 times a day for 3 days. Each group contained 7 mice.
(5)Evaluation of monomer and dimer anti-tumor of scFv/CHO Polypeptides with human myeloma model mice Tumor effect
The anti-tumor effect of scFv/CHO polypeptide monomers and dimers was evaluated with a human myeloma model mouse based on changes in the concentration of human IgG (M protein) in the mouse serum and the survival time of the mouse. Mouse sera were collected 24 days after the transplantation of KPMM2 cells, and changes in human IgG concentration were measured by ELISA method in (1) above. The amount of human IgG (M protein) in the serum of the PBS (-) dosed group (control) was increased to about 8500. mu.g/ml, whereas the amount of human IgG in the scFv/CHO dimer dosed group was significantly lower, equal to or less than one tenth of that of the control group. Therefore, this result indicates that the scFv/CHO dimer strongly inhibited KPMM2 cell growth (fig. 32). As shown in FIG. 33, the survival time observed in the scFv/CHO dimer-administered group was significantly prolonged as compared with the PBS (-) administered group.
As described above, it was confirmed that the scFv/CHO dimer had an antitumor effect on a human myeloma model mouse. It is believed that the anti-tumor effect of the scFv/CHO dimer, i.e., the altered antibody of the present invention, is due to the apoptosis-inducing effect of the altered antibody.
5.15 hemagglutination reaction experiment
According to Tokyo Kagaku Dojin, edited by the Japan society of biochemistry, Zoku-Seikagaku Jikken Koza "Immuno-Biochemical Investigation", a hemagglutination reaction test was performed and the hemagglutination reaction was determined.
Blood from healthy donors was taken using a heparin-treated syringe, washed three times with PBS (-) and then a 2% final concentration suspension of erythrocytes was prepared with PBS (-). The test samples include, MABL-2 antibody, monomer and dimer of single-chain Fv polypeptide produced by CHO cells,E.colimonomers and dimers of the resulting single chain Fv polypeptidesMouse IgG was used as a control (ZYMED). The hemagglutination effect was studied using round bottom 96-well plates purchased from Falcon. Mu.l of the above antibody sample and 50. mu.l of a 2% erythrocyte suspension were added to each well and mixed in the well. After incubation at 37 ℃ for 2 hours, the reaction mixture was stored overnight at 4 ℃ and the hemagglutination reaction was determined. The control was 50. mu.l PBS (-) per well, and the hemagglutination reaction test was performed in the same manner. The final antibody concentrations of mouse IgG and antibody MABL-2 used were 0.01, 0.1, 1.0, 10.0, or 100.0. mu.g/ml. Single-chain Fv were used at final concentrations of 0.004, 0.04, 0.4, 4.0, 40.0 or 80.0. mu.g/ml, but onlyE.coliIn the case of the produced polypeptide dimer, 160.0. mu.g/ml was additionally used. The results are shown in Table 2. For antibody MABL-2, hemagglutination was observed at concentrations greater than 0.1. mu.g/ml, whereas no hemagglutination was observed for both monomer and dimer of single-chain Fv.
TABLE 2 hemagglutination response test
| Control | 0.01 | 0.1 | 1 | 10 | 100 | μg/ml | |||
| mIgGMABL-2 (complete) | -- | -- | -+ | -+++ | -+++ | -++ | |||
| Control | 0.004 | 0.04 | 0.4 | 4 | 40 | 80 | μg/ml | ||
| scFv/CHO monomer scFv/CHO dimer | -- | -- | -- | -- | -- | -- | -- | ||
| Control | 0.004 | 0.04 | 0.4 | 4 | 40 | 80 | 160 | μg/ml | |
| scFv/E.coli monomer scFv/E.coli dimer | -- | -- | -- | -- | -- | -- | -- | - |
Example 6 altered antibody sc (fv) comprising 2H chain V regions and 2L chain V regions2And the antibody MABL-2scFv with a linker of different length.
6.1 construction of antibody MABL-2sc (fv)2Expression plasmid
The above pCHOM2, which contained DNA encoding scFv derived from the above MABL-2, was modified by the following PCR method, and the resulting DNA fragment was inserted into pCHOM2 to prepare a modified antibody [ sc (fv)2](which comprises two H chain V regions and two L chain V regions derived from antibody MABL-2).
The primers used for PCR include the EF1 primer (SEQ ID No: 30) which is a forward primer designed to hybridize with the EF 1. alpha. encoding DNA, the reverse primer (SEQ ID No: 19) designed to hybridize with the DNA encoding the C-terminus of the V region of the L chain and comprising a linker region encoding DNA sequence, and the VLLAS primer (SEQ ID No: 31) comprising a SalI restriction enzyme recognition site.
100 μ l PCR solution contained 10 μ l10 XPCR buffer #1, 1mM MgCl20.2mM dNTPs (dATP, dGIP, dCTP and dTTP), 5 units KOD DNA polymerase (Toyobo, Inc.), 1. mu.M of each primer, and 100ng of template DNA (pCHOM 2). The PCR solution was heated at 94 ℃ for 30 seconds, at 50 ℃ for 30 seconds and at 74 ℃ for 1 minute in this order. This temperature cycle was repeated 30 times.
The PCR product was purified using QIAquick PCR Purification kit (QIAGEN) and digested with SalI. Cloning of the resulting DNA fragment into pBluescript KS+Vector (Toyobo, Inc.). After DNA sequencing, a plasmid containing the DNA sequence of interest was digested with SalI, and the resulting DNA fragment was ligated with SalI-digested pCHOM2 using a Rapid DNA Ligation kit (BOEHRINGER MANNHEIM). After DNA sequencing, the plasmid containing the DNA sequence of interest was designated "pCHOM 2(Fv)2"(see fig. 34). Plasmid pCHOM2(Fv)2Antibody MABL-2sc (fv) contained in (1)2The nucleotide sequence and amino acid sequence of the region are shown in SEQ ID No. 32.
6.2 expression plasmids of antibody MABL-2scFv with different length linkers were prepared.
scFv were prepared as described below using cDNA encoding the H chain and L chain derived from MABL-2 as templates, which comprise linkers of different lengths and V regions, and were designed in the order of [ H chain ] - [ L chain ] (hereinafter referred to as "HL") or [ L chain ] - [ H chain ] (hereinafter referred to as "LH").
Utilizing pCHOM2(Fv)2As a template, HL scFv was constructed by PCR. In the PCR step, the primer pair CFHL-F1(SEQ ID No: 33) and CFHL-R2(SEQ ID N)o: 34) or primer pair CFHL-F2(SEQ ID No: 35) and CFHL-R1(seq id No: 36) and KOD polymerase. A PCR program was performed by repeating 30 times a temperature cycle of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds and 72 ℃ for 1 minute to produce H chain cDNA whose 5 'end contains a leader sequence or L chain cDNA whose 3' end contains a FLAG sequence. The resulting H chain and L chain cDNAs were mixed, PCR was performed, and temperature cycling was performed by 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 1 minute in this order, which was repeated 5 times, using the mixture as a template and KOD polymerase. The CFHL-F1 and CFHL-R1 primers were added to the reaction mixture, and then the above temperature cycle was repeated 30 times to perform a PCR reaction, to generate an adaptor-free HL-0 type cDNA.
To construct LH-type scFv, PCR reaction was carried out using pGEM-M2L and pGEM-M2H as templates, which contained cDNAs encoding the L chain V region and H chain V region, respectively, of antibody MABL-2 (see JP-appl. 11-63557). Primer pair T7(SEQ ID NO: 37) and CFLH-R2(SEQ ID NO: 38), or primer pair CFLH-F2(SEQ ID NO: 39) and CFLH-R1(SEQ ID NO: 40), and KOD polymerase (Toyobo Inc.) were used. The temperature cycle was repeated 30 times at 94 ℃ for 30 seconds, 60 ℃ for 30 seconds and 72 ℃ for 1 minute in this order to carry out PCR reaction to produce L chain cDNA whose 5 'end contains a leader sequence or H chain cDNA whose 3' end contains a FLAG sequence. The resulting L chain and H chain cDNAs were mixed, and PCR was performed by repeating 5 times using KOD polymerase in a temperature cycle comprising 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 1 minute in this order using the mixture as a template. The reaction mixture was added with T7 and CFLH-R1 primers, and the above temperature cycle was repeated 30 times. PCR was performed using the reaction product as a template, using a primer pair CFLH-F4(SEQ ID No: 41) and CFLH-R1, in a temperature cycle comprising 30 seconds at 94 ℃, 30 seconds at 60 ℃ and 1 minute at 72 ℃ in this order, repeated 30 times to generate a linker-free LH-0 type cDNA.
The resulting LH-0 and HL-0 type cDNAs were digested with EcoRI and BamHI restriction enzymes (Takara Shuzo), and the digested cDNAs were introduced into a mammalian expression plasmid INPEP4 using Ligation High (Toyobo Inc.), respectively. Transformation competence with each plasmidE.coliJM109(Nippon Gene), transformed with QIAGEN Plasmid Maxi kit (QUIAGEN)E.coliIsolating the plasmid of interest. Thus, plasmids pCF2LH-0 and pC were preparedF2HL-0。
To construct HL type expression plasmids containing linkers of different sizes, pCF2HL-0 was used as a template, the sense primer used was CFHL-X3(SEQ ID No: 42), CFHL-X4(SEQ ID No: 43), CFHL-X5(SEQ ID No: 44), CFHL-X6(SEQ ID No: 45) or CFHL-X7(SEQ ID No: 46), and the antisense primer was BGH-1(SEQ ID No: 47), which was complementary to the vector sequence. The PCR reaction was performed using KOD polymerase, and the temperature cycle consisting of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 1 minute was repeated 30 times in this order, and the reaction product was digested with restriction enzymes XhoI and BamHI (Takara Shuzo). The digested fragments were introduced between XhoI and BamHI sites of pCF2HL-0 using Ligation High (Toyobo Inc.), respectively. Transformation competence with each plasmidE.coliJM109, transformed with Qiagen Plasmid Maxi kitE.coliIsolating the plasmid of interest. Thus, expression plasmids pCF2HL-3, pCF2HL-4, pCF2HL-5, pCF2HL-6 and pCF2HL-7 were prepared.
To construct an expression plasmid transiently expressed in COS7 cells, plasmids pCF2HL-0, pCF2HL-3, pCF2HL-4, pCF2HL-5, pCF2HL-6 and pCF2HL-7 were digested with restriction enzymes EcoRI and BamHI (Takara Shuzo), and the resulting fragment of about 800bp was purified by agarose gel electrophoresis. The obtained fragments were introduced between EcoRI and BamHI sites of expression plasmid pCOS1 using Ligation High (Toyobo Inc.) for expression in mammalian cells, respectively. Transformation competence with each plasmidE.coliDH5 alpha (Toyobo Inc.), obtained from transformation using Qiagen Plasmid Maxi kitE.coliIsolating the plasmid of interest. Thus, expression plasmids CF2HL-0/pCOS1, CF2HL-3/pCOS1, CF2HL-4/pCOS1, CF2HL-5/pCOS1, CF2HL-6/pCOS1, and CF2HL-7/pCOS1 were prepared.
As a typical example of these plasmids, the construction of the plasmid CF2HL-0/pCOS1 is shown in FIG. 35, and the nucleotide sequence and amino acid sequence of MABL2-scFv < HL-0> contained in the plasmid are shown in SEQ ID No. 48. The nucleotide and amino acid sequences of the linker regions in these plasmids are also shown in FIG. 36.
To construct LH-type expression plasmids containing linkers of different sizes, pCF2LH-0 was used as a template, CFLH-X3(SEQ ID No: 49), CFLH-X4(SEQ ID No: 50), CFLH-X5(SEQ ID No: 51), CFLH-X6(SEQ ID No: 52) or CFLH-X7(SEQ ID No: 53) are sense primers, BGH-1 is an antisense primer, which is complementary to the vector sequence. PCR was performed using KOD polymerase by repeating 30 times a temperature cycle comprising 94 ℃ for 30 seconds, 60 ℃ for 30 seconds and 72 ℃ for 1 minute in this order, and the reaction product was digested with restriction enzymes XhoI and BamHI. The digested fragments were introduced between XhoI and BamHI sites of pCF2LH-0, respectively, using Ligation High. Each plasmid was transformation competentE.coliDH5 α (Toyobo Inc.), the Plasmid of interest was isolated from transformed e.coli using the Qiagen Plasmid Maxi kit. Thus, expression plasmids pCF2LH-3, pCF2LH-4, pCF2LH-5, pCF2LH-6 and pCF2LH-7 were prepared.
To construct COS7 cells transient expression plasmids, plasmids pCF2LH-0, pCF2LH-3, pCF2LH-4, pCF2LH-5, pCF2LH-6 and pCF2LH-7 were digested with restriction enzymes EcoRI and BamHI (Takara Shuzo), and the resulting approximately 800bp fragment was purified by agarose gel electrophoresis. The obtained fragments were introduced between XhoI and BamHI sites of the mammalian cell expression plasmid pCOS1 using Ligation High, respectively. Transformation competence with each plasmidE.coliDH-5 alpha (Toyobo Inc.), transformed from the Plasmid using Qiagen Plasmid Maxi kitE.coliIsolating the plasmid of interest. Thus, expression plasmids CF2LH-0/pCOS1, CF2LH-3/pCOS1, CF2LH-4/pCOS1, CF2LH-5/pCOS1, CF2LH-6/pCOS1, and CF2LH-7/pCOS1 were prepared.
As a typical example of these plasmids, the plasmid CF2LH-0/pCOS1 was constructed as shown in FIG. 37, and the nucleotide sequence and amino acid sequence of MABL2-scFv < LH-0> contained in the plasmid are shown in SEQ ID No. 54. The nucleotide and amino acid sequences of the linker regions in these plasmids are also shown in FIG. 38.
6.3 expression of scFv and sc (fv) in COS7 cells2
(1) Preparation of culture supernatant with serum-containing Medium
Transient expression of HL-type and LH-type scFv and sc (fv) in COS7 cells (JCRB9127, Japan Health Sciences Foundation)2. In CO2COS7 cells were subcultured at 37 ℃ in a gas incubator with DMEM medium (GIBCO BRL) containing 10% fetal bovine serum (HyClone). CF2HL-0, 3-7/pCOS 1 or CF2LH-0, 3-7/pCOS 1, or pCHOM2(Fv) prepared in example 6.2 were electroporated using a Gene Pulser apparatus (BioRad)2The vector was transfected into COS7 cells. The DNA (10. mu.g) and 0.25ml of 2X 10 DNA were added to the cuvette7DMEM medium with cells/ml, 10% FBS and 5mM BES (SIGMA). After a 10 minute hold, the mixture was pulsed with a 0.17kv, 950 muF capacitance. After 10 minutes of room temperature recovery, the electroporated cells were transferred to 75cm3DMEM medium (10% FBS) in culture flasks. After 72 hours of culture, the culture supernatant was collected and centrifuged to remove cell debris. The culture supernatant was filtered with a 0.22um Filter (FALCON) to obtain a culture supernatant (hereinafter referred to as "CM").
(2) Preparation of culture supernatant Using serum-free Medium
Cells transfected in the same manner as in (1) were transferred to 75cm3Flasks were incubated overnight in DMEM medium (10% FBS). After culturing, the supernatant was discarded, and the cells were washed with PBS, followed by addition of CHO-S-SFM II medium (GIBCO BRL). After 72 hours of culture, the culture supernatant was collected, centrifuged to remove cell debris, and filtered with a 0.22um Filter (FALCON) to obtain CM.
6.4 detection of scFv and sc (fv) in COS7CM2
Detection of various MABL2-scFv and sc (fv) in COS7CM prepared in example 6.3(2) above by Western blotting2。
SDS-PAGE analysis of CM from each COS7 was transferred to a REINFORCED NC membrane (Schleicher & Schuell). The membrane was blocked with 5% skim milk (Morinaga Nyu-gyo) and washed with TBS. anti-FLAG antibodies were then added thereto. The membranes were incubated and washed at room temperature. Peroxidase-labeled mouse IgG antibody (Jachson Immuno Research) was added. After incubation and washing at room temperature, the substrate solution (KirKegaard Perry Laboratories) was added for color development (FIG. 39).
6.5 flow cytometry
Using COS7 cell culture supernatant prepared in example 6.3(1), flow cytometry analysis was performed to determine MABL2-scFv and sc (fv)2Binding to human Integrin Associated Protein (IAP) antigens. Adding 2 × 10 to the culture supernatant to be tested or COS7 cell culture supernatant as control5Individual mouse leukemia cells expressing human IAP L1210. After ice-bath and washing, 10. mu.g/ml mouse anti-FALG antibody (SIGMA) was added, followed by incubation and washing of the cells. FITC-labeled anti-mouse IgG antibody (BECTON DICKINSON) was then added, and the cells were incubated and washed again. The fluorescence intensity was measured using a FACScan apparatus (BECTON DICKINSON). Flow cytometry results showed that in COS7 cell culture supernatant, MABL2-scFv and sc (fv) with different length linkers2Has high affinity for human IAPs (see figures 40a and 40 b).
6.6 in vitro apoptosis inducing Effect
The induction of apoptosis of COS7 culture supernatants prepared in example 6.3(1) were examined by Annexin-V staining (Boehringer Mannheim) using L1210 cells transfected with human IAP gene (hIAP/L1210).
To 5X 104To each hIAP/L1210 cell culture supernatant of COS7 cells transfected with each vector, or control COS7 cell culture supernatant, was added to the mixture at a final concentration of 10% and the mixture was incubated for 24 hours. Then Annexin-V/PI staining was performed, and the fluorescence intensity was measured using a FACScan apparatus (BEKTON DICHINSON). The results showed that, scFv in CM of COS7 cells<HL3、4、6、7，LH3、4、6、7>And sc (fv)2The death of hIAP/L1210 cells is remarkably induced. These results are shown in FIG. 41.
6.7 construction of vectors for expression of scFv and sc (fv)2 in CHO cells
To isolate and purify MABL2-scFv and sc (fv)2 from the culture supernatant, vectors for expression in CHO cells were constructed as follows.
pCF2HL-0, 3-7 prepared in example 6.2 and the like were mixed using Ligation HighAnd EcoRI-BamHI fragments of pCF2LH-0 and 3-7, and are introduced between EcoRI and BamHI sites of a CHO cell expression vector pCHO 1. With which competence is transformedE.coliDH5 α. From the transformation using QIAGEN Plasmodin Midi kit (QIAGEN)E.coliSeparating plasmids, and preparing expression plasmids pCHOM2HL-0 and 3-7 and pCHOM2LH-0 and 3-7.
6.8 preparation of expression MABL2-scFv<HL-0、3～7>、MABL2-scFv<LH-0、3～7>And sc (fv)2The CHO cell of (1), and a culture supernatant thereof was prepared
Each of the expression plasmids pCHOM2HL-0, 3-7 and pCHOM2LH-0, 3-7, and pCHOM2(Fv) constructed in example 6.7 was used2Vector, CHO cells were transformed to produce CHO cells that constantly expressed each of the altered antibodies. As a typical example thereof, constantly expressing MABL2-scFv<HL-5>Or sc (fv)2The CHO cells of (1) were prepared as follows.
Expression plasmids pCHOM2HL-5 and pCHOM2(Fv) were linearized by digestion with restriction enzyme PvuI2CHO cells were transfected by electroporation using a Gene Pulser apparatus (BioRad). To a cuvette, DNA (10. mu.g) and 0.75ml 1X 10 were added7Cells/ml PBS, treated with 1.5KV 25 uF capacitive pulses. After 10 minutes of recovery at room temperature, the electroporated cells were transferred to α -MEM medium containing nucleic acids (GIBCO BRL) containing 10% fetal bovine serum for culture. After overnight incubation, the supernatant was discarded. Cells were washed with PBS and nucleic acid-free α -MEM medium (GIBCO BRL) containing 10% fetal bovine serum was added. After two weeks of culture, cells were cultured in medium containing 10nM (final concentration) methotrexate (SIGMA), followed by 50nM and 100nM methotrexate. The resulting cells were cultured in CHO-S-SFM II serum-free medium (GIBCO BRL) in flasks. The culture supernatant was collected, centrifuged to remove cell debris, and filtered through a 0.22 μm pore filter to obtain CM, respectively.
As described above, CHO cells constantly expressing MABL2-scFv < HL-0, -3, -4, -6, -7> and < LH-0, -3, -4, -5, -6, -7> and CM thereof were obtained.
6.9 purification of MABL2-scFv<HL-5>Dimer and sc (fv)2
MABL2-scFv was purified from CM prepared in example 6.8 using the following two purification methods<HL-5>And sc (fv)2。
< purification method 1>
Purifying HL-5 and sc (fv) by anti-FLAG antibody affinity column chromatography and gel filtration using a FLAG sequence at the C-terminus of the polypeptide2. A column (7.9ml) was prepared using anti-FLAG M2 affinity gel (SIGMA), equilibrated with 50mM Tris-HCl buffer (TBS, pH7.5) containing 150mM NaCl, and 1 liter of CM obtained in 6.8 was loaded. After washing the column with TBS, the scFv was eluted by 0.1M glycine-HCl buffer (pH 3.5). The fractions obtained were analyzed by SDS-PAGE to confirm elution of scFv. The scFv fraction was mixed with Tween20 up to a final concentration of 0.01% and concentrated with Centricon-10 (MILIPORE). The TSKgel G3000SWG column (7.5X 600mM) was equilibrated with 20mM acetate buffer (pH6.0) containing 150mM NaCl and 0.01% Tween20, and the concentrate was loaded. The scFv was detected by absorbance at 280nm at a flow rate of 0.4 ml/min. The main component eluted at the dimer was HL-5, and the main component eluted at the monomer was sc (fv)2。
< purification method 2>
Three-step purification of HL-5 and sc (fv) Using ion exchange chromatography, hydroxyapatite and gel filtration2. Purification of HL-5 in ion exchange chromatography, Q Sepharose Fast Flow column (Pharmacia), purification of sc (fv)2SP-sepharose Fast Flow columns were used. During and after the second step, HL-5 and sc (fv) were treated with the same procedure2。
First step of HL-5
HL-5 CM was diluted 2-fold with 20mM Tris-HCl buffer (pH9.0) containing 0.02% Tween20, and then adjusted to pH9.0 with 1M Tris. After the Q sepharose fast flow column was equilibrated with 20mM Tris-HCl buffer (pH8.5) containing 0.02% Tween20, the solution was loaded. The bound polypeptides on the column were eluted by the same buffer containing a linear gradient of 0.1-0.55M NaCl. Monitoring the eluted fraction by SDS-PAGE, collecting the eluted fraction containing HL-5, and loading the eluted fraction onto the hydroxyapatite of the second step.
sc(Fv)2First step of
sc(Fv)2The CM was diluted 2-fold with 20mM acetate buffer (pH5.5) containing 0.02% Tween20, and adjusted to pH5.5 with 1M acetic acid. After the SP-sepharose fast flow column was equilibrated with 20mM acetate buffer (pH5.5) containing 0.02% Tween20, the solution was loaded. And eluting the polypeptide bound on the column by using a buffer solution containing a linear gradient of 0-0.5M NaCl. Monitoring the eluted fractions by SDS-PAGE and collecting the fractions containing sc (fv)2The component (b) is loaded into the hydroxyapatite of the second step.
Second step ofHL-5 and sc (fv)2Chromatography of hydroxyapatite
Hydroxyapatite column (type I, BIORAD) was equilibrated with 10mM phosphate buffer (pH7.0) containing 0.02% Tween20, and HL-5 and sc (fv) obtained in the first step were separately loaded2And (4) components. After washing the column with the same buffer, the bound polypeptides on the column were eluted using a linear gradient of phosphate buffer up to 0.5M. The eluted fractions were monitored by SDS-PAGE, and fractions containing the polypeptide of interest were collected.
The third stepHL-5 and sc (fv)2Is subjected to gel filtration
Each fraction obtained in the second step was concentrated separately with Centriprep-10(MILIPORE) and applied to a Superdex200 column (2.6X 60cm, Pharmacia) equilibrated with 20mM acetate buffer (pH6.0) containing 0.02% Tween20 and 0.15M NaCl. Eluting at the dimer to HL-5, sc (fv) HL-5 and sc (fv)2Eluted as a main peak at the monomer, respectively.
Both purification methods have difficulty detecting HL-5 monomer, and it has been confirmed that a single-chain Fv dimer is formed in high yield when the linker of the single-chain Fv comprises about 5 amino acids. Further, dimer of HL-5 and sc (fv)2Can be stably stored for one month at 4 ℃ after purification.
6.10 evaluation of purified scFv<HL-5>DimerizationBody and sc (fv)2Binding Activity to antigen
Using purified MABL2-scFv<HL-5>Dimer and purified sc (fv)2Flow cytometry analysis was performed to assess binding to human Integrin Associated Protein (IAP) antigens. 10. mu.g/ml of purified MABL2-scFv<HL-5>Dimer, purified sc (fv)2Positive control antibody MABL-2 or negative control mouse IgG (zymed), 2X 105Individual mouse leukemia L1210 cells expressing human IAP (hIAP/L1210), or L1210 cells transformed with pCOS1 (pCOS1/L1210) were used as controls. After ice-bath and washing, 10. mu.g/ml mouse anti-FLAG antibody (SIGMA) was added, followed by incubation and washing of the cells. To this was added FITC-labeled anti-mouse IgG antibody (becton DICKINSON), and the cells were incubated and washed again. The fluorescence intensity was then measured using a FACScan apparatus (BECTON DICKINSON).
Due to the purified MABL2-scFv<HL-5>Dimer and purified sc (fv)2Specific binding to hIAP/L1210 cells can confirm scFv<HL-5>Dimer and sc (fv)2Has high affinity for human IAP (see figure 42).
6.11 purified scFv<HL-5>Dimeric and sc (fv)2Apoptosis inducing Activity in vitro
Detection of purified MABL2-scFv by Annexin-V staining (Boehringer Mannheim) using L1210 cells into which human IAP genes were introduced (hIAP/L1210) and human leukemia cell line CCRF-CEM cells<HL-5>Dimer and purified sc (fv)2The apoptosis-inducing effect of (1).
Mixing purified MABL2-scF at different concentrations<HL-5>Dimer, purified MABL2-sc (fv)2Positive control antibody MABL-2 or negative control mouse IgG, 5X 104A hIAP/L1210 cell or 1X 105In CCRF-CEM cells. After 24 hours of incubation, Annexin-V staining was performed, and the fluorescence intensity was measured using a FACScan apparatus (BECTONDICKLNSON). As a result, MABL2-scFv<HL-5>Dimer and MABL2-sc (fv)2Significant induction of hIAP/L1210 and CCRF-CEM cell death in a concentration-dependent manner (see FIG. 1)Fig. 43). The results show that compared to the original antibody MABL-2, MABL2-scFv<HL-5>And MABL2-sc (fv)2Has enhanced efficacy in inducing apoptosis.
6.12 purified scFv<HL-5>Dimer and sc (fv)2Hemagglutination reaction test of
The procedure of example 5.15 was followed using different concentrations of purified scFv<HL-5>Dimer and purified sc (fv)2And performing a hemagglutination reaction test.
Hemagglutination was observed using antibody MABL-2 as a positive control, and for the single-chain antibody MABL2-sc (fv)2And MABL2-scFv<HL-5>No hemagglutination was observed. In addition, there was no substantial difference in hemagglutination reaction between the two buffers used for antibody MABL-2. These results are shown in table 3.
TABLE 3
Hemagglutination reaction test
| Diluent agent: PBS (μ g/ml) | ||||||||||||||||
| Control | 28.9 | 14.45 | 7.225 | 3.6125 | 1.8063 | 0.9031 | 0.4516 | 0.2258 | 0.1129 | 0.0564 | 0.0282 | 0.0141 | 0.0071 | 0.0035 | 0.0018 | |
| MABL2-sc(Fv) | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
| Control | 28.0 | 14.0 | 7.0 | 3.5 | 1.75 | 0.875 | 0.4375 | 0.2188 | 0.1094 | 0.0547 | 0.0273 | 0.0137 | 0.0068 | 0.0034 | 0.0017 | |
| MABL2-sc(Fv)<HL5> | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
| Control | 80 | 40 | 20 | 10 | 5 | 2.5 | 1.25 | 0.625 | 0.3125 | 0.1563 | 0.0781 | 0.0391 | 0.0195 | 0.0098 | 0.0049 | |
| MABL2 (complete) mlgG | -- | +- | +- | +- | +- | +- | +- | +- | +- | +- | ±- | -- | -- | -- | -- | -- |
| Diluent agent: acetate salt (mu g/ml) | ||||||||||||||||
| Control | 80 | 40 | 20 | 10 | 5 | 2.5 | 1.25 | 0.625 | 0.3125 | 0.1563 | 0.0781 | 0.0391 | 0.0195 | 0.0098 | 0.0049 | |
| MABL2 (complete) | - | + | + | + | + | + | + | + | + | + | + | + | - | - | - | - |
6.13 purified scFv<HL-5>Dimer and sc (fv)2Antitumor Effect against human myeloma model mice
Testing of scFv prepared and purified in examples 6.8 and 6.9<HL-5>Dimer and sc (fv)2The antitumor effect of (1). Using the human myeloma mouse model generated in example 5.1, the amount of M protein produced by human myeloma cells in mouse serum was measured by ELISA, and the survival time of mice was measured and tested. Then, the scFv was evaluated based on the change in the amount of M protein in mouse serum and the survival time of mice<HL-5>Dimer and sc (fv)2The antitumor effect of (1).
The tests used HL-5 and sc (fv) at 0.01, 0.1 or 1mg/ml2They were dissolved in a vehicle containing 150mM NaCl, 0.02% Tween and 20mM acetate buffer (pH6.0) and administered to mice at a dose of 0.1, 1 or 10 mg/kg. Control mice were given vehicle only.
26 days after the transplantation of human myeloma cells, mouse serum was collected and the amount of M protein in the serum was measured by ELISA as described in example 5.14. As a result, HL-5 dimer and sc (fv) were administered2The amount of serum M protein in both groups of mice decreased in a dose-dependent manner (see fig. 44). Furthermore, administration of HL-5 (FIG. 45) and sc (fv) was observed as compared with the control group administered with vehicle2Both groups (fig. 46) had significantly longer survival times. These results show thatHL-5 and sc (fv) of the present invention2Has excellent performanceIn vivoAnti-tumor effect.
Example 7
Single chain Fv comprising H chain V region and L chain V region of human antibody 12B5 against human MPL
The V region of human monoclonal antibody 12B5 encoding anti-human MPL was constructed as follows:
7.1 construction of a Gene encoding the V region of chain 12B5H
The gene encoding the H chain V region of human antibody 12B5 that binds to human MPL was designed by ligating a leader sequence (SEQ ID NO: 56) derived from a human antibody gene (Eur. J. Immunol.1996; 26: 63-69) at the 5' end through the nucleotide sequence (SEQ ID NO: 55) of the gene. The designed nucleotide sequence was divided into four oligonucleotides (12B5VH-1, 12B5VH-2, 12B5VH-3, 12B5VH-4) each having an overlapping sequence of 15 bp. 12B5VH-1(SEQ ID NO: 57) and 12B5VH-3(SEQ ID NO: 59) were synthesized in the sense direction, and 12B5VH-2(SEQ ID NO: 58) and 12B5VH-4(SEQ ID NO: 60) were synthesized in the antisense direction, respectively. After each synthetic oligonucleotide was assembled according to the respective complementarity, the full-length gene was amplified by adding the outer primers (12B5VH-S and 12B5 VH-A). Respectively, 12B5VH-S (SEQ ID NO: 61) was designed to hybridize to the 5' end of the leader sequence by a forward primer and has a Hind III restriction enzyme recognition site and a KoZak sequence, while 12B5VH-A (SEQ ID NO: 62) was designed to hybridize to the nucleotide sequence encoding the C-terminus of the H chain V region by a reverse primer and has a splice donor sequence and a Bam HI restriction enzyme recognition site.
100 μ l PCR solution contained 10 μ l10 XPCR Gold Buffer II, 1.5mM MgCl20.08mM dNTPs (dATP, dGTP, dCTP and dTTP), 5 units of DNA polymerase AmpliTaq Gold (all from PERKIN ELMER), 2.5pmol of each synthesized oligonucleotide (12B5VH-1 to-4), an initial temperature of 94 ℃ for 9 minutes, 94 ℃ for 2 minutes, 55 ℃ for 2 minutes and 72 ℃ for 2 minutes. After repeating this cycle 2 times, 15pmol each of the outer primers 12B5VH-S and 12B5VH-A was added. The mixture was subjected to a temperature of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 1 minuteThe cycle was 35 times and heated at 72 ℃ for an additional 5 minutes.
The PCR product was purified using 1.5% low melting temperature agarose gel (Sigma), digested with restriction enzymes BamHI and Hind III, and cloned into the human H chain expression vector HEF-g.gamma.1. After DNA sequencing, the plasmid containing the correct DNA sequence was designated HEF-12B 5H-g.gamma.1.
HEF-12B 5H-g.gamma.1 was digested with restriction enzymes EcoRI and BamHI to generate the gene encoding 12B5VH, which was then cloned into the human Fab H chain expression vector pCOS-Fd to generate pFd-12B 5H. A human Fab H chain expression vector was constructed by amplifying DNA (SEQ ID NO: 63) containing an intron region between the human antibody H chain V region and the gene encoding the constant region, and the human H chain partial constant region by PCR, and inserting the PCR product into an animal cell expression vector pCOS 1. Using HEF-g.gamma.1 as a template, a forward primer G1CH1-S (SEQ ID NO: 64) (designed to hybridize with the 5 '-terminal sequence of intron 1 and to have restriction enzyme recognition sites EcoRI and BamHI), and a reverse primer G1CH1-A (SEQ ID NO: 65) (designed to hybridize with the 3' -terminal DNA of the human H chain constant region CH1 domain and to have a sequence encoding a part of the hinge region, 2 stop codons, and a restriction enzyme recognition site Bgl II), were used to amplify the human H chain constant region gene under the same conditions as described above.
Plasmids HEF-12B 5H-gGamma 1 and pFd-12B5H contain a reconstituted variable region of 12B5H, the nucleotide sequence and amino acid sequence of which are shown in SEQ ID NO: as shown at 66.
7.2 construction of a Gene encoding the V region of chain 12B5L
The gene encoding the L chain V region of human antibody 12B5 that binds human MPL was designed by ligating a leader sequence (SEQ ID NO: 68) derived from human antibody gene 3D6(Nuc. acid Res.1990: 18; 4927) at the 5' end through the nucleotide sequence (SEQ ID NO: 67) of the gene. The nucleotide sequences designed in the same manner as above were divided into four oligonucleotides (12B5VL-1, 12B5VL-2, 12B5VL-3, 12B5VL-4) each having an overlapping sequence of 15bp, and synthesized separately. 12B5VL-1(SEQ ID NO: 69) and 12B5VL-3(SEQ ID NO: 71) have sense sequences and 12B5VL-2(SEQ ID NO: 70) and 12B5VL-4(SEQ ID NO: 72) have antisense sequences, respectively. After each synthetic oligonucleotide was assembled according to the respective complementarity, it was mixed with external primers (12B5VL-S and 12B5VL-A) to amplify the full-length gene. 12B5VL-S (SEQ ID NO: 73), respectively, was designed to hybridize to the 5' end of the leader sequence by a forward primer and has a Hind III restriction enzyme recognition site and a Kozak sequence. 12B5VL-A (SEQ ID NO: 74) was designed to hybridize with the nucleotide sequence encoding the C-terminus of the L chain V region by a reverse primer, and to have a splice donor sequence and a Bam HI restriction enzyme recognition site.
PCR was performed as described above, and the PCR product was purified using 1.5% low melting temperature agarose gel (Sigma), digested with restriction enzymes BamHI and Hind III, and cloned into the human L chain expression vector HEF-g κ. After DNA sequencing, the plasmid containing the correct DNA sequence was designated HEF-12B5L-g κ. The plasmid HEF-12B 5L-gka comprises a reconstructed 12B5L chain V region, and the nucleotide sequence and the amino acid sequence of the plasmid are shown as SEQ ID NO: shown at 75.
7.3 Generation of reconstituted 12B5 Single chain fv (scFv)
The reconstructed 12B5 antibody single chain Fv was designed as a 12B5 VH-linker-12B 5VL sequence with a FLAG sequence (SEQ ID NO: 76) at the C-terminus for ease of detection and purification. By using a compound of (Gly)4Ser)3The reconstructed 12B5 single-chain Fv (sc12B5) was constructed from the linker sequence of 15 amino acids.
(1) Generation of a reconstituted 12B5 Single chain Fv with a linker sequence consisting of 15 amino acids
The H chain V region of 12B5, the linker region and the L chain V region of 12B5 were amplified and ligated by PCR, respectively, to construct a gene encoding a reconstituted 12B5 antibody single chain Fv comprising a linker sequence consisting of 15 amino acids. The method is illustrated schematically in fig. 47. Six primers (A-F) were used to prepare a reconstituted 12B5 single-chain Fv. Primers A, C and E have sense sequences and primers B, D and F have antisense sequences.
The forward primer 12B5-S (primer A, SEQ ID No.77) of the H chain V region was designed to hybridize to the 5' end of the H chain leader sequence and has an EcoRI restriction enzyme recognition site. The reverse primer HuVHJ3 (primer B, SEQ ID No.78) of the H chain V region was designed to hybridize to the coding DNA at the C-terminus of the H chain V region.
The forward primer RHUJH3 (primer C, SEQ ID No.79) of the linker was designed to hybridize to the coding DNA at the N-terminus of the linker and overlap with the coding DNA at the C-terminus of the H chain V region. The reverse primer RHUVK1 (primer D, SEQ ID No.80) of the linker was designed to hybridize to the coding DNA at the C-terminus of the linker and overlap with the coding DNA at the N-terminus of the V region of the L chain.
The forward primer HuVK1.2 (primer E, SEQ ID No.81) for the L chain V region was designed to hybridize to the coding DNA at the N-terminus of the L chain V region. The reverse primer 12B5F-A (primer F, SEQ ID No.82) of the L chain V region was designed to hybridize with the DNA coding for the C-terminus of the L chain V region and has a FLAG peptide coding sequence (Hopp T.P. et al, Bio/Technology, 6, 1204-1210, 1988), two stop codons and a NotI restriction enzyme recognition site.
In the first PCR step, three reactions A-B, C-D, E-F were performed, and the three PCR products obtained in the first PCR step were assembled according to their respective complementarity. After addition of primers A and F, the full length DNA encoding the reconstituted 12B5 single-chain Fv with a 15 amino acid linker was amplified (second PCR). In the first PCR, plasmid HEF-12B 5H-g.gamma.1 (see example 7.1) encoding the reconstructed 12B5H chain V region, plasmid pSCFVT7-hM21 (humanized ONS-M21 antibody) (Ohtomo et al, Anticancer Res.18(1998), 4311-Buchner 4316) comprising a linker region consisting of Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser GlyGly Gly Gly Gly Gly Gly Gly Ser (Huston et al, Proc. Natl. Acad. Sci. USA, 85, 5879-5883, 1988), and plasmid HEF-12B 5L-g.kappa (see example 7.2) encoding the reconstructed 12B5L chain V region were used as templates, respectively.
The first 50. mu.l of PCR reaction solution contained 5. mu.l of 10 XPCR Gold Buffer II, 1.5mM MgCl20.08mM dNTPs, 5 units of DNA polymerase AmpliTaq Gold (all from PERKIN ELMER), 100pmol of each primer and 100ng of each template DNA. The PCR solution is startedThe mixture was heated at 94 ℃ for 9 minutes, and at 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute. After this temperature cycle was repeated 35 times, the reaction mixture was heated for a further 5 minutes at 72 ℃.
The second step was used to assemble PCR products A-B, C-D and E-F. 98. mu.l of the second-step PCR mixture solution contained 1. mu.l of the first-step PCR products A-B, 0.5. mu.l of the PCR products C-D and 1. mu.l
PCR product E-F as template, 10. mu.l 10 XPCR Gold Buffer II, 1.5mM MgCl20.08mM dNTPs, 5 units of DNA polymerase AmpliTaq Gold (all from PERKINELMER), heated at an initial temperature of 94 ℃ for 8 minutes, at 94 ℃ for 2 minutes, at 65 ℃ for 2 minutes, and at 72 ℃ for 2 minutes. After repeating this cycle for 2 times, 100pmol each of primers A and F was added, and after repeating a cycle consisting of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute 35 times, the reaction mixture was heated at 72 ℃ for 5 minutes.
The DNA fragment prepared by the second PCR was purified using 1.5% low melting temperature agarose gel, digested with EcoRI and NotI, and cloned into pCHO1 vector and pCOS1 vector (Japanese patent application No. 8-255196). The expression vector pCH01 was constructed by removing the antibody gene from DHFR-. DELTA.E-rvH-PM 1-f (see WO92/19759) by digestion with EcoRI and SmaI, and ligating an EcoRI-NotI-BamHI linker (TAKARA SHUZO). After DNA sequencing, the plasmids encoding the DNA fragments containing the reconstructed single-chain Fv correct amino acid sequence of 12B5 were designated pCHO-sc12B5 and pCOS-sc12B 5. The nucleotide and amino acid sequences of the reconstituted 12B5 single-chain Fv contained in plasmids pCHO-sc12B5 and pCOS-sc12B5 are shown in SEQ ID NO: as shown at 84.
7.4 expression of antibody 12B5(IgG, Fab) and Single chain Fv Polypeptides by animal cells
COS-7 cells or CHO cells were used to express antibody 12B5(IgG, Fab) and single chain Fv derived from antibody 12B 5.
Transient expression using COS7 cells was performed as follows. Transfection was performed by the electroporation method using a Gene Pulser apparatus (BioRad). In COS-7 cells (1X 10) suspended in 0.8ml PBS7Cells/ml) added to express antibody 12B5(IgG)10. mu.g each of the above expression vectors HEF-12B 5H-g.gamma.1 and HEF-12B 5L-g.kappa.were added to 10. mu.g each of pFd-12B5H and HEF-12B 5L-g.kappa.for expression of 12B5Fab fragment, and 10. mu.g of pCOS-sc12B5 was added to expression of single chain Fv. The mixture in the cuvette was treated with 1.5KV, 25 μ FD capacitance pulses. After 10 minutes of room temperature recovery, the electroporated cells were added to DMEM medium containing 10% fetal bovine serum (GIBCO BRL) for culture. After overnight incubation, the cells were washed once with PBS and added to serum-free medium CHO-S-SFM II for 2 days. The culture medium was centrifuged to remove cell debris, and filtered through a 0.22 μm filter to prepare a culture supernatant.
To establish a CHO cell line stably expressing single-chain Fv (polypeptide) derived from antibody 12B5, an expression vector pCHO-sc12B5 was introduced into CHO cells as follows.
The expression vector was introduced into CHO cells by the electroporation method using a Gene Pulser apparatus (BioRad). Linearized DNA (100. mu.g) was obtained by digestion with restriction enzyme PvuI and combined in a cuvette with CHO cells (1X 10) suspended in 0.8ml PBS7Cells/ml), left on ice for 10 minutes and pulsed with 1.5KV, 25 μ FD capacitance. After 10 minutes of room temperature recovery, the electroporated cells were added to CHO-S-SFM II (GIBCO BRL) containing 10% fetal bovine serum for culture. After 2 days of culture, culture was continued in CHO-S-SFM II (GIBCO BRL) containing 5nM methotrexate (SIGMA) and 10% fetal bovine serum. From the clones thus obtained, clones with high expression rate were selected as a 12B5 single-chain Fv-producing cell line. After culturing in serum-free medium CHO-S-SFM II (GIBCO BRL) containing 5nM methotrexate (SIGMA), cell debris was centrifuged to obtain culture supernatant.
7.5 purification of Single chain Fv derived from 12B5 produced by CHO cells
The culture supernatant of the 12B5 single-chain Fv-expressing CHO cell line obtained in 7.4 was purified using an anti-FLAG antibody column and a gel filtration column.
(1) anti-FLAG antibody column
Culture supernatants were added to PBS equilibrated anti-FLAG M2 affinity gel (Sigma). After the column was washed with the same buffer, the protein adsorbed on the column was eluted by 0.1M glycine-hydrochloric acid buffer (pH 3.5). The eluted fractions were immediately neutralized by adding 1M Tris-HCl buffer (pH 8.0). The eluted fractions were analyzed by SDS-PAGE, and fractions confirmed to contain single-chain Fv were concentrated using Centricon-10 (MILLIPORE).
(2) Gel filtration
(1) The concentrated solution obtained in (1) was added to a Superdex200 column (10X 300mm, AMERSHAM PHARMACIA) equilibrated with PBS containing 0.01% Tween 20. The product sc12B5 eluted as 2 peaks (A, B) (see fig. 48). Fractions A and B were analyzed using a 14% -SDS-polyacrylamide gel. The samples were electrophoresed according to the Laemmli method in the presence or absence of a reducing agent, and stained with Coomassie Brilliant blue after electrophoresis. As shown in FIG. 49, both components A and B produced a single band with an apparent molecular weight of about 31kD, regardless of the presence of the reducing agent. Fractions A and B were analyzed by gel filtration using Superdex200PC 3.2.2/3.0 (3.2X 300mm, AMERSHAMPHARMACIA), fraction A producing an eluted product with an apparent molecular weight of about 44kD, and fraction B producing a 22kD product (see FIGS. 50a and B). This result indicates that component A is a non-covalent dimer of sc12B5 single-chain Fv and component B is a monomer.
7.6 measurement of TPO-like agonist Activity of various Single chain Fvs
TPO-like activity of the anti-MPL single-chain antibody was evaluated by measuring proliferation activity on Ba/F3 cells (BaF/MPL) expressing human TPO receptor (MPL). After washing BaF/Mpl cells twice with RPMI1640 medium (GIBCO) containing 10% fetal bovine serum (GIBCO), the cells were treated at 5X 105The cell density of cells/ml is suspended in the culture medium. Dilution of anti-MPL Single chain antibody and human TPO (R) with Medium&D Systems). Add 50. mu.l of cell suspension and 50. mu.l of diluted antibody or human TPO to 96-well microplate (flat bottom) (Falcon), CO2In heat preservation box (CO)2Concentration: 5%) for 24 hours. After incubation with the addition of 10. mu.l of WST-8 reagent (for determining the number of primitive cell SF: Nacalai Tesque), the absorbance was measured immediately using a fluorescence absorptiometer SPECTRA Fluor (TECAN), measuring the wavelength at 450nm, as a referenceThe wavelength is 620 nm. In CO2In heat preservation box (CO)2Concentration: 5%) and after 2 hours of incubation, the absorbance was measured again using SPECTRA Fluor, the measurement wavelength was 450nm, and the reference wavelength was 620 nm. Since the WST-8 reagent produces a color reaction at a wavelength of 450nm depending on the number of living cells, the proliferation activity of BaF/Mpl was evaluated based on the change in absorbance after 2 hours by ED50 calculated as follows. In the proliferation reaction curve, the absorbance was plotted on the ordinate and the antibody concentration on the abscissa, and the absorbance in the plateau phase was set as the 100% reaction rate. Based on the plotted values near 50% reactivity, a straight line fitting (straight line fitting) method was used to obtain a fitting formula, and the antibody concentration at 50% reactivity was calculated as ED 50.
Agonist activity to MPL was measured using COS-7 cell culture supernatants expressing various 12B5 antibody molecules, and the results shown in FIG. 51 indicate that 12B5IgG having a bivalent antigen binding site had an increase in absorbance in a concentration-dependent manner and has TPO-like agonist activity (ED 50; 29nM), while 12B5Fab having a monovalent antigen binding site had very weak agonist activity (ED 50; 34, 724 nM). In contrast, a single chain Fv with a monovalent antigen binding site, such as Fab (sc12B5), showed a strong level of agonist activity, with an ED50 of 75 nM. However, it is known that the variable regions of single-chain Fv H-chains and L-chains are joined by non-covalent bonds, and thus the respective variable regions are separated in solution and can be combined with variable regions of other molecules to form multimers, such as dimers. When the molecular weight of the gel filtration-purified sc12B5 was measured, the presence of molecules identified as monomers and dimers was confirmed (see fig. 48). The sc12B5 monomer and sc12B5 dimer (see fig. 50) were isolated and their agonist activity against MPL was determined. As shown in FIGS. 51 and 52, ED50 of the sc12B5 monomer was 4438.7nM, which confirmed that the agonist activity was decreased compared to the results using COS-7 cell culture supernatant. In contrast, single-chain Fv with bivalent antigen-binding sites (sc12B5 dimer), showed about 400-fold higher agonist activity than monovalent sc12B5 (ED 50; 10.1 nM). Furthermore, bivalent single chain Fv showed agonist activity equal to or higher than that of human TPO and 12B5 IgG.
Example 8
Construction of Gene encoding variable region of human antibody 12E10 against human MPL
The variable region gene of human monoclonal antibody 12E10 encoding anti-human MPL was constructed as follows:
8.1 construction of a Gene encoding the V region of chain 12E10H
SEQ ID NO: 86 is a gene coding for the H chain V region of human antibody 12E10 that binds to human MPL designed based on the amino acid sequence (SEQ ID NO: 85) described in WO 99/10494. The full-length amino acid sequence was designed such that its 5' end was ligated with a leader sequence (SEQ ID NO: 87) derived from a human antibody gene (GeneBank accession No. AF 062252). The designed nucleotide sequence was divided into four oligonucleotides (12E10VH1, 12E10VH2, 12E10VH3, 12E10VH4) each having 15bp overlapping sequences. 12E10VH1(SEQ ID NO: 88) and 12E10VH3(SEQ ID NO: 90) were synthesized in the sense orientation, and 12E10VH2(SEQ ID NO: 89) and 12E10VH4(SEQ ID NO: 91) were synthesized in the antisense orientation, respectively. After assembly of each synthetic oligonucleotide according to the respective complementarity, the full-length gene was amplified by adding the outer primers (12E10VHS and 12E10 VHA). Respectively, 12E10VHS (SEQ ID NO: 92) was designed to hybridize to the 5' end of the leader sequence by a forward primer and has a Hind III restriction enzyme recognition site and a Kozak sequence, while 12E10VHA (SEQ ID NO: 93) was designed to hybridize to the nucleotide sequence encoding the C-terminus of the H chain V region by a reverse primer and has a splice donor sequence and a Bam HI restriction enzyme recognition site.
100 μ l PCR solution contained 10 μ l10 XPCR Gold Buffer II, 1.5mM MgCl20.08mM dNTPs (dATP, dGTP, dCTP and dTTP), 5 units of DNA polymerase AmpliTaq Gold (all from PERKIN ELMER), 2.5pmol of each of the oligonucleotides synthesized (12E10VH-1 to-4), an initial temperature of 94 ℃ for 9 minutes, 94 ℃ for 2 minutes, 55 ℃ for 2 minutes and 72 ℃ for 2 minutes. After repeating this cycle 2 times, 100pmol each of the outer primers 12E10VHS and 12E10VHA was added. The mixture was subjected to a cycle consisting of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 1 minute 35 times and heated at 72 ℃ for an additional 5 minutes.
The PCR product was purified using 1.5% low melting temperature agarose gel (Sigma), digested with restriction enzymes BamHI and Hind III, and cloned into the human H chain expression vector HEF-g.gamma.1. After DNA sequencing, the plasmid containing the correct DNA sequence was designated HEF-12E 10H-g.gamma.1.
HEF-12E 10H-g.gamma.1 was digested with restriction enzymes EcoRI and BamHI to generate the gene encoding 12E10VH, which was then cloned into the human Fab H chain expression vector pCOS-Fd to generate pFd-12E 10H. A human Fab H chain expression vector was constructed by amplifying DNA (SEQ ID NO: 63) containing an intron region between the V region of a human antibody H chain and the gene encoding the constant region and a part of the human H chain constant region by PCR, and inserting the PCR product into an animal cell expression vector pCOS 1. Using HEF-g.gamma.1 as a template, a forward primer G1CH1-S (SEQ ID NO: 64) (designed to hybridize with the 5 '-terminal sequence of intron 1 and to have restriction enzyme recognition sites EcoRI and BamHI), and a reverse primer G1CH1-A (SEQ ID NO: 65) (designed to hybridize with the 3' -terminal DNA of the human H chain constant region CH1 domain and to have a sequence encoding a part of the hinge region, 2 stop codons, and a restriction enzyme recognition site BglII), the human H chain constant region gene was amplified under the same conditions as described above.
The nucleotide sequence and amino acid sequence of the reconstructed 12E10H variable region contained in plasmids HEF-12E 10H-gGamma 1 and pFd-12E10H are shown as SEQ ID NO: as shown at 94.
8.2 construction of the Gene encoding chain V region of 12E10L
SEQ ID NO: 96 is a gene coding for the L chain V region of human antibody 12E10 that binds to human MPL, which is designed based on the amino acid sequence (SEQ ID NO: 95) described in WO 99/10494. The design was further carried out by ligating a leader sequence (SEQ ID NO: 97) derived from a human antibody gene (mol. Immunol.1992; 29: 1515-1518) to the 5' end thereof. The nucleotide sequences designed in the same manner as above were divided into four oligonucleotides (12E10VL1, 12E10VL2, 12E10VL3, 12E10VL4) each having an overlapping sequence of 15bp, and synthesized separately. 12E10VL1(SEQ ID NO: 98) and 12E10VL3(SEQ ID NO: 100) have sense sequences and 12E10VL2(SEQ ID NO: 99) and 12E10VL4(SEQ ID NO: 101) have antisense sequences, respectively. After each synthetic oligonucleotide was assembled according to the respective complementarity, it was mixed with external primers (12E10VLS and 12E10VLA) to amplify the full-length gene. 12E10VLS (SEQ ID NO: 102) was designed to hybridize to the 5' end of the leader sequence by a forward primer and has an EcoR I restriction enzyme recognition site and a Kozak sequence. 12E10VLA (SEQ ID NO: 103) was designed to hybridize with a nucleotide sequence encoding the C-terminus of the L chain V region by a reverse primer and has a BlnI restriction enzyme recognition site.
PCR was performed as described above, and the PCR product was purified using 1.5% low melting temperature agarose gel (Sigma), digested with restriction enzymes EcoRI and BlnI, and cloned into pUC19 containing the human lambda chain constant region gene. After DNA sequencing, a plasmid containing the correct DNA sequence was digested with EcoR I to generate a gene encoding the 12E10L chain V region and the human lambda chain constant region, which was then inserted into the expression vector pCOS-1. The plasmid having the 12E10L chain gene (SEQ ID NO: 104) was designated pCOS-12E 10L.
8.3 Generation of reconstructed 12E10 Single chain Fvs
The reconstructed 12E10 antibody single chain Fv was designed as a 12E10 VH-linker-12E 10VL sequence with a FLAG sequence (SEQ ID NO: 105) at the C-terminus for ease of detection and purification. By using a compound of (Gly)4Ser)315 amino acids represented by (Gly)4Ser)1The reconstructed 12E10 single-chain Fv (sc12E10 and db12E10) was constructed with a linker sequence of 5 amino acids as represented.
(1) Generation of a reconstituted 12E10 Single chain Fv with a linker sequence consisting of 5 amino acids
Will be (Gly)4Ser)1The nucleotide sequence of the linker was introduced into the 3 'end of the gene encoding the 12E10H chain V region and the 5' end of the gene encoding the 12E10L chain V region, and each of the genes thus obtained was amplified separately by PCR and the amplified genes were ligated to construct a gene encoding a reconstituted 12E10 single-chain Fv comprising a linker sequence consisting of 5 amino acids. Using four PCR primers (A-D)) A reconstituted 12E10 single-chain Fv was prepared. Primers A and C have sense sequences, and primers B and D have antisense sequences.
The forward primer for the H chain V region was 12E10S (primer A, SEQ ID No. 106). The reverse primer DB2 (primer B, SEQ ID No.107) of the H chain V region was designed to hybridize with the DNA encoding the C terminus of the H chain V region and has a linker (Gly4Ser)1And the coding nucleotide sequence of the N-terminus of the V region of the L chain.
The forward primer DB1 (primer C, SEQ ID No.108) of the L chain V region was designed to hybridize to the DNA encoding the N-terminus of the L chain V region and has a linker (Gly4Ser)1And a nucleotide sequence encoding the C-terminus of the V region of the H chain. The reverse primer 12E10FA (primer D, SEQ ID No.109) for the L chain V region was designed to hybridize with the DNA coding for the C-terminus of the L chain V region and has a FLAG coding sequence and a NotI restriction enzyme recognition site.
In the first PCR step, two reactions A-B, C-D were performed, and the two PCR products obtained in the first PCR step were assembled according to their respective complementarity. After addition of primers A and D, the full length DNA encoding the reconstituted 12E10 single-chain Fv with a linker consisting of 5 amino acids was amplified (second step PCR). First step PCR with respectively encoded reconstructed 12E10
Plasmid HEF-12E 10H-g.gamma.1 of the H chain V region (see example 8.1) and plasmid pCOS-12E10L encoding the reconstituted 12E10L chain V region (see example 8.2) were used as templates.
50 μ l of the solution of the first PCR reaction contained 5 μ l of 10 XPCR Gold Buffer II, 1.5mM MgCl20.08mM dNTPs, 5 units of DNA polymerase AmpliTaq Gold (from PERKIN ELMER), 100pmol of each primer and 100ng of each template DNA. The PCR solution was heated at an initial temperature of 94 ℃ for 9 minutes, at 94 ℃ for 30 seconds, at 55 ℃ for 30 seconds, and at 72 ℃ for 1 minute. After this temperature cycle was repeated 35 times, the reaction mixture was heated for a further 5 minutes at 72 ℃.
The products A-B (429bp) and C-D (395bp) were assembled using a second PCR. The second PCR mixture solution (98. mu.l) contained the first PCR products A-B and C-D eachMu.l of the seed was used as a template, 100pmol of each primer, 10. mu.l of 10 XPCR Gold Buffer II, 1.5mM MgCl20.08mM dNTPs, 5 units of DNA polymerase AmpliTaq Gold (from PERKIN ELMER) were reacted under the same conditions as described above.
The 795bpDNA fragment generated by the second PCR was purified using a 1.5% low melting temperature agarose gel, digested with EcoRI and NotI, and cloned into pCHO1 vector or pCOS1 vector. The expression vector pCHO1 was constructed by digesting DHFR-. DELTA.E-RVH-PM 1-f (see WO92/19759) with EcoRI and SmaI, removing the antibody gene, and ligating an EcoRI-NotI-BamHI linker (TAKARA SHUZO). After DNA sequencing, the plasmids encoding the DNA fragments comprising the reconstructed 12E10 single-chain Fv correct amino acid sequence were designated pCHO-db12E10 and pCOS-db12E 10. The nucleotide and amino acid sequences of the reconstituted 12E10 single-chain Fv contained in plasmids pCHO-db12E10 and pCOS-db12E10 are set forth in SEQ ID NO: 110.
(2) Generation of a reconstituted 12E10 Single chain Fv with a linker sequence consisting of 15 amino acids
Will linker (Gly)4Ser)3The nucleotide sequence of (a) was introduced into the 3 'end of the gene encoding the 12E10H chain V region and the 5' end of the gene encoding the 12E10L chain V region, and each of the genes thus obtained was amplified separately by PCR and the amplified genes were ligated to construct a gene encoding a reconstituted 12E10 antibody single chain Fv comprising a linker sequence consisting of 15 amino acids. Four PCR primers (A-D) were used to prepare a reconstituted 12E10 single-chain Fv. Primers A and C have sense sequences, and primers B and D have antisense sequences.
The forward primer for the H chain V region was 12E10S (primer A, SEQ ID No. 106). The reverse primer sc4.3 (primer B, SEQ ID No.111) for the H chain V region was designed to hybridize to the DNA coding for the C terminus of the H chain V region and has a linker (Gly4Ser)3And the coding nucleotide sequence of the N-terminus of the V region of the L chain.
The forward primer sc1.3 (primer C, SEQ ID No.112) for the L chain V region was designed to hybridize to the DNA encoding the N-terminus of the L chain V region and has a linker (Gly4Ser)3And a nucleotide sequence encoding the C-terminus of the V region of the H chain. The reverse primer 12E10FA (primer D, SEQ ID No.109) for the L chain V region was designed to hybridize with the DNA coding for the C-terminus of the L chain V region and has a FLAG coding sequence and a NotI restriction enzyme recognition site.
In the first PCR step, two reactions A-B, C-D were performed, and the two PCR products obtained in the first PCR step were assembled according to their respective complementarity. After addition of primers A and D, the full length DNA encoding the reconstituted 12E10 single-chain Fv with a 15 amino acid linker was amplified (second PCR). In the first PCR, the plasmid pCOS-db12E10 (see example 8.3(1)) encoding the reconstituted 12E10 single-chain Fv was used as template.
A50. mu.l solution of the first PCR reaction contained 5. mu.l of 10 XExTaq Buffer, 0.4mM dNTPs, 2.5 units of DNA polymerase TaKaRa ExTaq (from TAKARA), 100pmol of each primer and 10ng of each template DNA. The PCR solution was heated at an initial temperature of 94 ℃ for 30 seconds, at 94 ℃ for 15 seconds and at 72 ℃ for 2 minutes, and this cycle was repeated 5 times. After a cycle of 94 ℃ for 15 seconds and 72 ℃ for 2 minutes was repeated 28 times, the reaction mixture was heated at 72 ℃ for a further 5 minutes.
The second step was used to assemble PCR products A-B (477bp) and C-D (447 bp). The second PCR mixture solution (98. mu.l) contained 1. mu.l each of the first PCR products A-B and C-D as a template, 100pmol each of the primers A and D, 5. mu.l of 10 XExTaq Buffer, 0.4mM dNTPs, and 2.5 units of DNA polymerase TaKaRa ExTaq (from TAKARA) and reacted under the same conditions as described above.
The 825bpDNA fragment generated by the second PCR was purified using a 1.0% low melting temperature agarose gel and digested with EcoRI and NotI. The DNA fragment thus obtained was cloned into pCHO1 vector or pCOS1 vector. After DNA sequencing, the plasmids encoding the DNA fragments comprising the reconstructed single-chain Fv correct amino acid sequence of 12E10 were designated pCHO-sc12E10 and pCOS-sc12E 10. The nucleotide sequence and amino acid sequence of the reconstituted 12E10 single-chain Fv comprised in plasmids pCHO-sc12E10 and pCOS-sc12E10 are shown in SEQ ID NO: 113, respectively.
8.4 expression of antibody 12E10(IgG, C),Fab) and single chain Fv polypeptides
COS-7 cells or CHO cells were used to express antibody 12E10(IgG, Fab) and single chain Fv derived from antibody 12E10 (linker sequence 5 amino acids, 15 amino acids).
Transient expression was performed using COS7 cells as follows. Transfection was performed by the electroporation method using a Gene Pulser II apparatus (BioRad). In COS-7 cells (1X 10) suspended in 0.8ml PBS7Cell/ml), 10. mu.g each of the above expression vectors HEF-12E 10H-g.gamma.1 and pCOS-12E10L was added for expression of antibody 12E10(IgG), 10. mu.g each of pFd-12E10H and pCOS-12E10L was added for expression of 12E10Fab fragment, and 10. mu.g of pCOS-sc12E10 (10. mu.g) or pCOS-db12E10 (10. mu.g) was added for expression of single-chain Fv. The mixture in the cuvette was treated with 1.5KV, 25 μ FD capacitance pulses. After 10 minutes of room temperature recovery, the electroporated cells were added to DMEM medium containing 10% fetal bovine serum (GIBCOBRL) for culture. After overnight culture, cells were washed once with PBS and cultured for 3 days in serum-free medium CHO-S-SFM II (GIBCO BRL). The culture supernatant was centrifuged to remove cell debris and filtered through a 0.22 μm filter.
To establish a CHO cell line stably expressing single-chain Fv (polypeptide) derived from antibody 12E10, expression vectors pCHO-sc12E10 or pCOS-ds12E10, respectively, were introduced into CHO cells.
Each expression vector was introduced into CHO cells by the electroporation method using a Gene Pulser II apparatus (BioRad). Linearized DNA (100. mu.g) was obtained by digestion with restriction enzyme PvuI and combined in a cuvette with CHO cells (1X 10) suspended in 0.8ml PBS7Cells/ml), left on ice for 10 minutes and pulsed with 1.5KV, 25 μ FD capacitance. After 10 minutes of room temperature recovery, the electroporated cells were added to CHO-S-SFM II (GIBCO BRL) containing 10% dialyzed fetal bovine serum and nucleic acids for culture. After 2 days of culture, the culture was continued in nucleic acid-free CHO-S-SFM II medium (GIBCO BRL) containing 10% dialyzed fetal calf serum. From the clones thus obtained, clones with high expression rate were selected as a 12E10 single-chain Fv-producing cell line. After culturing in serum-free CHO-S-SFM II Medium (GIBCO BRL), the culture supernatant was centrifugedCell debris was removed and filtered through a 0.22 μm filter.
8.5 purification of Single chain Fv derived from 12E10 produced by CHO cells
Culture supernatants produced by the CHO cell lines expressing 12E10 single-chain Fv (sc12E10, db12E10) obtained in example 8.4 were purified using an anti-FLAG antibody column and a gel filtration column, respectively, to produce purified single-chain Fv.
(1) Purification using anti-FLAG antibody column
Each culture supernatant (sc12E10, db12E10) was added to an anti-FLAG M2 affinity gel column (Sigma) equilibrated with 50mM Tris-HCl buffer (pH 7.4) containing 150mM NaCl. After the column was washed with the same buffer, the protein adsorbed on the column was eluted by 100mM glycine buffer (pH 3.5). The eluted fractions were immediately neutralized by adding 1M Tris-HCl buffer (pH8.0) and analyzed by SDS-PAGE. Fractions confirmed to contain single-chain Fv were mixed and concentrated approximately 20-fold using Centricon-10 (AMICON).
(2) Gel filtration
(1) The concentrated solution obtained in (1) was added to a Superdex200 column HR (10X 300mm, AMERSHAM PHARMACIA) equilibrated with PBS containing 0.01% Tween 20. The chromatograms are shown in FIGS. 53 and 54. The product sc12E10 eluted as 2 peaks (A, B) (see fig. 53). The product db12E10 eluted as 2 peaks (C, D) (see FIG. 54). Each peak fraction was collected and electrophoresed using an electrophoretic sample with or without a reducing agent according to the Laemmli method, followed by staining with Coomassie Brilliant blue. As shown in FIG. 55, all fractions A, B, C and D produced a single band with an apparent molecular weight of about 31kD, regardless of the presence or absence of reducing agent. When these fractions were analysed by gel filtration using Superdex200HR, fraction A produced a product eluting at an apparent molecular weight of approximately 42kD, fraction B produced a product of 20kD (see FIG. 56), fraction C produced a product of 69kD, and fraction D produced a product of 41kD (see FIG. 57). This result suggests that component A derived from sc12E10 is a non-covalent dimer of a single-chain Fv, component B is a monomer of a single-chain Fv, component C derived from db12E10 is a non-covalent trimer of a single-chain Fv, and component D is a non-covalent dimer of a single-chain Fv.
8.6 measurement of TPO-like agonist Activity of various Single chain Fvs
TPO-like activity of the anti-MPL single-chain antibody was evaluated by measuring proliferation activity on Ba/F3 cells (BaF/MPL) expressing human TPO receptor (MPL).
After washing the BaF/mpl cells twice with RPMI1640 medium (GIBCO) containing 1% fetal bovine serum (GIBCO), the cells were treated at 5X 105The cell density of cells/ml is suspended in the culture medium. Dilution of anti-MPL Single chain antibody or human TPO (R) with Medium&DSystems). Add 50. mu.l of cell suspension and 50. mu.l of diluted antibody or human TPO to 96-well microplate (Flat bottom) (Corning) in CO2In heat preservation box (CO)2Concentration: 5%) for 24 hours. After incubation, 10. mu.l of WST-8 reagent (reagent for determining the number of primary cells SF: Nacalai Tesque) was added, and absorbance was immediately measured using an absorptiometer Benchmark Plus (BioRad), measuring wavelength 450nm, reference wavelength 655 nm. In CO2In heat preservation box (CO)2Concentration: 5%) and after 2 hours of incubation, the absorbance was measured again using a Benchmark Plus, measuring the wavelength 450nm, reference wavelength 655 Bm. Since the WST-8 reagent produces a color reaction at a wavelength of 450nm depending on the number of living cells, the proliferation activity of BaF/mpl was evaluated based on the change in absorbance after 2 hours.
Agonist activity to MPL was determined using COS-7 cell culture supernatants expressing various 12E10 antibody molecules, as shown in figure 58. Single chain Fv with 5 amino acid linker (ds12E10) and 15 amino acid linker (sc12E10) with increasing absorbance in a concentration-dependent manner, showed TPO-like agonist activity (ED 50; 9pM and 51pM, respectively), whereas 12E10IgG and 12E10Fab were not active.
It is known that single-chain Fv H-chains and L-chains are not only bound intramolecularly, but also intermolecularly to form multimers, e.g., dimers. Culture supernatants from CHO cells expressing 12E10 single-chain Fv were gel filtered and assayed for agonist activity against MPL. The results are shown in FIG. 59. The small amount of dimer contained in sc12E10 showed a TPO-like agonist activity (sc12E10 dimer, ED 50; 1.9pM) about 5000 times stronger than the monomer (sc12E10 monomer, ED 50; >10 nM). The activity was higher than that of TPO (ED 50; 27 pM). The dimer of db12E10 (db12E10 dimer, ED 50; 2.0pM) showed high activity comparable to the sc12E10 dimer. The trimer of db12E10 (the molecular weight deduced to be a trimer from gel filtration) (ED 50; 7.4pM) showed a high activity lower than that of db12E10 dimer. These results suggest that the bivalent antigen binding site (dimer) is important for the activity of agonist antibody 12E 10. Given the lack of activity of 12E10IgG, it is speculated that factors other than bivalent are important, such as the location, distance, or angle of the antigen binding site.
Drawings
FIG. 1 shows flow cytometry results, indicating that human IgG antibodies do not bind to human IAP-expressing L1210 cells (hIAP/L1210).
FIG. 2 shows flow cytometry results demonstrating that chimeric MABL-1 antibodies specifically bind to human IAP-expressing L1210 cells (hIAP/L1210).
FIG. 3 shows flow cytometry results demonstrating that chimeric MABL-2 antibodies specifically bind to human IAP-expressing L1210 cells (hIAP/L1210).
FIG. 4 is a schematic diagram showing the process of producing a single-chain Fv of the present invention.
FIG. 5 illustrates the structure of an expression plasmid which can be used inE.coliExpresses the DNA encoding the single-chain Fv of the present invention.
FIG. 6 illustrates the structure of an expression plasmid which can be used to express the DNA encoding the single-chain Fv of the present invention in mammalian cells.
FIG. 7 is a graph showing the results of Western blotting in example 5.4. Starting from the left, molecular weight markers (97.4, 66, 45, 31, 21.5 and 14.5kDa from above), COS7 cell culture supernatant introduced with pCHO1, and COS7 cell culture supernatant introduced with pCHOM 2. Indicating that recombinant single chain Fv comprising antibody MABL-2 (arrow) was contained in the culture supernatant of COS7 cells introduced with pCHOM 2.
FIG. 8 shows the results of flow cytometry, which indicates that the antibody in the culture supernatant of the control pCHO1/COS7 cells did not bind to the pCOS1/L1210 control cells.
FIG. 9 shows the results of flow cytometry, indicating that antibodies in the culture supernatant of MABL2-scFv/COS7 cells did not bind to pCOS1/L1210 control cells.
FIG. 10 shows the results of flow cytometry, indicating that the antibody in the control pCOS1/COS7 cell culture supernatant did not bind to hIAP/L1210 cells.
FIG. 11 shows flow cytometry results demonstrating that antibodies in the culture supernatant of MABL2-scFv/COS7 cells specifically bind to hIAP/L1210 cells.
FIG. 12 shows the results of competitive ELISA in example 5.6, in which the antigen-binding activity of single-chain Fv according to the invention (MABL2-scFv) was demonstrated in comparison with the control pCHO1/COS7 cell culture supernatant, as indicated by the inhibition of antigen binding by the mouse monoclonal antibody MABL-2.
FIG. 13 shows the results of the apoptosis-inducing effect in example 5.7, indicating that the antibody in the culture supernatant of pCHO1/COS7 cells as a control did not induce apoptosis of pCOS1/L1210 control cells.
FIG. 14 shows the results of the apoptosis-inducing effect in example 5.7, indicating that antibodies in the culture supernatant of MABL2-scFv/COS7 cells failed to induce apoptosis in pCOS1/L1210 control cells.
FIG. 15 shows the results of the apoptosis-inducing effect of example 5.7, indicating that the antibody in the culture supernatant of pCHO1/COS7 cells as a control did not induce apoptosis of hIAP/L1210 cells.
FIG. 16 shows the results of the apoptosis-inducing effect in example 5.7, indicating that antibodies in the culture supernatant of MABL2-scFv/COS7 cells specifically induce apoptosis of hIAP/L1210 cells.
FIG. 17 shows the results of the apoptosis-inducing effect in example 5.7, indicating that the antibody in the culture supernatant of pCHO1/COS7 cells as a control did not induce apoptosis of CCRF-CEM cells (final concentration: 50%).
FIG. 18 shows the results of the apoptosis-inducing effect in example 5.7, indicating that the antibody in the culture supernatant of MABL2-scFv/COS7 cells specifically induces apoptosis in CCRF-CEM cells (final concentration: 50%).
FIG. 19 shows the results of chromatography of single-chain Fv derived from antibody MABL-2 produced by CHO cell purification in example 5.9, showing the major peaks, component A and component B, obtained when the components derived from Blue-sepharose column were purified using hydroxyapatite column.
FIG. 20 shows the results of gel filtration purification of component A and component B obtained in example 5.9- (2), showing the major peaks (AI and BI, respectively) eluting for component A at an apparent molecular weight of about 36kD and component B at about 76 kD.
FIG. 21 is an SDS-PAGE analysis of fractions from single chain Fv derived from antibody MABL-2 produced by purified CHO cells from example 5.9, showing that a single band with a molecular weight of about 35kD is observed in both fractions.
FIG. 22 shows the results of analysis of the fractions AI and BI obtained by gel filtration purification of single-chain Fv derived from antibody MABL-2 from CHO cells, wherein fraction AI comprises monomers and fraction BI comprises dimers.
FIG. 23 illustrates the structure of an expression plasmid, which can be used inE.coliExpresses the DNA encoding the single-chain Fv of the present invention.
FIG. 24 shows gel filtration column purification of example 5.12E.coliThe results of the production of a crude single-chain Fv polypeptide derived from antibody MABL-2, in which each peak represents a respective peakE.coliMonomers or dimers of the resulting single-chain Fv.
FIG. 25 shows the results of the apoptosis-inducing effect of example 5.13, indicating that the control mouse IgG antibody was unable to induce apoptosis of hIAP/L1210 cells (final concentration 3. mu.g/ml).
FIG. 26 shows the results of the apoptosis-inducing effect in example 5.13, indicating that the CHO cell-produced MABL2-scFv dimer significantly induced apoptosis in hIAP/L1210 cells (final concentration 3. mu.g/ml).
FIG. 27 shows the results of the apoptosis-inducing effect in example 5.13, indicating thatE.coliThe produced MABL2-scFv dimer significantly induced apoptosis of hIAP/L1210 cells (final concentration of 3. mu.g/ml).
FIG. 28 shows the results of the apoptosis-inducing effect in example 5.13, indicating that the CHO cell-produced MABL2-scFv monomer induces apoptosis of hIAP/L1210 cells at the same level as the control (final concentration 3. mu.g/ml).
FIG. 29 shows the results of the apoptosis-inducing effect in example 5.13, indicating thatE.coliThe resulting MABL2-scFv monomer induced apoptosis of hIAP/L1210 cells at the same level as the control (final concentration 3. mu.g/ml).
FIG. 30 shows the results of the apoptosis-inducing effect of example 5.13, indicating that the control mouse IgG antibody, even when added with anti-FLAG antibody, did not induce apoptosis in hIAP/L1210 cells (final concentration 3. mu.g/ml).
FIG. 31 shows the results of the apoptosis-inducing effect in example 5.13, indicating that the MABL2-scFv monomer produced by CHO cells significantly induced apoptosis of hIAP/L1210 cells (final concentration 3. mu.g/ml) when anti-FLAG antibody was added.
Fig. 32 shows the results of quantitative measurement of human IgG in the serum of a mouse transplanted with the human myeloma cell line KPMM2, indicating the amount of human IgG produced by human myeloma cells in the mouse. Indicating that the scFv/CHO dimer significantly inhibited KPMM2 cell growth.
FIG. 33 shows the survival time of mice after tumor transplantation, indicating that the survival time of the scFv/CHO dimer-administered group was significantly prolonged.
FIG. 34 illustrates the structure of an expression plasmid that can express an altered antibody [ sc (fv) ] comprising two H chain V regions and two L chain V regions derived from the antibody MABL-22]。
FIG. 35 illustrates the structure of scFv (HL type) expressing plasmids in which the V regions are linked by [ H chain ] - [ L chain ] means without a peptide linker.
Fig. 36 illustrates the structure of HL type polypeptides and the amino acid sequence of peptide linkers.
FIG. 37 illustrates the structure of scFv (LH-type) expressing plasmids in which the V regions are linked by [ L chain ] - [ H chain ] means without a peptide linker.
FIG. 38 illustrates the structure of LH-type polypeptide and the amino acid sequence of the peptide linker.
FIG. 39 shows the results of Western blotting of example 6.4, showing that altered antibody sc (fv) comprising two H chain V regions and two L chain V regions is expressed2And MABL2-scFv with peptide linkers of different lengths.
FIGS. 40a and 40b show the flow cytometry results of COS7 cell culture supernatants prepared in example 6.3(1), showing MABL2-scFv and sc (fv) with peptide linkers of different lengths2Has high affinity for human IAPs.
FIGS. 41a and 41b show the results of the apoptosis-inducing effect of example 6.6, indicating scFv<HL3, 4, 6, 7, LH3, 4, 6 and 7>And sc (fv)2And the hIAP/L1210 cell death is obviously induced.
FIG. 42 shows the results of evaluation of the antigen-binding ability of example 6.10, indicating scFv<HL5>Dimer and sc (fv)2Has high affinity for human IAPs.
FIG. 43 shows example 6.11In vitroResults of apoptosis-inducing Effect, indicating scFv<HLS>Dimer and sc (fv)2hIAP/L1210 cells and CCRF-CEM cells were induced to apoptosis in a concentration-dependent manner.
FIG. 44 shows the results of quantitative measurement of M protein produced by the human myeloma cell line KPMM2 in the serum of a mouse into which human myeloma cells have been transplanted. Indicating scFv<HL5>Dimer and sc (fv)2The growth of KPMM2 cells is obviously inhibited.
Fig. 45 shows survival time (days) of mice after tumor transplantation, indicating that survival time was significantly prolonged for the scFv < HL5> administered group.
FIG. 46 shows the survival time (days) of mice after tumor transplantation, indicating sc (fv)2The survival time of the administered group was significantly prolonged.
FIG. 47 illustrates the method of constructing a reconstructed 12B5 single-chain Fv-encoding DNA fragment comprising a linker sequence consisting of 15 amino acids and its structure.
FIG. 48 shows the results of purification of each of the 12B5 single-chain Fv obtained in example 7.5(1) by gel filtration, which revealed that the sc12B5 was divided into two peaks (fractions A and B).
FIG. 49 shows the result of SDS-PAGE analysis of each of fractions A and B performed in example 7.5 (2).
FIG. 50 shows the results of Superdex200 column analysis of each of fractions A and B performed in example 7.5(2), showing that the main peak of fraction A elutes at an apparent molecular weight of about 44kD as shown in (a) and the main peak of fraction B elutes at an apparent molecular weight of about 22kD as shown in (B).
Fig. 51 shows the measurement results of the TPO-like agonist activity of sc12B5 and antibody 12B5(IgG, Fab), indicating that 12B5IgG and monovalent single chain Fv (sc12B5) exhibit TPO-like agonist activity in a concentration-dependent manner.
Fig. 52 shows the results of measurement of TPO-like agonist activity of sc12B5 monomer and dimer, indicating that single-chain Fv with bivalent antigen binding site (sc12B5 dimer) has approximately 400-fold higher agonist activity than monovalent sc12B5 and equal or higher potency than human TPO.
FIG. 53 shows the purification results of sc12E10 scFv by gel filtration chromatography using a Superdex200HR column, indicating that sc12E10 was separated into two peaks (fractions A and B).
FIG. 54 shows the purification results of db12E10 scFv by gel filtration chromatography using Superdex200HR column, indicating that db12E10 is divided into two peaks (fractions C and D).
FIG. 55 shows SDS-PAGE analysis of fractions A and B (sc12E10) and fractions C and D (db12E10) under reducing or non-reducing conditions.
FIG. 56 shows the results of analysis of fractions A and B by gel filtration chromatography using a Superdex200HR column, showing that (1) the major peak of fraction A elutes at an apparent molecular weight of about 42kD and (2) the major peak of fraction B elutes at an apparent molecular weight of about 20 kD. .
FIG. 57 shows the results of analysis of fractions C and D by gel filtration chromatography using a Superdex200HR column, showing that (1) the major peak of fraction C elutes at an apparent molecular weight of about 69kD and (2) the major peak of fraction D elutes at an apparent molecular weight of about 41 kD. .
Fig. 58 is a graph depicting the agonist activity of various 12E10 antibody molecules on MPL, indicating that single chain Fv (sc12E10, db12E10) show TPO-like agonist activity, while 12E10IgG and 12E10Fab do not.
Fig. 59 is a graph depicting agonist activity of MPL at sc12E10 monomers and dimers, and db12E10 dimers and trimers, indicating that sc12E10 dimers, and db12E10 dimers and trimers show TPO-like agonist activity higher than TPO.
Industrial applications
The altered antibodies of the invention have agonist effects capable of transducing signals into cells by cross-linking cell surface molecules, with the advantage of high permeability to tissues and tumors due to their reduced molecular size compared to the parent antibody molecule (complete IgG). The present invention provides altered antibodies with significantly higher agonist activity compared to the native ligand (e.g., TPO) and the parental antibody (complete IgG). Even if the parent antibody has no agonist activity, it is possible to provide an altered antibody having higher agonist activity than the native ligand. This is due to the fact that the altered antibody is more closely shaped to the ligand than the original antibody. Thus, the altered antibodies can be used as signal transduction agonists to achieve apoptosis-inducing, cell proliferation-inducing, cell differentiation-inducing, cell division-inducing or cell cycle modulating effects. According to the present invention, the change of antibody molecules into altered antibodies results in the reduction of side effects caused by intercellular cross-linking, and provides a novel drug that induces only desired effects by cross-linking cell surface molecules. Pharmaceutical preparations comprising the altered antibody of the present invention as an active ingredient are useful as preventive and/or therapeutic agents for cancer, inflammation, hormone disorders, autoimmune diseases and hematological diseases such as leukemia, malignant lymphoma, aplastic anemia, myelodysplastic syndrome and polycythemia vera.
Sequence listing
<110>CHUGAI SEIYAKU KABUSHIKI KAISHA
<120> agonist antibody having reduced molecular weight
<130>FP1032
<141>2001-10-22
<150>JP2000-321821
<151>2000-10-20
<150>JP2000-321822
<151>2000-10-20
<150>PCT/JP01/01912
<151>2001-03-12
<150>PCT/JP01/03288
<151>2001-04-17
<150>JP2001-277314
<151>2001-09-12
<160>113
<210>1
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>1
ccatcctaat acgactcact atagggc 27
<210>2
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>2
ggatcccggg tggatggtgg gaagatg 27
<210>3
<211>28
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>3
ggatcccggg ccagtggata gacagatg 28
<210>4
<211>26
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>4
ggatcccggg agtggataga ccgatg 26
<210>5
<211>394
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(393)
<223> pGEM-M1L.1-57; signal peptide, 58-394; mature peptides
<400>5
atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct gcg 48
Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala
1 5 10 15
tcc agc agt gat gtt gtg atg acc caa act cca ctc tcc ctg cct gtc 96
Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30
agt ctt gga gat caa gcc tcc atc tct tgc aga tct agt cag agc ctt 144
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45
cta cac agt aaa gga aac acc tat tta caa tgg tac cta cag aag cca 192
Leu His Ser Lys Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro
50 55 60
ggc cag tct cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct 240
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
65 70 75 80
ggg gtc cca gac agg ttc agt ggc agt gga tca ggg aca gat ttc aca 288
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
ctc aag atc agc aga gtg gag gct gag gat ctg gga gtt tat ttc tgc 336
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
100 105 110
tct caa agt aca cat gtt ccg tac acg tcc gga ggg ggg acc aag ctg 384
Ser Gln Ser Thr His Val Pro Tyr Thr Ser Gly Gly Gly Thr Lys Leu
115 120 125
gaa ata aaa c 394
Glu Ile Lys
130
<210>6
<211>409
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(408)
<223> pGEM-M1H.1-57; signal peptide, 58-409; mature peptides
<400>6
atg gaa tgg agc tgg ata ttt ctc ttc ctc ctg tca gga act gca ggt 48
Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
gtc cac tcc cag gtc cag ctg cag cag tct gga cct gac ctg gta aag 96
Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys
20 25 30
cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc 144
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
gtt aac cat gtt atg cac tgg gtg aag cag aag cca ggg cag ggc ctt 192
Val Asn His Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
50 55 60
gag tgg att gga tat att tat cct tac aat gat ggt act aag tac aat 240
Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
65 70 75 80
gag aag ttc aag ggc aag gcc aca ctg act tca gag aaa tcc tcc agc 288
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Glu Lys Ser Ser Ser
85 90 95
gca gcc tac atg gag ctc agc agc ctg gcc tct gag gac tct gcg gtc 336
Ala Ala Tyr Met Glu Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
100 105 110
tac tac tgt gca aga ggg ggt tac tat agt tac gac gac tgg ggc caa 384
Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Ser Tyr Asp Asp Trp Gly Gln
115 120 125
ggc acc act ctc aca gtc tcc tea g 409
Gly Thr Thr Leu Thr Val Ser Ser
130 135
<210>7
<211>394
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(393)
<223> pGEM-M2 L.1-57; signal peptide, 58-394; mature peptides
<400>7
atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct ggt 48
Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phc Trp Ile Pro Gly
1 5 10 15
tcc agc agt gat gtt gtg atg acc caa agt cca ctc tcc ctg cct gtc 96
Ser Ser Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val
20 25 30
agt ctt gga gat caa gcc tcc atc tct tgc aga tca agt cag agc ctt 144
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45
gtg cac agt aat gga aag acc tat tta cat tgg tac ctg cag aag cca 192
Val His Ser Asn Gly Lys Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
50 55 60
ggc cag tct cca aaa ctc ctg atc tac aaa gtt tcc aac cga ttt tct 240
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
65 70 75 80
ggg gtc cca gac agg ttc agt ggc agt gga tca gtg aca gat ttc aca 288
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Val Thr Asp Phe Thr
85 90 95
ctc atg atc agc aga gtg gag gct gag gat ctg gga gtt tat ttc tgc 336
Leu Met Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
100 105 110
tct caa agt aca cat gtt ccg tac acg ttc gga ggg ggg acc aag ctg 384
Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
gaa ata aaa c 394
Glu Ile Lys
130
<210>8
<211>409
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(408)
<223> pGEM-M2 H.1-57; signal peptide, 58-409; mature peptides
<400>8
atg gaa tgg agc tgg ata ttt ctc ttc ctc ctg tca gga act gca ggt 48
Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
gtc cac tcc cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag 96
Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc 144
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
gct aac cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt 192
Ala Asn His Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
50 55 60
gag tgg att gga tat att tat cct tac aat gat ggt act aag tat aat 240
Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
65 70 75 80
gag aag ttc aag gac aag gcc act ctg act tca gac aaa tcc tcc acc 288
Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr
85 90 95
aca gcc tac atg gac ctc agc agc ctg gcc tct gag gac tct gcg gtc 336
Thr Ala Tyr Met Asp Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
100 105 110
tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa 384
Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln
115 120 125
ggc acc act ctc aca gtc tcc tca g 409
Gly Thr Thr Leu Thr Val Ser Ser
130 135
<210>9
<211>32
<212>DNA
<213> Artificial sequence
<220>
<223> PR primer
<400>9
cccaagcttc caccatgaag ttgcctgtta gg 32
<210>10
<211>32
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>10
cccaagcttc caccatggaa tggagctgga ta 32
<210>11
<211>34
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>11
cgcggatcca ctcacgtttt atttccagct tggt 34
<210>12
<211>34
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>12
cgcggatcca ctcacctgag gagactgtga gagt 34
<210>13
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>13
catgccatgg cgcaggtcca gctgcagcag 30
<210>14
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>14
accaccacct gaggagactg tgagagt 27
<210>15
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>15
gtctcctcag gtggtggtgg ttcgggt 27
<210>16
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>16
cacaacatcc gatccgccac cacccga 27
<210>17
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>17
ggcggatcgg atgttgtgat gacccaa 27
<210>18
<211>57
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>18
ccggaattct cattatttat cgtcatcgtc tttgtagtct tttatttcca gcttggt 57
<210>19
<211>45
<212>DNA
<213> Artificial sequence
<220>
<223> linker amino acid sequence and nucleotide sequence
<400>19
ggt ggt ggt ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg 45
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
5 10 15
<210>20
<211>828
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(822)
<223>pscM1.MABL1-scFv
<400>20
atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc gct 48
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
gcc caa cca gcc atg gcg cag gtc cag ctg cag cag tct gga cct gac 96
Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Asp
20 25 30
ctg gta aag cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga 144
Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
35 40 45
tac acc ttc gtt aac cat gtt atg cac tgg gtg aag cag aag cca ggg 192
Tyr Thr Phe Val Asn His Val Met His Trp Val Lys Gln Lys Pro Gly
50 55 60
cag ggc ctt gag tgg att gga tat att tat cct tac aat gat ggt act 240
Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr
65 70 75 80
aag tac aat gag aag ttc aag ggc aag gcc aca ctg act tca gag aaa 288
Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Glu Lys
85 90 95
tcc tcc agc gca gcc tac atg gag ctc agc agc ctg gcc tct gag gac 336
Ser Ser Ser Ala Ala Tyr Met Glu Leu Ser Ser Leu Ala Ser Glu Asp
100 105 110
tct gcg gtc tac tac tgt gca aga ggg ggt tac tat agt tac gac gac 384
Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Ser Tyr Asp Asp
115 120 125
tgg ggc caa ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg 432
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa 480
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln
145 150 155 160
act cca ctc tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct 528
Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser
165 170 175
tgc aga tct agt cag agc ctt cta cac agt aaa gga aac acc tat tta 576
Cys Arg Ser Ser Gln Ser Leu Leu His Ser Lys Gly Asn Thr Tyr Leu
180 185 190
caa tgg tac cta cag aag cca ggc cag tct cca aag ctc ctg atc tac 624
Gln Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
195 200 205
aaa gtt tcc aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt 672
Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
210 215 220
gga tca ggg aca gat ttc aca ctc aag atc agc aga gtg gag gct gag 720
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu
225 230 235 240
gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg 768
Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr
245 250 255
tcc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac 816
Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp Asp
260 265 270
gat aaa taatga 828
Asp Lys
<210>21
<211>31
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>21
acgcgtcgac tcccaggtcc agctgcagca g 31
<210>22
<211>18
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>22
gaaggtgtat ccagaagc 18
<210>23
<211>819
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(813)
<223>pCHOM1.MABL1-scFv
<400>23
atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
gtc gac tcc cag gtc cag ctg cag cag tct gga cct gac ctg gta aag 96
Val Asp Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys
20 25 30
cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc 144
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
gtt aac cat gtt atg cac tgg gtg aag cag aag cca ggg cag ggc ctt 192
Val Asn His Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
50 55 60
gag tgg att gga tat att tat cct tac aat gat ggt act aag tac aat 240
Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
65 70 75 80
gag aag ttc aag ggc aag gcc aca ctg act tca gag aaa tcc tcc agc 288
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Glu Lys Ser Ser Ser
85 90 95
gca gcc tac atg gag ctc agc agc ctg gcc tct gag gac tct gcg gtc 336
Ala Ala Tyr Met Glu Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
100 105 110
tac tac tgt gca aga ggg ggt tac tat agt tac gac gac tgg ggc caa 384
Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Ser Tyr Asp Asp Trp Gly Gln
115 120 125
ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt 432
Gly Thr Thr Lou Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa act cca ctc 480
Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr Pro Leu
145 150 155 160
tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct tgc aga tct 528
Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser
165 170 175
agt cag agc ctt cta cac agt aaa gga aac acc tat tta caa tgg tac 576
Ser Gln Ser Leu Leu His Ser Lys Gly Asn Thr Tyr Leu Gln Trp Tyr
180 185 190
cta cag aag cca ggc cag tct cca aag ctc ctg atc tac aaa gtt tcc 624
Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser
195 200 205
aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tca ggg 672
Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
210 215 220
aca gat ttc aca ctc aag atc agc aga gtg gag gct gag gat ctg gga 720
Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly
225 230 235 240
gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg tcc gga ggg 768
Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr Ser Gly Gly
245 250 255
ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac gat aaa taa 816
Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp Asp Asp Lys
260 265 270
tga 819
<210>24
<211>828
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(822)
<223>pscM2.MABL2-scFv
<400>24
atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc gct 48
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
gcc caa cca gcc atg gcg cag gtc cag ctg cag cag tct gga cct gaa 96
Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Glu
20 25 30
ctg gta aag cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga 144
Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
35 40 45
tac acc ttc gct aac cat gtt att cac tgg gtg aag cag aag cca ggg 192
Tyr Thr Phe Ala Asn His Val Ile His Trp Val Lys Gln Lys Pro Gly
50 55 60
cag ggc ctt gag tgg att gga tat att tat cct tac aat gat ggt act 240
Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr
65 70 75 80
aag tat aat gag aag ttc aag gac aag gcc act ctg act tca gac aaa 288
Lys Tyr Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys
85 90 95
tcc tcc acc aca gcc tac atg gac ctc agc agc ctg gcc tct gag gac 336
Ser Ser Thr Thr Ala Tyr Met Asp Leu Ser Ser Leu Ala Sor Glu Asp
100 105 110
tct gcg gtc tat tac tgt gca aga ggg ggt tac tat act tac gac gac 384
Ser Ala Val Tyr Tyr Cys Ala Arg GLy Gly Tyr Tyr Thr Tyr Asp Asp
115 120 125
tgg ggc caa ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg 432
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa 480
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln
145 150 155 160
agt cca ctc tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct 528
Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser
165 170 175
tgc aga tca agt cag agc ctt gtg cac agt aat gga aag acc tat tta 576
Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Lys Thr Tyr
180 185 190
cat tgg tac ctg cag aag cca ggc cag tct cca aaa ctc ctg atc tac 624
His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
195 200 205
aaa gtt tcc aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt 672
Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
210 215 220
gga tca gtg aca gat ttc aca ctc atg atc agc aga gtg gag gct gag 720
Gly Ser Val Thr Asp Phe Thr Leu Met Ile Ser Arg Val Glu Ala Glu
225 230 235 240
gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg 768
Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr
245 250 255
ttc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac 816
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp Asp
260 265 270
gat aaa taatga 828
Asp Lys
<210>25
<211>819
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(813)
<223>pCHOM2.MABL2-scFv
<400>25
atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
gtc gac tcc cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag 96
Val Asp Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc 144
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
gct aac cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt 192
Ala Asn His Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
50 55 60
gag tgg att gga tat att tat cct tac aat gat ggt act aag tat aat 240
Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
65 70 75 80
gag aag ttc aag gac aag gcc act ctg act tca gac aaa tcc tcc acc 288
Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr
85 90 95
aca gcc tac atg gac ctc agc agc ctg gcc tct gag gac tct gcg gtc 336
Thr Ala Tyr Met Asp Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
100 105 110
tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa 384
Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln
115 120 125
ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt 432
Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa agt cca ctc 480
Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Leu
145 150 155 160
tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct tgc aga tca 528
Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser
165 170 175
agt cag agc ctt gtg cac agt aat gga aag acc tat tta cat tgg tac 576
Ser Gln Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu His Trp Tyr
180 185 190
ctg cag aag cca ggc cag tct cca aaa ctc ctg atc tac aaa gtt tcc 624
Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser
195 200 205
aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tca gtg 672
Asn Arg Phc Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Val
210 215 220
aca gat ttc aca ctc atg atc agc aga gtg gag gct gag gat ctg gga 720
Thr Asp Phe Thr Leu Met Ile Ser Arg Val Glu Ala Glu Asp Leu Gly
225 230 235 240
gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg ttc gga ggg 768
Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly
245 250 255
ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac gat aaa taa 816
Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp Asp Asp Lys
260 265 270
tga 819
<210>26
<211>456
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(450)
<223> pCHO-shIAP soluble human IAP
<400>26
atg tgg ccc ctg gta gcg gcg ctg ttg ctg ggc tcg gcg tgc tgc gga 48
Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly
1 5 10 15
tca gct cag cta cta ttt aat aaa aca aaa tct gta gaa ttc acg ttt 96
Ser Ala Gln Leu Leu Phe Ash Lys Thr Lys Ser Val Glu Phe Thr Phe
20 25 30
tgt aat gac act gtc gtc att cca tgc ttt gtt act aat atg gag gca 144
Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala
35 40 45
caa aac act act gaa gta tac gta aag tgg aaa ttt aaa gga aga gat 192
Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp
50 55 60
att tac acc ttt gat gga gct cta aac aag tcc act gtc ccc act gac 240
Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp
65 70 75 80
ttt agt agt gca aaa att gaa gtc tca caa tta cta aaa gga gat gcc 288
Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala
85 90 95
tct ttg aag atg gat aag agt gat gct gtc tca cac aca gga aac tac 336
Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr
100 105 110
act tgt gaa gta aca gaa tta acc aga gaa ggt gaa acg atc atc gag 384
Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu
115 120 125
cta aaa tat cgt gtt gtt tca tgg ttt tct cca aat gaa aat gac tac 432
Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Asp Tyr
130 135 140
aag gac gac gat gac aag tgatag 456
Lys Asp Asp Asp Asp Lys
145 150
<210>27
<211>46
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>27
ggaattccat atgcaagtgc aacttcaaca gtctggacct gaactg 46
<210>28
<211>31
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>28
ggaattctca ttattttatt tccagcttgg t 31
<210>29
<211>741
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(735)
<223>pscM2DEm02.MABL2-scFv
<400>29
atg caa gtg caa ctt caa cag tct gga cct gaa ctg gta aag cct ggg 48
Met Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly
1 5 10 15
gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc gct aac 96
Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asn
20 25 30
cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt gag tgg 144
His Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp
35 40 45
att gga tat att tat cct tac aat gat ggt act aag tat aat gag aag 192
Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys
50 55 60
ttc aag gac aag gcc act ctg act tca gac aaa tcc tcc acc aca gcc 240
Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Thr Ala
65 70 75 80
tac atg gac ctc agc agc ctg gcc tct gag gac tct gcg gtc tat tac 288
Tyr Met Asp Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr
85 90 95
tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa ggc acc 336
Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln Gly Thr
100 105 110
act ctc aca gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt ggt tcg 384
Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
ggt ggt ggc gga tcg gat gtt gtg atg acc caa agt cca ctc tcc ctg 432
Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
130 135 140
cct gtc agt ctt gga gat caa gcc tcc atc tct tgc aga tca agt cag 480
Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
145 150 155 160
agc ctt gtg cac agt aat gga aag acc tat tta cat tgg tac ctg cag 528
Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu His Trp Tyr Leu Gln
165 170 175
aag cca ggc cag tct cca aaa ctc ctg atc tac aaa gtt tcc aac cga 576
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg
180 185 190
ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tca gtg aca gat 624
Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Val Thr Asp
195 200 205
ttc aca ctc atg atc agc aga gtg gag gct gag gat ctg gga gtt tat 672
Phe Thr Leu Met Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr
210 215 220
ttc tgc tct caa agt aca cat gtt ccg tac acg ttc gga ggg ggg acc 720
Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr
225 230 235 240
aag ctg gaa ata aaa taatga 741
Lys Leu Glu Ile Lys
245
<210>30
<211>18
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>30
cagacagtgg ttcaaagt 18
<210>31
<211>72
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>31
cgcgtcgacc gatccgccac cacccgaacc accaccaccc gaaccaccac caccttttat 60
ttccagcttg gt 72
<210>32
<211>1605
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(1599)
<223>pCHOM2(Fv)2.MABL2-sc(Fv)2
<400>32
atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
gtc gac tcc cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag 96
Val Asp Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc 144
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
gct aac cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt 192
Ala Asn His Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
50 55 60
gag tgg att gga tat att tat cct tac aat gat ggt act aag tat aat 240
Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
65 70 75 80
gag aag ttc aag gac aag gcc act ctg act tca gac aaa tcc tcc acc 288
Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr
85 90 95
aca gcc tac atg gac ctc agc agc ctg gcc tct gag gac tct gcg gtc 336
Thr Ala Tyr Met Asp Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
100 105 110
tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa 384
Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln
115 120 125
ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt 432
Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa agt cca ctc 480
Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Leu
145 150 155 160
tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct tgc aga tca 528
Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser
165 170 175
agt cag agc ctt gtg cac agt aat gga aag acc tat tta cat tgg tac 576
Ser Gln Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu His Trp Tyr
180 185 190
ctg cag aag cca ggc cag tct cca aaa ctc ctg atc tac aaa gtt tcc 624
Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser
195 200 205
aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tca gtg 672
Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Val
210 215 220
aca gat ttc aca ctc atg atc agc aga gtg gag gct gag gat ctg gga 720
Thr Asp Phe Thr Leu Met Ile Ser Arg Val Glu Ala Glu Asp Leu Gly
225 230 235 240
gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg ttc gga ggg 768
Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly
245 250 255
ggg acc aag ctg gaa ata aaa ggt ggt ggt ggt tcg ggt ggt ggt ggt 816
Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
260 265 270
tcg ggt ggt ggc gga tcg gtc gac tcc cag gtc cag ctg cag cag tct 864
Ser Gly Gly Gly Gly Ser Val Asp Ser Gln Val Gln Leu Gln Gln Ser
275 280 285
gga cct gaa ctg gta aag cct ggg gct tca gtg aag atg tcc tgc aag 912
Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys
290 295 300
gct tct gga tac acc ttc gct aac cat gtt att cac tgg gtg aag cag 960
Ala Ser Gly Tyr Thr Phe Ala Asn His Val Ile His Trp Val Lys Gln
305 310 315 320
aag cca ggg cag ggc ctt gag tgg att gga tat att tat cct tac aat 1008
Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn
325 330 335
gat ggt act aag tat aat gag aag ttc aag gac aag gcc act ctg act 1056
Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr
340 345 350
tca gac aaa tcc tcc acc aca gcc tac atg gac ctc agc agc ctg gcc 1104
Ser Asp Lys Ser Ser Thr Thr Ala Tyr Met Asp Leu Ser Ser Leu Ala
355 360 365
tct gag gac tct gcg gtc tat tac tgt gca aga ggg ggt tac tat act 1152
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr
370 375 380
tac gac gac tgg ggc caa ggc acc act ctc aca gtc tcc tca ggt ggt 1200
Tyr Asp Asp Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly
385 390 395 400
ggt ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gat gtt gtg 1248
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val
405 410 415
atg acc caa agt cca ctc tcc ctg cct gtc agt ctt gga gat caa gcc 1296
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala
420 425 430
tcc atc tct tgc aga tca agt cag agc ctt gtg cac agt aat gga aag 1344
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Lys
435 440 445
acc tat tta cat tgg tac ctg cag aag cca ggc cag tct cca aaa ctc 1392
Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu
450 455 460
ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca gac agg ttc 1440
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe
465 470 475 480
agt ggc agt gga tca gtg aca gat ttc aca ctc atg atc agc aga gtg 1488
Ser Gly Ser Gly Ser Val Thr Asp Phe Thr Leu Met Ile Ser Arg Val
485 490 495
gag gct gag gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt 1536
Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val
500 505 510
ccg tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa 1584
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys
515 520 525
gac gat gac gat aaa taatga 1605
Asp Asp Asp Asp Lys
530
<210>33
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>33
tgaggaattc ccaccatggg atg 33
<210>34
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>34
cacgacgtca ctcgagactg tgagagtggt gccttggccc 40
<210>35
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>35
agtctcgagt gacgtcgtga tgacccaaag tccactctcc 40
<210>36
<211>31
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>36
gactggatcc tcattattta tcgtcatcgt c 31
<210>37
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>37
cgcgtaatac gactcactat ag 22
<210>38
<211>46
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>38
gcaattggac ctgttttatc tcgagcttgg tcccccctcc gaacgt 46
<210>39
<211>45
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>39
gctcgagata aaacaggtcc aattgcagca gtctggacct gaact 45
<210>40
<211>60
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>40
gactggatcc tcattattta tcgtcatcgt ctttgtagtc tgaggagact gtgagagtgg 60
<210>41
<211>32
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>41
gactgaattc ccaccatgaa gttgcctgtt ag 32
<210>42
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>42
cagtctcgag tggtggttcc gacgtcgtga tgacccaaag 40
<210>43
<211>43
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>43
cagtctcgag tggtggtggt tccgacgtcg tgatgaccca aag 43
<210>44
<211>46
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>44
cagtctcgag tggtggtggt ggttccgacg tcgtgatgac ccaaag 46
<210>45
<211>49
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>45
cagtctcgag tggtggtggt ggtggttccg acgtcgtgat gacccaaag 49
<210>46
<211>52
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>46
cagtctcgag tggtggtggt ggtggtggtt ccgacgtcgt gatgacccaa ag 52
<210>47
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>47
ggccgcatgt tgtcacgaat 20
<210>48
<211>780
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(768)
<223>CF2HL-0/pCOS1.MABL2-scFv<HL-0>
<400>48
atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt gtc 51
MET Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val
5 10 15
gac tcc cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag cct ggg 102
Asp Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly
20 25 30
gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc gct aac cat 153
Ala Ser Val Lys MET Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asn His
35 40 45 50
gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt gag tgg att gga 204
Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly
55 60 65
tat att tat cct tac aat gat ggt act aag tat aat gag aag ttc aag gac 255
Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Asp
70 75 80 85
aag gcc act ctg act tca gac aaa tcc tcc acc aca gcc tac atg gac ctc 306
Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Thr Ala Tyr MET Asp Leu
90 95 100
agc agc ctg gcc tct gag gac tct gcg gtc tat tac tgt gca aga ggg ggt 357
Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly
105 110 115
tac tat act tac gac gac tgg ggc caa ggc acc act ctc aca gtc tcg agt 408
Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
120 125 130 135
gac gtc gtg atg acc caa agt cca ctc tcc ctg cct gtc agt ctt gga gat 459
Asp Val Val MET Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp
140 145 150
caa gcc tcc atc tct tgc aga tca agt cag agc ctt gtg cac agt aat gga 510
Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly
155 160 165 170
aag acc tat tta cat tgg tac ctg cag aag cca ggc cag tct cca aaa ctc 561
Lys Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu
175 180 185
ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca gac agg ttc agt 612
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser
190 l95 200
ggc agt gga tca gtg aca gat ttc aca ctc atg atc agc aga gtg gag gct 663
Gly Ser Gly Ser Val Thr Asp Phe Thr Leu MET Ile Ser Arg Val Glu Ala
205 210 215 220
gag gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg 714
Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr
225 230 235
ttc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac gat 765
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp Asp Asp
240 245 250 255
aaa taa tga gga tcc 780
Lys
<210>49
<211>45
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>49
caagctcgag ataaaatccg gaggccaggt ccaattgcag cagtc 45
<210>50
<211>48
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>50
caagctcgag ataaaatccg gaggtggcca ggtccaattg cagcagtc 48
<210>51
<211>51
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>51
caagctcgag ataaaatccg gaggtggtgg ccaggtccaa ttgcagcagt c 51
<210>52
<211>54
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>52
caagctcgag ataaaatccg gaggtggtgg tggccaggtc caattgcagc agtc 54
<210>53
<211>57
<212>DNA
<213> Artificial sequence
<220>
<223> PCR primer
<400>53
caagctcgag ataaaatccg gaggtggtgg tggtggccag gtccaattgc agcagtc 57
<210>54
<2111>780
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(768)
<223>CF2LH-0/pCOS1.MABL2-scFv<LH-0>
<400>54
atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct ggt tcc 51
MET Lys Leu Pro Val Arg Leu Leu Val Leu MET Phe Trp Ile Pro Gly Ser
5 10 15
agc agt gat gtt gtg atg acc caa agt cca ctc tcc ctg cct gtc agt ctt 102
Ser Ser Asp Val Val MET Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu
20 25 30
gga gat caa gcc tcc atc tct tgc aga tca agt cag agc ctt gtg cac agt 153
Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
35 40 45 50
aat gga aag acc tat tta cat tgg tac ctg cag aag cca ggc cag tct cca 204
Asn Gly Lys Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro
55 60 65
aaa ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca gac agg 255
Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg
70 75 80 85
ttc agt ggc agt gga tca gtg aca gat ttc aca ctc atg atc agc aga gtg 306
Phe Ser Gly Ser Gly Ser Val Thr Asp Phe Thr Leu MET Ile Ser Arg Val
90 95 100
gag gct gag gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg 357
Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro
105 110 115
tac acg ttc gga ggg ggg acc aag ctc gag ata aaa cag gtc caa ttg cag 408
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gln Val Gln Leu Gln
120 125 130 135
cag tct gga cct gaa ctg gta aag cct ggg gct tca gtg aag atg tcc tgc 459
Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys MET Ser Cys
140 145 150
aag gct tct gga tac acc ttc gct aac cat gtt att cac tgg gtg aag cag 510
Lys Ala Ser Gly Tyr Thr Phe Ala Asn His Val Ile His Trp Val Lys Gln
155 160 165 170
aag cca ggg cag ggc crt gag tgg att gga tat att tat cct tac aat gat 561
Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp
175 180 185
ggt act aag tat aat gag aag ttc aag gac aag gcc act ctg act tca gac 612
Gly Thr Lys Tyr Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp
190 195 200
aaa tcc tcc acc aca gcc tac atg gac ctc agc agc ctg gcc tct gag gac 663
Lys Ser Ser Thr Thr Ala Tyr MET Asp Leu Ser Ser Leu Ala Ser Glu Asp
205 210 215 220
tct gcg gtc tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg 714
Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp
225 230 235
ggc caa ggc acc act ctc aca gtc tcc tca gac tac aaa gac gat gac gat 765
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Asp Tyr Lys Asp Asp Asp Asp
240 245 250 255
aaa taa tga gga tcc 780
Lys
<210>55
<211>351
<212>DNA
<213> human
<220>
<221>CDS
<222>(1)...(351)
<223>12B5HV.1-351 peptides
<400>55
cag gtg cag ctg gtg cag tct ggg gga ggc ttg gtc cgg ccc ggg ggg 48
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Arg Pro Gly Gly
1 5 10 15
tcc ctg agt ctc tcc tgt gca gtc tct gga atc acc ctc agg acc tac 96
Ser Leu Ser Leu Ser Cys Ala Val Ser Gly Ile Thr Leu Arg Thr Tyr
20 25 30
ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
gca ggt ata tcc ttt gac gga aga agt gaa tac tat gca gac tcc gtg 192
Ala Gly Ile Ser Phe Asp Gly Arg Ser Glu Tyr Tyr Ala Asp Ser Val
50 55 60
cag ggc cga ttc acc atc tcc aga gac agt tcc aag aac acc ctg tat 240
Gln Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Thr Leu Tyr
65 70 75 80
ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt 288
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
gcg aga gga gca cat tat ggt ttc gat atc tgg ggc caa ggg aca atg 336
Ala Arg Gly Ala His Tyr Gly Phe Asp Ile Trp Gly Gln Gly Thr Met
100 105 110
gtc acc gtc tcg agt 351
Val Thr Val Ser Ser
115
<210>56
<211>57
<212>DNA
<213> human
<220>
<221>CDS
<222>(1)...(57)
<223> leader sequence
<400>56
atg gag ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt tta aga ggt 48
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly
5 10 15
gtc cag tgt 57
Val Gln Cys
<210>57
<211>115
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VH-1
<400>57
atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60
gtgcagctgg tgcagtctgg gggaggcttg gtccggcccg gggggtccct gagtc 115
<210>58
<211>115
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VH-2
<400>58
aaggatatac ctgccaccca ctccagcccc ttgcctggag cctggcggac ccagtgcatg 60
ccgtaggtcc tgagggtgat tccagagact gcacaggaga gactcaggga ccccc 115
<210>59
<211>115
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VH-3
<400>59
ggcaggtata tcctttgacg gaagaagtga atactatgca gactccgtgc agggccgatt 60
caccatctcc agagacagtt ccaagaacac cctgtatctg caaatgaaca gcctg 115
<210>60
<211>108
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VH-4
<400>60
actcgagacg gtgaccattg tcccttggcc ccagatatcg aaaccataat gtgctcctct 60
cgcacagtaa tacacagccg tgtcctcggc tctcaggctg ttcatttg 108
<210>61
<211>32
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VH-S, PCR primers
<400>61
ttcaagcttc caccatggag tttgggctga gc 32
<210>62
<211>34
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VH-A, PCR primers
<400>62
ttgggatcca ctcaccactc gagacggtga ccat 34
<210>63
<211>588
<212>DNA
<213> human
<220>
<221>CDS
<222>(236)...(558)
<223> 1-235; intron, 236-558; human IgG constant region (part)
<400>63
gaattcgtga gtggatccca agctagcttt ctggggcagg ccaggcctga ccttggcttt 60
ggggcaggga gggggctaag gtgaggcagg tggcgccagc caggtgcaca cccaatgccc 120
atgagcccag acactggacg ctgaacctcg cggacagtta agaacccagg ggcctctgcg 180
ccctgggccc agctctgtcc cacaccgcgg tcacatggca caacctctct tgca gcc 237
Ala
1
tcc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc 285
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
5 10 15
acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc 333
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
20 25 30
ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc 381
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
35 40 45
gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc 429
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
50 55 60 65
agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac 477
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
70 75 80
atc tgc aacgtg aat cac aag ccc agc aac acc aag gtg gac aag aaa 525
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
85 90 95
gtt gag ccc aaa tct tgt gac aaa act cac aca 558
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
100 105
<210>64
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223> G1CH1-S, PCR primers
<400>64
tgagaattcg tgagtggatc ccaagct 27
<210>65
<211>60
<212>DNA
<213> Artificial sequence
<220>
<223> G1CH1-A, PCR primers
<400>65
aaaagatctt tatcatgtgt gagttttgtc acaagatttg ggctcaactt tcttgtccac 60
<210>66
<211>432
<212>DNA
<213> human
<220>
<221>CDS
<222>(12)...(419)
<223> HEF-12B5H-g gamma.12-419 peptide
<400>66
aagcttccac c atg gag ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt 50
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu
1 5 10
tta aga ggt gtc cag tgt cag gtg cag ctg gtg cag tct ggg gga ggc 98
Leu Arg Gly yal Gln Cys Gln Val Gln Leu Val Gln Ser Gly Gly Gly
15 20 25
ttg gtc cgg ccc ggg ggg tcc ctg agt ctc tcc tgt gca gtc tct gga 146
Leu Val Arg Pro Gly Gly Ser Leu Ser Leu Ser Cys Ala Val Ser Gly
30 35 40 45
atc acc ctc agg acc tac ggc atg cac tgg gtc cgc cag gct cca ggc 194
Ile Thr Leu Arg Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly
50 55 60
aag ggg ctg gag tgg gtg gca ggt ata tcc ttt gac gga aga agt gaa 242
Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Phe Asp Gly Arg Ser Glu
65 70 75
tac tat gca gac tcc gtg cag ggc cga ttc acc atc tcc aga gac agt 290
Tyr Tyr Ala Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Ser
80 85 90
tcc aag aac acc ctg tat ctg caa atg aac agc ctg aga gcc gag gac 338
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
95 100 105
acg gct gtg tat tac tgt gcg aga gga gca cat tat ggt ttc gat atc 386
Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala His Tyr Gly Phe Asp Ile
110 115 120 125
tgg ggc caa ggg aca atg gtc acc gtc tcg agt ggtgagtgga tcc 432
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
130 135
<210>67
<211>321
<212>DNA
<213> human
<220>
<221>CDS
<222>(1)...(321)
<223>12B5LV.1-321 peptide
<400>67
gac atc cag atg acc cag tct cct tcc acc ctg tct gca tct att gga 48
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly
1 5 10 15
gac aga gtc acc atc acc tgc cgg gcc agc gag ggt att tat cac tgg 96
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Gly Ile Tyr His Trp
20 25 30
ttg gcc tgg tat cag cag aag cca ggg aaa gcc cct aaa ctc ctg atc 144
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
tat aag gcc tct agt tta gcc agt ggg gcc cca tca agg ttc agc ggc 192
Tyr Lys Ala Ser Ser Leu Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly
50 55 60
agt gga tct ggg aca gat ttc act ctc acc atc agc agc ctg cag cct 240
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
gat gat ttt gca act tat tac tgc caa caa tat agt aat tat ccg ctc 288
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu
85 90 95
act ttc ggc gga ggg acc aag ctg gag atc aaa 321
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210>68
<211>66
<212>DNA
<213> human
<220>
<221>CDS
<222>(1)...(66)
<223> leader sequence
<400>68
atg gac atg agg gtc ccc gct cag ctc ctg ggg ctc ctg ctg ctc tgg 48
MET Asp MET Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
5 10 15
ctc cca ggt gcc aaa tgt 66
Leu Pro Gly Ala Lys Cys
20
<210>69
<211>110
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VL-1
<400>69
atggacatga gggtccccgc tcagctcctg gggctcctgc tgctctggct cccaggtgcc 60
aaatgtgaca tccagatgac ccagtctcct tccaccctgt ctgcatctat 110
<210>70
<211>110
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VL-2
<400>70
ggagtttagg ggctttccct ggcttctgct gataccaggc caaccagtga taaataccct 60
cgctggcccg gcaggtgatg gtgactctgt ctccaataga tgcagacagg 110
<210>71
<211>110
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VL-3
<400>71
aagcccctaa actcctgatc tataaggcct ctagtttagc cagtggggcc ccatcaaggt 60
tcagcggcag tggatctggg acagatttca ctctcaccat cagcagcctg 110
<210>72
<211>103
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VL-4
<400>72
tttgatctcc agcttggtcc ctccgccgaa agtgagcgga taattactat attgttggca 60
gtaataagtt gcaaaatcaat caggctgcag gctgctgatg gtg 103
<210>73
<211>32
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VL-S, PCR primers
<400>73
ttcaagcttc caccatggac atgagggtcc cc 32
<210>74
<211>35
<212>DNA
<213> Artificial sequence
<220>
<223>12B5VL-A, PCR primers
<400>74
tctaggatcc actcacgttt gatctccagc ttggt 35
<210>75
<211>415
<212>DNA
<213> human
<220>
<221>CDS
<222>(12)...(398)
<223> HEF-12B5H-g kappa.12-398 peptide
<400>75
aagcttccac c atg gac atg agg gtc ccc gct cag ctc ctg ggg ctc ctg 50
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu
1 5 10
ctg ctc tgg ctc cca ggt gcc aaa tgt gac atc cag atg acc cag tct 98
Leu Leu Trp Leu Pro Gly Ala Lys Cys Asp Ile Gln Met Thr Gln Ser
15 20 25
cct tcc acc ctg tct gca tct att gga gac aga gtc acc atc acc tgc 146
Pro Ser Thr Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile Thr Cys
30 35 40 45
cgg gcc agc gag ggt att tat cac tgg ttg gcc tgg tat cag cag aag 194
Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln Lys
50 55 60
cca ggg aaa gcc cct aaa ctc ctg atc tat aag gcc tct agt tta gcc 242
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Ala
65 70 75
agt ggg gcc cca tca agg ttc agc ggc agt gga tct ggg aca gat ttc 290
Ser Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
80 85 90
act ctc acc atc agc agc ctg cag cct gat gat ttt gca act tat tac 338
Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr
95 100 105
tgc caa caa tat agt aat tat ccg ctc act ttc ggc gga ggg acc aag 386
Cys Gln Gln Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys
110 115 120 125
ctg gag atc aaa cgtgagtgga tcctaga 415
Leu Glu Ile Lys
<210>76
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> FLAG tag sequence
<400>76
gac tac aag gat gac gac gat aag 24
Asp Tyr Lys Asp Asp Asp Asp Lys
5
<210>77
<211>31
<212>DNA
<213> Artificial sequence
<220>
<223>12B5-S, PCR primers
<400>77
atagaattcc accatggagt ttgggctgag c 31
<210>78
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> HuVHJ3, PCR primers
<400>78
tgaagagacg gtgaccattg tccc 24
<210>79
<211>28
<212>DNA
<213> Artificial sequence
<220>
<223> RhuJH 3PCR primers
<400>79
ggacaatggt caccgtctct tcaggtgg 28
<210>80
<211>32
<212>DNA
<213> Artificial sequence
<220>
<223> RhuVK1, PCR primers
<400>80
ggagactggg tcatctggat gtccgatccg cc 32
<210>81
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> HuVK1.2, PCR primers
<400>81
gacatccaga tgacccagtc tcc 23
<210>82
<211>59
<212>DNA
<213> Artificial sequence
<220>
<223>12B5F-A, PCR primers
<400>82
attgcggccg cttatcactt atcgtcgtca tccttgtagt ctttgatctc cagcttggt 59
<210>83
<211>45
<212>DNA
<213> Artificial sequence
<220>
<223> linker amino acid sequence and nucleotide sequence
<400>83
ggt ggt ggt ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg 45
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
5 10 15
<210>84
<211>823
<212>DNA
<213> human
<220>
<221>CDS
<222>(12)...(809)
<223> sc12B5, single-chain Fv
<400>84
aagcttccac c atg gag ttt ggg ctg agc tgg gtt ttc crc gtt gct ctt 50
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu
1 5 10
tta aga ggt gtc cag tgt cag gtg cag ctg gtg cag tct ggg gga ggc 98
Leu Arg Gly Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Gly Gly
15 20 25
ttg gtc cgg ccc ggg ggg tcc ctg agt ctc tcc tgt gca gtc tct gga 146
Leu Val Arg Pro Gly Gly Ser Leu Ser Leu Ser Cys Ala Val Ser Gly
30 35 40 45
atc acc ctc agg acc tac ggc atg cac tgg gtc cgc cag gct cca ggc 194
Ile Thr Leu Arg Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly
50 55 60
aag ggg ctg gag tgg gtg gca ggt ata tcc ttt gac gga aga agt gaa 242
Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Phe Asp Gly Arg Ser Glu
65 70 75
tac tat gca gac tcc gtg cag ggc cga ttc acc atc tcc aga gac agt 290
Tyr Tyr Ala Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Ser
80 85 90
tcc aag aac acc ctg tat ctg caa atg aac agc ctg aga gcc gag gac 338
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
95 100 105
acg gct gtg tat tac tgt gcg aga gga gca cat tat ggt ttc gat atc 386
Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala His Tyr Gly Phe Asp Ile
110 115 120 125
tgg ggc caa ggg aca atg gtc acc gtc tcg agt ggt ggt ggt ggt tcg 434
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg acc cag 482
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
145 150 155
tct cct tcc acc ctg tct gca tct att gga gac aga gtc acc atc acc 530
Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile Thr
160 165 170
tgc cgg gcc agc gag ggt att tat cac tgg ttg gcc tgg tat cag cag 578
Cys Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln
175 180 185
aag cca ggg aaa gcc cct aaa ctc ctg atc tat aag gcc tct agt tta 626
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu
190 195 200 205
gcc agt ggg gcc cca tca agg ttc agc ggc agt gga tct ggg aca gat 674
Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
210 215 220
ttc act ctc acc atc agc agc ctg cag cct gat gat ttt gca act tat 722
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr
225 230 235
TAC TGC CAA CAA TAT AGT AAT TAT CCG CTC ACT TTC GGC GGA GGG ACC 770
Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr
240 245 250
aag ctg gag atc aaa gac tac aag gat gac gac gat aag tgataagcgg c 820
Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp Asp Asp Lys
255 260 265
cgc 823
<210>85
<211>114
<212>PRT
<213> human
<400>85
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Ser Ser Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Ser Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg Gly Thr Met Val Thr Val
100 105 110
Ser Ser
<210>86
<211>342
<212>DNA
<213> human
<400>86
caggtgcagc tgcagcagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60
acctgcactg tctctggtga ctccatcagt agttactact ggagctggat tcggcagccc 120
ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180
ccctccctca agagtcgagt caccatatca gtagacacgt ccaagagcca gttctccctg 240
aagctgagct ctgtgaccgc cgcagacacg gccgtgtatt actgtgcgag agggcggtac 300
ttcgatgtct ggggccgtgg caccatggtc actgtctcct ca 342
<210>87
<211>57
<212>DNA
<213> human
<220>
<221>CDS
<222>(1)...(57)
<223> leader sequence
<308>GenBank No.AF062252
<400>87
atg aaa cat ctg tgg ttc ttc ctt ctc ctg gtg gca gct ccc aga tgg 48
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp
l 5 10 15
gtc ctg tcc 57
Val Leu Ser
<210>88
<211>110
<212>DNA
<213> Artificial sequence
<220>
<223>12E10Vtt1
<400>88
atgaaacatc tgtggttctt ccttctcctg gtggcagctc ccagatgggt cctgtcccag 60
gtgcagctgc agcagtcggg cccaggactg gtgaagcctt cggagaccct 110
<210>89
<211>110
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VH2
<400>89
acccaatcca ctccagtccc ttccctgggg gctgccgaat ccagctccag tagtaactac 60
tgatggagtc accagagaca gtgcaggtga gggacagggt ctccgaaggc 110
<210>90
<211>110
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VH3
<400>90
tggagtggat tgggtatatc tattacagtg ggagcaccaa ctacaacccc tccctcaaga 60
gtcgagtcac catatcagta gacacgtcca agagccagtt ctccctgaag 110
<210>91
<211>114
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VH4
<400>91
tgaggagaaca gtgaccatgg tgccacggcc ccagacatcg aagtaccgcc ctctcgcaca 60
gtaatacaacg gccgtgtctg cggcggtcac agagctcagc ttcagggaga actg 114
<210>92
<211>32
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VHS, PCR primers
<400>92
ttcaagcttc caccatgaaa catctgtggt tc 32
<210>93
<211>34
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VHA, PCR primers
<400>93
ttgggatcca ctcacctgag gagacagtga ccat 34
<210>94
<211>426
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(12)...(417)
<223>12E10H, H chain V region
<400>94
aagcttccac c atg aaa cat ctg tgg ttc ttc ctt ctc ctg gtg gca gct 50
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala
1 5 10
ccc aga tgg gtc ctg tcc cag gtg cag ctg cag cag tcg ggc cca gga 98
Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly
15 20 25
ctg gtg aag cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggt 146
Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
30 35 40 45
gac tcc atc agt agt tac tac tgg agc tgg att cgg cag ccc cca ggg 194
Asp Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
50 55 60
aag gga ctg gag tgg att ggg tat atc tat tac agt ggg agc acc aac 242
Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn
65 70 75
tac aac ccc tcc ctc aag agt cga gtc acc ata tca gta gac acg tcc 290
Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser
80 85 90
aag agc cag ttc tcc ctg aag ctg agc tct gtg acc gcc gca gac acg 338
Lys Ser Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
95 100 105
gcc gtg tat tac tgt gcg aga ggg cgg tac ttc gat gtc tgg ggc cgt 386
Ala Val Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg
110 115 120 125
ggc acc atg gtc act gtc tcc tca ggtgagtgga tcccaa 426
Gly Thr Met Val Thr Val Ser Ser
130
<210>95
<211>110
<212>PRT
<213>Mus
<400>95
Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Thr Arg
85 90 95
Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>96
<211>330
<212>DNA
<213>Mus
<400>96
tcctatgtgc tgactcagcc accctcggtg tcagggtctc ctggacagtc gatcaccatc 60
tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacag 120
cacccaggca aagcccccaa actcatgatt tatgagggca gtaaacggcc ctcaggggtt 180
tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240
caggctgagg acgaggctga ttattactgc agctcatata caaccagaag cactcgggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>97
<211>57
<212>DNA
<213> human
<220>
<221>CDS
<222>(1)...(57)
<223> leader sequence
<310>
<400>97
atg gcc tgg acc gtt ctc ctc ctc ggc ctc ctc tct cac tgc aca ggc 48
Met Ala Trp Thr Val Leu Leu Leu Gly Leu Leu Ser His Cys Thr Gly
1 5 10 15
tct gtg acc 57
Ser Val Thr
<210>98
<211>110
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VL1, PCR primers
<400>98
atggcctgga ccgttctcct cctcggcctc ctctctcact gcacaggctc tgtgacctcc 60
tatgtgctga ctcagccacc ctcggtgtca gggtctcctg gacagtcgat 110
<210>99
<211>62
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VL2, PCR primers
<400>99
tcatgagttt gggggctttg cctgggtgct gttggtacca ggagacatag ttataaccac 60
caacgtcact gctggttcca gtgcaggaga tggtgatcga ctgtccagga 110
<210>100
<211>110
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VL3, PCR primers
<400>100
cccccaaact catgatttat gagggcagta aacggccctc aggggtttct aatcgcttct 60
ctggctccaa gtctggcaac acggcctccc tgaccatctc tgggctccag 110
<210>101
<211>102
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VL4, PCR primers
<400>101
taggacggtc agcttggtcc ctccgccgaa cacccgagtg cttctggttg tatatgagct 60
gcagtaataa tcagcctcgt cctcagcctg gagcccagag at 102
<210>102
<211>31
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VLS, PCR primers
<400>102
atcaagcttc caccatggcc tggaccgttc t 31
<210>103
<211>36
<212>DNA
<213> Artificial sequence
<220>
<223>12E10VLA, PCR primers
<400>103
ctaggatccg ggctgaccta ggacggtcag cttggt 36
<210>104
<211>387
<212>DNA
<213>Mus
<220>
<221>CDS
<222>(1)...(387)
<223>12E10L, L chain V region
<310>
<400>104
atg gcc tgg acc gtt ctc ctc ctc ggc ctc ctc tct cac tgc aca ggc 48
Met Ala Trp Thr Val Leu Leu Leu Gly Leu Leu Ser His Cys Thr Gly
1 5 10 15
tct gtg acc tcc tat gtg ctg act cag cca ccc tcg gtg tca ggg tct 96
Ser Val Thr Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ser
20 25 30
cct gga cag tcg atc acc atc tcc tgc act gga acc agc agt gac gtt 144
Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val
35 40 45
ggt ggt tat aac tat gtc tcc tgg tac caa cag cac cca ggc aaa gcc 192
Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
50 55 60
ccc aaa ctc atg att tat gag ggc agt aaa cgg ccc tca ggg gtt tct 240
Pro Lys Leu Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser
65 70 75 80
aat cgc ttc tct ggc tcc aag tct ggc aac acg gcc tcc ctg acc atc 288
Asn Arg Phe Ser Gly Ser Lys Ser Gly Asr Thr Ala Ser Leu Thr Ile
85 90 95
tct ggg ctc cag gct gag gac gag gct gat tat tac tgc agc tca tat 336
Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr
100 105 110
Aca acc aga agc act cgg gtg ttc ggc gga ggg acc aag ctg acc gtc 384
Thr Thr Arg Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val
115 120 125
cta 387
Leu
<210>105
<211>24
<212>DNA
<213> Artificial sequence
<220>
<221>CDS
<222>(1)...(24)
<223> FLAG, leader sequence
<400>105
gac tac aag gat gac gac gat aag 24
Asp Tyr Lys Asp Asp Asp Asp Lys
<210>106
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223>12E10S, PCR primers
<400>106
tatgaattcc accatgaaac atctgtggtt 30
<210>107
<211>38
<212>DNA
<213> Artificial sequence
<220>
<223> DB2, PCR primers
<400>107
taggagctac cgcctccacc tgaggagaca gtgaccat 38
<210>108
<211>44
<212>DNA
<213> Artificial sequence
<220>
<223> DB1, PCR primers
<400>108
gtctcctcag gtggaggcgg tagctcctat gtgctgactc agcc 44
<210>109
<211>59
<212>DNA
<213> Artificial sequence
<220>
<223>12E10FA, PCR primer
<400>109
attgcggccg cttatcactt atcgtcgtca tccttgtagt ctaggacggt cagcttggt 59
<210>110
<211>792
<212>DNA
<213> Artificial sequence
<220>
<221>CDS
<222>(11)...(778)
<223>12E10, single-chain Fv
<400>110
gaattccacc atg aaa cat ctg tgg ttc ttc ctt ctc ctg gtg gca gct 49
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala
1 5 10
ccc aga tgg gtc ctg tcc cag gtg cag ctg cag cag tcg ggc cca gga 97
Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly
15 20 25
ctg gtg aag cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggt 145
Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
30 35 40 45
gac tcc atc agt agt tac tac tgg agc tgg att cgg cag ccc cca ggg 193
Asp Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
50 55 60
aag gga ctg gag tgg att ggg tat atc tat tac agt ggg agc acc aac 241
Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn
65 70 75
tac aac ccc tcc ctc aag agt cga gtc acc ata tca gta gac acg tcc 289
Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser
80 85 90
aag agc cag ttc tcc ctg aag ctg agc tct gtg acc gcc gca gac acg 337
Lys Ser Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
95 100 105
gcc gtg tat tac tgt gcg aga ggg cgg tac ttc gat gtc tgg ggc cgt 385
Ala Val Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg
110 115 120 125
ggc acc atg gtc act gtc tcc tca ggt gga ggc ggt agc tcc tat gtg 433
Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ser Tyr Val
130 135 140
ctg act cag cca ccc tcg gtg tca ggg tct cct gga cag tcg atc acc 481
Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr
145 150 155
atc tcc tgc act gga acc agc agt gac gtt ggt ggt tat aac tat gtc 529
Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val
160 165 170
tcc tgg tac caa cag cac cca ggc aaa gcc ccc aaa ctc atg att tat 577
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr
175 180 185
gag ggc agt aaa cgg ccc tca ggg gtt tct aat cgc ttc tct ggc tcc 625
Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser
190 195 200 205
aag tct ggc aac acg gcc tcc ctg acc atc tct ggg ctc cag gct gag 673
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu
210 215 220
gac gag gct gat tat tac tgc agc tca tat aca acc aga agc act cgg 721
Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Thr Arg Ser Thr Arg
225 230 235
gtg ttc ggc gga ggg acc aag ctg acc gtc cta gac tac aag gat gac 769
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Asp Tyr Lys Asp Asp
240 245 250
gac gat aag tgataagcgg ccgc 792
Asp Asp Lys
255
<210>111
<211>62
<212>DNA
<213> Artificial sequence
<220>
<223> sc4.3, PCR primers
<400>111
ggtggctgag tcagcacata ggacgatccg ccaccacccg aaccaccacc acccgaacca 60
cc 62
<210>112
<211>61
<212>DNA
<213> Artificial sequence
<220>
<223> sc1.3, PCR primers
<400>112
gcaccatggt cactgtctcc tcaggtggtg gtggttcggg tggtggtggt tcgggtggtg 60
g 61
<210>113
<211>822
<212>DNA
<213> Artificial sequence
<220>
<221>CDS
<222>(11)...(807)
<223> sc12E10, single-chain Fv
<400>113
gaattccacc atg aaa cat ctg tgg ttc ttc ctt ctc ctg gtg gca gct 49
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala
1 5 10
ccc aga tgg gtc ctg tcc cag gtg cag ctg cag cag tcg ggc cca gga 97
Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly
15 20 25
ctg gtg aag cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggt 145
Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
30 35 40 45
gac tcc atc agt agt tac tac tgg agc tgg att cgg cag ccc cca ggg 193
Asp Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
50 55 60
aag gga ctg gag tgg att ggg tat atc tat tac agt ggg agc acc aac 241
Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn
65 70 75
tac aac ccc tcc ctc aag agt cga gtc acc ata tca gta gac acg tcc 289
Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser
80 85 90
aag agc cag ttc tcc ctg aag ctg agc tct gtg acc gcc gca gac acg 337
Lys Ser Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
95 100 105
gcc gtg tat tac tgt gcg aga ggg cgg tac ttc gat gtc tgg ggc cgt 385
Ala Val Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg
110 115 120 125
ggc acc atg gtc act gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt 433
Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
ggt tcg ggt ggt ggc gga tcg tcc tat gtg ctg act cag cca ccc tcg 481
Gly Ser Gly Gly Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser
145 150 155
gtg tca ggg tct cct gga cag tcg atc acc atc tcc tgc act gga acc 529
Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr
160 165 170
agc agt gac gtt ggt ggt tat aac tat gtc tcc tggtac caa cag cac 577
Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His
175 180 185
cca ggc aaa gcc ccc aaa ctc atg att tat gag ggc agt aaa cgg ccc 625
Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Glu Gly Ser Lys Arg Pro
190 195 200 205
tca ggg gtt tct aat cgc ttc tct ggc tcc aag tct ggc aac acg gcc 673
Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala
210 215 220
tcc ctg acc atc tct ggg ctc cag gct gag gac gag gct gat tat tac 721
Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr
225 230 235
tgc agc tca tat aca acc aga agc act cgg gtg ttc ggc gga ggg acc 769
Cys Ser Ser Tyr Thr Thr Arg Ser Thr Arg Val Phe Gly Gly Gly Thr
240 245 250
aag ctg acc gtc cta gac tac aag gat gac gac gat aag tgataagcgg 818
Lys Leu Thr Val Leu Asp Tyr Lys Asp Asp Asp Asp Lys
255 260 265
ccgc 822
Claims (16)
1. A method of increasing agonist effect by modifying an antibody to an altered antibody comprising two or more H chain V regions and two or more L chain V regions of the antibody to crosslink cell surface molecules or intracellular molecules, wherein the altered antibody is:
(a) a dimer of a single-chain Fv comprising an H chain V region and an L chain V region; or
(b) A single-chain polypeptide comprising two H chain V regions and two L chain V regions,
and wherein the altered antibody has greater agonist activity than the parent antibody.
2. The method of claim 1, wherein the H chain V region and the L chain V region are connected by a linker.
3. The method of claim 2, wherein the linker is a peptide linker comprising at least one amino acid.
4. The method of any one of claims 1-3, wherein the altered antibody consists of a tetramer, trimer or dimer of a single chain Fv.
5. The method of claim 4, wherein the altered antibody consists of a dimer of single chain Fv.
6. The method of claim 1, wherein the altered antibody further comprises an amino acid sequence for peptide purification.
7. The method of claim 1, wherein the altered antibody has been purified.
8. The method of claim 1, wherein the H chain V region and/or the L chain V region is an H chain V region and/or an L chain V region derived from a human antibody.
9. The method according to claim 1, wherein the H chain V region and/or the L chain V region is a humanized H chain V region and/or L chain V region.
10. The method of claim 1, wherein the cell surface molecule or intracellular molecule is a hormone receptor, cytokine receptor, tyrosine kinase receptor, or nuclear receptor.
11. The method of claim 1, wherein the cell surface molecule or intracellular molecule is an erythropoietin receptor, a granulocyte colony stimulating factor receptor, a macrophage colony stimulating factor receptor, a granulocyte macrophage colony stimulating factor receptor, a tumor necrosis factor receptor, an interleukin-1 receptor, an interleukin-2 receptor, an interleukin-3 receptor, an interleukin-4 receptor, an interleukin-5 receptor, an interleukin-6 receptor, an interleukin-7 receptor, an interleukin-9 receptor, an interleukin-10 receptor, an interleukin-11 receptor, an interleukin-12 receptor, an interleukin-13 receptor, an interleukin-15 receptor, an interferon-alpha receptor, an interferon-beta receptor, a, Interferon-gamma receptor, growth hormone receptor, insulin receptor, blood stem cell proliferation factor receptor, vascular endothelial cell growth factor receptor, epithelial cell growth factor receptor, nerve growth factor receptor, fibroblast growth factor receptor, platelet-derived growth factor receptor, transforming growth factor-beta receptor, leukocyte migration inhibitory factor receptor, ciliary neurotrophic factor receptor, oncostatin M receptor, Notch family receptor, E2F, E2F/DP1, or TAK1/TAB 1.
12. The method of claim 1, wherein the agonist effect is induction of apoptosis, induction of cell proliferation, induction of cell differentiation, induction of cell division or cell cycle regulation.
13. The method of claim 1, wherein the altered antibody is a monospecific altered antibody.
14. The method of claim 1, wherein the altered antibody is a multispecific altered antibody.
15. The method of claim 14, wherein the altered antibody is a bispecific altered antibody.
16. The method of claim 15, wherein the L chain V region and the H chain V region are from the same monoclonal antibody.
Applications Claiming Priority (10)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000321822 | 2000-10-20 | ||
| JP2000-321821 | 2000-10-20 | ||
| JP2000321821 | 2000-10-20 | ||
| JP2000-321822 | 2000-10-20 | ||
| JPPCT/JP01/01912 | 2001-03-12 | ||
| PCT/JP2001/001912 WO2001066737A1 (en) | 2000-03-10 | 2001-03-12 | Polypeptide inducing apoptosis |
| PCT/JP2001/003288 WO2001079494A1 (en) | 2000-04-17 | 2001-04-17 | Agonist antibodies |
| JPPCT/JP01/03288 | 2001-04-17 | ||
| JP2001-277314 | 2001-09-12 | ||
| JP2001277314 | 2001-09-12 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1085932A1 HK1085932A1 (en) | 2006-09-08 |
| HK1085932B true HK1085932B (en) | 2008-08-08 |
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