WO1995014041A1 - Anticorps humain reconstitue contre des cellules medulloblastomateuses humaines - Google Patents

Anticorps humain reconstitue contre des cellules medulloblastomateuses humaines Download PDF

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WO1995014041A1
WO1995014041A1 PCT/JP1994/001763 JP9401763W WO9514041A1 WO 1995014041 A1 WO1995014041 A1 WO 1995014041A1 JP 9401763 W JP9401763 W JP 9401763W WO 9514041 A1 WO9514041 A1 WO 9514041A1
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chain
region
ser
gly
amino acid
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PCT/JP1994/001763
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English (en)
French (fr)
Japanese (ja)
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Toshihiko Ohtomo
Koh Sato
Masayuki Tsuchiya
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Chugai Seiyaku Kabushiki Kaisha
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Priority to EP94930341A priority Critical patent/EP0729976A1/en
Priority to AU79493/94A priority patent/AU7949394A/en
Priority to US08/646,265 priority patent/US6214973B1/en
Publication of WO1995014041A1 publication Critical patent/WO1995014041A1/ja
Priority to US10/839,799 priority patent/US7563599B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3053—Skin, nerves, brain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a human / mouse chimeric antibody comprising a variable region (V region) of mouse monoclonal antibody 0 NS-M21 against human medulloblastoma cells and a constant region (C region) of human antibody.
  • V region variable region
  • C region constant region
  • the complementarity determining regions (CDRs) of the human light chain (L chain) V region and the human heavy chain (H chain) V region show that the mouse monoclonal antibody ONS—M21 against human medulloblastoma cells It relates to reshaped human antibodies that have been replaced by CDRs and the L chains or H chains that constitute those antibodies.
  • the present invention further provides a DNA encoding the above antibody, particularly the V region thereof.
  • the present invention further relates to a vector comprising said DNA, especially an expression vector, and a host transformed with said vector.
  • the present invention further provides a method for producing chimeric antibodies against hepatic medulloblastoma cells and a method for producing reshaped human antibodies against human medulloblastoma cells.
  • the present invention also relates to a single-chain FV region formed by linking the H chain V region of a reshaped human antibody to human medulloblastoma cells and the L chain V region of the antibody.
  • the present invention also provides a DNA encoding the single-chain FV region, a recombinant vector comprising the DNA, a host transformed with the recombinant vector, and the host using the host.
  • the present invention relates to a method for producing a main chain FV region.
  • Medulloblastoma together with glioma accounts for 40% of brain tumors It is one of the ectodermal tumors, and it is a malignant brain tumor that frequently occurs in children under the age of 10, especially in the age of 5 to 10.
  • This tumor is a typical undifferentiated tumor that mimics the morphology and sequence pattern of undifferentiated cells that make up the medullary epithelium and stroma layer during embryonic development, and may differentiate into both neurons and glial cells (Tamura, K, et al., Cancer Res., 49: 53800-5384 (19989)). Because these malignant brain tumors are sensitive to radiation and chemotherapeutic agents, radiation therapy and chemotherapy, as well as surgery, are used as treatment.
  • Mouse monoclonal antibodies are highly immunogenic (sometimes referred to as "antigenic") in humans, and for this reason their therapeutic value in humans is limited. In addition, mouse antibodies cannot be administered as frequently without eliciting an immune response that not only interferes with the intended effect, but also poses a risk of adverse allergic response in the patient.
  • Mouse antibodies can be humanized in two ways. A simpler method is to make a chimeric antibody in which the variable regions are derived from the original mouse monoclonal antibody and the constant regions are derived from a suitable human antibody. The resulting chimeric antibody contains the complete variable region of the original mouse antibody and can be expected to bind to the antigen with the same specificity as the original mouse antibody 0
  • chimeric antibodies have a lower proportion of protein sequences derived from sources other than human. It is expected to be substantially reduced and therefore less immunogenic than the original mouse antibody. Chimeric antibodies bind well to antigens and are less immunogenic, but may still produce an immune response to the mouse variable region (Lo Buglio, AF et al., Proc. Nat. 1 .A cad .S ci .USA, 8 8, 4 2 2 0-4 2 2 4, 1 9 8 9) o
  • the second method for humanizing mouse antibodies is more complex-but it further reduces the potential immunogenicity of the mouse antibodies much more.
  • a complementarity-determining region (cmplememntaritindydetermineinggreegion; CDR) from the variable region of a mouse antibody is transplanted into a human antibody variable region to prepare a "reconstituted" (resshaped) human antibody variable region.
  • CDR complementarity-determining region
  • these reshaped human variable regions are linked to a human antibody constant region.
  • Only the CDR and a very small portion of the framework (FR) are derived from the protein sequence other than human in the finally reconstructed human antibody.
  • CDR is composed of a hypervariable protein sequence. They do not show species-specific sequences. For these reasons, a reshaped human antibody bearing mouse CDR should no longer be more immunogenic than a native human antibody containing human CDR.
  • reshaped human antibodies are expected to be useful for therapeutic purposes, but no reshaped human antibodies against human medulloblastoma cells are known. Furthermore, there is no method for producing reshaped human antibodies that is universally applicable to any particular antibody. Therefore, various measures are required to prepare a reshaped human antibody having sufficient activity against a specific antigen (for example, Sato, K. et al. Cancer Res. ⁇ 3, 1— 6 (1993).
  • the present inventors have developed a medulloblastoma cell line (ONS-7) from the cerebellum of medulloblastoma patients.
  • the present invention relates to a human monoclonal antibody ONS-M21 reshaped human antibody against human medulloblastoma cells.
  • the present invention also provides a human mouse chimeric antibody useful in the process of producing the reshaped human antibody.
  • the present invention further relates to a genetic engineering method for the production of a reshaped human antibody of mouse monoclonal antibody 0NS-M21 and a quinula antibody.
  • a human antibody a light chain comprising an L chain C region; and an L chain V region of a mouse monoclonal antibody ON S-M21 against the human medulloblastoma cell;
  • Human medulloblastoma comprising a human antibody H chain C region and a mouse monoclonal antibody against human medulloblastoma cells 0 NS—an H chain comprising the H chain V region of M21 Chimeric antibodies against cells, and
  • a reconstituted human light chain V region comprising:
  • the present invention also relates to L-chain or H-chain polypeptides constituting the various antibodies, and DNAs encoding them.
  • the present invention relates to an expression vector comprising the above-mentioned DNA and a host transformed with the expression vector.
  • the present invention further relates to a method for producing chimeric antibodies against human medulloblastoma cells and a method for producing reshaped human antibodies against human medulloblastoma cells.
  • the present invention also relates to a single-chain Fv region formed by linking the H chain V region of a reshaped human antibody to human medulloblastoma cells and the L chain V region of the monoclonal antibody.
  • the present invention further relates to a DNA encoding the single-stranded FV region, a recombinant vector comprising the DNA, and a host transformed by the recombinant vector.
  • the present invention still further relates to a method for producing a single-chain FV region, which comprises culturing the above-mentioned host and collecting a single-chain Fv region from the culture.
  • FIG. 1 shows the expression plasmids HE F—V L—g and HE F—VH—1 that comprise the human E F 1- ⁇ promoter Z enhancer, respectively, useful for the expression of L and H chains.
  • FIG. 2 is a graph showing the binding ability of the chimera ONS-M21 antibody (ChM21) to the human medulloblastoma cell line ONS-76.
  • FIG. 3 is a diagram showing the preparation of the first version (version “a”) of the reshaped human ONS—M21 antibody L chain V region.
  • Figure 4 shows a diagram of the production of the reshaped human ONS—M21 antibody V region.
  • FIG. 5 is a graph showing the binding ability of an antibody comprising a reconstituted human H chain and a chimeric L chain to the human medulloblastoma cell line ONS-76.
  • FIG. 3 is a graph showing the binding ability of the human medulloblastoma cell line ONS-76 to a chimeric antibody (ChM21).
  • FIG. 7 shows one of the versions “i”, “j”, “1”, “m”, “o” and “p” of the reshaped human H chain and the reshaped human L chain of the present invention.
  • the ability of the six reconstituted human 0N S-M21 antibodies to bind to the human medulloblastoma cell line ONS_76 was determined by using the chimeric antibody (ChM21) and the reconstituted L chain version.
  • FIG. 4 shows a comparison with an antibody having “k” and “n”.
  • FIG. 8 is a diagram schematically showing a process for preparing a single-stranded FV region of the present invention.
  • FIG. 9 shows the structure of an example of an expression plasmid used to express DNA encoding the single-chain FV region of the present invention.
  • FIG. 10 shows that the antigen binding activity of the single-chain FV region (scFV-hM21) of the present invention was determined by the reconstituted human 0NS-M21 antibody and the antigen binding of the Fab fragment of the antibody.
  • FIG. 3 is a graph showing, as an index, inhibition of antigen binding of mouse monoclonal antibody 0NS-M21 as compared to activity.
  • mRNA To clone DNA encoding the V region of a mouse monoclonal antibody against human medulloblastoma cells, mRNA must be prepared from mouse monoclonal antibody-producing cells, and then double-stranded by a known method. It is obtained by converting to cDNA and amplifying the target DNA using the polymerase chain reaction (PCR) method. As a source of this mRNA, it is necessary to produce a hybridoma that produces a monoclonal antibody against human medulloblastoma cells.
  • PCR polymerase chain reaction
  • the hybridoma cells were disrupted, treated with guanidine thiosinate, and then subjected to cesium chloride density gradient centrifugation (Chir gwin, JM et al., Biochemistry, 1 8, 5 2 9 4-5 2 9 9, 1 9 7 9)
  • Chir gwin, JM et al., Biochemistry, 1 8, 5 2 9 4-5 2 9 9, 1 9 7 A powerful method already used for cloning genes of other proteins, for example, the presence of ribonuclease inhibitors such as vanadium complex
  • Use a surfactant treatment and a phenol treatment below Boerger, S.L. et al., Biochemistry, 18, 51 143-51 149, 197 9). Can be.
  • the total RNA is made into type III, and the oligo (d) complementary to the p01yA chain at its 3 'end is obtained.
  • T) is treated with reverse transcriptase as a primer to synthesize single-stranded DNA (cDNA) complementary to all RNAs (Larrik, JW et al., Bio / Technology, ⁇ , 934). -9 3 8, 1 9 8 9).
  • a random primer may be used.
  • the cDNA was analyzed using the polymerase chain reaction (PCR) method. Perform specific amplification of the mouse antibody V region.
  • PCR polymerase chain reaction
  • primers described in Jones, ST. Et al., BioZ Technology, 9, 88-89, 1991 can be used.
  • H and L chains were typed to determine the primers used for cloning. It is necessary to decide the type.
  • the ON S—M 21 antibody was typed using a mouse monoclonal antibody isotyping kit (Amersham International p1c). The antibody was found to have a C-type light chain and a 7-1 C-type heavy chain. ⁇ Typing of NS—M21 is described later in Reference Example 2.
  • oligonucleotide primer Mae Kapa Variable; MKV
  • MKC oligonucleotide primer
  • the MKV primer hybridizes with a DNA sequence encoding a mouse kappa L chain leader sequence
  • the MK C primer hybridizes with a DNA sequence encoding a mouse kappa L chain C region.
  • oligonucleotide primers for amplification of the H chain V region of the mouse monoclonal antibody, 12 kinds of oligonucleotide primers (Mouse Heavy Variable; MHV) shown in SEQ ID NOS: 13 to 24 and SEQ ID NO: 25 Oligonucleotide primers (Mouse Heavylo Constant; MHC) is used as the 5'-end primer and 3 'one-end primer, respectively.
  • the 5'-terminal primer contains the sequence GTCGAC providing a restriction enzyme S a1I cleavage site near the 5'-terminal
  • the 3'-terminal primer is the 5'-terminal primer.
  • the one containing the nucleotide sequence CCCGGG that provides a restriction enzyme XmaI cleavage site near the end was used.
  • These restriction enzyme cleavage sites may be other restriction enzyme cleavage sites as long as they are used for subcloning the DNA fragment encoding the variable region into the cloning vector.
  • the amplified product was digested with restriction enzymes Sa1I and XmaI, and then subjected to low melting point agarose or column. Separate and purify using a kit for PCR product purification (such as .QIAGEN) and a kit for DNA purification (such as GENECLAN II).
  • a kit for PCR product purification such as .QIAGEN
  • a kit for DNA purification such as GENECLAN II.
  • an appropriate cloning vector such as a plasmid pUC19 is digested with the same restriction enzymes SaII and XmaI, and the DNA fragment is ligated to the pUC19 to obtain a mouse monoclonal antibody.
  • CDR Complementarity determining region
  • variable regions on the L and H chains form an antigen binding site.
  • the variable regions on the L and H chains are linked by four common, relatively conserved frameworks and three hypervariable or complementarity determining regions (CDRs). (Kabat, EA, et al., "Sequences of Proteinsof Immunological Internest," USD ept. Healthand Human Services 1983).
  • Most of the four framework regions (F R) have a —sheet structure, so that the three CDRs form a loop.
  • CDR may, in some cases, form part of a sheet structure.
  • the three CDRs are held sterically very close to each other by the FRs, and together with the three CDRs of the paired region contribute to the formation of the antigen binding site.
  • these CDR regions can be identified by Kabat, E.A., "Sequencesof This can be found from the empirical rule of "Proteins of Imm unological Interest", and is specifically described in Example 3.
  • Monoclonal antibody ONS Once the DNA fragments encoding the mouse L chain and H chain V regions of M21 have been cloned, these mouse V regions are combined with the DNA encoding the human antibody constant region. Linked and expressed Thus, a chimeric anti-human medulloblastoma cell antibody is obtained.
  • the basic method for producing chimeric antibodies is to combine the mouse leader sequence and V region sequence present in the cloned cDNA with the human antibody C already present in mammalian cell expression vectors. Linking the region to the coding sequence.
  • the human antibody C region can be any human L chain C region and human H chain C region, and examples thereof include human L chain C or H chain 11C and 7-4C, respectively. be able to.
  • DNA encoding a mouse L chain V region and a human L chain C region under the control of an expression control region such as an enhancer promoter system is used.
  • an expression vector comprising a DNA encoding a mouse H chain V region and a human H chain C region under an expression control region such as the Enhancer Z promoter system. I do.
  • host cells such as mammalian cells are co-transformed with these expression vectors, and the transformed cells are cultured in vitro or in vivo to produce quinula antibodies (eg, WO91).
  • the vector is transformed into a host cell using the vector, and the transformed host is cultured in vivo or in vitro to produce the desired chimeric antibody.
  • Mouse ONS DNA that encodes the M21 / c-type L chain Ligu region and V region is subcloned using PCR, and DNA that encodes the human genome L chain C c chain region is obtained.
  • the mouse ONS-M21 antibody y1 type H chain leader and V region linked to the expression vector The cDNA to be encoded is subcloned using the PCR method and ligated to an expression vector containing the genomic DNA encoding the human 7-1C region.
  • cDNAs encoding the V region of mouse ONS-M21 were introduced with appropriate nucleotide sequences at their 5'- and 3'-ends, They are designed to be easily inserted into an expression vector and to function properly in the expression vector (for example, the present invention is designed to increase the transcription efficiency by introducing a Kozak sequence).
  • the V region of mouse ONS-M21 obtained by PCR amplification using these primers was inserted into a HEF expression vector (Fig. 1) already containing the desired human C region. .
  • HEF expression vector Fig. 1
  • These vectors are suitable for transient (transient) or stable expression of engineered antibodies in various mammalian cell lines.
  • the chimera ONS—M21 antibody showed activity to bind to human medulloblastoma cells. Thus, it was shown that the correct mouse V region was cloned and sequenced.
  • the CDRs of the mouse monoclonal antibody have been transplanted into the human antibody.To produce reshaped human antibodies, there is high homology between the mouse monoclonal antibody FR and the human antibody FR. It is desirable. Therefore, the V regions of the L and H chains of the mouse ONS-M21 antibody were compared with the V regions of all known antibodies whose structures were elucidated using .ProteinDatBannk.
  • Mouse 0 NS—M21 L chain V region is most similar to the consensus sequence of human L chain V region subgroup IV (HSG IV), with 61, 9% homology .
  • Mouse L chain V region of S-M21 antibody is compared with known human antibody L chain V region, and human L chain V region is a member of subgroup I of human L chain V region.
  • the REI showed a 60.4% similarity. Therefore, REI FR was used as a starting material for the construction of the reshaped human ONS-M21 antibody L chain V region.
  • Tables 1 and 2 show the amino acid sequence of the L chain V region of mouse ON S-M21 antibody, FR and 16 versions of REI 0 NS-M21 antibody L chain V region. Show.
  • RVLp The underlined amino acid in REI FR is the amino acid sequence of human REI (Palm, W. et al., Hoppe — Seyler's, Z. Physiol. Chem. , 3556, 1667 — Indicates the position of amino acid different from 191, 1975).
  • the H-chain V region of mouse ONS-M21 is most similar to the consensus sequence of subgroup I (HSGI) of the human H-chain V region, with 57.9% homology.
  • the H-chain V region of the mouse ONS-M21 antibody was compared with known human antibody H-chain V regions, and FR1 through FR3 were members of the human H-chain V region subgroup I.
  • a certain human anti It was very similar to the H chain V region of the body Eu (Cunningham, B. J., et al., Biochemistry_9_, 3161, 1970).
  • the size of the CDRs was very similar between the mouse ON S-M21 antibody and the human antibody Eu.
  • the FR of the human antibody Eu was used as a starting material for the production of the V region of the H chain of the reshaped human 0NS-M21 antibody.
  • amino acid sequence of FR4 of human antibody Eu differs from the subgroup I consensus sequence of human antibody.
  • Human antibody ND in which the H chain V region belongs to subgroup I Keren, JH et al. Proc. Nat 1. Acad. Sci. USA, 79, 66, 61-66) It was decided to use the amino acid sequence of FR 4 of No. 5 (1982)).
  • the H chain V region of the reconstituted human 0 NS—M 21 antibody was designed. Amino acids at positions 27, 28, 29, 30 in human FR 1 and 94 in FR 3 were identical to those in mouse ONS-M21 .
  • an L chain comprising: a human L chain FR; and a mouse monoclonal antibody against human medulloblastoma 0 NS—an L chain V region of M 21 L chain V region comprising CDR; (B) (1) human H chain C region, and
  • H chain comprising a human H chain FR and an H chain V region comprising a mouse monoclonal antibody ONS-M 21 H chain V region CDR against human medulloblastoma;
  • the amino acid at position 46 of FR 2 in the L chain V region is proline, or the amino acid at position 42, 43, 46 is Those having an amino acid derived from mouse FR such as glutamine, serine, and proline are preferred, and more preferably, RVL i, RVL j, RVL 1, and RVL 1 in Tables 1 and 2 are preferable. It has the amino acid sequence of RVLm, RVLo, RVLp.
  • the human light chain C region can be any human light chain C region, eg, a human ⁇ C region; and the human heavy chain C region is any human heavy chain C region.
  • a toxin or radioisotope may be bound instead of the human L chain C region and / or the human H chain C region.
  • reshaped antibodies For the production of reshaped antibodies, two types of expression vectors, including DNA encoding the reshaped human light chain as defined above under the control of an expression control region, such as the Enhancer Z promoter system, are included. The resulting expression vector and another expression vector comprising DNA encoding a reshaped human H chain as defined above under an expression control region such as the Enhancer Z promoter system are prepared. Next, co-transformation of host cells, such as mammalian cells, using these expression vectors The transformed cells are then cultured in vivo or in vitro to produce the reshaped human antibody (eg, W091—16992).
  • an expression control region such as the Enhancer Z promoter system
  • the DNA encoding the reconstituted light chain and the DNA encoding the reconstituted human heavy chain are introduced into a single expression vector, and the vector is used to transform a host.
  • the transformed host cells are cultured in vivo or in vivo to produce the desired reshaped human antibody.
  • Fab or Fv, or single-chain FV linked to Fv can be produced in an appropriate host and used for the aforementioned purposes (for example, Bird et al., TIBTECH, 9_). , 1 3 2 — 1 3 7, (1 9 9 1)).
  • the transformant transformed with the gene encoding the desired chimeric antibody or humanized antibody is cultured, and the produced chimeric antibody or humanized antibody is intracellularly or intracellularly produced. It can be separated from outside the cell and purified to homogeneity.
  • the chimeric antibody or humanized antibody as the target protein of the present invention can be separated and purified using a protein A agarose column.
  • other separation / purification methods used for ordinary proteins may be used, and there is no limitation.
  • chimeric antibodies or humanized antibodies can be separated and purified by appropriately selecting and combining various types of chromatography, ultrafiltration, salting, dialysis and the like.
  • any expression system such as eukaryotic cells, such as animal cells, such as established mammalian cell lines, filamentous fungal cells , And yeast cells, as well as prokaryotic cells, such as bacterial cells, for example, E. coli cells.
  • eukaryotic cells such as animal cells, such as established mammalian cell lines, filamentous fungal cells , And yeast cells, as well as prokaryotic cells, such as bacterial cells, for example, E. coli cells.
  • the chimeric or reshaped antibodies of the invention are expressed in mammalian cells, such as COS cells or CH0 cells.
  • HCMV human cytomegalovirus promoter
  • promoters for gene expression in mammalian cells include retrovirus, polyoma virus, adenovirus, and simian virus 40 (SV).
  • a promoter derived from a mammalian cell such as a viral promoter such as 40
  • a polypeptide * such as a polypeptide, a chain, a fermentation factor, or a factor 1 (HEF-1) may be used.
  • the SV40 promoter when used, the method of Mulligan, RC et al. (Nature 2771 08-114 (19779)) or HEF-1
  • a promoter it can be easily carried out by following the method of Mizushima, S. et al. (Nucleic Acids Research, 18, 532, (1990)).
  • the origin of replication those derived from SV40, poliovirus, adenovirus, bovine papillomavirus (BPV) and the like can be used.
  • gene copy in a host cell system can be used. Because of the number amplification, the expression vector was selected as a selection marker for the phosphotransferase AP H (3 ') II or I (neo) gene, thymidine kinase (T K) gene, Escherichia coli xanthine-guanine phosphorolibosyltransferase (Ecogpt) gene, dihydrofolate reductase (DHFR) gene, and the like.
  • the present invention also provides a single-chain FV region formed by linking the H-chain V region and the L-chain V region of a reshaped human antibody to human medulloblastoma cells.
  • the H chain V region and the L chain V region are preferably linked via a linker, preferably via a peptide linker.
  • the H chain V region in the single chain F V region may be any of those described above as the H chain V region of the reshaped human antibody.
  • This H chain V region has an amino acid sequence defined below:
  • CDR2 Arglie Asp Pro Ala Asp Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gin Gly
  • the L chain V region has an amino acid sequence defined as follows:
  • an H chain V region consisting of amino acid sequences of amino acid numbers 1 to 116 in the amino acid sequence described in SEQ ID NO: 80, and SEQ ID NO: 4 0, 43, 46, 47, 50, 51, 54, 55, 58, 61, 62, 63, 66, 69, 70 or 73
  • a single-chain FV region comprising an L-chain V region consisting of an amino acid sequence of amino acids 1 to 106 in the amino acid sequence described in Crab.
  • amino acid sequence of SEQ ID NO: 80 is described.
  • H-chain V region consisting of amino acid sequences from 1 to 1 16 in the amino acid sequence and amino acid sequences 1 to 1 in the amino acid sequence described in SEQ ID NO: 73
  • a single-chain FV region comprising an L-chain V region consisting of up to 06 amino acid sequences.
  • V regions are preferably connected by a peptide linker.
  • Peptide linkers include, for example, any single-chain FV region consisting of 12 to 19 amino acids. Specific examples include Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly A peptide fragment consisting of Gly Gly Ser (SEQ ID NO: 90) is used.
  • SEQ ID NO: 89 An amino acid sequence as an example of the single-chain FV region is shown in SEQ ID NO: 89.
  • the single-chain FV region having this amino acid sequence is referred to as sc FV-hM21 in the present invention. This will be described in detail in Example 6.
  • DNA encoding the main chain FV region comprises DNA encoding the H chain or H chain V region of the reshaped human ONS—M21 antibody, which has already been described in detail.
  • Reshaped human ONS The DNA encoding the L chain or L chain V region of the M21 antibody is used as the chain type, and the DNA portion encoding the desired amino acid sequence in those sequences. Is obtained by PCR using a primer pair defining both ends thereof (in Example 6, a single strand comprising an H chain V region and an L chain V region version p).
  • the method for preparing DNA encoding the FV region is specifically described.
  • the method for preparing the amino acid sequence of purge ions a to o of the L chain V region and the DNA encoding the same are described in detail.
  • DNA can be the to produce that.
  • DNAs encoding single-chain FV regions are produced, an expression vector containing them and transformation by the expression vector are performed.
  • the transformed host can be obtained according to a conventional method, and a single-chain FV region can be obtained using the host according to a conventional method.
  • sc FV Humanized antigen-binding activity of hM21 ON S-M21 antibody and Fab fragment
  • scFv—hM21 showed the same binding inhibition ability as the Fab fragment.
  • the single-chain FV region has better translocation to tissues and tumors than wh01eIgG, and the scFV-hM21 constructed this time will be used for imaging by RI labeling in the future, and By combining with toxins and RI, it is expected to be used as a therapeutic agent.
  • the DNA encoding the variable region of mouse monoclonal antibody ONS-M21 against human medulloblastoma cells was cloned as follows. 1. Messenger RNA (mRNA) preparation
  • MRNA from Neubrydorma ONS—M21 was adjusted using FastTraccmRAIsolattiotKitVers 3.2 (manufactured by Invitrogen).
  • THECOPYKIT (Invit Rogen) was used to synthesize double-stranded cDNA.
  • the PCR method was performed using ThermalCycler (PerkInElmerCeTs).
  • the primers used in the PCR method are as follows: SEQ ID NOS: 1 to 11 that hybridize with the mouse kappa-type L chain leader sequence: ⁇ ⁇ (Mouse K appa Variable) primer (Jones, ST. B io T echnology, _9_, 8 8 — 8 9,
  • PCR solution 100 0 1 is 10 mM Tris — HC 1 (H 8.3), 50 mM KC 1, 0.1 mM d NTP s (d ATP, d GTP, d CTP, d TTP) , l. 5 mM M g C 1 2, 5 units of D NA poly main hydrolase Am pli T aq (P erkin E lmer C etus Co.), MK V primer shown in SEQ ID NO: 1 to 1 1 0. 1 M
  • One and 0.4 M of the MK C primer shown in SEQ ID NO: 12 and the double-stranded cDNAO.1 / g were amplified separately for each of the MK C 1-22 primers.
  • MHV Mae Heavy Variable primers 1 to 12 shown in SEQ ID NOS: 13 to 24 and MH C—Gl (Mouse H eavy Constant) Primer (J ones, ST The authors used Bio / Technology, _9_, 88-89, (1991)).
  • Amplification of cDNA was performed as described in 3. (1) above, except that amplification was performed using a mixture of 0.25 / M of each MHV primer and 2.5 / M of MHC-G1 primer.
  • the DNA fragment amplified by the PCR method as described above was purified by low-melting point agarose (manufactured by Sigma), and 10 mM Tris containing 10 mM MgCl 2 and 1 mM disthreitol was used.
  • Digestion was carried out in HC1 (H7.9) at 37 ° C for 3 hours using 5 units of restriction enzyme XmaI (New England Bio Labs).
  • the DNA fragment was digested with 40 units of the restriction enzyme Sa1I (Takara Shuzo) at 37 ° C. for 2 hours, and the resulting DNA fragment was digested with 1.5% low melting point agarose (Sigm a) (agarose gel electrophoresis).
  • a piece of agarose containing a DNA fragment of about 450 bp length was cut out, melted at 65 ° C for 5 minutes, and the same volume of 2 mM EDTA and 200 mM NaCl was added.
  • the contained 20 mM Tris-HCl (pH 7.5) was added.
  • the mixture was extracted with phenol and black hole form, and the DNA fragment was recovered by ethanol precipitation and dissolved in 10 mM Tris-HCl (pH 7.5) containing ImM EDTA.
  • DNA fragment containing a gene encoding a mouse brim-type L chain variable region and a DNA fragment containing a gene encoding a mouse H chain variable region were obtained. All of the above DNA fragments have a S a 1 I cohesive end at their 5'-end and an Xma I end at their 3'-end. Has cohesive ends.
  • the transformant was cultured overnight at 37 ° C. in 10 ml of 2 ⁇ YT medium containing 50 / g of ampicillin, and the culture was subjected to an anorecalization method (Molecular C 1). oning: AL aboratory Manual, Sambrook et al., Old Spring Harbor L aboratory Press, Plasmid DNA was prepared according to (1989).
  • a plasmid containing a gene encoding the mouse H chain V region derived from the hybridoma ONS—M21 was generated from the Sa1I-XmaI DNA fragment, and UC—M 2
  • the nucleotide sequence of the cDNA coding region in the plasmid was determined by using an automatic DNA sequencer (Appl ied B i os sy s t e m I n c) and T aq D y e D e x y T e r m i n a t o r
  • the nucleotide sequence was determined using CyclESecncingKit (manufactured by ApIiedBiosystemInc) according to the protocol specified by the manufacturer.
  • SEQ ID NO: 26 shows the nucleotide sequence of the gene encoding the L chain V region of the mouse ONS-M21 antibody contained in the plasmid pUC-M21-VL. Moreover, mice 0 NS contained in the positive Mi de p UC M 2 1-V H - shown in M 2 1 antibody H chain V region co one de the nucleotide sequence SEQ ID NO: 2 7 genes.
  • the overall structures of the V regions of the L and H chains are similar to each other, with each of the four framework parts being linked by three hypervariable regions, the complementarity determining regions (CDRs). ing.
  • the amino acid sequence of the framework is relatively well conserved, while the amino acid sequence in the CDR region is extremely variable (Kabat, EA, et al., “S'e quencesof P roteinsof I mmu no 1 ogica 1 Internest ”USD ept. Healthand Human Services, 1983).
  • the amino acid sequence of the variable region of the mouse monoclonal antibody against human medulloblastoma cells was applied to the amino acid sequence database of the antibody created by Kabat et al.
  • the CDR regions were determined as shown in Table 4 by examining the homology.
  • Back primer for L chain V region 0 NS—L722 S (SEQ ID NO: 28) and back primer for H chain V region ON S—H3.2S (SEQ ID NO: 29) are hybridized to the DNA encoding the beginning of the leader sequence of each V region and have a Kozak consensus sequence (Kozak, M. et al., J. Mo 1. Biol. 196.99.47- 950, 198 7) and a HindIII restriction site.
  • Forward primer for NS V region 0 NS—L722A (SEQ ID NO: 30) and forward primer for NS V region 0 NS -H3.2A (SEQ ID NO: 31) was designed to hybridize to the DNA sequence encoding the end of the J region and have a splice donor sequence and a BamHI restriction site.
  • the PCR product was purified using a 1.5% low melting point agarose gel, digested with Hind III and BamHI, and the L chain V region was converted to the HEF expression vector HEF—VL—g / c and the H chain The V region was cloned into the HEF expression vector HEF- VH -g, respectively.
  • the plasmids containing the DNA fragments having the correct DNA sequence were named HEF-M21L-g and HEF-M21H-gr1, respectively.
  • the expression vectors were tested in C0S cells to observe the transient expression of the Chimera ONS-M21 antibody.
  • ⁇ Cells were co-transformed by electroporation using HEF-M21L-g ⁇ and HEF-M21H-gylGene Pulser device (BioRad). Each D NA (1 0 g), was added to ⁇ Li Coat of 0. 8 ml of 1 X 1 0 7 cells Zml in PBS, and subjected to pulses of 1, 9 0 0 V, 2 5 F capacity .
  • Electroporation after a 10 minute recovery period at room temperature The cells thus obtained were added to a DMEM culture solution (manufactured by GIBCO) containing 10% ⁇ -globulin free fetal serum. 72 After incubation for 2 hours, the culture supernatant was collected, cell debris was removed by centrifugation, and a protein A agarose column (Affi-Gel) equilibrated with 5 volumes of binding buffer. Protein A MAPS II kit, BioRad). The column was washed with 15 volumes of binding buffer and then eluted with 5 volumes of elution buffer. The eluate was concentrated, and the buffer was changed to PBS using a microconcentrator (Centricon 100, manufactured by Amicon).
  • the Ce11-ELISA plate for antigen binding measurement was prepared as follows. 9 6-well plates in 1 0% ⁇ shea you containing calf serum R PM I culture by 1 X 1 0 6 human medulloblastoma cell lines ON S- 7 were adjusted to cells / ml 6 (T amu raeta 1., C ancer
  • Reconstituted human 0NS-M21 antibody L chain was prepared by CDR-grafting by the PCR method. This method is schematically shown in FIG. Eight PCR primers were used to generate a reshaped human antibody 0NS-M21 (version "a") having a FR from human antibody REI. External primers A (SEQ ID NO: 32) and H (SEQ ID NO: 33) were designed to hybridize with the DNA sequence of the HEF expression vector HEF—VL—gk.
  • CDR—Grafting primers B (SEQ ID NO: 34), C (SEQ ID NO: 35) and D (SEQ ID NO: 36) have a sense DNA sequence
  • CDR—Grafting primer E (SEQ ID NO: 37), F (SEQ ID NO: 38) and G (SEQ ID NO: 39) have antisense DNA sequences and correspond to the 5'-end DNA sequences of primers B, C and D, respectively. It has a complementary DNA sequence (23-35 bp).
  • PCR products A-E (2 18 bp), B-F (101 bp), C_G (13 1 bp) and D-H (147 bp) were converted to 1.5% low melting point agarose. It was purified using a gel and assembled in a second PCR. In the second PCR, 1 ⁇ g of the product of each first PCR and 98 ⁇ l of the PCR mixture containing 5 u of AmpIi Taq were added at 94 ° C for 2 minutes. Two cycles of incubation at 55 ° C for 2 minutes and at 72 ° C for 2 minutes were performed, and then 100 pmole of each external primer (A and H) was added. The PCR tube was covered with 50 nl of mineral oil and 30 cycles of PCR were performed under the same conditions as above.
  • the 5 16 bp DNA fragment generated by the second PCR was purified on a 1.5% low-melting point agarose gel, digested with BamHI and HindIII, and the resulting DNA fragment was subjected to HEF expression vector HEF. — V L — Cloned to gk. After DNA sequencing, reconstituted human ONS—M21 antibody Plasmid containing a DNA fragment encoding the correct amino acid sequence of the L chain V region of the HEF—RV L—M21a — Named gc. The amino acid sequence and base sequence of the L chain V region contained in this plasmid HEF-RVL-M21a-g are shown in SEQ ID NO: 40.
  • the version “b” of the reshaped human ONS—M21 antibody L chain V region was prepared by a mutagenesis method using PCR.
  • the mutagenic primers FTY-1 (SEQ ID NO: 41) and FTY-2 (SEQ ID NO: 42) were designed to mutate phenylalanine at position 71 to tyrosine.
  • Plasmid HEF—RV L—M21a_gc was type III, amplified using the above primers, purified, and digested with BamHI and Hindll. The obtained DNA fragment was cloned into a HEF expression vector HEF-VL-gc to obtain plasmid HEF-RVL-M21b-g / c.
  • SEQ ID NO: 43 shows the amino acid sequence and base sequence of the L chain V region contained in this plasmid HEF—RVL—M21b-gc.
  • Version “c” was designed to be a mutagenic primer, in which the tyrosine at position 87 was designed to mutate to phenylalanine; M21M2S (SEQ ID NO: 44) and M21M Using 2A (SEQ ID NO: 45), the plasmid HEF—RVL—M21a-g «was used as type I DNA and amplified by PCR, and plasmid HEF—RVL—M 2 1 c—g was obtained.
  • the amino acid sequence and base sequence of the L chain V region contained in this plasmid HE F—R V L—M 21 c-1 g c are shown in SEQ ID NO: 46.
  • Version "d” uses FTY-1 and FTY-2 and M21M2S and M21M2A as mutagenic primers, and plasmid HE F-RVL-M2 1a- gc Was used as type I DNA to obtain plasmid HEF-RVL-M2 1d-gc.
  • SEQ ID NO: 47 shows the amino acid sequence and the base sequence of the L chain V region contained in this plasmid HEF—RV L-M 21 d—g c.
  • Version “f” is plus HEF—RVL—M 21 c-g / c for Bam HI and H inf I, plus plus HEF—RVL—M 21 e—gc for Hindll And H inf I to obtain DNA fragments of 152 bp and 250 bp, respectively. These DNA fragments were separated and purified using a 15% low-melting point agarose gel, ligated, inserted into the HEF expression vector HEF-RVL-g /, and added to the plasmid HEF-RVL-M21f-. g / c was obtained. The amino acid sequence and the base sequence of the L chain V region contained in this plasmid HEF—RVL-M21f-1 g / c are shown in SEQ ID NO: 51.
  • Version “g” is M21M4S (SEQ ID NO: 52) and M21M4 designed as mutagenic primers so that the phenylalanine at position 73 is mutated to oral.
  • A SEQ ID NO: 53
  • SEQ ID NO: 54 was used to amplify the plasmid HEF — RVL—M21f1 g / c as type I DNA.
  • SEQ ID NO: 54 shows the amino acid sequence and base sequence of the L chain V region contained in this plasmid HEF-RVL-M21g-g.
  • Version “h” uses M21M4S and M21M4A as mutagenic primers and converts plasmid HEF—RVL—M21a-g ⁇ to type D Amplification was performed to obtain plasmid HEF-RVL-M21h-g / c.
  • This plasmid HEF — RVL— M 21 h — g / c contains the amino acid sequence and nucleotide sequence of the L chain V region in SEQ ID NO: 55 Shown in
  • Version "i” is a mutagenic primer in which lysine at position 42 is glutamine, alanine at position 43 is serine, and leucine at position 46 is proline.
  • plasmid M21M5S SEQ ID NO: 56
  • M21M5A SEQ ID NO: 57
  • SEQ ID NO: 58 shows the amino acid sequence and base sequence of the L chain V region contained in this plasmid HEF—RVL—M21i—g / c.
  • Version “k” uses M21M6S and M21M6A as mutagenic primers and converts plasmid HEF—RV L—M21-g ⁇ to type D Amplified as ⁇ to obtain plasmid HEF-RVL-M2 1k-1 g / c.
  • the amino acid sequence and base sequence of the L chain V region contained in this plasmid HE F—R V L—M 2 lk—g c are shown in SEQ ID NO: 62.
  • Version “1” is for plasmid HEF—RVL—M 21 a-g / c for BamHI and Sfa NI, and for plasmid HEF—RV L—M21 i—gc for Hind. After digestion with Ill and SfaNI, DNA fragments of 227 bp and 1669 bp were separated and purified. D obtained The NA fragment was ligated and inserted into the expression vector HEF-RVL_g / c to obtain plasmid HEF-RVL-M21_g / c. The amino acid sequence and the base sequence of the V region of the L chain contained in this plasmid HEF-RV L-M211 — g are shown in SEQ ID NO: 63.
  • Version “m” means that M21M5S, M21M5A and proline at position 8 are glutamic acid and serine at position 9 as mutagen primers.
  • M21M7S SEQ ID NO: 64
  • M21M7A SEQ ID NO: 65
  • Plasmid HEF-RVL-M21a-g was amplified as type I DNA to obtain plasmid HEF-RVL-M21m-g.
  • the amino acid sequence and base sequence of the L chain V region contained in this plasmid HEF—RVL—M2lm—gc are shown in SEQ ID NO: 66.
  • Version “n” was designed as a mutagenic primer so that lysine at position 42 is mutated to glutamic acid, and alanine at position 43 is mutated to serine. : 67) and M21M8A (sequence number: 68), and plasmid HEF—RVL—M21a—g was used as type II DNA to obtain plasmid HEF—RVL—M2. I n— gc was obtained. The amino acid sequence and base sequence of the L chain V region contained in this plasmid HEF—RVL—M2In—g / c are shown in SEQ ID NO: 69.
  • Version “oJ is for plasmid HEF—RVL—M2 lb—g for BamHI and BsrI, and for plasmid HEF—RVL—M21a—g / c for Hindlll and Bsr.
  • Each DNA fragment was separated and purified from each of the plasmids, and the resulting DNA fragments were ligated together and inserted into the expression vector HEF-VL-gc.
  • the plasmid HEF—RVL—M2lo—gc was obtained, and the L chain V region contained in this plasmid HEF—RVL—M21 o—g / c
  • the amino acid sequence and base sequence of the region are shown in SEQ ID NO: 70.
  • Plasmid HEF-RVL-M21a-g ⁇ was used as type I DNA using M9A (SEQ ID NO: 72), and plasmid HEF-RVL-M21p-gc was used. Obtained.
  • the amino acid sequence and the nucleotide sequence of the L chain V region contained in this plasmid HEF—RVL-M21p—g / c are shown in SEQ ID NO: 73.
  • the DNA encoding the reshaped human ONS-M21 antibody H chain V region was designed as follows. DNA sequences encoding FRs 1-3 of human antibody Eu and FR4 of human antibody ND were converted to V regions (Kabat, EA, et al., US Dept. Health and Human Services). , U S. G overnment P rinting • Employees, designed based on “codonusage” in 1991, and connecting to the mouse ONS-M21 antibody H chain V region CDR coding DNA sequence. As a result, a full-length DNA encoding the reshaped human 0 NS—M 21 antibody H chain V region was designed.
  • the DNA sequence designed in this way is divided into four oligonucleotides. Then, in the oligonucleotides that may interfere with the assembly of these oligonucleotides. A secondary analysis of the secondary structure was performed.
  • oligonucleotide sequences are shown in SEQ ID NOs: 74-77. These oligonucleotides have a length of 11 to 13 bases and an overlap region of 23 to 26 bp.
  • VH2 SEQ ID NO: 74
  • RVH4 SEQ ID NO: 75
  • R R
  • VH3 (SEQ ID NO: 77) has an antisense DNA sequence.
  • Figure 4 shows a method for assembling these four oligonucleotides by the PCR method. (See Fig. 4)
  • a 98 81 PCR mixture containing 10 ng of each of the four oligonucleotides and 5 u of AmpIiTaq was subjected to an initial denaturation at 94 ° C for 2 minutes. Thereafter, two cycles of incubation at 94 ° C for 2 minutes, 55 ° C for 2 minutes and 72 ° C for 2 minutes were performed ⁇ 100 pmole RH P After adding 1 (SEQ ID NO: 78) and RHP2 (SEQ ID NO: 79) as external primers, cover the PCR tubes with 50 a1 mineral oil and at 94 ° C. After an initial denaturation of 1 minute, perform 38 cycles of 1 minute at 94, 1 minute at 55 ° C and 1 minute at 72 ° C, and then at 72 ° C. Incubated for 10 minutes.
  • HEF- V H - cloned g ⁇ 1 did.
  • HEF-RVH-M21-r1 The amino acid sequence and base sequence of the H chain V region contained in M21-gr1 are shown in SEQ ID NO: 80.
  • VH-M2 1-gr1 and chimera ONS—M2 1 antibody light chain The COS cells were trans-transfected as described above with the current HEF-M21L-gr1 and the antibody product was recovered as described above. And subjected to antigen binding assay.
  • Fig. 5 shows the results. As shown in Fig. 5, antigen binding between the chimeric antibody (ChM21) as a positive control and the antibody (ChL / RVH) consisting of the reconstituted H chain and chimeric L chain It was confirmed that there was no difference in gender.
  • the antibodies with iJ, j, 1J, m, o, and PJ L chain versions are the chimera ONS—M21 antibodies that are positive controls.
  • (ChM21) showed a good antigen-binding property comparable to that of (ChM21), suggesting that this combination forms a functional antigen-binding site in the human antibody. No antigen-binding activity was found for the antibodies having "" and "n” (see Figure 7).
  • Example 6_ Creation of single-chain FV (scFv) region of reconstituted human ONS-M21 antibody
  • DNA encoding a linker region consisting of Gly Gly Gly Gly Ser was designed as follows. DNA sequence encoding the linker region (Huston, J.S. et al., Proc. Natl. Acad. Sci. USA 85, 587-9-5 883, (19) 8 8)) A 5 bp DNA sequence encoding the 5 amino acid residue at the C-terminus of FR 4 of the H chain V region on the 5'- A 15 bp DNA sequence encoding the 5 amino acid residues at the N-terminus of the FR1 region was added to the 5'-side and 3 ' 'A HindIII recognition site and an EcoRI recognition site were added to one side.
  • the two oligonucleotide sequences are shown in SEQ ID NOs: 81 and 82, respectively. These oligonucleotides are 84 bases in length and have an 81 bp overlap region.
  • the samples were incubated at 7 ° C overnight, and annealing was performed.
  • the DNA annealed as described above was ligated to a PUC19 vector prepared by digesting with HindHI and EcoRI. After DNA sequencing, the plasmid containing the DNA fragment with the correct DNA sequence was named pUC-scFv-5.
  • a single-chain FV region of the reconstituted human ONS—M21 antibody was prepared as follows. Reconstituted human 0 NS—M21 antibody H chain V region and linker region, and reconstituted human ONS—M21 antibody L chain V region are amplified by PCR and ligated. As a result, a single-chain FV region of reconstituted ONS—M21 antibody was prepared. This method is schematically shown in FIG. Six PCR primers (AE) were used to generate the single chain Fv region of the reconstituted human 0NS-M21 antibody. Primers A, C and E have a sense sequence and primers B, D and F have an antisense sequence.
  • the backward primer for the heavy chain V region, SCP1, hybridizes to DNA encoding the N-terminus of the heavy chain V region and has an Ncol recognition site. Designed to.
  • the forward primer SCP 2 (primer B, SEQ ID NO: 84) for the H chain V region hybridizes to the DNA encoding the C-terminus of the H chain V region and overlaps with the linker Designed to be.
  • the backward primer for the linker, SCP 3, hybridizes to the DNA encoding the N-terminus of the linker and binds to the C-terminus of the H chain V region. Designed to overlap with the DNA to be loaded.
  • the forward primer SCP4 (primer D, SEQ ID NO: 86) for the linker hybridizes to the DNA encoding the C-terminus of the linker and encodes the N-terminus of the V region of the light chain. Over DN A It was designed to wrap.
  • the posterior primer SCP 4 (primer E, SEQ ID NO: 87) for the L chain V region hybridizes to the DNA encoding the C-terminus of the linker and binds the N-terminus of the L chain V region. It was designed to overlap the coding DNA.
  • the forward primer WS4-2 (primer F, SEQ ID NO: 88) for the L chain V region hybridizes to the DNA encoding the C terminal of the L chain V region and encodes the FLAG peptide. (Hopp, T.P. et al., Bio / Technology, 6, 1204-122, 198 88), with two transcription stop codons and an Ec0RI recognition site. It was designed as follows.
  • the first PCR stage three reactions, AB, CD, and EF, were performed, and each PCR product was purified.
  • the three PCR products obtained from the first PCR were assembled by their own complementarity.
  • primers A and F were added to amplify the full-length DNA encoding the single-chain FV region of the reconstituted human ONS-M • 21 antibody (second PCR.).
  • plasmid HEF—RVH—M21-g71 (see Example 5) encoding the reshaped human ONS—M21 antibody H chain V region was used.
  • the PCR products A—B (382 bp), C—D (92 bp), and E_F (366 bp) were purified using a 1.5% low melting point agarose gel and subjected to a second PCR. Assembled. In the second PCR, 1 ⁇ g of each of the products of the first PCR as a ⁇ type, and a 98 z1 PCR mixture containing 5 u of Amp1i Taq were added at 94 ° C. Two cycles of incubation for 2 minutes, 2 minutes at 55 ° C and 2 minutes at 72 ° C, then 10Opmole of primers A and F, respectively, were added.
  • the PCR tube was covered with 50 a1 of mineral oil, and 30 cycles of PCR were performed at 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes ( No. 1).
  • the 7667 bp DNA fragment generated by PCR was purified on a 1.5% low melting point agarose gel, digested with NcoI and Ec0RI, and the obtained DNA fragment was cloned into the expression vector PSCFVT7.
  • the expression vector pSCFVT7 is a pe 1B signal sequence (Lei, SP et al., J. B acterio 1 ogy, 1669, 4379, 4379-43) suitable for the E. coli periplasmic secretion expression system.
  • the reconstituted human ONS-M21 antibody contains a DNA fragment that encodes the correct amino acid sequence of the single-chain FV region.
  • the plasmid was named p SCFVT 7 — h M 21. (See Figure 9.) Reconstituted human 0 NS—M 21 antibody single chain contained in this plasmid PSCFVT 7 — h M 21 Amino acid sequence of FV region And the base sequence is shown in SEQ ID NO: 89.
  • This transformant was transformed into an LB medium containing 1% glucose and 50 ng / ml of ampicillin (Molecular Cloning: AL aboratory Manual, Sambrook, Old Spring Harbor Laboratory). The cells were cultured overnight at 37 ° C. in 30 ml of ress, (1 899). Next, it was diluted 100-fold with LB medium containing 50 g / ml of ampicillin, and cultured at 37 ° C. 6 When the absorbance at 50 nm reaches about 0.3, add is 0 pr 0 py 1 thio 1-thio-yS-D-ga 1 actoside. (IP TG) so that the final concentration becomes 0.5 mM. And induced expression of the T7 promoter.
  • Reconstituted human ONS-M21 antibody single-chain FV region using mouse monoclonal monoclonal 0N S—M21 antibody to inhibit antigen binding The original binding activity was measured. After blocking the Ce11-ELISA plate prepared as described above, reconstituted human NS with serial dilutions with mouse monoclonal ONS-M21 antibody at a concentration of 500 ngZml. Single-chain FV region of M21 antibody, reconstituted human ONS- M21 antibody and Fab fragment prepared from reconstituted human ONS-M21 antibody are added to each well, and incubated and washed at room temperature. After that, alkaline phosphatase-conjugated goat anti-mouse IgG antibody (ZYMED) was added. After incubation and washing, the substrate solution was added, and then the absorbance at 405 nm was measured.
  • ZYMED alkaline phosphatase-conjugated goat anti-mouse IgG antibody
  • the single-chain FV region of reconstituted human ONS-M21 antibody was inhibited by about 1Z10 compared to the reconstituted human ONS-M21 antibody (hM21). Although the activity was reduced, it showed the same level of inhibitory activity as the Fab fragment (hM21-Fab) prepared from the reconstituted human ONS-M21 antibody (see Fig. 10). .
  • Escherichia coli having the plasmid HEF—RVL—M21p—g is Escherichiacoli DH5a (HEF—RVL—M21p—gc) and plasmid HEF—RVH—M21—.
  • E. coli containing gr1 was submitted to Escherichiacoli DH5a (HEF-RVH-M21-g ⁇ l) at the Institute of Biotechnology and Industrial Technology (1-3-1-3 Higashi, Tsukuba-shi, Ibaraki Prefecture). On January 18, 2013, they were deposited under the Budapest Treaty as FE RM BP-44472 and FE RM BP-44471, respectively.
  • Hypridoma which produces anti-human medulloblastoma cell monoclonal antibodies, was obtained from spleen cells of mouse and myeloma cells P3U1 which were immunized with human medulloblastoma cells ONS-76. was prepared by fusing with a conventional method using polyethylene glycol. Screening was performed using the activity of binding to the medulloblastoma cell line ONS-76 as an index to establish Hypridoma 0 NS-M21 (Moriuchi, S. et al., Br. J, Cancer , 6 8, 8 3 1-8 3 7 (1 9 9 3))).
  • Sequence type nucleic acid
  • Sequence type nucleic acid
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  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid Number of chains: single strand
  • Sequence type nucleic acid
  • Sequence type nucleic acid
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  • V N ⁇ 3 ⁇ S 0 ⁇ 31 ⁇ Army: one ⁇ 4 ⁇ ⁇ : 0 m: ourn
  • GAA CAG GGC CTG GAG TGG ATT GGA AGG ATT GAT CCT GCG
  • GAT GGT 225 Glu Gin Gly Leu Glu Trp lie Gly Arg lie Asp Pro Ala Asp Gly
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid Number of chains: single strand
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  • ACATTAGTAC CCACATTCTG ACTGGCCTTA CAGGTGATGG TCAC 44 SEQ ID NO: 3 8
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Organism name mouse and human
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Organism name mouse and human
  • AGC GCC AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCC AGT 135 Ser Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Lys Ala Ser
  • AAG GCT CCA AAG CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT 225 Lys Ala Pro Lys Leu Leu lie Tyr Ser Ala Ser Tyr Arg Tyr Ser
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Organism name mouse and human
  • AGC GCC AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCC AGT 135 Ser Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Lys Ala Ser
  • AAG GCT CCA AAG CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT 225 Lys Ala Pro Lys Leu Leu lie Tyr Ser Ala Ser Tyr Arg Tyr Ser
  • Sequence type nucleic acid
  • Organism name mouse and human
  • Sequence type nucleic acid.
  • Sequence type nucleic acid
  • Organism name mouse and human
  • AGC GCC AGC GTG GGT GAC AGA GTG TCC GTC ACC TGT AAG GCC AGT 135 Ser Ala Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser
  • AAG GCT CCA AAG CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT 225 Lys Ala Pro Lys Leu Leu lie Tyr Ser Ala Ser Tyr Arg Tyr Ser
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Organism name mouse and human
  • Amino acid 8 9 — 9 7 CDR 3
  • Amino acid 9 8 — 10 7 FR 4
  • AGC GCC AGC GTG GGT GAC AGA GTG TCC GTC ACC TGT AAG GCC AGT 135 Ser Ala Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser
  • AAG GCT CCA AAG CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT 225 Lys Ala Pro Lys Leu Leu lie Tyr Ser Ala Ser Tyr Arg Tyr Ser
  • Sequence type nucleic acid
  • Organism name mouse and human
  • AGC GCC AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCC AGT 135 Ser Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Lys Ala Ser
  • AAG GCT CCA AAG CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT 225 Lys Ala Pro Lys Leu Leu lie Tyr Ser Ala Ser Tyr Arg Tyr Ser
  • Sequence type nucleic acid
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PCT/JP1994/001763 1993-11-19 1994-10-19 Anticorps humain reconstitue contre des cellules medulloblastomateuses humaines WO1995014041A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP94930341A EP0729976A1 (en) 1993-11-19 1994-10-19 Reconstituted human antibody against human medulloblastomatous cell
AU79493/94A AU7949394A (en) 1993-11-19 1994-10-19 Reconstituted human antibody against human medulloblastomatous cell
US08/646,265 US6214973B1 (en) 1993-11-19 1994-10-19 Reshaped human antibody to human medulloblastoma cells
US10/839,799 US7563599B2 (en) 1993-11-19 2004-05-06 Reshaped human antibody to human medulloblastoma cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP5/291078 1993-11-19
JP29107893 1993-11-19

Related Child Applications (3)

Application Number Title Priority Date Filing Date
US08/646,265 A-371-Of-International US6214973B1 (en) 1993-11-19 1994-10-19 Reshaped human antibody to human medulloblastoma cells
US08646265 A-371-Of-International 1994-10-19
US09/749,873 Division US20030023045A1 (en) 1993-11-19 2000-12-29 Reshaped human antibody to human medulloblastoma cells

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WO1995014041A1 true WO1995014041A1 (fr) 1995-05-26

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PCT/JP1994/001763 WO1995014041A1 (fr) 1993-11-19 1994-10-19 Anticorps humain reconstitue contre des cellules medulloblastomateuses humaines

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US (3) US6214973B1 (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)
EP (1) EP0729976A1 (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)
AU (1) AU7949394A (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)
TW (1) TW526206B (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)
WO (1) WO1995014041A1 (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998009651A1 (fr) * 1996-09-03 1998-03-12 Chugai Seiyaku Kabushiki Kaisha COMPLEXES D'ANTICORPS ANTI-INTEGRINE α3
WO2009088032A1 (ja) 2008-01-10 2009-07-16 Shionogi & Co., Ltd. ＰｃｒＶに対する抗体
WO2015115656A1 (ja) 2014-02-03 2015-08-06 独立行政法人国立がん研究センター 抗Ｔｉｓｓｕｅ Ｆａｃｔｏｒモノクローナル抗体
WO2019230856A1 (ja) 2018-05-31 2019-12-05 塩野義製薬株式会社 Ｎａｖ１．７モノクローナル抗体
WO2020138489A1 (ja) 2018-12-27 2020-07-02 塩野義製薬株式会社 新規抗ｃｃｒ８抗体
WO2022004760A1 (ja) 2020-06-30 2022-01-06 塩野義製薬株式会社 抗ｃｃｒ８抗体と化学療法剤の併用
WO2023074888A1 (ja) 2021-11-01 2023-05-04 塩野義製薬株式会社 新規なＮａｖ１．７モノクローナル抗体
WO2023234406A1 (ja) 2022-06-03 2023-12-07 塩野義製薬株式会社 カンナビノイド１型受容体に結合する抗体

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CN1314803C (zh) * 2001-06-22 2007-05-09 中外制药株式会社 含有抗磷脂酰肌醇蛋白聚糖3抗体的细胞生长抑制剂
ATE543509T1 (de) * 2001-08-13 2012-02-15 Univ Florida Material und verfahren zur förderung der reparatur von nervengewebe
EP2808032B1 (en) * 2005-12-12 2018-08-01 AC Immune S.A. A beta 1-42 specific monoclonal antibodies with therapeutic properties
CL2007002070A1 (es) 2006-07-14 2008-02-08 Ac Immune S A Genentech Inc Anticuerpo quimerico o humanizado, o fragmentos de ellos, que se adhieren especificamente a por lo menos un epitopo en la proteina beta-amiloide; molecula de acido nucleico que lo codifica; composicion que lo comprende; su uso para tratar enfermedade
US8048420B2 (en) * 2007-06-12 2011-11-01 Ac Immune S.A. Monoclonal antibody
RU2567151C2 (ru) * 2007-06-12 2015-11-10 Ац Иммуне С.А. Гуманизированные антитела к амилоиду бета
US8613923B2 (en) 2007-06-12 2013-12-24 Ac Immune S.A. Monoclonal antibody
ES2612788T3 (es) * 2007-10-05 2017-05-18 Genentech, Inc. Métodos y composiciones para el diagnóstico y tratamiento de la amiloidosis
SG10201505369QA (en) 2007-10-05 2015-08-28 Genentech Inc Use of anti-amyloid beta antibody in ocular diseases
BRPI0818623A2 (pt) * 2007-10-05 2017-05-23 Ac Immune Sa composição farmacêutica, e, métodos para reduzir a carga da placa na camada de célula de gânglio retinal de um animal, para reduzir a quantidade de placas na camada de célula de gânglio retinal de um animal, para diminuir a quantidade total de amilóide-beta solúvel na camada de célula de gânglio retinal de um animal, para prevenir, tratar e/ou aliviar os efeitos de uma doença ocular associada com anormalidades patológicas/mudanças no tecido do sistema visual, para monitorar doença ocular residual mínima associada com anormalidades patológicas/mudanças nos tecidos do sistema visual, para predizer responsividade de um paciente, e para reter ou diminuir a pressão ocular nos olhos de um animal
MY164579A (en) 2010-07-30 2018-01-15 Ac Immune Sa Safe and functional humanized antibodies
MA40764A (fr) 2014-09-26 2017-08-01 Chugai Pharmaceutical Co Ltd Agent thérapeutique induisant une cytotoxicité

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JPS6147500A (ja) * 1984-08-15 1986-03-07 Res Dev Corp Of Japan キメラモノクロ−ナル抗体及びその製造法

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HU218140B (hu) * 1991-04-25 2000-06-28 Chugai Seiyaku Kabushiki Kaisha Humán interleukin-6-receptorral szembeni átalakított humán antitest
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JPS6147500A (ja) * 1984-08-15 1986-03-07 Res Dev Corp Of Japan キメラモノクロ−ナル抗体及びその製造法

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Title
BRITISH JOURNAL OF CANCER, Vol. 68, No. 5, (November 1993), S. MORIUCH et al., "Characterization of a New Mouse Monoclonal Antibody (ONS-M21), Reactive with Both Medullo Blastomas and Gliomas", p. 831-837. *
PROC. NATL. ACAD. SCI. USA, Vol. 85, (August 1988), J.S. HUSTON et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia Coli", p. 5879-5883. *
See also references of EP0729976A4 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998009651A1 (fr) * 1996-09-03 1998-03-12 Chugai Seiyaku Kabushiki Kaisha COMPLEXES D'ANTICORPS ANTI-INTEGRINE α3
WO2009088032A1 (ja) 2008-01-10 2009-07-16 Shionogi & Co., Ltd. ＰｃｒＶに対する抗体
WO2015115656A1 (ja) 2014-02-03 2015-08-06 独立行政法人国立がん研究センター 抗Ｔｉｓｓｕｅ Ｆａｃｔｏｒモノクローナル抗体
WO2019230856A1 (ja) 2018-05-31 2019-12-05 塩野義製薬株式会社 Ｎａｖ１．７モノクローナル抗体
WO2020138489A1 (ja) 2018-12-27 2020-07-02 塩野義製薬株式会社 新規抗ｃｃｒ８抗体
WO2022004760A1 (ja) 2020-06-30 2022-01-06 塩野義製薬株式会社 抗ｃｃｒ８抗体と化学療法剤の併用
WO2023074888A1 (ja) 2021-11-01 2023-05-04 塩野義製薬株式会社 新規なＮａｖ１．７モノクローナル抗体
WO2023234406A1 (ja) 2022-06-03 2023-12-07 塩野義製薬株式会社 カンナビノイド１型受容体に結合する抗体

Also Published As

Publication number Publication date
US20030023045A1 (en) 2003-01-30
AU7949394A (en) 1995-06-06
EP0729976A4 (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html) 1996-10-16
TW526206B (en) 2003-04-01
US20050249726A1 (en) 2005-11-10
US7563599B2 (en) 2009-07-21
US6214973B1 (en) 2001-04-10
EP0729976A1 (en) 1996-09-04

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