WO1994002627A1 - Recombinant antibody against human interleukin-1 - Google Patents

Recombinant antibody against human interleukin-1 Download PDF

Info

Publication number
WO1994002627A1
WO1994002627A1 PCT/JP1993/000941 JP9300941W WO9402627A1 WO 1994002627 A1 WO1994002627 A1 WO 1994002627A1 JP 9300941 W JP9300941 W JP 9300941W WO 9402627 A1 WO9402627 A1 WO 9402627A1
Authority
WO
WIPO (PCT)
Prior art keywords
human
region
antibody
chain
antibody molecule
Prior art date
Application number
PCT/JP1993/000941
Other languages
French (fr)
Japanese (ja)
Inventor
Yoshikatsu Hirai
Raymond John Owens
Yasukazu Omoto
Tsutomu Nishida
Original Assignee
Otsuka Pharmaceutical Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Otsuka Pharmaceutical Co., Ltd. filed Critical Otsuka Pharmaceutical Co., Ltd.
Priority to AU45140/93A priority Critical patent/AU4514093A/en
Publication of WO1994002627A1 publication Critical patent/WO1994002627A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244Interleukins [IL]
    • C07K16/245IL-1
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide

Definitions

  • the present invention relates to an antibody against IL-11 (inter! Ikin-1), particularly to a recombinant human'mouse antibody.
  • IL-1 was reported by Gery et al. In 1972 as a mouse thymocyte growth-promoting factor present in human monocyte culture supernatants [Gery, I. et al., J. Bxp. Med., 139, 128 (1972) 3.
  • the IL-11 is produced by many cells such as monocytes and macrophages, keratinocytes, NK cells, T cells, B cells, vascular endothelial cells, and neutrophils, and acts on many cells. It has been reported to show biological activity and is involved in reactions in the body such as immunity, inflammation, hematopoiesis, endocrine, cranial nerves, and homeostasis [Oppenhim, JJ et al., Immunol.
  • IL-11 is translated from mRNA as a precursor with amino acids around 270 and a molecular weight of 30 KD, and this precursor is cleaved at amino acid 110 and finally C-terminal A mature form of 17 KD consisting of about 150 amino acids on the side.
  • human IL-11 ⁇ is composed of 159 amino acids
  • human IL-11) 9 is composed of 153 amino acids.
  • Various applications of IL-1 have been studied as pharmaceuticals such as antitumor agents and thrombocytopenia, while various diseases associated with abnormal production of IL-11, such as rheumatoid arthritis (RA), Chronic inflammatory diseases such as thyroiditis, hepatitis, nephritis, arteriosclerosis, Kawasaki disease (MCLS), etc.
  • RA rheumatoid arthritis
  • Chronic inflammatory diseases such as thyroiditis, hepatitis, nephritis, arteriosclerosis, Kawasaki disease (MCLS), etc.
  • Abnormal production of vasculitis, generalized intravascular coagulation syndrome (DIC :), blood cancer, etc. has also been reported.
  • the enhanced biological activity of IL-1 is suppressed or neutralized.
  • human-human fusion cells are not suitable for excellent fusion partners such as mouse myeloma cell line. It still has a lot of issues and humanitarian issues.
  • variable region (V) region having the antigen-binding activity of an antibody is derived from a mouse hybridoma and the constant region (C) region having an immunological activity is derived from a human.
  • V variable region
  • C constant region
  • the chimeric antibody since the chimeric antibody has its V region still at 100% in mouse and remains antigenic, the chimeric antibody has also recently been used to reduce antigenicity to humans in this region.
  • Recombinant antibody derived only from the complementarity determining region (CDR) of the V region Production of a reshaped antibody (Reshaped antibody) has also been attempted [Jones, PT et al., Nature, 321, 522 (1986): Riechmann et al., Nature, 332, 323 (1988)].
  • a gene for expressing such an antibody that is, a recombinant gene in which a part of the DNA sequence of a human antibody is substituted with the DNA sequence of a mouse antibody is hereinafter referred to as "human-mouse grafting”.
  • human-mouse grafting The recombinant gene obtained in this way is called a “required gene”.
  • an object of the present invention is to provide a chimeric antibody, a recruitment antibody, and a combination antibody thereof (hereinafter, collectively referred to as "recombinant antibody”) against human IL-11.
  • Another object of the present invention is to provide a recombinant antibody against human IL-11 that can be used for immunoassay of human IL-11.
  • Another object of the present invention is to provide a recombinant antibody against human IL-11 as a drug that can be administered and administered for the purpose of suppressing or neutralizing the action of IL-11 in various diseases accompanied by abnormal production of IL-11. Is to provide.
  • Another object of the present invention is the labeled recombinant antibody, which has low antigenicity to humans and can be used for administration to humans to perform diagnostic imaging such as the site of IL-11 production in the body and its identification. Is to provide.
  • Still another object of the present invention is to provide a technique for producing the above-mentioned recombinant antibody. Disclosure of the invention
  • the L chain has the C region of a human antibody molecule and the V region of a mouse anti-human IL-11 antibody molecule having the amino acid sequence described in Sequence Listing: 1
  • the H chain has A recombinant antibody against human IL-1 having the C region of the human antibody molecule and the V region of the mouse anti-human IL-1 antibody molecule having the amino acid sequence set forth in SEQ ID NO: 2
  • the L chain is a human antibody A human-mouse graft V region containing the CDR region of the mouse anti-human IL-11 antibody molecule having the amino acid sequence described in SEQ ID NO: 3 and the H chain having the C chain of the human antibody molecule.
  • Region and SEQ ID NO: 2 mouse anti-human amino acid sequence
  • a recombinant antibody against human IL-11 having a CDR region and a human * mouse graft V region is provided.
  • mouse anti-human IL one 1 antibody L chain V region (V L) that encoding a sequence Shigeru No.: DN A having the nucleotide sequence represented by 1, mouse anti-human IL- 1 antibody H Sequence encoding the V region (V H ) of the chain Ban: DNA having the nucleotide sequence represented by 2, human / mouse graph antibody L chain containing CDR region of mouse anti-human IL-11 antibody molecule DNA having the nucleotide sequence represented by SEQ ID NO: 3 encoding the V region ( VL ), and the V region of the H chain of a human * mouse-grafted antibody that includes the CDR region of a mouse anti-human IL-11 antibody molecule A DNA having the base sequence represented by SEQ ID NO: 4 that encodes (V H ) is provided.
  • a recombinant antibody comprising DNA encoding the C region of a human antibody molecule and DNA encoding the V region of a mouse anti-human IL-1 antibody molecule having the amino acid sequence of SEQ ID NO: 1.
  • Human IL-11 antibody L chain expression vector for example, pAC53, a mouse anti-human IL-11 antibody having DNA encoding the C region of the human antibody molecule and the amino acid sequence of SEQ ID NO: 2
  • a recombinant anti-human IL-11 antibody heavy chain expression vector having a DNA encoding the V region of the molecule, e.g., pAC52 and pAC77, a DNA encoding the C region of the human antibody molecule
  • a recombinant anti-human IL-11 antibody L chain expression vector having the amino acid sequence of SEQ ID NO: 3 and a DNA encoding a human / mouse graft V region including a CDR region of a mouse anti-human IL-1 antibody
  • Human ⁇ mouse comprising CDR regions of mouse anti-human IL one 1 antibody Ryo amino acid sequence
  • the present invention also relates to a recombinant anti-human IL having a C region of a human antibody molecule and a human / mouse graft V region having a CDR region of a mouse anti-human IL-11 antibody having the amino acid sequence described in Sequence Listing: 3.
  • the present invention also provides a recombinant anti-human IL-11 anti-expression vector having a DNA encoding the heavy chain of a recombinant anti-human IL-11 antibody having a region, for example, PAC79.
  • the recombinant antibody of the present invention has specific reactivity to human IL-11, which is a new immunoassay (Immunoassay method) capable of measuring human IL-11 with high sensitivity, high accuracy, and simple operation.
  • the present invention can be advantageously used for a technique for specific purification of human IL-11 by a technique such as affinity chromatography or the like.
  • the recombinant antibody of the present invention has a characteristic of low antigenicity to humans, and thus is suitable for administration to human bodies.
  • the recombinant antibodies of the present invention also include antibodies of a type having a neutralizing activity against the biological activity of human IL-11.
  • Such antibodies include various diseases associated with abnormal production of IL-11, For example, in chronic inflammatory diseases such as rheumatoid arthritis, thyroiditis, hepatitis and nephritis, arteriosclerosis, vasculitis such as Kawasaki disease, generalized intravascular coagulation syndrome, blood cancer, etc.
  • the present invention is useful for suppressing or neutralizing one biological activity, and the present invention can bring out a highly valuable drug for treating such various diseases.
  • the labeled recombinant antibody obtained by labeling the recombinant antibody of the present invention can be used to administer it to humans, thereby enabling diagnostic imaging of the IL-11 production site in the body and its identification, and the like. According to the present invention, such an image diagnostic method can also be provided.
  • FIG. 1 is a schematic diagram of the mouse H chain cDNA cDNA clone pIL068.
  • FIG. 2 is a schematic diagram of mouse L chain cDNA clone pIL086.
  • FIG. 3 is a schematic diagram of preparation of a chimeric H chain IgG 4 ANOC-301 expression vector PAC 52 and a chimeric H chain IgG, ANOC-301 expression vector p AC 77.
  • FIG. 4 shows the DNA sequences of primers R2391 (forward primer) and R2390 (reverse primer).
  • FIG. 5 shows the characteristics and restriction map of the vector pAC52.
  • FIG. 6 is a schematic diagram of construction of the chimeric L chain ANOC-301 expression vector pAC53.
  • FIG. 7 shows the DNA sequences of primers R2429 (forward ⁇ primer) and R2430 (reverse 'primer).
  • FIG. 8 shows the characteristics and restriction map of the expression vector pAC53.
  • FIG. 9 is a schematic diagram showing the construction of CDR-grafted L chain ANOC-301 expression vector pAL31.
  • FIG. 10 shows the DNA sequence of the human 'mouse graft VL gene and the corresponding amino acid sequence.
  • FIG. 11 shows the characteristics and restriction map of the expression vector pAL31.
  • FIG. 12 is a schematic diagram of production of CDR-graft H chain Ig G4ANOC-30 1 expression vector PAL34.
  • FIG. 13 shows the DNA sequence of the human'mouse graft VH gene and the corresponding amino acid sequence.
  • FIG. 14 shows the characteristics and restriction map of the expression vector pAL34. .
  • FIG. 15 is a schematic diagram showing the production of CDR-graft ANOC-301 expression vector pAC79.
  • FIG. 16 shows the DNA sequence of an oligonucleotide for CDR-graft L chain gene construction.
  • FIG. 17 shows the DNA sequence and the corresponding amino acid sequence between B st B I -S p ⁇ I of the CDR graph L chain gene.
  • FIG. 18 shows the DNA sequence of an oligonucleotide for CDR-graft L chain gene construction.
  • FIG. 19 shows the DNA sequence of the oligonucleotide for construction of the CDR-graft H chain gene.
  • a cDNA library of a mouse anti-human IL-11 monoclonal D-null antibody-producing cell was used to construct a cDNA library of the antibody, and its H chain (heavy-chain) and L chain ( Clones were performed by rearranging the DNA sequence encoding the variable region (V region) of each variable region (light chain, light chain), and taking into account the promoter region and enhancer region.
  • H chain heavy-chain
  • L chain L chain
  • a DNA base sequence modified from the human antibody-derived V region to include the complementarity determining region (CDR) in the V region of the DNA sequence encoding the anti-human IL is obtained. 11 Including CDR region of antibody molecule
  • the DNA nucleotide sequence preparative mouse graft V region code be coupled with DN A sequence encoding the C region of antibody derived from a human to produce a recombinant gene (Risheibuto gene).
  • Each of the H-chain and L-chain chimeric 73 ⁇ 4-residue genes thus prepared is inserted alone or in combination of two types into an appropriate vector to prepare an expression vector for each gene.
  • a desired recombinant antibody of the present invention can be obtained by introducing the vector into a suitable cell to express the above genes.
  • the mouse anti-human IL-11 monoclonal antibody-producing cells used in the present invention are generally prepared using human IL-1 as an immunogen, or using recombinant human IL-1 produced by a gene recombination technique as an immunogen. It can be prepared according to the method for producing a monoclonal antibody described above. Specific examples of the mouse anti-human IL-11 monoclonal antibody-producing cells include, for example, those of human IL-11 and human IL-19 described in JP-A-63-258595. An antibody-producing hybridoma for each of them can be exemplified. All of these hybridomas have been deposited with the Institute of Biotechnology and Industrial Technology, the Agency for Industrial Science and Technology.
  • the above-mentioned antibody-producing hybridoma against human IL-11 ⁇ is As KOCO 30 1 (FBRM BP-1554), antibody-producing hybridomas against human IL-1 j9 were KOC02 03 (FBRM BP-1551), KOCO 205 (FBRM BP-1552) KOCO 206 (FBRM BP -1553).
  • hybridomas can be used for producing the recombinant antibody of the present invention by preparing the gene as described below. That is, as the gene fragment encoding the V region of the antibody of the present invention, a gene sequence that has been shared and analyzed from mouse anti-human IL-11 monoclonal ⁇ -nal antibody producing cells as described above can be used. it can. Single cone and cloning of genes (V L and V H ) encoding a V region specific for the mouse anti-human IL-11 antibody of interest can be carried out mainly according to two methods.
  • One of them is to digest the chromosomal DNA of antibody-producing cells with appropriate restriction enzymes, fractionate by sucrose density gradient centrifugation, ligate to one arm of phage vector, and perform in-vitro packaging. From the chromosome library 1, the H region and the L chain 3'-terminal probes are used to carry out Southern hybridization, etc., to clone the V region gene of the desired antibody. Methods can be mentioned. This method can be performed in more detail with reference to, for example, the method of Oy et al., See CVernon T. Di et al., Proc. Natl. Acad. Sci. U.S.A., 80, 825 (1983)].
  • a method of extracting mRNA from mouse anti-human IL-11 antibody-producing cells according to a conventional method and screening from a cDNA library [MS Neuberger, EMBD J., 2, 1373 (1983)] was used.
  • a probe for cloning the V region gene from the above cDNA library was used as a probe for the previously reported nucleotide sequence of the mouse immunoglobulin gene [for example, BB Max et al., J. Biol. Chem., 256, 5116 ( 1981)] and an example of a method of synthesizing and screening two screening DNA probes complementary to the H chain and the L chain C region of mouse immunoglobulin (Ig). it can.
  • the probe can be easily synthesized by a commercially available automatic oligonucleotide synthesizer, and its 5 ′ end can be labeled using [r- 32 P] ATP using T4 polynucleotide kinase.
  • Cloning in this latter method involves obtaining poly (A) + RNA from antibody-producing cells and then using Gubler and Hoffman (Gubler, U. & Hoffman, B.,
  • Escherichia coli for example, HB101 strain, LE392 strain, NM539 strain, Y109 strain strain, etc. can be used as a host for transformation, and thus a desired cDNA library can be constructed. it can.
  • pSP64 see International Publication WO89 / 019974
  • a probe synthesized from the obtained cDNA library can be used.
  • Positive clones containing each of the desired VL and VH genes were identified by colony hybridization by the method of Hanahan et al. [Hanahan, RA et al, Gene, 10, 63 (1980)].
  • a cDNA library is constructed by, for example, gt10, gt11, or the like by the invito ⁇ packaging method, and a probe synthesized from this cDNA library is used.
  • the target VL and VH genes by plaque hybridization by the method of Penton and Davis [Benton, W. and Davis, R., Science, 196, 383-394 (1977)]. Can be cloned.
  • the DNA of the region containing the V region of interest is subjected to PCR [Polymerase ⁇ -chain reaction] Olymerase chain reaction, Rosana, Q. et al., Pro Natl. Acad. Sci. USA, 86, 3833 (1989)], and a method in which a DNA fragment amplified and amplified is incorporated into a vector.
  • PCR Polymerase ⁇ -chain reaction
  • a method in which a DNA fragment amplified and amplified is incorporated into a vector.
  • the desired VL gene and VH gene can be purified, and the size of the DNA of the clone hybridized with the probe can be determined by PCR using the vector-side primer.
  • the recombinant antibody of the present invention can be expressed by incorporating the amplified V region genes (! ⁇ Gene and !! gene) into an Ig expression vector.
  • V region genes ! ⁇ Gene and !! gene
  • clone V L gene and V H genes were cloned above can be single ⁇ purified by Agaro Sugeru electrophoresis.
  • a suitable vector e.g. pUC 1 8, such as PBR 322 and a suitable restriction enzyme, for example E This can be performed using c 0 RI or the like.
  • the method of Sanger et al. [Sanger, F. et al., Pro Natl. Acad. Sci. USA, 74, 5463 (1977)] was used. By using such methods, the nucleotide sequence of the cDNA region of the target VL and VH genes can be determined.
  • the nucleotide sequences of the VL gene and VH gene of the mouse anti-human IL-11 antibody for producing the recombinant antibody of the present invention obtained as described above are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • Specific examples of plasmids containing these genes include plasmid pIL068 (including the H chain gene of mouse anti-human IL-1 antibody) and plasmid pIL086 (mouse (Including the L chain gene of the human IL_1 antibody).
  • C L recombinant antibodies
  • H chain C region utilizing any of various derived from a human antibody molecule
  • the derived common human immune globulin also the it can.
  • C region genes of the human immune globulin, i.e. C L gene and C H gene, for example, RE I production cell lines [human myeloma protein:... BPP , 0. et al, Bur J. Biochem, 45, 513-524 (1974)] can be isolated by the same method as described above.
  • human immunoglobulin which has already been reported can be used as a probe for cloning the target gene.
  • DNA synthesized with reference to the nucleic acid base sequence [Ellison, JW et al., Nuc. Acids Res., 10, 4071 (1982)]: Heiter, PA et al., Cell, 22, 197 (1980), etc. Probes and the like can be used.
  • Various human C region genes are already known, and plasmid vectors having various human C region genes have already been established. Therefore, in the present invention, it is convenient and advantageous to use the ones possessed by these vectors, even if the genes are not individually prepared from the antibody-producing cells as described above.
  • the C region gene for example, in the case of the H chain, typically r, genes such as ⁇ r 4 chains, or other in; "chain, contains genes for each strand of the chain, when the L-chain
  • Examples of the plasmid include a ⁇ ⁇ ⁇ : chain and a (chain) gene, all of which can be used in the present invention.
  • plasmid vector having the C region of human immunization group ⁇ -prin and an enhancer include: For example, ⁇ 104 CP EE 6 having a human IgG 4 ⁇ chain C region (P. Stephens and. Cockett, Nucl. Acids Res., 17, 7110 (1989): Prot.
  • Gene for the production of chimeric antibody against human IL- 1 of the present HatsuTomo includes a gene selected from the VL gene and VH genes of the above-mentioned various gene fragments, i.e. mice, C L gene from human and a gene selected from C H gene, is constructed by bringing together each binding.
  • the recombinant human of the present invention obtained by binding the mouse-derived VL gene and the 7-derived 1_ gene.
  • An outline of the construction of an I-11 antibody L chain expression vector is, for example, as shown in FIG.
  • a specific example of the expression vector thus obtained is pAC53. This is obtained by digesting the BstBI and Sp1I digested fragments of pIL086 with the same restriction enzyme as the plasmid pE1081, which has a DNA sequence encoding the light chain C region of human IgA: It was constructed by inserting it into a fragment.
  • the outline of the construction of the recombinant human IL-11 antibody H chain expression vector of the present invention obtained by binding the mouse-derived VH gene and the human-derived CH gene is, for example, as shown in FIG.
  • Specific examples of the expression vector thus obtained include pAC52 and pAC77.
  • H i ⁇ dl [and Ap a I cleavage fragment of p IL 0 6 8, having a DNA sequence encoding the H chain C region of a human I gG 4 PAC 52 is constructed by inserting it into the same restriction enzyme digested fragment of plasmid pE1001 which has a DNA sequence coding for plasmid pE1004 and human IgG G! 0H chain C region.
  • the antibody against human IL-11 of the present invention includes, in addition to the chimeric antibody described above, a recombinant antibody, that is, at least one of the L chain and the H chain covers a CDR region of a mouse anti-human IL-11 antibody molecule.
  • An antibody having a human-mouse graft V region is included.
  • the gene (grafted receive gene, in particular, the graft VL gene and the graft VH gene) for producing the ricin X antibody of the present invention include, for example, the mouse-derived VL gene or the VL gene obtained as described above.
  • VH genes that respond morphism human antibody molecule.
  • a gene can be chemically synthesized by an automatic oligonucleotide synthesizer with reference to the designed DNA sequence, and a part of the designed DNA sequence can be obtained by various methods described above. It can also be produced and amplified according to the PCR method using genes.
  • graft gene examples include the darafft VL gene shown in SEQ ID NO: 3 and the graft VH gene shown in SEQ ID NO: 4.
  • FIG. 9 Schematic construction of expression vectors of the graph preparative V L gene and Rishiyui but-antibody L chain is constructed by coupling the C L gene from human is as shown in FIG. 9, thus as a specific example of the expression vector obtained Can exemplify PAL 31.
  • This can be obtained by the two-stage method shown in the examples below. That is, a DNA sequence containing a part of the graft VL gene of SEQ ID NO: 3 (BstBI and Sp1I cleavage fragments excluding the CDR1 to CDR3 regions, amplified by PCR) was added to plasmid.
  • the fragment was inserted into the same restriction enzyme-cleaved fragment of pE1081 (step 1), and the CDR1 to BstEH-KpnI present between the BstBI and Sp1I of the obtained plasmid were separately synthesized. It was constructed by inserting (step 2) the DNA sequence of the CDR3 region (BstEII and ⁇ I cleavage fragment), and the features are as shown in FIG.
  • the Rishi constructed by coupling the C H genes from Gurafudo V H gene and human
  • the outline of the construction of the expression vector for the Ito antibody H chain is as shown in FIG. 12, and the expression vector thus obtained specifically includes PAL34.
  • the V region and C region genes isolated from the chromosomal DNA are mainly used, ie, the V region and C region genes isolated from the chromosomal DNA, depending on the generally obtained method for isolating each gene. It is necessary to take into consideration the combination of V region and the combination of V region and C region isolated from cDNA. For example, when a V region gene isolated from mouse chromosomal DNA is linked to a C gene isolated from human chromosomal DNA, the mouse V region gene must have an expression regulatory region such as the promoter Jenhansa required for expression. It is preferable to include in advance. However, it is not necessary that the pi motor Jenhansa etc. be derived from a mouse, and may be derived from a human.
  • the promoter is located 5 ′ upstream of the V region, and the enhancer is preferably located between the V region gene and the C region gene, but the enhancer is not necessarily limited to this position.
  • the binding site is determined using an appropriate restriction enzyme site and, if necessary, an appropriate linker.
  • the amino acid sequence encoded by the V region gene and the amino acid sequence encoded by the C region gene do not shift, and the V region amino acid sequence and the C region amino acid sequence do not change.
  • the thus prepared chimeric antibody gene or receive antibody gene of the present invention can be used, for example, with the vector plasmids PEE6, PE101, pE104, pE1081, etc. SV 2 gpt [RC Mullgan et al., Pro. NAS. USA, 78, 2027 (1981)], pSV2- ⁇ eo CP. J. Southern et al., J. ol. APPI. Genet., 1, 327 (1982) 3, etc.
  • Plasmids in plasmids or host cells By integrating the H-chain gene and L-chain gene separately or simultaneously into a vector plasmid containing a part of a viral gene (such as papilloma virus) Desired chimeric antibody L chain expression plasmid, chimeric antibody H chain expression plasmid, received antibody L chain expression plasmid and recruited antibody H chain expression plasmid can be constructed, and a combination of the above L chain and H chain Thus, the recombinant antibody expression plasmid of the present invention can be constructed.
  • a viral gene such as papilloma virus
  • the plasmid into which the H chain gene and the L chain gene have been separately incorporated is transformed into a host cell using the L chain expression plasmid and the H chain expression plasmid.
  • the desired antibody-expressing transformant of the present invention can be obtained.
  • the plasmid integrated simultaneously with the H chain gene and the L chain gene can be used alone to transform host cells to obtain a desired transformant expressing the antibody of the present invention.
  • the plasmid which is simultaneously incorporated with the H-chain gene and the L-chain gene for producing the above-mentioned antibody-expressing transformant of the present invention include, for example, PAC obtained by a method described in detail in Examples below. 7 9 can be exemplified. More specifically, as shown in FIG. 15, on the other hand, from the plasmid PAL34, a gene containing polyA in the H chain V region and the C region, which contains the human CMV promoter, is isolated. The SV40Zgpt gene fragment of pEl004 is ligated and inserted into the plasmid pAL31 appropriately, and the plasmid thus obtained is the target H chain gene and L chain gene. It has a selection marker together with its offspring and is useful as an expression vector for the recombinant antibody of the present invention. The characteristics of the thus obtained plasmid pAC79 are also shown in FIG.
  • the production of the anti-human IL-11 recombinant antibody of the present invention comprises preparing a host animal cell using a plasmid containing the antibody gene or the H chain or L chain gene produced as described above. It can be carried out by transforming and culturing the transformant.
  • the host animal cells used for this transformation include COS cells [Nu Acids Res., 12, 5707-5717 (1988); ATCC CCL70; CV-1 cells; Russ-transformed cell line], CHO cells [: Chinese hamster ovary cell; ATCC CCL61: CHO-KK WO 89/017883],: BHK cells [baby hamster kidney cell; ATCC CCL1] 0: BHK-21 (C- 13), WO 89/01783] and B-lymphoid cells, for example, P3X63 Ag8.653 (ATCC CRL 1580). Transformed cell tumor such as P3X63Ag8U ⁇ 1 (ATCC CRL 1597).
  • Methods of transforming cells with DNA include DEAE-dextran method CNeuberger, S., BBD J., 2, 1317 (1983)], calcium phosphate coprecipitation method CChen, C. and Okayama H., ol. Cell. Biol., 7, 2745-2752 (1987) 3, Puguchi toplast fusion method [0i, VT and Morrison, S., Bio Techniques, 4 (3), 214-221 (1986)], Elect. Mouth poration method [Potter. H. et al., Proc.
  • the transformed cells obtained as described above can be cultured and grown under the same appropriate conditions as those used for culturing hybridomas, such as normal animal cells, in a suitable medium, a standard culture medium commonly used, for example, Dulbecco's Modification. It is performed in Eagle's medium (DMEM) or RPMI 1640 medium, which can be supplemented with mammalian serum, if necessary, such as fetal calf serum, or trace elements and growth-sustaining capture elements.
  • DMEM Eagle's medium
  • RPMI 1640 medium which can be supplemented with mammalian serum, if necessary, such as fetal calf serum, or trace elements and growth-sustaining capture elements.
  • the medium is supplemented with the above-mentioned selection medium, for example, G418 or a medium containing xanthine, hypoxanthine, thymidine, and mycophenolic acid. preferable.
  • the anti-human IL-11 recombinant antibody of the present invention is secreted and produced.
  • Can be Techniques for mammalian cell culture under these tissue culture conditions are obvious in the art.
  • the supernatant of the cell culture thus obtained can be used in general, such as the fluorescence-labeled antibody method using Enzyme Immunoassay (EIA), Dot-At-Assy, FITC, or Radioimmunoassay (RIA), latex agglutination, hemagglutination, etc.
  • EIA Enzyme Immunoassay
  • FITC Dot-At-Assy
  • RIA Radioimmunoassay
  • latex agglutination hemagglutination
  • a desired anti-human IL-11 recombinant monoclonal antibody can be screened using a suitable screening method.
  • the cells producing the desired anti-human IL-1 recombinant monoclonal antibody thus obtained can be subcultured in the above-mentioned ordinary medium, and can be stored for a long time in liquid nitrogen.
  • the anti-human IL-11 recombinant monoclonal antibody of the present invention can be collected from the antibody-producing cells by culturing the cells according to a conventional method, and using the culture supernatant or the cells as a peritoneal cavity of a mammal compatible therewith. Can be used to obtain the ascites from the cells.
  • the immunoglobulin in the culture supernatant is concentrated by, for example, precipitation with ammonium sulfate, dialysis against a hygroscopic substance such as PEG, or filtration through a selective membrane. If necessary or desired, the antibody is concentrated by a conventional chromatography method such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose or column chromatography using cellulose. And the like.
  • the antibody of the present invention thus obtained can be used to purify human IL-11 simply and specifically by a conventional purification means such as immunoprecipitation or affinity chromatography.
  • human IL-11 in a sample can be specifically measured by an immune reaction.
  • the method include a conventional competition method, a radioimmunoassay method (RIA) by a sandwich method, an enzyme immunoassay method.
  • RIA radioimmunoassay method
  • the test substance is reacted with the insolubilized antibody of the present invention to form a human IL-11-insoluble antibody complex.
  • the complex is allowed to react with a fixed amount of the labeled antibody, then the conjugate of the complex and the labeled antibody and the unbound labeled antibody are separated, and the labeling activity of one of them is measured to obtain a sample.
  • human IL-11 can be quantified.
  • the body fluid used as a specimen in the above-mentioned assay include, for example, blood, cell tissue fluid and the like. Among them, blood, particularly serum or plasma is preferable.
  • the labeling substance of the present invention an antibody, Darko amylase, bar oxidase, alkaline phosphatase, various enzymes, such as one galactosyl Tokishidaze, 125 1, 1 3, radioactive substances such as tritium, and the like.
  • the labeling method may be a conventional one [see Nature, 194, 495 (1962): Acta. Bndocrliol. Suppl., 168, 206 (1972)].
  • An insolubilized antibody is produced by chemically or physically binding human IL-11 or the antibody of the present invention to an insoluble carrier.
  • insoluble carriers include Cell D-powder, Sephadex, Sepharose, polystyrene, filter paper, carboxymethylcellulose, ion-exchange resin, dextran, plastic film, plastic tubes, nin, glass beads, silk and poly
  • examples include a copolymer of min-methyl vinyl ether-maleic acid, a copolymer of amino acid, and a copolymer of ethylene-maleic acid.
  • the insolubilization is carried out by the dithiol method, peptide method, alkylation method as a covalent bond method, carrier binding method using a cross-linking reagent (using glutaraldehyde, hexamethylene isocyanate, etc. as a cross-linking reagent), and Ug1 reaction. It is carried out by a chemical reaction such as a carrier binding method; or an ion binding method using a carrier such as ion exchange resin; and a physical adsorption method using a porous glass such as glass beads as a carrier.
  • the reaction is usually carried out under 45 £ i, preferably at a temperature of 4 to 40, for several hours to 24 hours.
  • human IL-11 in a sample can be measured simply and accurately.
  • the antibody of the present invention has an action of suppressing or neutralizing the action of IL-11.
  • diseases associated with abnormal production of human IL-11 typically chronic inflammatory diseases such as rheumatoid arthritis, thyroiditis, nephritis, vasculitis such as arteriosclerosis, Kawasaki disease, and DIC It is possible to treat and diagnose various diseases such as blood cancer.
  • the antibody of the present invention has low antigenicity and can be administered to the human body. For example, by labeling it and administering it to the human body, tissue sites associated with abnormal production of IL-11 in the human body, etc. It can also be used for diagnostic imaging.
  • the present invention includes not only recombinant antibodies, but also methods for producing the same, cloning vectors, expression vectors, and transfected cell lines used in the methods, and human IL-11 purification systems for the antibodies of the present invention.
  • the present invention also encompasses the use of the present invention in a measurement system, and a method of applying a therapeutic or diagnostic agent containing the antibody as an active ingredient, and the like.
  • the immunogen is recombinant human IL-11 ⁇ , the isotype of the resulting antibody is IgG, and the affinity of the antibody is 0.8-2.0 X 1 O- ⁇ M ⁇ . See JP-A-63-258595.
  • FCS fetal calf serum
  • ImM glutamin and 50 units of Zm ⁇ venicillin-streptomycin (p / s, Gibco (GIBC0) Co., Ltd.) using RPMI-1640 medium containing, a 5% C0 2 / air gas were cultured at 37 in Rabotoru were fed
  • Poly (A) was prepared using Pharmacia's mRNA Purification Kit (Cat. No. 27-92258-01) based on a pre-packed span column with oligo (dT) -cell ⁇ -cells. + RNA was prepared. The resulting poly (A) + RNA is 50
  • CDN ⁇ was synthesized from 5; / g of poly (A) + RNA using a cDNA synthesis kit (Cat. No. RPN I256Y) manufactured by Amersham.
  • a phosphorylated Bst XI adapter (125 picomoles) having an internal EcoRI site was ligated to 0.5 picomoles of cDNA, and the cDNA with the adapter was converted to ACA34 columns. And purified by gel filtration.
  • the cDNA with the adapter was converted into a single cone Xba from the modified vector of pSP64 containing the Bst XI site (Vector-CDM8 (B. Seed, Nature, 329, 84 (H1987))).
  • Oligonucleotide probe The 5 'end with T 4 polynucleotide kinase as shown in Table 1 [r- 32 [rho] with O oligo nucleotide probe labeled with ATP, about 1 than c DNA library obtained in the above 2 0 0 0 The colonies were screened for the respective cDNAs of the L chain and H chain of the antibody.
  • R498 is complementary to the sequence of 4658-8-4677 in the mouse Cc gene sequence [Max et al., J. Biol. Chera., 256, 5116 (1981)].
  • R 627 corresponds to the complementary strand of the sequence 115-133 of the C H1 domain of the mouse Cr! Gene [Honjo et al., Cell, 18, 559 (1979)]. I do.
  • oligonucleotide which had been lapel at 32 P, was added to a solution having the same composition as the prehybridization solution so as to have a concentration of 5 ng / m £, and the mixture was adjusted to 65: and then released at room temperature for 16 hours or more. Was placed. The filter was washed twice at room temperature in a 6 XS SC-0.1% SDS solution and then three times at 45 in a 6 XS SC-0.1% SDS solution.
  • the base sequences of the oligos R8883 and R1767 are shown in Table 2 below.
  • the nucleotide region of each V region of the two H chain cDNA clones (pIL068pIL072) and the two L chain cDNA clones (pIL086, pIL089) was determined. The sequence was examined.
  • the outline of the H chain cDNA clone pIL068 is as shown in FIG. 1, and the outline of the L chain cDNA clone pIL086 is as shown in FIG.
  • SP6 RNAp01P indicates the promoter region of SP6 RNA polymerase, and Amp R indicates the ampicillin resistance gene.
  • both PIL068 and pIL072 clones had full-length mouse IgG! H chain gene cDNA.
  • the light chain cDNA clones of pIL086 and pIL089 differed in the V region.
  • pIL089 had the L chain gene because it was derived from the fusion partner of the hybridoma, and pIL086 had the mouse kappa chain gene of ANOC-301.
  • the nucleotide sequence of the V region of the H chain cDNA clone PIL068 is as shown in SEQ ID NO: 1.
  • the nucleotide sequence of the V region of the L chain cDNA clone pI L086 is as shown in Sequence Listing: 2.
  • the above pIL086 lacks the first three amino acid residues of the leader sequence. Therefore, when constructing an expression vector, methionine is used based on the known leader sequence of the power chain as shown below. , Arginine and valine sequences (ATGAGGGTC).
  • V H is a mouse H chain V region gene
  • G4 is the C region of the human I gG 4
  • G1 is human I &, of (: the area
  • hCMV is human Tosai Tomega o virus of MI E (major immediate early )
  • ⁇ motor gpt
  • xanthinguanine phosphoribosyltransferase gene pA
  • poly A addition signal sequence
  • SVori replication origin of SV40 virus, respectively.
  • the V region gene was excised and inserted into the vector pE1001 between Hi TidlE and Ap aI.
  • the desired pAC77 was obtained.
  • VL indicates the L chain V region gene of ANOC-301 (mouse)
  • C k also indicated as C kappa or C c
  • AmpR and hCMV are the same as above. It is.
  • V L mouse L chain variable region gene
  • R 242 forward primer
  • R 243 0 reverse • primer
  • the VL gene amplified by the PCR method is digested with the restriction enzymes BstBI and Sp1I, and this is cut into the vector pE1081CpE1004 having the human kappa chain C region. It was prepared based on pEE6 in the same manner as described above. Was inserted between BstBI and Sp1I to obtain pAC53.
  • FIG. 8 shows the characteristics and restriction enzyme map of pAC53 thus obtained.
  • CDR-grafting was carried out using oligonucleotides in accordance with the method described by Daughterty et al. (Nucl. Acids Res., 19, 2471 (1991)) using oligonucleotides according to steps 1 and 2 below.
  • the VL gene was obtained.
  • a universal VL cassette encoding the leader sequence, framework 1 and framework 4 of the human antibody REI power chain was constructed. Specifically, a PCR reaction was performed using the oligonucleotides shown in FIG. The reaction mixture consisted of 50, 10 picomoles each of 3 'and 5'—short ⁇ Oligonucleotide ( ⁇ -20, L-RBIVBC 3), 1 picomole of each * internal * oligonucleotide 1, L-RBIVBC 1, L-RBIVBC 2), 1 OmM tris monohydrochloride (pH8. 3), 1. 5m MgC 2. 0. 01% (w / v) gelatin, 5 OmMKCi 0.
  • a PCR reaction was performed using the oligonucleotides shown in Fig. 18 in the same manner as in step 1, and finally the DNA fragment between BstEH and KplI was obtained. Obtained. This was inserted between BstEII and KpnI of the universal VL cassette pAL29 obtained in step 1 previously cut with the restriction enzymes BstEII and KpnI to obtain pAL31.
  • the obtained CDR-graft VL gene DNA sequence and corresponding amino acid sequence are as shown in SEQ ID NO: 3.
  • FIG. 11 shows the characteristics and restriction enzyme map of the target PAL31 into which BstBI and Sp1I fragment (FIG. 10) were inserted.
  • CDR graft heavy chain IgG 4 ANOC—301 expression vector
  • this expression vector PAL 34 is as shown in FIG. 12, and the vector is a human antibody KOL CB.
  • the CDR-grafted V H chain gene (the DNA sequence of which is the same as that described in (9) above) is obtained in one step by the PCR method using an oligonucleotide (FIG. 19). ).
  • the obtained gene was digested with restriction enzymes Hindlll and ApaI to obtain a DNA fragment meeting the human / mouse graft VH gene. It has the DNA sequence and the corresponding amino acid sequence shown in FIG.
  • the above-mentioned Hi Ti dm-Apa I fragment was inserted between HindE and Ap aI of vector pE1004 to obtain a desired pAL34.
  • FIG. 14 shows the characteristics and restriction map of pAL34 thus obtained.
  • a single DNA fragment was prepared as a Sail DNA fragment, which was inserted into the Sal1 site of the CDR-grafted L chain ANOC-301 expression vector pAL31. Further, the hCMVZCDR IgG 4 cassette was inserted into the BamHI site of the obtained vector to prepare the desired expression vector PAC79. The outline of the operation is as shown in Fig. 15 o
  • CHOL 761h cells [MI Cockett et al., Nucl. Acids Res., 19, 319 (1991)] cultured in Dulbecco's modified Eagle's medium containing 10% FCS, ImM glutamine, and 50% nicillin trebutomycin were used. Approximately 2 ⁇ 10 1 cells were seeded on a Falcon 175 flask, and 50 ⁇ g of each plasmid DNA was transfused by the calcium phosphate method.
  • Plasmid DN A was used, the above (6) to (11) obtained as or a combination thereof in, that expression of the chimeric H chain I gG 4 ANOC- expression vector p AC 52 301 and a chimeric L chain ANOC- 301 Combination of vector pAC53 and chimera chain ANOC-301 expression vector pAC53 and chimera H chain IgdANOC-301 expression vector pAC77 combination and chimera L chain ANOC-301 expression vector P AC 53 and CDR- grafting H chain I gG 4 ANOC- l 0 3 expression base Kuta - combination of PAL 34, CDR- grafted L chain I gG 4 ANOC- 3 0 1 expression vector PAL31 the chimeric H chain I gG 4 ANOC -301 expression vector
  • the medium was recovered 72 hours after transfection.
  • the expressed recombinant antibodies are as follows.
  • the matrix is washed with the equilibration buffer (3 column volumes, 66 mL), and then 50 mM sodium acetate buffer (pH 4.5) (3 column volumes, At 66 mi), bovine IgG in the culture supernatant was eluted. Thereafter, the chimeric IgGIANOC-301 was eluted with a 0.1 M glycine hydrochloride (PH2.5) solution, and the eluate was immediately neutralized with 2 M ris • hydrochloric acid (PH8.0).
  • the fraction containing IgGiANOC-301 was dialyzed against phosphate-buffered saline containing 0.05% timezal, and stored at 0-10.
  • I gG 4 ANOC-3 0 1 of purification was also carried out in the above-mentioned chimeric I gG! ANOC- 3 0 1 the same way.
  • the chimeric I g G 4 ANOC- 3 0 1 is 2.4 in the culture supernatant from 3 2 5 jug
  • chimeric L chain graft H chain I gG 4 AN_ ⁇ _C one 3 0 1, 2. 1 ⁇ [delta] 8200 jug from the culture supernatant of the above
  • grafted L chain Z chimeric H chain IgG 4 ANOC-301 was 94.5 tg from the culture supernatant of 1.1
  • Pre-packed PD-10 column (manufactured by Pharmacia) equilibrated with PBS (phosphato buffered saline) containing 0.05% thimerosal (manufactured by Sigma) ) And counted for each fraction (1 ml / fraction), and the fraction containing 125 IL-1 ⁇ was stored at 4.
  • PBS phosphato buffered saline
  • thimerosal manufactured by Sigma
  • ANOC-301 and the recombinant antibody were prepared in PBS (hereinafter referred to as antibody diluent) containing 3% normal goat serum and 0.05% thimerosal (Sigma), respectively. Dilute two times serially to 5 ngZm £, put each 10 into an Assay tube (glass tube, 10 mmX75, manufactured by Corning), and further add 100 / ⁇ (10 (Ocpm) 125 IL-1 ⁇ and an antibody diluent of 300 ⁇ were added and mixed, and the mixture was allowed to stand at 4 for 48 hours. 500 / ⁇ £ 25% PEG (polyethylene glycol, manufactured by Wako Pure Chemical Industries) was added, stirred, and left for 30 minutes at t :. After centrifugation (300 rpm, 15 min, 4 t :), the supernatant was removed, and the force of the precipitate was measured with an r-counter.
  • PBS normal goat serum
  • thimerosal thimerosal
  • the concentration of the antibody showing 50% B / T (B: bound count, T: total count) is shown in Table 3 as the titer.
  • a measurement method by an immunoassay method capable of accurately measuring human IL-11 in a sample having a low concentration of human IL-11 such as a clinical sample it is possible to provide a pharmaceutical for the purpose of suppressing or neutralizing the action of IL-11 against various diseases accompanied by abnormal production of IL-11, an imaging diagnostic agent for a tissue site of abnormal production of IL-11 in a human body, and the like.
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • ATC TAC TAC ACA TCA AGA TTA CAC TCA GGA GTC CCA TCA AGG TTC AGT 240 l ie Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser
  • Sequence type nucleic acid

Abstract

A chimeric antibody against human IL-1, and other human-mouse chimeric and reshaped antibodies, wherein the L chain has the constant region of human antibody molecules and the variable region of mouse antihuman IL-1 antibody molecules having the amino acid sequence described in the sequence identification no. 1 and the H chain has the constant region of human antibody molecules and the variable region of mouse antihuman IL-1 antibody molecules having the amino acid sequence described in the sequence identification no. 2. This antibody is utilizable in treating various diseases accompanied by the abnormal production of IL-1 and conducting diagnostic imaging of IL-1 producing sites in the body.

Description

明 細 書 ヒ トイ ンター αィキン一 1に対する組換え抗体  Remarks Recombinant antibody against human interferon α-1
技術分野 Technical field
本発明は I L一 1 (ィ ンター !□ィキン一 1) に対する抗体、 殊にヒ ト 'マウス 組換え抗体に関する。 背景技術  The present invention relates to an antibody against IL-11 (inter! Ikin-1), particularly to a recombinant human'mouse antibody. Background art
I L— 1は、 1 9 7 2年にゲリ一 (Gery) らによりヒト単球培養上清中に存在 するマウス胸腺細胞増殖促進因子として報告された 〔Gery, I. et al., J. Bxp. Med. , 139, 128(1972)3 。 また該 I L一 1は単球やマクロファージをはじめケラ チノサイ ト、 NK細胞、 T細胞、 B細胞、 血管内皮細胞、 好中球など多くの細胞 から産生され、 多くの細胞に作用し、 多種多様の生物活性を示し、 免疫、 炎症、 造血、 内分泌、 脳神経、 生体恒常性など生体における反応にかかわつていること が報告されている [Oppenhim, J. J. et al. , Immunol. Today, 7, 45(1986)] 。 動物種を問わず、 I L一 1はアミノ酸 2 7 0前後、 分子量 3 0 K Dの前駆体とし て mRNAから翻訳され、 該前駆体はアミノ酸 1 1 0番合で切断されて最終的に は C末端側約 1 5 0個のアミノ酸からなる 1 7 KDの成熟型となる。 また、 I L 一 1はその等電点の違いによって or型 (p 1 =5) と β型 (ρ I =7-8) の 2 つの型に分類されており、 それらの一次構造も明らかにされ、 さらにはそのポリ ぺプチドもしくはその前駆体をコ一ドする遺伝子の存在も報告されている 〔Proc. Natl. Acad. Sci., 81, 7907-7911 (1984) : Nature, 315, 641 (1985) : J. BXP. Med., 164, 237(1986) : Nuc. Acids Res., 13(16) 5869(1985)) 。  IL-1 was reported by Gery et al. In 1972 as a mouse thymocyte growth-promoting factor present in human monocyte culture supernatants [Gery, I. et al., J. Bxp. Med., 139, 128 (1972) 3. The IL-11 is produced by many cells such as monocytes and macrophages, keratinocytes, NK cells, T cells, B cells, vascular endothelial cells, and neutrophils, and acts on many cells. It has been reported to show biological activity and is involved in reactions in the body such as immunity, inflammation, hematopoiesis, endocrine, cranial nerves, and homeostasis [Oppenhim, JJ et al., Immunol. Today, 7, 45 (1986) ]. Regardless of the animal species, IL-11 is translated from mRNA as a precursor with amino acids around 270 and a molecular weight of 30 KD, and this precursor is cleaved at amino acid 110 and finally C-terminal A mature form of 17 KD consisting of about 150 amino acids on the side. In addition, IL-11 is classified into two types, or-type (p 1 = 5) and β-type (ρ I = 7-8), depending on the difference in isoelectric point, and their primary structures have been clarified. Furthermore, the existence of a gene encoding the polypeptide or its precursor has been reported [Proc. Natl. Acad. Sci., 81, 7907-7911 (1984): Nature, 315, 641 (1985). ): J. BXP. Med., 164, 237 (1986): Nuc. Acids Res., 13 (16) 5869 (1985)).
上記報告によれば、 ヒト I L一 1 αは 1 5 9個のアミノ酸からなり、 ヒト I L 一 1 )9は 1 5 3個のアミノ酸からなると記されている。 上記 I L— 1については、 抗腫瘍剤や血小板減少改善剤などの医薬品としての応用が種々研究されている一 方で、 I L一 1の異常産生を伴う各種疾患、 例えば慢性関節リウマチ (RA) 、 甲状腺炎、 肝炎、 腎炎等の慢性炎症性疾患、 動脈硬化、 川崎病 (MCLS) 等の 血管炎、 汎発性血管内凝固症候群 (D I C:) 、 血液ガン等において、 その異常産 生も報告されており、 これらの各疾患においては、 亢進した I L— 1の生物活性 を抑制乃至中和するための医薬としての抗 I L一 1抗体の開発が望まれている。 本出願人らも先に I L一 1 α及び I L— 1 )9のそれぞれに特異反応性を有するヒ ト I L一 1に対するマウスモノク σ—ナル抗体を開発し、 該抗俅に係わる発明を 特許出願した 〔特開昭 6 3— 2 5 8 5 9 5号公報参照〕 。 According to the above report, human IL-11α is composed of 159 amino acids, and human IL-11) 9 is composed of 153 amino acids. Various applications of IL-1 have been studied as pharmaceuticals such as antitumor agents and thrombocytopenia, while various diseases associated with abnormal production of IL-11, such as rheumatoid arthritis (RA), Chronic inflammatory diseases such as thyroiditis, hepatitis, nephritis, arteriosclerosis, Kawasaki disease (MCLS), etc. Abnormal production of vasculitis, generalized intravascular coagulation syndrome (DIC :), blood cancer, etc. has also been reported. In each of these diseases, the enhanced biological activity of IL-1 is suppressed or neutralized. It is desired to develop an anti-IL-11 antibody as a medicament for achieving this. The present applicants have previously developed a mouse monoclonal σ-nal antibody against human IL-11 having specific reactivity to each of IL-11α and IL-1) 9, and filed a patent application for an invention relating to the antibody. [Refer to Japanese Patent Application Laid-Open No. 63-2585995].
しかして、 1 9 7 5年にマウスの抗体産生細胞と骨髄腫細胞とを融合させた細 胞の増殖法によるモノクローナル抗体の作製法が確立されて以来、 今日までに上 記出願にかかわる抗体に限らず、 多くのモノク ーナル抗体が作製されてきてお り 〔Kohler G. & Mi lstein C. , Nature, 256, 495 (1975) ] 、 今日、 特に医学の 分野においてこれらのモノクローナル抗体は、 イ ンビボ診断、 イ ンビトロ診断、 さらに治療への応用が試みられるようになってきている。 しかるに、 ヒ トへの投 与を考慮した場合、 これらのマウス由来のモノク ーナル抗钵よりも、 ヒ ト由来 のモノク D—ナル抗体 ほうが、 抗体自体の抗原性や抗体の免疫活性の面から、 当然に好ましいと考えられ、 従って、 近年ヒト型モノク D—ナル¾¾钵の作製も試 みられつつあるが、 ヒト · ヒト融合細胞は、 マウスにおける骨髄腫細胞株のよう な優れた融合のパートナーがないことや、 人道的な面でなおかなり多くの問題を 有している。  Since the establishment of a monoclonal antibody production method by cell proliferation in which mouse antibody-producing cells and myeloma cells were fused in 1975, the antibody involved in the above-mentioned application has been established to date. Many, but not limited to, monoclonal antibodies have been produced [Kohler G. & Milstein C., Nature, 256, 495 (1975)], and today, especially in the field of medicine, these monoclonal antibodies are used in vivo. Diagnosis, in vitro diagnosis, and further therapeutic applications are being attempted. However, in consideration of administration to humans, human monoclonal D-nal antibody is more suitable than human monoclonal monoclonal antibody in terms of the antigenicity of the antibody itself and the immunological activity of the antibody. Naturally, it is considered preferable. Therefore, although the production of human D-nal II has been attempted in recent years, human-human fusion cells are not suitable for excellent fusion partners such as mouse myeloma cell line. It still has a lot of issues and humanitarian issues.
そこで、 これらの問題を解決するために、 抗体の抗原結合活性を有する可変部 (V ) 領域をマウスハイプリ ドーマ由来とし、 且つ免疫活性を有する定常部 (C ) 領域をヒト由来とした組換え抗体、 所謂キメラ抗体の作製が遺伝子組換え技術を 用いて試みられ、 1 9 8 4年のモリソン (Morrison) 以来、 さまざまな特異性を もつキメラ抗体が作製されてきた Dlorrison S. し. et al., Proc. Natl. Acad. Sci. U. S. A. , 81, 6851 (1984) : Sharon J. et al. , Nature, 309, 364 (1984): Neuberger, . S. et al. , Nature, 312, 604 (1984) : Boul ianne, G. し. et al. , Nature, 312, 634 (1984) 等参照〕 。  In order to solve these problems, a recombinant antibody in which the variable region (V) region having the antigen-binding activity of an antibody is derived from a mouse hybridoma and the constant region (C) region having an immunological activity is derived from a human. Attempts have been made to produce so-called chimeric antibodies using genetic recombination techniques, and since Morrison in 1984, chimeric antibodies with various specificities have been produced by Dlorrison S. et al. Et al. Natl. Acad. Sci. USA, 81, 6851 (1984): Sharon J. et al., Nature, 309, 364 (1984): Neuberger,. S. et al., Nature, 312, 604 (1984). ): Boul ianne, G. Shi. Et al., Nature, 312, 634 (1984), etc.]).
更に、 上記キメラ抗体は、 その V領域がなおマウス 1 0 0 %のままであり、 抗 原性が残っているため、 該部分でのヒトへの抗原性をも低減させるために、 最近、 上記 V領域の相補性決定領域 (C D R) のみをマウス由来とした組換え抗体、 即 ちリシ イブト抗体 (Reshaped抗体) の作製も試みられている 〔Jones, P. T. et al. , Nature, 321, 522 (1986) : Riechmann し. et al. , Nature, 332, 323 (1988)] 。 このような抗体を発現させるための遺伝子、 即ちヒト抗体の DN A配 列の一部をマゥス抗体の D N A配列と置換させた組換え遺伝子の創製を以下 「ヒ ト ·マウスグラフト」 といい、 またかくして得られる組換え遺伝子を 「リシエイ ブト遺伝子」 という。 Furthermore, since the chimeric antibody has its V region still at 100% in mouse and remains antigenic, the chimeric antibody has also recently been used to reduce antigenicity to humans in this region. Recombinant antibody derived only from the complementarity determining region (CDR) of the V region Production of a reshaped antibody (Reshaped antibody) has also been attempted [Jones, PT et al., Nature, 321, 522 (1986): Riechmann et al., Nature, 332, 323 (1988)]. The creation of a gene for expressing such an antibody, that is, a recombinant gene in which a part of the DNA sequence of a human antibody is substituted with the DNA sequence of a mouse antibody is hereinafter referred to as "human-mouse grafting". The recombinant gene obtained in this way is called a “required gene”.
上記のようにキメラ抗体ゃリシユイブト抗体等の組換え抗体は従来報告されて いるが、 ヒト I L一 1に対するこれら組換え抗体についての報告は未だにない。 従って、 本発明の目的は上記ヒ ト I L一 1に対するキメラ抗体、 リシユイブト 抗体及びこれらの組合せ抗体 (以下これらを総称して 「組換え抗体」 という) を 提供することにある。  As described above, recombinant antibodies such as chimeric antibody and peroxidase antibody have been reported so far, but no report has been made on these recombinant antibodies against human IL-11. Accordingly, an object of the present invention is to provide a chimeric antibody, a recruitment antibody, and a combination antibody thereof (hereinafter, collectively referred to as "recombinant antibody") against human IL-11.
また本発明の目的はヒト I L一 1の免疫学的測定法に利用できるヒト I L一 1 に対する組換え抗体を提供することにある。  Another object of the present invention is to provide a recombinant antibody against human IL-11 that can be used for immunoassay of human IL-11.
また本発明の他の目的は I L一 1の異常産生を伴う各種疾患において、 該 I L 一 1の作用を抑制乃至中和を目的として投与適用され得る医薬品としてのヒ ト I L一 1に対する組換え抗体を提供することにある。  Another object of the present invention is to provide a recombinant antibody against human IL-11 as a drug that can be administered and administered for the purpose of suppressing or neutralizing the action of IL-11 in various diseases accompanied by abnormal production of IL-11. Is to provide.
また本発明の他の目的はヒトに対する抗原性が低く、 ヒトへの投与適用によつ て、 体内における I L一 1産生部位やその特定等の画像診断を行ない得る標識さ れた上記組換え抗体を提供することにある。  Another object of the present invention is the labeled recombinant antibody, which has low antigenicity to humans and can be used for administration to humans to perform diagnostic imaging such as the site of IL-11 production in the body and its identification. Is to provide.
更に本発明の他の目的は上記組換え抗体の製造技術を提供することにある。 発明の開示  Still another object of the present invention is to provide a technique for producing the above-mentioned recombinant antibody. Disclosure of the invention
本発明によれば、 ( 1 ) L鎖がヒト抗体分子の C領域と配列審号: 1に記載の アミノ酸配列のマウス抗ヒト I L一 1抗体分子の V領域とを有し、 且つ H鎖がヒ ト抗体分子の C領域と配列番号: 2に記載のアミノ酸配列のマウス抗ヒト I L— 1抗体分子の V領域とを有するヒ ト I L—1に対する組換え抗体、 (2 ) L鎖が ヒト抗体分子の C領域と配列番号: 3に記載の了ミノ酸配列のマウス抗ヒト I L 一 1抗体分子 C D R領域を含むヒト ·マウスグラフト V領域とを有し、 且つ H 鎖がヒゝ抗体分子の C領域と配列番号: 2に記載の了ミノ酸配列のマウス抗ヒト I L一 1抗体分子の V領域とを有するヒト I L—1に対する組換え抗体、 (3 ) L鎖がヒト抗体分子の C領域と配列番号: 1に記載のアミノ酸配列のマウス抗ヒ ト I L一 1抗体分子の V領域とを有し、 且つ H鎖がヒト抗体分子の C領域と配列 蕃号: 4に記載のアミノ酸配列のマウス抗ヒト I L一 1抗体分子の C D R領域を 含むヒト ·マウスグラフ ト V領域とを有するヒト I L—1に対する組換え抗体、 及び (4 ) L鎖がヒ ト抗体分子の C領域と配列番号: 3に記載の了ミノ酸配列の マウス抗ヒト I L一 1抗体分子の C D R領域を舍むヒト ·マウスグラフト V領域 とを有し、 且つ H鎖がヒト抗体分子の C領域と配列番号: 4に記載の了ミノ酸配 列のマウス抗ヒ卜 I L一 1抗体分子の C D R領域を舍むヒト *マウスグラフト V 領域とを有するヒト I L一 1に対する組換え抗体が提供される。 According to the present invention, (1) the L chain has the C region of a human antibody molecule and the V region of a mouse anti-human IL-11 antibody molecule having the amino acid sequence described in Sequence Listing: 1, and the H chain has A recombinant antibody against human IL-1 having the C region of the human antibody molecule and the V region of the mouse anti-human IL-1 antibody molecule having the amino acid sequence set forth in SEQ ID NO: 2, (2) the L chain is a human antibody A human-mouse graft V region containing the CDR region of the mouse anti-human IL-11 antibody molecule having the amino acid sequence described in SEQ ID NO: 3 and the H chain having the C chain of the human antibody molecule. Region and SEQ ID NO: 2 mouse anti-human amino acid sequence A recombinant antibody against human IL-1 having a V region of an IL-11 antibody molecule; and (3) a mouse anti-human IL-11 having an amino acid sequence represented by SEQ ID NO: 1 as the C region of a human antibody molecule having a light chain. A human / mouse graft having the V region of the antibody molecule and the H chain comprising the C region of the human antibody molecule and the CDR region of the mouse anti-human IL-11 antibody molecule having the amino acid sequence described in Ban No. 4: A recombinant antibody against human IL-1 having a V region; and (4) a mouse anti-human IL-11 antibody molecule in which the L chain has the C region of the human antibody molecule and the amino acid sequence described in SEQ ID NO: 3. A human anti-mouse IL-11 antibody molecule having a human-mouse graft V region having a CDR region, and a H chain having a C region of a human antibody molecule and the amino acid sequence of the amino acid sequence of SEQ ID NO: 4; A recombinant antibody against human IL-11 having a CDR region and a human * mouse graft V region is provided.
本発明によれば、 マウス抗ヒト I L一 1抗体 L鎖の V領域 (VL) をコードす る配列蕃号: 1で表わされる塩基配列を有する DN A、 マウス抗ヒト I L— 1抗 体 H鎖の V領域 (VH) をコードする配列蕃号: 2で表わされる塩基配列を有す る DN A、 マウス抗ヒト I L一 1抗体分子の C D R領域を舍むヒト ·マウスグラ フ ト抗体 L鎖の V領域 (VL) をコードする配列番号: 3で表わされる塩基配列 を有する DN A、 及びマウス抗ヒト I L一 1抗体分子の C D R領域を舍むヒト * マウスグラフ ト抗体 H鎖の V領域 (VH) をコードする配列番号: 4で表わされ る塩基配列を有する D N Aが提供される。 According to the present invention, mouse anti-human IL one 1 antibody L chain V region (V L) that encoding a sequence Shigeru No.: DN A having the nucleotide sequence represented by 1, mouse anti-human IL- 1 antibody H Sequence encoding the V region (V H ) of the chain Ban: DNA having the nucleotide sequence represented by 2, human / mouse graph antibody L chain containing CDR region of mouse anti-human IL-11 antibody molecule DNA having the nucleotide sequence represented by SEQ ID NO: 3 encoding the V region ( VL ), and the V region of the H chain of a human * mouse-grafted antibody that includes the CDR region of a mouse anti-human IL-11 antibody molecule A DNA having the base sequence represented by SEQ ID NO: 4 that encodes (V H ) is provided.
更に本発明によれば、 ヒト抗体分子の C領域をコードする DN Aと配列番号: 1に記載のアミノ酸配列のマウス抗ヒト I L—1抗体分子の V領域をコードする D N Aとを有する組換え抗ヒ ト I L一 1抗体 L鎖発現べクタ一、 例えば p A C 5 3、 ヒ ト抗体分子の C領域をコードする DN Aと配列番号: 2に記載のァミノ 酸配列のマウス抗ヒト I L一 1抗体分子の V領域をコードする DN Aとを有する 組換え抗ヒト I L一 1抗体 H鎖発現べクタ一、 例えば p A C 5 2及び p A C 7 7、 ヒト抗体分子の C領域をコードする DN Aと配列番号: 3に記載の了ミノ酸配列 のマウス抗ヒト I L— 1抗体の C D R領域を含むヒト ·マウスグラフト V領域を コードする D N Aとを有する組換え抗ヒト I L一 1抗体 L鎖発現ベクター、 例え ば P A L 3 1、 及びヒト抗体分子の C領域をコードする D N Aと配列審号: 4に 記載の了ミノ酸配列のマウス抗ヒト I L一 1抗体の C D R領域を含むヒト ♦マウ スグラフト V領域をコードする D N Aとを有する組換え抗ヒ ト I L— 1抗体 H鎖 発現ベクター、 例えば p A L 3 4が提洪される。 Further, according to the present invention, there is provided a recombinant antibody comprising DNA encoding the C region of a human antibody molecule and DNA encoding the V region of a mouse anti-human IL-1 antibody molecule having the amino acid sequence of SEQ ID NO: 1. Human IL-11 antibody L chain expression vector, for example, pAC53, a mouse anti-human IL-11 antibody having DNA encoding the C region of the human antibody molecule and the amino acid sequence of SEQ ID NO: 2 A recombinant anti-human IL-11 antibody heavy chain expression vector having a DNA encoding the V region of the molecule, e.g., pAC52 and pAC77, a DNA encoding the C region of the human antibody molecule; A recombinant anti-human IL-11 antibody L chain expression vector having the amino acid sequence of SEQ ID NO: 3 and a DNA encoding a human / mouse graft V region including a CDR region of a mouse anti-human IL-1 antibody; For example, PAL31 and the DNA encoding the C region of the human antibody molecule and described in Sequence Listing: 4 Human ♦ mouse comprising CDR regions of mouse anti-human IL one 1 antibody Ryo amino acid sequence A recombinant anti-human IL-1 antibody H chain expression vector having DNA encoding the Sgraft V region, for example, pAL34, is introduced.
また本発明は、 ヒト抗体分子の C領域と配列審号: 3に記載のアミノ酸配列の マウス抗ヒト I L一 1抗体の C D R領域を舍むヒト ·マウスグラフト V領域とを 有する組換え抗ヒト I L一 1抗体 L鎖をコードする DN A、 及びヒト抗体分子の C領域と配列蕃号: 4に記載のァミノ酸配列のマウス抗ヒト I L一 1抗体分子の C D R領域を舍むヒト ·マウスグラフト V領域とを有する組換え抗ヒト I L一 1 抗体 H鎖をコードする D N Aを有する組換え抗ヒト I L一 1抗^発現べクタ一、 例えば P A C 7 9をも提供するものである。  The present invention also relates to a recombinant anti-human IL having a C region of a human antibody molecule and a human / mouse graft V region having a CDR region of a mouse anti-human IL-11 antibody having the amino acid sequence described in Sequence Listing: 3. A human-mouse graft V containing a CDR region of a mouse anti-human IL-11 antibody molecule having the amino acid sequence described in SEQ ID NO: 4 and a DNA region encoding the antibody 1 L chain, and the C region of the human antibody molecule V The present invention also provides a recombinant anti-human IL-11 anti-expression vector having a DNA encoding the heavy chain of a recombinant anti-human IL-11 antibody having a region, for example, PAC79.
本発明組換え抗体は、 ヒ ト I L一 1に特異反応性を有するものであり、 これは ヒト I L一 1を髙感度、 高精度でしかも簡便に測定可能な新しい免疫検定法 (ィ 厶ノアツセィ法) やァフィ二ティークロマトグラフィ一等の手法によるヒト I L 一 1の特異的精製技術に有利に利用できる。  The recombinant antibody of the present invention has specific reactivity to human IL-11, which is a new immunoassay (Immunoassay method) capable of measuring human IL-11 with high sensitivity, high accuracy, and simple operation. The present invention can be advantageously used for a technique for specific purification of human IL-11 by a technique such as affinity chromatography or the like.
殊に、 本発明組換え抗体は、 上記特異性に加えて、 ヒトに対する抗原性が低い という特徴を有しており、 従ってこれはヒト体内への投与適用に適している。 また本発明組換え抗体には、 ヒト I L一 1の生物活性に対して中和活性を有す るタイプの抗体が包含され、 かかる抗体は、 I L一 1の異常産生を伴う各種の疾 患、 例えば慢性関節リウマチ、 甲状腺炎、 肝炎、 腎炎等の慢性炎症性疾患、 動脈 硬化、 川崎病等の血管炎、 汎発性血管内凝固症候群、 血液ガン等において、 その 異常産生に基づく亢進された I L一 1の生物活性を抑制乃至中和するために有用 であり、 本発明はかかる各種疾患の治療上極めて価値ある医薬を提洪することが できる。  In particular, in addition to the above specificity, the recombinant antibody of the present invention has a characteristic of low antigenicity to humans, and thus is suitable for administration to human bodies. The recombinant antibodies of the present invention also include antibodies of a type having a neutralizing activity against the biological activity of human IL-11. Such antibodies include various diseases associated with abnormal production of IL-11, For example, in chronic inflammatory diseases such as rheumatoid arthritis, thyroiditis, hepatitis and nephritis, arteriosclerosis, vasculitis such as Kawasaki disease, generalized intravascular coagulation syndrome, blood cancer, etc. The present invention is useful for suppressing or neutralizing one biological activity, and the present invention can bring out a highly valuable drug for treating such various diseases.
更に、 本発明組換え抗体を標識して得られる標識組換え抗体は、 これをヒト钵 内に投与適用することによって、 体内における I L一 1産生部位やその特定等の 画像診断が可能であり、 本発明によればかかる画像診断法をも提供することがで きる。 図面の簡単な説明  Furthermore, the labeled recombinant antibody obtained by labeling the recombinant antibody of the present invention can be used to administer it to humans, thereby enabling diagnostic imaging of the IL-11 production site in the body and its identification, and the like. According to the present invention, such an image diagnostic method can also be provided. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 マウス H鎖 c DN Aクローン p I L 0 6 8の概略図である。 図 2は、 マウス L鎖 c DNAクローン p I L 0 86の概略図である。 FIG. 1 is a schematic diagram of the mouse H chain cDNA cDNA clone pIL068. FIG. 2 is a schematic diagram of mouse L chain cDNA clone pIL086.
図 3は、 キメラ H鎖 I gG4ANOC— 30 1発現ベクター P AC 52及びキ メラ H鎖 I gG,ANOC-30 1発現ベクター p AC 77作製の概略図である。 図 4は、 プライマー R 2 39 1 (フォワード ·プライマー) 及び R 2 39 0 (リバース ·プライマー) の DN A配列を示す。 FIG. 3 is a schematic diagram of preparation of a chimeric H chain IgG 4 ANOC-301 expression vector PAC 52 and a chimeric H chain IgG, ANOC-301 expression vector p AC 77. FIG. 4 shows the DNA sequences of primers R2391 (forward primer) and R2390 (reverse primer).
図 5は、 ベクター p A C 5 2の特徴及び制限酵素地図を示す。  FIG. 5 shows the characteristics and restriction map of the vector pAC52.
図 6は、 キメラ L鎖 ANOC— 3 0 1発現ベクター pAC 5 3作製の概略図で あ too  FIG. 6 is a schematic diagram of construction of the chimeric L chain ANOC-301 expression vector pAC53.
図 7は、 プライマー R 2 4 2 9 (フォワード♦プライマー) 及び R 2 4 3 0 (リバース 'プライマー) の DN A配列を示す。  FIG. 7 shows the DNA sequences of primers R2429 (forward ♦ primer) and R2430 (reverse 'primer).
図 8は、 発現べクター p A C 5 3の特徴及び制限酵素地図を示す。  FIG. 8 shows the characteristics and restriction map of the expression vector pAC53.
図 9は、 CDR—グラフト L鎖 ANOC— 30 1発現ベクター pAL 3 1作製 の概略図である。  FIG. 9 is a schematic diagram showing the construction of CDR-grafted L chain ANOC-301 expression vector pAL31.
図 1 0は、 ヒト 'マウスグラフト VL遺伝子の DN A配列及び対応するアミノ 酸配列を示す。 - 図 1 1は、 発現べクター p A L 31の特徵及び制限酵素地図を示す。 FIG. 10 shows the DNA sequence of the human 'mouse graft VL gene and the corresponding amino acid sequence. FIG. 11 shows the characteristics and restriction map of the expression vector pAL31.
図 1 2は、 CDR—グラフ ト H鎖 I g G4ANOC-30 1発現べクタ一 P AL 34作製の概略図である。  FIG. 12 is a schematic diagram of production of CDR-graft H chain Ig G4ANOC-30 1 expression vector PAL34.
図 1 3は、 ヒト 'マウスグラフト VH遺伝子の DNA配列及び対応するァミノ 酸配列を示す。 FIG. 13 shows the DNA sequence of the human'mouse graft VH gene and the corresponding amino acid sequence.
図 1 4は、 発現ぺクター p A L 34の特徵及び制限酵素地図を示す。 .  FIG. 14 shows the characteristics and restriction map of the expression vector pAL34. .
図 1 5は、 CDR—グラフト AN OC— 3 0 1発現べクタ一 p AC 7 9作製の 概略図である。  FIG. 15 is a schematic diagram showing the production of CDR-graft ANOC-301 expression vector pAC79.
図 1 6は、 CDRグラフ ト L鎖遺伝子構築のためのォリゴヌクレオチ ドの DN A配列を示す。  FIG. 16 shows the DNA sequence of an oligonucleotide for CDR-graft L chain gene construction.
図 1 7は、 CDRグラフ ト L鎖遺伝子の B s t B I -S p Γ I間の DNA配列 及び対応ァミノ酸配列を示す。  FIG. 17 shows the DNA sequence and the corresponding amino acid sequence between B st B I -S p ΓI of the CDR graph L chain gene.
図 1 8は、 CDRグラフ ト L鎖遺伝子構築のためのォリゴヌク レオチ ドの DN A配列を示す。 図 1 9は、 C D Rグラフ ト H鎖遺伝子構築のためのォリゴヌク レオチ ドの DN A配列を示す。 発明を実施するための最良の形態 FIG. 18 shows the DNA sequence of an oligonucleotide for CDR-graft L chain gene construction. FIG. 19 shows the DNA sequence of the oligonucleotide for construction of the CDR-graft H chain gene. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明組換え抗体の製造法につき詳述する。  Hereinafter, the method for producing the recombinant antibody of the present invention will be described in detail.
まずマウス抗ヒト I L一 1モノク D—ナル抗体産生細胞由来の抗惊を利用して、 該抗体の c DN Aライブラリーを構築し、 その H鎖 (重鎖, heavy - chain) 及び L鎖 (軽鎖, light- chain) の各可変部領域 (V領域) をコードする DN A配列 をそれぞれ再配列させた遺伝子をクローニングし、 プロモータ一領域及びェンハ ンサー領域などを考慮して、 これらをそれぞれヒ ト由来の抗体の定常部領域 (C 領域) をコードする D N A配列と結合させて組換え遺伝子 (キメラ遺伝子) を作 製するか、 又は上記マウス抗ヒト I L一 1モノクローナル抗体の H鎖及び L鎖を コードする DN A配列の V領域中の相補性決定領域 (C D R) を含むようにヒ ト 由来の抗体の V領域を修飾した DN A塩基配列を作製し、 かくして得られるマウ ス抗ヒ ト I L一 1抗体分子の C D R領域を含むヒ ト ·マウスグラフト V領域をコ ードする DNA塩基配列を、 ヒト由来の抗体の C領域をコードする DN A配列と 結合させて組換え遺伝子 (リシェイブト遺伝子) を作製する。  First, a cDNA library of a mouse anti-human IL-11 monoclonal D-null antibody-producing cell was used to construct a cDNA library of the antibody, and its H chain (heavy-chain) and L chain ( Clones were performed by rearranging the DNA sequence encoding the variable region (V region) of each variable region (light chain, light chain), and taking into account the promoter region and enhancer region. To create a recombinant gene (chimeric gene) by combining with a DNA sequence encoding the constant region (C region) of the antibody derived from the mouse, or the H and L chains of the mouse anti-human IL-11 monoclonal antibody described above. A DNA base sequence modified from the human antibody-derived V region to include the complementarity determining region (CDR) in the V region of the DNA sequence encoding the anti-human IL is obtained. 11 Including CDR region of antibody molecule The DNA nucleotide sequence preparative mouse graft V region code, be coupled with DN A sequence encoding the C region of antibody derived from a human to produce a recombinant gene (Risheibuto gene).
このようにして作製された H鎖及び L鎖のそれぞれのキメラ 7¾至リシユイブト 遺伝子を、 単独で又は 2種組合せて適当なベクターに組込んで、 各遺伝子の発現 ぺクタ一を作製し、 これらべクタ一を適当な細胞に導入して上記各遺伝子を発現 させることにより、 所望の本発明組換え抗体を得ることができる。  Each of the H-chain and L-chain chimeric 7¾-residue genes thus prepared is inserted alone or in combination of two types into an appropriate vector to prepare an expression vector for each gene. A desired recombinant antibody of the present invention can be obtained by introducing the vector into a suitable cell to express the above genes.
本発明に用いるマウス抗ヒ ト I L一 1モノクローナル抗体産生細胞は、 ヒ ト I L— 1を免疫原として、 或いは遺伝子組換え技術に従い製造される組換え型ヒ ト I L— 1を免疫原として、 通常のモノクローナル抗体の製造法に従い作製する ことができる。 該マウス抗ヒト I L一 1モノクローナル抗体産生細胞の具体例と しては、 例えば特開昭 6 3— 2 5 8 5 9 5号公報に記載の、 ヒト I L一 1 及び ヒ ト I L一 1 9のそれぞれに対する抗体産生ハイプリ ドーマを例示できる。 これ らのハイプリ ドーマはいずれも工業技術院生命工学工業技術研究所に寄託されて いる。 即ち上記ヒ ト I L一 1 αに対する抗体産生ハイブリ ドーマは、 KOCO 30 1 (FBRM BP-1554) として、 ヒト I L— 1 j9に対する抗体産生ハイ プリ ドーマは、 KOC02 0 3 (FBRM BP-1551) 、 KOCO 2 0 5 (FBRM BP- 1552) KOCO 20 6 (FBRM BP- 1553) として寄託されている。 The mouse anti-human IL-11 monoclonal antibody-producing cells used in the present invention are generally prepared using human IL-1 as an immunogen, or using recombinant human IL-1 produced by a gene recombination technique as an immunogen. It can be prepared according to the method for producing a monoclonal antibody described above. Specific examples of the mouse anti-human IL-11 monoclonal antibody-producing cells include, for example, those of human IL-11 and human IL-19 described in JP-A-63-258595. An antibody-producing hybridoma for each of them can be exemplified. All of these hybridomas have been deposited with the Institute of Biotechnology and Industrial Technology, the Agency for Industrial Science and Technology. That is, the above-mentioned antibody-producing hybridoma against human IL-11α is As KOCO 30 1 (FBRM BP-1554), antibody-producing hybridomas against human IL-1 j9 were KOC02 03 (FBRM BP-1551), KOCO 205 (FBRM BP-1552) KOCO 206 (FBRM BP -1553).
これらのハイプリ ドーマは、 以下の如き遺伝子調製を行なうことによって、 本 発明組換え抗体の製造に利用することができる。 即ち、 本発明抗体の V領域をコ ードする遺伝子断片としては、 上記のようなマウス抗ヒト I L一 1モノク σ—ナ ル抗体産生細胞より分雜され、 解析された遺伝子配列を用いることができる。 目 的のマウス抗ヒト I L一 1抗体に特異的な V領域をコードする遺伝子 (VL及び VH) の単錐及びクローニングは、 主として 2種の方法に従い実施することがで き 。 These hybridomas can be used for producing the recombinant antibody of the present invention by preparing the gene as described below. That is, as the gene fragment encoding the V region of the antibody of the present invention, a gene sequence that has been shared and analyzed from mouse anti-human IL-11 monoclonal σ-nal antibody producing cells as described above can be used. it can. Single cone and cloning of genes (V L and V H ) encoding a V region specific for the mouse anti-human IL-11 antibody of interest can be carried out mainly according to two methods.
その一つとしては、 抗体産生細胞の染色体 D N Aを適当な制限酵素により消化 させた後、 ショ糖密度勾配遠心法により分画し、 ファージぺクタ一アームにライ ゲーシヨンさせて、 イ ンビト口パッケージング法等に従って、 染色体ライブラリ 一から、 H鎖及び L鎖のそれぞれの 3' 末端側プローブを用いて、 サザンハイブ リダィゼーション法等を実施して、 所望抗体の V領域遺伝子をク o—二ングする 方法を挙げることができる。 この方法はより詳しくは、 例えばオイらの方法を参 照して実施することができる CVernon T. Di et al., Proc. Natl. Acad. Sci. U.S.A., 80, 825 (1983)参照〕 。  One of them is to digest the chromosomal DNA of antibody-producing cells with appropriate restriction enzymes, fractionate by sucrose density gradient centrifugation, ligate to one arm of phage vector, and perform in-vitro packaging. From the chromosome library 1, the H region and the L chain 3'-terminal probes are used to carry out Southern hybridization, etc., to clone the V region gene of the desired antibody. Methods can be mentioned. This method can be performed in more detail with reference to, for example, the method of Oy et al., See CVernon T. Di et al., Proc. Natl. Acad. Sci. U.S.A., 80, 825 (1983)].
もう一つの方法としては、 マウス抗ヒト I L一 1抗体産生細胞から常法に従い mRNAを抽出し、 cDNAライブラリーからスクリーニングする方法 〔M. S. Neuberger, EMBD J. , 2, 1373(1983)〕 を採用して、 上記 cDNAライブラリ一 から V領域遺伝子をクローニングするためのプローブを、 すでに報告されている マウス免疫グロプリン遺伝子の塩基配列 〔例えば、 B. B. Max et al., J. Biol. Chem. , 256, 5116(1981)] 等を参考にして、 マウスの免疫グ ブリン (I g) の H鎖と L鎖の C領域に相補的な 2つのスク リーニング用 D N Aプロ一ブを合成 し、 スク リーニングする方法を例示できる。  As another method, a method of extracting mRNA from mouse anti-human IL-11 antibody-producing cells according to a conventional method and screening from a cDNA library [MS Neuberger, EMBD J., 2, 1373 (1983)] was used. In addition, a probe for cloning the V region gene from the above cDNA library was used as a probe for the previously reported nucleotide sequence of the mouse immunoglobulin gene [for example, BB Max et al., J. Biol. Chem., 256, 5116 ( 1981)] and an example of a method of synthesizing and screening two screening DNA probes complementary to the H chain and the L chain C region of mouse immunoglobulin (Ig). it can.
該プローブは市販の自動オリゴヌクレオチド合成機により容易に合成でき、 さ らに T4ポリヌクレオチドキナーゼを用いて、 〔r— 32P〕 ATP等を用いて、 その 5' 末端を標識することができる。 この後者の方法におけるク ロ一ニングは、 抗体産生細胞よりポ リ (A) +RNAを得た後、 グブラーとホフマン 〔Gubler, U. & Hoffman, B. , The probe can be easily synthesized by a commercially available automatic oligonucleotide synthesizer, and its 5 ′ end can be labeled using [r- 32 P] ATP using T4 polynucleotide kinase. Cloning in this latter method involves obtaining poly (A) + RNA from antibody-producing cells and then using Gubler and Hoffman (Gubler, U. & Hoffman, B.,
Gene, 25, 263-268(1983)] の方法に従い、 1本鎖 cDNAを合成し、 さらに二 本鎖 cDN Aを合成した後、 これをプラスミ ドベクターやファージベクター等に 挿入し、 コンビテントな状態にある大腸菌、 例えば HB 1 0 1株、 LE 39 2株、 NM 539株、 Y1 0 9 0株などを宿主として、 形質転換させることにより実施 でき、 かくして所望の cDNAライブラリーを構築することができる。 Gene, 25, 263-268 (1983)], synthesize single-stranded cDNA, synthesize double-stranded cDNA, insert this into a plasmid vector or phage vector, etc. Escherichia coli, for example, HB101 strain, LE392 strain, NM539 strain, Y109 strain strain, etc. can be used as a host for transformation, and thus a desired cDNA library can be constructed. it can.
上記プラスミ ドベクターを用いる場合は、 例えば p SP 64 (国際公開 WO 8 9 / 0 1 9 7 4号公報参照) 等を有利に使用することができ、 得られる c DNAライブラリーから合成したプローブを用いて、 ハナハンら 〔Hanahan, R. A. et al, Gene, 10, 63(1980)] の方法によるコロニー ·ハイブリダィゼー シヨンにより、 目的の VL遺伝子及び VH遺伝子のそれぞれを含む陽性クローンを ク D—二ングすることができる。 When the above-described plasmid vector is used, for example, pSP64 (see International Publication WO89 / 019974) can be advantageously used, and a probe synthesized from the obtained cDNA library can be used. Positive clones containing each of the desired VL and VH genes were identified by colony hybridization by the method of Hanahan et al. [Hanahan, RA et al, Gene, 10, 63 (1980)]. Can be
またファージベクターを用いる場合、 該ベクターとしては、 例えばス g t 1 0、 g t 1 1などを利用して、 ィンビト σパッケージング法等により cDNAライ ブラリーを構築し、 この cDNAライブラリーから合成したプローブを用いて、 ペントンとデービス 〔Benton, W. and Davis, R. , Science, 196, 383-394(1977) 〕 らの方法によるプラーク ·ハイブリダィゼーシヨンにより、 目的の VL遺伝子及 び V H遺伝子のそれぞれを含む陽性クローンをクローニングすることができる。 When a phage vector is used, a cDNA library is constructed by, for example, gt10, gt11, or the like by the invito σ packaging method, and a probe synthesized from this cDNA library is used. And the target VL and VH genes by plaque hybridization by the method of Penton and Davis [Benton, W. and Davis, R., Science, 196, 383-394 (1977)]. Can be cloned.
更に、 上記クローユングの別法としては、 例えばポリ (A) +RNAから得ら れた一本鎖 cDNAより、 目的とする V領域を含む領域の DNAを PCR 〔ポリ メラ一" ε ·チェイン · リアクション; olymerase chain reaction, Rosana, Q. et al., Pro Natl. Acad. Sci. U.S.A., 86, 3833(1989)〕 により増幅し、 増 幅した DN A断片をベクターに組込む方法を採用することもでき、 これにより所 望の V L遺伝子及び V H遺伝子のク ϋ一ニングが可能である。 またプローブとハイ ブリダィズしたク σ—ンについて、 ベクタ一側ブライマ一を用いて P C Rにより、 その DNAの大きさを確認することができる。 更に、 上記増幅した V領域遺伝 子 ( !^遺伝子及び !!遺伝子) を I g発現ベクターに組込むことによって、 本発 明の組換え抗体を発現させることもできる。 尚、 上記でクローニングした VL遺伝子及び VH遺伝子のクローンは、 ァガロー スゲル電気泳動により単雜♦精製することができる。 Further, as another method of the above-mentioned clawing, for example, from a single-stranded cDNA obtained from poly (A) + RNA, the DNA of the region containing the V region of interest is subjected to PCR [Polymerase ε-chain reaction] Olymerase chain reaction, Rosana, Q. et al., Pro Natl. Acad. Sci. USA, 86, 3833 (1989)], and a method in which a DNA fragment amplified and amplified is incorporated into a vector. Thus, the desired VL gene and VH gene can be purified, and the size of the DNA of the clone hybridized with the probe can be determined by PCR using the vector-side primer. Furthermore, the recombinant antibody of the present invention can be expressed by incorporating the amplified V region genes (! ^ Gene and !! gene) into an Ig expression vector. Note that clone V L gene and V H genes were cloned above can be single雜♦ purified by Agaro Sugeru electrophoresis.
上記で得られた VL遺伝子及び VH遺伝子のそれぞれのク口一二ングベクタ一へ の導入は、 一般的方法に従って、 適当なベクター例えば pUC 1 8、 P B R 322など及び適当な制限酵素、 例えば E c 0 R Iなどを用いて実施できる。 得 られる組換え体プラスミ ドの作製後、 サンガーら 〔Sanger, F. et al., Pro Natl. Acad. Sci. U.S.A., 74, 5463(1977)] のデォキシ♦チヱ一ン .タ一ミネ ーション法などを利用して、 目的とする V L及び V H遺伝子の c D N A領域の塩基 配列を決定することができる。 Introduction into each click slot twelve Ngubekuta one V L gene and V H genes obtained above, according to the general method, a suitable vector e.g. pUC 1 8, such as PBR 322 and a suitable restriction enzyme, for example E This can be performed using c 0 RI or the like. After the production of the resulting recombinant plasmid, the method of Sanger et al. [Sanger, F. et al., Pro Natl. Acad. Sci. USA, 74, 5463 (1977)] was used. By using such methods, the nucleotide sequence of the cDNA region of the target VL and VH genes can be determined.
上記のごとくして得られる本発明組換え抗体作製用のマウス抗ヒト I L一 1抗 体の VL遺伝子及び VH遺伝子の各塩基配列は、 配列番号: 1及び配列番号: 2と して示した通りである。 またこれら各遺伝子を含むプラスミドの具体例としては、 それぞれ後述する実施例に示すプラスミ ド p I L 0 68 (マウス抗ヒト I L— 1 抗体の H鎖遺伝子を含む) 及びプラスミ ド p I L 086 (マウス抗ヒト I L_ 1 抗体の L鎖遺伝子を含む) を例示できる。 一 The nucleotide sequences of the VL gene and VH gene of the mouse anti-human IL-11 antibody for producing the recombinant antibody of the present invention obtained as described above are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. As expected. Specific examples of plasmids containing these genes include plasmid pIL068 (including the H chain gene of mouse anti-human IL-1 antibody) and plasmid pIL086 (mouse (Including the L chain gene of the human IL_1 antibody). one
—方、 本発明組換え抗体の L鎖 C領域 (CL) 及び H鎖 C領域 (CH) としては ヒト抗体分子由来の各種のもの、 例えば一般的なヒト免疫グロプリン由来のも のを利用できる。 該ヒト免疫グロプリンの C領域遺伝子、 即ち CL遺伝子及び CH 遺伝子は、 例えば RE I産生細胞株 〔human myeloma protein : BPP, 0. et al. , Bur. J. Biochem., 45, 513-524(1974)] のようなヒト抗体産生細胞から、 前記 と同様の方法により単離することができ、 その際、 目的遺伝子のクローニングの ためのプローブとしては、 すでに報告されているヒト免疫グロブリ ンの核酸塩基 配列 [Ellison, J. W. et al., Nuc. Acids Res., 10, 4071 (1982)] : Heiter, P. A. et al., Cell, 22, 197(1980)等〕 を参照して合成した DN Aプローブ等を利 用することが可能である。 - How, as the L chain C region of the present invention recombinant antibodies (C L) and H chain C region (C H) utilizing any of various derived from a human antibody molecule, for example, the derived common human immune globulin also the it can. C region genes of the human immune globulin, i.e. C L gene and C H gene, for example, RE I production cell lines [human myeloma protein:... BPP , 0. et al, Bur J. Biochem, 45, 513-524 (1974)] can be isolated by the same method as described above. In this case, as a probe for cloning the target gene, human immunoglobulin which has already been reported can be used. DNA synthesized with reference to the nucleic acid base sequence [Ellison, JW et al., Nuc. Acids Res., 10, 4071 (1982)]: Heiter, PA et al., Cell, 22, 197 (1980), etc. Probes and the like can be used.
また上記ヒト C領域遺伝子としては、 既に各種のものが知られており、 各種 © C領域遺伝子を有するプラスミ ドベクターが既に確立されている。 従って、 本発 明では該遺伝子を特に上記のように抗体産生細胞より単雜して調製せずとも、 こ れらベクターの保有するものを利用するのが簡便であり有利である。 かかる公知 の C領域遺伝子には、 例えば H鎖の場合は、 代表的には r ,〜r 4鎖などの遺伝子 が、 またその他に; "鎖、 鎖の各鎖の遺伝子が含まれ、 L鎖の場合は、 Λ:鎖、 ( 鎖などの遺伝子が包含され、 これらはいずれも本発明に利用できる。 上記ヒト免 疫グ σプリ ンの C領域及びェンハンサーを舍有するプラスミ ドベクターの具体例 としては、 例えばヒ ト I gG4の Η鎖 C領域を有する ρΕ 1 0 0 4 CP EE 6 (P. Stephens and . Cockett, Nucl. Acids Res., 17, 7110(1989) : Prot. Eng. , 1(6) 499-505 (1987)に基づいて作製されてもの〕 、 ヒ ト I 01の11鎖〇領域を 有する P E l 0 0 1 (上記 pE l 0 0 4と同様にして p EE 6に基づいて作製さ れたもの〕 、 ヒ ト I g /cの L鎖 C領域を有する p E 1 0 8 1 〔上記と同様に EE 6に基づいて作製されたもの〕 等を例示できる。 更に p S V 2厶 Hg p t, p S V 1 84 ΔΗη e o 〔0i, V. T. and Morrison S. し, Biotechnique, 4 (3) , 214-221(1980)] 等も上記ヒト免疫グロプリンの C領域遺伝子を有するプラス ミドベクターに包含される。 Various human C region genes are already known, and plasmid vectors having various human C region genes have already been established. Therefore, in the present invention, it is convenient and advantageous to use the ones possessed by these vectors, even if the genes are not individually prepared from the antibody-producing cells as described above. Such known The C region gene, for example, in the case of the H chain, typically r, genes such as ~r 4 chains, or other in; "chain, contains genes for each strand of the chain, when the L-chain Examples of the plasmid include a 有 す る: chain and a (chain) gene, all of which can be used in the present invention. Specific examples of the plasmid vector having the C region of human immunization group σ-prin and an enhancer include: For example, ρΕ 104 CP EE 6 having a human IgG 4 Η chain C region (P. Stephens and. Cockett, Nucl. Acids Res., 17, 7110 (1989): Prot. Eng., 1 (6 ) 499-505 (1987) to those are fabricated on the basis], based on the PE l 0 0 1 (p EE 6 by the same way as the pE l 0 0 4 with 11 chain 〇 region of human I 0 1 Produced, and pE1081 having an L chain C region of human Ig / c [produced based on EE6 in the same manner as described above]. Hg pt, p SV 1 84 ΔΗη eo [0i, VT and Morrison S. et al., Biotechnique, 4 (3), 214-221 (1980)] and the like are also included in the above plasmid vector having the human immunoglobulin C region gene.
本発朋のヒト I L— 1に対するキメラ抗体の作製のための遺伝子は、 上記した 各種の遺伝子断片、 即ちマウス由来の V L遺伝子及び V H遺伝子から選ばれる遺伝 子と、 ヒ ト由来の CL遺伝子及び CH遺伝子から選ばれる遺伝子とを、 それぞれ結 合させることにより構築される。 Gene for the production of chimeric antibody against human IL- 1 of the present HatsuTomo includes a gene selected from the VL gene and VH genes of the above-mentioned various gene fragments, i.e. mice, C L gene from human and a gene selected from C H gene, is constructed by bringing together each binding.
上記マウス由来の VL遺伝子と七ト由来の 1_遺伝子とを結合させて得られる本 発明の組換えヒト. I丄一 1抗体 L鎖発現ベクターの構築の概略は、 例えば図 6に 示す通りであり、 かくして得られる発現ベクターの具体例としては p AC 5 3を 例示できる。 これは、 p I L 0 8 6の B s t B I及び S p 1 I切断断片をヒ ト I g A:の L鎖 C領域をコードする DNA配列を持つプラスミ ド pE 1 0 8 1の同 制限酵素切断断片に挿入して構築されたものであり、 その特徴は図 8に示す通りThe recombinant human of the present invention obtained by binding the mouse-derived VL gene and the 7-derived 1_ gene. An outline of the construction of an I-11 antibody L chain expression vector is, for example, as shown in FIG. And a specific example of the expression vector thus obtained is pAC53. This is obtained by digesting the BstBI and Sp1I digested fragments of pIL086 with the same restriction enzyme as the plasmid pE1081, which has a DNA sequence encoding the light chain C region of human IgA: It was constructed by inserting it into a fragment.
^める o ^ O
また、 上記マウス由来の VH遺伝子とヒト由来の CH遺伝子とを結合させて得ら れる本発明の組換えヒト I L一 1抗体 H鎖発現ベクターの構築の概略は、 例えば 図 3に示す通りであり、 かくして得られる発現ベクターの具体例としては p AC 5 2及び p AC 7 7を例示できる。 これらは、 p I L 0 6 8の H i τι dl [及び Ap a I切断断片を、 ヒト I gG4の H鎖 C領域をコードする DNA配列を持つ プラスミ ド pE 1004及びヒト I g G!0H鎖 C領域をコードする DNA配列 を持つプラスミ ド pE 1001の同制限酵素切断断片にそれぞれ挿入して構築さ れたものであり、 PAC 52の特徴は図 5に示す通りである。 In addition, the outline of the construction of the recombinant human IL-11 antibody H chain expression vector of the present invention obtained by binding the mouse-derived VH gene and the human-derived CH gene is, for example, as shown in FIG. Specific examples of the expression vector thus obtained include pAC52 and pAC77. These and H i τι dl [and Ap a I cleavage fragment of p IL 0 6 8, having a DNA sequence encoding the H chain C region of a human I gG 4 PAC 52 is constructed by inserting it into the same restriction enzyme digested fragment of plasmid pE1001 which has a DNA sequence coding for plasmid pE1004 and human IgG G! 0H chain C region. As shown in FIG.
また、 本発明のヒト I L一 1に対する抗体には、 上記キメラ抗体の他に、 リシ ヱイブト抗体、 即ち L鎖及び H鎖の少なくとも一方がマウス抗ヒト. I L一 1抗体 分子の CDR領域を舍むヒト ·マウスグラフト V領域を有する抗体が包含される。 かかる本発明リシ Xイブト抗体の作製のための遺伝子 (グラフトされたリシエイ ブト遺伝子、 特にグラフト VL遺伝子及びグラフト VH遺伝子) としては、 例えば 上記のごとくして得られるマウス由来の VL遺伝子又は VH遺伝子の DNA配列中 の CDR領域の DNA配列を参照して、 この配列部分を例えばヒト抗体分子の射 応する V L遺伝子又は V H遺伝子と置換させた D N A配列を有するものを利用する。 かかるグラフ ト遺伝子は、 上記設計された DNA配列を参照して、 その全遺伝子 を自動オリゴヌクレオチド合成機により化学合成することもでき、 また上記設計 された DN A配列の一部は、 前述した各種遺伝子を利用して、 PCR法に従って 作製、 増幅することもできる。 In addition, the antibody against human IL-11 of the present invention includes, in addition to the chimeric antibody described above, a recombinant antibody, that is, at least one of the L chain and the H chain covers a CDR region of a mouse anti-human IL-11 antibody molecule. An antibody having a human-mouse graft V region is included. Examples of the gene (grafted receive gene, in particular, the graft VL gene and the graft VH gene) for producing the ricin X antibody of the present invention include, for example, the mouse-derived VL gene or the VL gene obtained as described above. with reference to the DNA sequence of the CDR regions of the DNA sequence of the V H genes, utilizing the sequence portions such as those having the DNA sequence was replaced with VL gene or VH genes that respond morphism human antibody molecule. Such a gene can be chemically synthesized by an automatic oligonucleotide synthesizer with reference to the designed DNA sequence, and a part of the designed DNA sequence can be obtained by various methods described above. It can also be produced and amplified according to the PCR method using genes.
上記グラフト遺伝子の具体例としては、 配列番号: 3に示すダラフト VL遺伝 子及び配列番号: 4に示すグラフト VH遺伝子を例示できる。 Specific examples of the above-mentioned graft gene include the darafft VL gene shown in SEQ ID NO: 3 and the graft VH gene shown in SEQ ID NO: 4.
グラフ ト VL遺伝子とヒト由来の CL遺伝子とを結合させて構築されるリシユイ ブト抗体 L鎖の発現ベクターの構築の概略は図 9に示す通りであり、 かくして得 られる発現ベクターの具体例としては PAL 31を例示できる。 これは、 後記実 施例に示した 2段階法により得られる。 即ち、 配列番号: 3のグラフ ト VL遺伝 子の一部を含む DN A配列 (CDR 1〜CDR 3領域を除く B s t B I及び Sp 1 I切断断片, PCRにより増幅したもの) を、 プラスミ ド pE 1081の 同制限酵素切断断片に挿入し (ステップ 1) 、 次いで得られるプラスミ ドの上記 B s t B I -S p 1 I間に存在する B s tEH-Kpn I間に、 別途合成した CDR 1〜CDR 3領域の DNA配列 (B s tEII及び Κριι I切断断片とした もの) を挿入 (ステップ 2) して構築されたものであり、 その特徴は図 11に示 す通りである。 Schematic construction of expression vectors of the graph preparative V L gene and Rishiyui but-antibody L chain is constructed by coupling the C L gene from human is as shown in FIG. 9, thus as a specific example of the expression vector obtained Can exemplify PAL 31. This can be obtained by the two-stage method shown in the examples below. That is, a DNA sequence containing a part of the graft VL gene of SEQ ID NO: 3 (BstBI and Sp1I cleavage fragments excluding the CDR1 to CDR3 regions, amplified by PCR) was added to plasmid. The fragment was inserted into the same restriction enzyme-cleaved fragment of pE1081 (step 1), and the CDR1 to BstEH-KpnI present between the BstBI and Sp1I of the obtained plasmid were separately synthesized. It was constructed by inserting (step 2) the DNA sequence of the CDR3 region (BstEII and ΚριιI cleavage fragment), and the features are as shown in FIG.
またグラフド VH遺伝子とヒト由来の CH遺伝子とを結合させて構築されるリシ イブト抗体 H鎖の発現ベクターの構築の概略は図 1 2に示す通りであり、 かく して得られる発現ベクターには、 具体的には PAL 34が包含される。 これは、 後記実施例に示した方法により得られる配列番号: 4のグラフト VH遺伝子を舍 む DN A配列 Hi II dm及び Ap a I切断断片 (P C Rにより増幅したもの) を、 プラスミド pE 1 004の同制限酵素切断断片に挿入して構築され ものであり、 その特徴は図 1 4に示す通りである。 The Rishi constructed by coupling the C H genes from Gurafudo V H gene and human The outline of the construction of the expression vector for the Ito antibody H chain is as shown in FIG. 12, and the expression vector thus obtained specifically includes PAL34. This was accomplished by combining the DNA sequence HiII dm containing the graft VH gene of SEQ ID NO: 4 obtained by the method described in the Examples below and the ApaI digestion fragment (amplified by PCR) with the plasmid pE1004. It is constructed by inserting it into the same restriction enzyme cleavage fragment, and its characteristics are as shown in FIG.
尚、 上記各発現ベクターの構築の際には、 一般に得られる各遺伝子の単離法に 応じて、 主として次の 2つの結合の組合せ、 即ち、 染色体 DN Aから単離した V 領域と C領域遺伝子の組合せ及び c D N Aから単離した V領域と C領域の組合せ に考慮をはらう必要がある。 例えば、 マウス染色体 DNAから単離した V領域遺 伝子をヒト染色体 DNAから単離した C遺伝子と結合させる場合は、 マウス V領 域遺伝子には発現に必要なプロモーターゃェンハンサ一等の発現調節領域を予め 含ませておくのが好ましい。 但し、 プ πモーターゃェンハンサ一等はマウス由来 である必要がなくヒト由来であってもかまわない。 上記プロモーターは V領域の 5' 上流域に位置し、 ェンハンサ一は V領域遺伝子と C領域遺伝子の間に位置す るのが好ましいが、 ェンハンサーについては必ずしもこの位置に限定されるもの ではない。 他方、 マウス c DN Aから単錐した V領域遺伝子をヒト cDNAから 単雜した C領域遺伝子と結合させる場合、 その結合部分は適当な制限酵素サイ ト や、 必要であれば適当なリンカ一を用いて、 V領域遺伝子のコードしているアミ ノ酸配列と C領域遺伝子のコ一ドしているァミノ酸配列がずれないように、 また V領域ァミノ酸配列と C領域了ミノ酸配列が変化しないように結合しなければな らず、 さらに勖物細胞中で発現を可能にするために適当なプロモータ一やェンハ ンサ一等の発現調節領域を遺伝子の 5' 上流域に付加してやる必要がある。 これ らの各操作は、 いずれも常法に従い実施することができ、 必要に応じて利用され る発現調節領域、 制限酵素サイ ト作製のためのリンカ一等は公知のそれらを参照 して適宜決定することができる。  When constructing each of the above expression vectors, the following two combinations are mainly used, ie, the V region and C region genes isolated from the chromosomal DNA, depending on the generally obtained method for isolating each gene. It is necessary to take into consideration the combination of V region and the combination of V region and C region isolated from cDNA. For example, when a V region gene isolated from mouse chromosomal DNA is linked to a C gene isolated from human chromosomal DNA, the mouse V region gene must have an expression regulatory region such as the promoter Jenhansa required for expression. It is preferable to include in advance. However, it is not necessary that the pi motor Jenhansa etc. be derived from a mouse, and may be derived from a human. The promoter is located 5 ′ upstream of the V region, and the enhancer is preferably located between the V region gene and the C region gene, but the enhancer is not necessarily limited to this position. On the other hand, when a V region gene single-cone of mouse cDNA is ligated to a C region gene multiplexed from human cDNA, the binding site is determined using an appropriate restriction enzyme site and, if necessary, an appropriate linker. The amino acid sequence encoded by the V region gene and the amino acid sequence encoded by the C region gene do not shift, and the V region amino acid sequence and the C region amino acid sequence do not change. In order to enable expression in animal cells, it is necessary to add an appropriate expression regulatory region such as a promoter or enhancer to the 5 ′ upstream region of the gene. Each of these operations can be carried out according to a conventional method, and the expression control region to be used as necessary, a linker for preparing a restriction enzyme site, and the like are appropriately determined with reference to known ones. can do.
このようにして作製された本発明のキメラ抗体遺伝子乃至リシ イブト抗体遺 伝子を、 例えば上記べクタ一プラスミ ド PEE 6、 P E 1 0 0 1、 pE 1 0 04、 pE 1 08 1等や p S V 2 g p t 〔R. C. Mullgan et al., Pro. NAS. U.S.A., 78, 2027 (1981)] 、 p S V 2 -τι e o CP. J. Southern et al., J. ol. APPI. Genet. , 1, 327(1982)3 等の選択マーカーの付いた適当なベクタープラスミ ド或 いは宿主細胞内でプラスミ :'ド状態で増殖できるウィルス遺伝子の一部 (パピロー マウィルスなど) を持ったベクタープラスミ ドに H鎖遺伝子と L鎖遺伝子を別々 に、 あるいは同時に組み込むことにより、 所望のキメラ抗体 L鎖発現プラスミ ド、 キメラ抗体 H鎖発現プラスミ ド、 リシ イブト抗体 L鎖発現ブラスミ ド及びリ シ ユイブト抗体 H鎖発現プラスミ ドを構築でき、 また上記 L鎖と H鎖との組合せで ある本発明組換え抗体発現プラスミ ドを構築することができる。 The thus prepared chimeric antibody gene or receive antibody gene of the present invention can be used, for example, with the vector plasmids PEE6, PE101, pE104, pE1081, etc. SV 2 gpt [RC Mullgan et al., Pro. NAS. USA, 78, 2027 (1981)], pSV2-τιeo CP. J. Southern et al., J. ol. APPI. Genet., 1, 327 (1982) 3, etc. Plasmids in plasmids or host cells : By integrating the H-chain gene and L-chain gene separately or simultaneously into a vector plasmid containing a part of a viral gene (such as papilloma virus) Desired chimeric antibody L chain expression plasmid, chimeric antibody H chain expression plasmid, received antibody L chain expression plasmid and recruited antibody H chain expression plasmid can be constructed, and a combination of the above L chain and H chain Thus, the recombinant antibody expression plasmid of the present invention can be constructed.
殊に、 上記 H鎖遺伝子と L鎖遺伝子とを別々に組込んだプラスミ ドは、 その内 の L鎖発現プラスミドと H鎖発現プラスミ ドとを用いて宿主細胞を形質転換させ ることにより、 本発明の所望抗体発現形質転換体とすることができる。 また上記 H鎖遺伝子と L鎖遺伝子と同時に組込んだプラスミ ドは、 これを単独で用いて宿 主細胞を形質転換させることによって、 所望の本発明抗体発現形質転換体を収得 できる。  In particular, the plasmid into which the H chain gene and the L chain gene have been separately incorporated is transformed into a host cell using the L chain expression plasmid and the H chain expression plasmid. The desired antibody-expressing transformant of the present invention can be obtained. In addition, the plasmid integrated simultaneously with the H chain gene and the L chain gene can be used alone to transform host cells to obtain a desired transformant expressing the antibody of the present invention.
上記本発明抗体発現形質転換体の作製のための H鎖遺伝子と L鎖遺伝子と同時 に組込んだプラスミ ドの具体例としては、 例えば後記実施例に詳述する方法によ り得られる P AC 7 9を例示できる。 これはより詳しくは図 1 5に示すように、 —方で前記プラスミ ド PAL 34より、 ヒト CMVプロモーターを舍む H鎖 V領 域及び C領域にポリ Aを含む遺伝子を単雜し、 他方で pE l 0 04の SV4 0Z g p t遺伝子断片を単雜し、 之等を前記プラスミ ド pAL 3 1に適当に挿入する ことにより得られ、 かくして得られるプラスミ ドは目的の H鎖遺伝子及び L鎖遺 伝子と共に選択マーカ一を保有しており、 本発明組換え抗体の発現べクタ一とし て有用である。 かくして得られるプラスミド pAC 7 9の特徴を図 1 5に併記す る。  Specific examples of the plasmid which is simultaneously incorporated with the H-chain gene and the L-chain gene for producing the above-mentioned antibody-expressing transformant of the present invention include, for example, PAC obtained by a method described in detail in Examples below. 7 9 can be exemplified. More specifically, as shown in FIG. 15, on the other hand, from the plasmid PAL34, a gene containing polyA in the H chain V region and the C region, which contains the human CMV promoter, is isolated. The SV40Zgpt gene fragment of pEl004 is ligated and inserted into the plasmid pAL31 appropriately, and the plasmid thus obtained is the target H chain gene and L chain gene. It has a selection marker together with its offspring and is useful as an expression vector for the recombinant antibody of the present invention. The characteristics of the thus obtained plasmid pAC79 are also shown in FIG.
本発明の抗ヒト I L一 1組換え抗体の製造は、 上記のようにして 製された抗 体遺伝子又はその H鎖もしくは L鎖遺伝子のそれぞれを含むプ.ラスミ ドを用いて、 宿主動物細胞を形質転換し、 この形質転換体を培養することにより実施できる。 この形質転換に使用される宿主動物細胞としては、 COS細胞 〔Nu Acids Res., 12, 5707-5717(1988) ; ATCC CCL 7 0 ; CV— 1細胞を S V 4 0ゥィ ルスで形質転換した細胞株〕 、 CHO細胞 [: Chinese hamster ovary cell; ATCC CCL 6 1 : CHO -K K WO 89/0 1 7 83号公報〕 、 : BHK 細胞 〔baby hamster kidney cell; AT CC CCL 1 0 : BHK- 2 1 (C- 1 3 ) 、 WO 8 9 / 0 1 7 8 3号公報〕 や B リ ンパ系細胞铢、 例えば P 3X 63 Ag 8 · 6 5 3 (ATCC CRL 1 580 ) . P 3 X 63 A g 8 U · 1 (ATCC CRL 1 5 9 7 ) . P 3/N S 1 /1 -Ag 4 - 1 (ATCC CRL 1 8) 等の形質転換細胞腫 [: Riechmann, L. et al., Nature, 332, 323 (1988) :特開平 2— 2 3 5 2号公報等参照〕 やハイプリ ドーマを例示 できる。 The production of the anti-human IL-11 recombinant antibody of the present invention comprises preparing a host animal cell using a plasmid containing the antibody gene or the H chain or L chain gene produced as described above. It can be carried out by transforming and culturing the transformant. The host animal cells used for this transformation include COS cells [Nu Acids Res., 12, 5707-5717 (1988); ATCC CCL70; CV-1 cells; Russ-transformed cell line], CHO cells [: Chinese hamster ovary cell; ATCC CCL61: CHO-KK WO 89/017883],: BHK cells [baby hamster kidney cell; ATCC CCL1] 0: BHK-21 (C- 13), WO 89/01783] and B-lymphoid cells, for example, P3X63 Ag8.653 (ATCC CRL 1580). Transformed cell tumor such as P3X63Ag8U · 1 (ATCC CRL 1597). P3 / NS1 / 1 / Ag4-1 (ATCC CRL18) [: Riechmann, L. et al., Nature, 332, 323 (1988): See Japanese Patent Application Laid-Open No. 2-3522] and hybridomas.
DN Aによる細胞の形質転換方法としては、 DE AE—デキス トラン法 CNeuberger, . S., B BD J., 2, 1317 (1983)〕 、 リン酸カルシウム共沈降法 CChen, C. and Okayama H., ol. Cell. Biol., 7, 2745-2752(1987)3 、 プ口 トプラスト融合法 〔0i, V. T. and Morrison, S. し, Bio Techniques, 4(3), 214-221(1986)〕 、 エレク ト口ポレーシヨン法 〔Potter. H. et al., Proc.  Methods of transforming cells with DNA include DEAE-dextran method CNeuberger, S., BBD J., 2, 1317 (1983)], calcium phosphate coprecipitation method CChen, C. and Okayama H., ol. Cell. Biol., 7, 2745-2752 (1987) 3, Puguchi toplast fusion method [0i, VT and Morrison, S., Bio Techniques, 4 (3), 214-221 (1986)], Elect. Mouth poration method [Potter. H. et al., Proc.
Natl. Acad. Sci., U.S.A., 81, 7161-7163(1984)] 等の方法があり、 いずれの 方法でもよい。 H鎖と L鎖のキメラ抗体遺伝子を同時に持つプラスミ ドで形質転 換を行なう場合には、 選択マーカーは一種類でよいが、 H鎖及び L鎖を別々に形 質転換させる場合には、 2種類のマーカーが必要である。 この場合においては、 まず一つのプラスミ ドで形質転換を行なった後に、 さらに他のプラスミ ドで形質 転換を行なう二重形質転換法を用いるのが好ましい。 Natl. Acad. Sci., U.S.A., 81, 7161-7163 (1984)], and any of these methods may be used. When performing transformation with a plasmid having both the H-chain and L-chain chimeric antibody genes at the same time, only one type of selectable marker may be used, but when transforming the H-chain and L-chain separately, 2 Different types of markers are needed. In this case, it is preferable to use a double transformation method in which transformation is first performed with one plasmid, followed by transformation with another plasmid.
上記のようにして得られた形質転換細胞の培養、 増殖は、 通常の動物細胞ゃハ イブリ ドーマを培養すると同じ適当な条件下で、 適当な培地、 常用の標準的な培 地、 例えばダルベッコ改良イーグル培地 (DMEM) 又は RPMI 1 64 0培地 中で行なわれ、 これは必要に応じ哺乳類の血清、 例えばゥシ胎児血清、 又は微量 要素及び増殖維持捕捉要素などを補充することもできる。 また、 抗体産生細胞が 選択マーカーを保有している場合は、 該培地は上記選択培地、 例えば G4 1 8又 はキサンチン、 ヒポキサンチン、 チミジン及びミコフヱノ一ル酸を含有する培地 で補充されるのが好ましい。 上記のようにして細胞から通常のハイプリ ドーマの 産生する抗体を同様にして、 本発明の抗ヒト I L一 1組換え抗体を分泌産生させ ることができる。 これら組織培養条件下での哺乳類の細胞培地のための技術は、 当業界において自明である。 The transformed cells obtained as described above can be cultured and grown under the same appropriate conditions as those used for culturing hybridomas, such as normal animal cells, in a suitable medium, a standard culture medium commonly used, for example, Dulbecco's Modification. It is performed in Eagle's medium (DMEM) or RPMI 1640 medium, which can be supplemented with mammalian serum, if necessary, such as fetal calf serum, or trace elements and growth-sustaining capture elements. When the antibody-producing cell has a selection marker, the medium is supplemented with the above-mentioned selection medium, for example, G418 or a medium containing xanthine, hypoxanthine, thymidine, and mycophenolic acid. preferable. In the same manner as described above for the antibody produced by a normal hybridoma from cells, the anti-human IL-11 recombinant antibody of the present invention is secreted and produced. Can be Techniques for mammalian cell culture under these tissue culture conditions are obvious in the art.
かくして得られる細胞培養物の上清は、 ェンザィムィムノ了ッセィ (E I A) 、 ドッ ト一アツセィ、 F I T Cなどを用いる蛍光標識抗体法又はラジオィムノアツ セィ (R I A) 、 ラテックス凝集反応、 血球凝集反応など、 通常使用されるスク リーニング法を用いて、 所望する抗ヒト I L一 1組換えモノクローナル抗体につ いてスクリーニングすることができる。  The supernatant of the cell culture thus obtained can be used in general, such as the fluorescence-labeled antibody method using Enzyme Immunoassay (EIA), Dot-At-Assy, FITC, or Radioimmunoassay (RIA), latex agglutination, hemagglutination, etc. A desired anti-human IL-11 recombinant monoclonal antibody can be screened using a suitable screening method.
かくして得られる所望の抗ヒト I L—1組換えモノク ϋーナル抗体を産生する 細胞は、 上記した通常の培地で継代培養でき、 また、 液体窒素中で長期間保存可 能である。  The cells producing the desired anti-human IL-1 recombinant monoclonal antibody thus obtained can be subcultured in the above-mentioned ordinary medium, and can be stored for a long time in liquid nitrogen.
該抗体産生細胞からの本発明抗ヒト I L一 1組換えモノクローナル抗体の採取 は、 該細胞を常法に従って培養し、 その培養上清として、 或いは該細胞をこれと 適合性のある哺乳動物の腹腔内に投与して増殖させ、 その腹水として得る方法等 を採用できる。  The anti-human IL-11 recombinant monoclonal antibody of the present invention can be collected from the antibody-producing cells by culturing the cells according to a conventional method, and using the culture supernatant or the cells as a peritoneal cavity of a mammal compatible therewith. Can be used to obtain the ascites from the cells.
該組換え抗体の単雜には、 培養上清中の免疫グロプリンを、 例えば硫酸了ンモ ニゥムによる沈澱、 P E Gのような吸湿性物質に対する透析、 選択膜を通す濾過 等により、 濃縮する。 また、 必要に応じ、 又は所望により濃縮された該抗体を通 常のクロマトグラフィー法、 例えばゲル濾過、 イオン交換クロマトグラフィー、 D E A E—セルロース上でのクロマトグラフィ一又はプ αティン Αを用いるカラ ムクロマトグラフィ一等の通常の精製手段により精製することができる。  The immunoglobulin in the culture supernatant is concentrated by, for example, precipitation with ammonium sulfate, dialysis against a hygroscopic substance such as PEG, or filtration through a selective membrane. If necessary or desired, the antibody is concentrated by a conventional chromatography method such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose or column chromatography using cellulose. And the like.
かくして得られる本発明抗体は、 これを利用して、 例えば免疫沈降法、 ァフィ 二ティクロマトグラフィー等の通常の精製手段により、 ヒト I L一 1を簡便且つ 特異的に精製することが可能である。  The antibody of the present invention thus obtained can be used to purify human IL-11 simply and specifically by a conventional purification means such as immunoprecipitation or affinity chromatography.
上記の如くして得られる本発明抗体の利用によれば、 検体中のヒト I L一 1を 免疫反応により、 特異的に測定することができる。 該方法としては、 通常の競合 法、 サンドィツチ法によるラジオィムノ了ッセィ法 (R I A) 、 酵素免疫測定法 According to the use of the antibody of the present invention obtained as described above, human IL-11 in a sample can be specifically measured by an immune reaction. Examples of the method include a conventional competition method, a radioimmunoassay method (RIA) by a sandwich method, an enzyme immunoassay method.
(E L I S A) 、 凝集法等の通常免疫学的手法が挙げられ、 これらの方法の提作、 手順等は、 常法と変わるところはない。 より具体的には、 例えば競合法を採用す る場合、 測定しょうとする検体中のヒト I L一 1と一定量の不溶化されたヒ ト I L一 1とを、 標識剤で標識させた本発明の抗体の一定量と競合反応させ、 次い で、 不溶化ヒト I L一 1と標識抗体との結合体及び非結合標識抗钵を分離し、 そ の何れか一方の標識活性を測定することにより、 またサンドイ ッチ法を採用する 場合は、 測定物質と不溶化された本発明抗体とを反応させて、 ヒト I L一 1不溶 化抗体複合体を形成させ、 この複合体に標識抗体の一定量を反応させ、 次いで複 合体と標識抗体との結合体及び非結合標識抗体を分離し、 その何れか一方の標識 活性を測定することにより、 それぞれ検体中のヒト I L一 1を定量できる。 上記 検定法において検体として用いられる体液としては、 例えば血液、 細胞組織液等 を例示でき、 これらのうちでは血液、 特に血清又は血漿が好ましい。 Conventional immunological techniques such as (ELISA) and agglutination methods can be mentioned, and the proposal and procedure of these methods are not different from those of ordinary methods. More specifically, for example, when a competitive method is used, human IL-11 in a sample to be measured and a certain amount of insolubilized human IL-11 is subjected to a competitive reaction with a fixed amount of the antibody of the present invention labeled with a labeling agent, and then a conjugate of the insolubilized human IL-11 and the labeled antibody and a non-bound labeled antibody are separated. By measuring the labeling activity of one of them, or when the sandwich method is adopted, the test substance is reacted with the insolubilized antibody of the present invention to form a human IL-11-insoluble antibody complex. The complex is allowed to react with a fixed amount of the labeled antibody, then the conjugate of the complex and the labeled antibody and the unbound labeled antibody are separated, and the labeling activity of one of them is measured to obtain a sample. Of human IL-11 can be quantified. Examples of the body fluid used as a specimen in the above-mentioned assay include, for example, blood, cell tissue fluid and the like. Among them, blood, particularly serum or plasma is preferable.
本発明抗体の標識物質としては、 ダルコアミラーゼ、 バーオキシダーゼ、 アル カリフォスファターゼ、 一ガラク トォキシダーゼ等の各種の酵素、 125 1、 1 3 、 トリチウム等の放射性物質等が挙げられる。 該標識化法は、 常法に従え ばよい [Nature, 194, 495 (1962) : Acta. Bndocrliol. Suppl. , 168, 206 (1972) 等参照〕 ο The labeling substance of the present invention an antibody, Darko amylase, bar oxidase, alkaline phosphatase, various enzymes, such as one galactosyl Tokishidaze, 125 1, 1 3, radioactive substances such as tritium, and the like. The labeling method may be a conventional one [see Nature, 194, 495 (1962): Acta. Bndocrliol. Suppl., 168, 206 (1972)].
不溶化抗体は、 ヒト I L一 1又は本発明抗体を、 不溶性担体に化学的又は物理 的に結合させることにより製造される。 不溶性担体としては、 セル D—ス粉末、 セフアデッタス、 セファロース、 ポリスチレン、 濾紙、 カルボキシメチルセル口 ース、 イオン交換樹脂、 デキス トラン、 プラスチックフイルム、 プラスチックチ ニューブ、 ナイ ン、 ガラスビーズ、 絹、 ポリ了ミ ンーメチルビニルエーテル一マ レイン酸共重合体、 ァミノ酸共重合物、 エチレン一マレイン酸共重合物等が挙げ られる。 不溶化は、 共有結合法としてのジ了ゾ法、 ペプチド法、 了ルキル化法、 架橋試薬による担体結合法 (架橋試薬としてグルタルアルデヒ ド、 へキサメチレ ンイソシアナ一ト等を用いる) 、 U g 1反応による担体結合法等の化学反応;あ るいはィォン交換榭脂のような担体を用いるィォン結合法;ガラスビーズ等の多 孔性ガラスを担体として用いる物理的吸着法によって行なわれる。 上記測定法に おいて反応 (免疫反応) は、 通常 4 5 £i下、 好ましくは 4〜4 0での温度で、 数時間〜 2 4時間程度で行なわれる。 かくして、 本発明抗体を用いれば、 簡便に、 高精度に、 検体中のヒト I L一 1を測定することができる。  An insolubilized antibody is produced by chemically or physically binding human IL-11 or the antibody of the present invention to an insoluble carrier. Examples of insoluble carriers include Cell D-powder, Sephadex, Sepharose, polystyrene, filter paper, carboxymethylcellulose, ion-exchange resin, dextran, plastic film, plastic tubes, nin, glass beads, silk and poly Examples include a copolymer of min-methyl vinyl ether-maleic acid, a copolymer of amino acid, and a copolymer of ethylene-maleic acid. The insolubilization is carried out by the dithiol method, peptide method, alkylation method as a covalent bond method, carrier binding method using a cross-linking reagent (using glutaraldehyde, hexamethylene isocyanate, etc. as a cross-linking reagent), and Ug1 reaction. It is carried out by a chemical reaction such as a carrier binding method; or an ion binding method using a carrier such as ion exchange resin; and a physical adsorption method using a porous glass such as glass beads as a carrier. In the above assay, the reaction (immune reaction) is usually carried out under 45 £ i, preferably at a temperature of 4 to 40, for several hours to 24 hours. Thus, by using the antibody of the present invention, human IL-11 in a sample can be measured simply and accurately.
また、 本発明抗体は I L一 1の作用を抑制乃至中和する作用を有するものであ り、 その利用によれば、 ヒト I L一 1の異常産生を伴う疾患、 代表的には慢性関 節リウマチ、 甲状腺炎、 腎炎等の慢性炎症性疾患、 動脈硬化、 川崎病等の血管炎、 D I C、 血液癌等の各種疾患の治療及び診断を行なうことが可能である。 更に、 本発明抗体は抗原性が低く、 ヒト体内に投与することも可能であり、 例えばこれ を標識してヒト体内に投与することによって、 ヒト体内の I L一 1の異常産生を 伴う組織部位等の画像診断にも利用することができる。 Further, the antibody of the present invention has an action of suppressing or neutralizing the action of IL-11. According to its use, diseases associated with abnormal production of human IL-11, typically chronic inflammatory diseases such as rheumatoid arthritis, thyroiditis, nephritis, vasculitis such as arteriosclerosis, Kawasaki disease, and DIC It is possible to treat and diagnose various diseases such as blood cancer. Furthermore, the antibody of the present invention has low antigenicity and can be administered to the human body. For example, by labeling it and administering it to the human body, tissue sites associated with abnormal production of IL-11 in the human body, etc. It can also be used for diagnostic imaging.
以上のように、 本発明は組換え抗体のみならず、 その製造方法、 該方法に用い られるクロニーングベクター、 発現ベクター、 トランスフ クトされた細胞系等 や本発明抗体のヒト I L一 1精製系、 測定系への利用並びに該抗体を有効成分と する治療薬乃至診断薬及び之等の適用方法をも包含している。  As described above, the present invention includes not only recombinant antibodies, but also methods for producing the same, cloning vectors, expression vectors, and transfected cell lines used in the methods, and human IL-11 purification systems for the antibodies of the present invention. The present invention also encompasses the use of the present invention in a measurement system, and a method of applying a therapeutic or diagnostic agent containing the antibody as an active ingredient, and the like.
かかる本発明抗体を利用した精製系、'測定系の設定、 治療乃至診断分野への適 用、 それらの改変乃至応用は当業者にとり自明である。  The purification system using the antibody of the present invention, the setting of a measurement system, the application to the therapeutic or diagnostic fields, and the modification or application thereof are obvious to those skilled in the art.
実施例 Example
辺下、 本発明を更に詳しく説明するため、 実施例を挙げる。  In order to explain the present invention in more detail, examples will be given.
実施例 1 -Example 1-
(1) ANOC-301産生ハイプリ ドーマの培養 (1) Culture of ANOC-301-producing hybridoma
マウス抗ヒト I L一 1 モノクローナル抗体である ANOC— 301を産生す るハイプリ ドーマ細胞 〔KOCO 301 (FBRM BP-1554) 。 免疫原は組換えヒト I L一 1 αであり、 得られる抗体のイソタイプは I gG, で、 抗体の親和性は 0. 8〜2. 0 X 1 O—^M ^である。 特開昭 63— 258595号公報参照。 〕 を、 10%仔牛胎児血清 (以下 FCSと略す) (ハイプリマックス (Hybrimax) , シグマ (Sigma) 社製) 、 ImMグルタミ ン及び 50ュニッ ト Zm^ベニシリ ン一ス トレプトマイシン (p/s, ギブコ (GIBC0) 社製) を含む RPMI— 1640 培地を用いて、 5 %C02/空気ガスを供給した ーラボトルで 37でで培養し A hybridoma cell that produces ANOC-301, a mouse anti-human IL-11 monoclonal antibody [KOCO 301 (FBRM BP-1554). The immunogen is recombinant human IL-11α, the isotype of the resulting antibody is IgG, and the affinity of the antibody is 0.8-2.0 X 1 O- ^ M ^. See JP-A-63-258595. ] With 10% fetal calf serum (hereinafter abbreviated as FCS) (Hybrimax, Sigma), ImM glutamin and 50 units of Zm ^ venicillin-streptomycin (p / s, Gibco (GIBC0) Co., Ltd.) using RPMI-1640 medium containing, a 5% C0 2 / air gas were cultured at 37 in Rabotoru were fed
(2) mRNAの調製 (2) Preparation of mRNA
上述した方法により培養した AN OC— 301産生ハイプリ ドーマ細胞 3 X 108個 (1 £培養分) を遠心 (200 Orpm, 15分) により集めた後、 滅菌 した PBS (リン酸塩緩衝生理食塩水 (phosphate buffered saline) ) で一度 洗浄した。 上記細胞を、 5Mグァニジン 'チオシァネート、 5 0raMトリスー塩酸 (pH7. 5) 、 1 OmME DT A及び 8 %)9—メルカプトエタノールから成る 2 0m£の溶液に溶かした。 この細胞溶解液を 1 6 Gの注射針に 5回、 2 1 Gの注 射針に 20回通すことにより D N Aを切断し、 これに 7倍容量の 4 M塩化リチゥ ムを加えた。 4 tで一晩放置後、 遠心 (1 0 0 0 Orpm, 6 0分, 4で) により 沈澱を回収した。 沈澱を 2 M塩化リチウム一 4 M尿素溶液に再懸濁し、 再び遠心 ( 1 0 0 00 rpni, 3 0分) した後、 ImMEDTA及び 0. 1 %SDSを舍む 1 OroMトリス一塩酸 (PH7. 4) 溶液に最小容量 (約 2 ΰηέ) で溶かした。 この 溶液を凍結♦融解後、 等量のフヱノール:クロ口ホルム:イソアミルアルコール ( 2 4 : 2 4 : 1 ) 混液で 2回抽出し、 さらにクロ口ホルムで抽出した。 得られ た水層に 0. 5容量の 7. 5 Μ酢酸了ンモニゥムと 2容量のエタノールを加えて、 全 RNAを沈澱させた。 得られた全 RNAは 1. 8mgであった。 3 × 10 8 ANOC-301-producing hybridoma cells (1 £ culture) cultured by the method described above were collected by centrifugation (200 rpm, 15 minutes), and then sterilized PBS (phosphate buffered saline). (Phosphate buffered saline)) Washed. The cells were dissolved in a 20 ml solution of 5 M guanidine 'thiocyanate, 50 raM tris-HCl (pH 7.5), 10 mM EDTA and 8% 9-mercaptoethanol. The cell lysate was passed through a 16 G injection needle 5 times and a 21 G injection needle 20 times to cut the DNA, and a 7-fold volume of 4 M lithium chloride was added. After standing at 4 t overnight, the precipitate was collected by centrifugation (100 rpm, 60 min, 4). The precipitate was resuspended in a 2M lithium chloride-14M urea solution, centrifuged again (10000 rpni, 30 minutes), and then immobilized with ImMEDTA and 0.1% SDS. 1 OroM Tris-monohydrochloride (PH7. 4) Dissolved in the solution with the minimum volume (about 2 ΰηέ). After freezing and thawing this solution, it was extracted twice with an equal volume of a mixture of phenol: chloroform: isoamyl alcohol (24: 24: 4: 1), and further extracted with chloroform. To the resulting aqueous layer was added 0.5 volume of 7.5 mM ammonium acetate and 2 volumes of ethanol to precipitate total RNA. The obtained total RNA was 1.8 mg.
オリゴ (dT) —セル π—スを有するプレパックスパンカラムに基づいたファ ルマシア社製の mRN A精製キッ ト (Ca t. No. 27— 9 2 58— 0 1) を 用いて、 ポリ (A) +RNAを調製した。 得られたポリ (A) +RNAは、 5 0 Poly (A) was prepared using Pharmacia's mRNA Purification Kit (Cat. No. 27-92258-01) based on a pre-packed span column with oligo (dT) -cell π-cells. + RNA was prepared. The resulting poly (A) + RNA is 50
JU gでめった o JU g
(3) cDNAライブラリーの調製  (3) Preparation of cDNA library
5 ;/gのポリ (A) +RN Aよりアマ一シャム社製 c DN A合成キッ ト (C a t. No. RPN I 2 5 6 Y) を用いて c DN Αを合成した。 c DNA (0. 5ピコモル) に、 内部に E c oR I部位を有するリン酸化した B s t X I アダプター (1 2 5 ピコモル) を連結し、 アダプタ一の付いた c DNAを、 A C A 3 4カラ厶でゲル濾過を行なうことにより精製した。 該アダプタ一の付い た c DNAを、 B s t X I部位を含む p S P 6 4の修飾べクタ一 〔ベクタ一 CDM8 (B. Seed, Nature, 329, 84{H1987))〕 から単錐した X b a Iスタッフ ァ一断片 ( stuff er fragment) を p S P 6 4 (プロメガ社製 (Promegs Corporation, U.S.A.)製) の X b a I部位に挿入した修飾 p S P 6 4ベクターで あり、 Xb a Iスタッファー断片内に 2ケ所の Bs t X I部位を有する〕 に連結 させ、 大腸菌 HB 1 0 1の派生株である LM1 0 35細胞に形質転換した。  CDNΑ was synthesized from 5; / g of poly (A) + RNA using a cDNA synthesis kit (Cat. No. RPN I256Y) manufactured by Amersham. A phosphorylated Bst XI adapter (125 picomoles) having an internal EcoRI site was ligated to 0.5 picomoles of cDNA, and the cDNA with the adapter was converted to ACA34 columns. And purified by gel filtration. The cDNA with the adapter was converted into a single cone Xba from the modified vector of pSP64 containing the Bst XI site (Vector-CDM8 (B. Seed, Nature, 329, 84 (H1987))). This is a modified pSP64 vector in which the I stuffer fragment is inserted into the XbaI site of pSP64 (Promegs Corporation, USA), and is contained in the XbaI stuffer fragment. Has two Bst XI sites) and was transformed into LM1035 cells, a derivative strain of Escherichia coli HB101.
(4) オリゴヌクレオチドプローブ 表 1に示す T 4ポリヌクレオチドキナーゼで 5 ' 末端を 〔 r— 32Ρ〕 ATPを用いてラベルしたォリゴヌクレオチドプローブを用いて、 上記で得られ た c DNAライブラリーより約 1 2 0 0 0コロニーについて、 抗体の L鎖及び H 鎖の各 c DNAをスクリ一エングした。 (4) Oligonucleotide probe The 5 'end with T 4 polynucleotide kinase as shown in Table 1 [r- 32 [rho] with O oligo nucleotide probe labeled with ATP, about 1 than c DNA library obtained in the above 2 0 0 0 The colonies were screened for the respective cDNAs of the L chain and H chain of the antibody.
表 1  table 1
尚、 上記 R 4 9 8は、 マウス C c遺伝子配列 〔Max et al. , J. Biol. Chera. , 256, 5116(1981)] の内の 4 6 5 8— 4 6 7 7の配列の相補鎖に相当し、 R 6 2 7は、 マウス C r !遺伝子の CH1ドメイン 〔Honjo et al., Cell, 18, 559(1979)〕 の 1 1 5— 1 3 3の配列の相補鎖に相当する。 The above-mentioned R498 is complementary to the sequence of 4658-8-4677 in the mouse Cc gene sequence [Max et al., J. Biol. Chera., 256, 5116 (1981)]. R 627 corresponds to the complementary strand of the sequence 115-133 of the C H1 domain of the mouse Cr! Gene [Honjo et al., Cell, 18, 559 (1979)]. I do.
(5) AN0C-3 0 1の V領域の c DNAのクローニング  (5) Cloning of cDNA for V region of AN0C-301
上記表 1に示す 5' 末端をラペルしたォリゴヌクレオチドプ ーブを用いてス クリーニングを行なった。  Screening was performed using an oligonucleotide probe having a lapel at the 5 'end shown in Table 1 above.
プレハイブリダィゼーシヨンは、 6 X S S C ( 1 X S S C = 0. 1 5 M NaC£-0. 0 1 5 Mクェン酸ナトリウ厶溶液) 、 5 xDenhart's 溶液 ( 1 x Denhart' s溶液 = 0. 02 %ポリビニルピ リ ドン一 o . 02 %牛血清了ルブミ ン (BSA) -0. 0 2%フイコール) 、 0. 1%303及び1 0 0 jug/mg変 性サケ精子 DNAからなる溶液中で、 4 6でで 3時間行なった。 次に、 このプレ ハイプリダイゼーシヨン溶液と同組成の溶液中に32 Pでラペルしたォリゴヌクレ ォチドを 5ng/m£になるように加え、 6 5 :にした後、 室温にて 1 6時間以上放 置した。 フィルターの洗浄は 6 XS SC— 0. 1 %SDS溶液中で室温で 2回行 なった後、 6 X S SC— 0. 1 %SDS溶液中で 4 5でで 3回行なった。 Prehybridization is 6 XSSC (1 XSSC = 0.15 M NaC £ -0.015 M sodium citrate solution), 5 x Denhart's solution (1 x Denhart's solution = 0.02% Polyvinyl pyridone o. 02% bovine serum rubumin (BSA)-0.02% ficoll), 0.1% 303 and 100 jug / mg denatured salmon sperm DNA For 3 hours. Next, oligonucleotide, which had been lapel at 32 P, was added to a solution having the same composition as the prehybridization solution so as to have a concentration of 5 ng / m £, and the mixture was adjusted to 65: and then released at room temperature for 16 hours or more. Was placed. The filter was washed twice at room temperature in a 6 XS SC-0.1% SDS solution and then three times at 45 in a 6 XS SC-0.1% SDS solution.
その結果、 プ α—ブとハイブリダィゼーシヨンしたクローンについて、 ベクタ 一側のプライマーを用いた PCR 〔ポリメラ一ゼチヱインリ了クシヨン  As a result, PCR using the primer on one side of the clone [Polymer-
(polymerase chain reaction)] により c DNAの大きさを分析した。  (polymerase chain reaction)] to analyze the size of the cDNA.
上記 P C Rの反応条件は以下の通りである。 テンプレート DNA O. 1 u g, 1 0 xPCR緩衝液 〔5 0 OmM KC l O OmMト リスー塩酸 (PH8. 3 ) , 1 5 mM M g C ·β 2 , ゼラチン 0. 1 %The reaction conditions of the above PCR are as follows. Template DNA O. 1 ug, 10 x PCR buffer [50 OmM KCl O OmM Tris-HCl (PH8.3), 15 mM Mg C · β 2 , Gelatin 0.1%
(w/v) 〕 5 β £, 2. 5mMdNTP s (N = A, G, C T) 5 ju ·β , オリ ゴ R 8 8 3 1 0ピコモル, オリゴ R 1 7 6 7 1 0ピコモル及び T a qポリメ ラーゼ (l U/ £) 0. 2 ^ Mi (水で反応液を 5 0 /χ·βとする) 。 (w / v)] 5 β £, 2.5 mM dNTP s (N = A, G, CT) 5 ju · β, oligo R 8 8 3 10 picomoles, oligo R 17 6 7 10 picomoles and Taq Polymerase (lU / £) 0.2 ^ Mi (react the solution with water at 50 / χ · β).
反応は 9 4でで 1分間、 5 5でで 1分間、 7 2でで 1分間を 2 0サイクル行な ο  Perform 20 cycles of reaction at 94 for 1 minute, 55 at 1 minute, and 72 at 1 minute ο
尚、 オリゴ R 8 8 3及び R 1 7 6 7の塩基配列を下記表 2に示す。  The base sequences of the oligos R8883 and R1767 are shown in Table 2 below.
表 2  Table 2
その結果、 Η鎖 c DNAについては 8ク 一ンが全長と考えられる 1. 9 k b の c DNAを有しており、 L鎖 c DNAについては 6ク ーンが全長と考えられ る 1. 1 k bの c DNAを有していた。  As a result, 8 clones are considered to be 1.9 kb in full-length cDNA, and 6 clones are considered to be 1.9 kb in full-length cDNA 1.1. It had kb cDNA.
そこで上記 2つの H鎖 c DN Aクローン (p I L 0 6 8 p I L 0 7 2 ) と 2 つの L鎖 c DNAクローン (p I L 0 8 6, p I L 0 8 9 ) の各 V領域について、 塩基配列を調べた。 尚、 上記 H鎖 cDNAクローン p I L 0 6 8の概略は図 1に 示す通りであり、 また L鎖 c DNAクローン p I L 0 8 6の概略は図 2に示す通 りであり、 各図において、 S P 6 RNAp 0 1 Pは S P 6 RNAポリメラーゼの プロモーター領域を、 AmpRはアンピシリ ン耐性遺伝子を示す。 Therefore, the nucleotide region of each V region of the two H chain cDNA clones (pIL068pIL072) and the two L chain cDNA clones (pIL086, pIL089) was determined. The sequence was examined. The outline of the H chain cDNA clone pIL068 is as shown in FIG. 1, and the outline of the L chain cDNA clone pIL086 is as shown in FIG. SP6 RNAp01P indicates the promoter region of SP6 RNA polymerase, and Amp R indicates the ampicillin resistance gene.
その結果、 P I L 0 6 8及び p I L 0 7 2の両クローンは共に、 全長のマウス I gG!H鎖遺伝子の c DNAを有していた。 一方 p I L 0 8 6と p I L 0 8 9 の L鎖 c DNAクローンは、 V領域で異なっていた。 p I L 0 8 9は、 ハイプリ ドーマの融合パートナーから由来した為 L鎖遺伝子を有しており、 p I L 0 8 6 が ANOC— 3 0 1のマウスカッパ鎖遺伝子を有していた。  As a result, both PIL068 and pIL072 clones had full-length mouse IgG! H chain gene cDNA. On the other hand, the light chain cDNA clones of pIL086 and pIL089 differed in the V region. pIL089 had the L chain gene because it was derived from the fusion partner of the hybridoma, and pIL086 had the mouse kappa chain gene of ANOC-301.
H鎖 c DNAクローン P I L 0 6 8の V領域の塩基配列は配列番号: 1として 示した通りである。 L鎖 cDNAクローン p I L086の V領域の塩基配列は配列審号: 2として 示した通りである。 The nucleotide sequence of the V region of the H chain cDNA clone PIL068 is as shown in SEQ ID NO: 1. The nucleotide sequence of the V region of the L chain cDNA clone pI L086 is as shown in Sequence Listing: 2.
上記 p I L 086は、 リーダー配列の最初の 3了ミノ酸残基が欠けており、 そ のために発現ベクター作製時には、 以下に示すように既知の力ッパ鎖のリーダ一 配列に基づいてメチォニン、 アルギニン及びバリンの配列 (ATGAGGGTC) を付加した。  The above pIL086 lacks the first three amino acid residues of the leader sequence. Therefore, when constructing an expression vector, methionine is used based on the known leader sequence of the power chain as shown below. , Arginine and valine sequences (ATGAGGGTC).
(6) キメラ H鎖 I gG4ANOC— 301の発現ベクター p AC 52の作製 この発現ベクター P AC 52の作製の概略は図 3に示す通りである。 尚、 図中(6) Summary of the generation of chimeric H chain I gG 4 ANOC- 301 This expression vector P AC 52 Preparation of expression vector p AC 52 of is as shown in FIG. In the figure
VHはマウス H鎖 V領域遺伝子を、 G4はヒ ト I gG4の C領域を、 G1はヒ ト I &,の(:領域を、 hCMVはヒ トサイ トメガ oウィルスの MI E (major immediate early)ブ πモータ一を、 g p tはキサンチングァニンフォスホリボ シルトランスフユラーゼ遺伝子を、 p Aはポリ A付加シグナル配列を、 SVori は SV40ウィルスの複製起源をそれぞれ示す。 ^下の図においても同 様と ^>o V H is a mouse H chain V region gene, G4 is the C region of the human I gG 4, G1 is human I &, of (: the area, hCMV is human Tosai Tomega o virus of MI E (major immediate early ), Π motor, gpt, xanthinguanine phosphoribosyltransferase gene, pA, poly A addition signal sequence, SVori, replication origin of SV40 virus, respectively. And ^> o
まずマウス H鎖 V領域遺伝子 (VH) を含む P I L 068より、—図 4に示す 2 種のプライマー (フォワード 'プライマー (R 239 1) 及びリバース ·プライ マー (R 239 0 ) 。 それぞれ自動オリゴヌクレオチド合成機により化学合成し た。 ) を用いて、 上記 (5) で述べたと同様の条件で PC R法にて、 上記 VH遺 伝子を含む DN A断片を単雜した。 このものの塩基配列及び对応する了ミノ酸配 列は、 配列審号: 1として示す通りである。 First, from PIL 068 containing mouse H chain V region gene (V H ), two types of primers shown in FIG. 4 (forward 'primer (R 239 1) and reverse primer (R 239 0). The DNA fragment containing the VH gene was multiplexed by the PCR method under the same conditions as described in (5) above. The nucleotide sequence and the corresponding amino acid sequence are as shown in Sequence Listing: 1.
次いで、 上記 PCR法により増幅させた VH遺伝子を含む DNA断片を、 制限 酵素 Hi 11(1111と八 3 Iで切断し、 これをヒト I gG4の C領域を有するぺク ター PE1004 CpEE 6 (P. Stephens and M. Cockett, Nucl. Acids Res., 17, 7110(1989)) に基づいて作製した。 〕 の Hi ndlEと Apa I間に挿入して、 目的の p AC 52を得た。 得られた p AC 52の特徴及び制限酵素地図を図 5に 示す。 Then, a DNA fragment containing the V H gene was amplified by the PCR method, restriction enzyme Hi 11 (1111 and cut with eight 3 I, Bae click terpolymers PE1004 CpEE 6 having a C region of a human I gG 4 This ( P. Stephens and M. Cockett, Nucl. Acids Res., 17, 7110 (1989))] between HindlE and ApaI to obtain the desired pAC52. The characteristics and restriction map of the obtained pAC52 are shown in FIG.
(7) キメラ H鎖 I gG!ANOC— 301の発現べクタ一 p AC 77の作製 この発現ベクター P AC 77の作製の概略も図 3に併記する通りであり、 この ものはヒト I gG4の C領域を有するベクタ一 pE 1 0 04に代えてヒト I gGt の C領域を有するベクター P E 1 0 0 1 CP E 1 0 0 と同様に P EE 6に基づ いて作製した。 〕 を用いて、 上記 6と同様にして作製できるが、 この例では、 以 下の如くして作製した。 (7) Outline of producing a chimeric H chain I gG! ANOC- 301 This expression vector P AC 77 Preparation of expression base Kuta one p AC 77 also are as also shown in FIG. 3, of this compound human I gG 4 human I gG t instead of the vector one pE 1 0 04 having a C region The vector was prepared on the basis of PEE6 in the same manner as the vector PE1001CPE100 having the C region. ] Can be produced in the same manner as in the above item 6, but in this example, it was produced as follows.
即ち、 上記 (6) で作製したキメラ H鎖 I gG4ANOC— 30 1の発現べク ター p AC 5 2より、 制限酵素 H i n dmと Ap a Iで ANOC— 3 0 1のマウ ス H鎖 V領域遺伝子を切り出し、 これをベクター pE 1 0 0 1の H i Ti dlEと Ap a I間に挿入した。 かくして目的の p AC 77を得た。 That is, a chimeric H chain I gG 4 ANOC- 30 1 than the expression downy click coater p AC 5 2 of restriction enzymes H in dm and Ap a I in ANOC- 3 0 1 of mouse H chain prepared above (6) The V region gene was excised and inserted into the vector pE1001 between Hi TidlE and Ap aI. Thus, the desired pAC77 was obtained.
(8) キメラ L鎖 ANOC— 3 0 1の発現べクタ一 p AC 5 3の作製  (8) Construction of expression vector pAC53 for expression of chimeric L chain ANOC-301
この発現ベクター P AC 5 3の作製の概略は図 6に示す通りである。 尚、 図中 VLは ANOC— 3 0 1 (マウス) の L鎖 V領域遺伝子を、 Ck (Ckappa 又は C cとも表示する) はヒトカッパ鎖 C領域を示し、 AmpR及び hCMVは前記 に同じである。 The outline of construction of this expression vector PAC53 is as shown in FIG. In the figure, VL indicates the L chain V region gene of ANOC-301 (mouse), C k (also indicated as C kappa or C c) indicates the human kappa chain C region, and AmpR and hCMV are the same as above. It is.
即ち、 まずマウス L鎖可変領域遺伝子 (VL) を含む p I L 086より、 図 7 に示す 2種のプライマー (フォワード ·プライマ (R 242 9 ) 及びリバース •プライマー (R 243 0) 。 それぞれ自動オリゴヌクレオチド合成機により化 学合成した。 ) を用いて、 前記 (5) で述べたと同様の条件で PC R法にて、 上 記 V L遺伝子を含む D N A断片を単錐した。 このものの塩基配列及び対応する了 ミノ酸配列は、 配列番号: 2として示す通りである。 That is, from the p IL 086 first containing the mouse L chain variable region gene (V L), 2 types of primers (forward primer (R 242 9) and reverse • primer (R 243 0 shown in FIG. 7). Automatic respectively oligo Using a nucleotide synthesizer, a DNA fragment containing the VL gene was single-polished by the PCR method under the same conditions as described in (5) above. The nucleotide sequence and the corresponding amino acid sequence are as shown in SEQ ID NO: 2.
次いで、 上記 PC R法により増幅させた VL遺伝子を制限酵素 B s t B Iと S p 1 Iで切断し、 これをヒ トカッパ鎖 C領域を有するベクター p E 1 0 8 1 CpE 1 0 04の場合と同様に p EE 6に基づいて作製した。 〕 の B s t B Iと S p 1 I間に挿入して、 pAC 5 3を得た。 かくして得られた p AC 5 3の特徴 及び制限酵素地図を図 8に示す。 Next, the VL gene amplified by the PCR method is digested with the restriction enzymes BstBI and Sp1I, and this is cut into the vector pE1081CpE1004 having the human kappa chain C region. It was prepared based on pEE6 in the same manner as described above. Was inserted between BstBI and Sp1I to obtain pAC53. FIG. 8 shows the characteristics and restriction enzyme map of pAC53 thus obtained.
(9) CDR—グラフト L鎖 ANOC— 30 1発現べクタ一 p A L 3 1の作製 この発現ベクター PAL 3 1の作製の概略は図 9に示す通りである。 該ベクタ 一は、 ヒ ト抗体 R E I CB. A. abat et al. , (1987) in Sequences of Proteins of Immunological Interest. , 4th edition, (US Department of Health and Human Services)〕 のカッパ鎖 VLドメイン内 CDR領域のアミノ酸 配列を、 ANOC— 30 1の VLドメイン内の CDR領域のアミノ酸配列で置換 させたものであり、 以下の通り作製された。 (9) Construction of CDR—grafted L chain ANOC-30 1 expression vector pAL31 The outline of construction of this expression vector PAL31 is as shown in FIG. The vector is located in the Kappa chain VL domain of the human antibody REI CB. A. abat et al., (1987) in Sequences of Proteins of Immunological Interest., 4th edition, (US Department of Health and Human Services). Replace the amino acid sequence of the CDR region with the amino acid sequence of the CDR region in the VL domain of ANOC-301 It was produced as follows.
即ちまず、 ドウターティ (Daughterty) らの方法 (Nucl. Acids Res., 19, 2471 (1991)によって述べられた方法に準じて、 オリゴヌクレオチドを用いて、 下 記ステップ 1及びステップ 2に従い、 CDR—グラフト VL遺伝子を得た。 First, CDR-grafting was carried out using oligonucleotides in accordance with the method described by Daughterty et al. (Nucl. Acids Res., 19, 2471 (1991)) using oligonucleotides according to steps 1 and 2 below. The VL gene was obtained.
(ステップ 1)  (step 1)
ヒト抗体 RE Iの力ッパ鎖のリーダー配列、 フレームワーク 1の部分及びフレ ームワーク 4の部分をコードするユニバーサル VLカセッ トを構築した。 具抹的 には、 図 16に示すオリゴヌクレオチドを用いて PCR反応を行なった。 反応液 は 50 からなり各 10ピコモルの 3' 及び 5' —ショート ♦オリゴヌクレオ チド (ΒϋΝΒΜ-20, L-RBIVBC 3) 、 各 1ピコモルのィンターナル *ォリゴヌクレオ チド 1, L-RBIVBC 1, L-RBIVBC 2) 、 1 OmMトリス一塩酸 (pH8. 3) 、 1. 5m MgC 2. 0. 01% (w/v) ゼラチン、 5 OmMKCi 0. 25 mMdNTP s (N = A, G, T, C) 及び 0. 25ユニッ トの T a qポリメラー ゼの組成を有し、 94でで 1分間、 55でで1分間、 72 tで 1分間の 20サイ クルを行なった。 反応終了後、 フヱノール♦クロ口ホルムで 2回抽出し、 ェタノ ール沈澱を行ない、 その後、 水に溶解後、 制限酵素 B s t B Iと S p 1 Iで切断 し、 pE 1081の Bs tB Iと Sp 1 I間に挿入し、 ユニバーサル VLカセッ ト p A L 29を得た。 この B s t B Iと S p 1 I間の塩基配列は図 17に示す通 りである。 A universal VL cassette encoding the leader sequence, framework 1 and framework 4 of the human antibody REI power chain was constructed. Specifically, a PCR reaction was performed using the oligonucleotides shown in FIG. The reaction mixture consisted of 50, 10 picomoles each of 3 'and 5'—short ♦ Oligonucleotide (ΒϋΝΒΜ-20, L-RBIVBC 3), 1 picomole of each * internal * oligonucleotide 1, L-RBIVBC 1, L-RBIVBC 2), 1 OmM tris monohydrochloride (pH8. 3), 1. 5m MgC 2. 0. 01% (w / v) gelatin, 5 OmMKCi 0. 25 mMdNTP s ( N = A, G, T, C) and It had a composition of 0.25 units of Taq polymerase and performed 20 cycles of 1 minute at 94, 1 minute at 55, and 1 minute at 72 t. After completion of the reaction, the reaction mixture was extracted twice with phenol-black form, precipitated with ethanol, then dissolved in water, cut with restriction enzymes BstBI and Sp1I, and combined with BstBI of pE1081. It was inserted between Sp 1 I to obtain a universal VL cassette pAL29. The base sequence between BstBI and Sp1I is as shown in FIG.
(ステップ 2)  (Step 2)
CDR— 1〜CDR— 3の領域については、 図 18に示したォリゴヌクレオチ ドを用い、 ステップ 1と同様の方法で PC R反応を行ない、 最終的に Bs tEH と Kp l I間の DN A断片を得た。 これを、 予め制限酵素 Bs tEII及び Kpn Iで切断しておいた前記ステップ 1で得られたユニバーサル V Lカセット p A L 29の Bs tEIIと Kpn I間に挿入して、 pAL31を得た。  For the CDR-1 to CDR-3 regions, a PCR reaction was performed using the oligonucleotides shown in Fig. 18 in the same manner as in step 1, and finally the DNA fragment between BstEH and KplI was obtained. Obtained. This was inserted between BstEII and KpnI of the universal VL cassette pAL29 obtained in step 1 previously cut with the restriction enzymes BstEII and KpnI to obtain pAL31.
得られた CDR—グラフト VL遺伝子の DNA配列及び対応する了ミノ酸配列 は、 配列番号: 3として示す通りである。 The obtained CDR-graft VL gene DNA sequence and corresponding amino acid sequence are as shown in SEQ ID NO: 3.
Bs tBIと Sp 1 I断片 (図 10) を挿入された目的の PAL 31の特徴及 び制限酵素地図を図 11に示す。 ( 1 0 ) C DR—グラフ ト H鎖 I g G4ANOC— 301発現べクタ一 FIG. 11 shows the characteristics and restriction enzyme map of the target PAL31 into which BstBI and Sp1I fragment (FIG. 10) were inserted. (10) CDR—graft heavy chain IgG 4 ANOC—301 expression vector
AL 34の作製  Fabrication of AL 34
この発現ベクター PAL 34の作製の概略は図 12に示す通りであり、 該べク ターは、 ヒ ト抗体 KOL CB. abat et al. , (1987) in Sequences of Proteins of Immunological Interest. , 4th edition, (US Department of Health and Human Services)] の H鎖 VHドメイン内の 3ケ所の C DR領域のァ ミノ酸配列及びフレームワーク内の 12ケ所の了ミノ酸残基を ANOC— 301 の VHドメイン内の封応するアミノ酸残基で置換したものであり、 以下の通り作 製された。 The outline of the construction of this expression vector PAL 34 is as shown in FIG. 12, and the vector is a human antibody KOL CB. Abat et al., (1987) in Sequences of Proteins of Immunological Interest., 4th edition, (US Department of Health and Human Services)] and the amino acid sequences of three CDR regions in the heavy chain VH domain and 12 amino acid residues in the framework in the VH domain of ANOC-301. It was substituted with the corresponding amino acid residue within, and was produced as follows.
即ち、 上記 (9) で述べた方法と同様の方法で、 オリゴヌクレオチド (図 19) を用いた PC R法により一段階で、 CDR—グラフト VH鎖遺伝子 (その DNA 配列は配列審号: 4として示す) を得た。 得られた遺伝子を制限酵素 H i ndlll と Ap a Iで切断して、 ヒト ·マウスグラフト VH遺伝子を会む DN A断片を得 た。 このものは図 13に示す DN A配列及び対応アミノ酸配列を有している。 上記 H i Ti dm-Ap a I断片を、 ベクター pE 1 0 04の H i n dlEと Ap a I間に挿入して、 目的の p AL 34を得た。 かくして得られた p AL 34 の特徴及び制限酵素地図を図 14に示す。 That is, the CDR-grafted V H chain gene (the DNA sequence of which is the same as that described in (9) above) is obtained in one step by the PCR method using an oligonucleotide (FIG. 19). ). The obtained gene was digested with restriction enzymes Hindlll and ApaI to obtain a DNA fragment meeting the human / mouse graft VH gene. It has the DNA sequence and the corresponding amino acid sequence shown in FIG. The above-mentioned Hi Ti dm-Apa I fragment was inserted between HindE and Ap aI of vector pE1004 to obtain a desired pAL34. FIG. 14 shows the characteristics and restriction map of pAL34 thus obtained.
(11) CDR—グラフト AN OC— 301発現ベクター p AC 79の作製  (11) Construction of CDR-graft AN OC-301 expression vector pAC79
CDR—グラフトー H鎖 I gG4ANOC— 301発現ベクター p AL 34の hCMVプロモーターの 5' 末端の C l a I部位に、 BamHI認識部位を挿入 して、 hCMVZCDR I gG4カセッ トを、 B amH I DNA断片として単離 しブ The CDR- Gurafuto H chain I gG 4 ANOC- 301 expression 5 'end of the C la I site of hCMV promoter vector p AL 34, by inserting the BamHI recognition site, the hCMVZCDR I gG 4 cassettes, B AMH I DNA Isolated as fragments
—方、 P E 1 0 0 4から S V 4 0Zg p t遺伝子のカセッ トを、  -The cassette of the PE104 to SV400Zgpt gene was
S a i l DNA断片として単錐し、 これを CDR—グラフト L鎖 ANOC— 301発現ベクター pAL 31の S a 1 I部位に挿入した。 さらに、 得られたベ クタ一の BamH I部位に hCMVZCDR I gG4カセッ トを揷入して、 目的 の発現ベクター P AC 79を作製した。 その操作の概略は図 15に示す通りであ る o A single DNA fragment was prepared as a Sail DNA fragment, which was inserted into the Sal1 site of the CDR-grafted L chain ANOC-301 expression vector pAL31. Further, the hCMVZCDR IgG 4 cassette was inserted into the BamHI site of the obtained vector to prepare the desired expression vector PAC79. The outline of the operation is as shown in Fig. 15 o
かくして得られた p AC 79の特徴及び制限酵素地図を図 15に併記する。 (12) CHOL 761 h細胞を用いた組換え抗体の一過的発現 The features and restriction map of pAC79 thus obtained are also shown in FIG. (12) Transient expression of recombinant antibody using CHOL 761 h cells
10%FCS、 ImMグルタミ ン及び 50 ぺニシリ ンース トレブトマイ シ ンを含むダルベッコ修飾イーグル培地で培養された CHOL 761 h細胞 〔M. I. Cockett et al., Nucl. Acids Res., 19, 319(1991)] を、 ファルコン丁 175 フラスコに約 2 X 101細胞数で播き、 この細胞に 5 0 Λί gの各プラスミ ド DN Aをリン酸カルシウム法によってトランスフヱクシヨンした。 CHOL 761h cells [MI Cockett et al., Nucl. Acids Res., 19, 319 (1991)] cultured in Dulbecco's modified Eagle's medium containing 10% FCS, ImM glutamine, and 50% nicillin trebutomycin were used. Approximately 2 × 10 1 cells were seeded on a Falcon 175 flask, and 50 μg of each plasmid DNA was transfused by the calcium phosphate method.
用いたプラスミド DN Aは、 上記 (6) 〜 (11) で得られたもの又はそれら の組合せ、 即ちキメラ H鎖 I gG4ANOC— 301の発現ベクター p AC 52 とキメラ L鎖 ANOC— 301の発現べクタ一 p AC 53との組合せ、 キメラし 鎖 ANOC— 301の発現ベクター p AC 53とキメラ H鎖 I gdANOC- 301発現ベクター p AC 77の組合せ、 キメラ L鎖 ANOC— 301の発現べ クタ一 P AC 53と CDR—グラフト H鎖 I gG4ANOC— l 0 3発現べクタ - P A L 34との組合せ、 CDR—グラフト L鎖 I gG4ANOC— 3 0 1発現 ベクター PAL31とキメラ H鎖 I gG4ANOC-301の発現ベクター Plasmid DN A was used, the above (6) to (11) obtained as or a combination thereof in, that expression of the chimeric H chain I gG 4 ANOC- expression vector p AC 52 301 and a chimeric L chain ANOC- 301 Combination of vector pAC53 and chimera chain ANOC-301 expression vector pAC53 and chimera H chain IgdANOC-301 expression vector pAC77 combination and chimera L chain ANOC-301 expression vector P AC 53 and CDR- grafting H chain I gG 4 ANOC- l 0 3 expression base Kuta - combination of PAL 34, CDR- grafted L chain I gG 4 ANOC- 3 0 1 expression vector PAL31 the chimeric H chain I gG 4 ANOC -301 expression vector
P AC 5 2との組合せ及び C DR—グラフト H鎖及び L鎖を組合わせた発現ぺク ター P AC 7 9を用いた。 An expression vector PAC79 in combination with PAC52 and a CDR-grafted H chain and L chain was used.
トランスフヱクシヨン後、 7 2時間で培地を回収した。  The medium was recovered 72 hours after transfection.
発現される組換え抗体は以下の通りである。  The expressed recombinant antibodies are as follows.
'キメラ I gG4AN。C— 301 'Chimera IgG 4 AN. C—301
•キメラ I gG!ANOC— 301  • Chimera IgG! ANOC—301
•キメラ L鎖 グラフト H鎖 I gG4ANOC— 301 • Chimeric L chain Graft H chain IgG 4 ANOC—301
'グラフト L鎖 Zキメラ H鎖 I gG4ANOC— 301 '' Graft L chain Z chimera H chain IgG 4 ANOC— 301
•グラフト I gG4ANOC— 301 • Graft IgG 4 ANOC—301
(13) 組換え抗体の精製  (13) Recombinant antibody purification
キメラ I gG!ANOC— 301は、 プロテイン Α ·セファロ一ス ♦ ファース ト · フロー (FF) (フアルマシア社製) を使用して培養上清から精製した。 尚、 プロティン A ·セファ in—スカラム (22m£べッ ト容量) は、 あらかじめ 4. 3 M · NaC を含む 5 0 raMグリシン Zグリシン酸ナトリゥ厶緩衝液 (PH 8. 8) で平衡化した (5カラム容量, 110m£) 。 そして 480 jugのキメラ I gG! ANOC-3 0 1 (EL I S Aによる測定値) を含む 2. 4 £の培養上清を上記 カラムにかけた。 培養上清をカラムに乗せ *わった後、 マトリックスを平衡緩衝 液で洗い ( 3カラム容量, 6 6 m£) 、 その後 5 0 mM酢酸ナト リゥム緩衝液 (pH 4. 5) (3カラム容量, 6 6 mi) で培養上清中の牛 I gGを溶出した。 その 後 0. 1Mグリシン塩酸 (PH2. 5) 溶液でキメラ I gGiANOC— 3 0 1を 溶出し、 溶出液を 2M リス ·塩酸 (PH8. 0 ) ですぐに中和した。 キメラChimera IgG! ANOC-301 was purified from the culture supernatant using Protein II Sepharose ♦ First Flow (FF) (Pharmacia). The Protein A Sepha-in column (22 mB) was equilibrated in advance with 50 raM glycine Z sodium glycinate buffer (PH8.8) containing 4.3 M NaC (PH8.8). 5 column capacity, 110m £). And 480 jug chimera I gG! 2.4 £ of culture supernatant containing ANOC-301 (measured by ELISA) was applied to the column. After the culture supernatant is applied to the column, the matrix is washed with the equilibration buffer (3 column volumes, 66 mL), and then 50 mM sodium acetate buffer (pH 4.5) (3 column volumes, At 66 mi), bovine IgG in the culture supernatant was eluted. Thereafter, the chimeric IgGIANOC-301 was eluted with a 0.1 M glycine hydrochloride (PH2.5) solution, and the eluate was immediately neutralized with 2 M ris • hydrochloric acid (PH8.0). Chimera
I gGiANOC— 3 0 1を含む画分を、 0. 0 5 %チメ ザール含有リン酸緩 衝食塩水にて透析し、 一 Ί 0でで保存した。 得られたキメラ I gGiANOC—The fraction containing IgGiANOC-301 was dialyzed against phosphate-buffered saline containing 0.05% timezal, and stored at 0-10. The obtained chimera IgGiANOC—
3 0 1は 3 5 7jugであった。 3 0 1 was 3 5 7 jug.
キメラ I gG4ANOC— 3 0 1、 キメラ L鎖/グラフト H鎖 I gG4AN〇C 一 3 0 1、 グラフト L鎖 Zキメラ H鎖 I gG4AN〇C一 3 0 1及びグラフト Chimera IgG 4 ANOC—301, Chimera L chain / graft H chain IgG 4 AN〇C 131, Graft L chain Z Chimera H chain IgG 4 AN〇C 301 and graft
I gG4ANOC-3 0 1の精製も、 上記キメラ I gG!ANOC— 3 0 1と同 様にして行なった。 I gG 4 ANOC-3 0 1 of purification was also carried out in the above-mentioned chimeric I gG! ANOC- 3 0 1 the same way.
その結果、 キメラ I g G4ANOC— 3 0 1は 2. 4 の培養上清より 3 2 5 jug、 キメラ L鎖 グラフト H鎖 I gG4AN〇C一 3 0 1は、 2. 1 ·δの培養 上清より 8 2 0 jug、 グラフト L鎖 Zキメラ H鎖 I gG4ANOC-3 0 1は、 1. 1 の培養上清より 9 4. 5 tg、 グラフ ト I gG4AN〇C一 3 0 1は As a result, the chimeric I g G 4 ANOC- 3 0 1 is 2.4 in the culture supernatant from 3 2 5 jug, chimeric L chain graft H chain I gG 4 AN_〇_C one 3 0 1, 2. 1 · [delta] 8200 jug from the culture supernatant of the above, grafted L chain Z chimeric H chain IgG 4 ANOC-301 was 94.5 tg from the culture supernatant of 1.1, and the graft IgG 4 AN〇C-1 3 0 1
1. 3 £の培養上清より 1 4 0 0 jugで得られた。  It was obtained at 1400 jug from 1.3 £ of culture supernatant.
(1 4) ヒト組換え I L— 1 αの 1251標識体の作製 (1 4) Preparation of 125 1-labeled of human recombinant IL- 1 alpha
1. 5m£のポリプロピレン製ェッペンドルフ♦タイプのチューブに 2 0 ju ·2の ョードゲン溶液 〔ジクロロメタン或いはクロ口ホルムに溶かした 0. 2m Zm£の ョードゲン (1, 3, 4, 6—テトラク 口一 3 a, 6 a—ジフヱニルグリコ一 Jレリ Jレ (1, 3, 4, 6一 tetrachloro— 3a, 6a一 diphenylglycoluril, Pirerce Chemical Company, U.S.A.)) 〕 を加え、 減圧下で溶媒を除いた。 このようにして得られた ョードゲンをコーティングしたチューブに 1 0 £の0. 2 5Mリン酸ナトリウ 厶緩衝液 (pH7. 5) と 5 «·β ( 1 8. 5MB q) の 3. 7 GB q/m£N a · 1251 (了マーシャム社 (Amersham, code 1MS30)製) を加え、 さらに 1 0 « の 0. Img/m^ヒト '組換え I L— 1 (0. 0 5 Mリン酸ナトリウム緩衝液、 pH 7. 5に溶解) を加えた後、 ピぺッ トで反応液を混合し、 氷上に 3分間放置した。 0. 0 5Mリン酸ナトリウム緩衝液 (PH7. 5) を 2 5 0 加えた後、 あらか じめ 0. 1 % B S A (牛血清アルブミ ン (bovin serum albumin) , シグマ 社 (Sigma)製) と 0. 0 5%チメロザール (シグマ社製) を含む PBS (リン酸 塩緩衝生理食塩永 (phosphato buffered saline)) で平衡化したプレパック P D 一 1 0 (pre-packed PD-10) カラム (フアルマシア社製) にかけ、 各分画 ( 1 m£ /分画) についてカウントし、 125 I L— 1 αを含む画分を 4でで保存した。 1. Put 20 ju · 2 eodogen solution in a 5m £ polypropylene eppendorf ♦ type tube [0.2m Zm £ eodogen dissolved in dichloromethane or black-mouthed form (1,3,4,6-tetrakoku 1-3) a, 6a-diphenylglycol-Jr (1,3,4,6-tetrachloro-3a, 6a-diphenylglycoluril, Pirerce Chemical Company, USA)]] and the solvent was removed under reduced pressure. The thus obtained tube coated with lodogen was charged with 3.7 GB q of 10 £ of 0.25M sodium phosphate buffer (pH 7.5) and 5 «β (18.5 MB q). / m £ N a · 1 25 1 ( Ryo Marsham Inc. (Amersham, code 1MS30) Ltd.) was added, further 1 0 «0. Img / m ^ person 'recombinant IL- 1 (0. 0 5 M phosphoric acid The reaction mixture was mixed with a pipette and left on ice for 3 minutes. After adding 250 M of 0.05 M sodium phosphate buffer (PH7.5), it was added to 0.1% BSA (bovin serum albumin, Sigma) in advance. Pre-packed PD-10 column (manufactured by Pharmacia) equilibrated with PBS (phosphato buffered saline) containing 0.05% thimerosal (manufactured by Sigma) ) And counted for each fraction (1 ml / fraction), and the fraction containing 125 IL-1α was stored at 4.
(1 5) 1251 L— 1 αを用いた抗体の力価の測定 (1 5) Measurement of antibody titer using 125 1 L-1 α
ANOC-3 0 1及び組換え抗体をそれぞれ 3%正常ャギ血清と 0. 0 5 %チ メロザール (シグマ社製) を含む PB S (以後抗体希釈剤とする) で 1 m£から 1. 2 5ngZm£まで 2倍ずつ段階希釈し、 それぞれ 1 0 ずつをアツ セィチューブ (ガラスチューブ, 1 0mmX 7 5薩, コ一二ング (Corning)社製) に入れ、 さらに 1 0 0 /·β ( 1 0 0 0 Ocpm)の125I L— 1 αと 30 0 ^の抗 体希釈液を加えて混合した後、 4でで 48時間放置した。 500 /χ£の 2 5%P EG (ポリエチレングリコール, 和光純薬製) を加えて攪拌し、 3 0分間、 t: で放置した。 遠心 (30 0 Orpm, 1 5分, 4 t:) 後、 上清を除いて沈澱物の力 ゥントを r一カウンターで測定した。 ANOC-301 and the recombinant antibody were prepared in PBS (hereinafter referred to as antibody diluent) containing 3% normal goat serum and 0.05% thimerosal (Sigma), respectively. Dilute two times serially to 5 ngZm £, put each 10 into an Assay tube (glass tube, 10 mmX75, manufactured by Corning), and further add 100 / β (10 (Ocpm) 125 IL-1α and an antibody diluent of 300 ^ were added and mixed, and the mixture was allowed to stand at 4 for 48 hours. 500 / χ £ 25% PEG (polyethylene glycol, manufactured by Wako Pure Chemical Industries) was added, stirred, and left for 30 minutes at t :. After centrifugation (300 rpm, 15 min, 4 t :), the supernatant was removed, and the force of the precipitate was measured with an r-counter.
その結果、 5 0 %B/T (B:結合したカウント, T:全カウント) を示す抗 体の濃度を力価として、 表 3に示す。  As a result, the concentration of the antibody showing 50% B / T (B: bound count, T: total count) is shown in Table 3 as the titer.
表 3  Table 3
( 1 6) 抗体の親和性の測定 抗体の親和性を、 競合法による R I A (ラジオィムノアツセィ) により、 下 の通り測定した。 即ちヒト ·組換え I L一 1 を抗体希釈液で 10 jugZm£から 2. 5ngZ?n£まで 2倍ずつ段階希釈し、 それぞれ 100 ^をアツセィチューブ に入れ、 ついで各抗体を抗体希釈液で 50%BZTを示す付近の濃度まで希釈し た液を 100 χ·β、 1251— I L一 1 αを 100 ( 1000 Ocpm).抗体希 釈液を 200 / 加え、 全量を 500 ·2とし、 4でで 48時間反応させた。 そ の後、 500 At «2の 25 %P E G (ポリエチレングリコール, 和光純薬社製) を 加えて攪拌し、 30分間、 4でで放置した後、 遠心 (300 Orpm, 15分, 4 t) し、 上清を除いて沈澱物のカウントを 7"—カウンターで測定した。 (16) Measurement of antibody affinity The affinity of the antibody was measured by RIA (Radio Immunoassay) by the competition method as follows. That is, human / recombinant IL-11 is serially diluted twice from 10 jugZm £ to 2.5 ngZn £ with antibody diluent, and 100 ^ is placed in each tube, and each antibody is diluted with antibody diluent to 50 100 χβ, 125 1-IL-11α 100 (1000 Ocpm) diluted to a concentration near the% BZT, and 200 / diluted antibody diluent to make a total volume of 500 · 2. For 48 hours. Then, add 500 At «2 25% PEG (polyethylene glycol, manufactured by Wako Pure Chemical Industries), stir, leave at 4 for 30 minutes, and centrifuge (300 Orpm, 15 minutes, 4 t). Excluding the supernatant, the count of the precipitate was measured with a 7 "-counter.
その結果よりスカツチャードプロッ トを行ない、 親和性を算出した。 結果を表 4に示す。  Scatchard plots were performed based on the results, and the affinity was calculated. Table 4 shows the results.
表 4  Table 4
産業上の利用可能性 Industrial applicability
本発明の抗ヒト I L一 1組換え抗体を利用すれば、 臨床サンプル等の低濃度の ヒト I L一 1を舍有する検体中の該ヒト I L一 1を正確に測定可能な免疫検定法 による測定手法、 I L一 1の異常産生を伴う各種疾患に対する、 I L一 1の作用 を抑制乃至中和を目的とする医薬品、 ヒト体内 I L一 1の異常産生組織部位等の 画像診断剤等が提供できる。 配列表 Using the anti-human IL-11 recombinant antibody of the present invention, a measurement method by an immunoassay method capable of accurately measuring human IL-11 in a sample having a low concentration of human IL-11 such as a clinical sample In addition, it is possible to provide a pharmaceutical for the purpose of suppressing or neutralizing the action of IL-11 against various diseases accompanied by abnormal production of IL-11, an imaging diagnostic agent for a tissue site of abnormal production of IL-11 in a human body, and the like. Sequence listing
配列番号: 1 SEQ ID NO: 1
配列の長さ : 4 5 3 Array length: 4 5 3
配列の型:核酸 Sequence type: nucleic acid
鎖の数 -二本鎖 Number of chains-double strand
トポロジー:直線状  Topology: straight
配列の種類: c D N A t o mR N A Sequence type: c D N A t o mR N A
配列の特徴: Array features:
特徴を表す記号: C D S  Characteristic symbol: CDS
存在位置: 3 1 , 4 5 3  Location: 3 1, 4 5 3
特徵を決定した方法: E  How to determine the features: E
配列: Array:
GTTAGTCT TAGGCACCAC TTCTTAGACA TC ATG GCT TGG GTG TGG ACC TTG 51  GTTAGTCT TAGGCACCAC TTCTTAGACA TC ATG GCT TGG GTG TGG ACC TTG 51
Met Ala Trp Val Trp Thr Leu  Met Ala Trp Val Trp Thr Leu
-15 - -15-
CTA TTC CTG ATG GCA GCT GCC CAA AGT GCC CAA GCA CAG ATC CAG TTG 99 Leu Phe Leu Met Ala Ala Ala Gin Ser Ala Gin Ala Gin lie Gin Leu CTA TTC CTG ATG GCA GCT GCC CAA AGT GCC CAA GCA CAG ATC CAG TTG 99 Leu Phe Leu Met Ala Ala Ala Gin Ser Ala Gin Ala Gin lie Gin Leu
-10 -5 1  -10 -5 1
GTG CAG TCT GGA CCT GAG TTG AAG AAG TCT GGA GAG ACA GTC AAG ATC 147 Val Gin Ser Gly Pro Glu Leu Lys Lys Ser Gly Glu Thr Val Lys He  GTG CAG TCT GGA CCT GAG TTG AAG AAG TCT GGA GAG ACA GTC AAG ATC 147 Val Gin Ser Gly Pro Glu Leu Lys Lys Ser Gly Glu Thr Val Lys He
5 10 15 . 20 5 10 15. 20
TCC TGC AAG GCT TCT GGG TAT ACC TTC AGA AAC TAT GGA ATG AAC TGG 195 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Asn Tyr Gly Met Asn Trp  TCC TGC AAG GCT TCT GGG TAT ACC TTC AGA AAC TAT GGA ATG AAC TGG 195 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Asn Tyr Gly Met Asn Trp
25 30 35  25 30 35
GTG AAG CAG GCT CCA GGA AAG GGT TTA AAG TGG ATG GGC TGG ATA AGT 243 Val Lys Gin Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Tr l ie Ser  GTG AAG CAG GCT CCA GGA AAG GGT TTA AAG TGG ATG GGC TGG ATA AGT 243 Val Lys Gin Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Tr lie Ser
40 45 50  40 45 50
ACT TAC ACT GGA GAG CCA ACA TAT GCT GAT GAC TTC AAG GGA CGG TTT 291 Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe 55 60 65 ACT TAC ACT GGA GAG CCA ACA TAT GCT GAT GAC TTC AAG GGA CGG TTT 291 Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe 55 60 65
GCC TTC TCT TTG GAA ACC TCT GCC AGC ACT GCC TAT TTG CAG ATC AAC 339 Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gin l ie Asn  GCC TTC TCT TTG GAA ACC TCT GCC AGC ACT GCC TAT TTG CAG ATC AAC 339 Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gin lie Asn
70 75 80  70 75 80
AAT TTC AAA AAT GAG GAC ACG GCT ACA TAT TTC TGT GCA AGA GGG CTC 387 Asn Phe Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Leu  AAT TTC AAA AAT GAG GAC ACG GCT ACA TAT TTC TGT GCA AGA GGG CTC 387 Asn Phe Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Leu
85 90 95 100 85 90 95 100
TAT TAC TTC GGT AG.T AGC TAT GCT TTG GAC TAC TGG GGT CAA GGA ACC 435 Tyr Tyr Phe Gly Ser Ser Tyr Ala Leu Asp Tyr Trp Gly Gin Gly Thr  TAT TAC TTC GGT AG.T AGC TAT GCT TTG GAC TAC TGG GGT CAA GGA ACC 435 Tyr Tyr Phe Gly Ser Ser Tyr Ala Leu Asp Tyr Trp Gly Gin Gly Thr
105 110 115  105 110 115
TCA GTC ACC GTC TCC TCA 453 Ser Val Thr Val Ser Ser  TCA GTC ACC GTC TCC TCA 453 Ser Val Thr Val Ser Ser
120 配列蕃号: 2  120 Sequence Ban No .: 2
配列の長さ : 3 7 2 Array length: 3 7 2
配列の型:核酸 Sequence type: nucleic acid
鎖の数:二本鎖 Number of chains: double strand
トポロジー:直線状  Topology: straight
4 配列の種類: c DN A t 0 mR N A 8 配列の特徴:  4 Sequence type: c DN A t 0 mR N A 8 Sequence characteristics:
特徴を表す記号: C D S  Characteristic symbol: CDS
存在位置: 1 , 3 7 2  Location: 1, 3 7 2
特徴を決定した方法: E  How the features were determined: E
配列: Array:
TCT GCT CAG TTC CTT GGT CTC CTG TTG CTC TGT TTT CAA GGT ACC AGA  TCT GCT CAG TTC CTT GGT CTC CTG TTG CTC TGT TTT CAA GGT ACC AGA
Ser Ala Gin Phe Leu Gly Leu Leu Leu Leu Cys Phe Gin Gly Thr Arg Ser Ala Gin Phe Leu Gly Leu Leu Leu Leu Cys Phe Gin Gly Thr Arg
-15 -10 -5  -15 -10 -5
TGT GAT ATC CAG ATG ACA CAG ACT ACA TCC TCC CTG TCT GCC TCT CTG 96 Cys Asp He Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu TGT GAT ATC CAG ATG ACA CAG ACT ACA TCC TCC CTG TCT GCC TCT CTG 96 Cys Asp He Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu
1 5 10 10  1 5 10 10
GGA GAC AGA GTC ACC ATC ACT TGC AGG ACA AGT CAG GAC ATT AAC AAT 144 Gly Asp Arg Val Thr l ie Thr Cys Arg Thr Ser Gin Asp l ie Asn Asn  GGA GAC AGA GTC ACC ATC ACT TGC AGG ACA AGT CAG GAC ATT AAC AAT 144 Gly Asp Arg Val Thr lie Thr Cys Arg Thr Ser Gin Asp lie Asn Asn
15 20 25 15 20 25
AAT TTA AAC TGG TTT CAA CAG AAA CCA GAT GGA ACT GTT AAA CTC CTG 192 Asn Leu Asn Trp Phe Gin Gin Lys Pro Asp Gly Thr Val Lys Leu Leu AAT TTA AAC TGG TTT CAA CAG AAA CCA GAT GGA ACT GTT AAA CTC CTG 192 Asn Leu Asn Trp Phe Gin Gin Lys Pro Asp Gly Thr Val Lys Leu Leu
30 35 40  30 35 40
ATC TAC TAC ACA TCA AGA TTA CAC TCA GGA GTC CCA TCA AGG TTC AGT 240 l ie Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser  ATC TAC TAC ACA TCA AGA TTA CAC TCA GGA GTC CCA TCA AGG TTC AGT 240 l ie Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser
45 50 55  45 50 55
GGC AGT GGG TCT GGA ACA GAT TAT TCT CTC ACC ATT AGC AAC CTG GAA 288 Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr l ie Ser Asn Leu Glu  GGC AGT GGG TCT GGA ACA GAT TAT TCT CTC ACC ATT AGC AAC CTG GAA 288 Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr lie Ser Asn Leu Glu
60 65 70 75 60 65 70 75
GAA GAA GAT GCT GCC ACT TAC TTT TGC CAA CAG GGT AAA ACC CTT CCG 336 Glu Glu Asp Ala Ala Thr Tyr Phe Cys Gin Gin Gly Lys Thr Leu Pro  GAA GAA GAT GCT GCC ACT TAC TTT TGC CAA CAG GGT AAA ACC CTT CCG 336 Glu Glu Asp Ala Ala Thr Tyr Phe Cys Gin Gin Gly Lys Thr Leu Pro
80 85 90  80 85 90
TGG ACG TTC GGT GGA GGC ACC AAG CTG GAA TTC AAA 372 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Phe Lys  TGG ACG TTC GGT GGA GGC ACC AAG CTG GAA TTC AAA 372 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Phe Lys
95 100 配列番号: 3  95 100 SEQ ID NO: 3
配列の長さ : 3 9 4 Array length: 3 9 4
配列の型:核酸 Sequence type: nucleic acid
鎖の数:二本鎖 Number of chains: double strand
トポロジー:直線状  Topology: straight
配列の種類:他の核酸 Sequence type: other nucleic acids
配列の特徴: Array features:
特徴を表す記号: C D S 存在位置: 1 , 3 9 4 Characteristic symbol: CDS Location: 1, 3 9 4
特徴を決定した方法: Ε  How the features were determined: Ε
配列: Array:
ATG TCT GTC CCC ACC CAA GTC CTC GGT CTC CTG CTG CTG TGG CTT ACA 48 ATG TCT GTC CCC ACC CAA GTC CTC GGT CTC CTG CTG CTG TGG CTT ACA 48
Met Ser Val Pro Thr Gin Val Leu Gly Leu Leu Leu Leu Trp Leu Thr Met Ser Val Pro Thr Gin Val Leu Gly Leu Leu Leu Leu Trp Leu Thr
-20 -15 -10 -5  -20 -15 -10 -5
GAT GCC AGA TGT GAC ATT CAA ATG ACC CAG AGC CCA TCC AGC CTG AGC 96 GAT GCC AGA TGT GAC ATT CAA ATG ACC CAG AGC CCA TCC AGC CTG AGC 96
Asp Ala Arg Cys Asp l ie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Asp Ala Arg Cys Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser
1 5 10  1 5 10
GCA TCT GTA GGA GAC CGG GTC ACC ATC ACA TGT AGA ACA TCT CAG GAC 144 Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Arg Thr Ser Gin Asp  GCA TCT GTA GGA GAC CGG GTC ACC ATC ACA TGT AGA ACA TCT CAG GAC 144 Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Arg Thr Ser Gin Asp
15 20 25  15 20 25
ATC AAC AAC AAC CTG AAC TGG TAT CAG CAG ACA CCT GGA AAG GCT CCT 192 He Asn Asn Asn Leu Asn Trp Tyr Gin Gin Thr Pro Gly Lys Ala Pro  ATC AAC AAC AAC CTG AAC TGG TAT CAG CAG ACA CCT GGA AAG GCT CCT 192 He Asn Asn Asn Leu Asn Trp Tyr Gin Gin Thr Pro Gly Lys Ala Pro
30 35 40  30 35 40
AAG CTG CTG ATC TAC TAC ACA TCT CGT CTG CAT TCT GGA GTC CCT TCT 250 Lys Leu Leu He Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser  AAG CTG CTG ATC TAC TAC ACA TCT CGT CTG CAT TCT GGA GTC CCT TCT 250 Lys Leu Leu He Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser
45 50 55 60 45 50 55 60
AGA TTC TCT GGT TCT GGC TCT GGA ACA GAC TAC ACA TTC ACA ATC TCT 298 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr l ie Ser  AGA TTC TCT GGT TCT GGC TCT GGA ACA GAC TAC ACA TTC ACA ATC TCT 298 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr lie Ser
65 70 75  65 70 75
TCT CTC CAA CCT GAG GAC ATC GCT ACA TAC TAC TGC CAA CAG GGA AAG 346 Ser Leu Gin Pro Glu Asp lie Ala Thr Tyr Tyr Cys Gin Gin Gly Lys  TCT CTC CAA CCT GAG GAC ATC GCT ACA TAC TAC TGC CAA CAG GGA AAG 346 Ser Leu Gin Pro Glu Asplie Ala Thr Tyr Tyr Cys Gin Gin Gly Lys
80 85 90  80 85 90
ACA CTG CCT TGG ACA TTC GGA CAG GGT ACC AAA TTG CAG ATC ACA CGT 394 Thr Leu Pro Trp Thr Phe Gly Gin Gly Thr Lys Leu Gin l ie Thr Arg  ACA CTG CCT TGG ACA TTC GGA CAG GGT ACC AAA TTG CAG ATC ACA CGT 394 Thr Leu Pro Trp Thr Phe Gly Gin Gly Thr Lys Leu Gin lie Thr Arg
95 100 105  95 100 105
配列審号: 4 . Arrangement Board: 4.
配列の長さ : 4 2 3 配列の型:核酸 Array length: 4 2 3 Sequence type: nucleic acid
鎖の数:二本鎖 Number of chains: double strand
トポロジー:直線状  Topology: straight
配列の種類:他の核酸 Sequence type: other nucleic acids
配列の特徴: Array features:
特徴を表す記号: C D S  Characteristic symbol: CDS
存在位置: 1 , 4 2 3  Location: 1, 4 2 3
特徴を決定した方法: E  How the features were determined: E
配列: Array:
ATG GAA TGG AGC TGG GTC TTT CTC TTC TTC CTG TCA GTA ACT ACA GGA 48 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly  ATG GAA TGG AGC TGG GTC TTT CTC TTC TTC CTG TCA GTA ACT ACA GGA 48 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
-15 -10 - 5 -15 -10-5
GTC CAT TCT CAG GTG CAG CTG GTG GAG TCT GGA GGA GGA GTG GTG CAG 96 Val His Ser Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin GTC CAT TCT CAG GTG CAG CTG GTG GAG TCT GGA GGA GGA GTG GTG CAG 96 Val His Ser Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin
1 5 lo - 1 5 lo-
CCT GGA AGA TCT CTG AGA CTG TCT TGT AAG GCA TCT GGA TAC ACC TTC 144 Pro Gly Arg Ser Leu Arg Leu Ser Cys lys Ala Ser Gly Tyr Thr Phe CCT GGA AGA TCT CTG AGA CTG TCT TGT AAG GCA TCT GGA TAC ACC TTC 144 Pro Gly Arg Ser Leu Arg Leu Ser Cys lys Ala Ser Gly Tyr Thr Phe
15 20 25  15 20 25
AGA AAC TAC GGC ATG AAT TGG GTG AAA CAG GCA CCT GGA AAG GGA CTC 192 Arg Asn Tyr Gly Met Asn Trp Val Lys Gin Ala Pro Gly Lys Gly Leu AGA AAC TAC GGC ATG AAT TGG GTG AAA CAG GCA CCT GGA AAG GGA CTC 192 Arg Asn Tyr Gly Met Asn Trp Val Lys Gin Ala Pro Gly Lys Gly Leu
30 35 40 45 30 35 40 45
AAG TGG ATG GGA TGG ATT TCT ACA TAC ACA GGA GAG CCT ACG TAC GCA 240 Lys Trp Met Gly Trp l ie Ser Thr Tyr Thr Gly Glu Pro Thr Tyr Ala AAG TGG ATG GGA TGG ATT TCT ACA TAC ACA GGA GAG CCT ACG TAC GCA 240 Lys Trp Met Gly Trp lie Ser Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
50 55 60  50 55 60
GAC GAC TTC AAG GGA AGA TTC ACA TTT TCT CTG GAC ACA TCT GCA TCT 288 Asp Asp Phe Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Ala Ser  GAC GAC TTC AAG GGA AGA TTC ACA TTT TCT CTG GAC ACA TCT GCA TCT 288 Asp Asp Phe Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Ala Ser
65 70 75  65 70 75
ACA GCT TTC CTG CAG ATG GAC TCT CTG AGA CCT GAG GAC ACA GGA GTG 336 Thr Ala Phe Leu Gin Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val ACA GCT TTC CTG CAG ATG GAC TCT CTG AGA CCT GAG GAC ACA GGA GTG 336 Thr Ala Phe Leu Gin Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val
02ΐ 9Ιΐ Οΐΐ 02ΐ 9Ιΐ Οΐΐ
J9S J9S ΐ¾Λ m Λ JlU 3 "19 13 丄 ιί dsy 13 Ωί 9X9 V3V 9X9 X33 V3V V999V3 V999913VA 3V9J9S J9S ΐ¾Λ m Λ Jl U 3 "19 13 丄 ιί dsy 13 Ωί 9X9 V3V 9X9 X33 V3V V999V3 V999913VA 3V9
501 00T 96 ngq ^ιγュ 丄 JQS ias ^193Hd nsq ^ig 3jy V ] 8 d 丄 8S 9X3 V393VX丄 L 131 V99 dii 3VJ, 3Vi 9XD V99 V9V V3B 1913113VJ, 501 00T 96 ngq ^ ιγ 丄 JQS ias ^ 19 3 Hd nsq ^ ig 3jy V] 8 d 丄 8S 9X3 V393VX 丄 L 131 V99 dii 3VJ, 3Vi 9XD V99 V9V V3B 1913113VJ,
06 98 08 lf600/£6df/JDd 厶 Z O/W O  06 98 08 lf600 / £ 6df / JDd rum Z O / W O

Claims

請 求 の 範 囲 The scope of the claims
1 . L鎖がヒト抗体分子の定常部領域と配列審号: 1に記載のアミノ酸配列のマ ウス抗ヒト I L一 1抗体分子の可変部領域とを有し且つ H鎖がヒト抗体分子の 定常部領域と配列番号: 2に記載のァミノ酸配列のマウス抗ヒト I L一 1抗体 分子の可変部領域とを有することを特徴とするヒト I L一 1に対する組換え抗 体。 1. The L chain has the constant region of the human antibody molecule and the variable region of the mouse anti-human IL-11 antibody molecule having the amino acid sequence described in SEQ ID NO: 1, and the H chain is the constant region of the human antibody molecule. A recombinant antibody against human IL-11, which comprises a variable region of a mouse anti-human IL-11 antibody molecule having the amino acid sequence of SEQ ID NO: 2 and a variable region of the amino acid sequence of SEQ ID NO: 2.
2. L鎖がヒ ト抗体分子の定常部領域と配列審号: 3に記載の了ミノ酸配列のマ ウス抗ヒト I L一 1抗体分子の C D R領域を含むヒト ♦マウスグラフ ト可変部 領域とを有し且つ H鎖がヒト抗体分子の定常部領域と配列蕃号: 2に記載の了 ミノ酸配列のマウス抗ヒト I L一 1抗体分子の可変部領域とを有することを特 徴とするヒト I L一 1に対する組換え抗体。  2. The L chain contains the constant region of the human antibody molecule and the CDR region of the mouse anti-human IL-11 antibody molecule having the amino acid sequence described in 3 above. And a H-chain having a constant region of a human antibody molecule and a variable region of a mouse anti-human IL-11 antibody molecule having an amino acid sequence described in SEQ ID NO: 2. Recombinant antibody to IL-11.
3. L鎖がヒト抗体分子の定常部領域と配列蕃号: 1に記載の了ミノ酸配列のマ ウス抗ヒト I L一 1抗体分子の可変部領域とを有し且つ H鎖がヒト抗体分子の 定常部領域と配列蕃号: 4に記載のァミノ酸配列のマウス抗ヒト I L一 1抗体 分子の C D R領域を舍むヒト ·マウスグラフト可変部領域とを有することを特 徴とする組換え抗体。  3. The L chain has the constant region of the human antibody molecule and the variable region of the mouse anti-human IL-11 antibody molecule having the amino acid sequence described in Sequence No. 1, and the H chain is the human antibody molecule. And a human / mouse graft variable region comprising a CDR region of a mouse anti-human IL-11 antibody molecule having the amino acid sequence described in No. 4: .
4. L鎖がヒト抗体分子の定常部領域と配列番号: 3に記載の了ミノ酸配列のマ ウス抗ヒト I L一 1抗体分子の C D R領域を含むヒト ♦マウスグラフト可変部 領域とを有し且つ H鎖がヒト抗体分子の定常部領域と配列番号: 4に記載のァ ミノ酸配列のマウス抗ヒト I L一 1抗体分子の C D R領域を含むヒト 'マウス グラフ ト可変部領域とを有することを特徴とするヒト I L—1に対する組換え 4. The L chain has a constant region of a human antibody molecule and a human ♦ mouse graft variable region including a CDR region of a mouse anti-human IL-11 antibody molecule having the amino acid sequence described in SEQ ID NO: 3. And that the H chain has a constant region of a human antibody molecule and a human 'mouse graft variable region including a CDR region of a mouse anti-human IL-11 antibody molecule having the amino acid sequence of SEQ ID NO: 4. Characteristic recombination against human IL-1
■抗体。 ■ Antibodies.
5 . マウス抗ヒト I L一 1抗体 L鎖の V領域をコードする配列蕃号: 1で表わさ れる塩基配列を有する D N A。  5. A sequence encoding the V region of the mouse anti-human IL-11 antibody L chain VAN: DNA having the nucleotide sequence represented by 1.
6 . マウス抗ヒト I L—1抗体 H鎖の V領域をコードする配列審号: 2で表わさ れる塩基配列を有する D N A。  6. Sequence identification code encoding V region of mouse anti-human IL-1 antibody H chain: DNA having the nucleotide sequence represented by 2.
7 . マウス抗ヒト I L一 1抗体分子の C D R領域を舍むヒト ·マウスグラフト抗 体 L鎖の V領域をコードする配列番号: 3で表わされる塩基配列を有する 7. It has the nucleotide sequence of SEQ ID NO: 3, which encodes the V region of the L chain of the human / mouse grafted antibody that covers the CDR region of the mouse anti-human IL-11 antibody molecule.
8. マウス抗ヒト I L一 1抗体分子の C D R領域を含むヒト ·マウスグラフト抗 体 H鎖の V領域をコードする配列審号: 4で表わされる塩基配列を有する DNA。 8. Sequence identification code encoding the V region of the human / mouse graft antibody H chain including the CDR region of the mouse anti-human IL-11 antibody molecule: DNA having the nucleotide sequence represented by 4:
9 . ヒト抗体分子の C領域をコードする DN Aと、 配列番号: 1に記載のァミノ 酸配列を有するマウス抗ヒト I L一 1抗体分子の V領域をコードする DN Aと を有する組換え抗ヒト I L一 1抗体 L鎖発現ベクター。  9. A recombinant anti-human having a DNA encoding the C region of a human antibody molecule and a DNA encoding a V region of a mouse anti-human IL-11 antibody molecule having the amino acid sequence of SEQ ID NO: 1. IL-1 antibody L chain expression vector.
10. ヒト抗体分子の C領域をコードする D N Aと、 配列番号: 2に記載のァミノ 酸配列を有するマウス抗ヒト I L一 1抗体分子の V領域をコードする D N Aと を有する組換え抗ヒト I L— 1抗体 H鎖発現ベクター。  10. A recombinant anti-human IL comprising a DNA encoding the C region of a human antibody molecule and a DNA encoding the V region of a mouse anti-human IL-11 antibody molecule having the amino acid sequence set forth in SEQ ID NO: 2. 1 Antibody H chain expression vector.
11. ヒト抗体分子の C領域をコードする DN Aと、 配列蕃号: 3に記載の了ミノ 酸配列を有するマウス抗ヒト I L一 1抗体の C D R領域を含むヒト *マウスグ ラフ ト V領域をコードする DN Aとを有する組換え抗ヒト I L一 1抗体 L鎖発 現ベクター。  11. Encodes a DNA encoding the C region of the human antibody molecule and a human * mouse graph V region containing the CDR region of the mouse anti-human IL-11 antibody having the amino acid sequence described in SEQ ID NO: 3. And a recombinant anti-human IL-11 antibody light chain expression vector having DNA.
12. ヒ ト抗体分子の C領域をコードする D N Aと、 配列蕃号: 4に記載のァミノ 酸配列を有するマウス抗ヒト I L一 1抗体の C D R領域を舍むヒト ·マウスグ ラフト V領域をコードする DN Aとを有する組換え抗ヒト I L一 1抗体 H鎖発 現ベクター。  12. Encodes a DNA encoding the C region of the human antibody molecule and a human-mouse-graft V region covering the CDR region of a mouse anti-human IL-11 antibody having the amino acid sequence described in SEQ ID NO: 4. A recombinant anti-human IL-11 antibody H chain expression vector having DNA.
13. ヒ ト抗体分子の C領域と、 配列番号: 3に記載の了ミノ酸配列を有するマウ ス抗ヒト I L一 1抗体の C D R領域を舍むヒト ♦マウスグラフト V領域とを有 する組換え抗ヒト I L— 1抗体 L鎖をコードする DN A、 及びヒト抗体分子の C領域と、 配列審号: 4に記載の了ミノ酸配列を有するマウス抗ヒト I L一 1 抗体の C D R領域を含むヒト ·マウスグラフト V領域とを有する組換え抗ヒト I L一 1抗体 H鎖をコードする D N Aを有する組換え抗ヒト I L一 1抗体発現 ベクター。 約 13. Recombination having the C region of the human antibody molecule and the human ♦ mouse graft V region that carries the CDR region of the mouse anti-human IL-11 antibody having the amino acid sequence of SEQ ID NO: 3. A human comprising a DNA encoding an anti-human IL-1 antibody L chain, a C region of a human antibody molecule, and a CDR region of a mouse anti-human IL-11 antibody having the amino acid sequence described in SEQ ID NO: 4. · Recombinant anti-human IL-11 antibody having a mouse graft V region and a recombinant anti-human IL-11 antibody expression vector having DNA encoding the H chain. about
L鎖がヒト抗体分子の定常部領域と配列番号: 1に記載のァミノ酸配列のマウ ス抗ヒト I L一 1抗体分子の可変部領域とを有し且つ H鎖がヒト抗抹分子の定常 部領域と配列番号: 2に記載の了ミノ酸配列のマウス抗ヒト I L一 1抗体分子の 可変部領域とを有するヒト I L—1に対するキメラ抗体、 その他のヒト 'マウス キメラ乃至リシユイブト抗体。 The L chain has the constant region of the human antibody molecule and the variable region of the mouse anti-human IL-11 antibody molecule having the amino acid sequence set forth in SEQ ID NO: 1, and the H chain has the constant region of the human antibody molecule. A chimeric antibody against human IL-1 having a region and a variable region of a mouse anti-human IL-11 antibody molecule having the amino acid sequence of SEQ ID NO: 2, and other human'mouse chimeric or recruited antibodies.
本発明抗体を利用することにより、 特に I L一 1の異常産生を伴う各種疾患の 治療や体内 I L一 1産生部位等の画像診断を行なうことができる。  By using the antibody of the present invention, it is possible to perform treatment of various diseases accompanied by abnormal production of IL-11 and image diagnosis of the IL-11 production site in the body.
PCT/JP1993/000941 1992-07-16 1993-07-08 Recombinant antibody against human interleukin-1 WO1994002627A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU45140/93A AU4514093A (en) 1992-07-16 1993-07-08 Recombinant antibody against human interleukin-1

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP18924892A JPH0630788A (en) 1992-07-16 1992-07-16 Recombinant antibody for human interleukin-1
JP4/189248 1992-07-16

Publications (1)

Publication Number Publication Date
WO1994002627A1 true WO1994002627A1 (en) 1994-02-03

Family

ID=16238114

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1993/000941 WO1994002627A1 (en) 1992-07-16 1993-07-08 Recombinant antibody against human interleukin-1

Country Status (3)

Country Link
JP (1) JPH0630788A (en)
AU (1) AU4514093A (en)
WO (1) WO1994002627A1 (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7005561B2 (en) 2000-03-08 2006-02-28 University Of Georgia Research Foundation, Inc. Arabitol or ribitol as positive selectable markers
EP1992636A2 (en) 1999-11-12 2008-11-19 Amgen Inc. Process for correction of a disulfide misfold in Fc molecules
EP2002846A2 (en) 1996-12-06 2008-12-17 Amgen Inc. Combination therapy using an IL-1 inhibitor for treating IL-1 mediated diseases
EP2087908A1 (en) 2001-06-26 2009-08-12 Amgen, Inc. Antibodies to opgl
US7727528B2 (en) 2004-07-22 2010-06-01 Early Detection, Llc Methods for diagnosis using anti-cytokine receptor antibodies
EP2213685A1 (en) 2002-09-06 2010-08-04 Amgen Inc. Therapeutic anti-IL-1R1 monoclonal antibody
EP2366715A2 (en) 2005-11-14 2011-09-21 Amgen Inc. Rankl Antibody-PTH/PTHRP Chimeric Molecules
US10041044B2 (en) 2016-07-29 2018-08-07 Trustees Of Boston University Age-associated clonal hematopoiesis accelerates cardio-metabolic disease development
WO2020035482A1 (en) 2018-08-13 2020-02-20 Iltoo Pharma Combination of interleukin-2 with an interleukin 1 inhibitor, conjugates and therapeutic uses thereof

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2009218069B2 (en) 2008-02-27 2014-05-01 Fuso Pharmaceutical Industries, Ltd. Expression vector for mass production of foreign gene-derived protein using animal cell and use thereof
AU2009331326B2 (en) 2008-12-22 2014-09-18 Fuso Pharmaceutical Industries, Ltd. Expression vector for producing protein derived from foreign gene in large quantity using animal cells, and use thereof
EP3805396A4 (en) 2018-05-24 2022-03-23 National University Corporation Hokkaido University Novel vector and use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62296890A (en) * 1986-03-27 1987-12-24 メディカル リサーチ カウンスル Recombinant dna product and its production
JPS63258595A (en) * 1986-11-13 1988-10-26 Otsuka Pharmaceut Co Ltd Antibody against interleukin-1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62296890A (en) * 1986-03-27 1987-12-24 メディカル リサーチ カウンスル Recombinant dna product and its production
JPS63258595A (en) * 1986-11-13 1988-10-26 Otsuka Pharmaceut Co Ltd Antibody against interleukin-1

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2002846A2 (en) 1996-12-06 2008-12-17 Amgen Inc. Combination therapy using an IL-1 inhibitor for treating IL-1 mediated diseases
EP1992636A2 (en) 1999-11-12 2008-11-19 Amgen Inc. Process for correction of a disulfide misfold in Fc molecules
US7005561B2 (en) 2000-03-08 2006-02-28 University Of Georgia Research Foundation, Inc. Arabitol or ribitol as positive selectable markers
EP2087908A1 (en) 2001-06-26 2009-08-12 Amgen, Inc. Antibodies to opgl
EP3492100A1 (en) 2001-06-26 2019-06-05 Amgen Inc. Antibodies to opgl
EP2277543A1 (en) 2002-09-06 2011-01-26 Amgen, Inc Therapeutic anti-IL-1R1 monoclonal antibody
EP2213685A1 (en) 2002-09-06 2010-08-04 Amgen Inc. Therapeutic anti-IL-1R1 monoclonal antibody
EP3020414A1 (en) 2002-09-06 2016-05-18 Amgen, Inc Therapeutic anti-il-1r1 monoclonal antibody
US7727528B2 (en) 2004-07-22 2010-06-01 Early Detection, Llc Methods for diagnosis using anti-cytokine receptor antibodies
EP2366715A2 (en) 2005-11-14 2011-09-21 Amgen Inc. Rankl Antibody-PTH/PTHRP Chimeric Molecules
EP2816060A1 (en) 2005-11-14 2014-12-24 Amgen Inc. Rankl antibody-PTH/PTHRP chimeric molecules
US10041044B2 (en) 2016-07-29 2018-08-07 Trustees Of Boston University Age-associated clonal hematopoiesis accelerates cardio-metabolic disease development
WO2020035482A1 (en) 2018-08-13 2020-02-20 Iltoo Pharma Combination of interleukin-2 with an interleukin 1 inhibitor, conjugates and therapeutic uses thereof

Also Published As

Publication number Publication date
AU4514093A (en) 1994-02-14
JPH0630788A (en) 1994-02-08

Similar Documents

Publication Publication Date Title
CA2332183C (en) Humanized and chimeric monoclonal antibodies that recognize epidermal growth factor receptor (egf-r); diagnostic and therapeutic use
JP3452062B2 (en) CDR-transplanted antibody
US5888773A (en) Method of producing single-chain Fv molecules
CA2130436C (en) Cloning and expression of humanized monoclonal antibodies against human interleukin-4
AU683836B2 (en) Design, cloning and expression of humanized monoclonal antibodies against human interleukin-5
AU701342B2 (en) Reconstituted human antibody against human interleukin-8
AU618989B2 (en) Improvements in or relating to antibodies
EP0388151A1 (en) Modified antibodies
US6048972A (en) Recombinant materials for producing humanized anti-IL-8 antibodies
WO1994028159A1 (en) Reconstructed human antibody against human interleukin-6
US5958708A (en) Reshaped monoclonal antibodies against an immunoglobulin isotype
US5811522A (en) Recombinant humanized anti-FB5 antibodies
JP2002514421A (en) Antibodies to CD23, their derivatives, and their therapeutic use
US20090220520A1 (en) Recombinant method for the production of a monoclonal antibody to CD52 for the treatment of chronic lymphocytic leukemia
WO1992004380A1 (en) Specific binding agents
WO1994002627A1 (en) Recombinant antibody against human interleukin-1
US20230212273A1 (en) Chimeric antibodies for treatment of amyloid deposition diseases
AU675449B2 (en) Reshaped monocolonal antibodies against an immunoglobulin isotype
WO1995024481A2 (en) Humanized monoclonal antibodies against human interleukin-4
CZ85898A3 (en) Process for preparing humanized antibody, the antibody per se, dna, vector and host cell
BG60687B1 (en) Pecific binding agents
JPH06205693A (en) Granule bonding antibody construction and production and usethereof
JPH09191886A (en) Humanized antibody, semichimera antibody and chimera antibody against human high-affinity ige receptor
AU633284B2 (en) Anti-hcg antibodies
IE911808A1 (en) Recombinant dna and process for the production of chimeric¹antibodies

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA KR US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase