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WO2002006486A1 - Proteine fusionnee a effet antitumoral - Google Patents

Proteine fusionnee a effet antitumoral Download PDF

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Publication number
WO2002006486A1
WO2002006486A1 PCT/JP2000/004389 JP0004389W WO0206486A1 WO 2002006486 A1 WO2002006486 A1 WO 2002006486A1 JP 0004389 W JP0004389 W JP 0004389W WO 0206486 A1 WO0206486 A1 WO 0206486A1
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WO
WIPO (PCT)
Prior art keywords
antibody
cells
chain
polypeptide
variable region
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2000/004389
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English (en)
Japanese (ja)
Inventor
Izumi Kumagai
Toshio Kudo
Masanori Suzuki
Kouhei Tsumoto
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Tohoku Techno Arch Co Ltd
Original Assignee
Tohoku Techno Arch 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.)
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Publication date
Application filed by Tohoku Techno Arch Co Ltd filed Critical Tohoku Techno Arch Co Ltd
Priority to AU2000257080A priority Critical patent/AU2000257080A1/en
Priority to PCT/JP2000/004389 priority patent/WO2002006486A1/fr
Publication of WO2002006486A1 publication Critical patent/WO2002006486A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody

Definitions

  • the present invention relates to a diabody-type multispecific antibody related to cancer-specific therapy, a method for producing the same, and a use thereof.
  • the present invention relates to a fusion protein that specifically binds to both a cell (particularly a bad ttISS cell, that is, a cancer cell) and a cell having cytotoxicity or phagocytosis, and fusion polypeptides constituting the same.
  • the present invention relates to a method for its production and its use, particularly to a pharmaceutical use for antitumor treatment. Height
  • Cancer has been the leading cause of death in Japan since 1981 (evil is the leading cause of death in Japan, but there is no cure at this time. Surgical removal, chemotherapy, radiation therapy, and ⁇ Although marrow therapy is still in development, it has many potential sources, and further progress is expected.
  • Cancer-specific ⁇ 5 therapy refers to a treatment in which cytotoxicity only affects cancer cells. It binds an antibody to a drug that exhibits cytotoxicity and gives the drug its targetivity. In difficult cases, it is also called missile therapy. Difficult to target substances that are abnormally expressed in cancer cells or substances that undergo some changes as the cells become cancerous and that can exert the phagocytosis of antibodies with minimal side effects Research is ongoing. Such is called cancer-related ⁇ 3 ⁇ 43 ⁇ 4.
  • An antibody molecule typically has ⁇ ⁇ ⁇ as shown in FIG. 1, and its basic units are a heavy chain ( ⁇ chain) having two identical sequences and an mM. (Light chain) having two identical sequences. ) Consists of a total of four polypeptides, which are linked by disulfide bonds. Each of these polypeptide chains is folded to form a separate domain.
  • H chain One chain N_3 ⁇ 4i-side region (about 110 residues from N- * ⁇ ⁇ side have amino acid sequence that differs by each antibody Are called variable ⁇ ! Area (VL; V-domain).
  • the V ;! region of the H chain is represented as VH
  • the V region of the L chain is represented as VL.
  • V regions of the H and L chains approach each other to form the binding site.
  • specificity of an antibody is said to be determined by the V region.
  • Fv The smallest unit that consists of a chain of ⁇ : containing one VH and one VL and has the binding activity to is called Fv (X is the Fv domain).
  • VL is: in the L chain of the I chain, the region from amino acid 1 to 107, and in the L chain, in the region from amino acid 1 to 108, VH is the amino group. It is considered to be the 1st to 113th region of the bulging group (Kabat et al., Sequences of Proteins of Immunological Interest, Department of health and Human Services, Fourth Edition, US (1987)).
  • Bispecific antibodies which are one of the multispecific antibodies, can specifically bind to two different antibodies. Research is being actively conducted on the possibility that it can be used as a therapeutic drug with a significant anti-effect.
  • the diabody is the highest / J position of such a bispecific antibody, and utilizes the property that VH and VL derived from the same parent antibody form a heterodimer by non-hybridization with each other. It was done.
  • the present inventors have developed a dibody-type multispecific antibody capable of binding specifically to at least two different Cross-linking between two or more types of cells, specifically at least one of which recognizes the source on the tumor cells, and fc remarks the surface ⁇ of one effector cell involved in cellularity It was found that the design could be used as a therapeutic agent with specific effects.
  • the present inventors have further improved the above-mentioned advantage by imparting a super-diameter function to a diabody multispecific antibody that specifically binds to both cells and cells having cytotoxicity.
  • the present inventors have found something and have led to the present invention.
  • the present invention relates to a diabody-type multispecific antibody that specifically binds to both cells and cells having cytotoxicity.
  • the diadody bispecific antibody can be obtained as a recombinant fusion protein by making full use of fei engineering.
  • the present invention relates to: (i) a first peptide (lb) having the binding site (a) of the variable region of the H chain of the first antibody and the binding site (b) of the variable region of the L chain of the second antibody in the same peptide lb; (2) the ⁇ 3 ⁇ 43 ⁇ 4 binding site (c) of the variable region of the L chain of the first antibody and the ⁇ binding site ((1)) of the ⁇ ⁇ variable region of the second antibody.
  • An insect protein containing a second polypeptide (ii) having the same peptide lb, and having a binding activity at least for both a listening cell and a cell having a cell crane activity; (For example, a pharmaceutical composition), a method for producing the same, and a use such as a method for wounding cancer cells using the same.
  • the first antibody specifically binds to a substance which is expressed in ⁇ -cells or is abnormally expressed in cancer cells, and which undergoes some change with the transformation of cells or cancerous cells.
  • the second antibody specifically binds to ⁇ which is expressed on cells having phagocytosis or cell activity, and (1) the variable region of the ⁇ chain of the first antibody!
  • the present invention relates to (1) the binding site (b) between the super l3 ⁇ 4I (e), the variable region of the H chain of the first antibody (a) and the variable region of the L chain of the second antibody (b) ) On the same peptide chain as the first polypeptide (iii), and (2) the ⁇ binding position (c) of the variable region of the L chain of the first antibody and the H chain of the second antibody. It contains a second polypeptide (ii) having the same peptide at the source position (d) of the variable page area and has at least binding activity to both cells and cells having cytotoxic activity.
  • the present invention relates to a fusion protein whose main purpose is to have it, a composition containing the same (for example, a medical pirates), a method for producing the same, and a method for wounding cancer cells obtained by using it.
  • the present invention relates to a substance specifically expressed in a cell where the first antibody is expressed in a cell, a substance which is abnormally expressed in a cancer cell, or a substance which undergoes a slight change as a result of cell cancer.
  • the second antibody specifically binds to the second antibody expressed on cells having phagocytic or cytotoxic activity, and (1) super-T, (e) and the first A first polypeptide having the same peptide
  • a fusion protein comprising a second polypeptide (ii) having the same as above, and a composition containing the fusion protein.
  • the present invention has applications for the polypeptide constituting the fusion protein, a thread containing the same (for example, mmm, a method for the same, and a method for wounding cancer cells using the same).
  • the present invention relates to: (i) the modification of the H chain of an antibody that specifically binds to * expressed in at * cells: a position (a) in the! Region and the cell ii; A single-chain polypeptide that has the ability to bind to the variable region of the L chain of an antibody that specifically binds (b); The binding position (c) of the variable region of the L chain of the antibody to which it binds and the dangling position (d) of the variable region of the H chain of the antibody that specifically binds to the source expressed in cells having cellular activity (d ) And expressed in (3) Super or) (A) of the variable region of the H chain of the antibody that specifically binds to 53 ⁇ 4i and the variable region of the L chain of the antibody that specifically binds to ffi that is expressed in cells that have cellular activity
  • the present invention provides a single-chain polypeptide characterized by having a 3 ⁇ 43 ⁇ 4!
  • the present invention provides a nucleic acid (including DNA, RNA, etc.) encoding the above fusion protein and the above fusion polypeptide, a replicable cloning or expression vector containing the nucleic acid, and a nucleic acid containing the nucleic acid.
  • the present invention provides a host cell obtained by transposition in the vector, a method for producing the same, and a use thereof.
  • the present invention provides a method for constructing the above-described nucleic acid, transforming a host cell with the nucleic acid, expressing the nucleic acid in the transformed host cell, and preparing the above-mentioned fusion protein and / or the above-mentioned fusion polypeptide.
  • the method of recovering peptides shall be shared.
  • the present invention provides a method of dissociating a polypeptide obtained by a DNA recombination method or the like, re-associating the polypeptide in the next step, and separating and recovering a desired fusion protein.
  • a DNA recombination method or the like re-associating the polypeptide in the next step.
  • separating and recovering a desired fusion protein Provide a special method.
  • the present invention provides a medical device for excluding or eliminating tumor cells such as cancer cells containing the above-mentioned fusion protein and the above-mentioned fusion polypeptide together with a pharmaceutically acceptable carrier and the like. It is intended to provide a method for removing or killing rice spores and a method for treating cancer, which comprises administering a parent product, the above-mentioned fusion protein and the above-mentioned fusion polypeptide to the number of glues.
  • Fig. 1 schematically shows the structure of a typical antibody (immunoglobulin).
  • FIG. 2 depicts the concept of a diabody-type multispecific anti- # 3 ⁇ 4 assay of the present invention.o
  • FIG. 3 schematically shows cell-cell cross-linking by a diabody-type bispecific antibody having a MUSE11 variable region and a 0-3 variable region;
  • Fig. 4 schematically shows the state of intercellular cross-linking by a diabody-type multispecific antibody that has been modified with SEA and further modified with a diabody-type bispecific anti- # construct having a MUSE11 variable region and a 0KT3 variable region. is there.
  • FIG. 5 shows the structure of a vector for expressing a polypeptide constituting the dibody-type multispecific antibody of the present invention.
  • A mSEA-MHOL expression vector
  • B 0HML expression vector
  • Figure 6 shows SDS-polyacrylamide gel electrophoresis (A) and Western-blotting of the insoluble bacterial fraction obtained by expressing the newly constructed vector in E. coli.
  • FIG. 7 shows the results of measurement of cytotoxic activity in the mouth of indigo using a diabody-type multispecific antibody.
  • FIG. 8 shows the results of in vivo measurement of cytotoxic activity using a diabody-type multispecific antibody for mice.
  • the horizontal axis indicates elapsed time, and the vertical axis indicates ⁇ weight.
  • the meaning of the symbol ⁇ on the graph is added in the order of control, only LAK meniscus, diabody, mSEA-diabody. I have. BEST MODE FOR CARRYING OUT THE INVENTION
  • the present invention provides a diabody-type multispecific antibody related to cancer-specific salmon therapy, a fiber method, and a use thereof.
  • the present invention relates to a fusion protein that specifically binds to both mmm (especially bad glutinous cells, ie, cancer cells) and cells having cytotoxicity or phagocytosis, fusion polypeptides constituting the same, It provides manufacturing methods and their applications, especially g3 ⁇ 4 applications for.
  • polypeptide refers to peptides and proteins having two 3 ⁇ 4_h, typically about 10 or more amino acids. They are used herein in their entirety, regardless of whether they have been modified after translation of the mRNAs that encode them or not, and are intended to be used in their entirety. It may be secreted into the ground directly by the transformed cells.
  • the polypeptide of the present invention may be composed of one J3 ⁇ 4h subunit, but is preferably composed of two subunits. The subunits of the fiber may be encoded by the same DNA sequence, or may be encoded by separate DNA sequences.
  • antibody is used in the broadest sense, and in particular, so long as they exhibit the desired biological activity, single monoclonal antibodies, antibody compositions having multiple epitopes, and An antibody fragment (eg, Fv, etc.) may be included.
  • monoclonal antibody refers to an antibody obtained from a substantially homogenous anti-doubt. That is, the individual antibodies that make up the population are identical to each other, except that a small number of potential mutants may be present there. Monoclonal antibodies are highly specific and recognize a single site. In addition, each monoclonal antibody recognizes a single in contrast to a negative (polyclonal) anti-spill product, which typically includes different antibodies to different (epitopes). In addition to their specificity, monoclonal antibodies are synthesized by hybridoma cultivation and other ⁇ Gloplinka ⁇ has advantages in that it is not.
  • “Monoclonal” is meant to indicate an antibody tree as obtained from a substantially homogeneous population of antibodies, and requires production of the antibody by any particular method. Should not be interpreted.
  • the monoclonal antibodies used in accordance with the present invention can be obtained by the hybridoma method first described by Kohler and Milstein, Nature (London) 256: 495 (1975). It can be controlled by recombinant DNA techniques (see, eg, US Patent No. 4,816,567 (Cabilly et al.)).
  • the monoclonal antibodies used in the present invention are described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991). Techniques can also be used to isolate antibodies from phage libraries.
  • Monoclonal antibodies herein are those in which the heavy chain and Z or 3 ⁇ 4
  • the rest of the chain identical or homologous to the corresponding rooster j in the antibody, is identical to the corresponding sequence in the antibody from another species or belonging to another antibody class or subclass Or chimeric "chimeric" antibodies glopurin), as well as fragments of such antibodies (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA • 81). , 6851-6855 (1984)).
  • “Humanized” forms of non-human antibodies include chimeric glopurine, immun! Truncated glopurin chains, or fragments thereof (eg, Fv, Fab, Fab ', F (ab') 2 , or other binding sequences of an antibody), which are minimal sequences derived from non-human globin. Including. Most humanized antibodies contain non-human animals (donor antibodies), such as mice, rats, or egrets, which contain the residues of the complementarity-determining region (CDR) of the recipient. Human ⁇ 3 ⁇ 4globulin (recipient antibody) substituted by residues derived from a CDR having specificity, affinity, and ⁇ ⁇ .
  • donor antibodies such as mice, rats, or egrets
  • humanized antibodies include recipient antibodies and It may contain a system that is not found in any of the introduced CDRs or framework ij. These modifications are made to further refine or eliminate the antibody performance.
  • " ⁇ A humanized antibody comprises at least one, and typically substantially all, of the two variable domains, wherein all or substantially all of the CDR regions are non-human: ⁇ globulin
  • Jones et al. Nature 321, 522-525 (1986); Reichmann et al., Nature 3 32, 323-329 (1988); EP-B-239400; Presta, Curr Op. Struct. Biol. 2, 593-596 (1992); and ⁇ - ⁇ -451216.
  • “Fv” is a “small antibody fragment containing a unique recognition or binding site” ( Figure 2-B). This region consists of one heavy-chain variable domain, one «variable domain, and a The three CDE forces in each variable domain interact with each other to arrange the VH-VL dimer and form a bond on the VH-VL dimer. The six CDRs assemble and share the same binding specificity for the antibody, but only a single variable domain (or Fv containing only three CMs specific to the 3 ⁇ 4). Even though it is an affinity f lower than position 15, it has the ability to recognize, recognize, and bind to ⁇ 3 ⁇ 43 ⁇ 4.
  • Single-chain Fv or “scFv” antibody fragments contain the VH and VL domains of an antibody, wherein the domains comprise a single polypeptide chain. It points to something inside ( Figure 2-C).
  • the Fv polypeptide further contains a polypeptide linker between the VH and VL domains, making it possible to provide the required structure for attachment.
  • the term "diabody” refers to an antibody fragment having two 1 ⁇ 2 ⁇ -positions and a small fragment, and the fragment is located in the variable region (VH) of the ⁇ chain. It contains the variable region (VL) of the linked L chain in its same polypeptide chain (VH-VL).
  • VH variable region
  • VL variable region of the linked L chain in its same polypeptide chain
  • a typical diabody combines the complementarity of another strand by flipping a linker that is too short to form the pair between the two domains on the same strand. Vs. Domain Together, they create two antigen-binding sites.
  • the diabody and its manufacturing technology see US Pat. No. 4,704,692; US Pat. No. 4,946,778; US Pat. No. 5,990,275; US Pat.
  • a “multivalent antibody, multispecific antibody” is a molecule that has binding specificity for at least one type of 3 ⁇ 4. Usually, such a molecule binds to two antigens. Although more specific (ie, bispecific antibodies, BsAbs), antibodies with further specificity, such as trispecific antibodies, are also included in this expression. Among them, it may mean that it further contains a site such as a polypeptide domain capable of forming a specific bond, for example, a superantigen, etc. Among them, a certain form employed in the present invention is obtained by exchanging VH and VL of two different single-chain antibody scFvs respectively (FIGS. 2-D and 2-E).
  • linker or "linker”
  • linker region refers to an oligopeptide or polypeptide that functions to provide a fusion polypeptide by binding a functional polypeptide / domain to a functional polypeptide / domain as described below. pointing.
  • the linker is a peptide linker.
  • the peptide linker is not particularly limited as long as the two polypeptides can be functionally linked to give one fusion polypeptide, and examples thereof include those widely known in the art or It is possible to select and use known linkers modified.
  • the peptide linker may be, for example, a peptide consisting of 1 to about 50 amino acids, preferably a peptide consisting of about 2 to 30 amino acids, more preferably about 2 to 20 amino acids.
  • the term “functionally linked” refers to the proper folding of a polypeptide into the original protein (the polypeptide is Or derived from the original protein), for example, a fusion protein having a three-dimensional structure that can ⁇ S all or all of its functions, such as biological activity.
  • the length of the linker is not particularly limited as long as it gives a desired activity to the competing fusion polypeptide (or fusion protein) depending on the properties of the polypeptide to be bound.
  • the length of the linker should be long enough to allow the fusion polypeptide to be properly folded to obtain the desired biological activity.
  • the length of the linker can also be experimentally determined by testing fusion polypeptides of the desired biological activity, linked by linkers of various lengths. References can be made to documents cited in connection with the above-mentioned die body and its manufacturing technology.
  • the VL at the N-side is the same as that at the VL side, and the one at the VH side (VL-Linker-VH construct), In addition, it may be determined by any one of the phosphoric acids, followed by VL (VH-Linker-VL construct).
  • the Fv domain is typically selected from a monoclonal antibody.
  • Monoclonal antibodies are monoclonal antibodies derived from Bhufu Lfjj, such as mouse monoclonal antibodies and human monoclonal antibodies.
  • Preferred first monoclonal antibodies include: a surface on the surface of a cell, a cancer cell, and a substance or a cell that is abnormally expressed, and a slight change occurs as the cell becomes cancerous.
  • the power attached to a substance is mentioned.
  • the surface of the caffeine cells includes those appearing on the surface of malignant causal cells (cancer cells), and more preferably, human cancer surface or cancer-related.
  • mmmmm includes, for example, those appearing on the surface of adenocarcinoma cells, and more preferably expressed specifically in liver cancer, m, stomach cancer, lung cancer, kidney, bile duct cancer, 3 ⁇ 4i, egg ⁇ i, colon cancer, etc. $ m.
  • Satsuda vesicle surface examples include, for example, UC1 core protein, CEA and the like, and preferably include MUC1 core protein.
  • Representative first monoclonal antibodies include MUC1-specific antibodies, such as UUSE1 1 and so on.
  • Preferred second monoclonal antibodies include those against phagocytic or cytotoxic cell surfaces.
  • cells having phagocytosis or cytotoxicity include ⁇ cells such as “ ⁇ cells, macrophages, and T-LAK cells, and preferably, T-LAK cells that are used as cytotoxic ⁇ cells.
  • ⁇ cells such as “ ⁇ cells, macrophages, and T-LAK cells, and preferably, T-LAK cells that are used as cytotoxic ⁇ cells.
  • the surface of the return vesicle include CD2, CD3, CD4, CD5, CD6, CD8, CD16, CD28, and CD44, and preferably CD3.
  • Examples of the antibody include 0KT3, T3, Leu4, Til, 0KT11, Leu5b, NU-T1, T4, 0T4, Leu3a, NU-TH / I, T8, 0KT8, Leu2a, NU-Ts / c, and the like.
  • Examples of the fusion protein include 0KT3, T3, Leu4, etc.
  • the fusion protein of the present invention has a binding activity to both ⁇ cells and cells having a cell activity.
  • the ⁇ 3 ⁇ 4 ⁇ binding site of the variable region of the H chain of the first antibody (a) and the 3 ⁇ 4 junction of the variable region of the L chain of the second antibody (B) the first polypeptide having the same peptide as position (b) and (2) the variable region of the variable region of the L chain of the first antibody at the source position (c) and the H chain of the second antibody.
  • the second antibody specifically binds to an antigen, and the second antibody specifically binds to a cell expressed by a cell having cell i activity.
  • the first antibody The first peptide having the same peptide i: having the source region of the variable region of the H chain (a) and the second anti-Fan source binding site (b) of the variable H! Region of the human H chain And (2) the same peptide as the binding site (c) of the variable region of the light chain of the first antibody and the antigen binding site (d) of the variable region of the light chain of the second antibody.
  • the fusion protein of the present invention may comprise a superfan source in at least one of the polypeptides comprising it. (SAg) in the same peptide
  • a typical example is a substance capable of specifically activating T lymphocytes.
  • bacterial enterotoxins such as Staphylococcal enterotoxin or its derivatives (eg, SEA and SEB), Escherichia coli enterotoxin, cholera enterotoxin, and mutants and derivatives thereof.
  • Preferred superantigens for constituting the fusion protein of the present invention include, for example, a SEA mutant (mSEA) in which the locus of C class II is reduced to iffi.
  • SEA variants include SEA-D227A (L. Abrahmsen et al., The EMBO Journal 14 (13): 2978-2986 (1995)); U. Holzer et al., Iinmunology 90: 74-80. (1997))
  • the fusion protein thus typically comprises (1) a variable binding site (a) of the superdomain (e) and the variable region of the H chain of the first antibody and a variable region of the L chain of the second antibody.
  • the fusion protein typically comprises (1) the variable ⁇ !
  • the second antibody may contain a second polypeptide having on the same peptide as the abduction source binding site (d) of the variable region of the H chain of the second antibody.
  • a super-chain a variable region of the ⁇ chain of the first antibody, a variable region of the L chain of the second antibody, a variable region of the L chain of the first antibody, and
  • the variable region of the second chain of the second antibody is preferably located via the above linker.
  • the terms "cellular”, “cell line”, and “cell line” culture) "are used interchangeably with each other, and all such designations include their descendants. All descendants are old: by fc or by accidental mutation, It will be appreciated that the DNA content does not have to be exactly the same. It includes mutant progeny that have the same function or biological property as screened for in the originally transformed cell.
  • host cell is a host of a true cell or a host of a primary cell in terms of H3 ⁇ 4.
  • suitable host cells include those described below.
  • transformation refers to the introduction of DNA into an organism, making the DNA available as an extrachromosomal element or as an element integrated into a chromosome.
  • transfection refers to the incorporation of an expression vector by a host cell, whether or not the encoded sequence is actually obscured.
  • transfected host cell and “transformed” refer to the introduction of]) M into the cell. These cells are called “host cells (host ce 11)", even if they are the original machinery! It may be a rice spore. As representative sap cells of the original citric spores, there are various strains of E. coli. Typical host cells of the citrus sac include B. Fu-ho U.S.A., such as Cyanini-Hazhamus-egg fiber sleep sac (CH0 cells) and human saccharus.
  • the introduced DNA sequence can be from the same species as the host cell to a species different from the host cell, and is a hybrid DNA sequence containing foreign DNA and homologous DNA. Atsushi,
  • the terms "replicatable expression vector” and "expression vector” refer to a piece of DNA (usually double-stranded). Good, the DM can help insert foreign DNA fragments into it. Exogenous DNA is defined as MSDNA (heterologous DNA), which is MA that is not found naturally in target host cells. The vector is ffled to carry the foreign DNA or DNA into the appropriate host cell. Once in the host cell, the vector can replicate independently of the host chromosomal DNA, and the vector and several copies of the inserted DNA Can be In addition, vectors contain the elements essential to enable translation of foreign DNA into polypeptide. Therefore, many of the polypeptides encoded by NA) can be rapidly synthesized.
  • MSDNA heterologous DNA
  • vector refers to a DNA sequence that is operably linked to an appropriate control sequence so that it can be expressed in an appropriate host.
  • control sequences include a promoter for transcription, a sequence for any operator to control such transcription, a sequence encoding an appropriate mRNA liposome binding site, and transcription and translation. An array that controls the end of (translation).
  • the vector may be a plasmid, a phage particle, or simply a genomic insert. Once introduced into a suitable host in the form of a KI transfection, the vector may be made independent of or recognizable by the host genome. Further, the vector may be integrated into the genome.
  • Plasmid is the most powerful form of the vector, and is used in the present invention as long as it has a function equivalent to that of the vector. And also those known or those which have become known in the art.
  • control sequence refers to the DNA sequence required to express the coding sequence to which it is operably linked in a particular host.
  • the control sequences that are suitable for the prototype envelope include a promoter, an optional operator sequence, and a ribosome binding site.
  • true cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • isolated nucleus is one which has been immobilized and separated from at least one nucleic acid molecule that is normally associated with the nucleic acid source of the natural antibody.
  • the term refers to such a nucleic acid.
  • an isolated nucleus is one that is different from the form or state found in the world.
  • an isolated nucleon is one that can be distinguished from that present in the natural beta-envelope, for example, a chromosome that is different from the nucleus in the cells of the ⁇ .
  • the nucleus contained in the cell expressing the antibody in the cell may be included in the cell.
  • a nucleic acid is arranged to have a functional return with another nucleic acid sequence, so that the nucleic acid is said to be "operably linked.” This involves binding an appropriate (eg, translattonal activator protein) protein to a regulatory sequence, but in a form that allows expression of the gene. The purpose is to force-couple with the regulatory sequence.
  • an appropriate (eg, translattonal activator protein) protein to a regulatory sequence, but in a form that allows expression of the gene. The purpose is to force-couple with the regulatory sequence.
  • a DNA such as a propeptide sequence or a secretory leader sequence is operably linked to the polypeptide DNA.
  • the promoter sequence is operably linked to the code sequence, and for transcription of the DNA sequence or translation to amino acid, the liposome is added to the coding sequence.
  • the system binding site is operably linked.
  • operably linked means that the DNA sequence to which it is linked is located
  • Oligonucleotides are short length, single or chain polydeoxynucleotides, which can be synthesized by known methods (eg, phosphotribodies using solid phase technology as described in US Pat. No. 266,032). Synthesized chemically by the ester method, the phosphite method, or the phosphoramidite method, or by methods such as those described by Starbucks et al., Nucl. Acids Res. 14, 5399 (1986). Purified on polyacrylamide gel. Several techniques for antibodies are known in the art and have been described in the literature.
  • Such antibody techniques include, for example, the active hybridoma method for producing a monoclonal antibody, a thread-cloning technique for producing an antibody (for example, including a chimeric antibody such as the human antibody), antibody production in transgenic animals. And recently described phage display technology for "fully human” anti- # 1 production. These techniques are briefly described below.
  • a polyclonal antibody against the subject can be made to enter the body of the animal, typically by sizing the assay and adjuvant once, subcutaneously (sc) or intraperitoneally (ip).
  • the thigh is a fragment that contains the target amino acid sequence) and is a protein that is atrogenic in the animal species being transported, such as keyhole lysine, to mosaicin, serum albumin, thyroglobulin, or soybean trypsin inhibitor.
  • bifunctional or degrading agents such as maleimidobenzoylsulfosuccinimide ester (bonded through a cysteine residue), datalaldehyde, succinaldehyde, thionyl chloride, or ⁇ ONR (R and R 1 Is a different alkyl group).
  • the animals were prepared by intradermal injection of a solution of 1 g to 1 mg of the conjugate (for each egret or mouse) with 3 volumes of the complete adjuvant in the oral cavity, and then intradermally injecting the solution.
  • Sexual conjugates or ⁇ invitations ⁇ .
  • the animal was immunized with the conjugate solution in Freund's adjuvant by subcutaneously sizing the fiber using 1/5 to 1/10 the original volume. Is done.
  • the serum titers obtained from the animals are assayed for their antibody titer. Animals receive ⁇ 3 ⁇ 4 ⁇ until the titer reaches a plateau.
  • the animal receives the iiira ⁇ s by a protein that is bound to a different protein that is the same ⁇ and a conjugate that is bound via ⁇ or a different excipient.
  • the conjugate can also be prepared as a fusion protein during incubation from recombinant cells.
  • Consequences U can also be used to enhance delivery response.
  • Monoclonal antibodies may be obtained from a substantially homogeneous population of antibodies using the hybridoma method first described by Kohler & Milstein, Nature 256: 495 (1975), or may be recombinant. (U.S. Pat. No. 4,816,567).
  • mouse or other suitable host cells such as hamsters, are produced as described above and produce lymphocytes that produce antibodies that will specifically bind to the purified protein. Alternatively, it induces lymphocytes capable of expressing the antibody.
  • the lymphocyte cells may be immunized in vitro.
  • Linha is fused with myeloma cells by piercing a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59- 103, Academic Press, 1986)).
  • a suitable fusion agent such as polyethylene glycol
  • Hybridomas prepared in this manner preferably prevent the growth or survival of unfused parental myeloma cells
  • HGPRT hypoxanthanganin phosphoribosyltransferase
  • HPRT hypoxanthanganin phosphoribosyltransferase
  • the medium for the hybridoma will typically grow HGPRT-deficient cells. It will contain the inhibiting substances hypoxanthis aminobuterin and thymidine (HATi ground).
  • Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • the preferred myeloma fine line is the Salk Institute Cell Distribution Center, San
  • the specificity of the binding of the monoclonal antibody by the hybridoma cells is determined by sedimentation or by an in vivo binding assay such as radioimmunoassay (RIA) or medullary atsey (ELISA).
  • RIA radioimmunoassay
  • ELISA medullary atsey
  • the binding affinity of the monoclonal antibody can also be measured, for example, by Scatchard analysis in Munson & Polland, Anal. Biochem. 107: 220 (1980).
  • Hybridomas producing antibodies with the desired specificity, affinity and / or activity Once defined, the clones can then be subcloned by limiting dilution and grown by standard techniques.
  • Suitable culture media include Dulbecco's modified Eagle medium or RPMI-1640 medium.
  • hybridoma cells can be grown in vivo as a test of animal ascites
  • Monoclonal antibodies secreted by the subclone can be derived from medium, ascites, or serum, such as protein A.
  • 3 ⁇ 4g can be used to create transgenic animals (such as mice) that can reap a large repertoire of human antibodies without endogenous immunoglobulin.
  • transgenic animals such as mice
  • the deletion of the anti-species chain I region (JH) of a chimeric mutant mouse in the ⁇ I series was homozygously deleted, indicating that the endogenous antibody was completely inhibited. Have been.
  • Transfer of the human sequence of the immunoglobulin sequence into such live mutant mice will result in the development of human antibodies by antibody challenge (eg, Duchosal et al., Nature 355: 258 (1992). Natl. Acad. Sci.
  • Human antibodies are phage display Bryri (Marks et al., J. Mol. Biol. 222: 581-597 (1991); Hoogenboom et al., J. Mol. Biol. 227: 381 (1991); Hoogenboom et al. , Immunol. Rev. 130: 41-68 (1992); Vaughan et al., Nature Biotech. 14: 309 (1996)).
  • the DNA encoding the antibody can be easily isolated using a conventional method, and further, its sequence can be determined (for example, the weight of mouse antibody can be controlled by co-production). (R. Orlandi et al., Proc. Natl. Acad. Sci. USA 86: 3833-3837). (1993)).
  • the hybridoma cells described above serve as a preferred source of such DNA.
  • cDNA libraries can be isolated from the cDNA library based on sequences described in the literature, by hybridization, or by the polymerase chain reaction (PCR) technique. Once isolated, the DNA is placed in an expression vector, which is then transferred to E.
  • coli cells coli cells, COS cells, Chinese hamster egg bursa (CH0 cells), or a myelogen that does not produce immunoglobulin. It can be transfected into a host cell such as a mammary cell to synthesize a monoclonal antibody in the recombinant host cell.
  • the PCR reaction can be performed by a method known in the relevant field or a method substantially similar to or a modification thereof. For example, R Saiki, et al., Science, 230: 1350, 1985; R. Saiki, et al., Science, 239: 487, 1988; HA Erlich ed., PCR Technology, Stockton Press, 1989; DM Glover et al. ed., "DNA Cloning", 2nd ed., Vol.
  • PCR Protocols a guide to methods and app lications", Academic Press, New York (1990)
  • MJ McPherson, P. Quirke and GE Taylor (Ed.) PCR: a practical approach, IRL Press, Oxford (1991)
  • MA Frohman et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002 can modify or modify it according to the modified method.
  • the PCR method can be performed using a commercially available kit suitable for the PCR method, and the PCR method specified by a kit manufacturer or a kit manufacturer is used.
  • the antibody or antibody fragment is isolated from an antibody phage library using the techniques described in, for example, IfcCafferty et al., Nature, 348: 552-554 (1990). (Clackson et al., Nature, 352: 624-628 (1991) and ark et al., J, Mol. Biol. 222: 581-597 (1991)) describe mouse and human antibodies using phage libraries. There is a description of the isolation of each.) Selection of the appropriate antibody or antibody fragment can be performed using the title source.
  • high-affinity human antibodies are produced by the chain shuffling method (Mark et al., Bio / Technol. 10: 779-783 (1992)).
  • Combinatorial infection and in vivo recombination are also known as techniques for constructing extremely large phage libraries. I have. These techniques are viable alternatives to obtaining monoclonal antibodies by specific hybridoma technology for isolation of the monoclonal antibodies encompassed by the present invention.
  • the human method for non-human antibodies is well known in the art. "In the end, humanized antibodies have one amino acid R_h introduced from a non-human source.
  • Non-human amino expanded fibers are typically obtained from a variable region. Humanization is specifically performed, for example, in Jones et al., Ature 321: 522-525 (1986); Riechman et al., Nature 332: 323-327 (1988); Verhoeyen et al, Science 239: 1534-1536. (1988)) and the like, by substituting rodent CDRs or CDR sequences with the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567).
  • humanized antibodies are typically human antibodies in which several CDR residues have been replaced by residues from analogous sites in rodent antibodies.
  • the humanized antibody is prepared by analyzing the parent sequence and various target human proteins using the 3 ⁇ immunoglobulin model of the humanized sequence according to a preferred method. Force adjusted. Three-dimensional immunoglobulin models are well known to those skilled in the art. A computer program is available that exemplifies and displays the three possible configurations of the selected immunoglobulin roster. By observing these 3 ⁇ 4 ⁇ , the role of the residue in the immunoglobulin sequence of the immunoglobulin can be identified. It is possible to analyze the residues that appear in the combined results. In this way, the residues can be selected from the consensus sequence and the introduced sequence, and can bind, and can have the desired antibody characteristics, such as increased affinity for the target 3 ⁇ 4. For further details, 1) 92/22653 can be referred to.
  • An “original nucleic acid” can be modified to encode an i-fusion peptide (ie, an engineered force, mutagenized) and an interesting polypeptide of interest.
  • the original nucleic acid can be a naturally occurring nucleic acid or has been previously modified (eg, a humanized antibody fragment).
  • nucleic acids may contain nucleic acids.
  • the "" of a nucleic acid means that it is mutated by insertion, iron or substitution in at least one codon which encodes the target amino acid labile group in the original nucleic acid. Normally, the codon that codes for the original amino expansion is used for the codon that codes for the amino expansion to be introduced. This is done by substitution.
  • substitution Such techniques for artificially modifying DNA in such a manner are described in a review in Mutagenesis: a Practical Approach, MJ Mcpherson (Ed.), (IRL Press, Oxford, 11 (1991)). This includes, for example, the site-specific mutation method (site-specific mutation method), the force-set mutation method, and the polymerase chain reaction (PCR) mutation method.
  • the oligonucleotide-mediated mutagenesis method is a preferred method for making substitution mutations of DNA encoding a polypeptide.
  • This technique is well known in the art, as described by Adelman et al., DNA, 2: 183 (1983). Briefly, the method involves hybridizing an oligonucleotide encoding the desired mutation to a single-form DNA template of a plasmid or pacteriophage containing the unmodified or native DNA sequence of a given portion of the antibody.
  • the polypeptide is modified by soybean.
  • the DNA polymerase is used to incorporate the oligonucleotide primers and synthesize the entire phase capture chain of the template that will encode the selected DNA in the antibody or target fusion polypeptide DNA. Is displayed.
  • the cassette mutation method is described in, for example, Well et al., Gene 34: 315 (1985). This can be done by replacing the region of the paired DNA with a heterologous fragment made by annealing to a unique oligonucleotide.
  • the PCE mutagenesis method is also used to generate mutants of polypeptide DNA.
  • reference is made to DNA, but this technology can be applied to RNA as well.
  • PCR technology is described in ⁇ d in, for example, Erlich, Science, 252: 1643-1650 (1991).
  • Polypeptide linkers may be synthesized by synthetic techniques, such as, for example, recombinant techniques, in vitro peptide synthesis, techniques such as enzymatic or chemical introduction of amino swelling groups by peptide bonds, or any combination of these techniques. Allows for example to be introduced into the part between L and VH. Therefore, the introduced linker is
  • NA is expressed using recombinant techniques that are widely reversible in this field.
  • selected genes include mammalian LIII cell expression vectors and hosts so that the fusion polypeptide is properly glycosylated (eg, if it contains a glycosylated antibody region).
  • the recombinant Fv contains VH and VL, and is obtained by co-expressing two chains and secreting them from the product).
  • the host cell will contain DNA or multiple polypeptides encoding both the first and second polypeptides, either on a single vector or on a separate vector.
  • Form SI is replaced by other DNA encoding a polypeptide necessary for forming a multimer composed of It is also possible to express the first polypeptide and the second polypeptide in independent suspensions, and to bind the externally expressed polypeptide in vitro.
  • a nucleic acid eg, cDNA or genomic DNA
  • a vector vector typically includes, but is not limited to, one of the following Jt elements: signal sequence, origin of replication, one or more marker elements fe ?, enhancer element, promoter, and Transcription termination sequence.
  • the fusion polypeptide can be expressed as a signal polypeptide or a fusion polypeptide with a mature protein or another polypeptide having a truncated Dfg position specific to the N- of the polypeptide.
  • the signal sequence can be a vector, a component of DNA, or the DNA of a vector to be inserted into a vector.
  • Target to be selected The signal sequence is preferably one that is recognized and processed by the host cell (ie, cleaved by signal peptide).
  • the signal sequence may be, for example, a prokaryotic signal selected from the group consisting of alkaline phosphatase, penicillinase, lpp, or thermostable progenitor enterotoxin II.
  • the signal sequence may be, for example, yeast invertase leader, alpha factor leader (Saccharomyces and Kluyveromyces) one-factor leader (US Pat. No. 5,010,182), or It may be replaced by an acidic phosphatase leader, C. albinase dullcoamylase reader (EP362, 179), or a signal described in TO90 / 13646.
  • yeast invertase leader alpha factor leader (Saccharomyces and Kluyveromyces) one-factor leader (US Pat. No. 5,010,182)
  • It may be replaced by an acidic phosphatase leader, C. albinase dullcoamylase reader (EP362, 179), or a signal described in TO90 / 13646.
  • the natural signal sequence eg, the predecessor sequence of the antibody that normally governs the secretion of these from human cells in vivo
  • DM of such a precursor region is ligated in reading frame to DNA encoding a polypeptide forming a multimeric protein of interest.
  • Both cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence allows the vector to replicate independently of the host chromosome DM and includes the origin of replication or a self-replicating sequence.
  • Such sequences are well known for various bacteria, yeasts and viruses.
  • the replication origin from plasmid pBE322 is suitable for most Gram-negative bacteria, the 2 / plasmid origin is suitable for yeast, and various viral origins (SV40, poliooma, adenovirus, VSV or BPV) is useful for cloning vectors in mammalian cells.
  • the origin of the replication component is not required for the ftfj expression vector (the SV40 origin is typical because it contains a shelf promoter).
  • the source and cloning vector must contain a selection, also referred to as a selectable marker.
  • Typical selections include (a) substances such as ampicillin, neomycin, methotrexate or tetracycline or other substances; Conferring resistance to toxins, (b) attracting M members, or (C) obtaining from a complex medium, such as a feline encoding for example)) -alanine racemase from Bacillus. Encode proteins that supply important nutrients that are not available.
  • One example of a selection scheme shelves IJ to kit the growth of host cells. Those cells, which have been exposed behind them by difficulty, show proteins that confer drug resistance, and thus
  • neomycin Survive in selective culture. Examples of such vertical selection include neomycin (Sout hern et al., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid (Muligan et al., Science 209: 1422 (1980) )) Or hygromycin (Sugden et al., Hoi. Cell. Biol. 5: 410-413 (1985)).
  • the three examples above show the use of bacteria and tt under the control of difficulties, respectively, to confer resistance to the appropriate drug, G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin. You.
  • Another example of appropriate selection of mammalian LK cells is to identify cells capable of selectively picking up a given polypeptide nucleic acid, such as DHFR or thymidine kinase. It is assumed that. Transformation of the baby can be done by picking up the marker and selecting a unique adaptation for the transformant to survive.
  • the selection string is subjected to conditions in which the selection IJ key in the medium is sequentially changed, which leads to the amplification of both the selection gene and the DNA encoding the predetermined polypeptide. It is loaded by culturing the transformants.
  • the amplified amount of a given polypeptide is synthesized from the amplified]) NA.
  • amplifiable genes include metallothionein I and II, preferably transcribed metallothionein, adenosine deminase, orditin decarboxylase, and the like.
  • cells transformed by the DHFE selection method can be obtained by first culturing all of the transformed forms in a medium containing DHFE-treated methotrexate (MTX). Identified. Suitable host cells, employing natural 5 » DHFR activity prepared and grown as described in Urlaub and Chassin, Proc. Natl. Acad. Sci. USA 77: 4216 (1980). It is a CH0 cell that has high blood pressure. The cells transformed with form SI are then exposed to increasing 'S methotrexate.
  • MTX methotrexate
  • This amplification technique employs, for example, a mutant DHFR gene, which is highly resistant to MTX, regardless of the presence of endogenous DHFR, such as ATCC No. CCL61 CH0-K1. It can be used as a suitable host (EP117,060).
  • APH aminoglycoside 3'-phosphotransferase
  • Transformed host cells are cultured in a medium containing selection markers such as kanamycin, neomycin, or an aminoglycoside ⁇ substance such as G418. (See US Pat. No. 4,965,199).
  • ⁇ ffl in yeast is the trpl residue present in yeast plasmid Y3 ⁇ 4) 7 (Stinchcob et al., Nature 282: 39 (1979); Kingsman et al., Gene 7: 141 (1979); or Tschemper et al., Gene 10: 157 (1980)).
  • the trpl residue gives a selection marker for yeast varieties lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977 )) 0 Damage to trpl in the yeast host cell genome, in the absence of tryptophan power, provides a useful job for detecting transformation by growth.
  • Leu2-deficient yeast strains (ATCC 20, 622 or 38, 626) is I the Leu2 heritage fe ⁇ 1: is the trapped by plasmid of ⁇ to ⁇ .
  • vectors derived from the 1.6 circular plasmid pKDl can be used to transform Kluyvercces yeast (Bianchi et al., Curr. Genet. 12: 185 (1987)). More recently, a report has been published on a practice (K. lactis) for producing a recombinant calf chymosin as a model (Van den Berg, Bio / Technology 8: 135 (1990)). A stable multicopy expression vector for secretion of recombinant mature human serum albumin by an industrial strain of Kluyveromyces is also known (Fleer et al., Bio / Technology 9: 968-975 (1991)). ).
  • the cloning vector is recognized by the 3 A promoter operably linked to the peptide nucleic acid. There are many promoters that are recognized by various potential host cells. These promos
  • —Yuichi is functionally transferred to a fusion polypeptide-KDNA by removing the promoter from the source DNA by restriction digestion and inserting the isolated Promote-One sequence into the vector. .
  • Suitable promoters for ⁇ ffl with prokaryotic hosts are the lactose and lactose promoters (Chang et al., Nature 275: 615 (1978); and Goed del et al. , Nature 281: 544 (1979)), Alkaline phosphatase, Tributofane (trp) Promote overnight (Goeddel, Nucleic Acids Res. 8: 4057 (1980) and ⁇ 36,776) and tac promoter Hybrid promoters (deBoer et al., Proc. Natl. Acad. Sci. USA 80; 2125 (1983)).
  • lactose and lactose promoters Chang et al., Nature 275: 615 (1978); and Goed del et al. , Nature 281: 544 (1979)
  • Alkaline phosphatase Alkaline phosphatase
  • trp Tributofane Promote overnight
  • fusion polypeptide can be functionally linked to the coding MA (Siebenlist et al., Cell 20: 269 (1980)). Promoters found in bacterial systems encode fusion polypeptides.]) Shine-Dalgarno (SD) sequences operably linked to NA will also be included.
  • the power of a promoter arrangement is also used.
  • Virtually all eukaryotic remains, iS ⁇ have an AT-rich region located approximately 25-30 bases upstream from the transcriptional site.
  • Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is the CXCMT region (X can be any nucleotide).
  • the 3 '* i ⁇ of most eukaryotic f's is an MTAAA sequence, which can be a signal that activates a poly A till in the 3' of the coding system. All of these sequences are suitably inserted into eukaryotic expression vectors.
  • promoter overnight sequences for use by yeast hosts include the promoter for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073 (1980)) or other plasmids. (Hess et al., J. Adv. Enzyme Reg.
  • yeast promoters having an oral IJ point whose transcription is controlled by growth conditions, include inducible promoters such as alcohol dehydrogenase 2, isocytochrome, and acid phosphatase. And glyceraldehyde-3-glycophospho-dehydrogenase, as well as maltoses and galactoses (promoter domains involved in j). Suitable vectors and promoters for use in expression are described in Hitzeman et al., ⁇ 73, 657 ⁇ Yeast enhancers are also advantageously used with yeast promoters.
  • ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ . ⁇ . derived from viral genomes such as sipperpia virus, avian sarcoma virus, cytomegalovirus, retrovirus, B lysitis virus and most preferably Simian virus 40 (SV40), such as actin promoter or immunoglobulin promoter. It is controlled by a promoter obtained from one of the natural promoters or the heat shock promoter.
  • SV40 Simian virus 40
  • the early or late promoter of the SV40 virus is conveniently obtained as a restriction fragment of SV40, which also contains the SV40 viral replication origin (Fiers et al., Nature 273: 113 (1978); Mulligan and Berg, Science 209: 1422-). 1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci. USA 78: 7398-7402 (1981)).
  • the immediate 3 promoter of human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment (Greenaway et al., Gene 18: 355-360 (1982)). DNA generation in mammalian hosts using Dipapis monovirus as a vector is disclosed in US Pat. No. 4,419,446.
  • Examples include the cytomegalovirus promoter enhancer, the SV40 enhancer (bplOO-270) on the late side of the copy origin, the polio enhancer located on the late side of the copy origin, and the adenovirus enhancer.
  • the enhancer is the force that can be spliced into the vector at the 5 'or 3' position of the stomach-binding polypeptide ij, preferably at the 5 'site of the promoter.
  • Construction of an appropriate vector containing one of the above J3 ⁇ 4_h components uses standard concatenation techniques.
  • the isolated plasmid or DNA fragment is cleaved, paired, and religated in the form required to generate the required plasmid.
  • the transformed mixture was used to transform the 294 strain of E. coli K12 (ATCC 31, 446), followed by the successful transformation.
  • the recombinant is appropriate, select it for ampicillin or tetracycline resistance.
  • Plasmids were prepared from the transformants, analyzed by restriction endonuclease, and analyzed by the methods of Z or Messing et al., Nucleic Acids Res. 9: 309 (1981), or Maxam et al., Methods in Enzymol ogy 65. : 499 (1980).
  • ⁇ ⁇ expression involves the use of expression vectors that allow efficient replication in the host cell, and the host cell can burn multiple copies of the expression vector, Then, the desired polypeptide encoded by the expression vector is synthesized at a high level.
  • Temporary expression of i ⁇ with suitable expression vectors and host cells is convenient and allows identification of whether the polypeptide encoded by the cloned DNA is live or not, and provides the desired binding specificity. It enables rapid screening of fusion polypeptides having a Z-binding affinity.
  • fusion polypeptide is expressed as a peptide
  • a particularly useful plasmid for this is pRK5 (EP 307,247) or 3 ⁇ 168091/08291).
  • adenovirus EIA-plasmid transduction 293 human embryonic kidney cells can be transfected with pRK5-based vector by modification of the calcium phosphate method using pRK5-based vector, which is efficient. Allows expression of the fusion polypeptide.
  • CDM8-based vectors can be used to transfect COS cells by the DEAE-dextran method (Aruffo et al., Cell 61: 1303-1313 (1990); Zettmeissl et al. , DNA Cell Biol.
  • the desired fusion polypeptide can be expressed after stable transfection of the host cell, where the desired protein is desired.
  • pRK5-based vectors encode dihydrofolate reductase (DHFR) and can be introduced into CH0 cells in the presence of oral plasmids that confer resistance to G418.
  • G418 resistant clones can be selected for cultivation. When these clones are cultured under increasing amounts of DHF and the harmful agent methotrexate, the numbers of copies of DHFE and the fusion polypeptide sequence encoding both are amplified. The clone is selected there.
  • fusion polypeptide or ** contains a ** leader sequence, it will be cleaved and secreted by the transfected cells.
  • Expression of a fusion polypeptide with a more specific structure may require a uniquely adapted host cell.
  • components such as fiber or heavy chains may be provided by certain myeloma or hybridoma host cells (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84: 2936-2940). (1987) and "Martin et al., J. Viro 1.67: 3561-3568 (1993)).
  • Progenitors suitable for this purpose include cells, yeast, or other higher eukaryotic cells; true IE bacteria, such as Gram-negative or Gram-positive substances, eg, Enterobacteriaceae, eg, coli, etc.), Proteus, Proteus, Enterobacter, Klebshe Lale (Klebsiella), Erwinia (Erwinia), Salmonella 'Shimoleum (Salmone 11a 1; yphimurii), etc., Sanoremo shira, Serachi (Serachi), Serenosias (Serratia marces cans), etc., Serratia (Shigella), Bacillus subtilis subtilis and Bacillus licheniformis; Pseudomonas aeruginosa; Streptomyces; and Bacillus such as Bacillus licheniformis.
  • true IE bacteria such as Gram-negative or Gram-positive substances, eg, Enterobacteriaceae
  • E. coli M101 strain, coli 294 strain force is (ATC C 31, 446) E. coli B s E. coli X1776 (ATCC 31, 537), E. coli W3 110 ( ATCC 27, 325), E. coli XL-1 (Stratagene), ⁇ coli HB101, coli title 522, I coli NM538, K_ coli NM539, coli DH5, _ coli DH10B, K_ coli J109 and others. It is suitable. These examples are illustrative and not limiting.
  • E. coli strain W3110 is a preferred host strain for recombinant DNA fermentation, and is a particularly preferred host or parent host.
  • the host cells should secrete minimal prokaryotic enzymes.
  • the strain W3110 can be modified to produce a genetic mutation in the protein coding region, as in the case of the ⁇ coli W3110 27C7 strain (ATCC 55, 244). it can.
  • the ⁇ : na form of the 27C7 strain is tonA mu tpr3 phoAAE15 A (argF-lac) 169 ompT mu deg P41karf.
  • a coli strain having a mutated periplasmic protease disclosed in US Pat. No. 4,946,783 can be used.
  • cloning methods for PCR or other nucleic acid polymerase reactions are also suitable.
  • fungi such as filamentous fungi or yeast are also cloning and expression hosts for fusion polypeptide coding vectors.
  • Saccharomyces cerevisiae or normal / ⁇ . Yeast is the most commonly used ⁇ ! Among lower W host organisms.
  • Schizosaccharomyces pombe (Beach and Nurse, Nature 290: 140 (1981); EP 139, 383); Kluyveromyces host ( U.S. Pat. No. 4,943,529; Fleer et al., Bio / Technology 9: 968-975 (1991)), e.g., K. lactis (MI98-8C, CBS683, CBS4574; Louvencourt et. al., J.
  • K. drosophilarum ATCC 36, 906; Van den Berg, Bio / Technology 8: 135 (1990)
  • K. fragilis ATCC 12,424)
  • K. walti ATCC 56, 500
  • K. bulg aricus ATCC 16, 045
  • K. wickeramii ATCC 24, 178
  • K. K. thermotolerans and K. marxianus
  • yarrowia EP 402, 226
  • Pichia pastoris EP 183, 070; Sreekrishna et al., J. Basic Microbiol.
  • Host cells that are deficient in the expression of glycosylated fusion polypeptides are derived from the multipartite ⁇ -family. Such host cells are capable of difficult processing and glycosylation. In principle, any higher cell culture, whether spinal or invertible, can be used. Examples of invertebrate cells include plants and insects. Many baculovirus strains and variants and corresponding acceptable insect host cells, such as Spodoptera frugiper da (imomushi), Aedes albopictus (mosquito), Aedes aegypti aegypti) (mosquitoes), Drosophila melanogaster (Pa'e), and Bombyx mori have been strongly identified.
  • plant cell cultures such as cotton, corn, potato, soybean, petunia, tomato, and tobacco can be shelved.
  • plant cells are transfected by culturing with certain strains of the bacterium, Agrobacteriura tumefaciens, which has been previously engineered to contain the fusion polypeptide DNA.
  • Agrobacteriura tumefaciens which has been previously engineered to contain the fusion polypeptide DNA.
  • DNA encoding the fusion polypeptide is transferred to the plant host and transfected during the culture, and under appropriate conditions, the fusion polypeptide is transfected. Will express peptide DNA.
  • control and signal sequences suitable for plant cells are available (D-marker icker et al., J. Mol. Appi. Gen. 1: 561 (1982)).
  • DNA fragments isolated from the upstream region of T-DNA 780 can activate or increase plant-expressible transcript levels in recombinant DNA-containing plant tissues. (EP 321, 196).
  • a preferred host is a vertebrate cell, and it has recently become obsolete to grow vertebrate cells by culture (Kruse and Patterson (Ed.), Tissue Culture, Academic Press, (1973)).
  • Examples of useful honeymoon host Hozukito include monkey kidney cells (CV1, ATCC CCL70); monkey kidney CV1 transformed with SV40 (COS-7, ATCC CRL1651); (VER0-76, ATCC CRL-1587); human embryonic kidney system (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977). )); Fetal hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovum-DHFR (CH0, Urlaub and Chasin, Proc. Natl.
  • Mouse celery cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); mouse ⁇ ( ⁇ 060562, ATCC CCL51); dog kidney cells (MDCK, ATCC CCL34); (BRL3A, ATCC CRL1442); human cervical malignant cells (HeLa, ATCC CCL2); human lung cells (W138, ATCC CCL75); human liver cells (Itep G2, HB 8065); TRI cells (Mather et al.,
  • the host cell is transfected with the expression vector or cloning vector of the present invention to induce a promoter, to select a form of lightning complex, or to encode a desired sequence.
  • Culture is performed in a conventional ftt medium that has been appropriately modified for amplification. Transfection is performed using standard techniques appropriate for such cells, depending on the host cell to be treated. J. Sambrook, EF Fritzsch & T. Maniatis, 'Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) Use calcium chloride as described in section 1.82. Calcium treatment, or electroporation, used for prokaryotic cells or substantially thighs and other cells with ivy barriers. "Infection by Agrobacterium ummefaciens is used by Shaw et al. al.,
  • plants As described in Gene 23: 315 (1983) and WO 89/05859, it is shelved in certain types of planted cells. In addition, plants may be transfected through sonication, as described in WO 91/00358.
  • the mammalian host cells used to produce the fusion polypeptide polypeptides of the present invention can be cultured in various media.
  • Commercially available media such as HamF10 (Sigma), top, ⁇ 1 medium (MEM, Sigma), RPMI-1640 (Sigma), Dulbecco's ⁇ ⁇ ⁇ ⁇ (DMEM, Sigma) Ffil for host cell culture.
  • 4,560,655; W0 90/03430; W0 87/00195; U.S. Reexamination Patent No. 30,985 or U.S. Patent No. 5,122. , 69 can also be used as a culture medium for host cells (the disclosure of the above-mentioned documents is incorporated herein by reference thereto). ). If necessary, all of these media may contain hormones and / or other growth factors (insulin, transferrin, or long factor, (such as sodium salt, calcium, magnesium, and phosphorus), buffers (HEPES, Tris, etc.).
  • growth factors insulin, transferrin, or long factor, (such as sodium salt, calcium, magnesium, and phosphorus), buffers (HEPES, Tris, etc.).
  • Nucleosides such as adenosine and thymidine
  • substances such as gentamicin TM
  • m usually defined as inorganic compounds that are present at the end of the micromonetary range
  • glucose or equivalent energy sources Any other necessary insecticide may be included in the media as appropriate to the skilled artisan, and the culture conditions, such as pH, may vary depending on the sleep conditions selected for expression. The condition of the parcel has been done before and will be apparent to those skilled in the art.
  • the nucleic acids, polypeptides, proteins, etc., such as DNA and RNA described in this specification can be synthesized by applying a method widely known in the art.
  • the fusion polypeptide obtained by expression or the like can be recovered.
  • the fusion polypeptide is recovered from the medium as a polypeptide secreted into " ⁇ , but may be recovered from host cell lysates if it has been made directly without a secretion signal. If the fusion polypeptide is membrane-bound ft ⁇ r, remove the membrane from the membrane using a suitable detergent (eg, Triune-X100).
  • the fusion polypeptide When the fusion polypeptide is made in a recombinant cell other than a human-derived cell, it contains no human-derived protein or polypeptide. However, to obtain a homogenous preparation of the fusion polypeptide, the fusion polypeptide may be purified from recombinant cellular proteins or polypeptides. As a first step, medium or lysate is usually centrifuged to remove particulate cells.
  • the antibody fusion polypeptide is suitably purified by hydroxylapatite chromatography, gel electrophoresis, spinning, or affinity chromatography.
  • affinity chromatography is one of the preferred purification techniques.
  • ion exchange columns fractionation on ion exchange columns, ethanol ', reversed-phase HPLC, chromatography on silica, chromatography on heparin sepharose, anions
  • cationic resin chromatography such as a polyaspartic acid column
  • chromatofocusing SDS-PAGE
  • ammonium sulfate can also be used depending on the polypeptide to be recovered.
  • the carrier to which the affinity ligand is attached can be many: ti ⁇ agarose, and other carriers are available.
  • Conditionally stable carriers such as porous glass or poly (styrenedivinyl) benzene, allow for faster processing times and shorter processing times than agarose. Once a suitable ligand is selected, the desired fusion polypeptide can be directly and efficiently bound from the culture solution.
  • two polypeptides that associate to constitute a protein This can be used to separate domains into individual polypeptides, or to keep associated domains apart from each other.
  • This is referred to as “horn type” processing.
  • the dissociation process can be carried out preferably using a dissociating agent.
  • the return J include those widely known in the art, for example, alcohols such as ethanol, guanidine salts, and urea.
  • Other horns? Reasonable ones include surfactants or similar reagents that have the function of interfering with the ffit of protein conformation, and those skilled in the art are familiar with them.
  • a combination of guanidine salt and ethanol can be used as a dissociating agent.
  • 0-50 vol% ethanol, 0-2.0 mol / L (M) guanidine salt can be ⁇ ffl. More preferably, it is 10 to 30% by volume of ethanol and 0.5 to 1.0 mol / L guanidine salt ⁇ , and more preferably, 20% by volume of ethanol and 0.5M guanidine salt.
  • An example of a buffer for dissociation treatment is 0.5 g guanidine salt ⁇ , 20% by volume of ethanol, 0.05 M Tris and 0.01 M CaCl 2, and an aqueous solution of H 8.0. No.
  • the association process may mean returning the dissociated polypeptide or domain to the proper configuration.
  • the association treatment also has the meaning of returning polypeptides or domains to an associated state, and thus can be referred to as "reassociation", or reassociation in the sense of having a desired biological activity. It can also be called a configuration, and may be called refolding.
  • the associating treatment can be performed by bringing the polypeptides or the domains together in a suitable arrangement in a suitable buffer solution and associating with each other.
  • the buffer include an aqueous solution of pH 7.5 containing 0.04 M M0PS and 0.10 M calcium acetate.o
  • the buffer is suitable for re-associating VH and VL, Anything known to those skilled in the art can be used without limitation.
  • an “isolated” fusion polypeptide or fusion protein of the present invention refers to a fusion polypeptide or fusion protein that has been identified and isolated from and / or recovered from its native microbial culture.
  • Naturally occurring substances are substances that may interfere with the diagnostic or therapeutic conversion of the fusion polypeptides or fusion proteins of the invention, including proteins, hormones, and many proteinaceous or non-proteinaceous substances. Tan May contain parkinous solutes.
  • the fusion polypeptide or fusion protein may be (1) up to 95% or more, or most preferably more than 99%, in the protein difficulty at J ⁇ as measured by the Lowry method.
  • the N- N or internal amino acid sequence is at least 15 sq.m., or (3) Coomassie or Preferably, it will be purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions, preferably using IS3 ⁇ 4 color.
  • the fusion polypeptides of the present invention are purified to "substantially substantially homogeneous. Phrases such as” substantially homogeneous, “" substantially homogeneous form, “and” substantial homogeneity. " Are shown to indicate that they are substantially devoid of by-products or by-products from the combination of unwanted polypeptides. As represented by the key, substantial homogeneity means that the amount of by-products does not exceed 10% in percent, preferably less than 5%, more preferably less than 1%, most preferably 0.5%. %.
  • the active substance of the present invention comprises: (1) the same binding site (a) of the variable region of the H chain of the first antibody and the same 3 ⁇ 4 binding site (b) of the variable region of the L chain of the second antibody; (1) the first polypeptide contained in peptide 11 and (2) the source binding site (c) of the variable region of the L chain of the first antibody and the variable region of the H chain of the second antibody Containing a second polypeptide having the same binding site (d) as that of the same peptide lib, and having binding activity for both a cell and a cell having cytotoxic activity.
  • Protein or fusion thereof A fusion polypeptide constituting a protein, and a fusion polypeptide in which a super-polypeptide is bound to at least one peptide chain of the fusion polypeptide. Fusion proteins containing the polypeptides and the superantigen polypeptide I ⁇ j fused polypeptides, and salts thereof.
  • the active substance of the present invention may include the object of the present invention, the gist thereof, and all of the excellent properties obtained by applying the technology disclosed in the present specification. .
  • the active substance of the present invention has many therapeutic applications.
  • the active materials of the present invention can be used to kill or eliminate cells, and are useful as agents It is.
  • the active substance of the present invention includes a diapody having a binding activity to both a spore and a cell having a cell activity, and a so-called modified ⁇ diapody in which the function of the diapody is enhanced.
  • ⁇ Tasushi especially malignant cells
  • the purified fusion polypeptide of the invention is administered to a cancer patient to inhibit the survival or growth of cells, particularly malignant cells (or cancer cells), as described in Jih. I can do it.
  • the active substance of the present invention is preferably delivered to a site by local administration; the term “locally” as used herein means local to ⁇ cells, and is not essential. Nor does it refer to the application to ⁇ if, such as 3 ⁇ 4 ⁇ .
  • the actives of the present invention are usually capable of being mixed with a carrier and other ingredients such as Z or an adjuvant. Such other ingredients must be pharmaceutically acceptable and must also be effective for the intended administration, and may diminish or inhibit the efficacy of the active agents of the present invention. That it must not be activated k There is no restriction on its properties.
  • the active substance of the present invention is suitable for storage of flour by selecting a physiologically acceptable carrier, mk or stabilizing agent, etc., from a fusion protein having a desired ® and the like. It can be prepared in the form of dry powder or an aqueous solution (A. Osol (Ed.), Remington's Pharmaceutical Sciences, 16 Edition, (980)).
  • Acceptable carriers, agents or stabilizers in such therapeutic forms include those that are clear to the recipient at the time of administration and include, for example, phosphorus, quenching and other Buffering agents such as organic acids; antioxidants including ascorbic acid; low-abundance (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or imnoglobulin; ilzR such as polyvinylpyrrolidone
  • Amino acids such as glycine, glutamine, asparagine, arginine or lysine; simple, j and other carbohydrates containing glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; Inflatable ions such as sodium; and Z or Tween, Punole
  • the active substances of the present invention including nonionic surfactants such as Ronics or polyethylene glycol (PEG) +, can be used, for example, in microcapsules prepared by coacervate technology or by interfacial polymerisation (eg, respectively).
  • Hydroxymethyl cellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules) in colloidal form eg, ribosomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • colloidal form eg, ribosomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • A. Osol Ed.
  • the active substances of the invention used for in vivo administration must be sterile. Sterility is facilitated by filtration through a sterile filter membrane, preceded or subsequent to Azuma Tatsu and reconstitution.
  • the active substance of the present invention will be stored in a form or solution in the form of a fiber.
  • the therapeutic live organism of the present invention is placed in a container having a sterile access port, for example, a solution bag for intravenous administration, or a vial having a stopper into which a hypodermic injection needle can be inserted.
  • a container having a sterile access port for example, a solution bag for intravenous administration, or a vial having a stopper into which a hypodermic injection needle can be inserted.
  • the filtered solution may be frozen and fibered to obtain a powdered, sterile active substance of the present invention.
  • the method of administering the active substance of the present invention to a living body includes intravenous, intraperitoneal, intracerebral, intraspinal, intramuscular, intraocular, intraarterial, particularly intrabiliary, or intralesional fiber injection or intralesional fiber. , And sustained release systems (J.)
  • the active substances of the invention can be administered continuously by car key or by injection.
  • the export is generally in a form from which the active substance of the present invention can be released for a certain time, and a typical example of the export preparation is a solid containing protein. It includes a half-body of a hydrophobic polymer, the body being in the form of an object such as a film or a micro force cell.
  • the modified carrier include polyestenol, nydrogenol (Langer et al., J. Biomed. Master. Res. 15: 167-277 (1980) and Langer, Chem. Tech. 12: 98-105 (1982), poly (2-hydroxyethyl monomethacrylate) or a copolymer of poly (vinyl alcohol)) and borylactide (US Pat. No.
  • Some polymers such as ethylene-vinyl acetate and lactate-glycolic acid, are capable of releasing ⁇ ⁇ over a period of 100 days. Some hydrogels release proteins over a longer period of time. If the encapsulated proteins remain in the body for a period of time, they are exposed to moisture at 37 ° C, resulting in denaturation or aggregation, and biologically active 3 ⁇ 4 ⁇ or; ⁇ Rational strategies for protein stabilization can be devised, depending on the forces that can cause the changes to and the mechanisms involved.
  • the aggregation mechanism is a favorable S-S formation via thiodisulfide exchange, stabilization involves modification of sulfhydryl residues, freezing crane from acid f solution. , Moisture ⁇ * preparation, suitable additives ⁇ , specific polymer carrier yarn! It can be done by developing things.
  • the active agent compositions of the present invention in a canopy form for contact with H5K cells include those in liposomes.
  • a liposome containing the active substance of the present invention can be produced by a method known per se. For example, DE 3, 218, 121; Epstein et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); EP 52, 322; EP 36, 676; EP 88, 046; EP 143, 949; EP 142, 641; Japanese Patent Application Laid-Open No. 58-U8008; U.S. Pat. No. 4,485,045 and U.S. Pat. No. 4,544,545; I can do it.
  • ribosomes are tiny (about 200-800 angstroms) monolayers, and their S-potency ⁇ * is about 30% cholesterol, which is a powerful and selective ratio for optimal therapy. Can be adjusted.
  • the active substance of the present invention may be formulated with carriers and / or adjuvants, such as albumin non-ionic surfactants and other such as milk IJ. These other properties include that they must be acceptable to the effect, have no effect on the intended administration, and have a modest activity in the product. There are no restrictions, except that they are not powerless
  • the active substance of the present invention can be administered together with other pharmaceutical agents.
  • the active agents of the present invention may be capable of binding to IFN, eg, IFN-7, and other cytokines, if desired, or may be free of interferons such as IFN- ⁇ . If the forces, cytokines, etc. are species-specific, the appropriate cytokine or drug for the species being treated will be selected. According to the invention, animal or human power is provided. It is possible, but not preferred, to treat one animal species with another species of active substance.
  • the preferred active material for the shelf according to the present invention is soluble.
  • the therapeutic formulation comprises an active substance of the invention encapsulated in ribosomes or complexed with a liposome.
  • a ribosome containing the active substance of the present invention can be formed by using the active substance of the present invention that has been combined with a glycophosphatidylinositol moiety.
  • the therapeutic composition comprises cells that activate the active substance of the invention. Such cells can enter directly into the patient's tissue or can be forced into a perforated cavity and implanted into the patient with ⁇ . In the case of a patient whose primary function is to increase or decrease the amount of the active substance, the active substance of the present invention is delivered to a predetermined area in the body of the patient.
  • an expression vector containing DNA encoding an active agent of the present invention can be used for in vivo transformation of patient cells to achieve the same result.
  • the amount of ⁇ J of the active substance of the invention used for treatment will depend, for example, on the purpose of the treatment, the route of administration and the condition of the patient. Therefore, it may be necessary for the practitioner to determine the dose and change the route of administration necessary to achieve a * ⁇ therapeutic effect.
  • a typical daily dose will depend on the factors described above and, if possible, e.g., first in vitro, and then, if possible, using, for example, the method of Appropriate dose ranges may be determined by a suitable animal model which may deviate the dose range for human patients.
  • compositions described herein may be administered, for example, subcutaneously or intramuscularly.
  • the Fujisato activity of the active substance of the present invention can strengthen the intestine for a long time due to the sustained release effect of the silkworm of the present invention. Therefore, the frequency of administration can be reduced.
  • This composition can also be used as a fibrous material into the arteries controlling by direct injection.
  • the dose of the active substance of the present invention can be selected according to the type, site, size, and view of the active substance of the present invention.
  • the administration key can be appropriately selected according to the type of fiber and the administration authority. «Small in control arteries or to itself: ⁇ , frequent repeated injections are not required, and a single injection every 1 to 4 weeks may be sufficient for the therapeutic effect of longing obtain.
  • ⁇ ⁇ indicates specific operations and processing conditions in DNA cloning according to J. Sambrook, EF Fritsch & T. Maniatis, "Molecular Cloning", 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); Especially in the PCR method, HA Erlich ed., PCR Technology, Stockton Press, 1989; DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol.
  • MHL the main chain antibody
  • HyHELlO anti-chicken egg white lysozyme antibody
  • G3 Peptide tag and His-tag (Hisx6: histidine hexamer tag) force have been introduced into 3 ⁇ 4 ⁇ [J.
  • (GGGGS) 3 (hereinafter, “G3”) is used for a part of polypeptide linker of scFv.
  • the iauci antibody MUSE11 was purchased from MUSEll ⁇ B cell hybridoma with an antibody cloned by Kozo Imai of Sapporo Medical University, mRNA was extracted using Dynas Oligo-dT30, and cDNA was reverse-transcribed. Prepared. MUSEll VH (hereinafter “MH”) is amplified from this cDNA by PCR using an AB primer, and the remaining 5 ⁇ fragment encoding HyHEL-10 VH of plasmid pTZUT-3 by Nco I-Eag I digestion.
  • MH MUSEll VH
  • MUSEll VL (hereinafter referred to as TML) is amplified by PCR using CD primers, and then replaced with the fragment encoding the HyHELlO VL of pTZUT-3 by EcoR V-Sac II digestion. pTZUT-3-MHL-G3 continued. The remaining fei sequences were revealed using the dideoxy method.
  • the Mx3 diabody is composed of two components, MH0L (ffl-G31inker-0L) and 0HML (0H-G31inker-ML).
  • Expression vectors are pSNE4-MH0L-G3, pSNE4-OHML by digesting with Nco I-Eag I using pTZUT-3-MHL-G3 and pTZUT-3-0HL-G3-HClOOaS, respectively, and exchanging VH with each other. -The G3-HC100aS vector was deleted.
  • SEA Staphyloccous Enterotoxin A
  • SEA-producing Staphylococcus aureus strain obtained from Prof. Tadashi Kurokawa of the University of Agriculture and Technology of Tokushima University.
  • genomic DNA was extracted by a conventional method.
  • the SEA fragment was amplified by PCR using J-K primers and inserted into Novagen's pET20b vector by Nco I-Xho I digestion to produce pET-SEA.
  • the K-primer contains a linker sequence (GGGGS (hereinafter, “G1”)) necessary for the production of a fusion protein to SEA * i ⁇ .
  • [SEQ ID NO: 13] Code the fusion protein (mSEA-Mx3). 5) is based on the following already constructed remains. They are as follows. MJC1—Expression vector (pSNE4-MHL) containing a fe chain encoding the main chain antibody scFv (MUSE llscFv: “MHL”), anti-CD3—encoded scFv (0KT3 scFv: “0HL”) Expression vector (pSNE4 -0HL) containing the remaining fe, MH0UMUSE11 of which each VH was exchanged by digestion with EcoR V-SacII and VL of linker-0KT3 were combined with polypeptide (GGGGS) 3 ("G3").
  • VH 0KT3 H chain variable region
  • G1 polypeptide linker that binds the ⁇ variable region
  • the insoluble cell fraction after centrifugation is immersed in 6M guanidine hydrochloride / PBS overnight at 4 ° C to solubilize the protein, and then a metal chelate that specifically binds to His Tag in a denatured state Purification was performed by metal chelation affinity chromatography using a resin (TAL0NTM: manufactured by CL0NTECH). The results of SDS-PAGE of each fraction are shown in the figure. Highly ffl 0L and OHML were obtained in the elute fraction. Such an operation gave 2 mg of the target protein per liter of the form-induced nutrient solution in which expression was induced. Difficult case 5
  • the purified protein Since the purified protein is denatured by guanidine hydrochloride, an unwinding operation is required to obtain a protein having an active tertiary structure.
  • the purified protein solution was put into a dialysis membrane. Dilute the MH0L, 0HML at 280 nm to 0.2 (protein i! S approx. 7.5; wM), add 2-mercaptoethanol to a final concentration of 375, and leave at 4 ° C for 4 hours. (4) After RJS, the purified proteins were mixed in equal amounts, considering that MH0L and 0HML were produced by a 1: 1 association.
  • the denaturant was removed by a precipitation method in which the concentration of guanidine hydrochloride in the external solution was gradually reduced to 3 M, 2 M, 1 M, 0.5 M, and 0 M every other day.
  • oxidized glutathione was added as the acid 1J at 375 juM and L-arginine as the aggregation inhibitor was added to the 0.4 M external dialysis solution and kept at 4 ° C.
  • Such an operation gave a yield of about 1 ⁇ 3 ⁇ 4 (about 15 / g / mL) per 1 L of culture (MH0L: 500 mL, OHML.-500 mL).
  • the MTS assay was used to determine how much was performed by the cholangiocarcinoma cells TFK-1 cells. Perform cell count, adjust so as to be 10 4 cells per RPMI100 j L. Dispense 100 ⁇ l each into a 96-well plate, and leave it at 37 ° C. The target protein was diluted with RPMI to obtain the target fiber, and 50 L of the protein was dispensed into the plate prepared the day before. LA K cells are diluted with RPMI to achieve the desired E / T ratio and dispensed in 50 ⁇ increments ( ⁇ / ⁇ ratio: effector (LAK) / target (TFK-l)) 0 48 hours at 37 ° C culture. Remove the culture medium from the plate, wash with PBS, add TS, PMS, and RPMI and incubate at 37 ° C for 30-60 minutes. Measure 490 g of absorption with a plate reader.
  • mice 10 days after delivery of SCID mice, 5 x 10 8 TFK-1 cells were injected subcutaneously into mice 10 days after TFK-1 transplantation (about 4 to 6 mm), 2 x 10 7 T-LAK After mixing the cells with 20 g of mSEA-diabody, 3 ⁇ 4 ⁇ was passed through the mouse tail vein for 4 consecutive days. -The diameter of the hall was measured every time, and H »f was estimated from the major axis and minor axis. The experiment was terminated when the 11 mm diameter of the control mouth exceeded 20 thighs. The results are as shown in FIG. In the case of Diabody, the force mSEA-Mx3, which suppressed the growth of SHI spores in a certain model, also suppressed the growth of ⁇ spores for 8 weeks.
  • Staphylococcal Enterotoxin A is a protein with a molecular weight of 27. IKDa produced by staphylococci, and is known as the most prominent tongue. It is known that high doses can cause cytokine-dependent toxic shock ⁇ $ bow i. This is mainly due to the strong affinity of SEA for MHC classll, which is due to the accumulation of SEA force in MHC classll cells such as spleen and activation of T cells throughout the body. On the other hand, it has been reported that the D227A mutant of SEA can reduce the sensitivity to MHC classll without decreasing the affinity to TC1? (L.
  • MUC1 was selected as cancer-related.
  • MUC1 is abundantly found in glandular J :, and is a glycoprotein that works on Ser and Thr in the repetitive domain through a 0-glycosidic bond. Everything happens, and the core protein is exposed. Therefore, the MUC1 core protein is considered to be highly adenocarcinoma-specific, and is called adenocarcinoma-related i3 ⁇ 4M, and is one of the powerful targets.
  • CD3 which is expressed only on the surface of ⁇ cells and is used as the best method for detecting mature T cells in peripheral blood, was selected as a cell that binds to cells.
  • a diabody was constructed using the antibody Fv region of the MUC1-specific antibody MUSE11 and the CD3-specific antibody 0KT3. Further, a protein was constructed by fusing the SEA-D227A mutant to this diabody. An expression vector was constructed so that it could be prepared by an expression system using Escherichia coli, and its expression was controlled by SDS-PAGE and Western Blotting.
  • the tumor accumulation property of the SEA is fit, and it is possible to minimize the high side effects caused by force and SEA.
  • ⁇ There is no example of a recombinant protein that has been led to disappearance.
  • ⁇ ⁇ ⁇ (approximately 60,000) is small and easily penetrates into the tissue of a restaurant, and 3 ⁇ 4is low in humans 3 ⁇ 4 ⁇ genicity.
  • 3the expression of bispecific antibodies is Kakura has a stable supply: ⁇ .
  • Two bridges between cells, concrete Has a special effect by recognizing the surface of an effector cell such as
  • the dibody-type multispecific antibody of the present invention is excellent in the function of significantly bringing malignant cells and cytotoxic cells into close proximity, and is a therapeutic agent having an excellent W effect.

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Abstract

L'invention concerne un dianticorps à multispécificité concernant une immunothérapie spécifique au cancer ; son procédé de production et son utilisation ; une protéine fusionnée se liant spécifiquement à la fois à une cellule tumorale (en particulier, une cellule tumorale maligne, notamment une cellule cancéreuse) et à une cellule à cytotoxicité ou à phagocytose ; les polypeptides fusionnés la constituant ; un procédé de production de la protéine fusionnée ; et son utilisation notamment en médecine comme agent antitumoral.
PCT/JP2000/004389 2000-06-30 2000-06-30 Proteine fusionnee a effet antitumoral Ceased WO2002006486A1 (fr)

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AU2000257080A AU2000257080A1 (en) 2000-06-30 2000-06-30 Fused protein with antitumor effect
PCT/JP2000/004389 WO2002006486A1 (fr) 2000-06-30 2000-06-30 Proteine fusionnee a effet antitumoral

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EP1454917A3 (fr) * 2003-02-17 2005-04-06 Tohoku Techno Arch Co., Ltd. Anticorps bispécifique de type diabody
WO2007108152A1 (fr) 2006-03-23 2007-09-27 Tohoku University Anticorps bispecifique hautement fonctionnel
WO2011062112A1 (fr) * 2009-11-18 2011-05-26 国立大学法人東北大学 Mutant hautement fonctionnel de région variable d'anticorps anti-egfr humanisée
CN105367660A (zh) * 2015-12-22 2016-03-02 深圳市北科生物科技有限公司 一种抗cd16a抗原和抗muc1抗原的双特异性抗体

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* Cited by examiner, † Cited by third party
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EP1454917A3 (fr) * 2003-02-17 2005-04-06 Tohoku Techno Arch Co., Ltd. Anticorps bispécifique de type diabody
US7635475B2 (en) 2003-02-17 2009-12-22 Tohoku Techno Arch Co., Ltd. Diabody-type bispecific antibody
WO2007108152A1 (fr) 2006-03-23 2007-09-27 Tohoku University Anticorps bispecifique hautement fonctionnel
EP2522727A1 (fr) 2006-03-23 2012-11-14 Tohoku University Anticorps bispécifique hautement fonctionnel
WO2011062112A1 (fr) * 2009-11-18 2011-05-26 国立大学法人東北大学 Mutant hautement fonctionnel de région variable d'anticorps anti-egfr humanisée
US8921525B2 (en) 2009-11-18 2014-12-30 Tohoku University Highly-functional mutant of humanized anti-EGFR antibody variable region
CN105367660A (zh) * 2015-12-22 2016-03-02 深圳市北科生物科技有限公司 一种抗cd16a抗原和抗muc1抗原的双特异性抗体
CN105367660B (zh) * 2015-12-22 2018-12-21 深圳市北科生物科技有限公司 一种抗cd16a抗原和抗muc1抗原的双特异性抗体

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