CN1188149A - Multifunctional ligan system for cell-specific transfer of nucleic acid - Google Patents

Abstract

The invention relates to a multifunctional ligand system that is not immunogenic and that carries out cell-specific transfer of nucleotide sequences. The system comprises at least one target cell-specific ligand, at least one linker and at least one gene construct-specific ligand, with the gene construct-specific ligand advantageously comprising an antibody, or a part thereof, which binds directly or indirectly to the gene construct. The invention also relates to ligand systems for preparing a pharmaceutical or a vaccine for the treatment or prevention of a disease of the skin, of the mucous membranes, of the nervous system, of the internal organs, of blood coagulation, of the hemopoietic system, of the immune system, of the musculature, of the sustentacular tissue or of the joints.

Description

The cell-specific of nucleic acid shifts used multifunctional ligand system

The present invention relates to polynucleotide are transferred to the modification method and the agents useful for same of cell.

For with for transgenosis is to the cell, not enough and necessity even gene construct is attached to cell surface, also be essential.This combination is tight more, and gene construct is successfully admitted by cell and be then big more in the possibility of transit cell record.This bonded cell-specific is strong more, gene construct more may preponderate ground or combine with target cell individually and admitted by this cell.When except gene construct and target cell, also have the cell of another kind of type, but gene construct is when being admitted by target cell preferentially or fully, gene construct and target cell bonded specificity are even more important.

Developed the multiple gene construct that can make with cell-specific mode bonded technology, the something in common of all these technology is that they have utilized combining of (1) part and its acceptor, the latter is expressed on the cytolemma, or (2) antibody and its antigen or haptenic the combination, described antigen, haptens are exposed on the cytolemma.The example of these technology comprises as heregulin (Han etal., PNAS 92,9747 (1995)), erythropoietin (Kasahara et al., Science266,1373 (1994)) part, as strand Fv (Marin et al., J.Virol.70,2957 (1996) antibody fragment or as acceptor (the Dougherty etal. of Fc acceptor born of the same parents outside part, Transfusion Science 17,121 (1996) mixes the outer of retroviral vector and causes the specific recombination method of target cell by glycoprotein and with this method.

Also used the chemical process will be as asialoglycoprotein (Wu et al., J.Biol.Chem.269,1152 (1994) or this protein synthesis derivative (Marwin et al., Bioconjugate Chem.5,612 (1994)) part links to each other with poly-lysine, and or with the latter and gene construct complexing, perhaps with its Chemical bond to the coat protein of adenovirus carrier.Another kind method is that part is attached on the streptavidin, the latter transfers to combine with vitamin H, the phospholipid head groups of described vitamin H and liposome is puted together, liposome is by gene construct complexing (Redelmeir et al., Drug Deliv.J.Deliv.Targeting Therap.Agents 2,98, (1995)).First Fas lignand system is by Fominaya et al., J.Biol.Chem.271,10560,1996 provide, this Fas lignand system contains the antibody fragment of the ErbB2 acceptor that is specific on the tumour cell, pseudomonas fusogenic peptide exotoxin A and the proteic DNA-of yeast Gal-4 land, described land combines with the corresponding Gal-4 binding sequence that inserts the plasmid that contains metastatic gene.Although this Fas lignand system causes the transfection of target cell specificity, it still can run into the disadvantage of yeast Gal-4 protein immunization originality.

None can fully solve a difficult problem in conjunction with carrier in the specific mode of target cell these disclosed methods, lacks specific major cause and comprises: the weakening of the virus vector function that (1) is modified; (2) sizable complicacy of used Fas lignand system and size; (3) immunogenicity and the consistency of allos or modified protein or used streptavidin-vitamin H coupling system; (4) the bonded gene construct influences the deficiency of target cell specific cell transduction ability.

These circumscribed results are sought after being easy to preparation, and part useful on the function is to be used for that virus and non-virus carrier are attached to target cell.

Therefore, an object of the present invention is to provide the Fas lignand system that is used for target cell transspecific nucleotide sequence, the relative known system of described system, right and wrong are immunogenic, and easily preparation and use.Another purpose is to increase carrier and target cell bonded specificity, thereby improves effectiveness and the selectivity that nucleic acid is transferred to target cell.

In order to realize these and other purposes, one aspect of the present invention provides the multifunctional ligand system that is used for target cell transspecific nucleotide sequence, it contains at least one target cell ligands specific, at least one contains the gene construct ligands specific of antibody or antibody moiety, and the joint that connects these two parts, wherein said system right and wrong are immunogenic.In an advantageous embodiment, at least one part is humanized antibody or antibody fragment.In another advantageous embodiment, the composition of curing the disease is provided, it contains the multifunctional ligand system that is useful on target cell transspecific nucleotide sequence, described system contains at least one target cell ligands specific, at least one contains the gene construct ligands specific of antibody or antibody moiety, and the joint that connects these two parts, wherein said system right and wrong are immunogenic, and it is provided to be administered to patient in appropriate carriers.

In another advantageous embodiment, Fas lignand system contains the connector that two parts are linked together, and described connector contains two joints.The Fas lignand system that another advantageous embodiment provided contains (a) TS part, and it contains anti--NCAM recombinant single chain Fv fragment, wherein by short peptide sequence the segmental variable heavy chain of Fv is linked to each other with the light chain covalency; (b) contain the joint of fusogenic peptide, described fusogenic peptide has sequence GLFEALLELLESLWELLLEA (SEQ ID No.:1); (c) GS part, it contains N ^-6-The recombinant antibodies of-methyladenine.

In another advantageous embodiment, the present invention relates to the purposes that Fas lignand system according to the present invention is used to prepare medicine, described medicine can prevent or treat tetter, mucosal disease, nervous system disorders, internal's disease, the coagulation of blood disease, disease of hematopoietic system, disease of immune system, musculature disease and sustentacular tissue or joint disease.With Fas lignand system as vaccine topical application or injection give patient or, selectively, bestow the cell that derives from patient with prevention or treatment and by skin, mucous membrane, neural system, the internal, coagulation of blood, hemopoietic system, immunity system, musculature, the one or more relevant disease in the group of sustentacular tissue and joint composition.

According to the present invention, may be favourable by connecting cell specific ligand and gene construct.

From following detailed description, other purposes of the present invention, feature and benefit will be conspicuous.Yet should understand, indication the present invention more preferably the detailed description of embodiment and specific embodiment only in order to illustrate the present invention, because multiple change that obtains in from then on describing in detail and modification are within the spirit and scope of the present invention, this will be apparent to those skilled in the art.

Fig. 1 represents three selectable Fas lignand systems, is made up of target cell ligands specific and gene construct ligands specific.Wherein part is connected by " joint " (Fig. 1 a and 1b) or " connector " (Fig. 1 c).

The embodiment of Fig. 2 presentation graphs 1C Fas lignand system, wherein " connector " contains the hinge region of antibody.

Another embodiment of Fig. 3 presentation graphs 1C Fas lignand system, wherein " connector " contains the Gal80-land of Gal 80 albumen and Gal4.

Fusogenic peptide is used to connect two antibody fragments in the embodiment that Fig. 4 represents.

Fig. 5 represents useful in the present invention expression vector pHENIS and pAB1.

The inventor finds target cell ligands specific and gene construct ligands specific to be interconnected by joint shown in Figure 1, and can not form immunogenic mixture.And especially by using " connector " the constructed Fas lignand system shown in Fig. 1 C and Fig. 2 and 3 can overcome transducer cell immunogenicity in the prior art, poor specificity is renderd a service low defective.

This paper term " target cell ligands specific " (TS part) is and target cell surface determinant bonded molecule, promptly with the binding partner of target cell ligands specific, as acceptor or adhesion molecule bonded molecule.

" joint " is the molecule that connects target cell ligands specific and gene construct ligands specific in simple example, and advantageously has fusion characteristics, promptly allow new Fas lignand system to penetrate cytolemma and/or lysosome membrane, promptly enter the characteristic of kytoplasm from lysosome.In advantageous embodiment of the present invention, joint also has fusion characteristics.

" gene construct ligands specific " (GS part) is by antibody or its part is direct or indirect and gene construct bonded molecule.

In the suitable embodiment of the present invention, the TS part is connected by " connector " with the GS part, in addition, arbitrary or two parts can contain independently joint, according to used special GS part and TS part, utilize the embodiment of connector can take different forms (1 * joint for example, 2 * TS part, 1 * GS part or 1 * joint, 1 * TS part, 2 * GS part).

[technical description is seen Sedlacek et al.Contrib.to Oncol.32 by covalent attachment, 42-49 and 81-85, Karger Verlag Munich (1988)] or as can realize the TS part, the coupling between joint and the GS part based on the non--covalent manner (ionic bond) of charge differences.Yet, also can use recombinant technology that Fas lignand system is prepared into the form of fusion rotein, this point is existing the description in EP-Al-0 464 533.

The nucleotide sequence that is imported into target cell by Fas lignand system of the present invention can be RNA or the DNA that exposes, with non-virus carrier or viral blended naked rna or naked DNA.During use Fas lignand system of the present invention is mixed mutually with gene construct, the gained mixture is added in the cell of desiring to be transduceed, perhaps mixture can be bestowed patient.

The target cell ligands specific useful to the present invention, gene construct ligands specific, the example of joint and connector are listed in hereinafter to illustrate some possible combinations that thought over through the contriver.The TS part

In the context of the present invention, the TS part can be with target cell on active compound, the part of active compound or the analogue of active compound of receptors bind.Endogenous material, the material of endogenous material part and the one or more epi-positions of other simulation endogenous material is favourable, because compare with most of allogenic materials, their immunogenicity is lower in the time of in importing body.

The example of this active compound is: somatomedin, and as VEGF, PDGF, EGF, TGF α, TGF β, KGF, SDGF, FGF, IGF, HGF, NGF, BDNF, neurotrophin, BMF, bombesin, M-CSF, thrombopoietin, erythropoietin, SCF, SDGF, oncostatin, PDEGF and interior skin 1; Cytokine, as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14 and IL-15; Interferon alpha, β and γ; TNF (Tumor Necrosis Factor) alpha and TNF β; Chemokine, as RANTES, MCAF, MIP-1 α, MIP-1 β, NAP and β-thromboglobulin; Peptide hormone, as SRH, SIH, STH, MRH, MSH, PRH, PIH, prolactin antagonist, LH-RH, FSH-RH, LH/ICSH, FSH, TRH, TSH, CRH, ACTH; Angiotonin, phytokinin, or histamine or their homologue or analogue; Steroid hormone, as oestrogenic hormon, progestogen, male sex hormone, glucocorticosteroid or mineralocorticoid, or their homologue or analogue; And VITAMIN, as folic acid.

In the context of the present invention, the TS part also can be an adhesion molecule, the part of adhesion molecule or the analogue of adhesion molecule, they combine or combine with specificity another structure combination of the target cell of adhesion molecule with the corresponding adhesion molecule that is arranged in cytolemma.

The example of this adhesion molecule that can work as the TS part is Lewis X (for GMP-140), S-Lewis X (for ELAM-1), LFA-1 (for ICAM-1 and ICAM-2), MAC-1 (for ICAM-1), VLA-4 (for VCAM-1), PECAM (for PECAM), vitronectin (for Vitronectic receptor), GMP-140 (for Lewis X), S-Lewis X (for ELAM-1), ICAM-1, ICAM-2 is (for LFA-1, MAC-1), VCAM-1 (or VLA-4), fibronectin (for VLA-4), ln (for VLA-6), fibronectin, ln is (for VLA-1, VLA-2, VLA-3), fibronectin (for VLA-4), fibrinogen (for GPIIb-IIIa), B7 (for CD28), CD28 (for B7), CD40 (for CD40L) and CD40L (for CD40).

In the context of the invention, the TS part also can be born of the same parents' outside part (Dougherty et al., Trahsfusion Science 17,121 (1996)) of Fc acceptor, and the antibody that is specific to target cell is by its Fc part combination with it.

In the context of the invention, the TS part also can be VDL acceptor or ldl receptor (as ldl receptor, LDL-associated receptor albumen, the FC-acceptor of IgG (as Fc γ RII-B2, Fc γ RI); 88 KDa glycoprotein receptors, acetylize ldl receptor, oxidation ldl receptor or megalin) part.The part of this specific character has been described in detail in the document; they for example are: the Apolipoprotein B-100 or its fragment that contain the C-terminal part; apo E; proteolytic enzyme/inhibitor complexes; as the tPA/PAI-1 mixture; the 2-macroglobulin; the N-terminal district of thrombospondin or its heparin-binding (as amino acid/11-214); the LDL of oxidation; the acetylizad LDL of acetylizad LDL or ethanoyl, acetylizad Methionin, asialylated LDL; the LDL of mda-put together; the albumin of formaldehyde-processing or glutaraldehyde-handled, the albumin of maleylation, 39KDa acceptor-relevant protein or its fragment; for example those contain amino acid/11 8-112; the protein of 113-218 and 219-323 or amino acid/11-114 and 114-319, lactoferrin, aprotinin; lipoprotein lipase, amyloid precursor protein (proteolytic enzyme nexin 2) and Pseudomonas exotoxin.

In the context of the present invention, the TS part also can be the epi-position-bound fraction of antibody molecule or antibody molecule.

As use mouse monoclonal antibody, should preferably use its humanized form to limit its immunogenicity, humanization can (Nature 349 by Winter et al., 293 (1991)) and the described mode of Hoogenboom etal. (Rev.Tr.Trahsfus.Hemobiol.36,19 (1993)) realize.Can prepare antibody fragment according to the ready-made method in present technique field, as passing through Winter et al., Nature349,293 (1991), Hoogenboom et al.Rev.Tr.Transfus.Hemobiol.36,19 (1993), Girol, Mol.Immunol. 28,1379 (1991) or Huston et al., Int.Rev.Immunol.10,195 (1993) methods of describing.

Recombinant antibody fragment can directly prepare from ready-made hybridoma, or also can use phage-display technique (Smith, Science 228,1315 (1985)) (Winter et al., Annu.Rev.Immunol.12,433 (1994)) from mouse or people's antibody fragment library, separate, on gene level, directly use these antibody fragments then with further operation (for example merging) with other protein.

For antibody fragment by hybridoma preparation reorganization, by separating mRAN, reverse transcription RNA obtains cDNA, use polymerase chain reaction (Saiki et al. subsequently, Science230,1350 (1985)) and with variable segmental 5 ' and 3 ' terminal complementary oligonucleotide respectively (Orlandi et al., PNAS-USA 86,3833 (1989)) amplification can obtain antigen-land (VH, genetic information VL) of encoding antibody.Then with for example Fv fragment (Skerra﹠amp; Pluckthun, Science 240,1038 (1988)), and strand Fv fragment (sc-Fc) (Bird et al., Science 242,423 (1988), Huston et al., PNAS-USA 85,5879 (1988)) or Fab fragment (Better et al., Science 240,1041 (1988)) form with VH and VL fragment cloning in bacterial expression vector.

Also can use phage-display technique directly to separate new antibody fragment from the antibody library (immune library is used for the library of testing first) in mouse or people source.In the phage display of antibody fragment, antigen-land as with the fusion rotein of the g3P coat protein of filobactivirus, with scFv fragment (McCafferty et al., Nature 348,552 (1990)) or Fab fragment (Hoogenboom et al., Nucl.Acid Res.19,4133 (1991), Barbas et al., PNAS-USA 88.7978 (1991)) form, or be cloned into (McCafferyt al. in the phage genome, Nature 348,552 (1990)), or be cloned into (Breitling et al. in the phasmid carrier, Gene 104,147 (1991)).Be loaded with antigenic plastic containers (elutriation) (Marks et al., J.Mol.Biol.222,581 (1991)) on, at paramagnetic beads (the Hawkins et al. that puts together with antigen, J.Mol.Biol.226,889 (1992)) go up or select and antigen bonded phage by combine (Marks et al., Bio/Technol.11,1145 (1993)) with cell surface.

By pcr amplification derive from the humanization animal (Sastry et al., PNAS-USA 86,5728 (1989), Ward et al., Nature 341,544 (1989), Clackson et al., Nature352,624 (1991) or patient (Mullinax et al .PNAS-USA87,8095 (1990), Barbas et al., PNAS-USA 88,7978 (1991)) the variable antibody fragment of bone-marrow-derived lymphocyte can prepare immune library.Utilized for this purpose and be specific to mouse (Orlandiet al., PNAS-USA 86,3833 (1989), Sastry et al., PNAS-USA86,5728 (1989)) or human immunoglobulin gene (Larrick et al., BBRC160,1250 (1989)) or be specific to the combination of the oligonucleotide of human immunoglobulin gene family (Marks et al., Eur.J.Immunol.21,985 (1991)).

Can prepare natural gene library (Marks et al., J.Mol.Biol.222,581 (1991)) by using not as the source of immunoglobulin gene by the donor of immunization.In addition, can use the immune globulin white race is that gene prepares semisynthetic antibody repertoire, and the primer that uses degeneracy simultaneously is through the variable segmental complementation-determining area 3 (Hoogenboom﹠amp of pcr amplification; Winter, J.Mol.Biol.227,381 (1992), Barbas et al., PNAS-USA 89.4457 (1992), Nissim et al., EMBO J.13,692 (1994), Griffiths et al., EMBO J.13,3245 (1994)).Compare with immune library, these so-called lists-hit (single-pot) library have advantage, can separate the Chinese People's Anti-Japanese Military and Political College and measure antigenic antibody fragment (Nissim et al., EMBO J.13,692 (1994)) from a single storehouse.

By using phage-display technique still can further increase the affinity of antibody fragment, by random mutagenesis (Hawkins et al., J.Mol.Biol.226,889 (1992), Gram et al., PNAS-USA 89,3576 (1992)), mutagenesis (Glaser et al., J.Immunol.149,3903 (1992)) or site-directed mutagenesis (Balint﹠amp based on codon; Larrick, Gene 137,109 (1993)), derive from the fragment that is used to the repertoire of testing first by use and make each district's chain reorganization (Marks et al., Bio/Technol.10,779 (1992)) or use bacterium mutator (Low et al., J.Mol.Biol.260,359 (1996)), under stringent condition, reselect and isolate antibody fragment (Hawkins et al. with improved characteristics, J.Mol.Biol.226,889 (1992)) can prepare new library by existing antibody fragment.In addition, replace a variable region, select (instruct and select) can make murine antibody fragment humanization (Jespers et al., Bio/Technol.12,889 (1994)) with original antigen then by the repertoire of progressively choosing.In addition, also can make murine antibody humanization (Jones et al., Nature 321,522 (1987)) by the hypervariable region that replaces people's antibody with the respective area specificity of original murine antibody.

In the context of the invention, the TS part also can be by its envelope protein and the envelope protein of selected cell-specific bonded virus or the part of envelope protein.The selection of TS part is depended on and is desired the target cell of being transduceed by gene construct.

The material that the example of the useful part of the present invention is comprised can activate endotheliocyte, scavenger cell and lymphocyte; In conjunction with muscle cell, hematopoietic cell, synovial fluid cell and inflammatory cell; In conjunction with by the cell of virus infection; In conjunction with as hepatocellular histocyte; In conjunction with neurogliocyte; With in conjunction with the leukemia cell.Some representative example of only having summarized these parts herein.Activate the TS part of endotheliocyte

On meaning of the present invention, these parts comprise antibody or the antibody fragment that is directed to the endotheliocyte membrane structure, this point is described in for example Burrows et al., Pharmac.Ther.64,155 (1994), Hughes et al., Cancer Res., 49,6214 (1989) and Maruyama et al., PNAS-USA 87,5744 (1990).Specifically these parts comprise the antibody of anti-vegf receptor.

The TS part also comprises and endotheliocyte membrane structure or all active compounds of membrane receptor bonded.For example these parts are included in the material that terminal position contains seminose, but also comprise IL-1 or somatomedin, or their fragment or its partial sequence, the receptors bind that these parts can be expressed with endotheliocyte, described acceptor is for example PDGF, bFGF, VEGF or TGF β (Pusztain et al., J.Pathol.169,191 (1993)).

The TS part also comprises the part at ldl receptor, for example at the acetylize ldl receptor, and the ldl receptor of oxidation, LDL-associated receptor albumen (LRP), the part of the Fc acceptor of 88KDa glycoprotein receptor and IgG.The part of this character has a detailed description in the literature.

The TS part further comprises the endotheliocyte bonded adhesion molecule with activated and/or propagation, the adhesion molecule of this character such as Slex, LFA-1, MAC-1, LECAM-1, VLA-4 or vitronectin are described (sees Augustin-Voss et al., J.Cell.Biol.119,483 (1992), Pauli et al., Cancer Metast.Rev.9,175 (1990) and Honn etal., Cancer Matast.Rev.11,353 (1992)).On meaning of the present invention, the TS part comprises that particularly endotheliocyte is had the viral outer by glycoprotein of preferendum, these viral examples have: filovirus, for example have the marburg virus of its GP (glycoprotein) and sGP (second glycoprotein) coat protein or under every kind of occasion, all have the Ebola virus of its GP and sGP coat protein, has the proteic cytomegalovirus of its gB especially, people α scar exanthema virus 1 type, HIV-1 virus, Measles virus, Hantaan virus, the onychonosus toxogen, as cold Mu Liji forest virus, epidemic haemorrhagic fever virus, back of the body tephromyelitis virus, and as ECHO9, the enterovirus of ECHO12 or coxsackie B 3 viruses.The TS part of activating macrophage and/or activated lymphocyte

On meaning of the present invention, part can comprise and immunocyte surface specific bonded material that this material comprises antibody or the antibody fragment that is directed to the immunocyte membrane structure, for example Powelson et al., Biotech.Adv.11 states in 725 (1993).

The TS part further comprises above all parts of the existing ldl receptor of describing, the TS part also can further comprise by the mono-clonal of the variable part of its conjugated antigen and the Fc-y on the immunocyte or Fc-ε or Fc-μ receptors bind or polyclonal antibody or antibody fragment (Rojanasakul et al., Pharm.Res.11,1731 (1994)).

These parts also comprise the Fc fragment of human monoclonal or polyclonal immunoglobulin, the genetic manipulation method by for example using recombinant DNA or according to Haupt et al., Klin Wschr.47,270 (1969), Kranz et al., Dev.Biol.Standard 44,19 (1979); Fehr et al., Adv.Clin.Pharmac 6,64 (1974), Menninger et al., Immunochem.13,633 (1976) method can prepare the Fc fragment with this characteristic.

The TS part comprises further and the membrane receptor bonded all substances on immunocyte surface that these parts comprise cytokine, as IL-1, IL-2, IL-3, IL-4, IL-6, IL-10, TNF α, GM-CSF and M-CSF, and somatomedin are as EGF, TGF, FGF, IGF or PDGF or their fragment or its partial sequence, the receptors bind that described part and immunocyte are expressed.

The TS part further comprises and membrane structure, as seminose 6-phosphate receptor bonded adhesion molecule and other parts on spleen, liver, lung and other tissue macrophages.Perales et al., Eur.J.Biochem.226,255 (1994) are seen in the selection of these parts and membrane structure.

On meaning of the present invention, the TS part also comprises from the envelope glycoprotein that lymphocyte and/or scavenger cell is had the virus of preferendum.These viral examples that infect scavenger cell are: HIV-1, particularly those have the strain of sudden change in the V3 district of gP120, described sudden change can cause the combination of scavenger cell is increased, HIV-2, Hantaan virus, Punmala virus for example, cytomegalovirus, respiratory syncytial virus, simple scar exanthema virus and filovirus.

The example that infects lymphocytic virus is: people α simplexvirus 3 types (VZV), scar exanthema virus 6 types (HHV-6), rabies virus, HIV-1, HTLV-II, HTLV-I, influenza virus C is (because influenza virus C merges (HEF) albumen and N-ethanoyl-β-acetyl neuraminic acid (Neu5 by hemagglutinin-esterase, 9Ac) combination, this incident preferentially takes place on bone-marrow-derived lymphocyte, and on than low degree or on the T lymphocyte, do not take place); Locate to have the influenza virus C of sudden change at nucleotide position 872 (coding HEF aminoacid sequence 284), for example replace Threonine with Isoleucine, because have the HEF surface protein of this sudden change and comparing of wild-type virus, N-ethanoyl-9-O-acetyl neuraminic acid acceptor there is obviously stronger affinity; Influenza virus C HEF split product, it contains and N-ethanoyl-9-β-acetyl neuraminic acid bonded structure because this integrated structure is by catalytic triplet Serine 71, Histidine 368 or 369 and aspartic acid 261 determine; People γ scar exanthema virus 4 types, because EBV infects the B cell, people α herpes virus type 2, because HSV-2 infects the T cell, and Measles virus.The TS part of muscle cell

Comprise with the part of muscle cell surface bonding for example being directed to muscle cell, especially the antibody of smooth muscle cell membrane structure or antibody fragment.The example of the antibody of this characteristic is: antibody 10F3, anti-actin antibody, the antibody of the antibody of anti-angiogenic tonin II acceptor or antibiosis growth factor receptor body or be directed to for example EGF acceptor, the antibody of the antibody of pdgf receptor or FGF acceptor or anti-endothelium peptide A acceptor.

The TS part comprises further and muscle cell membrane structure or all active substances of membrane receptor bonded that these parts comprise for example somatomedin or its fragment or its partial sequence, the receptors bind that described part and smooth muscle cell are expressed, described acceptor such as PDGF; EGF; TGF β; TGF α; FGF and endothelium peptide A.

On meaning of the present invention, the TS part also comprises those viral envelope glycoproteins that muscle cell had preferendum, and these viruses comprise for example cytomegalovirus.The TS part of hematopoietic cell

The TS part comprises antibody or the antibody fragment that is directed to the acceptor of expressing on the undifferentiated relatively hemocyte, for example the antibody at this characteristic of following acceptor is described: the STEM CELL FACTOR acceptor, IL-1 acceptor (I type), IL-1 acceptor (II type), IL-3 acceptor α, IL-3 acceptor β, IL-6 acceptor and GM-CSF acceptor.

The TS part also can further comprise mono-clonal or polyclonal antibody or the antibody fragment by the Fc-γ receptors bind on its constant region and the immunocyte.The TS part also comprise with relative undifferentiated hemocyte on membrane structure or membrane receptor bonded all substances, these parts for example comprise the somatomedin of the receptors bind of expressing with hemocyte, as SCF, and IL-1, IL-3, IL-6 and GM-CSF or its fragment or its partial sequence.The TS part of synovial fluid cell and inflammatory cell

These parts comprise that the example of this membrane structure is a vimentin by the membrane structure bonded mono-clonal of its variable region and synovial fluid cell or inflammatory cell or polyclonal antibody or antibody fragment, fibronectin and Fc acceptor.These parts also comprise by the mono-clonal of its constant region and Fc receptors bind or polyclonal antibody or antibody fragment.These parts further comprise with synovial fluid cell on membrane structure or all active compounds of membrane receptor bonded, these compounds comprise for example cytokine or somatomedin or its fragment or its partial sequence, acceptor such as IL-1-RA that they and synovial fluid cell are expressed, TNF α, IL-4, IL-6, IL-10, IGF and TGF β combination.These parts further comprise composition being the TS part of terminal seminose, and it combines with seminose 6-phosphate receptor on the scavenger cell.By the TS part of the cell of virus infection

The TS part also can be aimed at by the antibody of the virus antigen of expressing on the cytolemma of the cell of virus infection or antibody fragment, for example, by HBV, HCV, HSV, HPV, HIV, the antibody of this characteristic of the cell of EBV and HTLV virus infection is described.Liver cell and other histiocytic TS parts

The TS part comprises and surface of hepatocytes membrane structure or membrane receptor bonded all substances, these parts for example comprise the somatomedin with the receptors bind of this type of cell expressing, as cytokine, EGF, TGF, FGF or PDGF or its fragment or its partial sequence, these parts further comprise the part with membrane structure bonded TS, and described membrane structure is optionally for particular tissues.For example:

Table 1 membrane structure TS part histocyte asialoglycoprotein takes off the sticking egg liver cell of sialic acid serum class

The polymeric immunoglobulin receptor neoglycoprotein

(neoglycoprotein)

Scavenger cell Fc-γ receptor immunoglobulin G reticuloendothelial system in semi-lactosi transferrin receptor transferrin liver and other histocyte insulin receptor Regular Insulin livers and other histocyte seminoses 6-phosphoric acid ester seminose spleen, liver, lung and its hetero-organization acceptor and other

Tissue

These parts and membrane structure are referring to Perales et al., Eur.J.Biochem.226,255 (1994).On meaning of the present invention, the TS part specifically comprises the envelope glycoprotein that selecting cell is had the virus of preferendum, as the glycoprotein of segmental bronchus cellular respiration road syncytial virus, liver cell hepatitis C virus, filovirus, the glycoprotein of hepatitis B virus and hepatitis D virus.For example, liver cell combines with marburg virus by the asialoglycoprotein acceptor or liver cell preferably combines with pre S2 and the pre S1 district of HBV by the asialoglycoprotein acceptor.Another example is the glycoprotein of liver hole shape cell hepatitis B virus, because HBV is combined by fibronectin.The TS part of neurogliocyte

These parts comprise by for example Mirsky et al., Coakham et al., with antibody that is directed to the neurogliocyte membrane structure or antibody fragment that Mckeeveret al very leads, these membrane structures further comprise the nerve adhesion molecule as N-CAM, especially its peptide C chain.

These parts also comprise and neurogliocyte membrane structure or all active compounds of membrane receptor bonded, for example, these parts are included in the material that terminal position carries seminose, described material combines with seminose 6-phosphate receptor, also comprise Regular Insulin and rhIGF-1 and PDGF, and with those fragments of these somatomedins of related film receptors bind.

On meaning of the present invention, the TS part specifically comprises those viral envelope glycoproteins that neurogliocyte had preferendum, and these viral examples are HIV-1 hypotype JRF1 and people α scar exanthema virus I type.Leukemia cell's TS part

These parts comprise and are directed to antibody or the antibody fragment that the leukemia cell goes up membrane structure, have described the monoclonal antibody that is used in a large number to diagnose with this characteristic of therapeutics and (have seen Kristensen, Danish Medical Bulletin 41,52 (1994); Schranz, TherapiaHungarica 38,3 (1990); Drexler et al., Leuk, Res.10,279 (1986); Naeim, Dis.Markers 7,1 (1989); Stickney et al., Curr., Opin, Oneol, 4,847 (1992); Drexler et al., Blut 57,327 (1988); Freedman et al., CancerInvest, 9,69 (1991)).According to leukemic type, following monoclonal antibody or itself and antigen bonded antibody fragment are suitable for use as part: (1) for the AML cell, membrane antigen CD13, CD14, CD15, CD33, CAMAL, Sialosyl-Le; (2) for B-CLL membrane antigen CD5, CD1c, CD23, the idiotype of membrane immunoglobulin and isotype; (3) for T-CLL membrane antigen CD33, M38, IL-2 acceptor, TXi Baoshouti; (4) for ALL membrane antigen CALLA, CD19 is non--Hodgkin lymphoma.

The TS part further comprises membrane structure or all active compounds of membrane receptor bonded with the leukemia cell, and these parts for example comprise somatomedin or its fragment or its partial sequence of the receptors bind of expressing with the leukemia cell.

The somatomedin of this characteristic is described (sees Cross et al., Cell64,271 (1991); Aulitzky et al., Drugs 48,667 (1994); Moore, Clin.CancerRes.1,3 (1995); With Van Kooten et al., Leuk.Lymph, 12,27 (1993)).For example under the situation of non--Hodgkin lymphoma IFN α; IL-2 is especially under the situation of T chronic myeloid leukemia; At the T cell, monocyte, marrow is FGF under the leukemic situation of red corpuscle and megakaryoblast; Under leukemic situation be TGF β and, under the leukemic situation of acute promyelocyte, be retinoids, as vitamin A acid.The TS part of tumour cell

These parts comprise the fragment of the antibody that is directed to the tumour cell membrane structure and these antibody, the antibody of this characteristic has been described in for example Sedlacek et al., Contrib, toOncol.32, Karger Verlag, Munich (1988) and Contrib.toOncol.43, Karger Verlag, Munich (1992).

The antibody of other examples is anti-: Sialyl Lewis; by the peptide on the tumour of T cell recognition; protein by oncogene expression; as GD3; GD2, GM2, the Sphingolipids,sialo of 9-0-ethanoyl GD3 and Fucosyl GM1; blood class antigen and their precursor, antigen on the multiform endothelium Saliva Orthana and the antigen on the heat shock protein(HSP).Joint

The selection of joint is depended on the chemical property of TS part and GS part and is used for by joint TS part and the interconnected method of GS part.If part is peptide or protein, then preferably use peptide or protein as joint, preferably utilize peptide bond that joint is interconnected TS part and GS part.The preferred recombinant DNA technology of using is prepared into fusion rotein with the TS part-joint-GS part or the TS part-GS part-linkers of this characteristic.Generally, favourable joint will be that the material of endogenous generation or similar endogenous material are to limit its immunogenicity.

If the TS part is not peptide or protein, the joint of simple form will be the structure that connects TS part and GS part.Be used for the amino group on molecule and the protein, oh group, the SH group, the different chemically conjugated method of carbonyl group or aldehyde radical bonded can obtain the structure of this characteristic and (see Sedlacek et al., Contrib.to Oncol.32,42-49 and 81-85, KargerVerlag, Munich (1988)).

In all cases, joint and GS part self also can be peptide or protein, at this moment, preferably by peptide bond with by using a chemically conjugated method to be connected being connected between realization joint and GS part with joint, especially at embodiment of the present invention C) in used this method.

The selection of joint depends on also that by the characteristic of GS part bonded gene construct RNA that if gene construct is or inclusive NAND virus vector compound is exposed separately or exposed DNA, joint is preferably the molecule with fusion characteristics.Thereby these fusion characteristics are convenient to gene construct and are passed cytolemma and break through lysosome and enter kytoplasm.

If gene construct is a virus, can select to have the molecule of fusion characteristics as joint.On meaning of the present invention, the preferred joint that uses with fusion characteristics.The fusion characteristics of joint can compensate because of the infringement that combine the virus envelope proteins fusion characteristics that cause of GS part with virus, or the fusion characteristics of enhanced virus coat protein.

On meaning of the present invention, virus or bacterial peptide or protein and synthetic peptide (for example forming those peptides of alpha-helix in the acid environment of endosome) can be used as the joint with fusion characteristics.Example with molecule of fusion characteristics is: peptide (Wels et al., Cancer Res.52,6310 (1992) of containing the displacement district (III district) of ETA; Fominaga et al., J.Biol.Chem.271,10560 (1996)), contain peptide GLFEALLELLESLWELLLEA (SEQ ID NO.:1)) peptide (Gottschalk et al.Gene Ther.3 448 (1996)), contain peptide AALAEA[LAEA] ₄LAAAAGC (SEQ ID No:2)) peptide (Wang etal., Technol. Advances in Vector SVst.For Gene Ther., May 6-7,1996, Coronado, IBC Conference), peptide (the Yeagle et al. that contains Measles virus fusion rotein peptide FAGVVLAGAALGVAAAAQI (SEQ ID NO.:3), Biochem.Biophys.Acta 1065,49 (1991)), peptide (the Luneberg et al. that contains influenza A virus HA2 protein peptide GLFGAIAGFIEGGWWGMIDG (SEQ ID NO.:4), J.Biol.Chem.270,27606 (1995)) peptide (the Burger et al. that, contains peptide GLFGAIAGFIENGWEGMIDGGLFGAIAGFIENGWEGMIDG (SEQ ID NO.:5), Biochem.30,11173 (1991)) or peptide GLFGAIAGFIE; (SEQ ID NO.:6), ALFGAIAGFIE; (SEQID NO.:7), LFLGAIAGFIE; (SEQ ID NO.:8), LLLGAIAGFIE; (SEQ ID NO.:9), LILGAIAGFIE; (SEQ IDNO.:10), GIFGAIAGFIE; (SEQ ID NO.:11), GLLGAIAGFIE; (SEQ ID NO.:12), GLFAAIAGFIE; (SEQID NO.:13), GLFEAIAGFIE; (SEQ ID NO.:14), GLFGAMAGFIE; (SEQ ID NO.:15) GLFGAIAGLIE (SEQ IDNO.:16) or peptide GLFGAIAGFIV (SEQ ID NO.:17) (Steinhauer etal., J.virol.69,6643 (1995)) or peptide GLFEAIAEFIEGGWEGLIEG (SEQ ID No.:18) or peptide GLLEALAELLEGGWEGLLEG (SEQ IDNO.:19) (Ishiguro et al., Biochem.32 9792 (1993)).

In the context of the present invention, further used the virus protein with fusion characteristics, a large amount of viruses have the coat protein of fusion, paramyxovirus for example, retrovirus and simplexvirus (Gaudin et al., J.Gen.Virol.76,1541 (1995)).A large amount of viruses also have the glycoprotein (Gaudin et al., J.Gen.Virol.76,1541 (1995)) that work are merged in virus absorption and cytolemma subsequently, and by as alphavirus, rhabdovirus and orthomyxovirus can form the protein of this characteristic.

Virus amalgamation protein on the meaning of the present invention has been described in Hughson, Curr.Biol.5,265 (1995); Hoekstra, J.Bioenergetics Biomembranes 22,675 (1990); White, Ann.Rev, Physiol.52,675 (1990)).The example of the fusion rotein on the meaning of the present invention is the hemagglutinin of first type or Influenza B virus, especially HA2 composition, the M2 albumen of influenza A virus, or separately or with influenza virus hemagglutinin or lack enzymic activity but bring the mutant of the influenza A virus neuraminidase of hemagglutinative function to unite use (Ohuchi et al., J.Virol.68,920 (1994)), the peptide analogs of influenza virus hemagglutinin, the HEF albumen of influenza virus C, the proteic fusion-activity of HEF is by being cracked into HEF1 with HEF0 and HEF2 subunit is activated, the transmembrane glycoprotein of filovirus, the transmembrane glycoprotein of marburg virus and Ou Baola virus for example, the transmembrane glycoprotein of rabies virus, the transmembrane glycoprotein of vesicular stomatitis virus (G), the fusion rotein of HIV virus, especially gp41 composition and merge composition, the fusion rotein of Sendai virus, especially 33 of the F1 composition amino terminal amino acids, the transmembrane glycoprotein of cold Mu Liji forest virus, especially E1 composition, the transmembrane glycoprotein of tick-brone encephalitis virus, the fusion rotein (especially gP37 composition) of human respiratory syncytial virus (RSV), the fusion rotein of hepatitis B virus (S albumen), the fusion rotein of Measles virus, the fusion rotein of Avian pneumo-encephalitis virus, the fusion rotein of Wei Sina virus, the fusion rotein of murine leukemia virus (especially p15E), the fusion rotein of fusion rotein of HTL virus (especially gp21) and simmian immunodeficiency viruses (SIV).

By Mannio et al., BioTechniques 6,682 (1988) described by means of the coat protein in stain remover (as β-D-octyl group glycopyranoside) the lytic virus concentrated solution and by centrifugation it, perhaps utilize molecular biology method well known by persons skilled in the art can obtain virus amalgamation protein.The preparation of following fusion rotein is described, influenza virus hemagglutinin for example, the fusion fragment of influenza virus hemagglutinin, the M2 albumen of Influenza B virus, the HEF albumen of influenza virus C, filovirus, the transmembrane glycoprotein of marburg virus and Ou Baola virus for example, the transmembrane glycoprotein of rabies virus; The transmembrane glycoprotein of vesicular stomatitis virus, the transmembrane glycoprotein of cold Mu Liji forest virus, the transmembrane glycoprotein of transmembrane glycoprotein of tick-brone encephalitis virus and HIV-1 virus.The gene construct ligands specific

In the context of the present invention, the GS part is direct or indirect and gene construct bonded structure, and it contains the fragment in conjunction with epi-position of whole antibody molecule or antibody.The preferred mouse monoclonal antibody of humanization form that uses is to limit its immunogenicity, humanized realization is described in " description of TS part " this joint, be with people such as Hoogenboom (Rev.Tr.Transfus.Hemobiol.36,19 (1993)) and people's (Nature 349,293 (1991) described modes such as Winter.Prepare the Fv fragment of antibody fragment and reorganization according to the method for having stated in methods known in the art and " description of TS part " this joint.

Whether use divalence or unit price fragment to depend on the selection of antibodies specific and gene construct, if selected antibody has damaged the fusion-activity of virogene construct coat protein then preferred monovalent antibody fragments (for example Ubol et al., J.Virol.69 1990 (1995) is described).

The specificity of antibody depends on the characteristic of used gene construct, if gene construct is the RNA that exposes or exposed DNA, or separately the inclusive NAND virus vector is compound, and the specificity that a new embodiment of the present invention is an antibody is aimed at those and has been introduced into epi-position among the DNA.

By one or more modifications of DNA, introduce or do not introduce the epi-position that external source group (foreign gene material) can produce this characteristic.This example comprises DNA and cisplatin crosslinked, and with as mustargen, the alkylating agent of melphalan or Chlorambucil makes the N7 alkylation of guanine, will duplex as the anthracene class intercalation of DNA of Zorubicin or daunomycin in.

The example of monoclonal antibody with binding specificity of anti-modified DNA epi-position comprises and is directed to methylate DNA, O ⁶-ethyl pancreatic desoxyribonuclease (behind ethylnitrosourea processing DNA), N ⁷-ethyl guanine, N ⁵-methyl-N ⁵-formyl radical-2,5,6-triamino-4-hydroxy pyrimidine, O ⁶-methyl-2 '-pancreatic desoxyribonuclease, O ⁶-ethyl-2 '-pancreatic desoxyribonuclease, O ⁶-just-and butyl-2 '-pancreatic desoxyribonuclease, O ⁶-sec.-propyl-2 '-pancreatic desoxyribonuclease, O ⁴-methyl-2 '-deoxythymidine, O ⁴-ethyl-2 '-deoxythymidine, the adduct of melphalan and DNA and anthracene class.Some useful epi-positions in the application's context are that those produce in DNA by methylating of DNA in the DNA metabolic process.

Known coli strain can make the plasmid DNA of having introduced in the bacterium methylate, and methylation occurs in the N of VITAMIN B4 ⁶Position (Winnacker, From Genes to Clones, p18/19, VCH Publisher, Weinheim (1987)).Bacterium has the DNA adenine methylase, and this enzyme makes N specifically in the bacterium reproduction process ⁶The VITAMIN B4 methylate (Hattman et al., J.Mol.Biol.126,367 (1978)) of position.Therefore, the present invention relates to particularly in new Fas lignand system, and the purposes of the monoclonal antibody of anti-methylate DNA relates more specifically to anti-methylated VITAMIN B4 N ⁶The purposes of monoclonal antibody.

If gene construct and non-virus carrier are compound, the specificity that another specific embodiment of the present invention is an antibody is the epi-position that is directed on the carrier, and these carriers comprise cationic polymers, peptide, protein, polyamines or cation lipoid are as cation lipoid and phosphatide.The example of the antibody of anti-this characteristic carrier is anti-spermidine, spermine, putrescine, poly-lysine, the antibody of albumin and phosphatide.

If gene construct is a virus, the specificity of antibody is the one or more identical or different epi-positions of antiviral coat protein.Because the joint in the used Fas lignand system is preferably fusogenic peptide or protein, also can use by combine the antibody of the fusion-activity of damaging cell adhesion and/or virus with coat protein.The example of the antibody of the coat protein of the anti-virus that can be used as carrier is: anti-murine leukemia virus, especially the antibody of anti-coat protein gp70 and p15, anti HIV-1 virus, adenovirus, simple scar exanthema virus, especially anti-this viral Glycoprotein B, glycoprotein h, the antibody of glycoprotein L and glycoprotein D, anti-cytomegalovirus, especially anti-Glycoprotein B (gpB), minute virus of mice, adeno-associated virus, especially anti-cap and rep albumen, sindbis virus, the antibody of especially anti-envelope protein E 2 or E and vaccinia virus.

In another advantageous embodiment of the present invention, the GS part is the part of extracellular Fc acceptor, and one of its antigen binding domain and direct or indirect bonded antibody of gene construct of utilizing mentioned above passes through its Fc part Fc receptors bind therewith.

In another advantageous embodiment, the GS part be can with gene construct compound cationic structural unit, as cationic amino acid, cationic peptide or protein or derive from biological amine.The example of these cationic structural units comprises Methionin, poly-lysine, arginine, poly arginine, Histidine, the poly Histidine contains at least one Methionin, the peptide and the polyamines of an arginine and/or a Histidine, as cadaverine, spermidine, spermine, agmatine or putrescine.

In another advantageous embodiment; the GS part is the acceptor with coat protein of genetically modified virus; the acceptor of this characteristic of following virus has been described: the HIV that relates to CD4 molecule (soluble or natural) and galactosyl ceramide; the HBV that relates to IL-6 acceptor and annexin or lipophorin; the HTLV that relates to IL-2 acceptor (β and γ chain); the Measles virus that relates to the CD46 molecule; the Friend leukosis virus that relates to erythropoietin receptor; the segmental people α of Fc scar exanthema virus 3 types that relate to immunoglobulin G while; the Sendai virus that relates to glycophorin; the influenza virus C that relates to N-ethanoyl-9-acetylaminohydroxyphenylarsonic acid 9-deoxidation neuraminic acid and 9-O-ethanoyl-N-n acetylneuraminic acid n; the foot and mouth disease virus that relates to beta 2 integrin alpha V β 3 relates to the EBV of complement receptor 2 (CD21) and relates to the simple scar exanthema virus of 275KDa seminose 6-phosphate receptor or 46KDa seminose 6-phosphate receptor.Joint as the mixture (" connector ") of at least two molecules

At particular embodiment c of the present invention) meaning on, connector is the joint that contains the specific type of at least two molecules or composition.The molecule or the composition of connector combine with at least one gene construct ligands specific, and another molecule of connector or composition combine with at least one target cell ligands specific.Mixture further advantageously contains at least one other " alternative joints ", and alternative joint can be a fusogenic peptide.Under the situation of more than a kind of joint, alternative joint chemically can be identical or different.Fusogenic peptide helps nucleic acid and enters target cell, and another alternative joint can be that the signal that produces as radioisotopic material enters the amount of the mixture of cell to allow detection in the context of the invention.

The example of favourable connector is the hinge region of antibody, two heavy chains of taking this antibody interconnect (Burbon, TIBS 15,64, (1990); Oi et al., Nature 307,136 (1984); Alt etal., Science 238,1079 (1987); Lorenz, degree dissertation:Konstruktionund Expression von rek.Antikorper-Enzym-Hvbridmolekulen fur dieTumortherapie[Construction and expression of rec.antibody/enzyme hybrid molecules for tumor therapy], Faeulty of HumanMedicine, Marburg University (1991)).

For example, the sketch C1 of Fig. 2) shown the arrangement that hinge region is new in, hinge region preferably with the form of fusion rotein by peptide bond and joint be connected with the TS part with GS, described fusion rotein is to use the recombinant DNA technology preparation.

Another example of connector is according to the sketch C2 among Fig. 3), with the Gal80 albumen (Leuther et al., Science 256,1333 (1992)) of the Gal80-land (Leuther et al., Science 256,1333 (1992)) of Gal4 associating.

The purpose of following embodiment is in order to illustrate rather than to limit the present invention, having shown the structure of multifunction system as shown in Figure 4 among the embodiment.

The preparation of embodiment 1TS part

Anti--the hybridoma of NCAM monoclonal antibody 575/100/2 is used as the initial substance (Jaques et al., Cancer 72,418 (1993)) of TS part.By centrifugation this hybridoma about 10 ⁷Individual cell, use Pharmacia mRNA to extract test kit and from these cells, extract mRNA, then by use cDNA synthetic agent box and at random the reverse transcription of six oligonucleotide (Pharmacia) this mRNA is transcribed into cDNA, this cDNA is used as by using specific primer (Clackson et al., Nature 352,624 (1991)) polymerase chain reaction (Saiki et al., Science 230,1350 (1985)) variable heavy chain of immunoglobulin (Ig) or the initial substance of variable light chain increase.Simultaneously, primer has been introduced restriction enzyme site so that fragment cloning (is derived from pHENI to bacterial expression vector pHENIS; Hoogenboom et al., Nucl.Acids Res.19,4133 (1991); See Fig. 5).This carrier contains the pelB signal sequence and secretes for outer pericentral siphon, the myc marker is for detecting with monoclonal antibody 9E10, the histidine mark thing also contains the short sequence of the glycine-Serine joint of the coding region of heavy chain and light chain and 14 amino acid longs of encoding with by immobilized metal affinity chromatography (IMAC) purifying.In addition, at phage display, should merge with g3 β albumen.With suitable Restriction Enzyme (VH SfiI and ShoI; VL is with ApaLI and NotI) digestion heavy chain and light chain and be cloned in the carrier successively, thus obtain containing by the covalently bound variable heavy chain of short peptide sequence and the recombinant single chain Fv fragment of light chain.

The preparation of embodiment 2GS part

By at N ⁶-methyladenine-BSA or N ⁶(889 (1968) select N from natural or semisynthetic antibody library for Beiser et al., Methods Enzymol.XII in biological elutriation on-methyladenine-thyroglobulin conjugate ⁶-methyladenine (Sigma) has specific recombinant antibodies (Nissim et al., EMBO J.13,692 (1994)).In by antigen coated microtiter plate, identify positive antibody fragment (Nissim et al., EMBOJ.13,692 (1994)) by ELISA.The antibody that derives from these libraries has been required strand Fv form, can be directly used in clone subsequently.

The preparation of embodiment 3 joints

Has aminoacid sequence GLFEALLELLESLWELLLEA (SEQ IDNO.:1, Gottschalk et al., 1996) fusogenic peptide is used as joint, and the DNA of this peptide of encoding is prepared to double-stranded synthetic oligonucleotide, in its terminal coupling suitable restricted cleavage site (Ascl and XbaI) is arranged.For this reason, explanation according to manufacturers, use T4 the more nucleoside monophosphate kinase (Gibco) makes two synthetic oligonucleotide O1 (5 ' GGCCGCAGGCTTATTTGAGGCCCTTCTGGAATTGCTAGAGAGCCTCTGGGAATTGC TTCTGGAGGCAT, SEQ ID No.:20) and O2 (5 ' CTAGATGCCTCCAGAAGCAATTCCCAGAGGCTCTCTAGCAATTCCAGAAGGGCCTC AAATAAGCCTG, SEQ ID No.:21) phosphorylation, be heated to 80 ℃ and reach 5 minutes, slowly be chilled to room temperature then, this double chain DNA fragment is directly used in clone subsequently.

The preparation of embodiment 4 multi-functional parts

Expression vector pAB1 (by and the pHENIS similar methods make up, but do not comprise with g3p and merging; See Fig. 5) in prepare complete Fas lignand system with the form of 3-fragment cloning.What being limited property enzyme SfiI and NotI cut resists-NCAM strand Fv fragment (TS part), contains joint and anti--N of cloning site NotI and XbaI ⁶-methyladenine strand Fv fragment (GS part) is used as initial substance.In order to clone, use respectively the primer that inserts restricted cleavage site XbaI and AscI at N-terminal and the C-terminal GS fragment that increases again, these fragments are cloned in the pAB1 carrier of being limited property enzyme SfiI and AscI cutting, construct is transformed among the bacterial isolates TG1, the expression of Fas lignand system is subjected to the adjusting of bacterium lacZ promotor, and come the expression of inducing ligand system (as McCafferty et al. by adding isopropyl-(IPTG), Appl.Biochem.Biotech.47,157 (1994) is described).According to Griffiths et al., EMBO J.13,3245 (1994) method is used IMAC, and purifying is through expressed protein from outer pericentral siphon goods, the molecular weight of gross protein is about 55,000 dalton, shows as single aggressiveness.

Embodiment 5 detects the running of multi-functional part

Use cell culture technology well known by persons skilled in the art in cell culture, to breed and express the tumour cell (SCBC) of NCAM and separate it.By in intestinal bacteria, breeding the N that plasmid (structure gene that contains beta-Glucuronidase is seen patent application WO96/06940) prepares VITAMIN B4 ⁶Methylated DNA.

With 20: 1 mol ratios the multifunctional ligand system is mixed with plasmid DNA, place 37 ℃ to be incubated 30 minutes down in mixture, check the combination of plasmid DNA by ELISA, the mixture that will contain multi-functional part and plasmid mixes with 10: 1 ratio mutually with tumour cell, mixture places 37 ℃ to be incubated 1 hour down, washing tumour cell part, check combining of mixture and these tumour cells by immunofluorescence technique, remaining tumour cell is incubated 24 hours again, successfully taken in cell, the release from endosome of joint mediation and the transcript and expression of effector by using 4-methyl Umbrella shape base-β-glucuronide can measure mixture as the enzymic activity of beta-glucuronidase in the substrate detection substratum.

Claims (26)

1, the multifunctional ligand system that is used for target cell transspecific nucleotide sequence, contain at least a target cell ligands specific, at least a gene construct ligands specific that contains antibody or antibody moiety, with the joint that is connected these two parts, wherein said system right and wrong are immunogenic.

2, as the desired Fas lignand system of claim 1, further contain described two connectors that part links together, wherein connector contains two joints.

3, as claim 1 or 2 desired Fas lignand systems, wherein said target cell ligands specific and target cell surface bonding.

4; as any desired Fas lignand system among the claim 1-3; wherein said target cell ligands specific is selected from the group of being made up of following material: somatomedin; cytokine, Interferon, rabbit, tumour necrosis factor; chemokine; peptide hormone; angiotonin, phytokinin, histamine; steroid hormone; adhesion molecule, the part of VDL acceptor, the part of ldl receptor; the part of the ldl receptor of oxidation; the proteic part of LDL associated receptor, the part of IgGFc acceptor, the part of 88KDa glycoprotein receptor; the part of acetylizad ldl receptor, megalin part and VITAMIN.

5, as any desired Fas lignand system among the claim 1-3, wherein said target cell ligands specific is and target cell bonded antibody or antibody fragment.

6, as the desired Fas lignand system of claim 5, wherein said antibody fragment is selected from the group of being made up of following material: F (ab) ₂Fragment, Fab fragment, double-stranded Fv fragment, strand Fv fragment and Fc fragment.

7, as any desired Fas lignand system among the claim 1-3, wherein said target cell ligands specific is the cell outskirt by the Fc acceptor of antibody Fc fragment identification.

8, as any desired Fas lignand system among the claim 5-7, wherein said antibody or antibody fragment to small part derives from the people.

9, as any desired Fas lignand system among the claim 1-8, wherein said gene construct ligands specific is selected from the group of being made up of following material: antibody, antibody fragment, the acceptor of cationic structural unit and virus envelope proteins.

10, as the desired Fas lignand system of claim 9, wherein said antibody fragment is selected from the group of being made up of following material: F (ab) ₂Fragment, Fab fragment, double-stranded Fv fragment and strand Fv fragment.

11, as any desired Fas lignand system among the claim 1-8, wherein said gene construct ligands specific is the cell outskirt by the Fc acceptor of antibody Fc fragment identification.

12, as any desired Fas lignand system among the claim 1-11, wherein said antibody or antibody fragment to small part derives from the people.

13, as any desired Fas lignand system among the claim 1-12, wherein said antibody or described antibody fragment with by allogenic material is combined with nucleic acid, nucleic acid methylated or make the nucleic acid alkylation and the nucleic acid epitope specificity combination introduced.

14, as any desired Fas lignand system among the claim 1-13, wherein said antibody or described antibody fragment combine with epi-position on the non-virus carrier.

15, as the desired Fas lignand system of claim 14, wherein said non-virus carrier is selected from the group of being made up of following material: cationic polymers, peptide, protein, the amine of biogenetic derivation, polyamines, lipoid and phosphatide.

16, as any desired Fas lignand system among the claim 1-15, wherein said antibody or described antibody fragment combine with the epi-position of virus envelope proteins.

17, as the desired Fas lignand system of claim 16, wherein said virus is selected from the group of being made up of following material: murine leukemia virus, HIV, adenovirus, hsv, cytomegalovirus, the microvirus of mouse, adeno-associated virus, sindbis virus and vaccinia virus.

18, as the desired Fas lignand system of claim 9, wherein said cationic structural unit separately or unite and contain at least one lysine residue, at least one arginine, at least one Histidine and at least one polyamines.

19, as any desired Fas lignand system among the claim 2-18, wherein said connector contains the moiety covalently bound with functional residue, and functional residue is selected from the group of being made up of following material: amino residue, hydroxyl residue, SH residue, carbonyl residue and aldehyde residue.

20, as any desired Fas lignand system among the claim 2-19, wherein at least one described joint is an integration material.

21, as the desired Fas lignand system of claim 20, wherein said integration material is selected from the group of being made up of following material: synthetic fusogenic peptide, bacterium is merged the fusion product between skin or protein and skin or protein and the virus.

22, as any desired Fas lignand system among the claim 2-21, wherein said connector contain the antibody hinge region or with the protein bound Gal80 albumen of Gal4.

23, as the desired Fas lignand system of claim 1, contain

(a) contain the segmental target cell ligands specific of anti-NCAM recombinant single chain Fv, wherein make the segmental variable heavy chain of Fv and light chain covalently bound by short peptide sequence;

(b) contain the joint of fusogenic peptide, described fusogenic peptide has sequence GLFEALLELLESLWELLLEA (SEQ ID No.:1): and

(c) contain N ⁶The gene construct ligands specific of the recombinant antibodies of-methyladenine.

24, as the desired Fas lignand system of claim 23, further contain gene construct.

25, as the desired Fas lignand system of claim 24, wherein said gene construct is selected from the group of being made up of following material: exposed RNA, and plasmid, with cationic polymers, peptide, protein, or exposed nucleic acid or the plasmid of lipoid bonded, and virus.

26, any desired Fas lignand system is used for prevention or treatment tetter in preparation among the claim 1-25, mucosal disease, nervous system disorders, internal's disease, the blood coagulation disease, disease of hematopoietic system, disease of immune system, the purposes in musculature disease and sustentacular tissue or the arthropathic medicine.

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