CN1157570A - Intracellular delivery of chemical agents to specific cell type - Google Patents
Intracellular delivery of chemical agents to specific cell type Download PDFInfo
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- CN1157570A CN1157570A CN95195037A CN95195037A CN1157570A CN 1157570 A CN1157570 A CN 1157570A CN 95195037 A CN95195037 A CN 95195037A CN 95195037 A CN95195037 A CN 95195037A CN 1157570 A CN1157570 A CN 1157570A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
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Abstract
A composition for specific intracellular delivery of a chemical agent into a CR2 receptor-bearing cell comprises a CR2 receptor binding and endocytosis inducing ligand (CBEL) coupled to the chemical agent. The CBEL binds to the CR2 receptor and elicits endocytosis of the composition. Optionally, the composition can include a spacer, which can be either biodegradable or non-biodegradable, for coupling the CBEL to the chemical agent. Chemical agents can include cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like. The composition can further comprise a carrier such as a water soluble polymer, liposome, or particulate. Methods of using these compositions for delivering a chemical agent in vivo or in vitro are also disclosed.
Description
Background of the present invention
The present invention relates to the cytotropic carrying of chemicals.More particularly, the present invention relates to be used for chemicals, promptly express the cell of CR2 receptor, the compositions and the method for carrying in the cell to particular cell types.
Arouse considerable attention with antigenic toxitherapy cancer on targeted cells surface receptor or the tumor cell.For example, I.Pastan﹠amp; D.FitzGerald, Recombinant Toxins for CancerTreatment, 254 Science 1173 (1991); People's such as Anderson United States Patent (USP) 5169933 and 5135736; People's such as Thorpe United States Patent (USP) 5165923; People's such as Jansen United States Patent (USP) 4906469; The United States Patent (USP) 4962188 of Frankel; People's such as Uhr United States Patent (USP) 4792447; People's such as Masuho United States Patent (USP) 4450154 and 4350626.These medicines comprise the part of the targeted cells that links to each other with plant or bacteriotoxin, as somatomedin or antigen-binding proteins.Therefore the mechanism of their cell killings is different from traditional amic therapy method, reduces effectively or has eliminated cross tolerance to traditional chemotherapeutics.
Membrane glycoprotein CR2 (being also referred to as CD21) swallows epithelial cell at ripe bone-marrow-derived lymphocyte (B cell) and specific epithelial cell as the people, produces on people's folliculus arborescent cell and the neck epithelial cell, and is the receptor of epstein-barr virus (EBV) and complement fragment C3d/C3dg.N.Miller&L.M..Hutt-Fletcher，66?J.Virol.，3409(1990)。This receptor is the membrane glycoprotein of 145KD, and except that its combined function, it also participates in B cell-stimulating approach.As G.R.Nemerow, Deng the people, Identification and Characterization of the Epstein-Barr virus receptor onhuman B lymphocytes and its relationship to the C3d complementreceptor (CR2), 55 J.virol 347 (1985).EBV infected B cell is to be combined with the CR2 receptor-selective by membrane glycoprotein by this viral gp350/220, then CR2 receptor and interiorization and receptor-cause in conjunction with the cell endocytic of virion.For example people such as Tedder (1986 (, Epstein-Barr Virus binding induces internalization of the C3dreceptor:a novel immunotoxin delivery system, 137J.Immunol.1387 (1986).The epithelial cell that contains the CR2 receptor is also in conjunction with EBV, but obvious described cell is to infect by the mechanism except that receptor-mediated cell endocytic.
People such as Nemerow (Identification of gp350 as the viral glycoproteinmediating attachment of Epstein-Barr virus (EBV) to the EBV/C3dreceptor of B cells:sequence homology of gp350 and C3 complementfragment C3d, 61J.Virol.1416 (1987)) identified amino acid sequence similarity between C3dg and the gp350/220, described sequence comprises near gp350/220 (Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu; SEQ ID NO:1) domain, it is corresponding to the sequence (Glu-Asp-Pro-Gly-Lys-Gln-Leu-Tyr-Asn-Val-Glu among the C3dg; SEQ ID NO:2).People such as Nemerow (Identification of an epitope in the major envelope proteinof Epstein-Barr virus that mediates viral binding to the B lymphocyteEBV receptor (CR2), 56 Cell 369 (1989)) have described with purification CE2 receptor and CR2-and have expressed all bonded synthetic tetradecapeptide that contains the aminoacid sequence that is accredited as SEQ ID NO:1 of B cell.Should synthetic peptide also block combining of CR2 receptor on reorganization gp350/220 or C3dg and the B cell, and the Infection in Vitro of similar synthetic peptide inhibition EBV.Truncate and replace the analysis showed that of peptide analogues, with CR2 in conjunction with relevant EBV epi-position in Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu sequence (SEQ ID NO:1).It is low to observe the bound water pancake with short peptide, although Glu-Asp-Pro-Gly (SEQ ID NO:3) peptide has kept significant CR2 in conjunction with activity.Contain the CR2 that replaces the peptide that the single amino acids of Pro replaces with Gly also significantly reduces in conjunction with activity in this zone.
From aforementioned, be appreciated that compositions and its using method of being used for the chemicals cell contains on the B cell of transporting to the CR2 receptor have significant advantage in the art.The object of the invention and summary
One aspect of the present invention provides and is used for selected chemicals to particular cell types, and the cell of promptly expressing the CR2 receptor carries out the compositions of carrying in the cell, will cause receptor-mediated endocytosis with combining of described cell.
Another object of the present invention provides preparation and is used for the method that selected chemicals carries out carrying in the cell to the cell of expressing CR2 such as receptor.
Another object of the present invention provides and is used for selected chemicals, as cytotoxin, and transformed nucleic acid, Gene regulation agent, labelling, antigen, compositions from carrying in the cell to the cell of expressing CR2 such as receptor and method that medicine etc. carry out.
Another object of the present invention provides and can adhere to selected chemicals so that described chemicals CR2 receptors bind and produce the peptide part of chemicals endocytosis.
By being provided, a kind of compositions can finish these and other purposes, described compositions is used for carrying chemicals in the cell specifically is with the CR2 receptor to cell population (comprising the cell of not being with the CR2 receptor) cell, described compositions contains and can and induce the part (CBEL) of the endocytosis of receptor-mediation and in the bonded chemicals of described part in conjunction with the CR2 receptor, wherein said chemicals can cause the effect of selection when contain the cell that is transported to band CR2 receptor through cell in the time.Ripe bone-marrow-derived lymphocyte is the cell of the band CR2 receptor of these compositions targeting.Can carrying comprise cytotoxin, transformed nucleic acid, Gene regulation agent, labelling, antigen and medicine to the chemicals of described cell.CBEL and chemicals can be bonded to each other and/or be basic by the interval with other funtion parts, and as some peptide combination, they can be biodegradable, or abiotic degradable.Described compositions can also contain and is selected from water-soluble polymer, liposome and particulate carrier model system.
By under certain conditions, external cell population is contacted with the compositions of effective dose used compositions, and wherein CR2 receptors bind and cell endocytic-inducing ligand (CBEL) are in conjunction with the CR2 receptor and cause the endocytosis of receptor-binding compositions.For using in the body, the compositions of systemic administration effective dose is induced the endocytosis of described compositions then so that CBEL contacts and be combined in the CR2 receptor on the ripe bone-marrow-derived lymphocyte.After entering cell, described chemicals just can cause the function of its selection.The detailed description of accompanying drawing
Figure 1A and 1B illustrate CBEL and have the chemicals chemical bond of free sulfhydryl groups to form compositions of the present invention.
Fig. 2 A-2D represents the step of the plasmid of construction expression fusion rotein, and described fusion rotein contains CBEL and chemicals peptide of the present invention.
Fig. 3 represents relatively cellular exposure in the CBEL-ricin A fusion rotein of the present invention of variable concentrations and to CR2
+B cell (black rod) and CR2
-The influence of T cell (light rod).Detailed description of the present invention
The carrying chemicals is to the present composition and method of specific cells in disclosure and description are used for cell, should understand and the invention is not restricted to particular disclosed herein, method step and material, and described embodiment, method step and material can some changes.Should also be understood that owing to only limiting the present invention, so term used herein only is used to describe the purpose of particular by appended claim and equivalent.
Must be noted that used singulative " " and " described " in this description and claim are unless clearly explanation in the article otherwise includes plural form.Therefore for example comprise 2 or more part with reference to the compositions that contains " a kind of part ", " a kind of chemicals " comprises one or more described chemicalses, the chemicals that they can be identical or different, and " one base " at interval comprises 2 or a plurality of intervals base.
In description of the invention and claim, use following term according to following definition.
" peptide " refers to the peptide of any length as used herein, comprises protein.Term used herein " polypeptide " and " oligopeptide " are without any the restriction of specific size, specific unless stated otherwise size.
As used herein, " CR2 receptors bind and cell endocytic-inducing ligand " or " CBEL " refer to can in conjunction with CR2 (CD21) receptor and by the endocytosis of receptor and receptor-bonded CBEL the compositions of induced internalization.According to the present invention, CBEL combines with the molecule of difference in functionality so that behind the CBEL cell endocytic, also interiorization by the band cell of CR2 of bonded various functional moleculars with it.
According to present understanding, CBEL can be derived from EBV gp350/220 glycoprotein, comprises SEQ ID NO:1 and flanking sequence; The C3dg peptide comprises SEQ ID NO:2 and lateral order row; Or with its homologous basically peptide.Go into used herein, " homology basically " refers to keep in conjunction with CR2 receptor and functional peptide of inducing the endocytosis of receptor-mediation, although they may comprise the flanking sequence of SEQ ID NO:1 or SEQ ID NO:2 or by truncate, deletion mutant or replace mutant.In conjunction with and to induce the minimum requirements of the endocytosis of receptors bind be the sequence that is accredited as SEQ ID NO:3.Replacing mutant is to contain conservative those that replace of one or more amino acid residues.Conservative replacement be an aminoacid replacement another, wherein kept the function of described peptide, in this case, be meant in conjunction with the CR2 receptor and cause the function of the endocytosis of receptor-bonded compositions.The amino acid residue that belongs to specific conservative replacement can replace another amino acid residue with identical group sometimes.A kind of described group is as follows: Pro; Ala, Gly; Ser, Thr, Asn, Gln; Asp, Glu; His; Lys, Arg; Cys; Ile, Leu, Met, Val; And Phe, Trp, Tyr.See M..Jimenez-Montano﹠amp; L.Zamora-Cortina, Evolutinary model for thegeneration of amino acid sequences and its application to the study ofmammal alpha-hemoglobin chains, Proc.VIIth Int ' l; BiophysicsCongress, Mexico City (1981).Think that homologous basically other mutants comprise the L-aminoacid that the D-aminoacid replacement is natural, as the amino acid derivativges that contains other side chain replaces, and non-standard amino acid, promptly rare or in protein non-existent a-amino acid replace.The prototype structure of CBEL is only limited by functional.
As used herein, " chemicals " refers to and comprises after entering bone-marrow-derived lymphocyte inside by endocytosis that any material of selection is arranged.The particular chemical medicine has physiological role when interiorization is in cell, as cytotoxicity or to the effect of Gene regulation, B cell." transformed nucleic acid " (RNA or DNA), in the time of in entering cell, can be in time multiplexed cell system and/or expression.Other nucleic acid can be with intracellular adjusting sequence or regulatory factor effect so that influence intracellular gene expression in the mode of selecting.The detectable label of carrying makes and can identify the cell of the interiorization present composition by detecting described label in the cell.Carrying can cause the immunoreation of described antigenic specificity to intracellular antigen.Can eliminate the imbalance of cause a disease influence or other types with medicine or pharmacologic activity chemical compound.Useful especially chemicals comprises polypeptide, and some described chemicals is the active fragment of biological activity protein, or the antigen fragment of antigen protein (as epi-position).Therefore, chemicals comprises cytotoxin, Gene regulation agent, transformed nucleic acid, labelling, antigen, medicine etc.
As used herein, " carrier " refers to water-soluble polymer, granule or liposome.Described carrier can contain one or more CBEL and/or chemicals can multidigit point bonded with it.Described carrier improved described compositions molecular size, and can provide the selectivity and/or the stability of increase.Described selective problems is can not diffuse into cell by passiveness because the compositions that contains carrier is too big, and therefore the cell endocytic by receptor-mediation enters cell and just is restricted.Can be with the carrier connection peptides part that contains water-soluble polymer, chemicals and other functional moleculars.Set up the ability of the carrier that is used for the carrying of target medicine and measured, as referring to J.Kopecek.5 Biomaterials 19 (1984); E.Schacht et al., Polysaccharides as Drug Carriers, in Controlled-ReleaseTechnology 188 (P.I.Lee﹠amp; W.R.Good, eds., 1987); F.Hudecz et al., Carrierdesign:cytotoxicity and immunogenicity of synthetic branchedpolypeptides with poly (L-lysine) backbone, 19 J.Controlled Release231 (1992); Z.Brich et al., Preparation and characterization of a watersoluble dextran immunoconjugate of doxorubicin and the monoclonalantibody (ABL364), 19 J.Controlled Release 245 (1992).Therefore, illustrative water-soluble polymer comprises glucosan, inuloid, and poly-(L-lysine) contains the synthetic polymer of first acrylamide etc.
As used herein, " medicine " or " pharmacologic activity reagent " refers to deliver medicine to any chemical material or the chemical compound that is suitable among the cell B of band CR2 in inside, and it has induced biology or pharmacological effect required in the cell.
" effective dose " is the amount that produces selection as used herein.For example, contain cytotoxin can kill selected ratio in interval when selected as the selection of the compositions of chemicals mature B cell.The effective dose of described compositions is the amount that reaches this selected result, and described amount can be determined by the method that this area professional knows.
Compositions of the present invention provides the cell of the band CR2 receptor to the cell population (comprising the cell of not being with the CR2 receptor) of carrying chemicals in the cell specifically, described compositions contain can in conjunction with the CR2 receptor and induce receptor-mediated endocytosis CBEL and with the bonded chemicals of CBEL, wherein said chemicals can cause the effect of selection when cell contains when being transported in the cell of band CR2 receptor.Described chemicals is selected from cytotoxin, transformed nucleic acid, Gene regulation agent, labelling, antigen, medicine etc.CBEL can (but being not limited to) pass through covalent bond, electrostatic interaction, hydrophobic interaction, physics tunica etc. with combining of chemicals.CBEL can be directly with combining of chemicals or pass through another funtion part.Therefore, described compositions can also contain and CBEL and described chemicals are bonded and place interval base between CBEL and the described chemicals.Described interval base can be biodegradable or not biodegradable, and peptide base at interval is preferred.Compositions of the present invention also can contain and is selected from water-soluble polymer, liposome and particulate carrier.When carrier was water-soluble polymer, the molecular formula of described compositions was:
[L-S
a]
b-C-[S
e-A]
fWherein L can and induce the part (CBEL) of its cell endocytic in conjunction with the CR2 receptor; A is a chemicals; S is basic at interval; C is a water-soluble polymer, and it has and part, chemicals and the basic at interval compatible functional group of covalent bond that forms; A and e are 0 or 1; B and f are at least 1 integers.Described water-soluble polymer is selected from glucosan, inuloid, and poly-(L-lysine) contains the polymer of first acrylamide etc.
Therefore according to the present invention, CBEL provides and has made CR2 receptors bind on described compositions and the mature B cell, therefore causes the method by the described compositions interiorization of cell endocytic.Chemicals provides the method that reaches selection in the B cell.Therefore, for example, chemicals contains cytotoxin, comprises being used for optionally killing presenting CR
2The cell of receptor or make the performance CR
2The cell of receptor loses the radionuclide of function; Be used for causing the immunoreactive antigen of selection; The nucleic acid that is used for gene transformation or the gene expression of adjusting B cell; Be used to reach the medicine or the other drug active agent of selected therapeutical effect; Be used to detect the labelling of the cell that has absorbed described compositions, comprise fluorescent agent, radio-labeled and magnetic labelling; Deng.
Compositions of the present invention also can contain the water-soluble polymeric carrier so that have selected functional a plurality of CBEL and/or chemicals to be bonded in the compound molecule.At least 1 chemicals and at least 1 CBEL combine with described carrier, with the preferred number of the bonded CBEL of chemicals be 1-about 1000.Described carrier has increased the molecular size of described compositions, therefore can improve selectivity and/or stability to the funtion part that just can reach without carrier.Advantageously, the combination of CBEL and/or chemicals be by biology at interval base or biological nondegradable interval base finish.Described interval base can be selected according to its suitable sensitivity or resistance to intracellular hydrolysis of B and/or enzymatic lysis.Whether described selectivity provides for the professional in this field to select the ability of base at interval, described interval base to be based on it to help making chemicals to keep in cell and the combining or discharge from carrier by the desmoenzyme activity and select of carrier most.Liposome and granule do not rely on the use of water-soluble polymer carrier as the use of carrier.
In certain embodiments, by CBEL and chemicals chemical bond are made up described compositions.With CBEL and chemicals " chemical bond ", as used herein, refer to directly or utilize bound fraction CBEL and chemicals covalent bond.In specific embodiment,, can between functional group on the CBEL and chemicals, form key with bound fraction if chemicals is a peptide.For example, sulfydryl on the chemicals and amino on the CBEL are combined by the isodigeranyl functional cross-link agent can form the compositions that contains CBEL and chemicals.Reaction icon by maleimide amine crosslinker chemical bond CBEL and ricin A for example has been described in Figure 1A and 1B.Except that chemical bond CBEL and the maleimide amine crosslinker of chemicals that contains sulfydryl, can also use the halo acetyl group, alkyl halide, alkyl sulfonic ester, α, β-undersaturated carbonyl or α, β-undersaturated sulfone is partly as cross-linking agent.For the chemicals of chemical bond CBEL and amino-contained, can be with active ester as cross-linking agent.Whether feasible based on it to the functional group on CBEL and the chemicals, can use other bound fractions.
Can be at genetically engineered biological, as producing the present composition in the escherichia coli as " fusion rotein ".That is, can make up the hybrid gene of nucleotide sequence that contains the CBEL that encodes and the nucleotide sequence of encoding the chemicals peptide by recombinant DNA technology.This hybrid gene can be inserted in the organism, so that express " fusion rotein " of encoding by described hybrid gene.Then, can use standard method, comprise the described fusion rotein of affinitive layer purification.
Also can make up the fusion rotein that contains CBEL of the present invention and chemicals peptide with active synthetic method.
If produce compositions of the present invention in the mode of fusion rotein, then described chemicals can be closely adjacent each other, and promptly the carboxyl terminal of CBEL can directly link to each other with the amino terminal of described chemicals, and vice versa.In addition, described fusion rotein can also comprise other amino acid residues between CBEL and the described chemicals, so that these other amino acid residue can be used as the interval base between CBEL and the described chemicals.Usually the small peptide part is preferred, because the easier operation of small peptide, and because the amino acid residue of the existence, particularly basic number of other amino acid residues can influence the peptide part in the function of inducing through cell endocytic aspect the described chemicals interiorization.
Compositions of the present invention can also comprise localized protease digestion site, so that after described compositions enters in the cell, by the proteolysis that digests the site described chemicals and CBEL is divided and open.Described digestion site is present in the adjacent gp350/220 glycoprotein of SEQ ID NO:1 fragment natural, and therefore, the CBEL part that can extend compositions easily is to comprise described site.Can add the interval base of described protease sensitivity, and need not consider that described compositions is expression of peptides chemosynthesis or in genetically engineered biological.Under latter event, can between CBEL-encode fragment and chemicals peptide encode fragment, insert the encoding proteins enzyme sensitivity nucleotide of base at interval by technology well known in the art.
Another aspect of the present invention relates in the CR2-express cell in being contained in non-CR2-express cell population specificity and brings into play required active method, comprise described cell population is contacted with the compositions that contains the CBEL that is combined with chemicals that described chemicals is controlled described intracellular reactive.Compositions of the present invention is optionally combined with CR2-express cell in the mixed population, induce subsequently described compositions cell endocytic in cell, described chemicals is brought into play its activity in the CR2-express cell.
Except that other explanation, the application is used to operate peptide and is used to operate the standard technique of the nucleic acid of expression of peptides.The technology that is used in conjunction with oligopeptide and oligonucleotide is known in the art, and for example at T.Zhu et al., 3 Antisense Res.Dev.265 (1993); T.Zhu et al., 89Proc.Natl.Acad.Sci.USA 7934 (1992); P.rigaudy et al. is described among the 49 Cancer Res.1836 (1989).
As mentioned above, the present invention relates to be used as the peptide of CBEL in the compositions that also contains chemicals, described chemicals can also be peptide or contain peptide.Can comprise that organic synthesis and recombinant DNA technology prepare peptide of the present invention by various technology.For example at B.Merrifield et al., 21Biochemistry 5020-31 (1982); Among the Houghten 82 Proc.Natl Acad.Sci.USA 5131-35 (1985) (being incorporated herein by reference) technology that is used for chemical synthesising peptide has been described.It is known in the art being used for peptide and the bonded technology of other molecular chemistries.
Can prepare fusion rotein of the present invention by the nucleic acid of in suitable host cells, expressing oligonucleotide (as above-mentioned) that contains the CBEL that encodes and the oligonucleotide of encoding the chemicals peptide.The described technology that is used to produce recombination fusion protein is well known in the art, and at for example J.Sambrook et al., molecular cloning: in the laboratory manual (the 2nd edition, 1989) (relevant portion is incorporated herein by reference) description is arranged.Used reagent in described technology also is that this area is extensively known as restriction endonuclease etc., and can obtains from any suppliers.
With reference to wherein CBEL and cell toxicant chemicals peptide-bonded specific embodiment of ricin A are described the compositions that structure contains CBEL of the present invention and chemicals peptide.Ricin is the toxicity glycoprotein that is produced by castor-oil plants (Ricinus communis).It is made up of two subunits that connect together through disulfide bond, A chain and B chain, and molecular weight is about 30KD.Ricin is synthesized larger precursor, then its processing is produced ripe ricin A chain and B chain subunit.The A chain is the enzyme of the glycosidic inkage in the cracking 28S ribosomal RNA, destroys the ability of ribosome synthetic protein thus.A chain per minute makes about 1500 ribosome inactivations, this means that the ricin A molecule of an interiorization just can make cell-lethal.S.Olsnes?et?al.，Ribosome?inactivation?by?thetoxic?lectins?abrin?and?ricin，60?Eur.J.Biochem.281(1975)。The B chain combines with galactose moiety on the cell surface, and this is the necessary process of ricin interiorization.Remove the B chain and just can prevent that the A chain from entering cell, therefore make A chain inactivation.Ricin A chain combined with CBEL can make ricin A chain enter cell, obtain the competent cell toxin by the cell endocytic of receptor-mediation.
In first embodiment, form described compositions by active combination; And in second embodiment, compositions is a recombination fusion protein.Embodiment 1 chemical bond CBEL and ricin A
In order to illustrate, press shown in Figure 1A and the 1B, contain CBEL and the cell toxicant chemicals peptide of aminoacid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID NO:1) with two step method chemical bond.
In the first step, basically by the description in supplier's description by with m-dimaleoyl imino benzoyl-N-hydroxy thiosuccinimide ester (" sulfo--MBS "; Pierce, Rockford, Illinois, USA) reaction activates CBEL (by PeptideInternational, Kentucky, USA makes), in brief, in room temperature, through 2 hours with PBS buffer (20mM sodium phosphate, 0.15MNaCl.PH7.0) with 1: 10 mol ratio CBEL is mixed with sulfo--MBS.Use Superose 12 (Pharmacia) by FPLC, by the bioactive peptide of standard method purification gained.
In second step, in room temperature, (Missouri USA) presses 1: 5 mol ratio and reacted 1 hour in the PBS buffer for Sigma Chemical Co., St.Louis with maleimide-activatory peptide and deglycosylated ricin A.By from CBEL-ricin A conjugate, removing unreacted peptide molecule by film dialyzed overnight with 6-8KD cutoff value at 4 ℃.
The molecular weight that is used in the last gel electrophoresis assessment of 10%SDS-PAGE gel (U.Laemmli, 227 Nature 680 (1970)) resulting composition is about 32KD.Therefore each the ricin A molecule of compositions according to the present embodiment preparation contains a CBEL.The three-dimensional computer simulation shows that CBEL most possibly combines with the Cys residue of ricin A molecule C-end, because the Cys residue of N-end is embedded in the inside of folding ricin A molecule.Embodiment 2 contains the recombiant protein of CBEL and ricin A
Form CBEL and the cell toxicant chemicals peptide that contains aminoacid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID NO:1), the fusion rotein of ricin A with recombinant DNA technology.In a word, be inserted into the fusion rotein for preparing in the coli expression carrier in the present embodiment by synthetic oligonucleotide, described synthetic oligonucleotide is coded in the CBEL in coding ricin A polynucleotide downstream, in escherichia coli with the high yield expressed fusion protein, then by the described fusion rotein of affinity chromatograph purification from cell lysates.
With reference now to Fig. 2 A-2D,, by the following fusion rotein that contains CBEL and ricin A by recombinant DNA technology production.With limiting acid endo enzyme NcoI and BamHI digestion coli expression carrier pTrcHis B (Invitrogen, San Diego, California) (Fig. 2 A), this carrier contains a multiple clone site (MCS) in the downstream of translation initiation site (ATG), with the T4DNA polymerase the sticking end of gained is transformed into flush end, and then is connected (Fig. 2 B) with pTrc A.From plasmid pAKG (from Robert Weaver, University of Kansas, Lawrenc obtains; Be described in R.C..Halling et al., 13 Nucleic Acid Res.8019 (1985)) middle 863 bp BamHI-KpnI fragments of separating coding ricin A, be cloned into then among the pTrc B of BamHI and KpnI digestion.With the construct of BamHI digestion gained, with the T4DNA polymerase the sticking end of gained is transformed into flush end, and then connects the correct reading frame (Fig. 2 C) that generation is used to translate clone gene.
Digest the pTrc B carrier that gained is modified with KpnI and EcoRI then, it contains ricin A-coded sequence, following synthetic oligonucleotide is coupled, described following synthetic oligonucleotide contain the CBEL that encodes nucleotide residue and with at the compatible sticky end of KpnI and EcoRI site clone.
(SEQ?ID?NO：4) 5′-?CA?AAT?TTT?AAT?GAA?GAT?CCT
(SEQ?ID?NO：5) 3′-?CAT?GGT?TTA?AAA?TTA?CTT?CTA?GGA
(SEQ?ID?NO：6) Asn?Phe?Asn?Glu?Asp?Pro
(SEQ?ID?NO：4) GGT?TTT?TTC?AAT?GTT?GAG?CAT?CAT
(SEQ?ID?NO：5) CCA?AAA?AAG?TTA?CAA?CTC?GTA?GTA
(SEQ?ID?NO：6) Gly?Phe?Phe?Asn?Val?Glu?His?His
(SEQ?ID?NO：4) CAT?CAT?CAT?CAT?TAA?G -3′
(SEQ?ID?NO：5) GTA?GTA?GTA?GTA?ATT?CTT?AA -5′
(SEQ?ID?NO：6) His?His?His?His
The carrier of gained (Fig. 2 D) contains the hybrid gene of coding ricin A-protease site-part-His 6 fusion rotein, wherein " protease site " refers to above-mentioned protease digestion site or protease sensitivity base at interval, " part " refers to CBEL, " His 6 " refer to the zone of 6 continuous His residues, hereinafter will describe its function.Use gained plasmid transformation escherichia coli cell then, the screening transformant is also grown J.Miller in the LB culture medium, Experiments in MolecularGenetics, Cold spring Harbor Laboratory, Cold spring Harbor, N.Y. (1972).Dissolve these cells, at resin (" PROBOND ", Invitrogen, San Diego, the affinitive layer purification recombination fusion protein on post California) of nickeliferous-electric charge.6 His at fusion rotein C-end combine with nickle atom static on " PROBOND " resin.Washing the resin that contains bonded fusion rotein then depollutes to remove.Destroy electrostatic bond then, with the elution buffer eluting fusion rotein that contains imidazoles, to replace the His residue from nickel electric charge resin.Finish described purification according to supplier's handbook.Embodiment 3
The CBEL and the cell toxicant chemicals peptide that contain aminoacid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID NO:1) with the recombinant DNA technology production among the embodiment 2, the fusion rotein of the fusion rotein of ricin A, but except that leaving out the zone that contains 6 continuous His residues.Therefore, behind the pTrc B carrier of modifying with EcoRI and KpnI digestion, following synthetic oligonucleotide is coupled, and described following synthetic oligonucleotide contains and nucleotide residue and sticky end at the KpnI coding CBEL compatible with EcoRI site clone.
(SEQ?ID?NO：7) 5′-?CA?AAT?TTT?AAT?ATC?CAT?CTC
(SEQ?ID?NO：8) 3′-?CAT?GGT?TTA?AAA?TTA?TAG?GTA?GAG
(SEQ?ID?NO：9) Asn?Phe?Asn?Ile?His?Leu
(SEQ?ID?NO：7) ACG?GGT?GAA?GAT?CCT?GGT?TTT?TTC
(SEQ?ID?NO：8) TGC?CCA?CTT?CTA?GGA?CCA?AAA?AAG
(SEQ?ID?NO：9) Thr?Glu?Glu?Asp?Pro?Gly?Phe?Phe
(SEQ?ID?NO：7) AAT?GTT?GAG?TAA?G -3′
(SEQ?ID?NO：8) TTA?CAA?CTC?ATT?CTT?AA -5′
(SEQ?ID?NO：9) Asn?Val?Glu
The carrier of gained contains the hybrid gene of coding ricin A-protease site-CBEL fusion rotein, and wherein " protease site " refers to above-mentioned protease digestion site or protease sensitivity base at interval.Use gained plasmid transformation escherichia coli cell then, the screening transformant is also grown in the LB culture medium.By at the 20mM sodium phosphate, with the lysozyme dissolved cell of 1mg/ml, sonication is 3 times then among the pH7.8, separates expressed protein in each 1 minute.Under these conditions, fusion rotein is insoluble, but most of Escherichia coli protein is soluble.At 9000rpm with centrifugal 30 minutes of lysate, the resuspended bead that contains the gained fusion rotein, sonication is 1 minute before recentrifuge.With resuspended, sonication and centrifugation step repeat 3 times.The final bead that will contain pure relatively fusion rotein goods is dissolved in the solution that contains 6M urea and 5M dithiothreitol, DTT.Pass through described dissolved fusion rotein successive to the 4M urea; 2M urea and 20mM sodium phosphate, 500mM NaCl.pH7.8 dialyses degeneration.Embodiment 4
The CBEL and the cell toxicant chemicals peptide that contain aminoacid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID NO:1) with the recombinant DNA technology production among the embodiment 2, the fusion rotein of ricin A, but except that importing to another Cys residue in the fusion rotein.This structure obtains the recyclable fusion rotein of high yield more than embodiment 2, because can form intramolecular disulfide bond with described new Cys residue rather than with Escherichia coli protein at the free Cys residue of ricin A chain C-end.(California), it is except that having a different reading frame, and is similar to the pTrcHis B among Fig. 2 A for Invitrogen, San Diego with restriction endonuclease digestion coli expression carrier pTrcHisA.To encode then 6 continuous His residues and NcoI is arranged and the synthetic DNA of BamHI sticky end links to each other with described carrier.With BamHI digested plasmid pAKG, behind agarose gel electrophoresis, reclaim the 893bp fragment that discharges by electroelution.This fragment is connected in the modification pTrcHis A carrier of BaMHI and the enzymic digestion of Intestinum Bovis seu Bubali alkaline phosphatase.With described connection mixture transformed into escherichia coli strain X L-1 (Stratagene, La Jolla, California).The screening transformant is by determining the direction of BamHI fragment in carrier with BglII digestion.The DNA that the next self-contained ricin A gene transformation body of correct translation direction is arranged with SacI and EcoRI digestion, following synthetic oligonucleotide is coupled, and described following synthetic oligonucleotide contains and nucleotide residue and sticky end at the compatible coding CBEL of KpnI and EcoRI site clone.
(SEQ?ID?NO：10) 5′- C?GAA?GAT?CCT?GGT?TTT
(SEQ?ID?NO：11) 3′-?TC GAG?CTT?CTA?GGA?CCA?AAA
(SEQ?ID?NO：1) Glu?Asp?Pro?Gly?Phe
(SEQ?ID?NO：10) TTC?AAT?GTT?GAG?TAA?G -3′
(SEQ?ID?NO：11) AAG?TTA?CAA?CTC?ATT?CTT AA?-5′
(SEG ID NO:1) Phe Asn Val Glu with the DNA that connects be transformed into expressive host coli strain BLR (Novagen, Madison, Wisconsin) in, the recA bacterial strain does not have ioN and ompT protease yet.
By existing under the ampicillin condition, make cell transformed grow separating recombinant proteins matter at 37 ℃.When cultivation reaches the optical density of 0.6-0.8 (600nm), add isopropylthiogalactoside (IPTG) and reach the final concentration of 1mM with abduction delivering.After the regrowth 3 hours, collecting cell is suspended in then and contains 10mMTHCl, and pH 7.6,100mM KCl, and 20mM EDTA, the 10mM2-mercaptoethanol, in the buffer of 0.05%Nonidet P-40 and 0.5mg/ml lysozyme, insulation is 15 minutes in ice bath.Under the condition that has 0.5mM phenyl methyl sulfuryl fluoride, the lysate of sonication gained, then 4 ℃ with 9000rpm centrifugal 30 minutes.Adding ammonium sulfate reaches 40% saturation with the precipitation soluble protein, the bead of dissolution precipitation, and then to 10mMTrisHCl, pH7.4,100mM KCl dialysis.The product of will dialysing load to use the equilibrated reinforcing YIN-essence ion exchange column of same buffer (Q Sepharose Fast Flow, Pharmacia) on.Under these conditions, nearly all bacterioprotein all combines with this post, and unconjugated part is the recombinant protein of purification basically.Pass through the 4M urea, 20mM sodium phosphate, 500mM sodium chloride, pH7.8 are dialysed and are further purified recombinant protein again.Make dialysis product " PROBOND " resin, use the 4M urea then, 20mM sodium phosphate, 500mM sodium chloride, 5mM imidazoles, pH6.0 washing 5 times by nickeliferous electric charge.6 His residues in recombinant protein make recombinant protein by affinity interaction and described resin-bonded.With same buffer this post is washed 2 times, except that imidazole concentration is raised to the 30mM.With same buffer eluting recombinant protein, except that the concentration of imidazoles is the 300mM.With the protein denaturation of eluting, to the 2M urea, then to the 20mM sodium phosphate, 500mM NaCl, pH7.8 dialyse folding again by earlier then.Embodiment 5 with the carrying of cytotoxin targeting in the B cell
In order to illustrate the carrying of chemicals targeting is expressed in the B cell to CR2-, with reference now to specific embodiment, describe the use of the compositions of CBEL of the present invention and chemicals, wherein will express the B cell to CR2-has the ricin A of specific cell toxic action and the outer carrying of composition of CBEL to become lymphoid cell to Raji B.
In preparation explanation, at 37 ℃, RPMI 1640 culture medium of 1ml (Hyclone, Logan, Utah) in, need not handle in addition; Contain in the ricin A-CBEL compositions of 20ug in the foregoing description 1 at 1ml; In only containing the 1ml culture medium of ricin A with 1 * 10
6Individual CR2
+Raji B becomes lymphoid cell to cultivate 24 hours.Determine the cell death situation with trypan blue staining insulation back, then by using reverse microscopical erythrocytometer counting cells.Trypan blue is absorbed by dead cell, and blueness is distributed in the dead cell.(Missouri) dilution is 2 times for Sigma Chemical Co., St.Louis, is counting preincubation 5 minutes then with 0.4% trypan blue staining for a cell.With the percentage calculation of living cells is that the undyed cell number of per unit volume is divided by painted and undyed total cellular score * 100.
Only cell and the control cells of handling with ricin A is healthy.Cell with ricin A-CBEL compositions-treated has 99% death (1% survival) approximately.Because it is Cytotoxic that ricin A is only when having only in entering cell, these results show that ricin A-CBEL compositions of the present invention can make cell toxicant chemicals interiorization.Embodiment 6
Press following experimental example 2 recombinant C BEL-ricin A fusion rotein to CR2
+People B becomes lymph sample (Raii) cell and CR2
-The effect of T (HSB2) cell.With 1 * 10
6Individual cell suspending liquid thoroughly mixes in the 1ml culture medium with the purification of Recombinant fusion rotein of variable concentrations, then 37 ℃ of insulations 24 hours.After this, by embodiment 5 by assessing cell survival rate with the trypan blue staining cell.As shown in Figure 3, CR2
+Cell relies on mode with dosage and conjugate is handled react (black post), and CR2
-The influence that cell (shallow post) is not subject to processing.When fusion rotein concentration is 50-μ g/ml, CR2
+The survival of B cell is lower than 10%, and CR2
-Cell is 100%.Only all not have influence with CBEL or both cells of only handling with reorganization ricin A, this support recombinant C BEL-ricin A fusion rotein is to CR2
+The toxic action of B cell is the interiorization that results from recombination fusion protein CR2 receptor on the B cell.Embodiment 7
Repeat the process of embodiment 6, remove by using tetrazolium compound (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxyl methoxyphenyl)-2-(4-thio-phenyl)-2H-tetrazolium, inner salt; MTS) and electron coupling agent (phenazine methosulphate; PMS) colorimetry is determined the percentage ratio of survivaling cell.MTS is reduced into soluble first in tissue culture medium (TCM) by cell biological.Can directly measure first in the absorption value of 490nm and do not need other step from 96 hole check-out consoles.Viable count direct in the first product amount that 490nm absorption place is measured by first and in the cultivation is directly proportional.(Madison Wisconsin) obtains being used for the reagent that MTS tests from Promega Corp..Substantially the same among the result who obtains with this method and the embodiment 6.Embodiment 8
Method according to embodiment 6 detects the recombinant C BEL-ricin A fusion rotein of embodiment 3 to CR2
+People B becomes lymph sample (Raji) cell and CR2
-The effect of people T (HSB2) cell.The gained result is substantially the same with embodiment's 6.Embodiment 9
Method according to embodiment 6 detects the recombinant C BEL-ricin A fusion rotein of embodiment 4 to CR2
+People B becomes lymph sample (Raji) cell and CR2
-The effect of people T (HSB2) cell.The gained result is substantially the same with embodiment's 6.
Can be with CBEL-chemicals compositions of the present invention with chemicals carrying specifically to the CR2-express cell, normally CBEL induces described compositions cell endocytic to the condition of CR2-express cell therein, and the CR2-express cell is contacted with described compositions.Described then chemicals acts on the target cell that makes described compositions interiorization or plays a role in described target cell, the required effect of active medicine can be limited to CR2
+In those cells of phenotype.
For example, selectivity in the CBEL-Cytotoxic drugs compositions acting body of the present invention can be killed CR2
+Thereby the B cell is as the effective antitumor medicine.Preferably, by systemic administration, normally by subcutaneous, intramuscular or intravenous injection or intraperitoneal are applied to described main body with described compositions.Can be used for the injection of described purposes with the conventionally form preparation, can be liquid solution or suspension or the solid form that is suitable for being prepared into solution or suspension before injection, or as Emulsion.Suitable excipient comprises for example water, salt, glucosan, glycerol, ethanol etc.; If desired, can add a spot of auxiliary substance, as wetting agent or emulsifying agent, buffer etc.
Can be in external or body and cells contacting with described compositions.The CR2-express cell can contain the subgroup of the mixed population of (and expecting that in most of the cases it contains) cell type; Peptide part of the present invention can make conjugate enter the CR2-express cell through the endocytosis of CR2 specific cell.
Described chemicals can have any various required effect in described target cell.As mentioned above, in some useful especially embodiments, have only when or mainly when described medicine enters in the cell, described chemicals just pair cell is effective.Embodiment 10CBEL-antigen-drug target is transported in the B cell
The present invention can be contained that CBEL and antigenic compositions are applied to Homoiotherm so that definitiveness ground causes at CR2
+Immunoreation in the cell.Particularly, CBEL makes the interiorization of CR2 mediation make antigen enter cell, can cause the antibody-dependent/non-dependent approach that is used for complement activation in target cell then.That is,, can induce target cell to cause the immunoreation of antigen to n cell according to the present invention.
Activation contains the chemical bond of the CBEL of the aminoacid sequence that is accredited as SEQ ID NO:2, and (Sigma Chemical Co., St.Louis Missouri) link to each other to press embodiment and lysozyme of chicken then.Then CBEL-lysozyme conjugate is administered to mice.C3dg CBEL (SEQ IDNO:2) provides the binding specificity with mouse B cell, and induces the receptor-mediated endocytosis of CR2 of conjugate.Described conjugate causes the immunoreation of epi-position on the target B cell resistive connection compound then, and described epi-position comprises the unique epi-position of conjugate lysozyme part.The result of present embodiment can reappear by making up the CBEL-lysozyme fusion protein.Embodiment 11
Treatment human B cell leukemic method comprises that (a) provides and comprises CBEL, as EBV CBEL (SEQ ID NO:1) or with its homologous basically peptide, and cytotoxin, as the compositions of the present invention of ricin A with the compositions of (b) giving individual systemic administration effective dose.For example can be by the described compositions of the foregoing description 3 described preparations.The ripe human B cell of EBV CBEL targeting, ricin A is all virose to any cell that it can enter.The described compositions of giving individual systemic administration effective dose is so that described compositions enters blood, and with the B cells contacting.CBEL makes the CR2 receptors bind on compositions and the B cell, brings out the interiorization through the compositions of endocytosis then.Ricin A comes cell killing by destroying ribosome then.This process has reduced individual intravital Malignant B cell number, aspect the treatment disease plus effect is being arranged thus.Embodiment 12
According to the method treatment autoimmune disease of embodiment 11, as the method for lupus erythematosus or rheumatoid arthritis.After in entering the B cell, therefore the cytotoxin cell killing has reduced and has produced from the B of antibody cell number, aspect the treatment disease plus effect is being arranged thus.Sequence table (1) physical data:
(i) applicant: Ramesh K.Prakash
(ii) invention exercise question: chemicals carrying in the cell of specific cells
(iii) sequence number: 11
(iv) appropriate address:
(A) addressee: Thorpe, North﹠amp; Western
(B) street: 9035 South, 700 East, Suite 200
(C) city: Sandy
(D) state: Utah
(E) country: the U.S.
(F) postcode: 84070
(v) computer-reader form:
(A) interface types: Diskette, 3.5 inches, the 720Kb internal memory
(B) computer: Toshiba Satellite T1800
(C) operating system: DOS 6.0
(D) software: Word Perfect 6.0
(vi) current application materials:
(A) application number:
(B) applying date:
(C) classification number:
(vii) application materials formerly:
(A) application number:
(B) applying date:
(viii) agent/act on behalf of data:
(A) name: Alan J.Howarth
(B) registration number: 36553
(C) reference/number of documents: T2361
(ix) communications data:
(A) phone: (801) 566-6633
(B) fax: the data of (801) 566-0750 (2) SEQ ID NO:1:
(I) sequence signature:
(A) length: 9 aminoacid
(B) type: aminoacid
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: the data of SEQ ID NO:1Glu Asp Pro Gly Phe Phe Asn Val Glu 15 (2) SEQ ID NO:2:
(I) sequence signature:
(A) length: 11 aminoacid
(B) type: aminoacid
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: the data of SEQ ID NO:2 Glu Asp Pro Gly Lys Asn Leu Tyr Asn Val Glu 15 10 (2) SEQ ID NO:3:
(I) sequence signature:
(A) length: 4 aminoacid
(B) type: aminoacid
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: the data of SEQ ID NO:3 Glu Asp Pro Gly (2) SEQ ID NO:4:
(I) sequence signature:
(A) length: 60 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: the data of SEQ ID NO:4:CAAATTTTAA TGAAGATCCT GGTTTTTTCA ATGTTGAGCA TCATCATCAT CATCATTAAG 60 (2) SEQ ID NO:5:
(I) sequence signature:
(A) length: 68 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: the data of SEQ ID NO:5:AATTCTTAAT GATGATGATG ATGATGCTCA ACATTGAAAA AACCAGGATC TTCATTAAAA 60TTTGGTAC 68 (2) SEQ ID NO:6:
(I) sequence signature:
(A) length: 18 aminoacid
(B) type: aminoacid
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: the data of SEQ ID NO:6:Asn Phe Asn Glu Asp Pro Gly Phe Phe Asn Val Glu His His 15 10 His His His His 15 (2) SEQ ID NO:7:
(I) sequence signature:
(A) length: 57 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: the data of SEQ ID NO:7:CAAATTTTAA TATCCATCTC ACGGGTGAAG ATCCTGGTTT TTTCAATGTT GAGTAAG 57 (2) SEQ ID NO:8:
(I) sequence signature:
(A) length: 65 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: the data of SEQ ID NO:8:AATTCTTACT CAACATTGAA AAAACCAGGA TCTTCACCCG TGAGATGGAT ATTAAAATTT 60 GGTAC 65 (2) SEQ ID NO:9:
(I) sequence signature:
(A) length: 17 aminoacid
(B) type: aminoacid
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: the data of SEQ ID NO:9:Asn Phe Asn Ile His Leu Thr Gly Glu Asp Pro Gly Phe Phe 15 10 Asn Val Glu 15 (2) SEQ ID NO:10:
(I) sequence signature:
(A) length: 32
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:10:
The data of CGAAGATCCT GGTTTTTTCA ATGTTGAGTA AG 32 (2) SEQ ID NO:11:
(I) sequence signature:
(A) length: 40
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:11:
AATTCTTACT?CAACATTGAA?AAAACCAGGA?TCTTCGAGCT 40
Claims (51)
1 is used for the chemicals specific cell contained to be transported to comprising non-CR
2Receptor carries the compositions of the cell of band CR2 receptor in the cell population of cell, it comprises and can and induce the part and the chemicals that links to each other with described part of receptor-mediated endocytosis with described CR2 receptors bind, wherein can cause selected effect when described chemicals cell contains in the cell that is transported to described band CR2 receptor.
The compositions of 2 claim 1, the cell of wherein said band CR2 receptor is a bone-marrow-derived lymphocyte.
The compositions of 3 claim 2, wherein said part comprise and are selected from SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:6, the aminoacid sequence of SEQ ID NO:9 and with its peptide of the sequence of homology basically.
The compositions of 4 claim 3, wherein said chemicals is selected from cytotoxin, transformed nucleic acid, Gene regulation agent, label, antigen and medicine.
The compositions of 5 claim 4 also contain covalent bond and be inserted in described part and described chemicals between the interval base.
The compositions of 6 claim 5, wherein said interval base is biodegradable.
The compositions of 7 claim 6, wherein said interval base comprises peptide.
The compositions of 8 claim 7, wherein said part have the peptide of the sequence of SEQ ID NO:1, and described chemicals is ricin A.
The compositions of 9 claim 5, wherein said interval base are biological nondegradable.
The compositions of 10 claim 4 also contains and is selected from water-soluble polymer, liposome and particulate carrier.
The compositions of 11 claim 10, wherein said carrier are water-soluble polymer and the described compositionss with following formula:
[L-S
a]
b-C-[S
e-A]
fWherein L can and induce the described part of its endocytosis in conjunction with described CR2 receptor; A is a chemicals; S is basic at interval; C has and described part, the water-soluble polymer of the functional group that chemicals is compatible with the basic at interval covalent bond that forms; A and e are 0 or 1; B and f are at least 1 integer.
The compositions of 12 claim 11, wherein C is selected from glucosan, inuloid, poly-(L-Lys) and contain the polymer of first acrylamide.
13 external specificity carrying chemicalses comprise step to the method that comprises the cell of band CR2 receptor in the cell population of not being with the CR2 recipient cell:
(a) provide the interior carrying chemicals of specific cell to the compositions that comprises the cell of band CR2 receptor in the cell population of not being with the CR2 recipient cell, it comprises and can and induce the part and the chemicals that links to each other with described part of receptor-mediated endocytosis in conjunction with described CR2 receptor, in the time of wherein in described chemicals cell contains the cell that is transported to band CR2 receptor, can cause selected effect; With
(b) the CR2 receptors bind on the cell of described therein part and band CR2 receptor and causing under the condition of endocytosis of described compositions contacts described cell population with the described compositions of effective dose.
The method of 14 claim 13, the cell of wherein said band CR2 receptor is a bone-marrow-derived lymphocyte.
The method of 15 claim 14, wherein said part contain and have the SEQ of being selected from ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, the aminoacid sequence of SEQ ID NO:9 and with its peptide of the sequence of homology basically.
The method of 16 claim 15, wherein said chemicals is selected from cytotoxin, transformed nucleic acid, Gene regulation agent, label, antigen and medicine.
The method of 17 claim 16, wherein said compositions also comprise covalent bond and be inserted in described part and described chemicals between the interval base.
The method of 18 claim 17, wherein said interval base is biodegradable.
The method of 19 claim 18, wherein said interval base comprises peptide.
The method of 20 claim 19, wherein said part have the peptide of the sequence of SEQ ID NO:1, and described chemicals is ricin A.
The method of 21 claim 17, wherein said interval base are biological nondegradable.
The method of 22 claim 16, wherein said compositions also contain and are selected from water-soluble polymer, liposome and particulate carrier.
The method of 23 claim 22, wherein said carrier are water-soluble polymer and the described compositionss with following formula:
[L-S
a]
b-C-[S
e-A]
fWherein L can and induce the described part of its endocytosis in conjunction with described CR2 receptor; A is a chemicals; S is basic at interval; C has and described part, chemicals and the basic at interval water-soluble polymer that forms the compatible functional group of covalent bond; A and e are 0 or 1; B and f are at least 1 integer.
The method of 24 claim 23, wherein C is selected from glucosan, inuloid, poly-(L-Lys) and contain the polymer of first acrylamide.
The intracellular method of 25 specificity carrying chemicalses band CR2 receptor to the homoiothermic animal comprises step:
(a) provide the interior carrying chemicals of specific cell to the compositions that comprises the cell of band CR2 receptor in the cell population of not being with the CR2 recipient cell, it comprises and can and induce the part and the chemicals that links to each other with described part of receptor-mediated endocytosis in conjunction with described CR2 receptor, in the time of wherein in described chemicals cell contains the cell that is transported to band CR2 receptor, can cause selected effect; With
(b) the CR2 receptors bind on the cell of described therein part and band CR2 receptor and causing under the condition of endocytosis of described compositions delivers medicine to described homoiothermic animal with the described compositions whole body of effective dose.
The method of 26 claim 25, the cell of wherein said band CR2 receptor is a bone-marrow-derived lymphocyte.
The method of 27 claim 26, wherein said part comprise having the SEQ of being selected from ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, the aminoacid sequence of SEQ ID NO:9 and with its peptide of the sequence of homology basically.
The method of 28 claim 27, wherein said chemicals is selected from cytotoxin, transformed nucleic acid, Gene regulation agent, labelling, antigen and medicine.
The method of 29 claim 28, described compositions also comprise covalent bond and be inserted in described part and described chemicals between the interval base.
The method of 30 claim 29, wherein said interval base is biodegradable.
The method of 31 claim 30, wherein said interval base comprises peptide.
The method of 32 claim 31, wherein said part are the peptide with sequence of SEQ ID NO:1, and described chemicals is ricin A.
The method of 33 claim 29, wherein said interval base are biological nondegradable.
The method of 34 claim 28, wherein said compositions also contain and are selected from water-soluble polymer, liposome and particulate carrier.
The method of 35 claim 34, wherein said carrier are water-soluble polymer and the described compositionss with following formula
[L-S
a]
b-C-[S
e-A]
fWherein L can and induce the described part of its endocytosis in conjunction with described CR2 receptor; A is a chemicals; S is basic at interval; C has and described part, chemicals and the basic at interval water-soluble polymer that forms the compatible functional group of covalent bond; A and e are 0 or 1; B and f are at least 1 integer.
The method of 36 claim 35, wherein C is selected from glucosan, inuloid, poly-(L-Lys) and contain the polymer of first acrylamide.
37 are used to transform host's recombinant vector, and described carrier comprises that coding contains the dna fragmentation of the fusion rotein of CR2 receptors bind and endocytosis-inducing ligand and chemicals.
The recombinant vector of 38 claim 37, wherein said carrier contains plasmid.
The wherein said part of the recombinant vector of 39 claim 38 comprises having the SEQ of being selected from ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, the aminoacid sequence of SEQ IDNO:9 and with its peptide of the sequence of homology basically.
The recombinant vector of 40 claim 39, wherein said part contains the aminoacid sequence that is accredited as SEQ ID NO:1.
The recombinant vector of 41 claim 40, wherein said chemicals comprises cytotoxin.
The recombinant vector of 42 claim 41, wherein said cytotoxin comprises ricin A.
The recombinant vector of 43 claim 40, wherein said chemicals comprises antigen.
The recombinant vector of 44 claim 40, wherein said chemicals is selected from the Gene regulation agent, label and medicine.
45 codings contain the dna fragmentation of the fusion rotein of CR2 receptors bind and endocytosis-inducing ligand and chemicals.
The dna fragmentation of 46 claim 45, wherein said part contain and have the SEQ of being selected from ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, the aminoacid sequence of SEQ IDNO:9 and with its peptide of the sequence of homology basically.
The dna fragmentation of 47 claim 46, wherein said part contains the aminoacid sequence that is accredited as SEQ ID NO:1.
The dna fragmentation of 48 claim 47, wherein said chemicals comprises cytotoxin.
The dna fragmentation of 49 claim 48, wherein said cytotoxin comprises ricin A.
The dna fragmentation of 50 claim 47, wherein said chemicals comprises antigen.
The dna fragmentation of 51 claim 47, wherein said chemicals is selected from the Gene regulation agent, label and medicine.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US30577094A | 1994-09-13 | 1994-09-13 | |
| US08/305,770 | 1994-09-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1157570A true CN1157570A (en) | 1997-08-20 |
Family
ID=23182273
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95195037A Pending CN1157570A (en) | 1994-09-13 | 1995-09-12 | Intracellular delivery of chemical agents to specific cell type |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP0781139A1 (en) |
| JP (1) | JPH10505835A (en) |
| KR (1) | KR970705404A (en) |
| CN (1) | CN1157570A (en) |
| AU (1) | AU697469B2 (en) |
| BR (1) | BR9508951A (en) |
| CA (1) | CA2198361A1 (en) |
| CZ (1) | CZ74797A3 (en) |
| HU (1) | HUT77263A (en) |
| MX (1) | MX9701860A (en) |
| PL (1) | PL319100A1 (en) |
| WO (1) | WO1996008263A1 (en) |
| ZA (1) | ZA957688B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616665B (en) * | 1997-11-19 | 2010-10-06 | 乔治敦大学 | Targeted liposome gene delivery |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6251866B1 (en) | 1997-08-05 | 2001-06-26 | Watson Laboratories, Inc. | Conjugates targeted to the interleukin-2 receptor |
| WO1999007324A2 (en) * | 1997-08-05 | 1999-02-18 | Watson Pharmaceuticals, Inc. | Conjugates targeted to the interleukin-2 receptor |
| DE19926154A1 (en) * | 1999-06-09 | 2000-12-14 | Ktb Tumorforschungs Gmbh | Process for the preparation of an injectable pharmaceutical preparation |
| GB9922554D0 (en) * | 1999-09-23 | 1999-11-24 | Microbiological Res Authority | Inhibition of secretion from non-neuronal cells |
| US10806803B2 (en) | 2014-07-17 | 2020-10-20 | Ohio State Innovation Foundation | Compositions for targeting macrophages and other CD206 high expressing cells and methods of treating and diagnosis |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5135736A (en) * | 1988-08-15 | 1992-08-04 | Neorx Corporation | Covalently-linked complexes and methods for enhanced cytotoxicity and imaging |
| US5331090A (en) * | 1989-09-08 | 1994-07-19 | California Institute Of Biological Research | CR2 ligand compositions and methods for modulating immune cell functions |
| US5165923A (en) * | 1989-11-20 | 1992-11-24 | Imperial Cancer Research Technology | Methods and compositions for the treatment of hodgkin's disease |
-
1995
- 1995-09-12 CN CN95195037A patent/CN1157570A/en active Pending
- 1995-09-12 WO PCT/US1995/011515 patent/WO1996008263A1/en not_active Application Discontinuation
- 1995-09-12 AU AU35507/95A patent/AU697469B2/en not_active Ceased
- 1995-09-12 JP JP8510273A patent/JPH10505835A/en active Pending
- 1995-09-12 EP EP95932472A patent/EP0781139A1/en not_active Withdrawn
- 1995-09-12 CA CA002198361A patent/CA2198361A1/en not_active Abandoned
- 1995-09-12 CZ CZ97747A patent/CZ74797A3/en unknown
- 1995-09-12 BR BR9508951A patent/BR9508951A/en not_active Application Discontinuation
- 1995-09-12 KR KR1019970701469A patent/KR970705404A/en not_active Application Discontinuation
- 1995-09-12 PL PL95319100A patent/PL319100A1/en unknown
- 1995-09-12 HU HU9701746A patent/HUT77263A/en unknown
- 1995-09-12 MX MX9701860A patent/MX9701860A/en unknown
- 1995-09-13 ZA ZA957688A patent/ZA957688B/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616665B (en) * | 1997-11-19 | 2010-10-06 | 乔治敦大学 | Targeted liposome gene delivery |
Also Published As
| Publication number | Publication date |
|---|---|
| AU697469B2 (en) | 1998-10-08 |
| PL319100A1 (en) | 1997-07-21 |
| HUT77263A (en) | 1998-03-02 |
| ZA957688B (en) | 1996-05-13 |
| AU3550795A (en) | 1996-03-29 |
| CZ74797A3 (en) | 1997-08-13 |
| WO1996008263A1 (en) | 1996-03-21 |
| MX9701860A (en) | 1997-06-28 |
| CA2198361A1 (en) | 1996-03-21 |
| JPH10505835A (en) | 1998-06-09 |
| KR970705404A (en) | 1997-10-09 |
| BR9508951A (en) | 1999-04-06 |
| EP0781139A1 (en) | 1997-07-02 |
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