JPH07508400A - Receptor internalization signals - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] can be, Xll, when present, is alanine or phenylalanine, where at least one of residues X1, X, and X is an aromatic amino acid, Furthermore, residues X1, X2, Xl, XIG, and X11 are on the core sequence side. It can only exist if the immediately adjacent residue is present, However, FXNPXY, GPLY, PPGY, and yxYXKV (in the array X represents any amino acid) A composition containing a peptide.

22, containing a nucleotide sequence encoding the peptide according to claim 21 composition.

23. Provide a host subject with a heterologous internal enzyme that can modulate the intracellular transport of the ligand. Introducing cells from the host subject that have been modified to contain a cation signal. A gene therapy method consisting of steps.

24. Injecting into the host subject's cells a heterologous interface that can modulate the transport of the ligand into the cell. Expression vector containing a nucleotide sequence encoding a naturalization signal A method of gene therapy consisting of the process of introducing.

Background of the invention The present invention provides internalization signals as well as intracellular delivery of ligands. on the use of internalization signals in regulating the transport of Ru.

2. Related technology Receptor-dependent endocytosis is the process by which a variety of nutrients, hormones, and growth factors are detected. It is a mechanism that specifically and efficiently transports into cells. receptor-dependent endocytosis The process of tosis is complex and consists of several distinct biochemical processes. en The process of docytosis usually involves (1) the recruitment of soluble coat proteins to the cell membrane; (2) Coat constituents gather and form coating pits. The bit grows, (3) specific receptors are captured within the growing coated bit, and (4 ) the cell membrane invaginates, (5) the coat closes, and (6) the coated bit exits inside. Proceeds through the process of membrane fusion at the point where it sprouts, breaks off, and forms a coated vesicle. .

The contents of the vesicle are ultimately transported to the end site. receptor-dependent endocytosis The key to the entire process of tosis is the interface present in the cytoplasmic tail of the receptor. It is a narration signal. Cytoplasmic tail during formation of coated bits interacts with soluble coat proteins.

Receptors such as transferrin receptor (TR) and low density lipoprotein The LDL receptors are present in constitutive clusters within the coated bits and are internalizes quickly in the presence as well as absence of the code. other receptors, For example, the epidermal growth factor (EGF) receptor binds to a ligand and then It is concentrated and internalized within the cut. From previous research, we have found that The cytoplasmic domain of the receptor used contains internalization signals. present and this internalization signal is present in the coated bit adapter. It interacts with proteins to promote highly efficient endocytosis. Something has become clear.

Modulating endocytosis using heterologous internalization signals Preferable if possible. At that time, for example, the interaction of toxic substances by tumor cells may be stimulating naturalization or inhibiting the uptake of essential nutrients. should be possible. However, until the applicant's invention, the The successful transfer of an ization signal from one receptor to another is could not. The present invention responds to these needs and provides a using localization signals and discovering previously unknown signals. It provides a means to infer sequence and structure.

Disclosure of invention The present invention involves introducing heterologous internalization signals into cells. We have discovered that the transport of ligands into cells can be regulated by signal may be introduced as a nucleotide sequence or as an encoded peptide. Inter transfer the naturalization signal from one receptor to another and still The ability to retain its activity means that it can be used in various scientific and medical applications, such as microorganisms. Important for controlling endocytosis during drug delivery to cells It has a meaning.

Therefore, the present invention provides methods for modulating receptor-dependent transport of ligands into cells. The process of introducing a foreign internalization signal into cells is The present invention provides a method for including.

The present invention also provides a mechanism for controlling the internalization of cell surface receptors. It also provides a way to identify columns. This method comprises: (a) having such a receptor; cell surface receptors in the presence or absence of sequences and, as appropriate, cell surface receptors. (b) in the presence of said sequence or comprising measuring internalization of said cellular receptor in the absence.

The invention also provides compositions that modulate the transport of ligands into cells.

The composition of the present invention has a tight turn conformation. and includes peptides having the predetermined amino acid sequences detailed below. The present invention , also provides nucleotide sequences encoding such peptides.

Additionally, the present invention provides a method of applying gene therapy to a host subject. It is. This method uses, for example, a heterologous protein that can modulate the transport of the ligand into the cell. Cells from a host subject that have been modified to contain a internalization signal are then can be carried out by introducing the host subject into a host subject.

The present invention provides methods for heterologous internalization that can modulate the transport of ligands into cells. ・Introducing an expression vector containing a nucleotide sequence encoding a signal into a host subject Also provided is a method of gene therapy comprising the step of introducing.

Brief description of the drawing The following drawing description describes the first successful heterogeneous internalization. This paper describes signal transplantation.

Figure 1. Human Tf by CEF expressing wild type or mutant human TR The amount of 1.9Fe taken in from. From human Tf by CEF expressing human TR The measurement of the uptake amount of 19Fe is shown in the diagram with the cell "Fe-Tf". By incubating the cells for a period of time, washing the cells, and measuring their radioactivity. I went. The results shown are for two representative experiments A and B. Each point represents 55 cells from three cultures of CEF expressing wild-type or mutant TR. It represents the average value of IFC uptake. Graph A shows wild type (mu) and 20 YTFR23 → NPVY (B), “LSYTRF” → FDNPVY (I I), “YTRF”-YKSV (○), “LSYTRF” → YKYSKV (・), and relative S I F for mutant TR of F23M (△) Indicates the amount of e taken in.

Graph B shows wild type (Mu) and Z　OY　T　RF　23→NPVY(E l), “LSYTRF”-FDNPVY (Maro), F23W (○), F23I (・), and relative 59 for mutant human TR of △3-59 (△) The amount of Fe uptake is shown.

Figure 2. Steady-state distribution (tablet) and human TR mutations expressed in CEF (TR Mutation at the carboxy-terminal aromatic residue of the internalization motif ) Comparison of 69pe uptake amount (b). The 23rd phenylalanine is methi Onine (F23M), Isoleucine (F23I), Tryptophan (F23W) , changed to either alanine (F23A) or glycine (F23G) did. Data for F23A and F23B mutants are shown in ColCo11 o, et al, Ce1l, 63: 1061-1072. From 1990 That's what I got. For steady-state analysis, CEF expressing human TR was After incubating at 37°C for 60 minutes with '2&tte-labeled Tf, Washed with buffer. using the acid cleaning technique described in the Materials and Methods section. We distinguished between surface-bound Tf and internalized Tf. Inter The rate of Narai Zenyong represents the average value of three experiments and is based on the wild type (Wt ) is shown as percentile standard error for TR.

Figure 3. Six-residue mannose-〇-phosphate receptor (Man-6-PR) and low density An protein with a sequence that matches the pattern of the LDLR Overlay of crystallographic turn structures of internalization motif analogs. . (A) Analogue of the internalizing soyon motif of Man-6-PR.

(B) Analogue of the internalizing motif of LDLR.

(C) Analogs of the internalization motif of Man-6-PR and L Overlay of analogs of the DLR internalization motif.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention involves the step of introducing a heterologous internalization signal into cells. A method of modulating receptor-dependent transport of a ligand into a cell, including.

The term "heterologous" is used to describe the internalization signals of the present invention. All internalizations introduced into the cells should be ・Refers to a signal.

The term "ligand" refers to any substance that can bind to a receptor on the surface of a cell. is called. The term "tight turn" refers to Reverse turns or helical turn conformations of amino acid residues in the signal Collawn, et al, Ce11.6 3: 1061.1990; CoCo11a.

et at, EMBOJ, 10: 3247.1991). ``Cell table The term "face receptor" refers to all receptors, whether or not they have a natural ligand. refers to molecules on the cell surface. The term "sequence" refers to an amino acid sequence and refers to a nucleic acid sequence. ``Internalization Motif'' The term refers to the internalization signal of amino acids with a tight turn structure. Names Naru. The term internalization motif and The terms internalization signals are used interchangeably. . The term “core” refers to the core involved in internalization at the cell surface. Refers to the minimum sequence of amino acid residues.

Amino acids referred to herein are abbreviated to the three-letter or one-letter abbreviations shown below. Therefore, it may be specified.

3 characters 1 character Amino acid abbreviation abbreviation L-alanine Ala A L-Arginine Arg R L-asparagine Asn N L-aspartic acid Asp D L-cystine Cys C L-glutamine Gin Q L-glutamic acid Glu E L-glycine cty G L-histidine His H L-isoleucine lie I L-Leucine Leu L L-lysine Lys K L-methionine Met M L-phenylalanine Phe F L-Proline Pro P L-serine Ser S L-threonine Thr T L-tryptophan Trp W L-Tyrosine Tyr Y L-valine Val V The heterologous internalization signal used in the present invention is already present in the cell. May be the same or different from existing internalization signals. stomach. When introducing DNA encoding a foreign signal into target cells, introduce The nucleotide sequence further encodes another nucleotide sequence that encodes a cell surface receptor. may be included in This cell surface receptor is already present on target cells It may be the same as or different from the receptor.

Thus, in the present invention, receptors containing internalization signals are cells with receptors that do not have an internalization signal. Embodiments for introducing heterologous internalization signals into cells with That's what I'm trying to do. In addition, internalization sequences can be integrated into cell surface receptors. When introduced as part of the body, this receptor is already present in the target cell. It may be the same or different from the receptor.

The introduction of heterologous internalization signals into target cells according to the present invention , can be done by several means.

For example, the signal can be linked to a peptide or a nucleolyte encoding this peptide. It can be introduced as a sequence. Also, the introduced array is adjacent to this array. It may further include an array. This additional flanking sequence is associated with cell surface receptors. or may be a cell surface receptor.

internalized signal peptide or a peptide that encodes this peptide. Introduction of nucleic acid sequences to cell surface receptors can be accomplished by several methods. These methods include the following: l) Residues in the receptor are Replace with a residue in the entered internalization sequence (1:1 residue/base) ). 2) between two residues/bases in an already existing receptor; Insert a optimization signal. Therefore, the receptor sequence becomes longer. 3 ) to replace some residues in the already existing receptor with a larger number of internal residues. Replace with the residue of the cation signal. Therefore, the receptor becomes longer. in Which method can be used to introduce the internalization signal peptide? The decision to select is made in each case by considering various structural and experimental conditions.

The preferred position for introducing internalization signals into the receptor sequence is , by determining the location of endogenous internalization signals. In addition, the receptor sequence has a tight turn conformation and a backing conformation. Determine the location that has interaction and can accommodate the introduced signal. It can be confirmed by Information regarding construction and backing can be found at Can be obtained by crystal structure analysis, NMRl or high resolution electron microscopy . In situations where such information is not available, commonly used and difficult to obtain bends as tight turns using easy-to-use algorithms for secondary structure estimation. It is a good idea to determine the positions of sequence regions that are likely to be affected. Internals of receptor array When replacing the internalization array, if the internalization array has an array The tight turn region of the most similar receptor is the preferred site for substitution.

This is because substitutions at these sites cause minimal structural instability. be. Sequence similarity can be calculated using matrices of mutation data and other sequence similarities. It can be determined by indicators such as residue size and polarity.

Introduction of a heterologous internalization signal can improve this reorganization of the ligand. Transport into cells with surface receptors reactive with Gund can be regulated.

Through these adjustments, what is selected as a heterologous internalization signal? Depending on the situation, an increase or decrease in endocytosis is induced. mentioned above As shown above, surface receptors hold the key to receptor-dependent endocytosis. This is an internalization signal present in the cytoplasmic tail of . cell surface Internalization signals control surface receptor uptake That's why.

Heterologous internalizers that can modulate cell surface receptor internalization Identification of this type of receptor activation signal will allow cells with this type of receptor to become internalized. constant in the presence or absence of a sequence that is thought to be a cation signal. It is necessary to incubate at a warm temperature. Cell culture was performed by Jing, et al., J. , Ce1l Bio+,.

110:283.1990. If necessary, constant temperature culture is carried out in the presence of the ligand, and cell surface receptors are detected in the presence or absence of the sequence. Measuring body internalization.

The present invention also provides methods for inhibiting internalization of cell surface receptors. It is something that This process involves the receptors being clustered together in the coating. Delivering an effective amount of a heterologous internalization signal peptide into cells This is carried out by inserting the foreign peptide into the adapter. - Receptor internalization signals are involved in binding to proteins. It will be competitive with the peptide of Le. The term "effective amount" refers to This refers to the amount at which the formation of cyto-cys vesicles is suppressed. Effective amount Delivery of internalization signal peptides can be achieved by the mechanisms described herein. for example, encapsulation in liposomes or other techniques well known in the art. It can be done by law.

Internalization signal peptides that can be used in the present invention has a tight turn, and the amino acid sequence: X I Xr Xh X 4 Xs It is characterized by having 　Xs　XI　X　X@Xi　XIoXz.

Here, X1Xs constitutes a core array, The residues X, -X, and X9Xl+ are present as necessary. ,and Xl, if present, is leucine or glutamic acid; X2, if present, is isoleucine, methionine, or proline. the law of nature, X, if present, is any amino acid residue, X4, if present is selected from alanine, polar amino acids, or aromatic amino acids; If present, at least one of the X3 or X residues is polar and χG is an aromatic amino acid if the residues X, -X, are absent, and at least There is also a residue of X, or (one or more) residues further upstream is selected from aromatic amino acids or polar amino acids, and X6 is a polar amino acid. It is an amino acid or alanine, and X is an X, -X residue is not present. has at least X4 residues selected from polar amino acids or alanine or if there are (one or more) residues further upstream, any is an amino acid residue, XI is selected from aromatic amino acids or hydrophobic amino acids, and X is present X to is serine or alanine, and X to is serine or alanine, if present. Ranine or leucine, Xll, when present, is alanine or phenylalanine, Here, at least one of the residues X, , X, and X is an aromatic amino acid , and furthermore, residues Xl, X2, Xl, X1o, and Xll are on the core sequence side It can only be present if there is a residue immediately adjacent to .

The first group of peptides suitable for internalization signals are amino Noic acid sequence: X5X6X7XI , where X5−X is as defined above, and Douche X6 and X7 cannot both be alanine.

Peptide of internalization signal having sequence X5Xs X7Xs In a group more suitable as a code, X5 in the sequence is phenylalanine or tyrosine. and X6 is alanine, arginine, glutamine, serine, or Onin, X, is alanine, arginine, aspartic acid, glycine, glutamic acid, stidine, lysine, or threonine, and X6 is isoleucine, leucine, methionine, phenylalanine, valine, a Rui is tryptophan.

an internalization signal peptide having the sequence x, x, x, x; The most suitable as a code are YTRM, YARF, YTRI, YQDL, YTKF. , YSKV, YTRWSYRHV, YSAFSYQTI, YTAF, YTGFS It is selected from the group consisting of YTEF, FTRF, and YTRF.

A second preferred group of internalization signal peptides is amino Noic acid sequence: x, x, x5x, x, Xs , where X3 XI is as defined above.

internalization sig with array X+X, XI Xa x, x A more preferable group as a null peptide is one in which X in the sequence is asparagine, leuko Syn, methionine, phenylalanine, proline, or tyrosine, and , but alanine, aspartic acid, glutamine, lysine, phenylalanine, R is serine, and XI is asparagine, glutamine, or tyrosine. can be, x6 is arginine, glycine, proline, serine, or toonine the law of nature, X is alanine, arginine, lysine, phenylalanine, or valine Yes, and x6 is isoleucine, leucine, methionine, phenylalanine, tryptophyl It is one that is phantom, tyrosine, or valine.

Internalizer X3Xt Xi X6X7Xs arrangement The most suitable peptides are YKY SKV, NFYRAL, LA. YTRFSPQQGFFSFDNPVY, MSYTRF, and LSYTRF. selected from a group of

The novel composition of the invention has a tight turn and has an amino acid sequence: XI X2 Xl X, Xi x6X, Xs Xl Xl, X,.

Xll is a peptide with alanine or phenylala when present This includes: Here, X, -X, constitute a core array, The residues X, -X, and X5Xz are present as necessary, and Xl, if present, is leucine or glutamic acid; X2, if present, is isoleucine, methionine, or proline. the law of nature, Xa, if present, is any amino acid residue; is selected from alanine, polar amino acids, or aromatic amino acids, and Xs is also present, at least one of the Xa or X4 residues is polar and X 5 is an aromatic amino acid when the residues X, -X, are absent, and at least In both cases, the X4 residue or one (one or more) residue further upstream is present. in case of X6 is selected from aromatic amino acids or polar amino acids; is an amino acid or alanine, and there is no residue of X, -X, In some cases, at least the X4 residue is selected from polar amino acids or alanine. Alternatively, if there are (one or more) residues further upstream, any It is an amino acid residue of meaning, Xl is selected from aromatic amino acids or hydrophobic amino acids; XIG, if present, is serine or alanine; Nin or leucine, ``Sensitivity'' is a receptor internalizenin that does not have a signal, wherein at least one of residues X, , X, and XI is an aromatic amino acid; And furthermore, residues x, ,x, ,x,,x,. , and Xll are the core arrays It can only exist if there are immediately adjacent residues on the side, However, FXNPXYSGPLYSPPGY, and YXYXKV (in the array X represents any amino acid).

As used herein, the term "polar amino acids" refers to glycine, serine, threo nin, histidine, tyrosine, proline, aspartic acid, asparagine, glu Contains tamic acid, glutamine, arginine, and lysine (Rose, etc. at, 5science, 229:834, 1985).

The term "aromatic amino acid" refers to any amino acid containing an unsaturated carbocyclic ring, Examples include phenylalanine, tyrosine, and tryptophan.

The term "hydrophobic amino acids" includes cysteine, valine, isoleucine, leucine, Contains methionine, methionine, phenylalanine, and tryptophan ( Rose, et al, 5science.

229:834.1985’). [The term large hydrophobic amino acid J is Valine, isoleucine, leucine, methionine, phenylalanine, and It refers to an amino acid selected from the group consisting of ptophan.

An internalizing signal of activity (here, “more than twice the activity efficiency ) is defined by having an efficiency of In addition to increasing endocytosis, the present invention also increases endocytosis. It also seeks to suppress the Inhibition of endocytosis is caused by l) already existing Signals that have been shown to become inactive when replaced with existing active signals. 2) introduce an active internalization peptide into the receptor. Use as an antagonistic inhibitor of internalization (i.e., This peptide competes with the intact receptor for binding to the receptor complex. (to become). Inert, which can be used according to the first method The sexual signals correspond to the core sequence X = Xl XtX, and ATRF, G TRF, ATRA, YTRGSFQDr, IGSY, NTLY, HLAF, PP There are sequences selected from the group consisting of GY, and NPVY.

The generation of inactive internalization signals is caused by and by mutating the aromatic residues at positions X to non-aromatic residues. It can be carried out.

Any nucleotide sequence encoding the above-mentioned peptides may be used in the present invention. Can also be done. These peptide-encoding nucleotide sequences are further It may have adjacent nucleotide sequences encoding cell surface receptors.

In the present invention, a nucleotide sequence encoding an internalization signal is used. The sequence can be introduced into host cells by using recombinant expression vectors. Ru. The term "recombinant expression vector" refers to plasmids, viruses, etc. This vector is a carrier known in the art. manipulated by inserting or incorporating a naturalization signal sequence. It's made by me. This type of expression vector usually ensures that the inserted sequences are efficiently expressed in the host. Contains a promoter sequence that promotes transcription. This type of expression vector is usually , also contains specific genes that allow phenotypic selection of transformed cells. There is.

In addition, the nucleotide sequence encoding the internalization signal is For example, by microinjection or transfection, It can also be introduced directly as a plasmid.

The present invention relates to receptor-dependent transport of ligands into cells, or cell surface receptors. It also provides treatments for diseases using gene therapy that modulates the internalization of It is something to do. This type of treatment targets the cells of a diseased subject with an internal enzyme. This can be done by introducing an oscillation signal. Inter Delivery of the signal is carried out using methods well known in the art. All you have to do is For example, if the internalization signal is recombinant expression vectors, e.g. A chimeric virus or a colloidal diffusion system can be used.

What can be used to introduce the internalization signal of the present invention? Various viral vectors that can be used include adenovirus, herpes virus, virus, vaccinia, or preferably an RNA virus such as a retrovirus. There is a Retroviral vectors are derived from mouse or avian retroviruses. Preferably, it is a derivative. Retroviral vector capable of inserting a single foreign gene Examples of agents include Moloney murine leukemia virus (MoMuLV), Mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV) ), and Rous sarcoma virus (R8V), but are not limited to these. It is not something that will be done. In addition to these, many retroviral vectors can contain genes. All of these vectors contain selectable markers. transduced cells can be identified and cultured. It is possible.

The polynucleotide encoding the internalization signal is By inserting it into a viral vector specific for the target, cation signals will be targeted and delivered to specific cells. Leto In order to make the virus vector specific for the target, it is necessary to Polynucleotides encoding substances that bind to surface targets are inserted into retroviral vectors. It may be contained in the tar. Targeted delivery uses antibodies and retroviral vectors. This can be conveniently carried out by delivering the vector to the target. A person skilled in the art If so, it is inserted into the genome of the retrovirus and transmits the internalization signal. enables target-specific delivery of retroviral vectors containing null polynucleotides. Are you aware of the unique polynucleotide sequence or have done excessive experimentation? You should be able to easily check this.

Recombinant retroviruses are defective, so they cannot be used to produce infectious virus particles. We need help. For this purpose, for example, the entire structural remains of a retrovirus must be A helper cell line containing the plasmid encoding the gene is ) may be used under the control of regulatory sequences within. This type of plasmid is nucleotides that allow the packaging machinery to recognize the RNA transcript Missing array. As a helper cell line lacking packaging signals Examples include tF2, PA317, and PA12; It is not limited. This type of cell line has a packaged genome. Since there is no pillion, it generates an empty pillion. Packaging signals are intact Nono, in which the structural gene has been replaced with another relevant gene, When a virus vector is introduced, the vector is packaged and Pillions can be generated. Vector pylori generated by this method On, a large amount of chimera can be obtained by infecting a tissue cell line, such as NIH3T3 cells. It can be used to generate torovirus spirions.

In addition, tissue culture cells such as NIH3T3 cells were analyzed using the structure of retroviruses. A plasmid encoding the genes gag, pol, and env, with normal phosphate Transfect directly by transfection with calcium You can also do that.

In this case, these cells are transfected with a vector plasmid containing the gene in question. will be affected. The resulting cells contain retroviruses in the culture medium. emit.

Another use of recombinant retroviral vectors is to introduce polynucleotides into the host. Introduction of the code sequence is performed by skin transplantation of cells containing the virus. powerful fibers when transcription is induced using housekeeping gene promoters. Using blast-derived cells to express foreign genes over long periods of time in the graft You can also For example, the dihydrofolate reductase (DHFR) gene protein A motor can be used. Cells, such as fibroblasts, are It is possible to specifically target the gene of the lysis signal. genes, such as tumor-associated antigens (TAA), as well as strong promoters and can be infected with pilions containing retroviral constructs containing both.

Infected cells are embedded in a collagen matrix and this matrix is It can be transplanted into the connective tissue of the target's dermis. Retroviruses multiply and matric As the retrovirus leaves the cell, it specifically infects target cell populations. Ru.

In this way, as a result of transplantation, the proteins produced in diseased cells are The amount of peptides in the internalization signal increases.

In addition, internalization signal peptides or internal When introducing the nucleotide encoding the peptide of the ization signal, Colloidal diffusion systems can also be used as targeted delivery systems. Ko Lloyd diffusion systems include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and and liposomes. A preferred colloid system for the present invention is liposomes.

Internalization signal peptides interact with cell types when they act. Because targeted delivery systems may not differentiate between non-specific Therapeutic applications are significantly improved compared to random injection of different liposomes. It will be good for you. Liposo containing internalization signals Polyclonal or monoclonal antibodies are covalently bonded to the lipoprotein. Numerous methods can be used to make somesomes specific for their targets. Can be done. Antibody-targeted liposomes efficiently connect epitopes on target cells. monoclonal or polyclonal antibodies, as long as they bind to or a fragment thereof, such as Fab, or F(ab')2. can do. Liposomes are receptive to hormones or other serum factors. Cells expressing the body can also be targeted.

Liposomes are useful as carriers for in vitro as well as in vivo delivery. It is an artificial membrane vesicle. Dimensions are 0. 2-4. Large single lamella small in the 0μm range The vesicle (LUV) is capable of enclosing a substantial proportion of an aqueous buffer containing large macromolecules. It has been shown that this can be done. RNA, DNA, intact pillions, and peptides Putide is encapsulated within the aqueous interior of liposomes and delivered to cells in biologically active form. (Fraley, et al.).

Trends Biochem, Sci, 6:77, 1981). lipo Somes contain polynucleotides not only in mammalian cells but also in plants, yeast, It has also been used for delivery into bacterial cells. Liposomes provide efficient transfer. To be a delivery carrier, the liposome must contain (+) the peptide or nucleotide of interest. can be encapsulated at a high rate without impairing its biological activity; It can selectively and substantially bind to target cells compared to non-target cells. , (3) the aqueous contents of the vesicles can be delivered at a high rate to the cytoplasm of target cells. (4) It is necessary to be able to express genetic information accurately and effectively (Ma nnino, et al, Biotechniques, 6:682, 1 988).

The composition of liposomes is usually a mixture of phospholipids, especially phospholipids with high phase transition temperatures. quality, usually in combination with steroids, especially cholesterol. other It is also possible to use phospholipids or other lipids. Physical characteristics of liposomes The properties depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in producing liposomes include phosphatidyl compounds. substances, such as phosphatidylglycerol, phosphatidylcholine, phosphatidyl Dirserine, phosphatidylethanolamine, sphingolipid, ceredoside , and gangliosides. Particularly useful is diacylphosphatidyl glycol. In cerol, the lipid moiety has 14-18 carbon atoms, especially 16-18 carbon atoms. It contains and is saturated. As a specific phospholipid, egg phosph Atidylcholine, dipalmitoylphosphatidylcholine, and distearoyl Contains phosphatidylcholine.

The surface of a targeted delivery system can be modified by a variety of methods. target When liposomes are used in a delivery system with a defined containing a target-directed binding substance to stably associate with the liposome. Can be done. When binding lipid chains with targeting binding substances, various methods are used. Linking groups can be used.

In general, targeted delivery systems require that the delivery system be directed to receptors on the cell surface. This allows the delivery system to find the desired cell and land on that cell. It is what it is. However, the delivery system can be It is also possible to induce any cell surface molecule selectively found on the cell surface. Yes, if the molecule can associate with the delivery system. The targeting of liposomes is Antibodies can be used to target different cell surface molecules. and For example, antibody-internalized signal-containing liposomes can be directly For targeted delivery to sexually transmitted tumors, proteins called tumor-associated antigens (TAAs), Certain antigens that are specifically expressed on tumor cells can be used.

The present invention identifies sequences associated with cell surface receptor internalization. therefore, it is important to design therapeutic or diagnostic protocols using this type of sequence. Is possible. Therefore, persistence of the disease state may lead to internalization of cell surface receptors. If related to internalization, the original internalization Using sequences to disrupt the function of the original internalization signal It is possible to design an array that Such an approach, for example, masking mRNA with antisense nucleic acids using antisense nucleic acids and ribozymes or cleavage with ribozymes to inhibit the translation of specific receptor mRNAs. Block.

Antisense nucleic acids are complementary to at least a portion of a specific mRNA molecule. NA or RNA molecules (Weintraub).

5cientific American, 262+40.1990), Antisense nucleic acids pair with the corresponding mRNA to form double-stranded molecules in cells. do.

Since cells do not translate double-stranded mRNA, antisense nucleic acids can be used to translate mRNA Translation of A is inhibited. Antisense oligomers of about 15 nucleotides are preferred. This is easy to synthesize, and when introduced into cells producing the target receptor, This is because they are thought to be less likely to cause problems than molecules larger than this. Antise Inhibiting the in vitro translation of genes using ance-based methods is Well known in the art (Marcus-Sakura, Anal.

Biochem, 172:289.1988).

Lipozymes, in a manner similar to DNA restriction endoncleases, can be used to synthesize other single-stranded RNases. It is an RNA molecule that has the ability to specifically cleave A. Coding these RNAs By modifying the nucleotide sequence that encodes a specific nucleotide in an RNA molecule, It is possible to create molecules that recognize and cleave the leotide sequence (Cech, J.

Amer, Med, As5n, 260: 3030.1988). child The main advantage of this approach is that ribozymes are sequence-specific; Only those mRNAs that have the following are inactivated.

Lipozymes come in two basic types: Tetrahymena (Hassel) Hoff, Nature, 334:585.1988) and “Hammer There is a J type. Tetrahymena-type gluebozymes recognize sequences with a length of 4 bases. In contrast, "hammerhead" ribozymes are 11-18 bases long. Recognizes the base sequence of The longer the recognition sequence, the more likely it is to reach the target mRNA species. There is a high probability that the sequence exists exclusively. Therefore, specific mRNA species In terms of inactivating Tetrahymena, hammerhead-type glue bozyme is better A recognition sequence of 18 bases is preferable to a type-based bozyme, and a recognition sequence of 18 bases has a shorter recognition sequence. It is more suitable than array.

As administration of a heterologous internalization signal polynucleotide antisense sequences can be administered for therapeutic purposes by the methods described above. a Targeted liposomes are particularly useful for delivering antisense sequences for therapeutic purposes. suitable.

(Margins below this page) material and method Transplanting internalization signals and analyzing their structure used the materials and methods described below.

A, oligonucleotide site-directed mutagenesis containing the entire coding region of human TR. The CIal fragment was transferred to the phagemid pBIuescript SK (Stratagy Stratagene, California, USA) (Jing, et al.

J, Ce1l Biol, Yu 10: 283-294.1990). O Ligonucleotide site-directed mutagenesis was performed on pBluescri of wild-type TR. Using the ptSK phagemid template, Kunkel's method (P roc, Natl.

Acad, Sci, USA, 82: 488-492.1985) So, Muta-gene Mutagenesis Kit (Bio- Using Rad (Bio-Rad, Suchmond, California, USA) and as previously described (Collawn, et al., Ce1 1, 63: 1061-1072, 1990). Limit mutants C1al encoding the mutant receptor. Cut out the fragment and create the retrovirus expression vector RCAS (Hughes, et al., J. Virolog)', 61: 3004-3012. (1987) or cloned into BH-RCAS. Confirmation of mutation is Sequenase kit (USB Cor p., Cleveland, OH, USA) according to the manufacturer's instructions. Retroviral constructs were analyzed using dideoxynucleotide sequencing (1977; Ta bor and Richardson, Proc, Natl, Acad, Sc i.LISA.

84:4767-4771.1987).

B, Cell culture and expression of human TR in CEFs at 9-day-old embryos (SPAFAS, Primary chicken embryo fibroblasts (CEF ), 1% (v/v) chicken serum, 1% (V/V) normal calf blood Hyclone (Logan, Utah, USA), 2% (v) Tryptose phosphate broth (Dirco, USA) Grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with I let it grow. This CEF was treated with the retroviral construct prepared in (A) to a confluency of approximately Using 30 μg of DNA per 10 cm of tissue culture plate with 40% of the cells, Ni-dimethyl sulfoxide method (Niawai and NiN15hiza, Mo1. Ce11. Biol, 4: 1172-1174゜1984 ) was transfected by using.

A helper-independent retroviral vector derived from Rous sarcoma virus. A BH-RCAS (Hughes, et all, J.

Virology, 61: 3004-3012.1987). Wild type and mutant human TR were stably expressed. transfection 1-2 weeks after infection, CEF cell surface Above, wild-type as well as mutant forms of the receptor were stably expressed. at the cell surface Expression inhibits binding to 125I) transferrin under steady-state conditions of 37°C. Confirmed by doing.

In order to select mutant human 1-TR with high expression rate, transfection After 10-14 days, cells were incubated with selective medium (3% (v/v) horse serum, 2% phosphorus). Acid tryptose broth, as well as human binding to 50 μg/ml of two irons. Transferrin (Tf)’ (Miles Scientific (MilesS) scientific), Naperville, Illinois, USA) DME29 4, 1990).

C,12'I-Tf bond Human transferrin (Tf), which is bound to two iron atoms, is transferred to enzyme beads ( Enzymobeads) (Piolad, Suchimo, California, USA) specific activity is 2-4 μCi/mg when used according to the manufacturer's instructions. was labeled with 126I. 24 hours before performing the binding assay, cells were incubated for 24 hours. Well of Caster tissue culture plate, 7.5 x 10' cells/well The cells were plated at a density of cm2. Cells were incubated at 37°C in serum-free DMEM for 1 hour. Incubate for an hour and then add 0.15M Na containing 0.1% serum albumin. c]-0. OIM sodium phosphate buffer (pH 7,4) (BSA-PBS) Washed once with water-cooled water. 0.15 of 125I-Tf (4μg/ml) Add ml of BSA-PBS solution to three wells and incubate at 4°C for 60 min. Cultured.

Next, cells were washed 3 times with 0.5 ml of water-cooled BSA-PBS, and 0.15 J Tl] was removed from the well with IMN aOH and the radioactivity was measured using a gamma counter. It was counted.

D. Analysis of transferrin internalization at steady state. To investigate the steady-state distribution of mutant and wild-type human TRs, TR CEF expressing mutants were cultured in three groups as described in the binding analysis section. plated in wells. Cells were first incubated for 1 hour at 37°C in serum-free DMEM. Incubate and then incubate at 4 u g/m I in 0.1% BSA/DMEM. The cells were incubated with Tf at 37°C for 1 hour. Remove the medium and transfer the cells to 1 ml Washed three times with ice-cold BSA-PBS. Cells were then added twice in 0.5 ml Incubate for 3 minutes with 0.2M acetic acid-0.5M NaCl (pH 2,4). , removed surface-bound 1261-Tf (tlopkins and Tr owbridge, J, Ce11. Biol.

97: 508-521.1983'). Next, cells were washed with IMNaOH. The radioactivity of the acid wash solution and cell lysate was measured. Pickling Extending the time of incubation with purified solution did not affect the radioactivity released (J ing, et al, J, Ce1l Biol, dent: 283-29 4, 1990).

In the steady state, k, , (TR), , = to, , [TR'l　, , and acceptance Assuming that the degradation rate within the cells of the body is not significant, the Tf-TR complex on the cell surface 1.1 to the rate of internalization of combined CTRIs, Externalization of the apoT f-TR complex [TR] 1.1 Equal to velocity (CoCo11a.

et al., Ce1l, 63: 1061-1072.1990). "Appointment The term Tf,+ also refers to transferrin without iron binding. It is.

Incorporation of Man-6-PR and LDLR 6-residue signal types E and “F” measurement of Human apoTf was prepared using nitrilotriacetate with a specific activity of 5-10 μci. /mg, "Fe (FeC1s), Amersham Co., Ltd. (Bat Corp, Arlington Heights, Illinois, USA). es and 5chlabach, J., Biol, Chem.

248:3228-3232.1973). 24 hours before assay, cells Cells were grown 7°5×10 in a 24-well Caster tissue culture plate. The cells were cultured at a density of 1,000 cells/cm2. The next day, the cells were prewarmed. (37°C) twice with serum-free DMEM, then 0.1% BSA and 4 0.1 u g/m 1 in DMEM containing Fe-Tf at 37°C. , 2.3, and 4 hours of constant temperature culture. After the above time period, remove the medium and Cells were washed three times with water-cooled BSA in 0.1% PBS. For each elapsed time Next, cells in three wells were removed with 0.5 ml of IMNaOH to remove radioactivity. It was counted with a Ma counter. Human expressed on various CEF populations for each experiment. The relative levels of TR were determined. Prepare for 1 hour at 37°C in serum-free DMEM. After constant incubation, cells in three wells were labeled with 4 μg/ml 1251. Incubate for 1 hour on a water bath with Tf and then incubate with 1 ml of ice-cold BSA-PB. The cells were washed three times with S and the radioactivity bound to the cells was measured.

F, Ratio of protein structural analogs of the 6-residue internalization motif. comparison Then, I layered it on top of the ideal ■-shaped turn.

The analogue was published in the Protein Data Bank (Protein D) in October 1990. ata Bank, P D B) (Bernstein, et al, J.Mol.

Biol., 193: 775-791.1977) about 550 tampers. The pattern of the internalizer matches the protein structure, and the last four residues are exposed. Tight turn (helical turn or reverse turn) exposed on the surface Identified by searching for sequences.

Search is a computer program. Leslie Kuhn, the heir of Searchwild Developed by Michael Pique and C011aWn, e t al.

Ce1l, 63: 1061-1072.1990), PDH The sequence is a 6-residue M a n -6-P R pattern (YSF) (X) (Y, F) (X) (polar amino acids K, R, H, N, Q, D, E) (large hydrophobic Amino acids: ■, L　LSM, F, Y, W) and the 6-residue LDLR pattern. (F, Y) (X) (N) (X) (X) (Y, FSW) (Ll deviation pattern I also searched on Twitter for "X cannot be Gly". homologous match, if Exclude matches that lack three-dimensional correlation of side chains and surface exposures at the last four positions. Structures with exposed tight turns can be examined using molecular graphics to The preferred position of the strands was confirmed. A molecular graph showing the backbone atoms at the turn positions of each analogue. Insight II (Insight II) is a graphics program. Iosym Chikunoro 7-z (Biosym Technologies), Sun Diego, California, USA) and Noricon Graphics (Silic) on Graphics) Iris 4D 310 VGX Wa The following examples are given to illustrate the invention. However, the present invention is not limited to these Examples. Example below The procedures shown are representative of those available; Alternatively, other procedures well known to those skilled in the art may be used.

Cell surface receptor internalization signals are interchangeable We conducted a study to determine whether or not. In this study, low-density lipoprotein receptor (LDLR) and mannose-6-phosphate receptor (Man-6-PR ) was transplanted into the TR. oligonucleo Using titanium-directed mutagenesis, we created 4-residue residues in the cytoplasmic tail of wild-type TR. The internalization signal YTRF is transmitted to LDLR and Man -6-PR signal 4-residue sequence and 6-residue TR sequence LS YTRF with the 6-residue LDLR and M a n -6-P R signals. (Table I).

(Margins below this page) Table I in the transferrin receptor construct. internalization array array of “When replacing 4 residues, YTRF, which is a TR signal, was replaced with bovine Ma For the n-6-PR signal, YSKV was used, and for the human LDLR signal, Therefore, it was changed to NPVY. When replacing 6 residues, the TR sequence LS Changed YTRF to YKYSKV or FDNPVY. shown above the array. The numbers (-2 to 4) are considered to be turn areas in TR and LDLR. Position (CoCo11a, et al, Ce1l, 63: 1061 -1072.1990) shown with the turn starting at position 1. It is. Residue numbers of TR are shown as superscript numbers. Suddenly changed to alanine The essential residues of the internalization sequence identified by differentiating , underlined TR, CoCo11a, et al.

Ce1l, 63: 1061-1072.1990, Man-6-PR, Ca nfield.

Chen, et al, J, Biol, Chem, 3116- 3123.1990), aromatic and large hydrophobic residues are shown in bold.

The 4-residue sequence of LDLR, NPVY, was investigated because the pattern of NPXY was Other transmembrane proteins, such as EGF and the cytoplasmic side of the insulin receptor It is also found in the tail, and therefore this pattern of arrangement is found in the tails of these proteins. May play a role in internalization1 990). The YSKV sequence of Man-6-PR was investigated. Mutation analysis of (Canfield, et al., J., Biol., C. hem.

," Tomo: 5682-5688.1991), this tetrapeptide is highly efficient. Complete internalization in promoting high endocytosis This is because it has been shown to have approximately the same efficiency as arrays.

Wild-type and mutant TRs are derived from Rous sarcoma virus helper BH-RCAS, an independent retroviral vector (Materials and Methods) (see section), it is possible to efficiently use chicken embryo fibroblasts (CEF). was expressed. Relative internalization of wild-type and mutant receptors The sorption efficiency is determined by the measured amount of Fe uptake as well as by the Fe uptake under steady-state conditions. It was determined from the amount accumulated in the cells.

Figure 1 shows two representative Fe samples containing all of the mutant TRs analyzed. Experiments with the amount of internalization are shown in Table II. The results are shown below. Asse of both sides B, the internalization efficiency of mutant TR against wild-type receptor Similar values were obtained for Fe uptake, and the mutant receptor mediates Fe uptake. The ability to do so was highly reproducible.

(Margins below this page) Table II W-Situation soil 1.9b (7) C1oo 34shi and (phrase 1■^3-9 “Tail deletion J '24.7±4.6 (2) 176±12 (5)' 18LDLRarn er 25.7: 0.9 (7) I B 8 Sat 0.6 (5) 2a LDL R6mer 40.9±1.6(7) 37 19±1.9(5) 56Man -6-P day 4mer 61.7 t 3.5 (4) 86 30 ±14 ( 3) 88Man-6-P Sun Grner 68.5 Sat L8 (4) 116 33 ±4.0 (3197FZ3M 70.9±''Z8(3) 130 37 Sat λ7 (3) 1 (To) FZ31 60.5 ±15 (3) 82 33 Sat 3.8 (3) 97F23W 5&7 ±5.7 (3) 76 27! 4. + (3) 79″ wild type 5 Mean ± standard error °Number of independent experiments Surprisingly, YKYS, the signal of 6 residues of M a n -6-PR Mutant TRs, including KV, are internalized as efficiently as wild-type receptors. It was izzed. Contains YSKV, which is a 4-residue signal of M a n -6-PR. Mutant forms of the receptor are also rapidly internalized, overlapping the wild-type receptor. It has an internalization efficiency of about 87% of the internalization efficiency. (Table II, Figure IA). Contains FDNPVY, a 6-residue signal of LDLR. The mutant TR is internalized at approximately 50% the rate of the wild-type receptor. , the 6-residue signal of LDLR showed significant activity upon transplantation to the cytoplasmic tail of TR. It was suggested that it has a sexual nature. In contrast, the 4-residue LDLR signal The mutant TR containing the null NPVY has approximately the same rate as the receptor lacking the tail. (Table IL, Figures IA and B). From these experimental results, , LDLR and M a n -6-PR internalization signal The signal replaces the signal in the TR, allowing highly efficient internalization of the TR. This suggests that it may be a mediator of the Sequence of 4 residues of transplanted LDLR NPV The lack of activity of Y suggests complete internalization of LDLR. - The signal is suggested to be the 6-residue sequence FDNPVY.

Internalization of 4 to 6 residues of Man-6-PR and LDLR The results of analyzing the mutant type of TR into which the signal was grafted showed activity and In the amino-terminal aromatic residue, however, large non-aromatic hydrophobic This indicates that the aromatic residue at the carboxy terminus may be substituted by a are doing. In order for the TR internalization signal to show activity, it is important to In order to investigate whether the aromatic residue at the carboxy terminus is required, the 23rd Phe was changed to Met, then to He, and then to Trp. Met the 23rd Phe Alternatively, functional analysis of a mutant TR changed to Ile reveals that the interaction between The rate of naturalization as well as the ability to mediate Fe uptake was significantly higher than that of wild-type T. It was equivalent to R (Table 11). Mutant type with 23rd Phe changed to Trp The TR of was also rapidly internalized (Figs. 1 and 2).

Internal alignment of 6 residues of LDLR and Man-6-PR transplanted into TR The fact that Zaneyon signal showed activity is due to the active conformation of these sequences. tion takes advantage of the internalization motif that TR originally had. This suggests that it is related to sexual conformation. 4 residue TR In addition, the LDLR motif tends to form a tight turn structure (Coll awn, et at, Ce1l, 63: 1061.1990), The sequence of 6-residue LDLR and Ma n -6-P R intersects TR. The internalization pattern Contains a 6-residue sequence that matches The three-dimensional arrangement of essential side chains in various crystallographic protein structures Examined.

Internalization of TR, LDLRl and Ma n -6-P R Both signals are α-helix in their original protein sequence. For secondary structures, the turn must be located before or at the start position of Algorithms (Chouand Fasman, Adv, Enzymol, , 47:45.1978). M　a　　　　-6-P　R Four structural analogs were identified for the LDLR signal, and four structural analogs were identified for the LDLR signal. Five analogs were identified (Figure 3).

FIG. 3A shows an example of M a n -6-PR that matches the arrangement pattern YXYXKV. Analogues of the internalization motif are shown (detailed in Materials and Methods section). ). Side chains are shown for essential residues at positions -2, 1, 3, and 4. Man- Analogs of the 6-PR internalization signal are blue, FTFSDY , Fab fragment of immunoglobulin 4-4-20 (PDB code 4FAB ), residues H27-H32, red, FEFEKF, phosphoglycerate kinase ( 3PGK), residues 340-345. Purple, F M F N Q F, Concanavali A (I CNI), residues A128-AI33; green, FAFIRLSD-key isomerase (4XIA), residues A377-A382. Position- 2 and 1 pairs of aromatic rings have similar, almost parallel orientations (same color) (see the pair of rings in Fig. 3A, bottom left) also suggests that mutagenesis As shown, even if one aromatic ring is lost, the other aromatic ring can compensate. It is suggested that The functionally important side chains at positions -2, ■, 3, and 4 are all can also be accessed from either side of the turn simultaneously, requiring rotation of at most a single bond. Only.

In the four M a n -6-P R analogues, aromatic side chains at positions -2 and 1 (Position 1 is the first position of the turn) were paired, and the rings had similar orientations (same color). (see ring, bottom left). Of the four analogues, three are reverse turns and one is α It was Helix's initial turn. C of the aromatic side chain at position -2 and 1 The average distance between β is 6.6 people, and this distance is equal to It is comparable to the distance between βs (5.8 people on average), and the necessary The pair of essential residues and the pair of essential residues at positions 1 and 4 are placed at similar distances from each other. It was TR motifs with similar sequences (Collawn, et al., C e1l, 63: 1061.1990), the polar side chain at position 3 It extends towards the front of the page (protrudes from the page) and is located at position 4. Hydrophobic side chains formed a sector perpendicular to this plane.

It has five alanine residues on the amino-terminal side of the internalization signal. A truncated mutant version of Man-6-PR that High activity in Canfield.

et al, J, Biol, Chem,, 266: 5682.199 1) The alanine residue should stabilize the reverse turn or hemi-curl turn. It's worth mentioning for some reason.

FIG. 3B shows an LDLR internal pattern that matches the LDLR pattern FXNPXY. Analogues of the lization motif are shown.

Side chains at positions -2, 3 and 4 are shown. LDLR identified by search query Analogs of yellow, YANLVF, tryptophan synthase (IWSY), residue A l02-A107. Red, F YNAAW, lectin (2LTN), residue Al2 3-Al 28, orange, FTNEFY, cytochrome C peroxidase (2CY P), residues 198-203, blue, FKNSKY, chymotrypsinogen a (2C GA), residues A89-A94, and green, YDNKYW.

Calcium-free phospholipase Ax (I PP2), residues Ll13-L1 It is 18. The aromatic groups at positions -2 and 4 and the side chain at position 3 are identical in structure. It was exposed on the same side.

Analogues of LDL contain three surface-exposed inverted turns and two α-helix imprints. It included Nishal Taan. Positions −2 and 4 for each of the analogs important aromatic groups were accessible from the same side of the structure. Asn at position 1 The side chain (shown in Figure C) of They were divided into two groups, located either in front or behind the main body. The side chain (position) of this Asn (l) stabilizes the turn by forming hydrogen bonds with adjacent backbone atoms. This suggests that this side chain has a structural role.

The first two residues of the LDLR tetrapeptide, Asn-Pro, are the most common Wilmot, e. tal, , J, Mo1. Biol, 203: 221.1988 ), Asn-Pro is also a potent helix initiator (R tchardson, et al, 5science, 240: 164 8.1988).

Figure 3C superimposes M a n -6-P R and LDLR analogs. It is a diagram. Analogs of M a n -6-P R in Figure A are shown in blue, and analogs of LDLR in Figure B are shown in blue. Analogs are shown in yellow and side chains are indicated for positions -2, ■, and 4. -2 position the aromatic group of Asn (LDLR) in position 1 extends towards the left side of the turn. The side chain as well as the aromatic (Man-6-PR) side chain is the aromatic (LDLR) at position 4. a right-angled sector similar to that seen in side chains as well as hydrophobic (Man-6-PR) side chains. is formed. Ser of Man-6-PR at position 2 and Pro of LDLR is the -order structure, and YSKV analogues of Man-6-PR (L, A, K, etc. and J, A, T, data not shown), the side chain of Ser is in the Pro conformation. (Wilmot, et al, Protein Engineering ring, 3:479.1990) to form a skeletal hydrogen bond similar to Ric Hardson, et al, Prediction of Protein 5structure and Pr1ciples of Protein Conformation.

G. D. Fasman, ed. (New York: Plenum P. publishing Carp, ), pp.

1-98.1989), the side chain of P r o stabilizes the turn through covalent bonds. It seems that The fact that this position plays such a structural role is due to the fact that These side chains in the group analogs are located at the back of the turn, far from the essential side chains. It is supported by the fact that there is a tendency to Man-6-PR and LDL In any of the R analogues, the side chains at positions -2, l, and 4 form the base of the turn. Forming a transverse line (...), the line of essential side chains of the LDLR analogue is at an angle of about 35° to the line of the essential side chain of the analogue of M a n -6-P I was doing it.

By superimposing the Man-6-PR analog and the LDLR analog ( Figure 3C), the overall structural pattern, i.e., the essential amino-terminal aromatic group Important carboxy-terminal aroma localized to the left and somewhat anterior of the turn groups and hydrophobic side chains form a single mass, and each structure pattern in which all essential side chains of the turn are simultaneously recognizable from one side of the turn. The sound surfaced. These analogs allow the side chain in position 2 to make a turn. It was speculated that it plays a structural role in stabilization, but this side chain is not necessary. This side chain is not directly recognized because it points away from the side chain of the It seemed unlikely. Regarding the pattern of essential residues, the tight turn structure is Self-sufficient (Self-co Although this can be explained by the fact that the It is difficult to discover how the chief was able to fit into each position within the β-sheet. Ru. In the broad β conformation, the side chains of essential residues are positioned on both sides of the secondary structure. and recognize them at the same time, making it difficult to recognize them at the same time. Structure should be required. This possibility has been confirmed by previous deletion mutants. It is considered impossible.

Example 3 Porting multiple internalization signals To investigate the effect of the endocytosis signal on the endocytosis of TR. conducted research. Chou and Fasm algorithm an, Adv.

Cytoplasmic domain of TR using Enzymol, 47:45.1978) Structural analysis of the domain reveals that this domain consists of a series of helices separated by multiple turns. one such turn (turn 2, see Table III). It is presumed that wild-type internalization signals exist in Ta.

Table III Turn l Turn 2 Turn 3 Turn 4MMDQAR5AIGGεP LSI engineering FIFSLA day 0VDGroN5HVεMKLAVroεEEdoA4! N TK^NVTIO'KR-TMf mutant Turn l YTRF YrRF Turn 3 x Go 1 Sea I Go Ejimi Turn 4 YTRF YTRF Transmembrane region As shown in Table III, at position 113, which is assumed to be a turn, or at position 4, induces additional copies of YTRF, the internalization signal for TR. The mutated TRs were prepared using oligonucleotide-directed mutagenesis. did. Each mutant had the wild-type turn 2 signal intact. Therefore, it included two internalization signals. mutation The somatic TR is determined as a steady-state distribution in chick embryo fibroblasts as described above. Investigated by essay. The analysis results are shown in Table IV. Additional signals can be identified from the analysis results. It was shown that the effects of LE are mixed and vary depending on location.

Table IV Definition of a mutant of human 1-TR containing two internalization signals Comparison of distribution in steady state Internalization in steady state Relative efficiency of human TR construct (X) 1■■■---Kyuha■---1,---■---Yo-1-■■■■---嵨--- One, Yui.

Wild type TR ["LSYTRF23] 66.0=L3" (11 houses 100 Tur n 1 [’ITRF12] 651 = 3.6 (3) %Tum 3 [” YTRF"] 79.9 = 0.8 (3J 205Tum 4 [47YTRF Clause 73.8: 1.4 (3) 145 “Standard error above the mean 1 Number of independent experiments The sequences shown in parentheses were generated using oligonucleotide-directed mutagenesis. This is an additional internalization signal that has been ported within the each mutation Both bodies contain a wild-type signal [:20YTRF”) in turn 2. . The superscript number indicates the position of the mutated sequence in the TR. Mutant TR portion The fabric exhibits radiolabeled Tf bound to the cell surface as well as intracellular proteins under steady-state conditions. It was determined from the label Tf of the wheel.

Even if an intercalization signal is added to turn I, the resulting TR remains unchanged. Indistinguishable from sex-type receptors (rate of internalization compared to wild type) (96%). However, an internalization signal was added to turn 3. This results in a mutant with twice the rate of internalization than the wild type ( 206% ± 30%, 3 experiments, standard error of the mean). Add signal to turn 4 Although the effect was not so pronounced, a TR mutant with 145% activity was generated. Ru. From these results, we port additional internalization signals. This illustrates the possibility of adjusting the rate of internalization. It is.

By transplanting a heterologous receptor sequence, which may be a signal, into the TR. Examined. When investigating, we assumed that it was an internalization signal. The sequences of other receptors are determined.

TR using oligonucleotide-directed mutagenesis as described above. I ported it and investigated. The signals assayed by this method are shown in Table V.

(Margins below this page) Table V The human TR construct internalizes at steady state. Relative efficiency (%) of human TR (X) 6aO=1.3"(11)" 100ASGPR1-11["YODL"+ 54. ey9(a) ezASGPRH2 ["FQOI"] ZL6f;j ( 5) 16 poly 19th [to SAF”l 5Z [d15 (5) 57EQFR[ ``We bite town, 6 (phrase 119EGFFI ['-PQQGFFS''l 5 4!1.5 (4) 59 Mannose Day [l@FεNTU’-Town 31.5 th2. ! (41ASLAP [shijin i momme 311.3 (5) πLAP [ "PPG dead 27.8W, 9 (3120LAMP-j (Tsukasa super tail deletion correct [^3-59] 24.7 soil 1.6 (2) 17” average 2 standard error Number of 5 independent experiments In Table V, the sequences shown in parentheses were determined using oligonucleotide-directed mutagenesis. This is a sequence assumed to be a signal, which was transplanted into the TR using the following method. superscript numbers indicates the position of the mutated sequence in the TR. The distribution of mutant TR is in the steady state Radiolabeled Tf bound to the cell surface as well as labeled Tf in the intracellular pool under conditions It was decided from.

In this system, some sequences, for example the asialoglycoprotein receptor ( H1 subunit of ASGPR, sequence YQDL), polymeric immunoglobulin (polymer Ig, sequence YSAF), epidermal growth factor receptor (EGFR1 sequence NFYRAL ), lysosomal acid phosphatase (LAP, sequence YRHV), lysosome The sequence of related membrane glycoprotein (LAMP-1, sequence YQTI) is internalized. functions very well as a fusion signal, whereas other sequences, e.g. H2 subunit of ASGPR (sequence/FQDI), mannose receptor (sequence F ENTLY), and LAP (sequence PPGY) are malfunctioning, and their activity is (Table V). Signal is on the cytoplasmic side When localized to a region within the tail, this method maps the precise region. It is very useful. For example, the sequence PPGY, which is the region near the tyrosine of LAP, In the analysis of RHV, the sequence YRHV performs well, whereas another sequence PPGY has been shown to not work well.

This strategy promotes internalization by ligand-induced internalization. internalization signals of receptors that are stimulated, such as EGFR. It is also useful when mapping. This type of receptor has a cytoplasmic domain. This type of internal Given that not a single ization signal has been clearly identified, , It is of great significance that such mapping is possible. in ligand-induced receptors. In identifying internalization signals, transfer using TR is recommended. Further explant experiments and parallel mutagenesis experiments in the receptor may need to be done. EG works extremely well when transplanted to TR NFYRAL, which is assumed to be a signal for FR, is a cation-independent manno It appears to have a sequence related to the receptor for su-6-phosphate. Such research This suggests that the sequence that may be a signal should be transplanted to the TR and examined. Ligand-induced receptors are also constitutively recycled receptors. Internalization signals similar to body internalization signals This means that there is a possibility that it has a negative signal.

The above description is for the purpose of illustrating the scope of the present invention, and the scope of the present invention is The invention is not limited by these descriptions.

Indeed, one skilled in the art can rely on the teachings herein without undue experimentation. It should then be easy to devise and implement other embodiments.

(Margins below this page) 0.0 1.0 2.0 3.0 4.00.0 1. o 2.0 3.0 4 ．． 0 hours (hours) FIG, IB Continuation of front page (51) Int, C1,6 identification symbol Internal office reference number C07K 14/705 8318-4HC12N 15109 //　C1,2N　5/10 (C12N 5/10 C12R1:91) (72) Inventor: Drawbridge, Ian Niss.

Lakewood, San Diego, California 92122, United States 6287 (72) Inventor Colone, James F.

United States 92131 California San Diego, Ridgewar Turlane 10312 I (C12N 5100B CI 2 R1:91) (72) Inventor Tyner, John A.

United States of America 92107 California San Diego, Niagara Avenue (72) Inventor: Kuhn, Leslie A.

USA 92024 California Encinitas, AP -, F208, South El Camino Real

Claims (24)

[Claims] 1. A method of modulating receptor-mediated transport of a ligand into a cell, the method comprising: A method comprising the step of introducing a heterologous internalization signal into a cell. 2. Introducing internalization signals as nucleotide sequences The method according to claim 1. 3. The above nucleotide sequence further comprises a brand encoding a cell surface receptor. 3. The method according to claim 2, comprising a King sequence. 4. The receptor encoded by the introduced nucleotide sequence is already present in the cell. The method according to claim 3, wherein the cell surface receptor is different from that present in the cell surface receptor. . 5. Claims introducing at least two disparate internalization signals The method described in item 1. 6. The receptor encoded by the introduced nucleotide is already present in the cell. Claims having substantially the same ligand specificity as existing cell surface receptors The method described in Section 3. 7. Internalization of the receptor encoded by the introduced nucleotide tion signals are internalized by cell surface receptors already present in the cell. 7. The method of claim 6, wherein the signal is different from a lysis signal. 8. The modulation results in increased transport of the ligand into the cell. The method described in Section 1. 9. The nucleotide sequence encoding the internalization signal is 3. The method according to claim 2, wherein the method is incorporated into a current vector. 10. 10. The method according to claim 9, wherein the expression vector is incorporated into a carrier system. 11. It is proposed that the internalization signal be introduced as a peptide. The method described in Scope 1. 12. 12. The method of claim 11, wherein the peptide is incorporated into a carrier system. 13. the internalization signal has a tight turn, and Amino acid sequence: It has X1X2X3X4X5X6X7X8X9X10X11, and the X5-X8 constitute the core array, Residues X1-X4 and X9-X11 are present as necessary. ri, and X1 is leucine or glutamic acid when present; X2, if present, is isoleucine, methionine, or broline. the law of nature, X3, if present, is any amino acid residue; X4, if present is selected from alanine, polar amino acids, or aromatic amino acids, and X3 is also If present, at least one of the X3 or X4 residues is polar; 5 is an aromatic amino acid when residues X1-X4 are absent, and at least In both cases, there is an X4 residue or (one or more) residues further upstream. is selected from aromatic amino acids or polar amino acids, and X6 is a polar amino acid. amino acid or alanine, and X7 is an amino acid or alanine, and X7 is selected from polar amino acids or alanine, and at least the X4 residue or if there are (one or more) residues further upstream, optional is an amino acid residue selected from aromatic amino acids or hydrophobic amino acids. Found out, X9, if present, is serine or alanine; X10, if present, is alanine or leucine; X11, when present, is alanine or phenylalanine, wherein at least one of residues X3, X5 and X8 is an aromatic amino acid, Furthermore, residues X1, X2, X3, X10, and X11 are on the core sequence side. can only exist if the immediately adjacent residue is present A method according to claim 1, characterized in that: 14. The internalization signal has the amino acid sequence: X5X6X7X 8 It has the characteristic that X6 and X7 cannot both be alanine. 14. The method according to claim 13. 15. X5 is phenylalanine or tyrosine, X6 is alanine, Arginine, glutamine, serine, or threonine, X7 is alanine, arginine, aspartic acid, glycine, glutamic acid, stidine, lysine, or threonine, and X8 is isoleucine, leucine, methionine, phenylalanine, valine, a 15. The method according to claim 14, wherein the fluorine is tryptophan. 16. The amino acid sequence is YTRM, YARF, YTRI, YQDL, YTKF, YSKV, YTRW, YRHV, YSAF, YQTI, YTAF, YTGF, Y 16. The method according to claim 15, which is TEF, FTRF, or YTRF. 17. The internalization signal has the amino acid sequence: X3X4X5X 6X7X8 14. The method according to claim 13, comprising: 18. X3 is asparagine, leucine, methionine, phenylalanine, bromine Phosphorus or tyrosine, and X4 is alanine, aspartic acid, glutamic acid. X5 is asparagine, lysine, phenylalanine, or serine. , glutamine, or tyrosine, X6 is arginine, glycine, broline, serine, or threonine , X7 is alanine, arginine, lysine, phenylalanine, or valine Yes, and X8 is isoleucine, leucine, methionine, phenylalanine, tributof 18. The method according to claim 17, which is valine, tyrosine, or valine. 19. Amino acid sequence is YKYSKV, NFYRAL, LAYTRF, PQQG FF, FDNPVY, MSYTRF, or LSYTRF The method according to item 18. 20. How to identify sequences that regulate cell surface receptor internalization The law is (a) cells containing said receptor, in the presence or absence of said sequence, as appropriate; incubate in the presence of a ligand for a cell surface receptor, and (b) internalization of cell surface receptors in the presence of the sequence or A method comprising the step of measuring in the absence. 21. Has a tight turn and amino acid sequence: X1X2X3X4X5X6 A peptide having X7X8X9X10X11 in the sequence X5-X8 constitute the core array, Residues X1-X4 and X9-X11 are present as necessary. ri, and X1 is leucine or glutamic acid when present; X2, if present, is isoleucine, methionine, or broline. the law of nature, X3, if present, is any amino acid residue; X4, if present is selected from alanine, polar amino acids, or aromatic amino acids, and X3 is also If present, at least one of the X3 or X4 residues is polar and the X5 is an aromatic amino acid if the residues X1-X4 are absent, and at least There is also an X4 residue or (one or more) residues further upstream is selected from aromatic amino acids or polar amino acids, and X6 is a polar amino acid. It is an amino acid or alanine, and X7 is when the residues X1-X4 are not present. has at least X4 residues selected from polar amino acids or alanine or if there are (one or more) residues further upstream, any is an amino acid residue, and X8 is selected from aromatic amino acids or hydrophobic amino acids. Re, X9, if present, is serine or alanine; X10, if present, is alanine or leucine; X11, when present, is alanine or phenylalanine, wherein at least one of residues X3, X5 and X6 is an aromatic amino acid, Furthermore, residues X1, X2, X3, X10, and X11 are on the core sequence side. can only exist if the immediately adjacent residue is present, However, FXNPXY, GPLY, PPGY, and YXYXKV (X in the array represents any amino acid) A composition containing a peptide. 22. Contains a nucleotide sequence encoding the peptide according to claim 21 composition. 23. A heterologous internal enzyme that can modulate the intracellular transport of ligands into the host subject. Introducing cells from the host subject that have been modified to contain a cation signal. A gene therapy method consisting of steps. 24. A host subject cell is provided with a heterologous interface that can modulate the transport of the ligand into the cell. Expression vector containing a nucleotide sequence encoding a naturalization signal A method of gene therapy consisting of the process of introducing.