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EP0672132A1 - Signaux d'incorporation de recepteur - Google Patents

Signaux d'incorporation de recepteur

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Publication number
EP0672132A1
EP0672132A1 EP93907023A EP93907023A EP0672132A1 EP 0672132 A1 EP0672132 A1 EP 0672132A1 EP 93907023 A EP93907023 A EP 93907023A EP 93907023 A EP93907023 A EP 93907023A EP 0672132 A1 EP0672132 A1 EP 0672132A1
Authority
EP
European Patent Office
Prior art keywords
present
internalization
alanine
residues
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93907023A
Other languages
German (de)
English (en)
Other versions
EP0672132A4 (fr
Inventor
Ian S. Trowbridge
James F. Collawn
John A. Tainer
Leslie A. Kuhn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scripps Research Institute
Salk Institute for Biological Studies
Original Assignee
Scripps Research Institute
Salk Institute for Biological Studies
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Scripps Research Institute, Salk Institute for Biological Studies filed Critical Scripps Research Institute
Publication of EP0672132A4 publication Critical patent/EP0672132A4/fr
Publication of EP0672132A1 publication Critical patent/EP0672132A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/06—Fusion polypeptide containing a localisation/targetting motif containing a lysosomal/endosomal localisation signal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide

Definitions

  • the invention relates to internalization signals and the use of these signals to modulate the transport of ligand into a cell.
  • Receptor-mediated endocytosis is the mechanism by which a variety of nutrients, hormones, and growth factors are specifically and efficiently transported into the cell.
  • the process of receptor mediated endocytosis is complex and involves several distinct biochemical steps. Typically, the process proceeds by: (1) recruitment of soluble coat proteins to the cell membrane and nucleation of coated pit formation, (2) assembly of coat constituents and growth of the coated pit, (3) acquisition of specific receptors into the growing coated pit, (4) invagination of the cell membrane, (5) coat closure; and (6) membrane fusion wherein the coated pit buds in and pinches off to form a coated vesicle. The contents of the vesicles are ultimately delivered to the endosomes.
  • the internalization signal present in the cytoplasmic tail of the receptor interacts with soluble coat proteins during formation of a coated pit.
  • Receptors such as the transferrin receptor (TR) and the low-density lipoprotein (LDL) receptor are constitutively clustered in coated pits and undergo rapid internalization in the presence and absence of ligand.
  • TR transferrin receptor
  • LDL low-density lipoprotein
  • Other receptors such as epidermal growth factor (EGF) receptor are only concentrated in coated pits and internalized after binding ligand.
  • EGF epidermal growth factor
  • heterologous internalization signals to regulate endocytosis. In so doing, it would be possible, for example, to stimulate the internalization of toxic substances by tumor cells or inhibit uptake of essential nutrients.
  • Applicants' invention there has not been a successful transplantation of a heterologous internalization signal from one receptor to another.
  • the present invention addresses this need and provides the means for utilizing heterologous internalization signals and predicting the sequence and structure of heretofore unknown signals.
  • transport of ligand into a cell can be modulated by introducing a heterologous internalization signal into the cell.
  • the signal is introduced as a nucleotide sequence or as the encoded peptide.
  • the ability to transplant internalization signals from one receptor to another and retain activity has important implications for control of endocytosis for scientific and medical purposes, including drug delivery to cells.
  • the present invention provides a method of modulating receptor mediated transport of ligand into a cell, which method comprises introducing a heterologous internalization signal into the cell.
  • the invention provides a method for identifying a sequence which modulates internalization of a cell surface receptor. This method comprises: (a) incubating cells having such receptors in the presence or absence of sequence and, optionally, in the presence of ligand for the cell surface receptor; and (b) measuring internalization of the cell receptor in the presence or absence of the sequence.
  • compositions for modulating transport of ligand into a cell comprise compositions for modulating transport of ligand into a cell.
  • Compositions embraced by the present invention comprise peptides having a tight turn conformation and a defined amino acid sequence described in greater detail below.
  • nucleotide sequences encoding such peptides are provided also are nucleotide sequences encoding such peptides.
  • the present invention provides a method of administering gene therapy to a host subject. This method can be accomplished, for example, by introducing into a host subject, cells derived from the subject which have been modified to contain heterologous internalization signal capable of modulating transport of ligand into a cell.
  • the present invention also provides a method of gene therapy comprising introducing into a host subject an expression vector comprising a nucleotide sequence encoding a heterologous internalization signal capable of modulating transport of ligand into a cell.
  • FIGURE 1 Uptake of 59 Fe from human Tf by CEF expressing wild-type or mutant human TRs. Uptake of 59 Fe from human Tf by CEF expressing human TRs was determined by incubating cells with 59 Fe-Tf for the times indicated and then washing the cells and determining their radioactivity. The results shown are for two representative experiments, A and B, and each point represents the average values of 59 Fe uptake by triplicate cultures of CEF expressing wild-type or mutant TRs. Panel A shows the relative
  • FIGURE 2 Comparisons of steady-state distributions ( ⁇ ) and 59 Fe uptake ( ⁇ ) of human TR mutants expressed in CEFs (mutations at the carboxy-terminal aromatic residue of the TR internalization motif, YTRF). Phenylalanine 23 was changed to either methionine (F23M), isoleucine (F23I), tryptophan (F23W), alanine (F23A), or glycine (F23G). The data for the
  • F23A and F23G mutants is from Collawn, et al., Cell, 63:1061-1072, 1990.
  • CEFs expressing human TRs were incubated with 125 l-labeled Tf for 60 min at 37o C then washed with buffer.
  • the acid wash technique described in Materials and Methods was used to distinguish surface-bound and internalized Tf.
  • the internalization rates represent the average of three experiments and are given as percentages ⁇ standard errors relative to the wild-type (Wt) TR.
  • FIGURE 3 Superimposed crystallographic turn structures for internalization motif analogs with sequences matching the six-residue mannose-6-phosphate receptor (Man-6-PR) and the low-density lipoprotein receptor
  • LDLR LDLR patterns.
  • A Man-6-PR internalization motif analogs.
  • B LDLR internalization motif analogs.
  • C Superimposed Man-6-PR and LDLR analogs.
  • the present invention relates to a method of modulating receptor mediated transport of ligand into a cell wherein the method comprises introducing a heterologous internalization signal into the cell.
  • heterologous when used to describe the internalization signal of the invention refers to any internalization signal that is introduced into the cell.
  • ligand refers to any substance capable of binding to or with a cell surface receptor.
  • tight turn refers to the reverse or helical turn conformation of amino acid residues within the heterologous internalization signal (Collawn, et al., Cell, 63:1061 , 1990; Collawn, et al., EMBO J.,
  • cell-surface receptor refers to any cell surface molecule whether or not it has a naturally occurring ligand.
  • sequence refers to amino acid sequences as well as nucleic acid sequences.
  • internalization motif refers to an amino acid internalization signal having a tight turn structure. The terms internalization motif and internalization signal are used interchangeably.
  • core refers to the smallest sequence of amino acid residues involved in cell surface internalization.
  • heterologous internalization signals utilized herein may be the same as or different from internalization signal already present in the cell.
  • the introduced nucleotide sequence may further comprise additional nucleotide sequence encoding a cell surface receptor. This surface receptor may be the same as or different from receptor already present in the target cell.
  • the present invention envisions embodiments wherein heterologous internalization signal is introduced into cells having receptors containing internalization signals as well as those having receptors without internalization signals.
  • the receptor may be the same as or different from a receptor already present in the target cell.
  • heterologous internalization signal into target cells according to the invention can be accomplished by several means.
  • a signal can be introduced as a peptide or as a nucleotide sequence encoding such a peptide.
  • the introduced sequence can further comprise additional flanking sequence.
  • additional flanking sequence can encode or can be a cell surface receptor.
  • Internalization signal peptide or nucleic acid sequence encoding the peptide may be introduced into a cell surface receptor by several methods including: 1) substituting residues in the receptor by residues in the introduced internalization sequence (one for one replacement of residues/bases); 2) inserting an internalization signal between two residues/bases in the resident receptor so that the receptor sequence becomes longer; 3) replacing some residues in the resident receptor with a. greater number of internalization signal residues such that the receptor becomes longer.
  • the method selected for introduction of internalization signal peptide will be dictated by structural and experimental considerations on a case by case basis.
  • Preferred locations for introduction of internalization signal into a receptor sequence can be ascertained by locating endogenous internalization signal and by locating positions in the receptor sequence having tight turn conformation and packing interactions that can accommodate introduced signal.
  • Structure and packing information can be obtained from crystallography, NMR, or high electron resolution microscopy, in situations where such information is not available, commonly used and available secondary structure prediction algorithms may be used to locate sequence regions that likely fold as tight turns.
  • substitution of internalization sequence for receptor sequence tight turn regions of the receptor which are most sequence similar to the internalization sequence are preferred sites for substitution since substitutions in such sites minimizes structural destablization. Sequence similarity can be determined by mutation data matrices and other measures of sequence similarity, such as residue size and polarity.
  • heterologous internalization signal serves to modulate the transport of ligand into a cell having a surface receptor reactive with that ligand. This modulation can induce either an increase or a decrease in endocytosis, depending upon the choice of heterologous internalization signal.
  • the key to receptor-mediated endocytosis is the internalization signal present in the cytoplasmic tail of the surface receptor. It is the internalization signal that regulates the uptake of cell surface receptor. Identification of a heterologous internalization signal which can modulate internalization of cell surface receptor is accomplished by incubating ceils having such receptors in the presence or absence of the sequence suspected of being an internalization signal. Cells are incubated according to the method of Jing, et al., J.
  • the invention also provides a method for inhibiting internalization of cell surface receptor. This is accomplished by introducing into a cell having a receptor that clusters in coated pits, an effective amount of heterologous internalization signal peptide, wherein the heterologous peptide competes with internalization signal peptide of the receptor for binding with adaptor protein.
  • the term "effective amount” refers to the amount of peptide which results in inhibition of endocytotic vesicle formation. Delivery of an effective amount of the internalization signal peptide can be accomplished by one of the mechanisms described herein, such as by encapsulation in liposo.mes, or other methods well known in the art.
  • X 1 when present, is leucine or glutamic acid
  • X 2 when present, is isoleucine, methionine or proline;
  • X 3 when present, is any amino acid residue
  • X 4 when present, is selected from alanine, polar amino acids, or aromatic amino acids, and when X 3 is also present, at least one of the X 3 or X 4 residues is polar;
  • X 5 is an aromatic amino acid when residues X 1 - X 4 are not present, or is selected from aromatic amino acids or polar amino acids when atleast residue X 4 or additional upstream residue(s) is present;
  • X 6 is a polar amino acid or alanine
  • X 7 is selected from polar amino acids or alanine when residues
  • X 1 - X 4 are not present, or is any amino acid residue when at least X 4 or additional upstream residue(s) is present;
  • X 8 is selected from aromatic amino acids or hydrophobic amino acids
  • X 9 when present, is serine or alanine
  • X 10 when present, is alanine or leucine
  • X 1 1 when present, is alanine or phenylalanine; wherein at least one of residues X 3 , X 5 , and X 8 is an aromatic amino acid and further wherein residues X 1 , X 2 , X 3 , X 10 , and X 1 1 can only be present when the next adjacent residue(s) relative to the core is present.
  • a first preferred group of internalization signal peptides is characterized as having the amino acid sequence:
  • a more preferred group of internalization signal peptides having the sequence X 5 X 6 X 7 X 8 are those wherein:
  • X 5 is phenylalanine or tyrosine
  • X 6 is alanine, arginine, glutamine, serine, or threonine
  • X 7 is alanine, arginine, aspartic acid, glycine, glutamic acid, histidine, lysine, or threonine;
  • X 8 is isoleucine, leucine, methionine, phenylalanine, valine, or tryptophan.
  • Most preferred internalization signal peptides having the X 5 X 6 X 7 X 8 sequence are selected from the group consisting of YTRM, YARF, YTRI,
  • YQDL YTKF
  • YSKV YTRW
  • YRHV YSAF
  • YQTI YTAF
  • YTGF YTEF
  • FTRF FTRF
  • X 3 X 4 X 5 X 6 X 7 X 8 wherein X 3 - X 8 are as defined previously.
  • a more preferred group of internalization signal peptides having the sequence X 3 X 4 X 5 X 6 X 7 X 8 are those wherein:
  • X 3 is asparagine, leucine, methionine, phenylalanine, proline, or tyrosine;
  • X 4 is alanine, aspartic acid, glutamine, lysine, phenylalanine, or serine;
  • X 5 is asparagine, glutamine, or tyrosine;
  • X 6 is arginine, glycine, proline, serine, or threonine
  • X 7 is alanine, arginine, lysine, phenylalanine, or valine;
  • X 8 is isoleucine, leucine, methionine, phenylalanine,
  • X 3 X 4 X 5 X 6 X 7 X 8 are selected from the group consisting of YKYSKV, NFYRAL, LAYTRF, PQQGFF, FDNPVY, MSYTRF, or LSYTRF.
  • Novel compositions of the present invention comprise peptides having a tight turn and the amino acid sequence: X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 1 0 X 11 where X 5 - X 8 constitutes the core sequence,
  • X 1 - X 4 residues and X 9 - X 11 residues are optional and wherein:
  • X 1 when present, is leucine or glutamic acid
  • X 2 when present, is isoleucine, methionine or proline;
  • X 3 when present, is any amino acid residue;
  • X 4 when present, is selected from alanine, polar amino acids, or aromatic amino acids, and when X 3 is also present, at least one of the X 3 or X 4 residues is polar;
  • X 5 is an aromatic amino acid when residues X 1 - X 4 are not present, or is selected from aromatic amino acids or polar amino acids when at least residue X 4 or additional upstream residue (s) is present;
  • X 6 is a polar amino acid or alanine
  • X 7 is selected from polar amino acids or alanine when residues
  • X 1 - X 4 are not present, or is any amino acid residue when at least X 4 or additional upstream residue(s) is present;
  • X 8 is selected from aromatic amino acids or hydrophobic amino acids
  • X 9 when present, is serine or alanine
  • X 10 when present, is alanine or leucine
  • X 11 when present, is alanine or phenylalanine; wherein at least one of residues X 3 , X 5 , and X 8 is an aromatic amino acid and further wherein residues X 1 , X 2 , X 3 , X 10 , and X 11 can only be present when the next adjacent residue(s) relative to the core is present, provided that sequences selected from the group consisting of FXNPXY, GPLY, PPGY, and YXYXKV, where X stands for any amino acid, are excluded.
  • polar amino acid includes glycine, serine, threonine, histidine, tyrosine, proline, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine. (Rose, et al., Science, 229:834, 1985).
  • aromatic amino acid refers to any amino acid containing an unsaturated carbocyclic ring including phenylalanine, tyrosine, and tryptophan.
  • hydrophobic amino acid includes cysteine, valine, isoleucine, leucine, methionine, phenylalanine, and tryptophan.
  • large hydrophobic amino acid refers to amino acids selected from the group consisting of valine, isoleucine, leucine, methionine, phenylalanine, and tryptophan.
  • the present invention also embraces inhibition of endocytosis. Inhibition is accomplished by 1) introducing signal shown to be inactive when substituted to an active resident signal or 2) using active internalization peptides as competitive inhibitors of receptor internalization (i.e., the peptide competes with intact receptor for binding to adaptor complex).
  • Inactive signals which can be used according to the first method include sequences corresponding to the core X 5 X 6 X 7 X 8 and selected from the group consisting of ATRF, GTRF, ATRA, YTRG, FQDI, IGSY, NTLY, HLAF, PPGY and NPVY.
  • Generation of inactive internalization signals can be accomplished by mutating aromatic residues occurring in positions X 3 , X 4 , X 5 and X 8 to non-aromatic residues.
  • the present invention can utilize any nucleotide sequence encoding peptides described above. These peptide encoding nucleotide sequences can further comprise flanking nucleotide sequence encoding a cell surface receptor.
  • nucleotide sequences encoding internalization signal may be introduced into a host cell by means of a recombinant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of internalization signal sequences.
  • Such expression vectors typically contain a promotor sequence which facilitates efficient transcription of the inserted sequence in the host.
  • the expression vector also typically contains specific genes which allow phenotypic selection of the transformed cells.
  • nucleotide sequences encoding internalization signal can be introduced directly as a plasmid, for example, by microinjection, or transfection.
  • the present invention also provides methods for the treatment of disease employing gene therapy that modulates receptor mediated transport of ligand into a cell or internalization of cell surface receptor.
  • Such therapy can be effected by introduction of internalization signal into cells of a subject having the disease. Delivery of internalization signal can be achieved using techniques well known in the art. For example, when an internalization signal is introduced by means of a nucleotide sequence, a recombinant expression vector, such as a chimeric virus, or a colloidal dispersion system can be employed.
  • RNA virus such as a retrovirus
  • retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • a number of additional retroviral vectors can incorporate multiple genes. All of these vectors can incorporate a gene for a selectable marker so that transduced ceils can be identified and cultured.
  • Retroviral vectors can be made target specific by including in the retroviral vector a polynucleotide encoding a substance which binds to cell surface target. Preferred targeting is accomplished by using an antibody to target the retroviral vector.
  • Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the internalization signal polynucleotide.
  • helper cell lines that contain plasmids encoding ail of the structural genes of the retrovirus under the control of regulatory sequences within the LTR (long terminal repeat). These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include, but are not limited to, ⁇ 2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged.
  • the vector can be packaged and vector virion produced.
  • the vector virions produced by this method can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions.
  • NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • An alternative use for recombinant retroviral vectors comprises the introduction of polynucleotide sequences into the host by means of skin transplants of cells containing the virus. Long term expression of foreign genes in implants, using cells of fibroblast origin, may be achieved if a strong housekeeping gene promoter is used to drive transcription.
  • the dihydrofolate reductase (DHFR) gene promoter may be used.
  • Cells such as fibrobiasts, can be infected with virions containing a retroviral construct containing the internalization signal gene of interest together with a gene which allows for specific targeting, such as tumor-associated antigen (TAA), and a strong promoter.
  • TAA tumor-associated antigen
  • the infected cells can be embedded in a collagen matrix which can be grafted into the connective tissue of the dermis in the recipient subject. As the retrovirus proliferates and escapes the matrix it will specifically infect the target cell population. In this way the transplantation results in increased amounts of internalization signal peptide being produced in cells manifesting the disease.
  • colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a liposome.
  • a targeted delivery system offers a significant improvement in therapeutic applications over randomly injected non-specific liposomes.
  • a number of procedures can be used to covalently attach either polyclonal or monoclonal antibodies to a liposome containing internalization signals to render the liposome target specific.
  • Antibody-targeted liposomes can include monoclonal or polyclonal antibodies or fragments thereof such as Fab, or F(ab') 2 , as long as they bind efficiently to an epitope on the target cells. Liposomes may also be targeted to cells expressing receptors for hormones or other serum factors.
  • Liposomes are artificial membrane vesicles which are useful as in vitro and in vivo delivery vehicles. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 urn can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA, intact virions and peptides can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides into plant, yeast and bacterial cells.
  • LUV large unilamellar vesicles
  • a liposome In order for a liposome to be an efficient transfer vehicle, it should be capable of: (1) encapsulation of peptides or nucleotides of interest at high efficiency without compromising biological activity; (2) preferential and substantial binding to target cells relative to non-target cells; (3) delivery of aqueous contents of vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, ef al., Biotechniques, 6:682, 1988).
  • the composition of the liposome is usually a mixture of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidyicholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • diacylphosphatidylglycerols where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phospholipids include egg phosphatidyicholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • specific lipid groups can be incorporated into the liposome in order to maintain the target directed binding substance in stable association with the liposome.
  • Various linking groups can be used for joining the lipid chains to the target directed binding substance.
  • the targeted delivery system will be directed to cell surface receptors thereby allowing the delivery system to find and "home in” on the desired cells.
  • the delivery system can be directed to any cell surface molecule preferentially found in the cell population for which treatment is desired provided that the cell surface molecule is capable of association with the delivery system.
  • Antibodies can be used to target liposomes to specific cell-surface molecules.
  • tumor-associated antigens For example, certain antigens expressed specifically on tumor cells, referred to as tumor-associated antigens (TAAs), may be exploited for the purpose of targeting antibody-internalization signal-containing liposomes directly to a malignant tumor.
  • TAAs tumor-associated antigens
  • the present invention identifies sequences involved in the internalization of cell surface receptors, it is possible to design therapeutic or diagnostic protocols utilizing these sequences.
  • the native internalization sequence can be utilized to design sequences which interfere with the function of the native internalization signal.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 262:40. 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded.
  • Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target receptor-producing cell.
  • the use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal.Biochem., 172:289, 1988).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech,
  • Tetrahymena-type ribozymes recognize sequences which are four bases in length, while
  • hammerhead-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that that sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
  • Antisense sequences can be therapeutically administered by techniques as described above for the administration of heterologous internalization signal polynucleotides.
  • Targeted liposomes are especially preferred for therapeutic delivery of antisense sequences.
  • A. Oligonucieotide Site-Directed Mutagenesis A Cla I fragment containing the entire coding region of the human TR was cloned into the phagemid pBluescript SK (Stratagene, La Jolla, CA) (Ji ⁇ g, et al., J. Cell Biol., 110:283-294, 1990). Oligonucleotide site-directed mutagenesis was performed with pBluescript SK phagemid templates of the wild-type TR by the method of Kunkel, (Proc. Natl. Acad. Sci.
  • CEF Primary chicken embryo fibroblasts (CEF) were obtained from nine day embryos (SPAFAS, Norwich, Conn.) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% (v/v) chicken serum, 1% (v/v) defined calf bovine serum (Hyclone, Logan, Utah), 2% (v/v) tryptose phosphate broth (Difco, Detroit; MI).
  • CEF were transfected with retroviral construct prepared in (A) using 30 micrograms DNA per 10cm tissue culture plate of ⁇ 40% confluent cells using the polybrene-dimethyl sulfoxide method (Kawai and Nishizawa, Mol.
  • mutant human TRs were grown in selection media (DMEM supplemented with 3% (v/v) horse serum, 2% tryptose phosphate broth and 50 micrograms/ml diferric human transferrin (Tf) (Miles Scientific, Naperville, IL)) starting 10-14 days after transfection
  • selection media DMEM supplemented with 3% (v/v) horse serum, 2% tryptose phosphate broth and 50 micrograms/ml diferric human transferrin (Tf) (Miles Scientific, Naperville, IL)
  • Diferric human transferrin (Tf) was labeled with 125 I to a specific activity of 2-4 ⁇ Ci/mg using Enzymobeads (Bio-Rad, Richmond, CA) according to the manufacturer's directions.
  • Cells were plated at a density of 7.5 ⁇ 10 4 cells/cm 2 in 24-well Costar tissue culture plates 24 hr prior to the binding assay.
  • Cells were incubated in serum-free DMEM for 1 hr at 37o C and then washed once with ice-cold 0.15M NaCI-0.01 M Na phosphate buffer (pH 7.4) containing 0.1% bovine serum albumin (BSA-PBS).
  • BSA-PBS bovine serum albumin
  • BSA-PBS removed from the wells with 0.15 ml 1 M NaOH, and the radioactivity counted in a gamma counter.
  • CEFs expressing TR mutants were plated in triplicate wells as described for the binding studies.
  • the cells were first incubated for 1 hr at 37o C in serum-free DMEM, and then incubated with 4 micrograms/ml 125 l-Tf in 0.1% BSA in DMEM for 1 hr at 37° C.
  • the media was removed and the cells were washed three times with 1 ml of ice-cold BSA-PBS.
  • the cells were then incubated twice for 3 min with 0.5 ml of 0.2 M acetic acid-0.5M
  • k int [TR] sur k ext [TR] int
  • the rate of internalization, k int , of cell surface Tf-TR complexes, [TR] sur equals the rate of externaiization of the internal pool of apoTf-TR complexes, [TR] int , assuming an insignificant rate of intracellular degradation of receptors (Collawn, et al., Cell, 63:1061-1072, 1990).
  • apoTf refers to transferrin having no bound iron.
  • LDLR low density lipoprotein receptor
  • Man-6-PR mannose-6-phospate receptor
  • Oligonucleotide-directed mutagenesis was used to replace the four-residue YTRF internalization signal from the cytoplasmic tail of the wild-type TR with the four-residue sequences from LDLR and Man-6-PR signals and the six-residue TR sequence LSYTRF by the six-residue LDLR and Man-6-PR signals (TABLE I).
  • the TR signal YTRF was changed to YSKV for the bovine Man-6-PR signal and NPVY for the human LDLR signal.
  • the TR sequence, LSYTRF was changed to YKYSKV or to FDNPVY. Numbers above the sequences (-2 through 4) refer to positions relative to the turn region implicated in TR and LDLR (Collawn, et al., Cell, 63:1061-1072, 1990) with position 1 starting the turn. TR residue numbers appear as superscripts.
  • the four-residue sequence, NPVY, from the LDLR was tested because the NPXY pattern is also found in the cytoplasmic tails of other transmembrane proteins including the EGF and insulin receptors and therefore may play a role in the internalization of these proteins (Chen, ef al., J. Biol. Chem., 265:3116-3123, 1990; Backer, et al., J. Biol. Chem., 2 ⁇ 5j 16450-16454, 1990).
  • the YSKV sequence from Man-6-PR was tested because mutational analysis of Man-6-PRs (Canfield, et al., J. Biol. Chem., 266:5682-5688, 1991) indicated that this tetrapeptide was almost as efficient as the complete internalization sequence in promoting high efficiency endocytosis.
  • Efficient expression of wild-type and mutant human TRs- in chicken embryo fibroblasts was achieved using a helper-independent retroviral vector, BH-RCAS, derived from Rous sarcoma virus (see Materials and Methods).
  • the relative internalization efficiencies of wild-type and mutant receptors were determined from Fe uptake measurements and their intracellular accumulation under steady-state conditions.
  • mutant TRs containing the Man-6-PR six-residue signal, YKYSKV were internalized as efficiently as wild-type receptors.
  • Mutant receptors containing the Man-6-PR four-residue signal, YSKV were also rapidly internalized and had an internalization efficiency of approximately 87% that of wild-type receptors (TABLE II, FIGURE 1A).
  • Mutant TRs containing the LDLR six-residue signal, FDNPVY were internalized at ⁇ 50% the rate of wild-type receptors, indicating that the LDLR six-residue signal had significant activity when transplanted into the TR cytoplasmic tail.
  • mutant TRs containing the four-residue LDLR signal, NPVY were internalized at essentially the same rate as tailless receptors (TABLE II, FIGURE 1 A and B).
  • the results of these experiments indicate that LDLR and Man-6-PR internalization signals can substitute for the TR signal and mediate high efficiency internalization of the TR.
  • the lack of activity of the transplanted LDLR four-residue sequence, NPVY implies that the complete LDLR internalization signal is the six-residue sequence, FDNPVY.
  • mutant TRs with transplanted four- or six-residue Man-6-PR and LDLR internalization signals show that an amino-terminal aromatic residue is required for activity, but that a large non-aromatic hydrophobic residue may substitute for an aromatic residue at the carboxy-terminal position.
  • Phe-23 was altered to Met, then lie, then Trp.
  • Functional analysis of mutant TRs with Phe-23 altered to Met or Ile indicated that their internalization rate and ability to mediate Fe uptake were identical to wiid-type TRs (TABLE II). Mutant TRs with Phe-23 altered to Trp were also rapidly internalized (FIGURES 1 and 2).
  • the TR, LDLR, and Man-6-PR internalization signals were all predicted by a secondary structure algorithm (Chou and Fasman, Adv. Enzymol., 47:45, 1978) to be turns before or at the start of an alpha helix.
  • Four structural analogs were identified for the Man-6PR signal and five for the LDLR signal ( Figure 3).
  • Man-6-PR internalization motif analogs matching the sequence pattern YXYXKV (detailed in Material and Methods) are shown. Side chains are shown for critical residues at positions -2, 1 , 3, and 4.
  • Man-6-PR internalization analogs are: blue, FTFSDY, immunogiobulin 4-4-20 Fab fragment (PDB code 4FAB), residues H27-H32; red, FEFEKF, phosphoglycerate kinase (3PGK), residues 340-345; purple, FMFNQF, concanavalin A (1 CN1 ), residues A128-A133; green, FAFIRL, D-xylose isomerase (4XIA), residues A377-A382.
  • LDLR internalization motif analogs matching the LDLR pattern FXNPXY are shown. Side chains at positions -2, 3, and 4 are shown. LDLR analogs identified by Sequery are: yellow, YANLVF, trypto phan synthetase (1WSY), residues A102-A107; red, FYNAAW, lectin (2LTN), residues A123-A128; orange, FTNEFY, cytochrome c peroxidase (2CYP), residues 198-203; blue, FKNSKY, chymotrypsinogen a (2CGA), residues A89-A94; and green, YDNKYW, calcium-free phospholipase A 2 (1 PP2), residues L113-L118.
  • the aromatic groups at positions -2 and 4 and the position 3 side chain were exposed on the same side of the structure.
  • the LDL analogs included three surface exposed reverse turns and two initial turns of alpha helix.
  • the Asn side chains in position 1 fell in two families, positioned in front or in back of the turn according to whether the incoming secondary structure was to the back or in front.
  • the Asn side chain (position 1) can stabilize the turn by forming hydrogen bonds to adjacent main chain atoms, suggesting that this side chain has a structural role.
  • Asn-Pro the first two residues in the LDLR tetrapeptide, are the most favored residues for positions 1 and 2 in the most prevalent type of reverse turn (Wilmot, et al., J. Mol. Biol., 203:221, 1988) and Asn-Pro is also a potent helix initiator (Richardson, et al., Science, 240:1648. 1988).
  • Figure 3C shows superimposed Man-6-PR and LDLR analogs.
  • the Man-6-PR analogs from A are blue and the LDLR analogs from B are yellow with side chains shown for positions -2, 1 and 4.
  • the -2 position aromatic groups extend to the left of the turn, and the position 1 Asn (LDLR) and aromatic (Man-6-PR) side chains form perpendicular fans like those found for the aromatic (LDLR) and hydrophobic (Man-6-PR) side chains in position 4.
  • Position 2 Ser in Man-6-PR and Pro in LDLR, appears primarily structural, with the Ser side chains in Man-6-PR in YSKV analogs (LA.K.
  • the side chains in positions -2, 1 , and 4 formed a line (........) across the base of the turn, with the line of critical side chains in LDLR analogs at an angle of ⁇ 35o to that for Man-6-PR.
  • mutant TRs containing an additional copy of the TR internalization signal YTRF at putative turn positions 1 , 3, or 4 were prepared using oligonucleotide-directed mutagenesis. Each mutant retained the wild- type signal at turn 2, and therefore contained two internalization signals. Mutant TRs were tested in steady-state distribution assays in chicken embryo fibroblasts as described. The results of the analysis are shown in Table IV and indicate that additional signals have variable effects and appear to be positionally dependent.
  • the sequence shown in brackets represents the additional internalization signal that was transplanted into the TR using oligonucleotide-directed mutagenesis.
  • Each of the mutants contains the wild-type signal at Turn 2 [ 20 YTRF 23 ].
  • the superscripts represent the sequence position in the TR where the changes were made.
  • the distribution of mutant TRs was determined from the surface bound and internal pools of radiolabeled Tf under steady-state conditions.
  • Addition of an internalization signal at turn 1 creates a TR that is indistinguishable from a wild-type receptor (96% internalization rate compared to wild-type).
  • Addition at turn 3 creates a mutant with twice the internalization rate of the wild-type (206% ⁇ 30%; 3 experiments, mean ⁇ S.E).
  • Addition of a signal at turn 4 has a less dramatic effect, but still creates a TR mutant with 145% activity. The results demonstrate that internalization rates can be modulated by transplantation of additional internalization signals.
  • sequences function very well as internalization signals, e.g., sequences from the asialoglycoprotein receptor (ASGPR H1 subunit, sequence YQDL), polymeric immunoglobulin (poly Ig, sequence YSAF), epidermal growth factor receptor (EGFR, sequence NFYRAL), lysosomal acid phosphatase (LAP, sequence YRHV), lysosome-associated membrane giycoprotein (LAMP-1 , sequence YQTI), while others, e.g., ASGPR H2 subunit (sequence FQDI), mannose receptor (sequence FENTLY), and LAP (sequence PPGY) function poorly, with activity similar to that of tailless receptors (TABLE V).
  • ASGPR H2 subunit sequence FQDI
  • mannose receptor mannose receptor
  • LAP sequence PPGY
  • this method is useful for mapping the exact site. For example, analysis of the region near the tyrosine of LAP, sequence PPGYRHV, shows that one sequence, YRHV, functions well and another, PPGY, does not.
  • This strategy is also useful for mapping internalization signals from ligand-induced internalized receptors such as the EGFR. This is significant since none of these internalization signals have been clearly identified simply because the large cytoplasmic domains of these receptors makes the interpretation of deletional analysis more difficult. Identifying an. internalization signal in a iigand-induced receptor may require additional transplantation experiments with the TR, and parallel mutagenesis experiments within the receptor in question may have to be performed.
  • the putative EGFR signal, NFYRAL which functions very well in the context of the TR, appears to have a sequence related to the cation-independent mannose-6-phosphate receptor. The implication from these studies is that potential signals can be tested in the TR and. that ligand-induced receptors may have internalization signals similar to those of the constitutively recycling receptors.

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Abstract

Signaux d'incorporation et leur utilisation dans la modulation de l'endocytose de récepteur de surface cellulaire.
EP93907023A 1992-03-03 1993-03-01 Signaux d'incorporation de recepteur Withdrawn EP0672132A1 (fr)

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JOURNAL OF BIOLOGICAL CHEMISTRY., vol.266, no.9, 25 March 1991, BALTIMORE US pages 5682 - 5688 W M CANFIELD ET AL. 'Localization of the signal for rapid internalization of the bovine cation-independent mannose-6-phosphate/Insulin-like growth factor-II receptor to amino acids 24-29 of the cytoplasmic tail' *
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