U.S. patent application number 12/335010 was filed with the patent office on 2009-04-16 for high efficiency tissue-specific compound delivery system using streptavadin-protein a fusion protein.
This patent application is currently assigned to New York University. Invention is credited to Brandi A. Levin, Daniel Meruelo, Kouichi Ohno.
Application Number | 20090098148 12/335010 |
Document ID | / |
Family ID | 24262817 |
Filed Date | 2009-04-16 |
United States Patent
Application |
20090098148 |
Kind Code |
A1 |
Meruelo; Daniel ; et
al. |
April 16, 2009 |
HIGH EFFICIENCY TISSUE-SPECIFIC COMPOUND DELIVERY SYSTEM USING
STREPTAVADIN-PROTEIN A FUSION PROTEIN
Abstract
The present invention relates to methods and compositions that
can be employed to introduce toxins and nucleic acids into the
cytoplasm or nucleus of a eukaryotic cell, particularly a cell of a
higher vertebrate. The invention particularly concerns the use of a
fusion protein of streptavidin and protein A sequences to form a
non-covalent complex of a toxin or nucleic acid and an
antibody.
Inventors: |
Meruelo; Daniel;
(Scarborough, NY) ; Ohno; Kouichi; (New York,
NY) ; Levin; Brandi A.; (Rego Park, NY) |
Correspondence
Address: |
DARBY & DARBY P.C.
P.O. BOX 770, Church Street Station
New York
NY
10008-0770
US
|
Assignee: |
New York University
New York
NY
|
Family ID: |
24262817 |
Appl. No.: |
12/335010 |
Filed: |
December 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10289921 |
Nov 6, 2002 |
|
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12335010 |
|
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08566421 |
Nov 30, 1995 |
6497881 |
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10289921 |
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Current U.S.
Class: |
424/178.1 ;
435/188; 530/391.1 |
Current CPC
Class: |
C07K 16/2863 20130101;
C07K 16/2896 20130101; A61K 47/6817 20170801; A61K 47/6898
20170801; B82Y 5/00 20130101; A61K 38/00 20130101; C07K 2319/00
20130101; C07K 16/2833 20130101 |
Class at
Publication: |
424/178.1 ;
530/391.1; 435/188 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; C12N 9/96 20060101
C12N009/96 |
Claims
1. A complex for transferring a compound to a cell produced by a
method which comprises the steps of: Incubating a. a
streptavidin-protein A fusion protein having an antibody binding
site and a biotin binding site, wherein said streptavidin-protein A
fusion protein forms tetramers; b. an antibody, bound to the
antibody binding site, in which the antibody is specific for a cell
surface protein, and in which the cell surface protein undergoes
endocytosis after binding with the antibody; and c. a biotinylated
compound to be transferred to said cell, bound to the biotin
binding site, thereby forming a complex wherein said complex is
capable of transferring said compound to said cell when contacted
with said cell after forming said complex, resulting in the
transfer of said compound into said cell.
2. The complex of claim 1 in which the biotinylated compound is
selected from the group consisting of biotinylated single stranded
nucleic acid, double stranded DNA that forms triplex structure with
a biotinylated single stranded nucleic acid having a homopurine or
homopyrimidine portion, biotinylated enzyme and biotinylated
protein of a pathological bacteria or virus.
3. The complex of claim 1 in which there are four antibody binding
sites and four biotin binding sites.
4. The complex of claim 1 in which the streptavidin component of
said streptavidin-protein A fusion protein has a modified RYD
sequence.
5. The complex of claim 1, wherein the antibody recognizes a
surface antigen selected from the group consisting of: (a) human
lymphocyte antigen (HLA-DR); (b) cluster of differentiation (CD33);
(c) cluster of differentiation (CD34); and (d) epidermal growth
factor (EGF) receptor.
6. The complex of claim I in which the antibody is an IgG
antibody.
7. A pharmaceutical composition, comprising: (a) the complex of
claim 1, and (b) a pharmaceutically acceptable carrier, which
composition is substantially free of biotinylated compound not
bound to the streptavidin-protein A fusion protein.
8. A complex for transferring an enzyme into a cell produced by a
method which comprises the steps of: Incubating (a) a
streptavidin-protein A fusion protein having an antibody binding
site and a biotin binding site, wherein said streptavidin-protein A
fusion protein forms tetramers; (b) an antibody, bound to the
antibody binding site, which antibody is specific for a cell
surface protein, and which cell surface protein undergoes
endocytosis after binding with the antibody; and (c) a biotinylated
enzyme bound to the biotin binding site, thereby forming a complex
wherein said complex is capable of transferring said enzyme to said
cell when contacted with the cell after forming said complex
resulting in expression of said enzyme activity inside said
cell.
9. A complex for transferring a protein of a pathological bacteria
or virus into a cell, produced by a method which comprises the
steps of: Incubating (a) a streptavidin-protein A fusion protein
having an antibody binding site and a biotin binding site, wherein
said streptavidin-protein A fusion protein forms tetramers; (b) an
antibody, bound to the antibody binding site, which antibody is
specific for a cell antigen presenting cell surface protein, and
which cell surface protein undergoes endocytosis after binding with
the antibody; and (c) a biotinylated protein of a pathological
bacteria or virus, bound to the biotin binding site, thereby
forming a complex wherein said complex is capable of transferring
said complex into said cell when contacted with said cell after
forming said complex, resulting in the transfer of said protein
into said cell.
Description
[0001] The present application is a continuation of application
Ser. No. 10/289,921, filed on Nov. 6, 2002, which is a divisional
of application Ser. No. 08/566,421, filed Nov. 30, 1995, now U.S.
Pat. No. 6,497,881. The prior applications are hereby incorporated
herein by reference in their entirety.
INTRODUCTION
[0002] The present invention relates to methods and compositions
that can be employed to introduce toxins and nucleic acids into the
cytoplasm or nucleus of a eukaryotic cell, particularly a cell of a
higher vertebrate. The invention allows for the efficient and
specific delivery of the toxins and nucleic acids into cells that
bind an antibody. The invention particularly concerns the use of a
fusion protein of streptavidin and protein A sequences to form a
non-covalent complex of a toxin or nucleic acid and an
antibody.
[0003] The invention provides a method of treatment of human
disease by the introduction of toxins or antisense nucleotides into
human cells, e.g., tumor cells, in vivo or ex vivo. The invention
also provides methods of conducting biological research and methods
useful in the production of biological products by introducing
exogenous duplex DNA molecules into cultured cells.
BACKGROUND OF THE INVENTION
[0004] The selective introduction of compounds into the cytoplasm
or nucleus of specific cells has been a valuable technique in
biological and medical research and in medical practice. Cell
specific targeting of cytotoxins has been accomplished by
complexing toxins with cell binding proteins that can
preferentially bind targeted cells. The cell-binding proteins of
the complex can be either antibodies, particularly monoclonal
antibodies, or protein ligands, e.g., /growth factors/ which
recognize the corresponding surface antigens or receptor. Complexes
of toxin and antibody have been termed immunotoxins.
[0005] Conventionally, the toxin and cell binding protein of the
complex have been linked covalently through either chemical
coupling or gene fusion. Conventional immunotoxins have been made
by chemically linking a toxin component to an antibody, typically a
monoclonal antibody, with a heterobifunctional cross-linking
reagent that is non-specific. Accordingly, this method yields a
heterogeneous product in which some toxin molecules block the
antibody's ability to bind antigen by linking to the F(ab) portion
of the antibody. Additionally, the coupling chemistry can partially
destroy the toxins activity.
[0006] Many of the problems associated with chemical conjugation
have been overcome through the generation of single-chain fusion
toxins using recombinant DNA technology. However, this technology
requires that a new recombinant toxin for each target cell. The
biological activity of each new recombinant toxin is
unpredictable.
[0007] Alternative techniques have been developed in which the
cytotoxin is non-covalently linked to the antibody or ligand. One
such technique exploits the specific interaction between
Staphylococcal aureus protein A and immunoglobulins to generate
antibody complexes with two specificities. According to this
technique, protein A is complexed with antibodies of two different
specificities: a toxin specific antibody and a cell surface
specific antibody. Such complexes have been used to deliver ricin
toxin into targeted cells (Laky, et al., 1986/1987, Immunology
Letters 14:127-132). In a second immunotoxin targeting system,
single chain antibodies are fused with streptavidin which has a
strong and specific binding affinity for biotin. Using this
construct, biotinylated toxin was delivered into a target cell
(Dubel, et al., 1995, Journal of Immunological Methods,
178:201-209).
[0008] Recently, Sano et al. described a fusion protein consisting
of streptavidin and one or two immunoglobulin G (IgG)-binding
domains of protein A in Escherichia coli. (U.S. Pat. 5,328,985,
issued Jul. 12, 1994, which is hereby incorporated by reference in
its entirety). The streptavidin-protein A (ST-PA) fusion protein
has functional biotin and IgG binding sites. Sano further described
complexes of the streptavidin-protein A fusion protein, a
monoclonal antibody to BSA, and biotinylated horseradish
peroxidase.
[0009] Sano also described a method of labeling cells using the
ST-PA fusion protein. Cells were incubated with an antibody to a
cell surface antigen, Thy-1. The chimeric protein-biotinylated
marker complex was subsequently added to the cell suspension. This
technique was used to deliver biotinylated FITC to the surface of
cells having Thy-1 antigen on their surface. However, Sano did not
describe or suggest the use of the ST-PA fusion protein to deliver
compounds into the cytoplasm or nucleus of specific cells.
[0010] Immunotoxins appear to enter the cell via receptor-mediated
endocytosis (Pastan et al., 1986, Cell 47:1-44 and Pirker et al.,
1987, Lymphokines 14:361-382). Binding of the antibody moiety of
the immunotoxin complex to the surface receptor is followed by,
first, clustering of the complex into coated pits and then by
internalization of the complex into endosomes or receptosomes
within the cell (Middlebrook et al., 1994, Microbiol. Rev.,
48:199-221; Morris et al., 1985, Infect. Immun. 50:721-727;
Fitzgerald et al., 1980, Cell 21:867-873). During the journey into
the cell, the complex may be transported through different
intracellular compartments that vary in pH and proteolytic enzyme
activity before the toxins are translocated across an intracellular
membrane and into the cell cytoplasm where they can cause cell
death.
[0011] A second area which has been developed concerns methods for
introducing nucleic acids into cells. The most widely used methods
employ calcium phosphate or DEAE-dextran to promote uptake of
nucleic acids. These methods appear to involve the steps of DNA
attachment to the cell surface, entry into the cytoplasm by
endocytosis, and subsequent transfer into the nucleus. Maniatis,
Laboratory Cloning Manual, volume 2, 16.30. Depending upon the cell
type, up to 20% of a population of cultured cells can take up DNA
using calcium phosphate or DEAF-dextran.
[0012] Electroporation is an alternative transfection method in
which an electric field is applied to open pores in the cell plasma
membrane. DNA appears to enter the cell through these pores.
[0013] Liposomes have also been used to introduce nucleic acids
into cells. According to this technique, artificial lipid-bilayer
vesicles containing cationic and neutral lipids mediate the
transfer of DNA or RNA into cells. The mechanism of
liposome-mediated transfection, is not well understood, but it
appears that negatively charged phosphate groups on DNA bind to the
positively charged surface of the liposome, and that the residual
positive charge binds to negatively charged sialic acid residues on
the cell surface.
[0014] Sano did not use the complex to introduce nucleic acid into
the cell. Sano et al. described DNA-antibody complexes with the
ST-PA fusion protein by incorporating a single biotin molecule at
one end of a linearized pUC 19 plasmid. In contrast to the methods
of transfecting nucleic acids into the cell which are described
above.
SUMMARY OF THE INVENTION
[0015] The invention relates to a method of delivering toxins or
nucleic acids into specific cell types and to the complexes for the
practice of the method. According to the invention, an antibody
that recognizes a cell surface antigen is non-covalently bound to
the antibody binding site of a ST-PA fusion protein; a biotinylated
toxin or nucleic acid is bound to the biotin-binding site. In an
alternative embodiment, the toxin or nucleic acid can be bound to a
third biotinylated molecule, an adapter, which is bound to the
biotin binding-site.
[0016] In one embodiment of the present invention, a nucleic acid
is delivered into a specific cell type. The nucleic acid can be a
biotinylated single stranded nucleic acid bound to the biotin
binding site of the ST-PA fusion protein. In an alternative
embodiment, the nucleic acid can be a duplex nucleic acid that
forms a complementary triplex with a biotinylated single stranded
nucleic acid, which is in turn bound to the biotin binding
site.
[0017] The method of the invention relates to the steps of forming
a complex between a streptavidin-protein A fusion protein; an
antibody, that is specific for a cell surface protein, which
undergoes endocytosis after binding with the antibody; and some
targeted material e.g. a biotinylated multidrug resistance (mdr)
gene product, prodrug, toxin or nucleic acid; isolating the complex
from toxin that is not bound to the biotin binding site; and
exposing the target cell, to the complex so that the targeted
material enters the cell.
Definitions
[0018] As used herein, toxin, refers to holotoxins, modified
toxins, catalytic subunits of toxins, or any enzymes not normally
present in a cell that under defined conditions cause the cell's
death. A biotinylated toxin refers to a toxin that is either
directly biotinylated or one that is bound to a biotinylated
adapter.
[0019] The term "antibody" refers to any molecule which contains
one or more functional antigen binding domains and an Fc domain
that specifically binds protein A.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1A. Schematic representation of immunotoxin or
recombinant toxin binding to the corresponding surface antigen or
receptor on the cell surface.
[0021] FIG. 1B. Schematic representation of the
streptavidin-protein A/biotinylated macromolecule complex binding
to the corresponding surface antigen or receptor on the cell
surface.
[0022] FIGS. 2A-C. Comparison of the delivery of
biotin-.E-backward. galactosidase into A431 cells of an
ST-PA/anti-EGFR mAB/biotin-.E-backward. galactosidase complex (B)
and ST-TGF/biotin-.E-backward.-galactosidase complex (C).
[0023] FIG. 3. Time course analysis of cell .beta.-galactosidase
staining after transfer of .beta.-galactosidase into the cells.
[0024] FIG. 4A. Cell lines and cell surface molecules targeted by
the ST-PA-biotin-.beta.galactosidase-antibody complex.
[0025] FIGS. 4B-G. Transfer of ST-PA/mAB/biotin-.E-backward.
galactosidase into human cells Daudi (B and E), HL-60 (C and F) and
KG1 (D and G), where the mAB is specific for HLA-DR, CD33, or CD34
cell surface molecules.
[0026] FIGS. 5A-C. Schematic of the pAT-.E-backward. galactosidase
expression vector construct (A) for forming triplex DNA with
biotinylated poly (dT) oligonucleotide (B-C).
[0027] FIG. 6A. Amino acid homology between fibronectin (SEQ ID NO:
1) and streptavidin (SEQ ID NO:2). Bold type indicates homologous
residues. The RGD and RYD domain of each protein is underlined. The
sequence shown for fibronectin begins at residue 1481, and the
streptavidin sequence begins at residue 49.
[0028] FIG. 6B. Oligonucleotide primers DES (SEQ ID NO:3) and DER
(SEQ ID NO:4) designed to modify the RYD sequence of the
streptavidin gene.
[0029] FIG. 6C. Schematic of the methodology to be used in
modifying the RYD sequence of streptavidin.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] The present invention provides a method of specific delivery
of targeted material, e.g., toxins, prodrugs, mdr gene products, or
nucleic acids into cells by a complex of the targeted material, an
antibody specific for a cell surface antigen on the cell, which
antigen is endocytosed when the cell is exposed to an effective
concentration of the antibody, and a ST-PA fusion protein.
[0031] The streptavidin-protein A fusion protein (ST-PA) binds,
non-covalently the cell specific antibody in the antibody binding
site and the biotinylated targeted material in the biotin binding
site. The manner and method by which the targeted material is
biotinylated is not critical; the invention includes the use of any
and all such methods and reagents.
[0032] In one embodiment, the invention provides a method for the
specific destruction of cells, e.g., the destruction of tumor cells
in a host or ex vivo in short term or long term culture. A toxin
can be selected from the group consisting of: thymidine kinase,
endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas
exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed
antiviral protein, alpha-sarcin and cholera toxin. The toxin is
biotinylated and the complex of PA-ST/Ab/biotinylated toxin is
formed. An effective amount of the complex can then be administered
to the host or to the ex vivo culture system.
[0033] In an alternative embodiment the invention provides a method
of using an antibody to a cell to introduce into the cell a
cytotoxic prodrug, i.e., a non-toxic compound that is converted by
an enzyme, normally present in the cell, into a cytotoxic compound.
The embodiment contemplates the use of a complex of the ST-PA
chimeric protein with a tumor selective monoclonal antibody and
prodrug. Compounds suitable as prodrugs include by way of example
glutamyl derivative of a benzoic acid mustard alkylating agent,
phosphate derivatives of etoposide or mitomycin C, and
phenoxyacetamide derivative of doxorubicin.
[0034] A further embodiment of the invention contemplates a complex
for the delivery of single stranded nucleic acid into cells. As
used herein, the term "single stranded nucleic acid" refers to both
naturally and non-naturally occurring nucleic acids. The nucleic
acid to be delivered can be biotinylated using any of the methods
currently available, such as, random incorporation, extension
reactions using DNA polymerase, and PCR with biotinylated primers.
Such nucleic acids can be used to specifically destroy mRNAs to
which they are complementary.
[0035] A further embodiment of the invention concerns the
introduction into a cell of a duplex DNA that can integrate into
the cell's genome or replicate episomally and that can be
transcribed. The direct incorporation of biotin into a duplex DNA
can interfere with both replication and transcription of the DNA.
To overcome these problems, the duplex DNA to be delivered into the
cell is modified to include a sequence that can form a region of
triplex nucleic acid with a single stranded nucleic acid.
[0036] Triplex DNA formations have been described and typically
consist of T-A-T and C-G-C nucleotide triads. The third strand of
the triplex occupies the major groove of an A-form DNA helix and
forms Hoogsteen base pairs with the homopolymeric duplex DNA.
Alternatively, a triplex can be formed with duplex DNA. As used
herein, the term "triplex" refers to a structure formed by
Hoogsteen base pairing between a duplex DNA and a single stranded
nucleic acid.
[0037] In this embodiment, the complex contains a duplex DNA
Hoogsteen paired to a single stranded nucleic acid, which is
biotinylated and complexed with the ST-PA/mAb complex. The duplex
DNA can be a linear duplex DNA, which is suitable for recombination
into the genome of a cell or, alternatively, the duplex can be a
circular or supercoiled circular DNA, which can episomally
replicate.
[0038] This embodiment of invention can be used under any
circumstances it is desired to introduce cloned DNA into a cell,
e.g., to express a product or to alter the phenotype of the cell,
to investigate the function of any cloned gene.
[0039] A further embodiment of the invention comprises a complex of
an antibody to a cell surface protein found on an antigen
presenting cell, the ST-PA fusion protein, and a biotinylated
protein of a pathological bacteria or virus, such a complex can be
used to localize the antigen to antigen presenting cells and
thereby enhance the immune response of CD4 positive T cells
relative to that of other lymphocytes.
[0040] The complexes of the present invention can be formed by
simply admixing ST-PA, a monoclonal antibody, and the biotinylated
material in the appropriate ratios. The components can be mixed in
any order.
[0041] The ST-PA fusion protein forms tetramers which bind up to
four biotinylated molecules and four IgG molecules, which are each
bivalent. Without limitation as to theory, the octovalent binding
of complexes of the invention to the cell surface is believed to
cause the complexes to have superior binding and internalization
properties compared to other immunotoxins and immunopharmaceutical
complexes.
[0042] Biotin-blocked streptavidin is capable of specifically
interacting with cell surfaces through an Arg-Tyr-Asp sequence
present in the protein (the "RYD site"). This site is distinct from
the biotin-binding cleft of the protein and bears high homology to
the RGD-containing cell binding domain of fibronectin which
mediates fibronectin-cell surface interactions (Alon et al, 1993,
Europ. J. Cell Biol. 60: 11). Studies have suggested that
streptavidin acts as a close mimetic of fibronectin (Alon et al,
1993, Europ. J. Cell Biol. 60: 1-11).
[0043] The conserved RYD and RGD domains of fibronectin and
streptavidin function as universal recognition sequences for
interactions with many membrane-bound receptors. In one embodiment
of the invention, the streptavidin component of the complex can be
modified to alter the RYD site. As used herein, the term "modified
RYD sequence" includes any alteration to the RYD site or flanking
region which eliminates the non-biotin binding site-related
interaction of streptavidin with cell surface proteins. One such
modification is the replacement of aspartic acid by glutamic
acid.
EXAMPLES
1.1 Materials and Methods
[0044] Plasmid, pTSAPA-2, described in U.S. Pat. No. 5,328,985,
issued Jul. 12, 1994, carries the chimeric gene of streptavidin and
protein A (region E and D). Expression and purification of the gene
fusion of ST-PA was carried out according to the methods that
follow.
Fusion-Protein-Preparation:
[0045] Bacterial Strain lysogen BL21 (DE3) (pLysS) was transformed
with the pTSAPA-2 streptavidin-protein A fusion expression vector.
The transformed strain was grown at 37.degree. C. in LB media
supplemented with 50 .mu.g/ml ampicillin, 34 .mu.g/ml
chloramphenicol and 0.2% glucose. When the absorbance at 600 nm of
the culture was between 0.8 and 1.0 OD, 100 mM isopropyl
.beta.-D-thiogalactopoyranoside (IPTG) dissolved in water was added
to a final concentration of 0.4 mM to induce the T7 RNA polymerase
gene placed under the lac UV5 promoter. After the induction, the
cells were incubated at 37.degree. C. with shaking for 2 hours.
[0046] Purification of streptavidin-protein A fusion chimeric
protein was carried out at 4.degree. C. or on ice unless otherwise
indicated. The culture (100 ml) of BL21 (DE3) (pLysS)(pTSAPA-2)
incubated for 2 hours after the induction was centrifuged at
2,900.times.g for 15 min. The cell pellet was suspended in 10 ml of
2 mM EDTA, 30 mM Tris-Cl (pH 8.0), 0.1% Triton X-100, 0.5 mM PMSF
to lyse the cells and the lysate was stored at -70.degree. C. until
used. To the thawed cell lysate, PMSF, leupeptin, and pepstatin A
were added to final concentrations of 0.5 mM, 1 .mu.M, and 1 .mu.M,
respectively. The lysate was then treated with 10 .mu.g/ml of
deoxyribonuclease I and 10 .mu.g/ml ribonuclease A in the presence
of 12 mM MgSO.sub.4 at room temperature for 20 minutes. The mixture
was centrifuged at 39,000.times.g for 15 minutes and the pellet was
dissolved in 100 ml of 7 M guanidine hydrochloride overnight at
4.degree. C. with stirring. After the pellet was dissolved the
protein was then dialyzed against 150 mM NaCl, 50 mM Tris-C1 (pH
7.5), 0.05% Tween 20, 0.1 mM PMSF, 1 .mu.M leupeptin, 1 .mu.M
pepstatin A, 0.02% NaN.sub.3. To achieve slow removal of the
guanidine hydrochloride, the dialysis bag containing the protein
solution was left overnight in the dialysis solution (.about.1,000
ml) without stirring, followed by 3 changes of the dialysis
solution and dialysis with stirring at 4.degree. C. The dialysate
was centrifuged at 39,000.times.g for 15 minutes, and the
supernatant was applied to an IgG Sepharose 6 Fast Flow column
(1.2.times.1.1 cm) previously washed with 5-10 bed volumes of TST
Buffer. The column was then equilibrated with 2-3 bed volumes of
each: 1) 0.5 M Acetic acid, pH 3.4 (pH adjusted with
NH.sub.4CH.sub.3COOH(NH.sub.4Ac); 2) 150 mM NaCl, 50 mM Tris-Cl (pH
7.5), 0.05% Tween 20 (TST Buffer); 3) 0.5 M Acetic Acid, pH 3.4;
and 4) TST. The sample was applied to the column and the unbound
protein was removed by washing the column with: 1) 10 bed volumes
of TST, and 2) 2 bed volumes of 5 mM NH.sub.4Ac, pH 5.0. Elution
was performed with 0.5 M Acetic Acid, pH 3.4. The eluate was
collected in 1-2 ml fractions, and the fractions having the
greatest OD at 280 were dialyzed against 1 M NaCl, 50 mM sodium
carbonate (pH 11.0). The dialysate was clarified by centrifugation
at 39,000.times.g for 15 minutes, and applied to a 2-iminobiotin
agarose column (1.2.times.1.2 c.m) previously equilibrated with 1 M
NaCl, 50 mM sodium carbonate (pH 11.0). After the unbound proteins
were removed with the same solution, the bound proteins were eluted
with 6 M urea, 50 mM ammonium acetate (pH 4.0). The eluted proteins
were dialyzed against Tris-buffered saline [TBS; 150 mM NaCl, 20 mM
Tris-Cl (pH 7.5)] containing 0.02% NaN.sub.3, and the dialysate was
stored at 4.degree. C. after filtration through a 0.22 .mu.m filter
(Millex-GV, Millipore).
Formation of the ST-PAS/mAb/biotinylated .beta.-Galactosidase
complex:
[0047] A mixture of .about.2 .mu.g of antibody, .about.28 .mu.g of
fusion protein, and .about.2 units of biotinylated
.beta.-Galactosidase was incubated at room temperature for at least
10 minutes. After this incubation, the complex is ready to use.
.beta.-Galactosidase Staining: (X-GAL Staining)
[0048] .beta.-Galactosidase staining of cells that adhere to the
plate was performed according to Sanes, et al., 1986, EMBO J. 5:
3133-3142.
[0049] The protocol of Molecular Probes, Inc. was used to detect
lacZ .beta.-Galactosidase gene expression in cells that grow in
suspension.
[0050] The above protocols were used to determine if the complex
that contains the antibody coupled to the fusion protein and the
biotinylated .beta.-Galactosidase enzyme was successfully
transduced into the cell of choice. If the transduction was
complete, the cells were blue after overnight incubation.
2) Modification of Streptavidin Protein RYD Sequence.
[0051] Biotin-blocked streptavidin binds specifically
(Kd=3.times.10.sup.8M) to cell surfaces, presumably via an RYD
containing sequence that is distinct from the biotin-binding cleft
of the protein.
[0052] Alternation of the RYD domain (sequence) to other amino acid
residues is expected to eliminate the non-biotin related specific
surface binding of a large variety of cells.
[0053] One way to modify the RYD sequence would be to change the
RYD sequence to RYE. The change of RYD sequence to RYE can be
achieved by introducing a point mutation using sequential PCR
steps. This can be achieved by designing two primers, DES (SEQ ID
NO: 3) and DER (SEQ ID NO: 4) (see FIGS. 6A-C) and using the
pTSAPA-2 expression vector as a template of the streptavidin gene.
First, PCR is carried out using two pairs of primers: T7
promoter/DER and T7 terminator/DES. Second, the two amplified DNA
fragments are then purified and pooled into one sample. A second
round of PCR is then performed using the pooled purified products
of the first round as templates and the T7 promoter and T7
terminator primers. The mutated RYE sequence in the streptavidin
gene component is confirmed through sequence analysis of the
product of the second round of PCR.
3) Delivery of Biotin-.beta.-Galactosidase into A431 Cells.
[0054] In order to study the ability of ST-PA to deliver compounds
into a cell, the delivery of this complex was compared with that of
a complex in which core streptavidin was covalently linked to the
TGF.alpha. receptor.
[0055] pTSA-TGF.alpha., an expression vector for
streptavidin-TGF-.alpha. (ST-TGF) was constructed by replacing the
gene of protein A in pTSAPA-2 with a mature human TGF-.alpha. gene
(amino acids 1-50).
[0056] To demonstrate the capability of ST-PA and ST-TGF.alpha.
fusion proteins to deliver biotinylated protein into specific cells
types, biotinylated .beta.-galactosidase was complexed with the
streptavidin component of the fusion protein. Delivery of
.beta.-galactosidase into A431 cells was quantitated using known
staining techniques and FACS analysis.
[0057] ST-PA/biotin .beta.-galactosidase was complexed with
anti-EGFR mAb and the resulting complex was then incubated with
A431 human epidermoid cells over-expressing epidermal growth factor
receptor (EGFR). Alternatively, ST-TGF was mixed with
biotin-.beta.-galactosidase and the resulting complex was
administered to A431 cells.
[0058] As shown in FIG. 2B, ST-PA fusion protein efficiently
delivered biotin-.beta.-galactosidase into A43 1 cells through EGFR
on its surface (positive cells >99%) (see FIG. 2C). The
ST-TGF.alpha. fusion protein also displayed efficient delivery of
biotin-.beta.-galactosidase into A431 cells (positive cells
>99%). Surprisingly, the amount of biotin-.beta.-galactosidase
delivered into each cell by the ST-TGF.alpha. fusion protein was
lower than that observed in the ST-PA delivery system (the mean
fluorescence activity observed was 214 and 2402, respectively).
[0059] The highly efficient delivery by ST-PA fusion protein of
biotin-.beta.-galactosidase into A431 cells may be due to the four
binding sites for biotin and IgG contained on each ST-PA tetramer
(FIG. 1B). In a time course experiment shown in FIG. 3, more than
99% of cells demonstrated positive staining for
.beta.-galactosidase up to 2 days after transfer of
biotin-.beta.-galactosidase into the cell.
4) Delivery of Biotin .beta.-Galactosidase into Cells Using mAbs of
Different Specificity.
[0060] The experimental procedure applied in studying the delivery
of biotin .beta.-galactosidase into A431 cells was also used to
study the delivery of the antibody/ST-PA/biotinylated
.beta.-galactosidase complex into other cell types. The study
utilized antibodies that recognize several different cell surface
molecules. The cell lines and cell surface molecules used in this
experiment are summarized in FIG. 4A. As shown in FIGS. 4B-G, the
ST-PA/mAb complex was highly efficient (positive cells >99%) in
transferring .beta.-galactosidase into the human cell types tested
by way of the HLA-DR, CD33 and CD34 molecules present on the
surface of these cells.
5) pAT-.beta.-Galactosidase Expression Vector.
[0061] Although biotin can be incorporated into DNA (e.g.
expression plasmids), random incorporation of biotin into DNA may
result in a loss of transcriptional activity. In order to retain
the transcriptional activity of the DNA to be introduced into the
cell, a new gene transfer system was developed which utilizes a
pAT-expression vector having a poly(dA)/poly(dT) tract downstream
of the expression cassette (FIG. 5A). According to this transfer
system, the DNA to be delivered into the cell is cloned into the
expression vector. The pAT-expression vector forms triplex DNA with
a biotinylated poly(dT) oligonucleotide (SEQ ID NOS: 5 and 6) which
binds to the biotin binding site of the ST-PA fusion protein (FIGS.
5B-C). The antibody bound to the antibody binding site of the ST-PA
fusion protein targets the cells into which the DNA is to be
delivered.
[0062] The protocol for triplex DNA formation and DNA transfer of
the .beta.-galactosidase gene into cells is as follows. The
.beta.-galactosidase gene is cloned into the expression cassette of
the pAT-expression vector (FIG. 5A). The mixture of 4 .mu.g of
pAT-.beta.-galactosidase expression vector and 20 pmol of
Biotinylated poly(dT) oligonucleotide (Promega) in TMN buffer (10
mM Tris, pH 8.0, 1 mM MgCl.sub.2, 50 mMNaC1), is incubated at
37.degree. C. for 1 hour. ST-PA fusion protein (0.5 .mu.g) and mAB
(1.0 .mu.g) are then added to the mixture and the resulting
admixture is incubated at room temperature for 30 minutes. The
triplex DNA-ST-PA-mAb complex is added to the cells
(1.times.10.sup.6) and incubated at 37.degree. C. for 48 hours.
.beta.-galactosidase activity is detected by FACS.
REFERENCES
[0063] Sano, T. and Cantor, C. R. 1991. A streptavidin-protein A
chimera that allows one-step production of a variety of specific
antibody conjugates. Bio/Technol. 9:1378-1381. [0064] Pastan, I.
and FitzGerald, D. 1991. Recombinant toxins for cancer treatment.
Science. 254:1173-1177. [0065] Goshom, S. C., Svensson, H. P.,
Kerr, D. E., Somerville, J. E., Senter, P. D. and Fell, H. P. 1993.
Genetic construction, expression, and characterization of a single
chain anticarcinoma antibody fused to b-lactamase. Cancer Res.
53:2123-2127. [0066] Siegall, C. B., Xu, Y. -H., Chaudhary, V. K.,
Adhya, S., FitzGerald, D. and Pastan, I. 1989. Cytotoxic activities
of a fusion protein comprised of TGF.alpha. and pseudomonas
exotoxin. FASEB J. 3:2647-2652. [0067] Ghetie, M. -A., Laky, M.,
Morara, T. and Ghetie, V. 1986. Protein A vectorized toxins-I.
Preparation and properties of protein A-Ricin toxin conjugates.
Mol. Immunol. 23:1373-1379. [0068] Kiyama, R., Nishikawa, N. and
Oishi, M. 1994. Enrichment of human DNAs that flank poly(dA).
poly(dT) tract by triplex DNA formation. J. Mol. Biol. 237:193-200.
[0069] Ito, T., Smith, C. L. and Cantor, C. R. 1992.
Sequence-specific DNA purification by triplex affinity capture.
Proc. Natl. Acad. Sci. USA 89:495-498. [0070] Alon R., E. Bayer, M.
Wilchek, 1993, Cell Adhesion to streptavidin via RGD-dependent
integrins, European Journal of Cell Biology 60: 1-11.
Sequence CWU 1
1
6120PRTHomo sapiens 1Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg
Gly Asp Ser Pro Ala1 5 10 15Ser Ser Lys Pro20221PRTStreptomyces
2Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro1 5
10 15Ala Thr Asp Gly Ser20318DNAArtificial Sequenceoligonucleotide
primer DES designed to modify the RYD sequence of the streptavidin
gene 3cgttacgaaa gcgccccg 18415DNAArtificial
Sequenceoligonucleotide primer DER designed to modify the RYD
sequence of the streptavidin gene 4gctttcgtaa cgggt
15547DNAArtificial Sequencebiotinylated poly(dT) oligonucleotide
5ctagatctaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aactgca
47639DNAArtificial Sequencebiotinylated poly(dT) oligonucleotide
6gttttttttt tttttttttt tttttttttt tttttagat 39
* * * * *