Note: Descriptions are shown in the official language in which they were submitted.
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Targeted Therapeutic or Diagnostic Agents
and Methods of ~ and Using Same
This invention was made with government support under Grants RO1 HL52475,
5 PO1 HL3 1950, R01 CA56483 and R01 AR42750 awarded by the National Tn~titutçs of
Health. The govellu,lc.,l has certain rights in the invention.
BACKGROUN~ OF TEIE INVI~NTION
Field of the Invention
The present invention relates to the field of targeting therapeutic or diagnostic
entities to a site in a subject. Thus the present invention relates to targeted delivery of
therapeutic agents, such as proteins and nucleic acids, among others, and to targeting
15 diagnostic agents to useful sites in a subject
Background Art
Development of the ability to create novel protein-protein interactions promisesto provide important new therapeutic agents, novel strategies to target existing20 therapeutic or diagnostic agents to specific sites, and unique tools and reagents for the
study of key biological processes. Such advances in protein engineering may alsoprovide seminal information about how proteins interact. One strategy for manipulating
protein/protein interactions is to employ random or nearly random (e.g., alaninesc~2nning) (1) site-directed mutagenesis to identify amino acid residues critical for
25 binding affinity and specificity. Cumbersome, large scale mllt~g~nesi.c efforts followed
by laborious, time con~l2mi2lg assays of individual mllt~ted proteins can somçtim~s
extend this approach to create new molecular interactions. For exarnple, every residue
in human growth hormone was scanned for activity prior tO re-engine~ring the molecule
to bind the prolactin receptor (2), and a similar strategy f~'ilit~ted the construction of a
30 variant of IL-3 that binds the monomeric a receptor with higher affinity than it binds the
IL-3 a,B receptor (3). An important current challenge, therefore, is to create more rapid
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and effiriPns strategies tO ~.~ne~or proL~ .s with new binding prop~,~,es. A recent
a~Jpl.~acl~ which can obviate the need to ~hru,lll large numbers of binding assays with
individual ml~trted plotLins is bio~n.-ing using phage-display systems ,~ltholl~h
on~ynolly conceived as a means of scrcLn,ng vast llul~be,~ of peptide motifs (4), it is
5 now aypal ~ll that whole pl )leins and domains within p~ ot~s can be maniruhted using
the phage s~Piection strategy (5). Phage display itself, ho..~ ,., is also often sub~ect to
severe l;.~ c that prevent it from being a completely general ap~n~ q-~, many
pro~s, for ~ k., ca mot be d;~ .,d on the surface of phage in a biologically
active form, and significant pr~ mc with 1)l oteolysis of proteins displayed on phage are
10 also frequently .~ ..cnccd.
Another strategy that has been used to modify protein/protein interactions is the
g~i~iition, t~Pletion, or s~bstit~SiQn of entire dorrqinc within proteins (6-12). Although it
is often s~Ccec~ this strategy provides an e,.~ llely low resQI Ition picture ofprotein/plot~ ,. cLions; cQnce~uPntly~ manipulation of entire don~qin~ is often
15 followed by the construction and analysis of numerous point ~ nls as desclibed
above. Severe 1; .;~ c also arise if a protein domain of interest carries more than one
illl~,o~ l biol~gir-s-l activity; ...~ ing one activity (e.g., fi~nrsionslly significant
domain/domain illh.a.,lions) while altering another (e.g., high afflslity binding to a co-
factor or I ~c~lor) can be probl~mstir.
The present invention is ~ec;~ed to ove, colllc both of these limit~tiQns. ln the
present mPtho~ !o~rslly active ~I~u~;lur~s are i~l~tified in one or more proteinc that are well behaved in phage display studies and then gra~ed into other
p~Ol~S of interest. Such a system would e~ e the need to create new libraries for
25 each protein studied. SFerifir~lly~ the present invention provides a method to transfer
between plo~,.ns not entire do.. ; -~ but rather defined structural P~ ; within
protein ~ c, or mimetirs thereof. The present invention de . ol..l~es grafting
flexible protein loop ~ ,lu,es (13) which often guide protein binding ph~,.lonl~,na
Flexible loops are found on the surface of most protein mn~ es and exist as stretches of
30 4-20 amino acids that conn~ct regions of defined seconA~ y structure. ~ltho~lgh
crystallographic and N~ studies show that these loops are usually less well defined
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than helices and ~-sheets, their conforrnational freedom is normally restricted
substantially compared to free peptides. Consequently, the binding activities of surface
loops in proteins usually differ significantly from those of the corresponding linear amino
acid sequence (14).
S To substantially increase the speed and efficiency by which protein engineering
can be used to confer novel binding activities to a selected protein, the present method
adopts a strategy that combines phage display and loop gra~ing. In one embodiment of
the present invention, amino acids in a surface loop within the epidermal growth factor
(EGF) domain of tissue type pl~minogen activator were replaced with residues forming
10 one CDR of a monoclonal antibody that was directed against the adhesive integrin
receptor a~lb~3 and had been subjected to mutagenesis and "affinity maturation" or
"optimization" using a phage display system. The resulting variant of t-PA, LG-t-PA,
bound aIlb~3 with nanomolar affinity, possessed full activity towards both synthetic and
natural substrates, and was stim~ ted normally by the co-factor fibrin. Because of its
15 novel integrin-binding properties, this new variant of t-PA may display enhanced
thrombolytic potency toward the platelet rich thrombi that precipitate acute myocardial
infarction.
The resulting variant of t-PA, LG-t-PA, provides an improved thrombolytic
20 agent for the ll eal",cnt of acute myocardial infarction and other thromboembolic
disorders. Efforts to target plasminogen activators to blood clots, by conjugating the
enzyme to antibodies or Fab fragments of antibodies directed against either fibrin or
platelet integrins, have been previously reported and have often demonstrated that this
strategy can çnh~nce the thrombolytic potency of the plasminogen activator both in vitro
2~ and in vivo. For example, chemical conjugation of Mab 7E3 (anti-IIb-IrIa) to urokinase
yielded a molecule that was 25-fold more potent in Iysing platelet rich clots than wild
type urokinase (36). LG-t-PA, therefore, due to its novel binding properties, can be
used for enh~nced potency towards the platelet rich arterial thrombi which precipitate
acute myocardial infarction.
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SUMMARY OF THE INVENTION
The present invention provides a targeted therapeutic or diagnostic agent
comprising (a) a therapeutic or diagnostic functional entity linked to (b) an isolated
5 peptide mimetic that specifically binds a selected target. The invention additionally
provides a targeted therapeutic or diagnostic agent comprising (a) a therapeutic or
diagnostic functional entity linked to (b) an isolated, optimized, high-affinity polyamino
acid that specifically binds a selected target. The invention further provides a targeted
therapeutic or diagnostic agent comprising (a) a therapeutic or diagnostic functional
10 entity linked to (b) an isolated protein surface loop that specifically binds a selected
target, wherein the protein surface loop is not endogenous~ or native, to the functional
entity.
The present invention additionally provides a recombinant targeting protein
15 comprising (a) a surface loop from a first protein having a surface loop that specifically
binds the target and (b) a functionalmotif or domain of a second protein.
The present invention provides a recombinant targeting protein wherein the
surface loop is the HCDR3 of monoclonal antibody Fab-9, the second protein is human
20 tissue type plasminogen activator (t-PA), and the target is platelet glycoprotein
GPIlb/IIIa (integrin a~ 3)- The present invention thus provides a platelet-targeting
tissue plasminogen activator.
The present invention provides a method of reducing a blood clot in a subject
25 comprising admini~t~ring to the subject a therapeutic amount of a protein comprising (a)
a surface loop from the HCDR3 of monoclonal antibody Fab-9 and (b) a functionalmotif
or domain of human tissue type plasminogen activator (t-PA), thereby binding theprotein to platelet glycoprotein GPIIb/IIIa (integrin a, b~3) on a platelet in a blood clot in
the subject and reducing the blood clot in the subject.
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The present invention filrther provides a method of preventing thrombosis or
promoting thrombolysis in a subject comprising ~(lministçring to the subject a
therapeutic amount of a protein con~ ;sillg (a) a surface loop from the HCDR3 ofmonoclonal antibody Fab-9 and (b) a fi~nctionalmotif or domain of human tissue type
5 plasminogen activator (t-PA), thereby binding the protein to platelet glycoprotein
GP~Ib/IIIa (integrin anb~3) on a platelet in a blood clot in the subject and preventing
thrombolysis in the subject.
The present invention further provides a method of treating or preventing
10 myocardial infarction in a subject comprising administering to the subject a therapeutic
amount of a protein comprising (a) a surface loop from the HCDR3 of monoclonal
antibody Fab-9 and (b) a functionalmotif or domain of human tissue type plasminogen
activator (t-PA), thereby binding the protein to a platelet in the subject and treating or
preventing myocardial infarction in the subject.
The present invention additionally provides a method of targeting a therapeutic
compound to a tumor in a subject comprising administering to the subject a therapeutic
agent comprising a targeted therapeutic or diagnostic functional entity linked to heavy
chain complementarity determining region 3 (HCDR3) of monoclonal antibody Fab-9,20 wherein the therapeutic or diagnostic entity is an anti-tumor therapeutic compound.
The present invention also provides a method of targeting a therapeutic protein
to a tumor in a subject comprising ~mini~t~ring to the subject a recombinant targeting
protein comprising an anti-tumor therapeutic protein linked to the HCDR3 of
25 monoclonal antibody Fab-9.
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BR~EF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a I epresentaLion of the NMR structure of murine epiderrnal growth
factor adapted from Kohda et. al (28). Residue 23 of the murine protein corresponds to
residue 65 of human t-PA. The ~-turn formed by residues 23 -28 of the murine protein
may be Pxt~nded in t-PA due to the occurrence of a three amino acid insertion, 6'YFS69,
at the location indicated by the large arrow. The primary sequence of the substituted
region of LG-t-PA and the corresponding region of wild type t-PA are indicated at the
bottom of the figure. Dashes in the LG-t-PA sequence indicate identity to the wild type
enzyme.
Figure 2 shows standard indirect chromogenic assay of plasminogen activation by wild
type and LG-t-PA in the presence of buffer (Cl), fibrin monomers (0), fibrinogen (A), or
cyanogen bromide fragments of fibrinogen (o).
15 Figure 3 shows the binding of LG-t-PA to platelet integrin aIIb,B3 which was measured
using purified integrin as described in the Methods. Non-specific binding was
determined by parallel incubation with 10 rnM EDTA. The absolute amount of LG-t-PA bound was calculated based upon the specific activity of LG-t-PA.
20 Figure 4 demonstrates that RGD-con~ining peptide blocks the binding of LG-t-PA to
platelet integrin aIIb~3 A binding assay was performed as described in the examples.
The binding of LG-t-PA to integrin aIIb,B3 was challenged with synthetic peptides of
sequence GRGDSP (O) or SPGDRG (0).
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DETAILED DESCRIPTION OF TE~E INVENTION
The present invention provides a targeted therapeutic or diagnostic agent
comprising (a) a therapeutic or diagnostic fimctional entity linked to (b) an isolated
5 targeting motif or domain that specifically binds a selected target and that is derived
from or based on a protein or peptide binding region. Such targeting motifs or domains
can include peptide mimetics or surface loops of proteins. Such targeting motifs or
domains can include polyarnino acids derived from binding regions of protein which
binding regions have been isolated from the native protein and optimized to a higher
10 affinity than the original binding region in its native protein "Targeting motif or
domain" is not intended to limit the motif or domain to any particular length or size or
that it imply any particular secondary structure or anv secondary structure at all, unless
indicated.
For example, the present invention provides recombinant proteins wherein a
surface loop has been gra~ed into a functional protein such that the protein retains its
function(s) and has ~tt~ined targeting capability of the surface loop that the surface loop
imparted to its native protein. A specific example of this recombinant protein is an
altered tissue type plasminogen activator, LG-t-PA, wherein the epidermal growth20 factor (EGF) domain of tissue type plasminogen activator was replaced with residues
forming one CDR of a monoclonal antibody that was directed against the adhesive
integrin receptor allb,B3 and had been subjected to mutagenesis and "affinity maturation"
or "optimization" using a phage display system. The present invention further provides
compositions comprising such recombinant proteins and nucleic acids encoding these
25 recombinant proteins. Furtherrnore, the present invention provides methods of making
and using these recombinant proteins, including methods of treating thrombolyticdisorders and treating or preventing acute myocardial infarction.
A. General Considerations
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An inherent limitation of many therapeutic and diagnostic agents is an inability to
deliver these entities to specific sites, tissues, or pathological lesions within the body.
Consequently, advances that f~ tflte precise and selective delivery of these entities,
such as the present invention, promise to çnh~nce the efficacy of many therapeutic and
5 diagnostic regim~n~. Specific targeting is achieved in the current invention by obtaining
and exploiting detailed inforrnation regarding protein-protein interactions.
Protein-protein interactions can be guided by contacts between surface loops
within proteins. The present invention demonstrates that novel protein-protein
10 interactions can be created using a strategy of "loop grafting" in which the amino acid
sequence of a biologically active, flexible loop on one protein is used to replace a
surface loop present on an unrelated protein.
To demonstrate this strategy, a surface loop within an epidermal growth factor
15 module was replaced with the complementarity dete~ ing region (CDR) of a
monoclonal antibody. Specifically, the HCDR3 from Fab-9, an antibody selected to bind
the ~3-integrins with nanomolar affinity (Smith, J.W., Hu, D., Satterthwait, A., Pinz-
Sweeney, S., and Barbas, C.F. (1994), J. Biol. Chem. 269, 32788-32795), was grafted
into the EGF-like module of human tissue type plasminogen activator (t-PA). The
20 res~ ing variant of t-PA bound to the platelet integrin al~b,B3 with nanomolar affinity,
retained full enzymatic activity, and was stim~ ted normally by the physiological co-
factor fibrin. Binding of the novel variant of t-PA to integrin allb~3 was dependent on
the presence of divalent cations and was inhibited by an RGD-cont~ining peptide,demonstrating that, like the donor antibody, the novel t-PA binds specifically to the
25 ligand binding site of the integrin.
These findings demonstrate that surface loops within protein modules can be
interchangeable and that phage display can be combined with loop gra~ing to direct
proteins, at high affinity, to selected targets. These targets can include not only other
30 proteins but also peptides, nucleic acids, carbohydrates, lipids, or even uncharacterized
markers of specific cell types, tissues, or viruses.
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The present examples demonstrate the feasibility of a protein loop grafting
strategy. The resulting variant of t-PA, LG-t-PA, provides an improved thrombolytic
agent for the treatment of acute myocardial infarction and other thromboembolic
5 disorders. Previous efforts to target plasrninogen activators to bJood clots have often
demonstrated that such targeting can enhance the thrombolytic potency of the
plasminogen activator both in vitro and in vivo. LG-t-PA, therefore, due to its novel
binding properties, can also be utilized for enh~nced potency compositions to dissolve
the platelet rich arterial thrombi which precipitate acute myocardial infarction.
B. Definitions
As used in the specification and in the claims, "a" can mean one or more,
depending upon the context in which it is used.
An amino aGid residue is an amino acid formed upon chemical digestion
(hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described
herein are preferably in the ~'L" isomeric form. However, residues in the "D" isomeric
form can be substituted for any L-amino acid residue, as long as the desired functional
20 property is retained by the polypeptide. In keeping with the standard polypeptide
nomenclature (described in J. Biol. Chem., 243:3552-59 (1969) and adopted at 37 CFR
1.822(b) (2)), abbreviations for amino acid residues are shown in the following Table
I:
TABLE I
SYMBOL AMINO ACID
1 - Letter 3 - Letter
Y Tyr tyrosine
G Gly glycine
F Phe phenylalanine
M Met methionine
A Ala alanine
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S Ser serine
Ile isoleucine
L Leu leucine
T Thr ~hl eolune
V Val valine
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TABLE I (continued)
SYM~,OL AMINO ACID
I - Letter 3 - Letter
P Pro proline
K Lys Iysine
H His histidine
Q Gln ghlt~mine
E Glu glutamic acid
Z Glx Glu and/orGln
W Trp tryptophan
R Arg arginine
D Asp aspartic acid
N Asn asparagine
B Asx Asn and/orAsp
C Cys cysteine
X Xaa Unknown or other
All arnino acid residue sequences are represented herein by formulae whose left and
right orientation is in the conventional direction of amino-terminus to carboxy-terminus.
20 Furthermore, it should be noted that a dash at the beginning or end of an amino acid
residue sequence indicates a peptide bond to a further sequence of one or more amino
acid residues.
A "polypeptide" refers to a linear series of amino acid residues connected to one
25 another by peptide bonds between the alpha-amino group and carboxy group of
contiguous amino acid residues, and can be a protein.
A "peptide" refers to a linear series of less than or equaJ to about ~0 amino acid
residues, connected one to the other as in a polypeptide. A synthetic peptide is a
30 chemically produced chain of amino acid residues linked together by peptide bonds.
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12
A "polyarnino acid" refers to a linear series of amino acid residues connected one to
the other as in a polypeptide.
A protein refers to a linear series of greater than about 50 amino acid residues', connected one to the other as in a polypeptide.
A "peptide mimetic" is defined to include a chemical compound, or an organic
molecule, or any other peptide mimetic, the structure of which is based on or derived
from a binding region of a protein. For example, one can model predicted chernical
10 structures to rnirnic the structure of a binding region, such as a protein surface loop.
Such modeling can be performed using standard methods. Alternatively, peptide
rnimetics can also be selected from combinatorial chemical libraries in much the same
way that peptides are. (Ostresh, J.M. et al., Proc Natl Acad Sci U S A 1994 Nov
8;91(23):11138-42, Dorner, B. etal., BioorgMed Chem 1996 May;4(5):709-15;
15 Eichler, J. et al., Med ~es Rev 1995 Nov; 15(6):481 -96; Blondelle, S.E. et al. Biochem
1996 Jan 1;313 ( Pt 1):141-7; Perez-Paya, E. et al., JBi~)l Chem 1996 Feb
23 ;271(8):4120-6)
A "surface loop" is defined as a binding, i.e., targeting, element of a protein which
20 element is a flexible loop structure in the native protein of about 2 to about 20 amino
acids, that either connects regions of defined secondary structure in the native protein or
connects a dornain of secondary structure and a terminus of the native protein, and
which element is selective for binding to one or more binding sites. A surface loop, as
used in the claims, has retained one or more of its binding characteristics upon insertion
25 into a non-native functional protein in a manner such that it remains as a surface loop,
e.g., such that it replaces a removed surface loop in the non-native functional protein or
such that it is inserted either between two regions of defined secondary structure in the
non-native functional protein or between a domain of secondar,v structure and a
terminus in the non-native functional protein.
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A ~'therapeutic or diagnostic functional entity" is defined as any medical, diagnostic,
pharm~ceutic~l or biological entity whose delivery to a targeted site in a subject has
therapeutic benefit and/or diagnostic value and which entity can be linked to a peptide
mimetic or polyamino acid. Examples of therapeutic or diagnostic functional entities
S include a nucleic acid; a protein; a peptide; a gene delivery vehiGle (such as a plasmid, a
virus, a liposome complex); a synthetic or naturally occurring enzyme (such as aprotease, a phosphatase, a kinase, P450, or other drug metabolizing enzymes,
superoxide dismutases or nitric oxide synthase); a thrombolytic agent (such as tissue
plasminogen activator, uPA, vampire bat tPA, staphylokinase, streptokinase, an acylated
10 streptokinase-plasminogen complex, or variants of any of the preceding); an
anticoagulant (such as an inhibitor of the members of the blood coagulation cascade,
antagonists of integrin adhesion receptors, protein C or activated protein C, tissue factor
pathway inhibitor, or variants of blood coagulation enzymes like Factor VII, factor X or
thrombin, inhibitors of platelet function, inhibitors of Factor XIII, compounds which
15 modulate the activity of a protein involved in blood coagulation (for example, heparin),
or any variant of the above); a chemotherapeutic agent (such as doxyrubicin (or an
equivalent)); an apoptotic agent; a pharm~ceutical; a chemical compound; a growth
factor; a cytokine, other ligands for cell surface receptors; a carbohydrate, a lipid,
uncharacterized markers and targets, drug delivery systems (e.g., mi~i~turized osmotic
20 drug delivery pump), im~ine agents, such as radiochemicals, fluorescence chemicals~
metal ions that can be detected externally.
A target for binding of the therapeutic or diagnostic agent can be any region;
tissue; organ; cell; virus; organelle; microorganism; a synthetic or naturally occurring
2~ molecule or macromolecule (such as a peptide, a protein, a lipid, a carbohydrate, a
nucleic acid, as well as a modified variant thereof, such as a glycoprotein, a
phosphoprotein, a glycolipid, a hormone); a protein (such as an enzyme and its
inhibitors, a transcription factor, a kinase, a phosphatase, any protein found in the blood,
an adhesive protein, a component of the extr~ce~ r matrix, a receptor or other cell
30 surface protein, albumins, IgG-like molecules and antibodies, a growth factor, a
cytokine and modulators of angiogenesis); a cell surface protein (such as an integrins, a
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14
cadherin, a growth factor receptor, a proteoglycan, an ion channel, a member of the
seven-membrane spanner protein family (such as odorant and taste receptors, the
acetylcholine receptor)); a biological entity (such as a cell, a virus, a blood clot, a tumor,
an component of a pathologic lesion (such as an aneurism, an atherosclerotic plaque), or
5 a naturally occurring molecule or macromolecule). For example, a target can be integrin
~~llt.~3~ integrin av~3 or any other cell surface receptor.
A functional domain of a protein, which domain can be the therapeutic or diagnostic
functional entity in an agent of this invention, is a region o~ or an entire protein that has
10 an activity desired to be targeted to a target in the subject. In some agents one activity
has been isolated from its native protein or altered from its activity in its native protein.
Additionally, functional domains derived from or isolated from more than one source
can be linked together in an agent of this invention with a single or multiple targeting
element.
By "optimized" as used in the claims is meant that a polyamino acid has been
manipulated to increase its natural affinity for its target binding site. The term
"optimized" is used to convey that the affinity and possibly the selectivity of the
polyamino acid has been increased using a variety of standard approaches in the art.
20 Optimization can also be referred to as "affinity maturation."
By "linked" is meant joined in a manner such that the function of the functionaldomain remains active and such that the targeting capability of the targeting motif or
domain is available to target the agent. For example, when a surface loop is the25 targeting motif or domain and the functional entity is another protein, the surface loop
can be inserted into the protein in a manner consistent with surface loops, i.e., either
between two regions of defined secondary structure in the non-native functional protein
or between a domain of secondary structure and a terminus in the non-native functional
protein. Linkages can also include any type of chemical bond, or peptide bonds.
30 Linkages can include covalent and/or noncovalent bonds.
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A composition comprising any agent, recombinant protein or nucleic acid can
include a pharm~ce-ltically acceptable carrier, for example, or any other selected
addition that does not interfere with the desired function of the agent, protein or nucleic
acid.
By arimini~t~ring an agent, protein, nucleic acid or composition is meant
admini~tering the agent, protein, nucleic acid or composition in a manner suitable for
delivery to the selected target, as will be apparent to the artisan.
10 C. Therapeutic or diagnostic a~ents and compositions thereof
The present invention provides a targeted therapeutic or diagnostic agent comprising
(a) a therapeutic or diagnostic fi~nctional entity linked to (b) an isolated targeting motif
or domain that specifically binds a selected target and that is derived from or based on a
15 protein or peptide binding region. At least the following three categories of targeting
motifs or domains, derived from an entity other than the functional entity to which they
are to be linked to forrn the therapeutic or diagnostic agent, can be utilized in this
invention: (1) a peptide mimetic, i.e., a substance modeled on a peptide binding, (2) an
isolated, optimized, high affinity polyamino acid, derived from a binding site from any
20 selected protein, and (3) a surface loop isolated from a protein.
Specifically, the present invention provides a targeted therapeutic or diagnostic
agent comprising (a) a therapeutic or diagnostic functional entity linked to (b) an
isolated peptide mimetic that specifically binds a selected target. The invention
25 additionally provides a targeted therapeutic or diagnostic agent comprising (a) a
therapeutic or diagnostic functional entity linked to (b) an isolated, optimi7e~17 high-
affinity polyamino acid that specifically binds a selected target. The invention further
provides a targeted therapeutic or diagnostic agent comprising (a) a therapeutic or
diagnostic functional entity linked to (b) an isolated protein surface loop that specifically
30 binds a selected target, wherein the protein surface loop is not native/endogenous to the
functional entity.
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16
The therapeutic or filnctional entity is selected based upon the desired effect,whether therapeutic or diagnostic, and the targeting motif or domain is selected based
upon the site to be targeted. By "specifically binds" is meant that the targeting motif or
domain predominantly binds the target site, with minim~l non-specific binding to other
5 targets, i.e., with minim~l background. Specific binding further means that the targeting
motif or domàin can be used to selectively remove the target from a sample comprising
the target. Specific binding by an antibody CDR means that the antibody can be used to
selectively remove the factor from serum or inhibit the factor's biological activity and
can readily be determined by radio immune assay (RlA), bioassay, or enzyme-linked
10 irnmunosorbant (ELISA) technology. Many targeting elements are known and can be
utilized in the present invention. When the targeting element is a peptide mimetic, the
mimetic can be de~i~ned to specifically bind a specific site by modeling the mimetic on
the conformation of the original binding peptide. These mimetics can be further
optimized for higher affinity binding if desired, by any selected means.
When the targeting element is an isolated, optimi7e~ high affinity polyamino acid,
the element, several standard approaches are useful in optimizing the binding affinity
and/or binding selectivity of a polyamino acid. For example, vast libraries comprising
many variants of the polyamino acid can be displayed by several techniques that are well
20 known in the art. These include, but are not limited to: (1) phage display (for reviews
see Bradbury, A., Cattaneo, A (1995) The use of phage display in neurobiology. Trends
inNeuroscience 18:243-249; Barbas, C.F. (]993) Recent advances in phage display.Current Opinion in Biotechnology 4:526-530); (2) polysome display (Matthe~ki~,l ..C.,
Bhatt,R.R.,Dower,W.J. (1994) An in vitro polysome display system for identifying25 ligands from very large peptide libraries. Proc. Na~l. Acad. .Sci. U.S.A. 91, 9022-9026);
(3) displaying peptides on plasrnids (Cull, M.G. Miller, J.F., Schatz, P.J. (1992)
Screening for receptor ligands using large libraries of peptides linked to the C-terrninus
ofthe lac repressor. Proc. Natl. Acad. Sci. U.S.A. 89:1865-1869); (4) synthetic peptide
or peptidometic libraries (Pinilla C; Appel JR; Houghten RA. Investigation of
30 antigen-antibody interactions using a soluble~ non-support-bound synthetic decapeptide
library composed of four trillion (4 x 10(12) sequences. Biochem J 1994 Aug 1 ;301 ( Pt
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WO 97/39021 PCT/US96/20577
3):847-53; Dooley CT; Chung NN; Wilkes BC; Schiller PW, Bidlack JM; Pasternak
GW; Houghten RA An all D-arnino acid opioid peptide with central analgesic activity
from a combinatorial library. .Science 1994 Dec 23;266(5193):2019-22). The optimal
polyamino acids can then be obtained from this library by standard biochemical or
5 biological selection. Alternatively, (5) the isolated polyamino acid could be optimized
by chemical modification. For ~ ~al,~ple, its structure could be modified using standard
techniques such as combinatorial chernistry and medicinal chemistry.
When the targeting element is a surface loop, the surface loop is also specific for its
10 target. Surface loops can be selected for desirable targeting characteristics. Many
surface loop regions of proteins are already known from databases which contain the
three dimensional structures of proteins, and such surface loops can readily be used in
the present invention. The position of protein surface loops within proteins can also be
predicted using structural algorithrns. For example, A complementarity deterrnining
15 region of an antibody, monoclonal or polyclonal, directed against a target molecule of
interest can be used as a surface loop. Furthermore, any protein having a targeting
characteristic of interest can be structurally analyzed by standard means to determine
various regions cont~ining a surface loop and the surface loop thus identified. Such
structural analysis includes N~, crystallography or predictive algorithm.
20 Additionally, if desired, the affinity of a surface loop for its target can be increased, by
standard means.
Additional targeting motifs or domains, particularly for an agent to target a nucleic
acid target, are binding domains derived from transcription factors, such as a steroid
25 receptor; basal transcription factors (e.g. TFIID, etc.), and sequence specific DNA
binding transcription factors (e.g., APl, AP2, SPl, NF1, etc). Additional transcription
factors are listed in, for example, computer databases such as that m~int~ined by the
National Center for Biotechnology Information (NCBI, Bethesda, MD) accessible
through the BLAST program (see item 19 (TFD) for transcription factors; item 20 for
30 eukaryotic promoter sequences).
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WO 97139021 PCTIUS96120577
18
The target can include a tissue, a organ, a cell, protein, a peptide, a nucleic acid, a
carbohydratel a lipid, any component of a pathological lesion, or uncharacterized
markers and targets. For example, one can target cells of a tumor, me~ct~tic cells, cells
of the v~c~ h1re (such as endothelial cells and smooth muscle cells), cells of the lungs,
5 muscle (such as smooth muscle cells, cardiac muscle, etc.), cells ofthe kidneys, blood
cells (such as T-cells), cells of the bone marrow, such as stem cells, cells of bone,
neurons and related neurological cells such as glial cells, brain cells, liver cells, or
precursors of any selected cell, etc. by selecting a targeting motif or domain specific for
the tissue or cells, such as a targeting motif or domain that specifically binds a cell
10 surface receptor expressed on the cells of the organ or tissue. For example the target
receptor can be integrin like av~33, av~35, aIIb,B3. The target receptor can be a growth
factor receptor, a hormone receptor, a cytokine receptor, and the like. The target
receptor can be a growth factor-dependent receptor (e.g., epidermal growth factor,
nerve growth factor, etc.). The target receptor can also be a ligand-dependent receptor
15 (such as a steroid receptor, thyroid hormone receptor, retinoic acid receptor, retinoid X
receptor, TCCD (dioxin) receptor, fatty acid activatable receptors, and the like) or a
stimulus-dependent receptor (such as peroxisome proliferator-activated receptor).
Many ofthese receptors or factors can be found listed in the book [Parker, M.G. (1993)
Steroid Hormone Action (Oxford University Press, New York, pp. 210)], in a recent
20 review article [Tsai, M.J. & O'Malley, B.W. (1994) Anml. Rev. B70chem. 63, 451-486],
and in the GenBank database, which will contain additional receptors as well as the
complete nucleotide sequences of the genes and cDNAs. The target can also be a
soluble protein like an antibody, a growth factor, or a serum protein. Nucleic acid
targets can include, for example, a nucleic acid encoding a protein of interest, which
2~ e~pression is desired to alter by binding of a functional entity to the regulatory
sequences or to the RNA encoded by the nucleic acid. For example, one can increase or
inhibit expression of a nucleic acid, or one can alter the time of expression of the nucleic
acid.
30 The functional entity can be selected from a wide array of such entities, and is
selected based upon the target and the selected effect of the functional entity to be
CA 022410~1 1998-06-19
WO 97/39021 PCTIUS96/20!;77
19
delivered to the target. For example, one might wish to target to the region of a blood
clot an enzyme, like t-PA, that will act to dissolve blood clots. Similarly, proteins, and
their inhibitors, that act within the h~most~tic clotting cascade could be targeted to
clots. Similarly, drugs and proteins which act to modify platelet function could be
5 targeted to platelets within the clot, or to circ~ tin~ platelets which may Illtim~tely
become constituents of the clot.
The functional entity can be a nucleic acid, such as for gene therapy. Such nucleic
acids can encode, for example, a therapeutic protein or peptide, or it can encode an
10 ~ntisen.~e RNA. The functional entity can also include a gene delivery vehicle carrying a
gene of interest. For example, one might wish to target gene delivery vehicles to
endothelial cells, tumor cells or osteoclasts, (all of which express the~Yv~3 integrin). One
such gene delivery vehicle might be a virus, which could be but is not limited to, an
adenovirus, an adeno-associated virus, an ~tt~.n--~ted HIV virus and retroviruses, or
15 another gene delivery vehicle such as a liposome or a lipid-based delivery vehicle. The
types of genes which might be delivered to these sites could include genes that encode
tumor suppressors, molecules which act to promote or prevent cell growth and/or cell
migration. These genes could also include growth factors, cytokines and other various
ligands for cell surface receptors which would exist in the imm~ te vicinity of the
20 target.
The functional entity can further be, for example, drugs which act as poisons or
apoptotic agents to cells to specific cell types. For example, one can target
chemotherapeutic agents such as doxyrubicin (or an equivalent) to tumors with the
25 intent of focusing the activity of the chemotherapeutic agent at the site of the tumor.
Furthermore, one can also target drugs that act as poisons, or apoptotic agents to
endothelial cells such that these poisons would kill endothelial cells in tumors, thereby
~limin~ting the blood and nutrient supply to the tumor and killing it. Additionally, for
example, one can also target a cell in the immlme system, for example a cytotoxic T cell
30 or a natural killer cell, to tumors, such that the cytotoxic T cell would recognize, bind to
and kill the tumor cell by Iysing it
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In another example, bone marrow transplantation, one can also target stem cells
(functional entity) to the bone marrow (target) such that the stem cells would "home"
to the bone marrow and establish resi(l~nce, thereby increasing the "take" of the
transplantation. In another example, one can also target drugs which act as bone5 sparing agents to the bone in order to prevent osteoporosis. One such example could be
the bis-phosphonates, which act as poisons to the osteoclast, the major bone-resorbing
cell.
Another type of functional entity which can be targeted to a site by the present10 method is a medical or diagnostic device. Such a device can include a drug delivery
system, such as a rniniaturized osmotic drug delivery pump, an im~in~; agent, such as a
radiochemical, which enables the im~ging of tumors or disease lesions using standard
radiology techniques, an agent which can be targeted to tumors and other disease lesions
such that it is detected externally by nuclear magnetic resonance or by magnetic15 resonance im~ing (such as a spin-labeled probe, for example a metal ion like
m~ng~nese) .
The present invention further provides a recombinant targeting protein comprising
(a) a surface loop from a first protein having a surface loop that specifically binds the
20 target molcule and (b) a functional domain of a second protein. The second protein,
supplying the functional domain of a recombinant, targeting protein of this invention,
can be any selected protein that has a functional characteristic desired to be imparted to
the recombinant protein, as described further herein. Placement of the surface loop into
the second protein can be achieved by any of several standard methods, such as
25 recon~billalll subcloning techniques to isolate the surface loop from a nucleic acid
encoding the first protein having the surface loop and to insert the isolated nucleic acid
into a nucleic acid encoding a functional protein. The region of DNA encoding the
native surface loop of the functional protein can be removed prior to or during insertion
ofthe non-native surface loop also by standard subcloning techniques and the non-native
30 surface loop inserted. The recombinant DNA can then be expressed to produce the
recombinant protein. Alternatively, the removed native surface loop of the functional
CA 022410~1 1998-06-19
WO 97139021 PCT/US96/20577
protein can be m~-t~ted to create a nucleic acid fragment corresponding in sequence to a
~eting surface loop ofthe first protein having the surface loop and this mu~ted
nucleic acid reinserted into the functional protein. Such mutations can be generated by
standard techniques such as site-directed mutagenesis.
The present invention specifically provides a recombinant targeting protein wherein
the surface loop is a complem~nt~rity dele~ ng region (CDR) of a monoclonal
antibody directed against the target. Any desired monoclonal antibody can be utilized
for this protein. The antibody can be directed against a cell surface protein, for example.
10 The cell surface protein can be an integrin, such as platelet glycoprotein GPIIblIIIa
(integrin allb,B3) . Further more, the CDR can be further optimized for higher affinity, if
desired, such as by methods described herein. In a specific example herein the
monoclonal antibody Fab-9 has a CDR altered to contain a surface loop, and further, the
surface loop has been optimized by phage display (the surface loop in the HCDR3 of
1~ monoclonal antibodyFab-9.).
The present invention additionally provides a recombinant targeting protein wherein
the functional domain is human tissue type plasminogen activator (t-PA). A useful
targeting motif or domain for the activator is a motif or domain that binds a receptor on
20 a cell in or near a blood clot in an individual. For example, the target can be platelet
glycoprotein GPIIb/IIIa (integrin a"b~3) or av~3 on surrounding vascular endothelial and
smooth muscle cells, and thus the surface loop selected for the targeting motif or
domain is one that specifically binds the receptor(s). For example, the present invention
provides a recombinant molecule-targeting protein wherein the surface loop is the
2~ HCDR3 of monoclonal antibody Fab-9, the second protein is human tissue type
plasminogen activator (t-PA), and the molecule targeted is platelet glycoproteinGPIIb/IIIa (integrin a~ 3). Thus, the present invention provides a platelet-targeting
tissue plasminogen activator. This targeting protein is a clot-specific thrombolytic agent
and can be used in therapies where dissolution of a clot is desirable.
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WO 97/39021 PCTIUS9612O577
In isolating and, if desired, optimi7ing, a surface loop for use in a targeted entity of
the present invention, a nucleic acid encoding the region of a selected loop can be
removed from the nucleic acid its native protein and inserted into a generic scaffold,
such as a phage, polysome, or plasmid. The surface loop can be assayed for binding
5 affinity to a target. If desired, the surface loop can be optimi7ed, such as by mutagenesis
and affinity maturation using, e.g., phage display ( e.g, Pharmacia Biotech, Inc. 's
Recombinant Phage Antibody System), polysome display, displaying peptides on
p!~rnirls, and using synthetic peptide libraries. These c,p~ ed biologically active
motifs are then grafted, by standard methods, into several ~;lirrere.ll positions of the
10 recipient functional domain, e.g., ~functional domain of a protein, to create a novel
variant with altered binding properties. In particular, a selected surface loop can be
inserted into a protein carrying a functional domain between two regions of defined
secondary structure or between a domain of secondary structure and a terminus of the
protein. Thus, the methods described herein can be used to modify the surface loops of
15 a preselected protein binding (i.e., targeting) motif or domain to form a high-affinity
targeting module. Thereafter the targeting module can be fused with a preselected
protein's functional domain module to form the targeting protein.
The present invention provides a composition comprising any therapeutic or
20 diagnostic agent, recombinant protein or nucleic acid of this invention. Such a
composition can include a pharm~ceutic~lly acceptable carrier, for example,
physiological saline, or any other selected addition that does not interfere with the
desired function of the agent, protein or nucleic acid.
25 Nucleic acids and cells
The present invention provides an isolated nucleic acid that functionally encodes any
recombinant protein of tnis invention. By "isolated" as used herein is meant
subst~nti~lly free of, or separated from, other nucleic acids. Depending upon the
context, it can mean substantially free of, or separated from, other nucleic acids naturally
30 occurring in an organism cont~ining the nucleic acid. Nucleic acids can be made by
standard recombinant methods (see. eg., Sambrook et al., Molecular Cloning. A
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WO 97/39021 PCT/US96/20577
LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York, 1989; DNA cloning: A Prac~ical Approach, Volumes I and II, Glover, D.M.
ed., IRL Press Limited, Oxford, 1985) and/or other standard methods, such as chemical
synthesis. Nucleic acids can be produced in cells and isolated therefrom, if desired to be
5 isolated from a cell. Alternatively, a cell can be used to produce the reco,nbinalll protein
encoded by the nucleic acid. The nucleic acid can be in a plasmid, a virus, a phage, a
cosmid, a yeast artificial chromosome or any other vector of convenience.
To functionally encode a protein, i.e., allow the nucleic acid to be expressed, the
10 nucleic acid can include, for example, expression control sequences, such as an origin of
replication, a promoter, an enhancer, and necessary information processing sites, such as
ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional
terrninator sequences.
15 Additionally provided by the invention are nucleic acids that specifically hybridize to
the nucleic acids encoding the recolllbinallt proteins under sufficient stringency
conditions to selectively hybridize to the nucleic acid. Thus, nucleic acids for use, for
example, as primers and probes to detect or amplify the nucleic acids encoding the
recombinant proteins are contemplated herein. Typically, the stringency of
20 hybridization to achieve selective hybridization is about 5~(~ to 20~C below the Tm (the
melting tel,lpe~ re at which half of the molecules dissociate from its partner).Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA
hybridizations. The washing temperatures can similarly be used to achieve selective
stringency, as is known in the art. (Sambrook et al., Mole~ular Cloning: A Laboratory
25 Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods En~ymol. 1987: ] 54:367, 1987).
The present invention additionally provides a cell contAining any nucleic acid of this
invention. The cell can be generated by standard nucleic acid transfer methods, such as
30 transfection, electroporation, calcium phosphate-mediated transfer, direct injection and
the like. The cell can contain a vector contAinin~ the nucleic acid. A cell contAinin~ a
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WO 97/39021 PCT/US96/20577
24
nucleic acid encoding a chimeric protein typically can replicate the DNA and, further,
typically can express the encoded protein. The cell can be a prokaryotic cell,
particularly for the purpose of producing quantities of the nucleic acid, or a eukaryotic
cell, particularly a m~mm~ n cell. The cell is preferably a m~mm~ n cell for theS purpose of expressing the encoded protein so that the resultant produced protein has
".~.,....~li~n protein procçs.qing modifications.
Methods of use
10 The present invention provides a method of reducing a blood clot in a subjectcomprising administering to the subject a therapeutic amount of a protein comprising (a)
a surface loop from the HCDR3 of monoclonal antibody Fab-~ and (b) a functional
domain of human tissue type plasminogen activator (t-PA), thereby binding the protein
to platelet glycoprotein GPIIb/IIIa (integrin a"b~33) on a platelet in a blood clot in the
15 subject and re~h~c.ing the blood clot in the subject. The ~mini~tered protein can
function to (1) block integrin anb~3, (2) localize and concentrate the t-PA and its activity
to the clot by binding to the platelet and (3) localize t-PA to the clot by binding to av~3
expressed on vascular endothelial and smooth muscle cells in the vicinity of the clot.
For such a method, intravenous or intraanerial administration of the protein can be
20 particularly beneficial. Additional therapeutic proteins can be made that have a surface
loop that binds specifically to other entities within the cardiovascular system, such as
other components of blood and other components of blood clots and other surface
receptors on platelets, vascular endothelial cells.
25 Thus, the present invention further provides a method of preventing thrombosis or
promoting throm~olysis in a subject comprising adrninistering to the subject a
therapeutic amount of a protein comprising (a) a surface loop from the HCDR3 of
monoclonal antibody Fab-9 and (b) a functional domain of human tissue type
plasminogen activator (t-PA), thereby binding the protein to platelet glycoprotein
30 GPIIb/IIIa (integrin a~ 3) on a platelet in a blood clot in the subject and preventing
CA 022410~1 1998-06-19
WO 97139021 PCIIUS96/20577
thrombosis or promoting thrombolysis in the subject. For such a method, intravenous or
intraarterial administration of the protein can be particularly beneficial.
The present invention further provides a method of treating or preventing
5 myocardial infarction in a subject comprising a-lminict~ring to the subject a therapeutic
amount of a protein comprising (a) a surface loop from the HCDR3 of monoclonal
antibody Fab-9 and (b) a functional domain of human tissue type plasrninogen activator
(t-PA), thereby binding the protein to a platelet in the subject and treating or preventing
myocardial infarction in the subject. Additionally, the present invention provides a
10 method oftreating or preventing myocardial infarction in a subject comprising~,~mini.~tering to the subject a therapeutic amount of a targeted therapeutic agent of this
invention having as the therapeutic functional entity any anti-platelet agent or anti-
coagulant, linked to any targeting agent selective, or at least partially selective, for
constituents of blood clots (for example, proteins and peptide mimetics that bind the
15 a~ 3 integrin or any agent which binds the platelet). Administration is preferably
performed by methods typical for ~rlministering therapeutics for myocardial infarction,
and particularly by methods typical for ~minictering t-PA, such as intravenously and
intraarterially .
20 By "treating" a disease or condition is meant reclucing or preventing any ofthe
clinical n,al~,r~ ations of the disease or condition. For example, for treating myocardial
infarction, the ",anife~alions are known in the art and include chest pains, elevated
circul~tinf~ heart isozymes, alteration of an electrocardiogram, from occlusion of a
coronary artery, and others, as known to the skilled artisan One can monitor the effect
25 of a therapeutic agent of this invention on the disease or condition being treated by
detecting and/or, when possible, measuring any change in a clinical manifestation of the
disease or condition by the appropriate means for detecting such change, many of which
are known in the art.
30 The present invention additionally provides a method of targeting a therapeutic
compound to a tumor in a subject comprising administering to the subject a therapeutic
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26
agent comprising a targeted therapeutic or diagnostic functional entity linked to heavy
chain complem~ont~rity determining region 3 (HCDR3) of monoclonal antibody Fab-9,
wherein the therapeutic or diagnostic entity is an anti-tumor therapeutic compound.
Anti-tumor therapeutic compounds can include apoptotic agents or poisons, such as
5 chemotherapeutic agents, e.g., doxyrubicin; tumor suppressors, e.g, p53; and
modulators of nucleic acid metabolism. The therapeutic agent comprising a targeted
therapeutic or diagnostic functional entity linked to heavy chain complementarity
determining region 3 (HCDR3) of monoclonal antibody Fab-9 can bind av,B3, which is
present on several tumor cells, inclurling melanoma, breast cancer, ovarioan cancer.
10 Such a method can be used to reduce or halt tumor growth and/or establishment and
thus treat cancer.
The present invention also provides a method of targetlng a therapeutic protein to a
tumor in a subject comprising administering to the subject a recombinant tumor-
15 targeting protein comprising an anti-tumor therapeutic protein linked to the HCDR3 of
monoclonal antibody Fab-9. Such a method can be used to reduce or halt tumor growth
and/or establi~hm~nt and thus treat cancer.
The present invention also provides a method of targetlng a therapeutic compound20 to an osteoclast in a subject comprising a-lminictering to the subject a targeted
therapeutic agent comprising a therapeutic or diagnostic functional entity linked to
heavy chain complementarity determining region 3 (HCDR3 ) of monocJonal antibodyFab-9, wherein the therapeutic or diagnostic entity is an anti-osteoporosis therapeutic
compound. Examples of anti-osteoporosis therapeutic compounds include bis-
25 phosphonates.
The present invention further provides a method of targeting a therapeutic protein toan osteoclast in a subject comprising ~dministering to the subject, a recombinant
osteoclast-targeting protein comprising an anti-osteoporosis therapeutic protein linked
30 to the HCDR3 of monoclonal antibody Fab-9.
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WO 97/39021 PCTIUS96/20577
The present invention additionally provides a method of targeting a therapeutic
compound to an endothelial cell which is in the process of angiogenesis in a subject
comprising ~lmini~tering to the subject an endothelial cell- targeted agent comprising a
therapeutic compound linked to a targeting motif or domain, such as a peptide mimetic,
5 an opLi~ Ged high-affinity polyamino acid, or isolated protein surface loop, which
targeting motif or domain is selective for the aV~3 integrin and in which the therapeutic
compound is an anti-angiogenic factor or a cellular poison. For example, the targeting
motif or domain can be the HCDR3 of monoclonal antibody Fab-9 or any other CDR of
a monoclonal antibody that specifically binds ~~V~3 integrin.
The present invention further provides a method of targeting a therapeutic
compound to a tumor or tumor cell expressing av~3 integrin in a subject comprising
a~lmini~tering to the subject a tumor-targeted agent comprising a therapeutic compound
linked to a targeting motif or domain, such as a peptide mimetic, an optimized high-
15 affinity polyamino acid, or isolated protein surface loop, which targeting motif ordomain is selective for the av,B3 integrin and in which the therapeutic compound is a cell
with anti-tumor activity and which cell bears (or displays on its surface) the targeting
motif or domain. The targeting motif or domain could be, for example, a targeting motif
or domain grafted into the T cell receptor or any cell surface protein that is a member of
20 the IgG protein family, or any cell surface protein which contains an epiderrnal growth
factor-like module, or any cell surface protein that contains a fibronectin type III
module. For example, HCDR 3 of Fab-9 can be grafted into the T cell receptor. Using
a T cell expressing this loop-grafted T cell receptor, one can target a T cell to bind to a
tumor cell expressing ~V~33 integrin .
The present invention further provides a method of targeting a therapeutic
compound to a vascular smooth muscle cell (SMC) which is contributing to vascular
stenosis in a subject comprising administering to the subject a SMC-targeted agent
comprising a therapeutic compound linked to a targeting motif or domain, such as a
30 peptide rnimetic, an optimized high-affinity polyamino acid. or isolated protein surface
CA 022410~1 1998-06-19
WO 97/39021 PCT/US96/20577
28
loop, which la~gelh~g motif or domain is selective for the ~v~3 integrin and in which the
therapeutic compound is a modulator of cell growth, or a cellular poison.
For any method of this invention, the subject can be any animal, preferably a
5 m~mm~l, such as a human, a veterinary animal, such as a cat, dog, horse, pig, goat,
sheep, or cow, or a laboratory animal, such as a mouse, rat, rabbit, or guinea pig. The
therapeutic agent is selected accordin~,ly to oplilnl~e the treatment for the subject being
treated.
10 Diagnostic or therapeutic agents of the present invention can be arlmini~tered to a
subject or an animal model by any of many standard means for ~r~minictering
therapeutics or diagnostics to that selected site or standard for a~mini~tering that type of
functional entity. For example, an agent can be administered orally, parenterally (e.g.,
intravenously), by intramuscular injection, by intraperitoneal injection, topically,
15 transdermally, or the like. Agents can be administered, e.g., as a complex with cationic
liposomes, or encapsulated in anionic liposomes. Compositions can include various
amounts of the selected agent in combination with a pharmaceutically acceptable carrier
and, in addition, if desired, may include other medicinal agents, pharmaceutical agents,
carriers, adjuvants, diluents, etc. Parental ~rlmini~tration, if used, is generally
20 characterized by injection. Injectables can be prepared in conventional forms, either as
liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid
prior to injection, or as emulsions. Depending upon the mode of a-lmini.ctration, the
agent can be optimized to avoid degradation in the subject, such as by encapsulation,
etc.
Dosages will depend upon the mode of administration, the disease or condition to be
treated, and the individual subject's condition, but will be that dosage typical for and
used in a~lmini~tration ofthe functional domain. For example, for ~lmini~t~.ring a
protein comprising (a) a surface loop from the ~CDR3 of monoclonal antibody Fab-9
30 and (b) a functional domain of human tissue type plasminogen activator (t-PA) to reduce
a blood clot or to prevent thrombosis or promote thrombolysis or to treat or prevent
CA 022410~1 1998-06-19
WO 97t39021 PCT/US96/20577
29
myocardial infarction, the protein can be ~ministçred in a dosage typical for t-PA.
Such dosages are well -known in the art. Because of the efficient targeting of the
Iecolllbinalll protein ofthe present invention, this dosage can likely be reduced.
Furthermore, the dosage can be adjusted according to the typical dosage for the specific
disease or condition to be treated. Therefore, to reduce a blood clot, a typical dosage
will be similar to or Jess than that ~-~minictçred when t-PA is a-lmini.ctçred to reduce a
blood clot. To prevent thrombosis or prevent thrombolysis, a typical dosage will be
similar to or less than that a~minict~red when t-PA is a~mini~tered to prevent
thrombosis or promote thrombolysis. To treat or prevent myocardial infarction a
10 typical dosage will be similar to or less than that administered when t-PA is a-lmini.~tPred
to treat or prevent myocardial infarction. Additionally, the dosage of a plasmid or virus
can be that dosage typical for and used in a-lministration of other plasmid or viral
vectors (see e.g., U.S. Pat. No.4,897,355). Furthermore, culture cells or biopsy of the
target cell type can be used to optimize the dosage for the target cells in vivo. Often a
15 single dose can be sufficient; however, the dose can be repeated if desirable. The
dosage should not be so large as to cause adverse side e~ects. Generally, the dosage will
vary with the age, condition, sex and extent of the disease in the patient and can be
determined by one of skill in the art. The dosage can also be adjusted by the individual
physician in the event of any complication.
Statement Concernin~ Utility
The present invention provides a targeted therapeutic or diagnostic agent comprising
(a) a therapeutic or diagnostic functional entity linked to (b) an isolated targeting motif
25 or domain that specifically binds a selected target and that is derived from or based on a
protein or peptide binding region. Such agents can had widespread utility for therapy
and diagnosis. The present invention can be adapted to any known treatment that
includes ~flministering a substance that functions in the subject at a site that can be
targeted by this method, to improve the effectiveness of the treatm~nt.
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WO 97/39021 rCTlUS96120~;77
The present compositions and examples of use demonstrate specifically that surface
loops within protein modules can be interchangeable and that phage display can be
combined with loop grafting to direct proteins, at high affinity, to selected targets. A
major finding of this study was that arnino acids forming a biologically active, flexible
5 surface loop on one protein could be grafted into another, unrelated protein and still
m~int~in their initial binding activity. The protein sequence of the grafted loop was
originally optin~ized by mllt~n~ci~ and affinity maturation of the donor protein,
monoclonal antibody Fab-9, using phage display.
10 This invention differs significantly both in approach, and in end result, from previous
methods, for example, previously reported studies where RGD sequences have been
inserted into non-adhesive proteins (33-35) to target cells expressing receptors that bind
RGD. Although these efforts have successfully targeted recipient proteins to integrins,
substantial q~l~ntit~tion of binding affinities has not been reported for these proteins nor
15 has the integrin target been identified. In these studies, an RGD sequence was inserted
into either Iysozyme or calpastatin, and the apparent kD of the resulting, mutated protein
for integrin appealed, based on cell adhesion or cell spreading assays, to be
approximately 400 nM or 50 nM, respectively. In the present invention, a targeting
molecule against integrin that possesses 50-400 fold higher affinity, with a kD
20 approaching one nanomolar, is prepared. The present invention shows that grafting the
amino acid sequence of HCDR3 of the optimized antibody Into the extended loop of an
EGF module can transfer nanomolar affinity for integrin to the EGF domain. The high
affinity of LG-t-PA for integrin almost certainly depends on maintenance of important
biophysical properties of the loop; both linear and cyclic synthetic peptides cont~ining
25 the amino acid sequence of the Fab-9 HCDR3 exhibit approximately one-hundred fold
lower affinity for integrins than LG-t-PA or Fab-9.
The development of a general method to accomplish the ab initio design of a three
dimensional structure and underlying amino acid sequence of a peptide motif, or mimetic
30 thereof, that can be placed into a chosen recipient protein, or other agent, and will bind,
at high affinity, to a selected target molecule will be of great benefit. The inventive
CA 022410~1 1998-06-19
WO 97/39021 PCTIUS96/2OS77
31
method allows one to endow proteins with new, useful binding properties without resort
to very large scale mutagenesis protocols followed by laborious and time con~lming
screening of individual variants of the protein of interest.
The present invention is more particularly described in the following examples which
are int~n~led as illustrative only since numerous modifications and variations therein will
be apparel1t to those skilled in the art.
1 0 I~XAMPLES
Site directed mutagenesis and construction of expression vectors encodin,~ variants
of t-PA.
Oligonucleotide directed site specific mutagenesis was performed by the method of
1~ Zoller and Smith ~15) as modified by Kunkel (16). To simplify later manipulations, we
used as a template for mutagenesis a cDNA encoding t-PA (Pennica, D et al (1983)Nature vol. 310 pgs. 214-221; Sambrook et al (1986) Mol. Biol. & Medicine. vol 3,
pgs 459-481 ) that contained a silent mutation at nucleotide 407 which created a new
Kpn I site (17). The 437 bp Hind III - Kpn I bp fragment of this cDNA was subcloned
20 into bacteriophage M13mp] 8 and site directed mutagenesis was performed using the
mutagenic primer 5'-
CGGGGGCACCTGCTCATTCGGAAGGGGAGACATTAGGAATGTGTGCCAGTG
CC - 3' (SEQ ID NO: 1).
2~ Following mutagenesis, ssDNA corresponding to the entire 437 bp Hind III - Kpn I
fragment was fully sequenced to assure the presence of the desired mutation and the
absence of any additional mutations. Replicative form (RF) DNA was prepared for
appropriate phage, and the ml1t~ted 437 bp fragment was recovered after digestion of
RF DNA with Hind II1 and Kpn I and electrophoresis of the digestion products on an
30 agarose gel. The isolated Hind III - Kpn I fragment was used to reconstruct a full length
CA 022410~1 1998-06-l9
WO 97/39021 PCT/US96120577
cDNA encoding LG-t-PA. The surface loop generated in HCDR 3 of Fab-g was
separately grafted into the T cell receptor.
Expression of enzymes by transient transfection of Cos cells.
5 cDNAs encoding t-PA and LG-t-PA were ligated into the transient expression
vector pSVT7 (18) and then introduced into Cos 1 cells by electroporation using a Bio
Rad Gene Pulser. 20 llg of cDNA, 100 ',lg of carrier DNA and approximately 10' Cos
cells were placed into a 0.4 cm cuvette, and electroporation was performed at 320 V,
960 ~FD, and Q - ~. Following electroporation, cells were incubated overnight at10 37~C in DMEM cont~ining 10% fetal calfserum and 5 rnM sodium butyrate. Cells
were then washed with serum free medium and incubated in DM:EM for 48 hours at 3 7~
C. A~ter the incubation with serum free media, conditioned media were coilected and
enzyme concentrations were determined by ELISA.
15 Purification and O~l~ntit~tion of Enzymes.
Conditioned media were dialyzed against 20 mM sodium phosphate (pH = 7.0), 100
rnM NaCl~ 20 mM arginine, and 0.05% Tween 80 and loaded onto a Iysine-Sepharose
(Pharmacia) colurnn. The column was washed with 20 volumes of loading buffer, and t-
PA was eluted with buffer cont~ining 20 mM sodium phosphate (pH = 7.0), 100 mM
20 NaCI, 200 mM arginine, and 0.05% Tween 80. Enzyme concentrations were measured
by ELISA.
Kinetic Analysis of Plasmino~en Activation using Indirect Chromogenic Assays.
Indirect chromogenic assays of t-PA utilized the substrates Iys-plasminogen (American
25 Diagnostica) and Spectrozyme PL (Arnerican Diagnostica) and were performed aspreviously described ( 18-20). Assays were performed in the presence of the co-factor
DESAFIB (American Diagnostica). DESAFIB, a prepa~ ~lion of soluble fibrin
monomers, was produced by digesting highly purified human fibrinogen with the
protease batroxobin. Batroxobin cleaved the Arg 16 - Gly 17 bond in the Aa-chain of
30 fibrinogen and consequently released fibrinopeptide A. The resulting des-AA-fibrinogen
CA 022410~1 1998-06-19
WO 97/39021 PCT/US96/20577
or fibrin I monomers are soluble in the presence of the peptide Gly - Pro - Arg - Pro.
The concentration of Iys-plasminogen was varied from 0.0125 - 0.2 ~IM.
Indirect Chromo~enic Assays in the Presence of Various Stimulators.
Standard indirect chromogenic assays were performed as previously described (18-20). Briefly, 0.25 - 1 ng of enzyme, 0.2 ~lM Iys-plasminogen and 0.62 rnM Spectrozyme
PL were present in a total volume of 100 ~l. Assays were perforrned either in the
10 presence of buffer, 25 ~Ig/ml DESAFIB, 100 llg/ml cyanogen brornide fragments of
fibrinogen (American Diagnostica) or 100 ~lg/ml fibrinogen Assays were performed in
rnicrotiter plates, and the optical density at 405 nm was read every 30 seconds for one
hour in a Molecular Devices Thermomax. Reactions were performed at 37~C.
15 Direct Chromo~enic Assays of t-PA activity usin~ activated substrates.
Direct assays of t-PA activity utilized the substrate Spec t-PA (American
Diagnostica) and were performed as previously described (21) except that 0.02% Tween
80 was included in the assay.
20 Integrin Purification and Inte~rin Binding Measurements
Integrin al~b~3 was purified from human platelets on RGD-peptide affinity columns
as previously described (22, 23). Integrin av~3 was purified using antibody affinity
chromatography with LM-609 conjugated to Sepharose as described (24). Both
integrins were greater than 90% pure as judged by Coomassie blue staining of
25 acrylamide gels. Binding of the novel t-PA to each integrin was measured with an assay
adapted from the ligand receptor binding assays previously reported (~5, 26). Purified
integrin was immobilized in Titertek 96-well plates in 20 mM Tris-HCl, pH 7.4, 150 mM
NaCl cont~inin~ 1 mM CaC12 and 1 mM MgC12. For coating, the integrin was diluted to
a concentration of S llg/ml. Integrin was allowed to coat plastic plates for l 8 hours at
30 4~C. Following coating, the non-specific protein binding sites on the plate were blocked
by incubation with 50 mM Tris-HCI, pH 7.4, 100 rnM NaCI Cont~ining 20 mg/ml of
CA 022410~1 1998-06-19
WO 97139021 PCT/US96/20577
34
BSA. For binding measurements, conditioned media cont~ining wild type or modified t-
PA was diluted to the appropriate enzyme concentrations in binding buffer (50 rnM Tris-
HCI, pH 7.4, 100 mM NaCI, 0.5 mM CaCI2 cont~ining I mg/ml of BSA). The t-PA
was applied to integrin immobilized in rnicrotiter wells and allowed to bind for 2 hours
5 at 3 ? ~ C . After binding, free t-PA was removed from the plate by three rapid washes
with binding buffer and bound t-PA was detected using an activity assay for t-PA as
previously described (21). Non-specific binding of t-PA was determined by the addition
of 5 mM EDTA to chelate divalent cations that are required for integrin ligand binding
function (23). In some cases the peptide GRGDSP (synthesized by Coast Scientific)
10 was used as a competitor. This indirect assay was used for measuring t-PA binding to
integrin because of technical limitations encountered using purified t-PA. Following
purification, t-PA is m~int~ined in solution with 200 mM arginine. We found that this
concentration of arginine interfered with all of the integrin binding assays and precluded
attempts to use direct methods of measuring binding affinity. Thus, the indirect binding
15 assay was chosen. This type of measurement is expected to give an underestimate of
the binding of loop-grafted t-PA to integrins because COS cells secrete other integrin
binding proteins that compete with LG-t-PA for binding to integrin.
The Use of an Antibody CDR as a Donor and an EGF Module as a Recipient in Loop
20 Graftin~
The objective of this study was to test the concept that biologically active surface loops,
at least in some cases, can be grafted between proteins of dirrel ent backbone structure.
As our donor we chose a CDR from Fab-9, a human antibody that we recently
engineered to bind the ligand binding pocket of the ~3-integrins (27). The HCDR3 loop
25 from this antibody contains the active sequence SFGRGDIRN and is bounded by two
cysteine residues. While Fab-9 binds to ,~3-integrins with nanomolar affinity, synthetic
peptides with the HCDR3 sequence of Fab-9 display at least one hundred fo}d-lower
affinity for integrin ( ~ 4). The criteria for a successfui grafting of the loop, therefore, is
the retention of high affinity for integrin. An EGF module was chosen as a recipient
30 because the structure of several proteins cont~ining this module are available and show
that EGF contains a large, exposed loop that contains a ,B-turn structure. This loop is
CA 022410~1 1998-06-19
WO 97139021 PCT/US96/20577
bounded by two disulfide pairs (28). Using oligonucleotide directed site specific
mutagenesis and conventional cloning techniques, we constructed a cDNA encoding a
variant of t-PA in which residues forming the exposed loop on the surface of the EGF
domain have been replaced by amino acids found in the active loop of Fab-9 (Fig. 1).
5 The Arg of the RGD motif was placed at residue 66 of t-PA, which, based upon NMR
structures of EGF modules (28), is near the apex of this disulfide bounded loop.Because the CDR se~uence of Fab-9 is one residue shorter than the EGF loop, we
m~int~ined the carboxy-terminal residue within the EGF loop as Val, the native residue
at this position in t-PA The new variant of t-PA is referred to as loop-grafted t-PA, or
10 LG-t-PA.
The Modified t-PA ~l~int~in~ Full Enzymatic Activity and is Stim~ te~ by the
Physiolo,~ical Co-factor Fibrin.
A kinetic analysis of the activity of wild type and LG-t-l'A toward the small
15 chromogenic substrate Spec t-PA is summarized in Table IIa The close correspondence
between values of Km, 4at, and kcatJKm for the two enzymes in this assay clearlydemonstrates that LG-t-PA m~int~inc full activity towards synthetic substrates.
Table Ila. Kinetic constants for cleava~e of the chromo~enlc substance Spec t-PA.
20 Enzyme kcat (s ) K,l, (mM) kC~ " (M-lS-')
t-PA 44 0.3 I.~x 105
LG-t-PA 45 0.3 1 5 x lOs
25 Table IIb presents the results of a kinetic assay of plasminogen activation, in the
presence of the co-factor fibrin, by t-PA and LG-t-PA. The kinetic constants of LG-t-
PA for pl~cminogen activation are very similar to those of wild type t-PA; kcatlKm
values for the two enzymes vary by approximately 10% in this assay. LG-t-PA,
therefore, m~int~ined full enzymatic activity not only toward small synthetic substrates
30 but also towards the natural protein substrate plasminogen
CA 022410~1 1998-06-19
WO 97/39021 PCT/US96t20!;77
36
Table IIb. Activation of plasminogen in the presence of fibrin.
Enzyme kCat (s~ M) kCa,/KI,~ (M-ls-l)
t-PA 0.10 0.02 ~.Ox lo6
LG-t-PA 0.09 0.02 4.5 x 10~
The activity of wild type t-PA is stim~ ted by fibrin, fibrinogen, and cyanogen
bromide fragments of fibrinogen, and we and others have reported that mutations
mapping to at least five distinct regions of the enzyme can di~re,llially affect10 stimlll~tion oft-PA by these distinct co-factors (29, 30). To exarnine whether mutations
present in LG-t-PA affected stiml~l~tion of the enzyme by any of these three co-factors,
we performed standard indirect chromogenic assays of the two enzymes in the presence
of each of the co-factors. The results of these assays are depicted in Figure 2 and
indicate that the mutations present in LG-t-PA have not significantly compromised
1~ interaction of the enzyme with any of the co-factors.
The role, if any, of the EGF domain of t-PA in me~ ting stimulation of the enzyme
by fibrin has generated controversy and conflicting reports (9, 12, 31). Although this
study will not resolve the issue, our results do argue strongly that residues 63 - 71,
which form part of an antiparallel ~-sheet and a ,B-turn on surface of the EGF domain,
20 do not play a role in stim~ tion of the enzyme by fibrin or fibrinogen.
LG-t~PA Binds to ,B3-Inte~rins with Nanomolar Affinity
Two integrins containing the ~3 subunit have been described. These are platelet integrin
aIIb,B3 and integrin ocv,B3 . Both of these integrins bind the antibody Fab-9 with
25 nanomolar affinity (14, 27). The ability ofthe modified t-PA, LG-t-PA, to bind the two
,B3-integrins was tested using purified integrin and culture supernatant from cells
transfected with the cDNA encoding LG-t-PA An indirect binding assay was used tomeasure binding affinity of the LG-t-PA for integrins because purified t-PA cannot be
m~int~ined at high concentrations in buffers that are compatible with integrin binding
30 assays (unpublished observation). Consequently, t-PA binding to integrin was measured
by deterrnining the amount of t-PA present in conditioned media from transfected COS
CA 022410~1 1998-06-l9
WO 97/39021 PCTIUS96120577
cells and using this media as a source of t-PA. t-PA that bound to purified integrin was
reported as t-PA activity and was measured using a standard indirect chromogenicassay. The binding isotherm shown in Fig. 3 shows that the LG-t-PA binds purified
integrin a~b,~3. Non-specific binding was assessed by in~ ling EDTA in the binding
5 study because ligand binding to integrins is dependent upon divalent cations (23, 32).
The binding of LG-t-PA to allb,~3 is specific and saturable, exhibiting a K" of
applo~imately 0.9 nM. This kD is the average of four similar expe,in.enls in which the
dissociation consL~Ill ranged from 0. 5 to 1. 3 nM. In the experiment shown in Figure 3,
the apparenl kD is 0. 5 nM. The average kl, for this interaction compares favorably with
10 the kD of Fab-9 for this integrin of 5 nM ( 14). Like the protein containing the parent
loop, Fab-9, the modified t-PA also bound to integrin av~Q3 The ~ffinity ofthe
modified t-PA for this integrin was also high, with an apparent kD of 1.8 nM, similar to
the kD of 1.7 nM exhibited by Fab-9. Thus, by contrast to linear and cyclic synthetic
peptides with sequences that are identical to the HCDR3 of Fab-9, LG-t-PA m~ins~ined
15 the high affinity for integrins exhibited by Fab-9.
Loop-grafted t-PA Binds to the Ligand Binding Site of ~3-lntegrins
One of the hallmarks of ligand binding to the ,B3-integrins is that small synthetic
peptides with the RGD sequence can block their binding. The ability of RGD peptides
20 to block LG-t-PA was tested by a simple competition assay. A concentration range of
RGD peptide was included during the binding reaction with the modified t-PA. As a
control, a synthetic peptide with the same composition, but random sequence was also
used as competitor. Bound t-PA was detected with the indirect activity assay described
in Methods. As shown in Fig. 4, the peptide with sequence GRGDSP blocked the
25 binding of LG-t-PA to integrin, but a peptide with random sequence had no effect on
binding. The data shown are for integrin allb~3. and nearly identical data were obtained
when RGD peptide was used to block binding of LG-t-PA to purified integrin C(V~B3
These data show that, like Fab-9, LG-t-PA binds to the ligand binding site of the ,~3-
lntegrlns.
CA 02241051 1998-06-19
wo 97/39021 Pc~rtuss6/20577
Throughout this application, various publications are referenced. The disclosures of
these publications in their entireties are hereby incolporated by reference into this
application in order to more fully describe the state of the art to which this invention
pertains.
s
Although the present process has been described with reference to specific details of
certain embo~lim~ntc thereof, it is not intended that such details should be regarded as
limitations upon the scope of the invention except as and to the extent that they are
included in the accompanying claims.
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CA 02241051 1998-06-19
W O 97139021 PcTrusg6no577
SEQUENCE LISTING
(l) GENERAL INFORMATION:
(i) APPLICANT: Madison, Edwln L.
Smith, Jeffrey W.
~ii) TITLE OF INVENTION: TARGETED THERAPEUTIC OR DIAGNOSTIC
AGENTS AND METHODS OF MAKING AND USING SAME
) NUMBER OF SEQUENCES: 1
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: NEEDLE ~ ROSENBERG, P.C.
(B) STREET: 127 Peachtree St., NE S~ite 1200
(C) CITY: Atlanta
(D) STATE: GA
(E~ COUNTRY- USA
(F) ZIP: 30303
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy dlsk
(B~ COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.0, Jerslon #1.30
(Vl) CURRENT APPLICATION DATA:
(A~ APPLICATION NUM~ER:
(B) FILING DATE: December l9, 1996
(C) CLASSIFICATION:
(Vlii ) ATTORNEY/AGENT INFORMATION:
(A) NAME: Selby, Elizabeth
(B) REGISTRATION NUMBER: 38,298
(C) REFERENCE/DOCKET NUMBER: l9l9l.000l
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 40~ 688 0770
(B) TELEFAX: 904 688 9880
CA 02241051 1998-06-19
WO 97/39021 PCTrUS96/20577
42
(2) INFORMATION FOR SEQ ID NO:l:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 ~ase palrs
(B) TYPE: nucle-c acld
(C) STRANDEDNESS: double
(D) TOPOLOGY: llnear
(il) MOLECULE TYPE: other nuclelc acid
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CGGGGGCACC TGCTCATTCG GAAGGGGAGA CATTAGGAAT GTGTGCCAGT GCC 53
