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Description  |
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TECHNICAL FIELD
The present invention relates to covalently-linked complexes (CLC) having
enhanced diagnostic or therapeutic properties and methods of using these
complexes. The CLC of the present invention has three components: (1) a
targeting protein or peptide; (2) a cytotoxic agent, such as a
radioisotope, a drug or a toxin; and (3) one or more enhancing moieties
capable of promoting CLC-target cell membrane interaction.
BACKGROUND OF THE INVENTION
Immunoconjugates consisting of antibody joined to a cytotoxic agent have
been used in attempts to achieve selective killing of particular target
cells, such as tumor cells. In theory, immunoconjugates or targeting
protein conjugates should effect specific cellular cytotoxicity. In
practice, however, in vivo administration of immunoconjugates has proven
less efficacious than anticipated.
Several disadvantages related to retention, internalization and
translocation of immunoconjugates have been identified. For instance,
optimal retention of isotope-antibody fragment conjugates within tumor
tissue after in vivo administration has not been demonstrated. Additional
problems associated with target cell internalization and translocation of
immunoconjugates have been recognized, particularly in regards to
translocation and internalization of A-chain (derived from plant o r
bacterial toxin) immunoconjugates.
Thus, there is a need in the art for improved: (1) retention of targeting
protein conjugates (especially antibody fragment conjugates) at target
cell plasma membranes; (2) internalization of targeting protein conjugates
into target cell endosomic vesicles; and (3) translocation of targeting
protein conjugates across target cell endosomic vesicular membranes into
the cytoplasm. Enhancement of the interaction of targeting protein
conjugates with plasma membranes and/or internal membranes of target cells
may improve the cytotoxicity of targeting protein conjugates administered
in vivo. The present invention fulfills this need and further provides
other related advantages.
SUMMARY OF THE INVENTION
The present invention describes a covalently-linked complex (CLC) for
targeting a defined population of cells, comprising a targeting protein or
a targeting peptide; a cytotoxic agent; and an enhancing moiety, wherein
the enhancing moiety is capable of promoting CLC-membrane interaction.
A method for enhancing in vivo cytotoxicity of a targeting protein
conjugate comprising administering to a tumor-bearing patient a
therapeutically effective amount of the covalently-linked complex of the
present invention is also disclosed.
In addition, a method for enhanced in vivo imaging of a tumor comprising
administering to a tumor-bearing patient a diagnostically effective amount
of the claimed covalently-linked complex is disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a helical net structure representing an advantageous
spatial arrangement of amino acids present in a translocating peptide of
the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention, it may be helpful to set forth
definitions of certain terms to be used within the disclosure.
Targeting protein or targeting peptide: A protein or peptide that binds to
a defined population of cells. The targeting protein or peptide may bind a
receptor, an enzymatic substrate, an antigenic determinant, or other
binding site present on the target cell population. Hereinafter, the term
"targeting protein" will be inclusive of both targeting proteins and
targeting peptides.
Translocating peptide: A peptide capable of insertion into membranes at
acidic pH (typically pH 5.0-5.5).
Anchoring peptide: A peptide capable of insertion into membranes at
physiological pH (typically pH 6.8-7.5).
Accessory peptide: A peptide or non-peptide molecule that serves as a
substrate for target cell enzymes, and promotes membrane retention or
translocation of an anchoring and/or translocating peptide.
Fusion protein: A hybrid protein generated by means of recombinant DNA
technology. A fusion protein is translated from messenger RNA as one
continuous polypeptide chain, with the protein or peptide components
joined together by peptide bonds.
Conjugate: A two-component hybrid molecule wherein the components are
joined by a covalent chemical linkage.
Targeting protein conjugate: A conjugate wherein one component is antibody
(i.e., an immunoconjugate) or, more generally, a targeting protein.
Typically, the second component of a targeting protein conjugate is a
cytotoxic agent, such as a drug, a toxin or a radionuclide. In contrast to
fusion proteins, recombinant DNA methods are not involved in the covalent
linkage of targeting protein conjugate components.
Covalently-linked complex (CLC): A three-component complex comprising (1) a
targeting protein; (2) a cytotoxic agent; and (3) an enhancing moiety;
wherein the three components of the CLC are joined together by covalent
bonds.
Enhancing moiety: A moiety capable of promoting membrane interaction.
Enhancing moieties of the present invention include translocating
peptides, anchoring peptides, accessory peptides and organic membrane
intercalators. In addition, an enhancing moiety may be fused to one or
more components of a fusion protein. One or more enhancing moieties may be
covalently attached to a targeting protein conjugate to form a CLC having
enhanced membrane interactive characteristics.
In general, three levels of targeting protein conjugate-membrane
interaction have been identified that may be important for optimal in vivo
diagnostic or therapeutic efficacy: (1) binding of the conjugate to the
target cell plasma membrane; (2) internalization of the conjugate into
endosomic vesicles; and (3) translocation of the conjugate from endosomic
vesicles into the cytoplasm, which gives a targeting protein conjugate
access to cytoplasmic or nuclear target sites. If any one of these
targeting protein conjugate-membrane interactive steps becomes
rate-limiting, targeting protein conjugate potency may be diminished.
Optimization of the three levels of target cell membrane interaction noted
above (i.e., retention, translocation, internalization) may enhance the
cytotoxicity of targeting protein conjugates. Different types of targeting
protein conjugates (for instance, targeting protein conjugated to either a
drug, toxin or radioisotope) may require different levels of targeting
protein conjugate-membrane interaction in order to achieve optimal
cytotoxicity in vivo.
More specifically, radioisotope-targeting protein conjugates require
binding and prolonged retention of the conjugate, either within the tumor
or at the tumor cell plasma membrane, for maximal cytotoxic efficacy.
Drug-targeting protein conjugates that are active at the plasma membrane
may require (1) binding of the targeting protein conjugate at the plasma
membrane, and (2) expression of cytolytic activity at the plasma membrane.
Drug-targeting protein conjugates that are not active at the target cell
plasma membrane additionally require internalization of the drug for
cytotoxicity. Drug conjugates of this latter type and toxin-targeting
protein conjugates require three levels cf membrane interaction for
cytotoxicity: (1) binding of the targeting protein conjugate at the plasma
membrane; (2) internalization of the conjugate within the target cell; and
(3) translocation of the conjugate from endosomic vesicles into the
cytoplasm.
The "targeting protein" component of the covalently-linked complex (CLC) of
the present invention directs a covalently-attached cytotoxic agent to a
target cell population, such as tumor cells. Preferred targeting proteins
useful in this regard include antibody and antibody fragments; peptides,
such as bombesin, gastrin-releasing peptide, RGD peptide, substance P,
neuromedin-B, neuromedin-C, and metenkephalin; and hormores, such as EGF,
.alpha.- and .beta.-TGF, estradiol, neurotensin, melanocyte stimulating
hormone, follicle stimulating hormone, luteinizing hormone, and human
growth hormone. Biotin, avidin, proteins corresponding to known cell
surface receptors (including low density lipoproteins, transferrin and
insulin), fibrinolytic enzymes, and biological response modifiers
(including interleukin, interferon, erythropoietin and colony-stimulating
factor) are also preferred targeting proteins. Analogs of the above-listed
targeting proteins that retain the capacity to bind to a defined target
cell population may also be used within the claimed invention. In
addition, synthetic targeting proteins and peptides may be designed.
Monoclonal antibodies have precise specificity for a particular epitope
present on a target cell population. When a cytotoxic agent, such as a
drug, toxin or radioisotope, is conjugated to a monoclonal antibody,
increased amounts of the cytotoxic agent may be administered in vivo (as
compared to the unconjugated form of the cytotoxic agent), due to the
selective targeting properties of the monoclonal antibody component of the
conjugate.
Types of cytotoxic agents useful herein include toxins, drugs and
radionuclides. Several of the potent toxins useful within the present
invention consist of an A and a B chain. The A chain is the cytotoxic
portion and the B chain is the receptor-binding portion of the intact
toxin molecule (holotoxin). Because toxin B chain may mediate non-target
cell binding, it is often advantageous to conjugate only the toxin A chain
to a targeting protein. However, while elimination of the toxin B chain
decreases non-specific cytotoxicity, it also generally leads to decreased
potency of the toxin A chain-targeting protein conjugate, as compared to
the corresponding holotoxin-targeting protein conjugate.
One possible explanation for the decreased potency of A chain-targeting
protein conjugates is that B chain is required for translocation of the A
chain across endosomic membranes into the target cell cytoplasm. In the
absence of translocation, the targeting protein conjugate remains in the
interior of an endosome, and is ultimately transported to a lysosome.
Within the lysosome, the targeting protein conjugate is degraded, and thus
the A chain cytotoxic agent fails to reach its cytoplasmic target site.
The decreased potency associated with toxin A chain-targeting protein
conjugates also accompanies the use of ribosomal inactivating
protein-targeting protein conjugates. Ribosomal inactivating proteins
(RIPs) are naturally occurring protein synthesis inhibitors that lack
translocating and cell-binding ability.
Within the present invention, preferred toxins include holotoxins, such as
abrin, ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin,
pertussis toxin and Shiga toxin; and A chain or "A chain-like" molecules,
such as ricin A chain, abrin A chain, modeccin A chain, the enzymatic
portion of Pseudomonas exotoxin A, Diphtheria toxin A chain, the enzymatic
portion of pertussis toxin, the enzymatic portion of Shiga toxin, gelonin,
pokeweed antiviral protein, saporin, tritin, barley toxin and snake venom
peptides.
Preferred drugs suitable for use herein include conventional
chemotherapeutics, such as vinblastine, doxorubicin, bleomycin,
methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine, cyclophosphamide
and cis-platinum, as well as other conventional chemotherapeutics as
described in Cancer: Principles and Practice of Oncology, 2d ed., V. T.
DeVita, Jr., S. Hellman, S. A. Rosenberg, J. B. Lippincott Co.,
Philadelphia, Pa., 1985, Chapter 14. A particularly preferred drug within
the present invention is a trichothecene.
Experimental drugs, such as mercaptopurine, N-methylformamide,
2-amino-1,3,4-thiadiazole, melphalan, hexamethylmelamine, gallium nitrate,
3% thymidine, dichloromethotrexate, mitoguazone, suramin,
bromodeoxyuridine, iododeoxyuridine, semustine,
1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea,
N,N'-hexamethylene-bis-acetamide, azacitidine, dibromodulcitol, Erwinia
asparaginase, ifosfamide, 2-mercaptoethane sulfonate, teniposide, taxol,
3-deazauridine, soluble Baker's antifol, homoharringtonine, cyclocytidine,
acivicin, ICRF-187, spiromustine, levamisole, chlorozotocin, aziridinyl
benzoquinone, spirogermanium, aclarubicin, pentostatin, PALA, protein
conjugates, only plasma membrane interaction is required, but effective
cytotoxicity requires prolonged retention of the conjugate at the target
membrane.
The present invention discloses compositions and methods that promote
interaction(s) of targeting protein conjugates with various target cell
membranes. More specifically, biochemical linkage of a targeting protein
conjugate and one or more enhancing moieties capable of promoting membrane
interaction (or construction of an analogous recombinant fusion protein)
results in a "covalently-linked complex" (CLC) having improved membrane
interactive properties.
Enhancing moieties useful within the present invention may be subdivided
into several categories. The first category of enhancing moieties is
designated "translocating peptides," and includes pH-dependent
membrane-binding peptides. The second category is designated "anchoring
peptides," and includes membrane soluble peptide sequences and analogs
thereof. Anchoring peptides are capable of binding to target cell
membranes at physiologic pHs. A third category, "accessory peptides," may
be used in conjunction with translocating or anchoring peptides to enhance
membrane retention and/or translocation. A fourth category of enhancing
moieties includes membrane permeation enhancers, designated "organic
membrane intercalators," such as fatty acids and analogs thereof, bile
salts, membrane anesthetics, phospholipids, medium chain glycerides and
fusidic acid.
In an alternative embodiment, one or more enhancing moieties may be
included in a fusion protein. For generation of a fusion protein that
contains an enhancing moiety, a first DNA sequence (corresponding to a
targeting protein, a cytotoxic agent or an enhancing moiety) is joined at
the DNA level through recombinant DNA technology to a similar or
dissimilar second (third, fourth, etc.) DNA sequence. The resultant fused
DNA carboplatin, amsacrine, caracemide, iproplatin, misonidazole,
dihydro-5-azacytidine, 4'-decoxy-doxorubicin, menogaril, triciribine
phosphate, fazarabine, tiazofurin, teroxirone, ethiofos,
N-(2-hydroxyethyl)-2-nitro-1H-imidazole-1-acetamide, mitroxantrone,
acodazole, amonafide, fludarabine phosphate, pibenzimol, didemnin B,
merbarone, dihydrolenperone, flavone-8-acetic acid, oxantrazole,
ipomeanol, trimetrexate, deoxyspergualin, echinomycin, and dideoxycytidine
(see NCI Investigational Drugs, Pharmaceutical Data 1987, NIH Publication
No. 88-2141, Revised November 1987) are also preferred.
Radionuclides useful within the present invention include gamma-emitters,
positron-emitters, Auger electron-emitters, X-ray emitters and
fluorescenceemitters, with beta- or alpha-emitters preferred for
therapeutic use. Radionuclides are well-known in the art and include
.sup.123 I, .sup.125 I, .sup.130 I, .sup.131 I, .sup.133 I, .sup.135 I,
.sup.47 Sc, .sup.72 As, .sup.72 Se, .sup.90 Y, .sup.88 Y, .sup.97 Ru,
.sup.100 Pd, .sup.101m Rh, .sup.119 Sb, .sup.128 Ba, .sup.197 Hg, .sup.211
At, .sup.212 Bi, .sup.212 Pb, .sup.109 Pd, .sup.111 In, .sup.67 Ga,
.sup.68 Ga, .sup.67 Cu, .sup.75 Br, .sup.76 Br, .sup.77 Br, .sup.99m Tc,
.sup.11 C, .sup.13 N, .sup.15 O and .sup.18 F. Preferred therapeutic
radionuclides include .sup.188 Re, .sup.186 Re, .sup.203 Pb, .sup.212 Pb,
.sup.212 Bi, .sup.109 Pd, .sup.64 Cu, .sup.67 Cu, .sup.90 Y, .sup.125 I,
.sup.131 I, .sup.77 Br, .sup.211 At, .sup.97 Ru, .sup.105 Rh, .sup.198 Au
and .sup.199 Ag.
As noted above, with toxin-targeting protein conjugates, the limiting
membrane interactive events appear to be the rate of internalization and
the rate of translocation. With respect to drug-targeting protein
conjugates that are not active at the plasma membrane, internalization of
the targeting protein conjugate is required, with release of the drug from
endosomic vesicles before the conjugate reaches the lysosome (i.e., is
degraded). With drug-targeting protein conjugates that are active at the
plasma membrane, internalization is not required, but a strong, prolonged
interaction of the drug conjugate at the plasma membrane is important for
cytotoxic efficacy. For radionuclide-targeting sequences are transcribed
and translated into a hybrid fusion protein. When an enhancing moiety is
incorporated into a fusion protein, the resultant fusion protein possesses
improved membrane interactive properties.
In general, according to the present invention, the targeting protein
component of a covalently-linked complex recognizes a binding site at the
target cell membrane surface. A primary target cell interaction mediated
by the targeting protein component of the CLC is followed by a secondary
interaction of the enhancing moiety component with the plasma membrane.
This secondary interaction between enhancing moiety and membrane
stabilizes the targeting protein at the membrane surface. In instances
where an antibody (or antibody fragment) is the targeting protein,
interaction of enhancing moiety and target cell membrane may also increase
the affinity of an antibody for its antigen.
Translocating Peptides
The first category of enhancing moiety consists of translocating peptides,
which exhibit pH-dependent membrane binding. When a translocating peptide
assumes a helical conformation at an acidic pH, the translocating peptide
acquires the property of amphiphilicity, e.g., it has both hydrophobic and
hydrophilic interfaces. More specifically, within a pH range of
approximately 5.0-5.5, a translocating peptide forms an alpha-helical,
amphiphilic structure that facilitates insertion of the translocating
peptide into a target membrane. An alphahelix-inducing acidic pH
environment may be found, for example, in the low pH environment present
within cellular endosomes.
In aqueous solution at physiological pH, a translocating peptide is
unfolded (due to strong charge repulsion among charged amino acid side
chains) and is unable to interact with membranes. Within the present
invention, it may be advantageous to position amino acid residues within
the translocating peptide sequence so that charged amino acid side chains
will stack one above the other when the peptide folds into an amphiphilic
alpha helix at reduced pH. FIG. 1 represents a helical net display that
illustrates an advantageous spatial arrangement of the charged side
chains.
Charged amino acids capable of stacking within a translocating peptide
sequence include glutamate, aspartate and histidine. A preferred
pH-dependent membrane-binding translocating peptide includes a high
percentage of helix-forming residues, such as glutamate, methionine,
alanine and leucine. In addition, a preferred translocating peptide
sequence includes ionizable residues having pKa's within the range of pH
5-7, so that a sufficient uncharged membrane-binding domain will be
present within the peptide at pH 5 to allow insertion into the target cell
membrane.
A particularly preferred pH-dependent membrane-binding translocating
peptide in this regard is aa1-aa2-aa3-EAALA(EALA).sub.4 -EALEALAA-amide,
which represents a modification of the peptide sequence of Subbarao et al.
(Biochemistry 26: 2964, 1987). Within this peptide sequence, the first
amino acid residue (aa1) is preferably a unique residue, such as cysteine
or lysine, that facilitates chemical conjugation of the translocating
peptide to a targeting protein conjugate. Amino acid residues 2-3 may be
selected to modulate the affinity of the translocating peptide for
different membranes. For instance, if both residues 2 and 3 are lys or
arg, the translocating peptide will have the capacity to bind to membranes
or patches of lipids having a negative surface charge. If residues 2-3 are
neutral amino acids, the translocating peptide will insert into neutral
membranes.
Yet other preferred translocating peptides include peptides of
apo-lipoprotein A-1 and B; peptide toxins, such as melittin, bombolittin,
delta hemolysin and the pardaxins; antibiotic peptides, such as
alamethicin; peptide hormones, such as calcitonin, corticotrophin
releasing factor, beta endorphin, glucagon, parathyroid hormone,
pancreatic polypeptide; and peptides corresponding to signal sequences of
numerous secreted proteins. In addition, exemplary translocating peptides
may be modified through attachment of substituents that enhance the
alpha-helical character of the translocating peptide at acidic pH.
An example of a modified peptide having translocating activity at acidic pH
is fully succinylated melittin. In this example, a peptide (melittin) that
normally binds to membranes at physiological pH is converted to a
pH-dependent translocating peptide through succinylation of lysines. Upon
succinylation, the peptide displays an amphipathic character only at
acidic pHs.
Within the present invention, translocating peptides may be designed and
synthesized to provide enhanced membrane interaction(s). For instance,
translocating peptides conforming to the helical net structure depicted in
FIG. 1 may be generated. More specifically, in a translocating peptide
designed according to FIG. 1, stacked glutamates may occupy positions in
the helical net that are designated as "E"; the remaining amino acid
residues may consist (entirely or predominantly) of strong neutral helix
formers, such as methionine, alanine or leucine.
Insertion of a translocating peptide into a target cell membrane may be
enhanced through stabilization of the amphiphilic alpha helix. Helix
stabilization may be achieved: (1) by adding repeating "EALA" units to
form a longer peptide; (2) by placing an amide at the C-terminus of the
peptide, in order to counteract the helical dipole; (3) by polymerizing
the peptide; (4) by substituting a neutral helix-former for one or more of
the stacked glutamates; or (5) by attaching the peptide to a targeting
protein conjugate through use of a longer crosslinking agent, in order to
provide sufficient distance between the translocating peptide and the
targeting protein conjugate to allow the peptide to contact and interact
with the target cell membrane.
The amino acid sequence of a pH-dependent, membrane-binding translocating
peptide of the claimed invention may be selected to include all L-amino
acids or all D-amino acids having a side chain pKa from 5.0 to 9.0.
D-amino acids may be advantageously used to form non-proteolyzable
peptides, because the D-amino acids are not metabolized within the cell.
Further, the translocating peptides of the present invention may include a
combination of L- and D-amino acids, wherein D-amino acids are substituted
for L-amino acids on either side of a proteolytic cleavage site. Yet
another preferred noncleavable translocating peptide incorporates peptide
bond analogs that are not susceptible to proteolytic cleavage by cellular
enzymes.
The pH-dependent, membrane-binding translocating peptides of the present
invention may be attached singly or may be polymerized. Chemical linkage
of one or more translocating peptides (single, or polymerized) may be
accomplished either: (a) by direct attachment of a translocating peptide
to a targeting protein conjugate; (b) by disulfide, thioether, reduced or
non-reduced Schiff base or peptide bond formation; or (c) by use of a
peptide spacer. Preferred peptide spacers in this regard correspond to two
or more amino acid residues that allow the translocating peptide to assume
an alpha-helical conformation independent of its interaction with the
targeting protein conjugate, and may allow sufficient distance for
translocating peptide access to the cell surface from the peptide
attachment site on the targeting protein.
Polymerization of translocating peptides may be accomplished by placing a
cysteine residue at each end of a translocating peptide, followed by
oxidation using dissolved oxygen or other mild oxidizing agent, such as
oxidized glutathione. The average length of the polymerized translocating
peptide may be controlled by varying the polymerization reaction
conditions.
Mere membrane intercalation of a translocating peptide may be sufficient
for translocation of the peptide across endosomic membranes. However,
translocation may be improved by attaching to the translocating peptide
moiety a substrate for intracellular enzymes (i.e., an accessory peptide,
to be discussed in more detail in a following section). It is preferred
that an accessory peptide be attached to a portion(s) of the translocating
peptide that protrudes through the endosomic membrane to the cytoplasmic
face.
Covalent linkage of an enhancing moiety and a targeting protein conjugate
(forming a CLC) may provide enhanced retention of the conjugate (complex)
at a target cell plasma membrane upon in vivo administration. In addition,
a covalently-linked complex may exhibit more rapid and efficient
internalization rates than the corresponding two-component targeting
protein conjugate, due to secondary interaction(s) of the enhancing moiety
with the plasma membrane. Inclusion of both anchoring and translocating
peptides within a CLC may further facilitate initial binding and enhanced
translocation of the CLC across endosomic membranes into the target cell
cytoplasm.
Anchoring peptides
A second category of enhancing moiety suitable for use within the present
invention consists of anchoring peptides. Typically, anchoring peptides
contain membrane soluble peptide sequences which are highly apolar and
characteristically form alpha helices when inserted into a membrane. When
incorporated into a CLC, membrane insertion of the anchoring peptide
component may help to secure the targeting protein conjugate component to
a target cell membrane, and may further promote internalization of the
targeting protein conjugate/CLC.
A model for anchoring peptide interaction with a plasma membrane is the
opiate form of beta endorphin. Beta endorphin has one region that is
responsible for receptor binding, and another region which can assume an
amphiphilic helix (anchoring portion). The amphiphilic helix of beta
endorphin is believed to be responsible for an initial membrane
interaction, which is followed by diffusion of the hormone through the
cell membrane. Diffusion through the membrane allows the receptor-binding
region of the molecule to find its appropriate receptor (binding site). In
theory, the reverse process may also occur ---- the receptor binding
portion (targeting portion) of beta endorphin interacts with its receptor,
followed by alpha-helix formation and membrane insertion of the anchoring
portion of the molecule.
Anchoring peptides suitable for use within the present invention may be (i)
chemically synthesized; (ii) made by recombinant DNA technology; or (iii)
isolated from viral fusion proteins or other proteins. Viral fusion
peptides, such as those described by Gallaher (Cell. 50: 327-28, 1987),
are exemplary of anchoring peptides of the claimed invention. Preferred
viral fusion peptide sequences in this regard may be derived from viral
proteins of measles virus, respiratory syncytial virus, Sendai virus,
murine mammary tumor virus, human or simian immunodeficiency virus, visna
virus, or simian retrovirus. In addition, analogs of viral fusion peptides
that retain the capacity to embed within a membrane may be suitable for
use within the claimed invention.
AVGAIGAMFLGFLGAAGSTMGAASL represents yet another preferred anchoring
peptide sequence that may be incorporated into a covalently-linked complex
according to the present invention. An anchoring peptide sequence that
includes one or more internal repeats of the sequence "-FLG-" or "-FLA-"
or combinations thereof may also be preferred. For some therapeutic
applications, the addition of one or more negatively charged residues to
the anchoring peptide may be preferred. The additional negatively charged
residues may decrease levels of non-specific binding mediated by the
peptide domain of the CLC.
In a preferred embodiment, the anchoring peptide sequence includes an N
terminal aa1-aa2-aa3 sequence, which is defined in the same manner as
"aa1-aa2-aa3" of the pH-dependent, membrane-binding translocating
peptides, as described above. In addition, variable length peptide spacers
may be added to either terminus of the anchoring peptide sequence. The
remainder of the anchoring peptide sequence includes amino acid residues
capable of fusing with membranes or lipid bilayers.
In another preferred embodiment of the present invention, an anchoring
peptide may be attached to the targeting protein conjugate component of a
CLC by means of variable length crosslinking agents. In certain instances,
longer crosslinker spacer arms between the enhancing moiety and the
targeting protein conjugate are preferred. The span of a longer
crosslinking agent permits an anchoring peptide to reach from the binding
site of the targeting protein component to the target cell membrane.
In addition, aa2 and aa3 of an anchoring peptide sequence may be
substituted with a peptide spacer consisting of 1-40 amino acids. The
entire anchoring peptide plus spacer may be produced chemically in one
synthetic reaction. In a preferred embodiment, the spacer does not assume
a beta sheet or helical shape, and may be retained in an extended
conformation at physiological pH by charge repulsion.
A preferred spacer in this regard is CDNDNDDNDDGGG. Alternatively, a
preferred peptide spacer would include predominantly polar (charged or
uncharged) residues to aid solubility and, for spacers having charged
residues, only like charges. The sequence CRQRQRRQRRGGG is exemplary of a
positively charged spacer. The peptide spacers of the present invention
typically have a unique N-terminal residue (such as cys, lys, asp, or glu)
useful for crosslinking to a targeting protein. The insertion of a peptide
spacer provides greater distance between the targeting protein binding
site and the anchoring peptide, thereby increasing the probability that
the anchoring peptide will reach the target cell membrane and insert. For
instance, a 10-mer peptide spacer, conformationally decoupled from a
helix-forming anchoring peptide by insertion of three glycine residues,
would span approximately 30-40 .ANG. in an extended conformation. This
type of peptide spacer may also be advantageously used with translocating
peptides of the present invention. Alternatively, polymeric forms of
anchoring peptides may be used to span the distance from a targeting
protein binding site to the target membrane.
In instances where an anchoring peptide has a propensity for non-specific
insertion into non-target cell membranes, it may be desirable to decrease
the probability of membrane insertion of the anchoring peptide. Anchoring
peptide insertion into a membrane could be made less probable (1) by
shortening the anchoring peptide; (2) by including weaker neutral helix
formers in non-glutamate positions within the peptide sequence (see FIG.
1); (3) by substituting aspartate for glutamate within the anchoring
peptide sequence; (4) by synthesizing an anchoring peptide with a C
terminal carboxylate group; or (5) by incorporating into the peptide
sequence uncharged amino acids that are slightly more hydrophilic than
residues of a strongly translocating/anchoring peptide. By implementing
such peptide modifications, anchoring peptide dissolution in membranes
would be predicted to occur only upon primary interaction of the targeting
protein component with its binding site.
In one preferred embodiment, a virus-derived anchoring peptide sequence is
covalently attached to a targeting protein conjugate, forming a
covalently-linked complex. Antibody fragments, as well as intact antibody
molecules, are preferred targeting proteins for anchoring peptide
attachment.
Upon in vivo administration of a CLC, a primary interaction of the
targeting protein component with its binding site is followed by a
secondary interaction of the anchoring peptide component with the target
cell plasma membrane. The anchoring peptide component of the CLC is
solubilized within the membrane, thereby anchoring the targeting protein
conjugate component into the target cell membrane. The anchoring peptide
component may also act to enhance translocation of the CLC into the target
cell.
Accessory peptides
A third category of enhancing moiety, the "accessory peptides," may be
advantageously attached to the carboxy terminus of a translocating or
anchoring peptide. An accessory peptide of the present invention may
contain one or more amino acid residues. In one embodiment, an accessory
peptide may provide a substrate for cellular phosphorylation (for
instance, the accessory peptide may contain a tyrosine residue).
To exemplify the use of an accessory peptide within the claimed invention,
a phosphorylatable accessory peptide is first covalently attached to the
C-terminus of an anchoring peptide and then incorporated into a
covalently-linked complex. The anchoring peptide component of the CLC
intercalates into the target cell plasma membrane and, as a result, the
accessory peptide is translocated across the membrane and protrudes into
the cytoplasm of the target cell. On the cytoplasmic side of the plasma
membrane, the accessory peptide is phosphorylated by cellular kinases at
neutral pH. Once phosphorylated, the accessory peptide acts to
irreversibly anchor the CLC into the membrane. Further, the accessory
peptide may enhance the translocation of the CLC into the cell cytoplasm.
Preferred accessory peptides in this regard include kinase-substrate
accessory peptides that incorporate serine. A kinase-substrate accessory
peptide may be particularly advantageous for enhancement of CLC
cytotoxicity of tumor target cells, which have increased levels of protein
kinase activity for serines or tyrosines. Increased levels of kinase
activity within tumor cells may be attributed to the presence of oncogene
products, such as H-ras, on the cytoplasmic side of tumor cell plasma
membranes.
Suitable accessory peptides also include peptides that are kinase
substrates and peptides that possess a single positive charge. The latter
type of accessory peptide may form an ion pair with a "glutamate-like"
residue of an attached or closely adjacent translocating peptide. In this
regard, it may be desirable to replace an accessory peptide lysine and/or
arginine residue(s) with histidine, in order to facilitate movement of a
more neutral peptide through a target membrane at acidic pH.
Particularly preferred accessory peptides may be derived from the following
proteins and include the indicated amino acid sequences:
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Peptide Source Sequence
______________________________________
EGF receptor DVVDADEYLIPQ
EGF fragment CMHIESLDSYTC
Kemptide RGYALG or RGYSLG
Glycogen synthetase
PLSRTLSVAA
Transferrin receptor
FSLAR
H1 histone ASGSFKL
Casein kinase II substrate
AAAAAASEEE or
AAAAAASDDD
Insulin receptor auto-
DIYETDYYR
phosphorylation substrate
______________________________________
Other accessory peptides may be advantageously connected to hydrophobic,
membrane-soluble anchoring peptides. These accessory peptides (or
accessory compounds) may bind to, or be the substrate of, a cellular
cytoplasmic protein or enzyme. Preferred accessory peptides in this regard
include the following:
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Compound Structure Reaction
______________________________________
Aspartate protease
VLPFFVL (both Bind protease
inhibitor D-leu)
Vitamin K-dependent
FALEEI, Carboxylation
carboxylating enzyme
FALEEL or of glutamates
substrate FALEEV to form
gamma-carboxy
glutamate
Glycerol Phosphorylation
Pyridoxal Phosphorylation
Tetrahydrofolate Bind folate-
requiring
enzymes
Pantothenic acid Bind co-enzyme A
synthesizing
enzymes
Thiamine Bind synthetic
enzymes for
thiamine
pyrophosphate
______________________________________
Organic membrane intercalators
The fourth class of enhancing moiety that may be used to enhance retention
of a targeting protein conjugate at cell surfaces includes organic
membrane intercalators. Organic membrane intercalators include compounds
having high lipophilicity, such as fatty acids and fatty acid analogs,
bile acids, membrane anesthetics, phospholipids and glycolipids. This
class of enhancing moiety provides improved cell surface retention that is
particularly desirable, for instance, with radioisotopic-targeting protein
conjugates.
Long-chain fatty acids may be attached to targeting protein conjugates by
first modifying fatty acid carboxyl groups to form active esters. The
active ester form of the fatty acid may subsequently be conjugated to
targeting protein lysines or sulfhydryl groups.
If trans-unsaturated fatty acids are used, it is preferred that the double
bond be situated near the middle of the fatty acid molecule. Exemplary
trans-unsaturated fatty acids in this regard include trans-vaccenic acid
and elaidic acid. Long-chain hydrocarbons that may be hydrolyzed to
produce carboxylates, phosphonates or phosphates are also preferred
organic membrane intercalators within the present invention.
As an example, the lipophilicity of an artibody Fab-conjugate, which can
readily diffuse into tumors, may be modulated through the covalent
attachment of long-chain fatty acids or fatty acid analogs. The degree of
lipophilicity of the fatty acid-targeting protein conjugate (CLC) may be
modified by alte | | |
