Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CELL INTERNALIZED PEPTIDE-DRUG CONJUGATES
SEQUENCE LISTING
A printed Sequence Listing accompanies this application, and has also been
submitted
with identical contents in the form of a computer-readable ASCII file on a
CRROM.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention is concerned with treating diseases involve ICAM-1 and
LFA-1
proteins such as leukemia, Chron's disease, inflammation, asthma, rheumatoid
arthritis, and other
leukocyte related diseases. More particularly, the present invention is
concerned with improving
efficacy and reducing toxicity of drugs normally used to treat leukemia,
Chron's disease,
inflammation, asthma, rheumatoid arthritis, and other leukocyte related
diseases. Still more
particularly, the present invention is concerned with the conjugation of drugs
with peptides which
bind to cell adhesion receptors on cell surfaces of leukocytes, endothelial
cells, and epithelial
cells and are internalized by these cells. Finally, and most particularly, the
invention is
concerned with the conjugation of drugs with intercellular adhesion molecule-1
(ICAM-1)
peptides and lymphocyte function-associated antigen-1 (LFA-1 ) peptides for
drug delivery to the
cytoplasmic domain of cells expressing ICAM-1 and LFA-1.
Description of the Prior Art
Leukocyte-related diseases often result from aberrant immune responses
including
reactions of leukocytes on "self' antigens. Such reactions contribute to
autoimmune diseases
including rheumatoid arthritis, insulin-dependent diabetes mellitus, and
multiple sclerosis.
Similarly, organtransplantation rej ection results from leukocyte attack,
specifically fiom T-cells.
Accordingly inhibition of T-cell actions and their subsequent destruction aids
in combating such
diseases.
One wayto modulate leukocyte immune response utilizes inhibitors ofthe ICAM-
1/LFA-
1 receptor interaction. For example, monoclonal antibodies (mAbs) to ICAM-1
and LFA-1 have
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been utilized to generate tolerance in immune response disorders such as
allograft rej ection (Nato
et al., 1996; Nakamura et al., 1996), rheumatoid arthritis (Davis et al.,
1995), and autoimmune
encephalomyelitis (Willenborg et a1.,1996). Despite the encouraging clinical
results in inducing
tolerance, such mAbs may be potentially immunogenic and trigger an
effectiveness-limiting
immunity. In addition, the formulation of antibodies is challenging and
costly. Another way to
modulate immune response utilizes small peptide fragments derived from ICAM-1
and LFA-1
sequences which inhibit ICAM-1/LFA-1 interaction (Ross et al., 1992; Fecondo
et al., 1993;
Benedict et al., 1994; Siahaan et al., 1996). These peptides may have a better
physicochemical
stability than antibodies and may not possess any immunogenic properties. It
has also been
shown that a cyclic peptide (cIBR) derived from the sequence of ICAM-1
inhibits ICAM-1 /LFA-
1 interactions (Siahaan et al., 1996).
Furthermore, despite the ability to inhibit ICAM-1/LFA-1 interactions and
attendant
leukocyte-related diseases through treatment with antibodies, such treatments
are typically
ineffective over the long term due to their transient nature. Additionally,
once the mAbs invoke
an immune response, their effectiveness is severely limited.
When toxic drugs are used to kill the leukocytes and combat leukocyte-related
diseases,
many adverse side effects are encountered. These side effects include the non-
selective killing
of cells in addition to targeted cells as well as the suppression of the
proliferation of healthy cells.
Therefore, new methods which selectively target drugs to cells involved in the
disease process
will be beneficial to patients. For example, selectively targeting cytotoxic
drugs to leukocytes
will reduce drug toxicity and increase drug efficacy.
SUMMARY OF THE INVENTION
The present invention overcomes the problems inherent in the prior art and
provides
peptides derived from ICAM-1 and LFA-1 sequences which bind to receptors on
leukocytes.
These peptides are conjugated with drugs which are effective in treating
leukocyte-related
diseases. Such peptides and their conjugates are subsequently internalized by
the leukocyte
wherein the drug portion of the conjugate exerts toxic effects. Thus, the
present invention
provides an effective means of drug delivery to the cytoplasmic domain of
leukocytes with
improved efficacy and reduced toxicity in comparison to conventional methods
of treatment.
Because these peptides bind to specific receptors on the surface of the
leukocytes, drug delivery
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is "targeted" to cells having such receptors. This greatly reduces toxic
effects on other cells
which, due to the non-selectivity of the drugs utilized, generally accompanies
treatment of
leukocyte-related diseases.
Similarly, these drug-peptide conjugates can be used to treat diseases related
to increased
expression of ICAM-1 on endothelial and epithelial cells such as inflammation,
astlnna, allergies
and Chron's disease.
Peptides useful in the present invention include peptides which bind to LFA-1
or ICAM
receptors on leukocytes. Preferably, the peptides are derived from ICAM-1 and
LFA-1 sequences
and from about 4-30 amino acid residues in length. Still more preferably, such
sequences will
include from about 8-15 amino acid residues. Most preferably, such sequences
will include from
about 10-12 amino acid residues. Such sequences are linear and cyclic peptides
derived from I-
domain of LFA-1, including LAB, cLAB.L, cLAB.C, and cLAB.R which inhibit
homotypic
aggregation of a certain type of leukocyte, namely T-cells. These sequences
are listed herein as
SEQ ID Nos. 1-4. Other sequences include sequences derived from an ICAM-1
sequence, IB,
listed herein as SEQ ID No. 5. The sequences derived from ICAM sequences are
cyclized and
include cIBL, cIBC, and cIBR, listed herein as SEQ ID Nos. 6-8, respectively.
Sequences including or having a sequence which has at least about 50% sequence
identity
with any one of SEQ ID Nos. 1-8 and which exhibits similar internalization
properties are within
the scope of the present invention. Preferably, such sequences will have at
least about 60%
sequence identity with any one of SEQ ID Nos. 1-8 and still more preferably at
least about 75%
sequence identity. Alternatively, sequences including or having a sequence
which has at least
about 50% sequence homology with any one of SEQ ID Nos. 1-8 and which exhibits
similar
internalization properties are embraced in the present invention. More
preferably, such
sequences will have at least about 60% sequence homologywith any one of SEQ ID
Nos. 1-8 and
still more preferably at least about 75% sequence homology. Additionally,
sequences which
differ from any one of SEQ ID Nos. 1-8 due to a mutation event but which still
exhibit similar
properties are also embraced in the present invention. Such mutation events
include but are not
limited to point mutations, deletions, insertions and rearrangements.
Furthermore, as it is well
known in the art, peptidomimetics may be developed which have the same
modulation properties
as the preferred peptides detailed herein. As these peptidomimetics require no
more than routine
skill in the art to produce, such peptidomimetics are embraced within the
present application.
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Notably, the side chains of these peptidomimetics will be very similar in
structure to the side
chains of the preferred peptides herein, however, their peptide backbone may
be very different
or even entirely dissimilar.
One major complementary signal for sustaining leukocyte activation is the
interaction
between ICAM-1(ICAM-1, also known as CD54)and LFA-1 (LFA-1, made up of a dimer
comprising CDlla/CD18). Furthermore, the combination of ICAM-1/LFA-1 and
B7/CD28
complexes modulates the duration and amplitude of leukocyte activation. These
complexes
induce the movement of actin molecules in the cytoskeleton, thereby producing
the accumulation
of surface receptors such as LFA-l and ICAM-1 at the interface between antigen
presenting cells
(APC) and T-cells (Wiilfmg and Davis, 1998). When LFA-1 interacts with ICAM-2
(predominantly expressed on resting endothelium), its interaction with LFA-1
is more important
for non-activated circulating T-cells. Meanwhile ICAM-3 is expressed by
monocytes and resting
lymphocytes and plays a major role in the initiation of the immune response
(de Fougerolles et
al., 1992).
LFA-lIICAM-1 interaction requires the activation of LFA-1 (Dustin and
Springer,1989)
which can be triggered by CD2, CD3, phorbol esters (i.e., phorbol 12-myristate-
13 acetate or
(PMA)) and MHC class II molecules by stimulating protein kinase C (PKC)
mechanisms
(Rothlein and Springer, 1986; van Kooylc, et al., 1993). PMA activates LFA-1
molecules by
directly activating PKC while anti-CD2 and anti-CD3 antibodies activate PKC by
stimulating
inositol phospholipid metabolisms (Fidgor et al., 1990). Divalent cations
contribute to LFA-
1/ICAM-1 interactions by enhancing the functional activity of the adhesion
molecules (van
Kooyk et al., 1993), and the ion type requirement relates to a specific domain
of LFA-1 and
ICAM-1 (Stanley and Hogg, 1998).
SEQ ID No. 2 was synthesized as a 12-residue peptide containing 10 amino acid
residues
(I1e23' - G1y246) from the "insert" (I)-domain of LFA-1 which is known to
contain residues for
ICAM-1 binding (Benedict et al., 1994). Penicillamine (Pen) and cysteine (Cys)
residues were
then added to the N- and C-termini (Benedict et al., 1994) to form cyclic
peptides via a disulfide
bond between the Penl and Cysl2 residues. The formation of this cyclic peptide
restricts the
peptide conformation to produce a conformational stability, thereby providing
better selectivity
for cell surface receptors than its linear counterpart (Siahaan et al., 1996).
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The resulting peptide, cLAB.L, inhibits T-cell aggregation by inhibiting ICAM-
lILFA-1
interaction (Siahaan et al., 1996). There are two potential inhibitory
mechanisms performed by
this cyclic peptide: (a) inhibition of T-cell adhesion by binding to ICAM-1
and/or (b) disruption
of a- and p-subunit dimerization by binding to the (3-subunit of LFA-1.
Because the
heterodimeric formation of a- and (3-subunits of LFA-1 is necessary for ICAM
binding,
disruption of this heterodimeric formation results in an inhibition of the
ICAM-1/LFA-1
interaction.
Investigations leading to the present invention have also determined and
characterized
the cLAB.L binding sites. It was discovered that cLAB.L binds to the D1-domain
of ICAM-1
as well as to rCAM-3 on the surface of T-cells. Binding of the cLAB.L to the D
1-domain occurs
more efficiently than its binding to ICAM-3. The binding characteristics of
cLAB.L were
evaluated using the FITC-labeled cLAB.L (FTIC-cLAB.L) on activated Molt-3
cells. The FITC-
cLAB.L binding to the cell surface-receptor is inhibited by the unlabeled-
cLAB.L suggesting that
the FITC-cLAB.la bound to the same receptor as unlabeled-cLAB.L. Moreover, the
binding of
FITC-cLAB.L exhibited bimodal cell-distribution suggesting the occurrence of
multiple and
dynamic states of activated ICAM receptors. Binding of the peptide to ICAMs
(e.g. ICAM-1,
ICAM-2, ICAM-3) was enhanced by the presence of a Caz+ and Mg2+ mixture,
thereby suggesting
the involvement of divalent canons during peptide-ICAM interactions.
Additionally, it was
discovered that this peptide was internalized by ICAM receptors on T-cells.
Proof of this
internalization was supported by the marked differences in peptide binding
between 4°C and
37°C as well as by the presence of the peptide in the cytoplasm when
observed by confocal
microscopy. This internalization of the peptide, when coupled with drug
conjugation, permits
delivery of drugs to the cytoplasmic domain of cells having ICAM receptors.
Thus, the invention
provides effective treatment of leukocyte-related diseases, because leukocytes
have ICAM
receptors.
It is known that the cyclic peptide (cIBR) derived from the sequence of ICAM-1
inhibits
ICAM-1 /LFA-1 interactions (Siahaan et al., 1996). It has now been discovered
that this peptide
binds to and is internalized by LFA-1 surface receptors of T-cells.
Furthermore, it has also been
determined that the conformation of this peptide plays an important role in
its selectivity for the
receptor. Such receptor internalization is believed to be a complementary
mechanism of the
inhibition of cell-cell adhesion.
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Due to the internalization of peptides which bind to receptors on leukocytes,
one such
peptide, SEQ ID No. 8, was selected for conjugation experiments. To perform
the conjugation
experiments SEQ ID No. 8 was conjugated with drugs in order to evaluate
resultant toxicity to
targeted and non-targeted cells. Conjugation was accomplished by dissolving a
quantity of drug
in water and adding in an amount of peptide prior to storing the resultant
solution overnight.
Such conjugates bound to leukocytes expressing LFA-1 thereby interfering with
ICAM/LFA-1
interactions. Binding to the LFA-1 receptors resulted in internalization of
the peptide-drug
conjugate into the cytoplasmic domain of cells wherein the drug portion of the
conjugate exerted
potent toxic affects. Advantageously, these toxic affects were localized to
cells expressing LFA-
1 on their surfaces, thereby delivering these drugs to a specific type of
cell. Because only
leukocytes express LFA-1 on their surfaces, drugs can be specifically
delivered to the surfaces
of leukocytes and internalized before exerting their toxic affects and without
significant toxicity
to other cells. The present invention therefore finds great utility in the
treatment of leukocyte-
related diseases. Preferably, diseases treatable by such peptide-drug
conjugates include Chron's
disease, asthma, inflammations, lupus, rheumatoid arthritis, multiple
sclerosis, ulceritive colitis,
pemphigus vulgaris, pemphigoid, allergies, HIV infections, and epidermolysis.
Thus, by conjugating peptides which are internalized with drugs,
internalization of the
conjugate by leukocytes contributes to targeted drug delivery with a high
degree of specificity
and toxicity for targeted cells and decreased toxicity for non-targeted cells.
In the case of
peptides derived from LFA-1 sequences which are conjugated with drugs, such
conjugates are
internalized by leukocytes and epithelial and endothelial cells which have
ICAM receptors on
their surface. In the case of peptides derived from ICAM sequences which are
conjugated with
drugs, such conjugates are internalized by leukocytes which have LFA-1
receptors on their
surface. Preferably, the peptides are conjugated with drugs effective in
destroying leukocytes.
Still more preferably, the conjugated peptides are LFA-1 peptides which can
modulate the
functions of epithelial and endothelial cells via mechanisms in the cytoplasm
such as the
mechanisms used by the asthma drugs albuterol and propidium. Preferably, the
drug is selected
from the group consisting of drugs classified as antiinflammatory agents,
antitumor agents (i.e.,
intercalating agents, tubulin assembly inhibitors, alkylating agents),
oligonucleotides, cytokines,
enzyme inhibitors (i.e., ACE inhibitors, HIV-protease inhibitors; viral
protease inhibitors), and
vasoregulator agents. Still more preferably, the drug is selected from the
group consisting of
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methotrexate, lovastatin, taxol, ajmalicine, vinblastine, vincristine,
cyclophosphamide,
fluorouracil, idarubicin, ifosfamide, irinotecan, 6-mercaptopurine,
mytomycins, mitoxantrone,
paclitaxel, taxol, pentostatin, plicamycin, topotecan, fludarabine, etoposide,
doxorubicin,
doxotaxel, danorubicin, albuterol, propidium. Therefore, the resultant peptide-
drug conjugates
both interfere with ICAM-1/LFA-1 interactions and destroy leukocytes once the
conjugates are
internalized. Furthermore, the internalized conjugates may regulate functions
of epithelial and
endothelial cells such as those present in long epithelial cells and vascular
endothelial cells. This
ability to regulate functions is due to the presence of ICAM-1 on epithelial
and endothelial cells
which bind to and internalize LFA-1 peptides.
Notably, the present invention reduces the toxicity of anti-tumor and
imrnunosuppressive,
and other drugs by increasing the selectivity of the drugs to the target
cells. This selectivity also
results in increased potency at lower dosage levels. As an example, a drug
currently used to treat
leukemia, methotrexate (MTX), was conjugated to the N-terminal of the cIBR
peptide to yield
a MTX-cIBR conjugate. The conjugate's potency and selectivity was then
compared to that of
MTX alone. The conjugate was shown to be more potent against leukemic cells
(which have
LFA-1 receptors) than MTX alone. Additionally, the conjugate was not effective
in killing
Madin-Darby Canine Kidney (MDCK) cells, which do not have LFA-1 receptors. The
potency
and selectivity of the conjugates represent a distinct advance in the state of
the art as current
treatments using MTX have severe side effects including suppression of the
proliferation of other
healthy cells. Due to the conjugates' increased potency and selectivity,
patients will be able to
receive lower dosage levels of drugs with fewer adverse side effects.
BRIEF DESCRIPTION OF THE DRAWINGS
The filing of this patent contains at least one drawing executed in color.
Copies of this
patent with color drawings) will be provided by the Patent and Trademark
Office upon request
and payment of the necessary fee.
Figure 1 is a graph illustrating blocking by cLAB.L peptide on the binding of
FITC-
conjugate antibodies to ICAM-1, ICAM-3 and LFA-1;
Fig. 2 is a graph illustrating the distribution of Molt-3 cell-population in
response to the
binding to FITC-cLAB.L whereby cells were preactivated with PMA for 1 hour
(a), and cells
were preactivated with PMA for 48 hours (b), while distribution of Molt-3 cell
population in
response to the binding of FITC-conjugate antibody to domain Dl of ICAM-1 is
shown in (c);
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Fig. 3 is a graph illustrating the binding specificity of FITC-cLAB.L on
population-1 and
-2 of Molt-3 cells, indicated by the saturation profile of the FITC-cLAB.L
binding (a), and the
ability of unlabeled cLAB.L to block the binding of FITC-cLAB.L in
concentration-dependent
manner (b);
Fig. 4a is a graph illustrating proportions of cell population-1 and -2 in
response to the
duration of PMA activation;
Fig. 4b is a graph illustrating the effect of cell activation on the binding
of 25 ~M FITC-
cLAB.L to cell population-1;
Fig. 5a is a graph illustrating time-temperature dependence-profiles of 25 gM
FITC-
cLAB.L binding on population-1;
Fig. 5b is a graph illustrating the population-2 of Molt-3 cells;
Fig. 5c is a graph illustrating the effect of cation addition on the intensity
of FITC-
cLAB.L binding to PMA-activated Molt-3 cells;
Fig. 6a is a confocal microscopy photograph illustrating microscopy of FITC-
cLAB.L
binding and internalization on a Molt-3 cell clump at 37°C;
Fig. 6b is a confocal microscopyphotograph ofFITC-cLAB.L binding and
internalization
on a single Molt-3 cell at 37°C;
Fig. 6c is a confocal microscopyphotograph ofFITC-cLAB.L binding and
internalization
on a single Molt-3 cell at 4°C'
Fig. 6d is a confocal microscopy photograph of surface projection of Molt-3
cell
following 37°C incubation;
Fig. 6e is a confocal miscoscopy photograph of the same area as in Fig. 6d and
showing
the distribution of the FITC-cLAB.L in the cytoplasm;
Fig. 6f illustrates a single confocal section showing a minimal distribution
of the FITC-
cLAB.L on the cell peripheral;
Fig. 7 is a schematic of two different forms of an MTX-cIBR conjugate in
accordance
with the present invention;
Fig. 8 is a schematic illustration ofthe internalizationprocess of an MTX-cIBR
conjugate
in accordance with the present invention;
Fig. 9 is a graph of an MTT assay comparing the toxicities of different
concentration of
MTX-cIBR conjugates and MTX alone in accordance with the present invention;
and
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Fig. 10 is a comparative graph illustrating metabolic activity after treatment
with MTX,
cIBR, and MTX-cIBR conjugates.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
As used herein, the following definitions will apply: "Sequence Identity" as
it is known
in the art refers to a relationship between two or more polypeptide sequences
or two or more
polynucleotide sequences, namely a reference sequence and a given sequence to
be compared
with the reference sequence. Sequence identity is determined by comparing the
given sequence
to the reference sequence after the sequences have been optimally aligned to
produce the highest
degree of sequence similarity, as determined by the match between strings of
such sequences.
Upon such alignment, sequence identity is ascertained on a position-by-
position basis, e.g., the
sequences are "identical" at a particular position if at that position, the
nucleotides or amino acid
residues are identical. The total number of such position identities is then
divided by the total
number of nucleotides or residues in the reference sequence to give % sequence
identity.
Sequence identity can be readily calculated by known methods, including but
not limited to,
those described in: Computational Molecular Biology, Lesk, A. N., ed., Oxford
University Press,
New York ( 1988); Biocomputing: Informatics and Genome Projects, Smith, D. W.,
ed., Academic
Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin,
A.M., and Griffin,
H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular
Biology, von
Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. et
al., eds., M.
Stockton Press, New York (1991); and Carillo, H., et al. Applied Math.,
48:1073 (1988), the
teachings of which are incorporated herein by reference. Preferred methods to
determine the
sequence identity are designed to give the largest match between the sequences
tested. Methods
to determine sequence identity are codified in publicly available computer
programs which
determine sequence identity between given sequences. Examples of such programs
include, but
axe not limited to, the GCG program package (Devereux, J., et al., Nucleic
Acids Research,
12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec.
Biol.,
215:403-410 (1990). The BLASTX program is publicly available from NCBI and
other sources
(BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul,
S. F. et
al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are
incorporated herein by
reference). These programs optimally align sequences using default gap weights
in order to
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produce the highest level of sequence identity between the given and reference
sequences. As
an illustration, by a polynucleotide having a nucleotide sequence having at
least, for example,
95% "sequence identity" to a reference nucleotide sequence, it is intended
that the nucleotide
sequence of the given polynucleotide is identical to the reference sequence
except that the given
polynucleotide sequence may include up to 5 point mutations per each 100
nucleotides of the
reference nucleotide sequence. In other words, in a polynucleotide having a
nucleotide sequence
having at least 95% identity relative to the reference nucleotide sequence, up
to 5% of the
nucleotides in the reference sequence may be deleted or substituted with
another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the reference
sequence may be
inserted into the reference sequence. These mutations of the reference
sequence may occur at
the 5' or 3' terminal positions of the reference nucleotide sequence or
anywhere between those
terminal positions, interspersed either individually among nucleotides in the
reference sequence
or in one or more contiguous groups within the reference sequence.
Analogously, by a
polypeptide having a given amino acid sequence having at least, for example,
95% sequence
identity to a reference amino acid sequence, it is intended that the given
amino acid sequence of
the polypeptide is identical to the reference sequence except that the given
polypeptide sequence
may include up to 5 amino acid alterations per each 100 amino acids of the
reference amino acid
sequence. In other words, to obtain a given polypeptide sequence having at
least 95% sequence
identity with a reference amino acid sequence, up to 5% of the amino acid
residues in the
reference sequence may be deleted or substituted with another amino acid, or a
number of amino
acids up to 5% of the total number of amino acid residues in the reference
sequence may be
inserted into the reference sequence. These alterations of the reference
sequence may occur at
the amino or the carboxy terminal positions of the reference amino acid
sequence or anywhere
between those terminal positions, interspersed either individually among
residues in the reference
sequence or in the one or more contiguous groups within the reference
sequence. Preferably,
residue positions which are not identical differ by conservative amino acid
substitutions.
However, conservative substitutions are not included as a match when
determining sequence
identity.
Similarly, "sequence homology", as used herein, also refers to a method of
determining
the relatedness of two sequences. To determine sequence homology, two or more
sequences are
optimally aligned as described above, and gaps are introduced if necessary.
However, in contrast
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to "sequence identity", conservative amino acid substitutions are counted as a
match when
determining sequence homology. In other words, to obtain a polypeptide or
polynucleotide
having 95% sequence homology with a reference sequence, 95% of the amino acid
residues or
nucleotides in the reference sequence must match or comprise a conservative
substitution with
S another amino acid or nucleotide, or a number of amino acids or nucleotides
up to 5% of the total
amino acid residues or nucleotides, not including conservative substitutions,
in the reference
sequence may be inserted into the reference sequence.
A "conservative substitution" refers to the substitution of an amino acid
residue or
nucleotide with another amino acid residue or nucleotide having similar
characteristics or
properties including size, charge, hydrophobicity, etc., such that the overall
functionality does
not change significantly.
"Isolated" means altered "by the hand of man" from its natural state., i.e.,
if it occurs in
nature, it has been changed or removed from its original environment, or both.
For example, a
polynucleotide or polypeptide naturally present in a living organism is not
"isolated," but the
same polynucleotide or polypeptide separated from the coexisting materials of
its natural state
is "isolated", as the term is employed herein. "Drug" means any natural or
artificially
made chemical for use in the diagnosis, cure, mitigation, treatment, or
prevention of illness or
disease.
"Targeted cell" means a cell of a specific type or class which exhibits
certain physical or
functional characteristics and which is of interest due to these
characteristics. In the case of
medical treatment, when certain types of cells are involved in a disease-type
process, treatment
can be improved if such cells can be selectively treated without significantly
affecting other cells
which are not involved in the disease process. Thus, offending cells would be
"targeted" and
subject to selective treatment while other cells were unaffected.
Similarly, a "non-targeted cell" means a cell which does not have a physical
or functional
characteristic of a "targeted cell." Preferably, non-targeted cells are not
significantly affected
during medical treatment.
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EXAMPLES
The following examples set forth preferred embodiments of the present
invention. It is
to be understood, however, that these examples are provided by way of
illustration and nothing
therein should be taken as a limitation upon the overall scope of the
invention.
EXAMPLE 1
This Example confirmed that a peptide derived from LFA-1 inhibited ICAM-1/LFA-
1
interaction by binding to ICAM sequences. Furthermore, it was determined that
this peptide was
internalized by leukocytes after binding to ICAM. Thus, peptides such as
cLAB.L may be used
as a convenient shuttle source to the interior of the leukocyte, thereby
providing new methods
of treating leukocyte-related diseases. In addition, these peptides can also
be used to shuttle
drugs to the other cells expressing ICAM-1 such as endothelial and epithelial
cells.
Materials and Methods:
Cell Culture and Cell activation. Molt-3 cells were obtained from the American
Type
Culture Collection (Rockville, MD) and maintained in suspension in RPMI1640
supplemented
with 10% heat-inactivated fetal bovine serum and 100 mg/L of
penicillin/streptomycin. Cells
were grown or activated in 75-cm2 tissue culture flasks (Corning) at
37°C in a saturating
humidified atmosphere of 95% air and 5% CO2. For the purpose of cell
activation, phorbol 12-
myristate-13-acetate (PMA) from Sigma (St. Louis, MO) or anti-CD3 antibody
from Chemicon
(Temecula, CA) was used in concentrations of 0.2 ~,M and 10 ~g/mL,
respectively. Incubation
with PMA was varied (1, 2, 4, 16, 24, 40 and 48 hours) to study the affect of
time on receptor
activation. In other cases, periods of PMA activation for 4 hours and 48 hours
were compared
in their effects on cLAB.L binding to the cells.
Peptide Labeling. 50 mg of cLAB.L peptide was dissolved in a minimal amount of
Milli-Q water and added with FITC at two times the molarity of the peptide.
The FITC is reacted
to the N-terminus of cLAB.L. The pH mixture was adjusted to 9.0 with 1 N NaOH
and the
reaction was run for 1 hour. The pH was then brought to 7.0 by adding 10%
acetic acid. The
mixture was lyophilized and purified using preparative reversed-phase (RP)
HPLC. The
molecular weight of the fraction containing FITC-cLAB.L was confirmed by fast
atom
bombardment (FAB) mass spectrometry to give M+1=1586.
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Peptide Binding Experiment. Activated or non-activated cells were centrifuged
at 2000
rpm for 5 minutes and resuspended in serum-free medium to reach a
concentration of 3.5 X
106/mL. Peptide stock solution was prepared in phosphate buffer saline (PBS).
Serum-free
medium of RPMI1640 was used for the binding experiment in a 48-well cell
culture cluster
(Corning) into which the mixture of cell suspension, medium, and peptide
solution was 400
~L/well. The mixture was incubated at either 4°C or 37°C. In
time-temperature dependence
experiments, a sample was taken every 30 minutes for up to 4 hours incubation
time. In a
concentration-dependence experiment, incubation was carried out for 2 hours.
In experiments to
block FITC-cLAB.L binding, cLAB.L was added to the cell suspension followed by
incubation
for 1 hour at 4°C. The FITC-cLAB.L was then added followed by another
hour of incubation.
Cell suspensions without peptide addition were used as controls throughout the
experiments.
At the end of incubation, cell suspensions were centrifuged at 3000 g for 3
minutes before
being decanted and rinsed with 10 mM Hepes/PBS. The cell pellet was fixed
using 4%
paraformaldehyde/PB S for 20 minutes at room temperature. The mixture was then
washed twice
with 10 mM Hepes/PBS and resuspended in PBS. Samples were analyzed with a flow
cytometer
(Becton Dickinson). As many as 10,000 cells were counted for every sample, and
each
experiment was done at least in triplicate. Peptide binding affinity was
represented by the average
of median values of fluorescence intensity (FI), or in some cases, by its
ratio to the relevant
standard condition/reference.
Antibody Binding Experiment. A one step-directmethod using FITC-labeled anti-
LFA-
1, anti-ICAM-1 or anti-ICAM-3 was applied to examine the ability of cLAB.L to
block antibody
binding to Molt-3 cells. The mAbs to LFA-1 tested were anti-human CD1 la clone
38 and anti-
human CD18 clone IB4 from Ancell {Bayport, MN). Another pair of clones (DF1524
and
YFCl 18.3 from Accurate (Westbury, NY)) of anti-LFA-1 mAb that have been
proven to inhibit
aggregation and adhesion were also tested. Clone 15.2 and clone 8.4A6 of anti-
ICAM-1 mAbs
(Ancell), that recognize Dl- and D2-domain of ICAM-1, respectively, were also
used in this
study. For ICAM-3, clone 186-2G9 which recognized the D1-domain was used to
identify the
peptide-ICAM-3 recognition. Peptide solution was incubated with Molt-3 cells
for 1 hour prior
to the addition of FITC-antibody. The flow cytometer analysis was carried out
as described for
the peptide binding experiment. A cyclic Arg-Gly-Asp (RGD) peptide (cyclo-
(2,10)-Ac-Gly-
Pen-Gly-His-Arg-Gly-Asp-Leu-Arg-Cys-Ala-NHZ) (SEQ ID No. 9) was used as a
control peptide
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throughout the antibody binding experiments because LFA-1 does not recognize
the tripeptide
sequence Arg-Gly-Asp (SEQ ID No. 10) unlike other integrins (i.e., (33
integrins: a~(33, a"b(33 and
(31 integrins). Furthermore, ICAM-1 does not contain an RGD sequence (Detmer
and Wright,
1988).
Fluorescence and Confocal Microscopy Studies. Samples from peptide-binding
experiments at 4°C and 37°C were photographed using fluorescence
microscope Nikon Eclipse
TE300 and confocal microscope (Biorad MRC100 Laser Scanning Confocal Imaging
System
connected to Nikon Diaphot 2000 microscope).
Inhibition of Antibody Binding to LFA-1, ICAM-1 and ICAM-3 by cLAB.L. To
determine whether the peptide binds to LFA-1, ICAM-1 or ICAM-3, the ability of
the cLAB.L
peptide to inhibit binding of FITC-labeled mAbs to LFA-1, ICAM-1 and ICAM-3
was evaluated
and a cyclic RGD peptide was used as a negative control (Fig. 1 ). Two peptide
concentrations,
80 and 160 wM, were used in this inhibition study. Two anti-ICAM-1 mAbs
directed toward the
D 1- and D2-domains and an anti-ICAM-3 antibody directed towaxd the D 1-domain
of ICAM-3
were used to evaluate the binding properties of cLAB.L. Two pairs of mAbs to
LFA-1 were also
tested in this experiment. One pair of mAbs was anti-CD 11 a clone 38 and anti-
CD-18 clone IB4,
neither of which inhibit cell adhesion. The other pair was anti-CDlla clone
DF1524 and anti-
CD 18 clone YFC 118.3, which both inhibit cell adhesion.
Fig. 1 shows the results for these studies. For a negative control, cyclic RGD
peptide at
80 and 160 qM had no effect on the binding of all the tested mAbs (see
controls as the
representatives). In the figure, LFA-1 sp indicates the use of antibody
specifically described by
the manufacturer as an inhibitor of cell adhesion/aggregation. Fluorescence
data were normalized
betweenfluorescence of FITG-antibodies in the presence of cyclic-RGD peptide
(control peptide)
and the fluorescence of cells only. Both control peptide and cLAB.L were
tested at two different
concentrations shown on Fig. 1 as * and * * for 80 and 160 ~M respectively.
The data shown
represent the mean ~ S.E. of six determinations. The use of blank bars is to
show that no
blocking was observed. Binding of anti-ICAM-1 mAbs to D1-domain was blocked by
cLAB.L
peptide in a concentration dependent manner. However, the cLAB.L peptide did
not inhibit the
binding of anti-ICAM-1 antibody to the D2-domain. This result indicates that
the cLAB.L
peptide binds to the D1-domain but not D2-domain of ICAM-1. Interestingly, the
cLAB.L
peptide can also bind to the D1-domain of ICAM-3 because it can block the
binding of anti-
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ICAM-3 binding to D1-domain. The cLAB.L peptide at a concentration of 160 ~M
blocks the
binding of anti-ICAM-1 (D1) and anti-ICAM-3 (D1) 59% and 74%, respectively.
Thus, cLAB.L
prefers binding to ICAM-1 over ICAM-3 thereby indicating that the peptide's
major mechanism
of activity is inhibition of the ICAM-lILFA-1 interaction. The cLAB.L peptide
can weakly
inhibit the binding of anti-CD 11 a DF 1524, presumably due to the recognition
of cLAB.L by this
anti-CDlla antibody. Furthermore, the cLAB.L peptide prevents the binding of
antibody to
CD 11 a on T-cells. In other words, the same sequence as cLAB.L in CD-11 a is
recognized by this
anti-CDlla DF1524 antibody. The cLAB.L peptide did not block the binding of
other anti-
CD 11 a and anti-CD-18 antibodies. This result indicates that cLAB.L may not
bind to CD-18 to
disrupt the CD 11 a-CD 18 dimerization.
Binding Specificity of FITC-cLAB.L. As illustrated in Figs. 2a and 2b, the
flow
cytometry experiments show that FITC-cLAB.L binds to two populations
(population-1 and -2)
of Molt-3 cells. For these experiments, the peptide concentration was 25 ~.M
and the antibody
concentration was used at the dilution suggested by the manufacturer. In
contrast, FITC-labeled
mAbs to ICAM-1 or LFA-1 show binding to only one cell population. This is
illustrated in Fig.
2c. For FITC-cLAB.L peptide, the first population (population-1 ) has a high
number of cells with
low fluorescence intensity while the second population (population-2) has low
number of cells
but high fluorescence intensity (Figs. 2a and b). This indicates that the
receptors in population-2
have a higher affinity than those in population-1.
In order to characterize receptor-mediated binding specificity of FITC-cLAB.L,
two
different experiments were performed. The first experiment tested
concentration-dependent
blocking of FITC-cLAB.L binding by the unlabeled cLAB.L. The second experiment
tested
FITC-cLAB.L binding saturation to the receptors on Molt-3 cells. Results of
these experiments
are given in Figs. 3 a and 3b. The FITC-cLAB.L binding on 4 hour-preactivated
cells approached
saturation at around 400 ~,M and 800 ~.M on cell populations -l and -2,
respectively. These
results are given in Fig. 3a. To examine the blocking ability of unlabeled
cLAB.L, two different
preactivated cells (i.e., 4 hour and 48 hour activation) were used. These
results are shown in Fig.
3b. In Fig. 3a, a comparison was made between cells preactivated with PMA for
4 hours and
48 hours. The percentage of blocking by unlabeled peptide in Fig. 3b was taken
as the relative
fluorescence values to the binding of 25 ~M FITC-cLAB.L without the addition
of unlabeled
peptide (ratio of unlabeled and labeled peptide = 0). Each value in Fig. 3a
represents the mean
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of fluorescence values relative to the reference (binding of 3.12 p,M FITC-
cLAB.L) ~ S.E. Four
determinations were carried out for both experiments which are illustrated in
Figs. 3a and 3b.
Error bars are missing when less than the size of a symbol. A blocking
experiment was carried
out by incubating the cells with unlabeled cLAB.L prior to the addition of
FITC-cLAB.L. This
reaction was run at 4°C to miW mize peptide internalization (see
below). The results show that
the unlabeled cLAB.L inhibits the binding of FITC-cLAB.L on cell populations -
1 and -2 in a
concentration-dependent fashion (Fig. 3b). For population-l, both cell
activation times at 4 hours
and 48 hours produce comparable inhibitory results. On the other hand, the
population-2 of 48
hour-preactivated cells were more prone to blocking by unlabeled peptide than
those of 4 hour
preactivation. These results indicate that the 4 hour activation provides a
higher affinity state of
the receptors.
Effect of Activation Time and Activators on FITC-cLAB.L Binding. The number of
cells and fluorescence intensities between the two populations were changed at
various time of
PMA activation. The fluorescence intensity in population-1 increased sharply
in the first four
hours of PMA activation, and then reached a steady state at 40 hours time of
activation. At the
same time, the cell number of population-2 increased with increasing
activation time while the
cell number in population-1 decreased proportionately (Fig. 4a). Each value
shown in Fig. 4a
represents the mean ~ S.E. of four determinations. The fluorescence intensity
from the binding
of FITC-cLAB.L (50 E.iM) to PMA-activated cells was three- to six-fold higher
than binding to
the non-activated cells. Such a difference indicates that cell activation is
crucial for peptide
binding.
The effect of temperature and different activators (i.e., PMA, anti-CD3 and
both Ca2+ and
Mg2+) on FITC-cLAB.L binding to the receptors on the activated Molt-3 cells
was evaluated. The
results forpopulation-1 are shown in Fig. 4b and similar observations were
found for population-
2. Binding of the FITC-cLAB.L to non-activated Molt-3 cells at 4°C and
37°C was used as a
negative control. The binding experiment was carried out in the presence of
activating molecules
anti-CD3, PMA, or divalent cations (Ca2++ Mgz~ at final concentration of 1.5
mM) at 4°C or
37°C. Peptide binding on non-activated cells at 4°C was used as
reference. The data shown
represent the mean of fluorescence values relative to the reference ~ S.E. of
four determinations.
Error bars are missing when less than the size of symbol. As shown in Figs. 5a
and 5b, peptide
binding at 4°C and 37°C was compared using cells preactivated
with PMA for 4 hours or 48
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hours. Again, each value represents the mean of fluorescence value ~ S.E. of
four determinations
and error bars are missing when less than the size of symbol. The binding
reactions were
monitored over a four hour period. PMA and CD3 activated Molt-3 cells bind to
the peptide
with higher affinity than the non-activated cells at 4°C and
37°C. The fluorescence intensities
increased over the four hour time period when the experiments were performed
at 37°C.
However, the binding of the peptide was suppressed at 4°C thereby
indicating that at 37°C, the
peptide was internalized by the receptors. In contrast, addition Ca2+ and Mg2+
mixture to the non-
activated cells was unable to promote peptide binding. These results indicate
that the divalent
cations only affected binding of the peptide to the activated cells as shown
in Fig. 5c. For this
experiment, the final concentrations of Caz+, Mga+, and Ca2++ Mgz+ were 1.5
mM. The control
was compared to the intensity of FITC-cLAB.L binding to PMA-activated cells
without added
canons. The data shown represent the mean of fluorescence values relative to
the control ~ S.E.
of three determinations. Error bars are not shown due to very small values.
Effects of Temperature and Divalent Cations on FITC-cLAB.L Binding Affinity.
The effect of activation times and temperature on the FITC-cLAB.L binding to
the Molt-3 cells
was also evaluated in order to study the relationship of the activated state
of the receptors (i.e.,
ICAM-1) and activation time. The cells were preactivated with PMA for 4 hours
and 48 hours.
Binding of FITC-cLAB.L to the cells was sampled every 30 minutes for 240
minutes at 4°C and
37°C. Fluorescence intensities of binding for 30 minutes and 240
minutes incubation time are
shown in Figs.Sa and Sb. There are two consistent patterns shown by both
population-1 and -2.
First, the fluorescence intensity of peptide binding was lower at 4°C
than at the 37°C incubation.
This result was also confirmed by fluorescence microscopy (Figs. 6a-c). For
each of the photos
in Fig. 6a-c, the fluorescence and confocal-fluorescence microscopes were used
to observe
surface binding and internalization respectively. Fig. 6a shows the
fluorescence microscopy of
Molt-3 cell-clump after incubation at 37°C. Cell population-1 was more
susceptible to
suppression by low temperature than is cell population-2. Furthermore, flow
cytometry of
samples taken every 30 minutes showed a more noticeable onset of saturation of
binding in
population-1 than population-2. These results provide support for the idea
that the peptide is
internalized by a receptor-mediated process, and population-1 is more
sensitive to the energy-
dependent internalization than population-2. Additionally, the peptide has a
better affinity to
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both populations at 4 hour activation compared to the 48 hour activation,
indicating that the
activated state of the receptors diminished with time.
ICAM-1/LFA-1-mediated cell adhesion is dependent on divalent cations,
temperature and
an intact microfilamentous cytoskeleton (Detmer and Wright, 1988). Therefore,
the effect of
divalent cations on binding of FITC-cLAB.L to the PMA-activated cells was
evaluated. These
results are shown in Fig. 5c. A mixture of Mgz+ and Ca2+ improved binding of
FITC-cLAB.L 3.4-
fold and 1.8-fold to population-1 and -2, respectively, thereby demonstrating
that divalent cations
have higher influence on the FITC-cLAB.L binding to the receptors in cell
population-1 than the
receptors in population-2. Similarly, addition of Ca2+ cations alone caused a
1.5-fold binding
enhancement of FITC-cLAB.L on population-l, while no effect was detected on
population-2.
No peptide binding was observed with the addition of Mg2+ alone. However, Mg2+
addition did
increase the cell numbers of population-2 (data not shown). These results
demonstrate that
binding of cLAB.L to the surface receptor is influenced by the synergistic
effects of Ca2+ and
Mg2+ canons.
Microscopy of Peptide Binding and Internalization. In addition to flow
cytometer
analysis, binding and internalization of FITC-cLAB.L at 4°C and
37°C was examined using
fluorescence microscope (Figs. 6a-c) and confocal-fluorescence microscope
(Figs. 6d-f). Surface
binding of FITC-cLAB.L on an aggregate of Molt-3 cells is indicated by the
green fluorescence
with fluorescence microscope (Fig. 6a), and by the white to grey color with
confocal-fluorescence
microscope (Fig. 6d). Observation on single cells clearly showed a higher
fluorescence on the
cell from the 37°C binding experiment (Fig. 6b) than that of the
4°C binding experiment (Fig.
6c). Sectional images of cells from the two different temperatures indicate
that peptide
internalization took place at 37°C (Fig. 6e), but was almost negligible
at 4°C (Fig. 6f).
Discussion
These experiments evaluated the mechanisms of action of cLAB.L peptide [cyclo-
(1,12)-
Pen-Ile-Thr-Asp-Gly-Glu-Ala-Thr-Asp-Ser-Gly-Cys-OH] (SEQ ID No. 2) in
inhibiting ICAM-
1/LFA-1-mediated homotypic T-cell adhesion. As mentioned above, there are two
possible
mechanisms of action for this peptide in inhibiting homotypic T-cell adhesion.
One possibility
is that the cLAB.L binds to ICAMs (ICAM-1 and ICAM-3) to inhibit ICAMs/LFA-1
interaction.
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The second possibility is that the cLAB.L peptide can also bind to the (3-
subunit (CD 18) of LFA-
1, thereby disrupting the integrity of the LFA-1 and inhibiting the ICAMs/LFA-
1 interactions.
From these experiments, it was determined that cLAB.L peptide primarily binds
to the
D1 domain of ICAM-1. To a lesser extent, the cLAB.L peptide also binds to the
D1 domain of
ICAM-3 (Fig. 1 ). These results are supported by the ability of this peptide
to inhibit anti-ICAM-
1 and anti-ICAM-3 antibodies binding to the D 1 domain but not D2 domain. A
weaker blocking
effect of cLAB.L for binding of anti-ICAM-3 compared to anti-ICAM-1 may be due
to the higher
sequence and/or conformational selectivity of cLAB.L for ICAM-1 than ICAM-3.
Although 52%
homology between ICAM-1 and ICAM-3 is largely contributed by domain D2 (77%),
many
studies have pointed out that the important residues for LFA-1 binding to ICAM-
3 is in domain
Dl (Hotness, 1995; Bell et al., 1998). Thus, it is the non-conserved residues
in D1-domain of
ICAM-3 which contribute to integrin binding. These results also emphasize that
the ICAM-
1/LFA-1 interaction serves a different function than the ICAM-3/LFA-1
interaction.
Results with cLAB.L were also consistent with the previous study of a 9-amino
acid
linear peptide (TDGEATDSG), provided herein as SEQ ID No. 11. This peptide has
sequence
similarity with cLAB.L and inhibited binding of MgZ+/EGTA-activated, but not
phorbol 12, 13-
dibutyrate-activated T-cells, to ICAM-1 soluble protein. This peptide was also
the most potent
peptide found from the I-domain (McDowall et al., 1998). This linear peptide
was shown to
reduce binding of T-cells to ICAM-1 to 70% with 2 niM of linear peptides which
is higher than
the concentrations (80 and 160 ~M) of cLAB.L used to inhibit the binding of
ICAM-1-antibody
in this work. This indicates that the conformational rigidity of the cyclic
peptide cLAB.L
contributes to its binding selectivity (Benedict et al., 1994) to ICAM-1.
The cLAB.L peptide was also found to inhibit binding of anti-LFA-1 antibody to
the I-
domain (Fig. 1 ). This was due to the recognition of cLAB.L by the anti-LFA-1
antibody because
this peptide was derived from the I-domain of the LFA-1. In contrast, this
peptide cannot inhibit
antibody binding to the [3-subunit and other a-subunit of LFA-1 (Fig. 1).
These results suggest
that the primary mechanism of binding of this peptide is via the ICAM-1 D1
domain while
weakly binding via the ICAM-3 D1 domain but not to LFA-1.
The characteristics ofblocking ofunlabeled cLAB.L (Fig. 3a), saturationprofile
(Fig. 3b),
and marked differences in binding between 4°C and 37°C (Fig. 5a
and Sb) of FITC-cLAB.L
support the validity of FITC-cLAB.L as a model to study the binding of cLAB.L.
The binding
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of FITC-cLAB.L featured a two-population distribution and responded to the
duration of cell
activation (Figs. 2 and 4a), thus suggesting the occurrence of multiple and
dynamic states of
ICAM-1 and/or ICAM-3. A similar bimodal expression was observed as a result of
LFA-1
activation on T-cells with a population ratio 2.5-to-1 (Kurzinger et al.,
1981; Sanders et al.,
1988). In another case of x.5(31 integrin, integrin activation produces two or
more different
conformational states reflected in distinct stages in adhesion signaling
(Fault et al., 1993; Garcia
et a1.,1998). A similar explanation may be used for FITC-cLAB.L binding to
ICAMs. The short
(4 hour) and long (48 hour) activation times produced two different activated
states of ICAMs
with different affinities for FITC-cLAB.L. Longer PMA activation time (48
hour) gave a higher
number of cell population-2 than a 4 hour PMA activation time. The activated
state of ICAMs
in population-2 has higher affinity than that shown in population-1.
Population-2 may have an
activated ICAM state which forms clusters with high affinities for FITC-cLAB.L
peptide. On the
other hand, population-1 has activated ICAMs which spread out through the cell
surface with
lower affinities for FITC-cLAB.L than for that of in population-2. Thus, a
longer activation time
gave a change for membrane receptor and cytoskeleton rearrangements.
There are at least two possible explanations for why PMA activation modulates
the
binding of LFA-1 peptide (cLAB.L) in this case despite the suggested
constitutive-avidity of
ICAM-1 to LFA-1. The first possible explanation is an indirect mechanism of
activation based
on the facts that cell-cell adhesion via adhesion molecules consists of a
multistep process
involving a cascade of recognition and conformational states. In relation to
this event, it is
apparent that PMA activation of Molt-3 cells triggers other cellular
mechanisms such as
redistribution of some components ofplasma membrane or release ofreceptors
from cytoskeleton
which subsequently potentiate the binding of cLAB.L to ICAM-1. Reference may
be made to the
observed redistribution of lipid packing of the plasma membrane (Smith et
a1.,1993; Del Buono
et al., 1996) and the postulation on the release of receptors from
cytoskeletal constraint (Kucik
et al., 1996) due to cell activation. The second possible explanation is that
an active LFA-1,
resulting from PMA stimulation, directly triggers a more avid ICAM-1 in Molt-3
cells. This
explanation may be justified by the generality that T-cell stimulation could
lead to induction of
ICAM-1 on antigen-presenting cells. The use of PMA has an effect in increasing
the binding of
JY and SKW3 cells to purified LFA-1 within 0-200 sites Emi z of LFA-1 (Dustin
and Springer,
1989), thus indicating a response of ICAM-1 on the surface of T-cells to PMA
stimulation.
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Others have also used PMA to induce ICAM-1 expression in epithelial cells
(Bloemen et al.,
1993) and shown that PMA stimulation may cause a rapid and transient
phosphorylation on
serine residue of ICAM-3 (Lozano et al., 1992).
Temperature and other activators can influence binding properties of FITC-
cLAB.L
peptide to T-cells. For example, anti-CD3 antibody produced the same effect as
PMA. However,
divalent cations alone did not activate binding between FITC-cLAB.L and ICAMs.
For PMA and
anti-CD3 antibody, the fluorescence intensity of FITC-cLAB.L is higher at
37°C than at 4°C in
both populations. Thus, FITC-cLAB.L was internalized by ICAM receptors because
low
temperature (4°C) suppressed the ATP-dependent receptor-
internalization. Using fluorescence
microscopy, the binding of the labeled peptide is higher at 37°C than
at 4°C (Figs. 6a-cs).
Furthermore, internalization of this peptide was confirmed by the sectional
images from confocal
microscopy (Figs. 6d-f). The internalization of ligand by ICAM receptors may
be a process of
receptor recycling to control the receptor activity on the cell surface.
Therefore, the receptor-
ligand binding followed by endocytosis is one of the mechanisms for the
termination of receptor
activity (Almenar-Queralt et a1.,1995). This receptor-ligand internalization
process is also found
in other cell adhesion molecules such as integrin (Raub and I~uentzel, 1989)
and VCAM-1
(Ricard et al., 1998).
Binding of FITC-cLAB.L to the Dl domain of ICAM-1 on both populations of PMA
activated Molt-3 T-cells was enhanced dramatically by the presence of a Ca2+
and Mg'+ ion
mixture (Fig. 5c). However, Caz+ or Mg2+ alone gave a less dramatic or no
change in binding of
FITC-cLABLto ICAMs, respectively. Ca2+ improved binding of FITC-cLAB.L to
population-1
better than population-2. These results were supported by the suggestion that
a section of I-
domain binds to the ICAM-1 via divalent cations. Important residues on I-
domain of LFA-1 for
ICAM-1 binding have been proposed including Met'4°, Glu'46, Thrz43 and
Serz45 (Huang and
Springer, 1995), and recently Leu2os and Gluzø' (Edwards et al., 1998).
Meanwhile, there are
distinct but partially overlapping binding sites in the I-domain of LFA-1 for
ICAM-1 and ICAM-
3 (Binnerts et al., 1996) but none of the reported residues are part of the
cLAB.L sequence. It was
shown using CD, NMR and molecular modeling that cLAB.L can bind to Ca2+ and
Mg2~. The
binding properties of cLAB.L were also evaluated using docking experiments
between cLAB.L
and the D 1-domain of ICAM-1 in the presence and absence of divalent cations.
The peptide can
bind to ICAM-1 D1-domain via residue numbers 1 (Pen), 2 (Ile), 6 (Glu), and 10
(Ser).
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Conclusion
In conclusion, direct binding between cLAB.L and ICAM-1 on the surface of Molt-
3 T-
cells is primarily via binding to ICAM-1, and to a lesser extent, via ICAM-3.
The FITC-labeled
peptide binding is expressed in bimodal distribution of cell population and it
is saturable. The
FITC-labeled peptide can be inhibited by the unlabeled cLAB.L, thereby
indicating a receptor
mediated binding and no interference of FITC conjugation on the selectivity of
peptide binding
to the receptors. The peptide binding is influenced by a Ca2~ and Mg2+mixture,
and the peptide
may also be internalized by ICAMs. Accordingly receptor-mediated
internalization of cLAB.L,
combined with other methods such as drug conjugation, may have useful
applications in targeting
leukocyte-related diseases.
EXAMPLE 2
This Example confirmed that peptides derived from ICAM-1 could be conjugated
with
drugs and bind with cells expressing LFA-1 surface receptors. Furthermore, the
conjugate was
internalized and cells which internalized the conjugate were killed in a
concentration-dependent
manner. Due to the peptide portion of the conjugate and its affinity for LFA-1
receptors, the drug
portion of the conjugate was ineffective at killing cells which did not have
LFA-1 receptors.
Thus, peptide-drug conjugates were toxic toward selected targeted cells (i.e.
cells expressing
LFA-1 receptors) while the conjugate rendered the drug less toxic to .non-
targeted cells.
Accordingly, similar conjugates will be effective in treating diseases related
to LFA-1 expressing
cells (T-cell-related disorders).
Materials and Methods:
Synthesis of MTX-cIBR conjugate:
Methotrexate (10 mg, 0.02 mmol, Sigma A6770) was dissolved in milliQ water and
pH
of the solution was adjusted to 7Ø EDC (0.02 mmol). Next, 10 mg (0.008 mmol)
cIBR peptide
(SEQ ID No. 8) (0.008 mmol) was added into the MTX solution and stored
overnight at room
temperature. The reaction was concentrated in vacuo and the crude product was
purified with
reversed-phase HPLC with C-18 column (12 ~,M, 300 ~, 25 cm ~ 21.5 mm i.d.) and
UV
detection at 220 nm. A flow rate of 10 ml/min was used using a gradient run
using solvent A
which consisted of 0.1% TFA in HZO:acetonitrile (95:5) and solvent B which
consisted of 100%
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WO 02/09649 PCT/USO1/24088
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acetonitrile. Fractions were collected and subjected to analytical HPLC in
order to determine
product purity. Fractions containing the desired product were then pooled,
concentrated and
lyophilzed. The identity of the conjugate (MTX-cIBR) was confirmed by mass
spectrometry
(M+1 = 1611.4) and NMR.
Toxicity studies:
Toxicity studies were done by MTT assay, using both activated and non-
activated MOLT-
3 T-cells. Activation of the MOLT-3 T-cells was accomplished by using PMA
(phorbol 12-
myristate-13-acetate) at a final concentration of 0.2~uM. Aliquots of 200~L of
cell suspensions
containing 2.92 x 105 cells/ml nonactivated, or 2.47 x 105 cells/ml activated
MOLT-3 cells with
80% viability were plated onto a 48-well plate. Graded amounts of MTX, MTX-
cIBR, and/or
cIBR dissolved in PBS were then added to the plates and incubated for 48 hours
at 37°C in a
humidified 5% COZ/95% air atmosphere. As a negative control, a treatment with
lOmM
iodoacetamide (IAA) for 48 hours was used to determine the minimal value of
metabolic activity.
A positive control of cells only was used to determine maximum metabolic
activity. At the end
of the incubation period, 40u1 MTT (Sigma, St. Louis, MO) solution (Smg/ml
PBS) was added
to each well and further incubated for 2 hours at 37°C. Following
incubation, cell suspensions
were transferred to microcentrifuge tubes and centrifuged for 3minutes at
3,000 RPM before
removing the supernatants. The formazan crystals produced were solubilized by
the addition of
lml of 0.04 N HCl in isopropanol and subsequently sonicated for 7-10 minutes.
The
microcentrifuge tubes were again spun for 3 minutes at 3,000 RPM and 100uL
aliquots of
supernatant placed onto a 96 well plate and the absorbance at 540-nm
wavelength was
immediately measured using a Titertek Multiskan MCC/340. Metabolic activity
was calculated
as follows, where O.D. is the measured optical density at 540nm.
Metabolic activity = - (O.D.~-O.D.Samp~e)/( O.D.MTT - O.D.,,,~,)
Toxicity studies using MDCK cells proceeded as follows. To a 48 well plate,
300~,L aliquots of
cell suspension (8 x lO5ce11/ml) were grown to a confluent monolayer. Once a
complete
monolayer was reached, treatment with graded amounts of MTX, or MTX-cIBR were
added and
incubated as described. MTT assay was followed as above with one distinction:
the cell
CA 02417885 2003-O1-31
WO 02/09649 PCT/USO1/24088
-24-
monolayer, which remained attached to the plate after treatment, was scraped
off and transferred
to the microcentrifuge tube.
Results:
Results from toxicity studies show that the MTX-cIBR conjugate is toxic in a
concentration dependent manner and is approximately 19 fold more toxic than
MTX alone at a
similar concentration. To demonstrate that this increase in toxicity was due
to the cIBR binding
to MOLT-3, it was shown that the toxicity of MTX-cIBR can be inhibited in a
concentration
dependent manner by adding increasing amounts of cIBR.
The specificity of this drug-peptide conjugate for cells expressing LFA-1 was
demonstrated by comparing the toxicity in MDCK epithelial cells which do not
express LFA-1.
Treating MDCK cells with concentrations of MTX and MTX-cIBR which produced
nearly
completely toxic effects in T-cells showed that MTX-cIBR was approximately 5
times less toxic
than MTX alone. Without LFA-1 receptors, the conjugate is not carried inside
of the cells (e.g.
it is not internalized). Therefore, this binding specificity provides a great
deal of selectivity
because only T-cells express LFA-1. Accordingly, T-cell-related diseases can
be treated with
such conjugates without the adverse side effects which generally
accompanytreatment with drugs
effective against such diseases. Additionally, dosage levels may be lower due
to the high potency
of the drugs after internalization by the targeted cells. .
Thus, the present invention can be used to conjugate drugs with peptides in
order to treat
leukocyte-related diseases such as asthma, inflammations, Chron's Disease,
rheumatoid arthritis,
multiple sclerosis, ulcerative collitis, pemphigus vulgaris, pephigoid,
allergies, HIV-infections,
and epidermolysis. Additionally, drugs effective in treating such diseases can
be made less toxic
to non-targeted cells as well as more potent against targeted cells by
conjugating them with the
peptides of the present invention. Another use of the present invention
involves delivering HIV
protease inhibitors to T-cells. This method would help to avoid drug
resistance against HIV
protease inhibitors, which has been rising in patients with AIDS. Because the
replication of HIV
is in T-cells, the use of peptides targeted to T-cells (i.e. peptides which
bind to LFA-1 receptors)
is appropriate for suppressing HIV replication. Additionally, the efflux pump
receptors may be
entirely avoided using the methods and peptide-drug conjugates of the present
invention.
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Procedures and teachings of the following references are hereby incorporated
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30406.ST25.txt
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