Note: Descriptions are shown in the official language in which they were submitted.
PEPTIDES WITH al INTEGRIN SUBUNIT DEPENDENT
CELL ADHESION MODULATIN ACTIVITY
Cross-Referenced to Related ARnlications
The present application claims priority to U.S. provisional application
Serial No. 601072,119 filed on 22 January 1998, entitled "Peptides with Beta
Integrin Subunit Dependent Cell Adhesion Modulating Activity"; U.S.
provisional
application Serial No. 60/096,212 filed on 12 August 1998 entitled "Peptides
with
(31 Integrin Subunit Dependent Cell Adhesion Modulating Activity"; and U.S.
provisional application Serial No. 60/096,211 filed on 12 August 1998 entitled
"Peptides with ail Integrin Subunit Dependent Cell Adhesion Modulating
Activity" , the disclosures of which are herein incorporated by reference.
Background of the Invention
Cellular recognition of the extracellular matrix ("ECM") proteins and of
other cells has a complex molecular basis, involving multiple distinct cell
surface
receptors. Integrins are a family of receptors that are fundamentally
important for
mediating cell adhesion to ECM proteins. Tumor cells adhere to variety of ECM
proteins and molecules on other cells as they invade and metastasize. These
interactions of tumor cells have a profound effect on their phenotype.
Although its
exact role is complex and not completely understood, a4(31 integrin has been
implicated in tumor cell arrest and/or extravasation and is involved in tumor
ceil
invasion and metastasis. This integrin is expressed on many hematopoietic ~,
malignancies and also on tumors such as melanomas. a4~31 integrin is unique
among integrins in that it binds to both ECM components (e.g. fibronectin) and
Ig
superfamily adhesion receptors (e.g., VCAM-1) which are expressed on activated
endothelial cells and other cell types. a4~i 1 integrin also binds to itself
and
promotes homotypic cell adhesion. Although a role for a4(31 integrin has been
established in modulating various aspects of tumor cell biology, the
mechanisms by
which the function of the a4(31 integrin is modulated are complex and not well
understood. Understanding the nature of such interactions may help to explain
cell-
type specific behavior on ECM proteins that are often observed with integrins.
There is, accordingly, a continuing need to identify peptides capable of
modulating
a4~31 dependent cell adhesion as a means of furthering the understanding of
the
complex interacti'.~ns involv=7o this integrin.
:: " . . i>. ~....s..:l.:.,n..wvhc.,.~.~ ;'~,'.;;:
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BNSDOCID: <E2 99012360K>
Summary of the Invention
The present invention relates to peptides capable of modulating (31 integrin
subunit dependent cell adhesion. The peptides include a C-terminal amino acid
residue having a side chain which includes an aromatic group ("-Ar-") and an
amino
acid residue with a lipophilic alkyl side chain group ("-Lip-") as the
penultimate C
terminal residue. This C-terminal dipeptide sequence is referred to herein as
a
"LipAr motif." For example, suitable peptides of the invention may include a C-
terminal tyrosine residue and an isoleucine residue as the penultimate C-
terminal
residue, i.e., a C-terminal "IY motif' (Ile-Tyr). While the present peptides
may
include a relatively large number of amino acid residues, e.g., up to about
100 amino
acid residues or more, as disclosed herein even very small peptides which
include
the LipAr motif, such as the dipeptide Ile-Tyr and the tripeptide Arg-Ile-Tyr,
are
capable of modulating (31 dependent adhesion. The present peptides typically
have
no more than about SO and, preferably, no more than about 25 amino acid
residues.
The LipAr C-terminated peptides are preferably capable of inhibiting the X31
integrin ~
subunit dependent adhesion of cells, such as the a4~i 1 integrin dependent
adhesion
of Ramos cells and the a5~31 integrin dependent adhesion of erythroleukemic
cells
(e.g., the erythroleukemic cell line K562).
brief Description of the Drawing
Figure 1 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of a number of alanine knockout
analogs of FN-C/H V+y. FN C/H V+Y and a scrambled variant lacking a C-
terminal IY motif ("sV"; RPQIPWARY (SEQ ID N0:2)) were included as controls.
Figure 2 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of a number of alanine knockout
i
analogs of FN-C/H V+y. FN C/H V+Y and its scrambled analog sV were included ~,
as controls.
Figure 3 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of a number of fibronectin
fragments
tagged with a C-terminal tyrosine residue. FN C/H V+Y and its scrambled analog
sV were included as controls.
.~.:..:,~:::::;_::.,...:.~::::~:::f;::~:~:~:x::
.:::;.t(~"'~CA 0 2 319 2 0 7 2 0 0 0 0 7 21 : .;::
....................................................... ....
BNSDOCID: <E2 990t2360K>
WO 99/37669 PCT/US99/01236
3
Figure 4 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of a number of fibronectin
fragments
tagged with a C-terminal tyrosine residue. FN C/H V+Y and its scrambled analog
sV were included as controls.
Figure 5 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of two "IY" C-terminated peptides
and their corresponding "des-Y" analogs. FN C/H V+y and its scrambled analog
sV
were included as controls.
Figure 6 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of another "IY" C-terminated
peptide
and its corresponding "des-Y" analogs. FN C/H V+y and its scrambled analog sV
were included as controls.
Figure 7 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of a number of truncated analogs
of
FN C/Ii V+y. Controls included FN C/H V+Y and its scrambled analog sV.
Figure 8 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of a number of truncated analogs
of
FN C/H V+y. FN C/H V+y and its scrambled analog sV were employed as
controls.
Figure 9 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of "IY" and its component single
amino acid residues. FN C/H V+y and its scrambled analog sV were employed as
controls.
Figure 10 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of a several C-terminal
penultimate
substitution variants of FN C/H V+y. FN C/H V+Y and its scrambled analog sV
were employed as controls.
Figure 11 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of a several C-terminal
penultimate
substitution variants of FN C/H V+y. FN C/H V+y and its scrambled analog sV
were employed as controls.
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WO 99/37669 PCT/US99/01236
4
Figure 12 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of a several C-terminal
substitution
variants of FN C/H V+Y. FN C/H V+y and its scrambled analog sV were employed
as controls.
Figure 13 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of "IY" positional variants of FN
C/H
V+Y. FN C/H V+Y, its scrambled analog sV, and untagged FN C/H V
(WQPPRARI (SEQ ID NO: 37)) were employed as controls.
Figure 14 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of a negatively charged LipAr
terminated peptide. FN C/H V+y and its scrambled analog sV were employed as
controls.
Figure 15 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of PRARIY (SEQ ID NO: 24) and
I S PRARI (SEQ ID NO: 39). FN C/H V+y and its scrambled analog sV were
employed as controls.
Figure 16 shows a graph of % adhesion of the a5 (31 integrin dependent Mn+2
stimulated adhesion of erythroleukemic K562 cells to fibronectin ("FN") as a
function of the concentration of PRARIY (SEQ ID NO: 24) and PRARI (SEQ ID
NO: 39). FN C/H V+Y, its scrambled analog sV, RGD and BSA (bovine serum
albumin) were employed as controls.
Figure 17 shows a graph of % adhesion of the a5 ~i 1 integrin dependent Mn+z
stimulated adhesion of erythroieukemic K562 cells to fibronectin ("FN") as a
function of the concentration of RIY. FN C/H V+Y, its scrambled analog sV,
RGD,
CS1 and BSA (bovine serum albumin) were employed as controls.
Figure 18 shows a graph of % adhesion of the a2(31, a3~1 integrin dependent
human melanoma M14#5 cell adhesion to laminin ("LM") and type IV collagen
("TIV") and bovine serum albumin ("BSA").
Figure 19 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
IIICS-GST as a function of the concentration of all D-FN C/H V+Y (SEQ ID NO:1
),
and a retro inverso form of FN C/H V+Y (SEQ ID N0:40) versus various controls.
CA 02319207 2000-07-21
WO 99/37669 PCT/US99/01236
Detailed Description of the Invention
The present invention relates to peptides capable of modulating ~1 integrin
subunit dependent cell adhesion. These peptides include a C-terminal LipAr
motif
and are typically capable of inhibiting ail integrin subunit dependent cell
adhesion
5 and, in particular, of inhibiting a4~1 integrin dependent cell adhesion. The
present
peptides typically are also capable of inhibiting x2(31, a3 (31 and/or a5 ~ 1
integrin
dependent cell adhesion. As used herein, the term "LipAr motif' refers to a
dipeptide sequence in which C-terminal "Ar" residue has a side chain which
includes an aromatic group. Examples of suitable amino acid residues having an
aromatic group include tyrosine ("Tyr"), phenylalanine ("Phe"), histidine
("His"),
and tryptophan ("Trp"). The penultimate C-terminal "Lip" residue is an amino
acid
residue which includes a lipophilic alkyl side chain group. The a-carboxyl
group of
the C-terminal amino acid residue of the present peptides is typically in the
form of
a carboxylic acid (-COZH). In a preferred embodiment of the invention, the
"Lip"
and "Ar" residues are L-amino acid residues.
Examples of amino acid residues which have a lipophilic alkyl side chain
group include leucine ("Leu"), isoleucine ("Ile"), and valise ("Vaf'~.
Typically, the
lipophilic alkyl side chain group has a SCDC (cyclohexane-water side chain
distribution coefficient calculated as -RT In KD and expressed in kcaUmol) of
at least
about 3.0 and, preferably, at least about 4Ø For the purposes of this
application,
SCDC is defined according to Radzicka et al., Biochemistry, 27, 1664 (1988).
Where the SCDC of a particular alkyl side chain group is not known, the SCDC
value may be determined by measurement of the distribution coefficient between
wet cyclohexane and water or by a comparison of a compound containing the same
alkyl side chain group with other similar compounds using a hydrophobicity
scale
derived from HPLC retention according to the method of Parker et al.,
Biochemistry,
25, 5425 ( 1986). Despite its similarity in some respects to lipophilic alkyl
side chain
groups such as leucine, isoleucine, and valise, insertion of a methionine
residue at
the penultimate position (i.e., an "MY" C-terminal motif) resulted in an
inactive
analog.
Four C-terminal tyrosine tagged peptides having sequences corresponding to
different fragments of the fibronectin C-terminal heparin binding domain have
been
CA 02319207 2000-07-21
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. ~: . .. .. .. .. ..
.. . . . ..- . . -..
.... . . . . .. - . . .
. - . . . . . . . - . . ... ...
..
6 ... .... .. .. .. ..
reported to inhibit the binding of peripheral blood mononuclear cells and
spleen cells
to fibronectin and endothelial cell monolayers (see, e.g., Wahl et al., J.
Clin. Invest.,
~, 655-662 ( 1994)). Two of these peptides, FN-C/H I+Y and FN-C/H V+Y,
contain a C-terminal LipAr motif. The amino acid sequence of FN-C/H I+Y is
YEKPGSPPREV-VPRPRPGVY (SEQ ID N0:42). The amino acid sequence of
FN-C/H V+Y is WQPPRARIY (SEQ ID NO:I). The other two Tyr-tagged
fibronectin C-terminal heparin binding domain related peptides do not contain
a C-
terminal LipAr motif (both peptides end in "TY" (Thr-Tyr)). The amino acid
sequences of the these other two fibronectin C-terminal heparin binding domain
fragments are KNNQKSEPLIGR-KKTY (fN-CIH II+Y; (SEQ ID N0:43)), and
SPPRRARVTY (FN-C/H IV+Y; (SEQ ID N0:44)). Although all four Y-tagged
fragments inhibit leukocyte adhesion to fibronectin in vitro, only three of
the four, I
FN-C/H I+Y, FN-C/H II+Y and FN-C/H V+Y, are reported to exhibit anti-
inflammatory properties in an in vivo rat model. One of the four, FN-C/H V+Y,
has ',
also been reported to have to inhibit adhesion to VCAM, another extracellular
matrix protein. The reported results suggest that the biological activity of
the Y-
tagged fibronectin C-terminal heparin binding domain fragments is a functional
of °
the specific sequence of each of the peptides.
Several analogs were prepared to examine whether the inhibition of the ~i 1
integrin dependent cell adhesion is effected by the chirality of the
inhibitor. The all
D-form of FN-C/H V+Y (SEQ ID NO:1 ) and the all L-form of retro inverso FN-C/H
'
V+Y (SEQ ID N0:40; the all L-form of YIRARPPQW, the reverse primary
sequence of FN-C/H V+Y ) were prepared and examined in the 8A2 stimulated
Ramos cell adhesion assay. Neither of these two compounds inhibited Ramos cell
binding, suggesting that the present peptides preferably include the C-
terminal
LipAr motif in the form of L-enantioneric amino acid residues.
It has surprisingly been discovered, however, that the alanine knockout
analogs of FN-C/H V+Y which preserve the C-terminal LipAr motif (i.e., retain
the
C-terminal Ile-Tyr dipeptide sequence) are capable of inhibiting (31 integrin
dependent cell adhesion. As used herein, the term "alanine knockout analog"
refers
to an analog of a peptide in which a single residue has beer_ substituted by
an alanine ',
residue. Two of the alanine knockout analogs of FN-C/H V+Y have an alanine
..,:. :. . . .. _ _ , ..:
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__ . _
residue substituted for one of the arginine residues in the "PRARI" motif (Pro-
Arg-
Ala-Arg-Ile {SEQ ID N0:39)) within FN-C/H V+Y which has previously
demonstrated to be the implicated in stimulated focal contact formation (see,
e.g.,
Woods et al., Molec. Biol. Cell, 4, 605-613 ( 1993)). These alanine knockout
analogs have the amino acid sequences WQPPRAAIY (SEQ ID NO: 8) and
WQPPAARIY (SEQ ID NO: 17). Two of the other alanine knockout analogs,
AQPPRARIY (SEQ ID NO: 3), WAPPRARIY (SEQ ID NO: 4), also differ from
FN-C/H V+y by a non-conservative amino acid substitution (Ala for Trp and Ala
for Gln respectively).
As the examples described herein demonstrate, peptides which differ from
FN-C/H V+Y by a non-conservative amino acid substitution but retain the C-
terminal LipAr motif can be capable of modulating X31 integrin subunit
dependent
cell adhesion even if the overall physical properties of the peptide differ
substantially from FN-C/H V+Y. For example, an FN-C/H V+Y analog in which ',
the two arginine residues have been replaced by aspartic acid residues
inhibits the
8A2 stimulated adhesion of Ramos cells at least as strongly as FN-C/H V+Y. The
analog, WQPPDADIY (SEQ ID NO: 38), exhibits this activity even though it has
an.
overall net charge of -2 (in contrast to the +2 net charge of FN-C/H V+Y). I
Even more surprising than the fact that non-conservative substitution variants
of FN-C/H V+Y retain the capability of inhibiting X31 integrin subunit
dependent cell'
adhesion, is the fact that other short Lip Ar C-terminated peptides with
little or no
sequence homology to FN-C/H V+Y also possess this type of biological activity.
The results disclosed herein establish that even peptides with less than 50%
i
homology with the corresponding C-terminal portion of FN-C/H V+Y or FN-C/H
I+Y exhibit the capability of inhibiting X31 integrin subunit dependent
adhesion.
Examples of such peptides include ARITGYIIY (SEQ ID N0:14), RARITGYIY
(SEQ ID N0:13), PRQAWRPIY (SEQ ID N0:18), and RPAPQRWIY (SEQ ID
N0:20).
As used herein, the term "% homology" refers to the percentage of amino
acid residues of a peptide which are either identical to that of an original
peptide
sequence or differ from the original peptide sequence solely as a result of a
conservative ami.~.o acid substi~ution. For example, the peptide PAIFDRSCGS
>_::.;: v:-.'~.ca ~o'i3'io~ Zooo o~ Zi ':'
- - :.>.:
BNSDOCID: cE2 990123BOK>
12-fll~~'~~9~' P~TI~~ ~~ ~12~~ ~3~~~P~tEt?
... .... ..... ... .; ~ ~>:~;:~.:.. 1I II 11 11 I_ I
1 11 I I I I 1 1 ~ 1 I I
. ~ ~ I ~ ~ I I I 1 1 1 I I I
~ 1 I I I ~ I I 1 ~ 111 111
~ 1 1 1 1111 I_ I
1 I 111 11.11 11 11 11 11
~.
(SEQ ID N0:41) has 40% identity and 80% homology with respect to the peptide
sequence PKVMERTCDS (SEQ ID N0:45).
For the purposes of this invention, conservative amino acid substitutions are
'
defined to result from exchange of amino acids residues from within one of the
following classes of residues: Class I: Ala, Gly, Ser, Thr, and Pro
(representing
small aliphatic side chains and hydroxyl group side chains); Class II: Cys,
Ser, Thr
and Tyr (representing side chains including an -OH or -SH group); Class III:
Glu,
Asp, Asn and Gln (carboxyl group containing side chains): Class IV: His, Arg
and
Lys (representing basic side chains); Class V: Ile, Val, Leu, Phe and Met
(representing hydrophobic side chains); and Class VI: Phe, Trp, Tyr and His
(representing aromatic side chains). The classes also include related amino
acids ~ '
such as 3Hyp and 4Hyp in Class I; homocysteine in Class II; 2-aminoadipic
acid, 2- ',
aminopimelic acid, y-carboxyglutamic acid, (3-carboxyaspartic acid, and the
corresponding amino acid amides in Class III; ornithine, homoarginine, N-
methyl
lysine, dimethyl lysine, trimethyl lysine, 2,3-diaminopropionic acid, 2,4-
diaminobutyric acid, homoarginine, sarcosine and hydroxylysine in Class IV;
substituted phenylalanines, norleucine, norvaline, 2-aminooctanoic acid, 2-
aminoheptanoic acid, statine and ~i-valine in Class V; and naphthylalanines,
substituted phenylalanines, tetrahydroisoquinoline-3-carboxylic acid, and
halogenated tyrosines in Class VI.
In another embodiment of the present invention, the peptides contain no
more than 10 amino acid residues and have a sequence which does not correspond
substantially to the amino acid sequence of FN-C/H V+Y. As used herein, the
sequence of a particlar peptide does not correspond substantially to a
reference
amino acid sequence, if the particular peptide sequence has less than about
80%
identity and preferably less than about 50% homology with the reference
sequence.
One group of particularly suitable peptides of the invention are those which
include a C-terminal "IIY" motif, i.e., the sequence of the three C-terminal
most
amino acid residues is Ile-Ile-Tyr. One such peptide contains 9 amino acid
residues
and has the sequence ARITGYIIY (SEQ ID N0:14).
From a variety of standpoints, including cost, ease of production and overall
efficiency, smaller versions of the present peptides can offer many distinct
.........::...............CA 02319207 2000 07 21
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WO 99/37669 PCT/US99/01236
9
advantages. Thus, one group of particularly advantageous peptides of the
invention
include the C-terminal IY motif and contain no more than ten and, preferably,
no
more than six amino acid residues. In addition to the dipeptide Ile-Tyr,
suitable
examples of this group include PRARIY (SEQ ID N0:24), RARIY (SEQ ID
N0:25), ARIY {SEQ ID N0:26) and RIY.
Synthesis of Peptides
The peptides of the invention may be synthesized by the solid phase method
using standard methods based on either t-butyloxycarbonyI (BOC) or 9-
fluorenylmethoxy-carbonyl (FMOC) protecting groups. This methodology is
described by G.B. Fields et al. in Synthetic Peptides: A User's Guide, W.M.
Freeman & Company, New York, NY, pp. 77-183 (1992), the disclosure of which is
herein incorporated by reference. Peptide structures and purity can be
analyzed by
HPLC, and amino acid analysis and sequencing.
The present peptides may also be synthesized via recombinant techniques
well known to those skilled in the art. For example, U.S. Patent 5,595,887,
the
- disclosure of which is herein incorporated by reference, describes methods
of
forming a variety of relatively small peptides through expression of a
recombinant
gene construct coding for a fusion protein which includes a binding protein
and one
or more copies of the desired target peptide. After expression, the fusion
protein is
isolated and cleaved using chemical and/or enzymatic methods to produce the
desired target peptide.
The peptides described in the examples herein were synthesized by a solid
phase method. Tables I and II show the amino acid sequences of the peptides
described in the experiments reported herein. The following standard single
letter
code abreviations are used to designate the amino acid residues in the
peptides: A -
alanine, C - cysteine, D - aspartate, E - glutamate, F - phenylalanine, G -
glycine, H -
histidine, I - isoleucine, K - lysine, L - leucine, M - methionine, N -
asparagine, P -
proline, Q - glutamine, R - arginine, S - serine, T - threonine, V - valine, W
-
tryptophan, Y - tyrosine.
CA 02319207 2000-07-21
WO 99/37669 PCT/US99/01236
Peptide Carrier Conjugates
The peptides of the present invention may be employed in a monovalent state
(i.e., free peptide or a single peptide fragment coupled to a carrier
molecule). The
peptides may also be employed as conjugates having more than one (same or
5 different) peptide fragment bound to a single carrier molecule. The Garner
may be a
biological carrier molecule (e.g., a glycosaminoglycan, a proteoglycan,
albumin or
the like) or a synthetic polymer (e.g., a polyalkyleneglycol or a synthetic
chromatography support). Typically, ovalbumin, human serum albumin, other
proteins, polyethylene glycol, or the like are employed as the carrier. Such
10 modifications may increase the apparent affinity and/or change the
stability of a
peptide. The number of peptide fragments associated with or bound to each
carrier
can vary, but from about 4 to 8 peptide fragments per carrier molecule are
typically
obtained under standard coupling conditions.
For instance, peptide/carrier molecule conjugates may be prepared by
treating a mixture of peptides and carrier molecules with a coupling agent,
such as a
carbodiimide. The coupling agent may activate a carboxyl group on either the
peptide or the carrier molecule so that the carboxyl group can react with a
nucleophile (e.g., an amino or hydroxyl group) on the other member of the
peptidelcarrier molecule, resulting in the covalent linkage of the peptide and
the
carrier molecule. Preferably, the conjugate includes at least one peptide
fragment
which is not linked to the carrier molecule through an amide bond with the a-
carboxyl group of the C-terminal aromatic amino acid residue of the LipAr-
terminated fragment.
For example, conjugates of a peptide coupled to ovalbumin may be prepared
by dissolving equal amounts of lyophilized peptide and ovalbunun in a small
volume of water. In a second tube, 1-ethyl-3-(3-dimethylamino-propyl)-
carboiimide
hydrochloride (EDC; ten times the amount of peptide) is dissolved in a small
amount
of water. The EDC solution was added to the peptide/ovalbumin mixture and
allowed to react for a number of hours. The mixture may then dialyzed (e.g.,
into
phosphate buffered saline) to obtain a purified solution of peptide/ovalbumin
conjugate. Peptide/carrier molecule conjugates prepared by this method
typically
contain about 4 to 5 peptide fragments per ovaibumin molecule.
CA 02319207 2000-07-21
The invention will be further described by reference to the following detailed
examples. The examples are meant to provide illustration and should not be
construed as limiting the scope of the present invention.
~,,~am les
Assay for Inhibition of a4~31 Dependent Cell Adhesion
The assay described below was performed to determine whether specific
peptides were capable of inhibiting (31 integrin subunit modulated cell
adhesion and, ,
in particular, of inhibiting a4~31 dependent Ramos cell adhesion to IIICS-GST,
an .
a4(31 ligand. IIICS-GST is recombinantly produced fusion protein which
contains a
fragment from the type III CS region ("IIICS") of plamsa fibronectin fused to
glutathione-S-transferase ("GST"). The fibronectin fragment corresponds to
fibronectin amino acid residues 1961 to 2039 (sequence numbering for
fibronectin as.
designated in U.S. Patent 4,839,464) and includes the . '
DELPQLVTLPHPNLHGPEILDVPST (SEQ ID N0:46) amino acid sequence
("CS1"; fibronectin residues 1961-1985). A synthetically prepared peptide
having
the CS1 sequence has been shown to interact with a4(31 integrin on human
lymphocytes and promote cell adhesion but does not bind to heparin. In the
assay, a
96-well plate was coated with the substrate IIICS-GST. Ramos cells stimulated
with!
the ~i 1 activating monoclonal antibody 8A2 ("Ab 8A2") were preincubated with
one
of the peptides to be evaluated for their ability to adhere to IIICS-GST.
The fusion protein can be constructed by first using PCR primers to amplify
the coding sequence for residues 1961-2039 of plama fibronectin. The PCR
product
can be introduced into a suitable bacterial expression vector in frame with
the gene
for GST. The resulting vector can be transformed and expressed in a suitable
host
cell, such as E.Coli, to produce the fusion protein. If desired, the fusion
protein can
be purified using a glutathione column. In control experiments in which GST
alone
was coated onto a 96-well plate, no adhesion of 8A2 activated Ramos cells was
observed.
A 96-well plate was coated in triplicate with 50 Tl/well of IIICS-GST diluted
;
to 3-5 Tg/ml in PBS containing 1mM CaCIZ, MgCl2 ("PBS/cations") and incubated
'
overnight at 37°C. The IIICS-GST solution was removed and the wells
were
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~:::: ~~x_~."~ca o 30 ~ 2 0 0 0 - o ~ - 2 i
BNSDOCID: <E2 99012360K>
WO 99137669 PCT/US99/01236
12
blocked with 150p1/well of PBS/caoons containing 0.3% BSA for 1-2 hours at
37°C.
During the assay each well contained 100 p,l of Ramos cells (10,000
cells/well) with
or without peptide. Ramos cells were washed 3 times in adhesion media {DMEM
without phenol red containing 20mM HEPES and 3 mg/ml BSA). Cells were
counted and resuspended at 200,000 cells/ml. Concentrated Ramos cells were
labeled for 20 minutes at 37°C with 50 N,g of the fluorescent label
BCECF
resuspended in 30 Nl of dimethylsulfoxide ("DMSO"). The labeled cells were
centrifuged and resuspended in adhesion media at a concentration of 200,000
cells/ml. The cells were activated with the activating Ab 8A2 at a
concentration of 2
pg/ml purified IgG or 1:1000 culture supernatant.
While the cells were being labeled, inhibiting peptide dilutions were
prepared. Lyophilized peptides were weighed and resuspended in adhesion media
at
a stock concentration of twice the maximal inhibitory concentration. If a
peptide
was dii~cult to get into solution, it was initially resuspended in 30 Erl of
DMSO. If
a peptide needed to be suspended in DMSO, all of the epodes in that particular
experiment (including the controls) were suspended in 30 I,il of DMSO. Of the
peptides studied in the examples described herein, only the dipepode "Ile-Tyr"
required the use of this technique. The dose-dependent diluoons of peptides
were
prepared using adhesion media to dilute the stock peptide. Labeled cells were
nuxed
with peptide diluoons for 5 minutes at 37°C at a final concentration of
100,000
cells/ml and appropriate final peptide concentrations.
The blocking solution was removed from the 96-well plate and the
cell/peptide mixture is added at 100p,1/well ( 10,000 cells/well) and
incubated for 30
minutes at 37°C. An aliquot of standard cells/peptide (1000 ltl) was
placed at 37°C
for quantitating adhesion. Using aspiration, non-adherent cells were removed
from
the plate. The standard cells were centrifuged and resuspended in 1000 N.l of
adhesion media. The standard cells were added to empty wells at 100, 80, 60,
40, 20
and 0111/well representing 100%, 80%, 60%, 40%, 20% and 0% adhesion,
respectively. The plate fluorescence was read at excitation 485 and emission
530.
Cell adhesion was represented as percent input cells remaining adherent and
was
determined by a standard curve of the fluorescence obtained with the standard
cells.
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The experimental fluorescence readings were extrapolated from the standard
curve
to obtain percent adhesion.
Example 1 - Alanine Knockout Analogs of FN-C/H V+Y
To determine which amino acid residues were required for the.a4(31
dependent cell adhesion inhibiting activity of FN C/H V+Y, a series of analogs
having a single individual residue substituted by alanine were examined. The
results
are shown in Figures 1 and 2. The only alanine substitution which resulted in
loss of
the ability to inhibit adhesion was substitution of alanine for the isoleucine
residue at
the penultimate C-terminal position. All of the other alanine knockout
peptides
showed cell adhesion inhibition comparable to that of FN C/H V+y. As a
control, a
scrambled version of the FN C/H V+y sequence having a C-terminal tyrosine was
also examined (RPQIPWARY (SEQ ID N0:2)). The scrambled sequence, which
lacked the C-terminal LipAr motif, did not inhibit cell adhesion.
Example 2 - C-Terminal Tyrosine Tagged Fibronectin Fragments
A number of other C-terminal tyrosine tagged fibronectin fragments were
- also examined. These peptides corresponded to tyrosine tagged 8 residue
fibronectin
fragments which were incrementally displaced by one amino acid residue towards
the C-terminus of fibronectin (SEQ ID NOs 10-16 in Table I). The results are
shown in Figures 3 and 4. Unexpectedly, only those peptides which included the
C-
terminal LipAr motif were active in inhibiting a4~i 1 integrin dependent cell
adhesion. The most active peptide as far as cell inhibiting activity ended
with a C-
terminal IIY sequence
(-Ile-Ile-Tyr-). The full sequence of this peptide was ARITGYIIY {SEQ ID
N0:14).
The sequences of the other two Y-tagged fibronectin fragments which exhibited
a4~31 integrin dependent cell adhesion inhibition was RARITGYIY (SEQ ID
N0:13). The Y-tagged fibronectin fragments with a C-terminal Thr-Tyr ("TY"),
Gly-Tyr ("GY"), Tyr-Tyr ("YY") or Lys-Tyr ("KY") motif did not inhibit a4~i1
integrin dependent adhesion of the Ramos cells.
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14
Example 3 - Scrambled IY Tagged Sequences
To examine the effect of the N-terminal seven amino acid sequence on
inhibition of a4(31 dependent cell adhesion, three Ile-Tyr C-terminated
scrambled
versions of FN C/H V+y were examined. The activity of the eight amino acid des-
tyrosine analogs of the two scrambled peptides were also examined as controls.
The
results shown in Figures 5 and 6 clearly demonstrate that only the "LipAr" C-
terminated peptides ARITGYIIY (SEQ ID N0:14), PRQAWRPIY (SEQ ID N0:18)
and RPAPQRWIY (SEQ ID N0:20) inhibited cell adhesion. In each instance, the
identical primary amino acid sequence lacking the C-terminal tyrosine residue
did
not inhibit Ramos cell adhesion. Although not conclusive, this result strongly
suggests that there is little or no requirement for the N-terminal portion of
the
sequence in order for a peptide with a C-terminal LipAr motif to inhibit ~i 1
integrin
subunit dependent cell adhesion.
Example 4 - Inhibition by Short IY Terminated Peptides
To establish the minimum size of IY-peptide required for inhibition of a4~1
dependent cell adhesion, a study was carried out on a series of truncated FN
C/H
V+y analogs in which the N-terminal residue was systematically deleted. The
results are shown in Figures 7 and 8. The data establish that the "IY"
dipeptide itself
is capable of inhibiting a4(3I integrin dependent cell adhesion. The activity
of the
dipeptide was less than that observed with a number of longer IY terminated
peptides. The cell adhesion inhibiting activity of a 6 residue peptide, PRARIY
(SEQ
ID N0:24), and a 5 residue peptide, RARIY (SEQ ID N0:25), was comparable on
an equimolar basis to that of the 9 residue peptide, Y-tagged FN C/H V. These
two
shortened peptides both contain two arginine residues ("R") and having a net
charge
of +2 at neutral pH. Other short IY-terminated peptides with the sequences
QPPRARTY (SEQ ID N0:22), PPRARIY (SEQ ID N0:23), ARIY (SEQ ID N0:26)
and RIY also exhibited a4~i 1 integrin dependent cell adhesion inhibition
activity.
The cell adhesion inhibition activity of ARIY (SEQ ID N0:26) and RIY was
comparable on an equimolar basis to that of Y-tagged FN C/H V.
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Example 5 - Inhibition of Ile-Tyr versus Ile and/or Tyr
As a control experiment, the activity of the single amino acids, isoleucine
and tyrosine, alone and as part of a mixture, was also examined in the cell
adhesion
inhibition activity. The results shown in Figure 9 establish that even a
mixture of
5 the individual amino acids isoleucine and tyrosine is insufficient to
inhibit cell
adhesion at anything close to the concentration where the dipepdde "Ile-Tyr"
is
active.
Example 6 - Inhibition by "Xaa-Tyr" Terminated Peptides
10 To examine the structural requirements of the "LipAr" motif, the inhibition
of a4(31 dependent ltamos cell adhesion was examined for a number of FN C/H
V+Y analogs with substitutions at the penultimate C-terminal amino acid
residue.
The results are shown in Figures 10 and 11. The two analogs with a lipophilic
aliphatic side chain residue (Leu or Val) substituted at the penultimate C-
terniinal
15 position, WQPPRARLY (SEQ ID N0:28) and WQPPRARVY (SEQ ID N0:29),
had cell adhesion inhibiting activity comparable to that of FN C/H V+Y. The
corresponding analogs with a basic residue (Lys), a hydroxy side chain residue
('IMr), a methionine residue (Met) or an alanine residue (Ala) in penultimate
C-
terminal position were substantially inactive in the assay.
Example 7 - Inhibition by C-Terminal Varaints
To examine the structural requirements of the "LipAr" motif, the inhibition
of a4~i1 dependent lZamos cell adhesion was examined for a number of FN C/H
V+Y analogs with substitutions at the C-terminal amino acid residue. The
results
are shown in Figure 12. The two analogs with a C-terminal amino acid residue
having a side chain which includes an aromatic group (Phe or Trp) at the C-
terminal
position, WQPPRARIF (SEQ ID N0:32) and WQPPRARIW (SEQ ID N0:33), had
cell adhesion inhibiting activity comparable to that of FN C/H V+Y.
Example 8 - Inhibition by C-Terminal Variants
The inhibition of a4(31 dependent Izamos cell adhesion for a number of FN
C/H V+Y analogs with differently positioned IY motifs was examined. The
results
CA 02319207 2000-07-21
12~1~;-~'~ X99 < PI~'1"S ~~ 01'~3~ ' 13~~P~M I~
::: ~ , , .. .. .. .. ..
. .. . . . .. . . ..
. ., , . .... . . . . .. . . .
. . . . . . . :.. ...
' . . . . ..
... .... .. .. .. ..
16
are shown in Figure 13. Peptides with the "IY" motif at the N-terminus,
IYWQPPRAR (SEQ ID N0:34), or in the middle of the peptide, WQPIYPRAR
(SEQ ID N0:35) were inactive in the assay. Switching the order of the Ile and
Tyr
residues at the C-terminus of an FN C/H V+Y analog, WQPPRARYI (SEQ ID
NO:36), also resulted in a peptide which was inactive in the a4~31 dependent
Ramos
cell adhesion inhibition assay. Finally, control peptide having,the tyrosine
tag
removed from the C-terminus of FN C/H V+Y, WQPPRARI (SEQ ID N0:35), was
also inactive in the assay.
Example 9 - Inhibition of Adhesion by a Negatively Char,,g " ' Ar" Peg
All the the LipAr terminated peptides described in the above examples which'
were active in the Ramos cell adhesion inhibition assay have a net positive
charge.
In order to determine whether a net positive charge is required for this
activity, an
FN-C/H V+y analog in which the 2 arginines (positively charged) were replaced
by ',,
aspartic acid residues (negatively charged) was evaluated. Importantly, the C-
terminal "LipAr" motif ("IY") was retained in this peptide, WQPPDADIY (SEQ ID
NO: 38}. Figure 14 clearly demonstrates that substitution of the arginines
with
aspartic acid residues does not alter the ability of the peptide to inhibit
(31 integrin
subunit dependent adhesion, thereby further demonstrating the importance of
the
"LipAr" motif to this activity.
Example 10 - Inhibition of adhesion ~y~R_ARIY versus PRARI
In an experiment which further demonstrated the correlation of a C-terminal
LipAr motif with (31 integrin subunit dependent adhesion adhesion, the peptide
PRARIY (SEQ ID NO: 24) and the corresponding sequence lacking the terminal
aromatic residue ("Tyr") were evaluated for their ability to inhibit adhesion
in the
Ramos cell assay. Consistent with the previous results demonstrating the
requirement for a C-terminal "LipAr"motif, PRARIY but not PRARI was able to
inhibit a4~ 1 mediated Ramos cell adhesion to IIICS-GST (see Figure 15).
:: . ~. . ;: . . r.'.i. ~.r.~.~ :.
.:.....:......~:::::.:r::~::r.~. . . . i:~ .1. ~..~. .:. ..
_ _ :. ::.
::::::............... 0 7 21
~..........~.CA~ 0 3 9 07 2000 ''v
BNSDOCID: <E2 990123BOK>
WO 99/37669 PCT/US99/O1Z36
17
Example 11 - Inhibition of a5~i 1 Integrin Dependent Adhesion
To determine whether inhibition of adhesion by C-terminal isoleucine-
tyrosine is restricted to a4(31 integrin adhesion, peptide RIY and peptides
ending in
isoleucine-tyrosine (PRARIY) and isoleucine (PRARI) were evaluated for the
ability
to inhibit a5(31 integrin-mediated cell adhesion. The cell adhesion assay was
carried
out as described above. K562 cells stimulated with 1mM MnCl2 were preincubated
with the indicated concentration of peptide and allowed to adhere to FN. The
results
are shown in Figures 16 and 17, where (V), (SV) represent peptides FN-C/H V-Y
and scrambled FN-C/H V-Y, respectively. Each data point represents the mean of
triplicate determinations and the error bars represent the standard deviation
of the
mean. The solid black line represents adhesion to the negative control
substrate,
BSA.
Adhesion of the erythroleukemic cell Iine K562, which expresses a5(31 but
not a4/31 integrin, to FN is completely inhibited following preincubation with
soluble RGD or FN-C/H V-Y at the maximal concentration tested, 0.84 mM
(Figures 16, 17). The half maximal inhibitory concentration for soluble
peptides
- RGD and FN-C/H V-Y was 0.1 mM and 0.2 mM, respectively. Furthermore,
addition to peptides RIY or PRARIY, but not PRARI, completely inhibited a5(31
dependent K562 adhesion to FN with at the maximal concentration tested, 0.84
mM.
The half maximal inhibitory concentration of both RIY and PRARIY was
approximately 0.2 mM, similar to that observed for RGD and FN-C/H V-Y. These
results demonstrate that, like peptide FN-C/H V-Y, the smallest, maximally
active
peptide RIY and peptides ending in isoleucine-tyrosine, but not isoleucine
(PRARI),
inhibit a5~31 (in addition to a4~31) integrin-mediated adhesion (see Figures
16 and
17).
Example 12 - Inhibition of a_2~~a_3~i1 Integrin Dependent Adhesion
An experiment was conducted to examine the ability of FN-C/H V+Y (SEQ
ID NO:1 ) to inhibit a2~ 1, a3 (i 1 integrin dependent cell adhesion using an
assay
based on human melanoma cells (M14#5).
Laminin, type IV collagen and BSA were coated overnight in a 96 well
microtiter plate at 10 pg/ml and blocked with 0.3% BSA. M14#5 cells were
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WO 99!37669 PCT/US99/01236
18
preincubated with 0.5 mg/ml of peptide (equivalent to 0.42 mM FN-C/H V and
scrambled FN-C/H V and 0.17 mM CSI) and allowed to adhere to substrates for 30
minutes.
Soluble peptide FN-C/H V inhibited human melanoma M14#5 cell adhesion
to laminin and type IV collagen coated substrates, whereas scrambled FN-C/H V
has
no effect (see Figure 18). This adhesion is dependent on a2~i1 and a3(31
integrin as
determined using specific anti-integrin blocking mAbs (data not shown).
Example 13 - Influence of Chirality on Inhibition of _a~l Integrin Dependent
Adhesion
The potential chiral dependence of (31 integrin dependent cell adhesion by
the present peptides was examined by preparing the all D-form of FN-C/H V+Y
(SEQ ID NO:1 ) and the all L-form of retro inverso FN-C/H V+Y (SEQ ID N0:40;
the all L-form of YIRARPPQW, the reverse primary sequence of FN-C/H V+Y ).
These two compounds were examined in the 8A2 stimulated Ramos cell adhesion
assay.
The results (shown in Figure 19) show that there is a chiral dependence on
the adhesion inhibitory activity of FN-C/H V-Y. This suggests that C-terminal
isoleucine-tyrosine should be in the L-enantiomeric form since D-amino acid FN-
C/H V-Y and a retro-inverso FN-C/H V-Y (consisting of the L-amino acids in
reverse primary sequence) are both unable to inhibit adhesion.
Racnos cells and the (31 integrin stimulatory mAb 8A2 were preincubated
with the indicated concentration of synthetic peptide prior to addition to rCS
1 coated
wells. (V), (sV) represent peptides FN-C/H V-Y and scrambled FN-C/H V-Y,
respectively. Each data point represents the mean of triplicate determinations
and
the error bars represent standard deviation of the mean. Background Ramos
adhesion to GST is represented in the solid black line.
Example 14 - Inhibition of _a1~2 Integrin Dependent Adhesion
To determine whether inhibition of adhesion by soluble FN-C/H V is specific
for bl integrins, the ability of this peptide to inhibit (32 integrin-
dependent adhesion
was also evaluated. For these studies the adhesion of the B-cell line M16B
(which
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WO 99/37669 PCT/US99/01236
19
both express functional a4(31 and al/32 integrin) to purified rCS 1 or
recombinatnt
ICAM in the presence of soluble FN-C/H V. As expected, a4(31 integrin-
dependent
Mn+2 stimulated M 16B adhesion to rCS 1 was completely inhibited by soluble FN
C/H V and CS 1. However, a 1 ~i2 (LFA-1 ) integrin dependent adhesion to rICAM
was not inhibited by soluble FN-C/H V, although this adhesion can be inhibited
by
an anti-~i2 integrin blocking mAb.
The invention has been described with reference to various specific and
preferred embodiments and techniques. However, it should be understood that
many
variations and modifications may be made while remaining within the spirit and
scope of the invention.
Table I - Peptide Sequences
SEQ ID NO: Amino Acid Sequence Net Charge
2 RPQIPWARY +2
3 AQPPRARIY +2
4 WAPPRARIY +2
5 WQAPRARIY +2
6 WQPAR.ARIY +2
7 WQPPAARIY +1
8 WQPPRAAIY +1
9 WQPPRARAY +2
10 QPPRARITY +2
11 PPRARITGY +2
12 PRARITGYY +2
13 RARITGYIY +2
14 ARITGYIIY +1
15 RITGYIIKY 0
16 ITGYIIKYY -1
17 PRQAWRPI +2
18 PRQAWRPIY +2
19 RPAPQRWI +2
20 RPAPQRWIY +2
21 ARITGYII +1
22 QPPRARIY +2
23 PPRARIY +2
24 PRARIY +2
25 RARIY +2
26 ARIY +1
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Table I (coat.) - Peptide Sequences
SEQ ID NO: Amino Acid Sequence Net Charge
27 WQPPRARKY +1
5 28 WQPPRARLY +2
29 WQPPRARVY +2
WQPPItARTY +2
31 WQPPRARMY +2
10 32 WQPPRARIF +2
33 WQPPRARIW +2
34 IYWQPPRAR +2
WQPIYPRAR +2
36 WQPPRARYI +2
15 37 WQPPRARI +2
38 WQPPDADIY 2
39 PRARI +2
YIRARPPQW +2
CA 02319207 2000-07-21
