CA2319207A1

CA2319207A1 - Peptides with .beta.1 integrin subunit dependent cell adhesion modulating activity

Info

Publication number: CA2319207A1
Application number: CA002319207A
Authority: CA
Inventors: James B. Mccarthy; Leo T. Furcht; Angela Brienzo
Original assignee: Individual
Current assignee: University of Minnesota
Priority date: 1998-01-22
Filing date: 1999-01-21
Publication date: 1999-07-29
Also published as: JP2002501082A; WO1999037669A1; EP1049710A1

Abstract

Peptides capable of modulating .beta.1 integrin subunit dependent cell adhesion which includes a C-terminal aromatic amino acid residue and an amino acid residue having a lipophilic alkyl side chain as the penultimate Cterminal residue are provided. These "LipAr" C-terminated peptides are typically capable of modulating the .beta.1 integrin subunit dependent adhesion of cells, such as Ramos cells.

Description

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.
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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.
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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.

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.

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 12 ~8~9 ~~'1"~S 4~ 41~~ )a3E~~~'A~1I~
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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
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(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 BNSDOCID: <E2 99012360K>

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.

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.

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 .: :::::.~'~'f'::.:' A::ii*~71:: . . . ~~ : .:
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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.

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.

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.

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 12~1~;-~'~ X99 < PI~'1"S ~~ 01'~3~ ' 13~~P~M I~
::: ~ , , .. .. .. .. ..
. .. . . . .. . . ..
. ., , . .... . . . . .. . . .
. . . . . . . :.. ...
' . . . . ..
... .... .. .. .. ..

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>

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 WO 99!37669 PCT/US99/01236 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 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 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 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 39 PRARI +2 YIRARPPQW +2

Claims

WHAT IS CLAIMED IS:

1. A peptide having no more than six amino acid residues which comprises a C-terminal LipAr motif.

2. The peptide of claim 1 comprising a penultimate C-terminal Lip residue selected from the group consisting of Ile, Val and Leu.

3. The peptide of claim 1 comprising a C-terminal Ar residue selected from the group consisting of Tyr, Phe, His and Trp.

4. The peptide of claim 1 comprising a C-terminal motif selected from the group consisting of Ile-Tyr, Ile-Phe, Ile-Trp, Val-Tyr and Leu-Tyr.

5. The peptide of claim 1 comprising a C-terminal Ile-Ile-Tyr motif.

6. The peptide of claim 1 having the sequence Pro-Arg-Ala-Arg-Ile-Tyr (SEQ
ID NO:24), Arg-Ala-Arg-Ile-Tyr (SEQ ID NO:25), Ala-Arg-Ile-Tyr (SEQ ID
NO:26), Arg-Ile-Tyr or Ile-Tyr.

7. The peptide of claim i wherein said peptide is capable of modulating .beta.1 integrin subunit dependent adhesion.

8. The peptide of claim 7 wherein said peptide is capable of inhibiting .beta.1 integrin subunit dependent adhesion.

9. The peptide of claim 7 wherein said peptide is capable of modulating .alpha.4.beta.1 integrin dependent adhesion.

10. The peptide of claim 9 wherein said peptide is capable of inhibiting .alpha.4.beta.1 integrin dependent cell adhesion.

11. The peptide of claim 10 wherein said peptide is capable of inhibiting .alpha.4.beta.1 integrin dependent adhesion of Ramos cells to .alpha.4.beta.1 integrin binding fibronectin fragments.

12. A peptide having no more than about 10 amino acid residues which comprises a C-terminal LipAr motif and has no more than about 80%
identity with WQPPRARIY (SEQ ID NO:1), wherein said peptide does not contain a D-amino acid residue.

13. The peptide of claim 12 comprising a C-terminal sequence selected from the group consisting of ARITGYIIY (SEQ ID NO:14), RARITGYIY (SEQ ID
NO:13), PRQAWRPIY (SEQ ID NO:18), RPAPQRWIY (SEQ ID NO:20), and WQPPDADIY (SEQ ID NO:38)).

14. The peptide of claim 12 having no more than about 50% homology with WQPPRARIY (SEQ ID NO:1).

15. A peptide having no more than about 50 amino acid residues which comprises a C-terminal sequence selected from the group consisting of AQPPRARIY (SEQ ID NO:3), WAPPRARIY (SEQ ID NO:4), WQAPRARIY (SEQ ID NO:5), WQPARARIY (SEQ ID NO:6), WQPPAARIY (SEQ ID NO:7), WQPPRAAIY (SEQ ID NO:8), ARITGYIIY (SEQ ID NO:14), RARITGYIY (SEQ ID NO:13), PRQAWRPIY (SEQ ID NO:18), RPAPQRWIY (SEQ ID NO:20), WQPPRARLY (SEQ ID NO:28), WQPPRARVY (SEQ ID NO:29), WQPPRARIF (SEQ ID NO:32), WQPPRARIW (SEQ ID NO:33), and WQPPDADIY (SEQ ID NO: 38).

16. The peptide of claim 15 having the sequence AQPPRARIY (SEQ ID NO:3), WAPPRARIY (SEQ ID NO:4), WQPPAARIY (SEQ ID NO:7) or WQPPRAAIY (SEQ ID NO:8).

17. The peptide of claim 15 having the sequence WQAPRARIY (SEQ ID NO:5) or WQPARARIY (SEQ ID NO:6).

18. The peptide of claim 15 having the sequence ARITGYIIY (SEQ ID NO:14), or RARITGYIY (SEQ ID NO:13).

19. The peptide of claim 15 having the sequence PRQAWRPIY (SEQ ID
NO:18), or RPAPQRWIY (SEQ ID NO:20).

20. The peptide of claim 15 having the sequence WQPPRARLY (SEQ ID
NO:28), WQPPRARVY (SEQ ID NO:29), WQPPRARIF (SEQ ID NO:32), or WQPPRARIW (SEQ ID NO:33).

21. The peptide of claim 15 having the sequence WQPPDADIY (SEQ ID NO:
38).

22. The peptide of claim 15 having no more than about 15 amino acid residues.

23. A method for modulating the adhesion of cells to a substrate comprising:
combining a peptide with a suspension of said cells to form a modified cell suspension, wherein the peptide has no more than about 6 amino acid residues and comprises a C-terminal LipAr motif; and contacting the modified cell suspension with the substrate.