CA2148383A1 - Peptide inhibitors of fibronectin and related collagen-binding proteins - Google Patents

Abstract

Peptides derived from the second type 1 repeat of human endothelial cell thrombospondin which bind to the gelatin-binding domain of fibronectin have been isolated and synthetically produced. The peptides can be used to bind to fibronectin or other related collagen-binding proteins to inhibit fibronectin-dependant cell adhesion to collagen matrices and to inhibit interactions with collagen of other proteins that share homologies with the gelatin-binding domain of fibronectin.

Description

WO94/11395 21 ~ 8 3 8 3 PCT/US93/11104 ) PEPTIDE INHIBITORS OF FIBRONECTIN AND RELATED COLLAGEN-BINDING PROTEINS
FIELD OF ~E IN~N~ION

The present in~ention relates to peptides ~eri~ed ~rom a domaln of throm~ospondin mediating interaction of thrombospondin with fi~ronec~in, which peptldes bind :~-specif Lcally to fi~ronectin and inhibit fibronectin bindLng to collagen, as well as to pharmaceutical -compositions containi~g~these peptLdes, and methods for inhibiting fibronec~in bindLng to collagen using the~e peptide~

~ GRO~D OF T3E I~VENTIO~
,~, - ~hrombo3pondin i~ a multi-functional protein capable I of interacti~g ~ith numerou~ macromolecules, e~g.
fi~ronecti~ (Lahav~ et al., 1984, Cell 31:2~3-262; Lahav, : :
et aI. 1984, Eur. J. Biochem. 1451:151-1~6), hep~rin ~ (Lawler, et al. 1981, Thromb. Res. 22:267-279) and ~:~ collagen (Laha~ et al. 1984, Cell 3l:253-262, ~umby, et al., 1984 J. Cell. Biol. 98:646-652l. All of these 1~ ~molecules~are~constituents of the extracellula~ matrix, ¦: suggesting that ~hrombo~pondin fonms comp~exes~with other ~ 20` l ~ matrix``components following its deposltion Lnto the ~ ` :
mat-ix.
Gelder and ~ Brown : reported that thrombospondin inhibits interactions of: fibronectin with gelatin (Gelder, F.B. and Brown, S.T. 1987, J. Lab.~: Clin. ~ed. -~
110:548-557). Platelet ~hrombospondin and an :
~ ~ unidentifie:d but ~iologically sLmilar plasma protein were :
: ~ : :

W~94/11395 PCTIU593/11~04 21 ~ 8~3 i - ~ :
; 1 2 shown to inhibit the gelatin-binding acti~ity o~
fibronectin; howe~er, the domains or sequences of throm~ospondin responsible for the~interaction remained unknown. The interactive region has been implicated to be a different site~on fibronectin, than the fibrin nding domain~(Homandberg,~ G.A. and Kramer-Bjerke, J.
1987, Thromb. Res. 48:329-33~) and at least two disti~ct ~ ~ .
domains of thrombospondin ha~e ~been shown to bind ~ibronectLn (Dardik,~ R.~ and~Lahaw, J. 1989, Eur. J.
~10 Biochem. 185:581-~5~88;).~ Fibronectin and heparin co~pete ~for binding; to~the 27~kDa fragment of thrombospondin suggesting tha~ th~se two protein~ share a common or closely~oriented binding site ~within the N-terminal domain of thrombospondin.; Thrombospondin has also been ~shown to disrupt focal~contact adheslons of endo~helial ~::
cells~attached~to a~fibronectln matrLx~(Murphy-Ullrich, ;J.E.,~ and~Hook, ~. 1989, J.~Cell. Biol. 109:~1309-1319)~.

The~mechanism~of~thls~effect~ls unknown, but lnhibition ` of ~ the~ acti~ity~ of ~thrombospondln~ by sulfated;

20 ~ polysaccharides~suggèst~ed that~the heparln-bLnding domain of~th~o~ospondin~ s~involved.

A ~n ~ er~of bio~logically~actlve~ peptldes ~from ; thrombospondin~have been~identlf~ied~and~ lsolated.~ Two ; peptides~,~; ~ gGl~spAl~a~ Lawler,~J and~H~es~, RØ 1986, 25~ J;.~ell Blol~ 03:16~35-1~641);~and~ValThrCysGly (Prater, et~

al~ ;l9~91,~ J~.Cell~Biol~. 112:~`1031-1040) are~proposed~o be~ gand~ or~;the LnteracLlo~ of -hromb~osp-ndin~with 2 1 ~ 8 3 ~ 3 PCT/U~93/11104 -! 3 protein receptors on the ~ell surface. The thrombospondin peptide TrpSerProTrpSer ~Guo, et al. 1992, Proc. NatO Acad. Sci. USA, 89:3040-3044) binds to heparin and sulfatide. However, there are no known thrombospondin peptides that are capable of binding to ibrone~tin or related collagen binding proteins.
Fibronectin has been implicated in a variety of cell contact processes ~ including cell attachment and migration. Fi~ronectln interac~s with collasen through th~ "gelatin bindlng domain~' of fibr~nectin and this interaction bekween collagen and fibronectin is fundamental ~o the organization of extracellular ma~rices : and the beh vior of these cells on substra~es (Vaberir et a},, ~378, Proc. Natl. Acad. Scio USAr 75 4944-4948)~
Fibronectin is essential for ~h~ at~achment and migration of many cells, including various tumor and cancer cells.
Aocordi~gly, there is a need for inhibitcrs of :: .
fibronectin that will bLnd to fibronectin or related collagen-binding pro~ins wi~h high affinity. There is ~-,:
particularly a need for such inhibitors which will bind to fibronectin or related collagen-binding proteins to :preYent the fibronectln-dependent cell adhesion to col}agen matrices and to inhibit interaction with coIlagens of other proteins that share homologies with : 25 ~ the gelatln-binding domaln of fibrsnectin. -~
:
:: ~: It is, therefore,:an object of the present invention ~ ~ to pro~ide highly effective peptides havin~ sequences : - .

.
,.

3~5 PCT/US93/11104 ! ; . 2 1 4 8 3 8 3 which bind specifically and with specific affinity to fibxonectin or other proteins ha~ing homologies with the fibronectin gelatin-binding domain so as to inhibit fibronec~in-dependent cell adhesion to collagen matrices and to inhibit interaction of other proteins ha~ing homologies with the fibronectin gelatin-binding domain with collagens. ~
It is:a further object of the preaent Ln~ention to pro~ide pharmaceutical compositions containing at leas~
one peptide which has a high affinlty to fibron~ctin or other protein having homologies with ~he fibronectin : .:
gelatin-binding domain. :
It is yet another object of the pre~ent Lnvention to ~ ~-: provid~ a :method for bindlng; fibronectin or other ; 15 proteins havi~g~homologies with the.:fibronectin gelatin-binding domain i~ a patient in need thereof. : .
: ~ These and~other objects of the lnvention will becomP~
more; appare~t from the: following description and ..
preferred embodlments and the aid of the accompanylng .
~:: 20 drawings.

~`.'.1 ~094/1~395 PCT/US93/11i04 ,, '21~

BRIgF D~SCRIPTION OF ~E DRA~I~GS .
Figure 1 is a graph of the binding of fibroneckin to various immobilized thrombospondin pep~ides, i.e. ~-SerHisTrpSerProTrpSerSer (SEQ ID NO: 8) (o~;
LysArgPhe~ysGlnAspGlyGlyTrpSerHisTrpSerProTrpSerSer(SEQ
. ID NO:7)(o); LysArgPheLysGlnAspGlyGlyTrpSerHisTrpSerPro (SEQ ID NO: S) ~); GlyGlyTrpSerHisTrp (SEQ ID NO. 1) (~); and GlyGlyTrpSerHisTrpSerPro (SEQ ID NO: 4) (~).
Figure 2 is a graph of the specificity of inhibition . .
of fihronec~in binding to the thro~bospondin peptide GlyGlyTrpSerHisTrp (SEQ ID NO: 1) by ~y~thetic peptides. .
In Panel A the peptides are GlyGlyTrpSerHi5Trp (SEQ ID
NO~ ); GlyGlyTrpSerLysTrp (SEQ ID NO: 9) (~
GlyGlyTyrSerHisTrp (SEQ ID NO: 10~ (~); and GlyGlyTrpSerHisTyr (S Q ID NO: l}) (-); Panel B^
Gl~GlyTrpSerHisTrp (SEQ ID NO: l~ ; GlyGlyTrpThr~isTrp ~.
(SEQ ID NO: 12) (-~; GlyGlyTrpAlaHisTrp (SEQ ID NO: 13 (~); GlyTrpSerHisTrp (SEQ ID NO: 14) (+) and AspG~y~rpSerHisTrp (SEQ ID NO: 15) (~).
Fîgure 3 is a graph of the inhibition of fibronecti~
.
binding to thrombospondin peptide GlyG~y~rrpSerHisTrp by :. ~
:
soluble fibronectin, thrombospon~in and peptide ~ GlyGlyTrpSerHisTrp (SEQ ID NO: 1), GlyGlyTrpSerHisTrp : (SEQ I~ N~ ), fibronectin (~) and thrombospondin `~ 25 (~
: . Figure 4 lS a graph of the inhibltion of fibronectin binding to thrombospondin peptide GlyGlyTrpSerHisTrp by - - :

WO 94~11395 PCT/US~3/11104 21~8383 . . 6 proteoiytic fragments of fibronec~in: 30 kDa gela~in-binding domain (~), 31 kDa fibrin-binding domain (O), 40 kDa heparin-binding domain (-), 120 kDa cell-binding domain (~) and intact fibronectin (~
Figure 5 is a graph of the inhibition of fibronectin binding to gelatin by peptlde GlyGlyTrpSerHisTrp (SEQ ID
NO~
Figure 6~Aj is a~bar graph of the inhibition of ; ., fibronectin-mediaked melanoma cell ~2058 adhe~ion to : : 10 gelatLn ~y peptides G1yGlyTrpSerHisTrp (SEQ ID~NO~

(); GlyG1yTrpSerLysTrp (SEQ ID NO: ~9)~ or :~ ~rpSer~isTrpSerPro :(SEQ ID NO: 2).

:~ ;Figure 6(B) is a bar graph of the inhibition of ~direct adhesion o~ A2058:ce:lls to immobilized fibronectin ..

15 ~ by the:peptides of this Ln~entlon.

Figure~ 7 is ~ a bar graph of the inhibi`tion of ibronectin-m:ediated carcinoma cell M~A 4355~adhesion to gelatin by paptldes~GlyGlyTrpSerHisTrp (S~Q ID NO~

G1yG1yTrpSerLysTrp (SEQ I;D NO: 9) or T ~ SerHisTrpSerPro - ..

; ; (5EQ ID NO:~2)~

~ igure~7;(B)~ ls a bax graph of the inhibition o~

:~ direct~adhesion of MDA:435s cells to fib~onectin ~y the :

; peptides.~

Figure~ 8~ is ~ a::~graph of the ~ lnhibition~ of 25;:~fibronectin-mediated adheslon:of breast~carclnoma cells .. ~"

: :MDA 435s::~ to nati~e type -l collagen by peptide .~' GlyGlyTrpserHis~Trp;~(sEQ ID~ NO:~ 1); GlyGlyT~pSer~ysTrp :: ~ , WO94tl~395 PCT/US93/11104 2~

(SEQ ID NO: 9); TrpSerHisTrpSerPro (SEQ ID NO: 2) or GlyArgGly~spSer (SEQ ID NO: l6).
Figure 9 is a bax graph of the inhibition of gelatinase activity by pep~ide (SEQ ID NO: l).

s~MMaRy OF T~E INVENTION
The pr~sent inventors have isola~ed, purified and ch~racterized or synthesized and chararterized biologically active peptldes ha~ing a specific binding affinity for the gelatin-binding domain of fibronectin, which peptides have a sequence of at least five amino acids in~luding the sequence HisTrp and at least one : other tryptophan residue. In a pr~ferred embodiment of the in~ention ~he peptide inc~udes ~he sequence XaaHisT~p, wherein Xaa is an amino acid se~ected from serine, threonine and alanine. More prefera~ly, the peptide has a sequence including the hexapeptide GlyGlyTrp~aaHisTrp, whereln~Xaa is defined abo~e. In a particularly preferred embodiment of the in~ention, ~he peptide is a peptide having one of the sequences SEQ ID
: 20 NO:l, SEQ ID NO: :2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID
NO:5,`SEQ ID NO:6f SEQ ID NO:7 and SEQ ID NO:8. Most preferred is the hexapeptide GlyGlyTrp~erHisTrpj(SEQiID
~0 ~
In another :aspect of the invention, there is :~ 25 provided~ a pharmaceutical composi~ion for binding ' fibronectin or other related collagen-binding proteins : ~

WO ~4/11395 `, 2 1 ~ 8 3 ~ 3 ` PCT/US93/11104 ~.
. .~ `

which includes an effecti~e iamount of at least one peptide of the in~ention and a pharmaceutically acceptable excipient or c~rrier. In a preferred embodiment of the invention, the pharmaceutical ;;:~
5composition includes an effective amount o~ one of the peptides selected from SEQ ID NO: 1, SEQ ID NO. 2, SEQ ID
~0: 3, SEQ ID NO: 4, 5EQ ID NO: ~, SEQ ID NO: 6 ~ SEQ ID NO: 7 and SEQ ID NO:8 and a pharmaceutically acceptable carrier or excipient. ~ost preferably, the pharmaceutical lOcomposi~ion of the invention lS the hexapeptide ha~ing sequence SEQ ID NO :1 . ;
In ano~her aspe~t of the in~ention, ~here is i.
provided a method for binding fibronectin or other ~
collagen-binding proteins in a patient in need of such -....
15treatment wherein an effective amount of peptide having `~
: : a specific binding affinlty far the selatin-binding domain of fibronectin and which includes at least 5 ~ no acids, lncluding the se~uence:HisTrp and at least one ..
~ other txyptophan residue, is a~ministered ~o such . i/~
:~ 20 patient. In a~preferred embodiment of the invention '.
: method, the peptide is a peptlde having a sequence -;
elected from SEQ ID NO~ SEQ ID NO. 2, SEQ ID NO: 3, ~-SEQ ID NOo41 SEQ ID NO:5, SEQ .ID~ NO::6, SEQ ID ~0i:7 a~d SEQ TD NO::8~. Most preferred is a me~hod whexein ~he : ~ 25 peptide is the:hexapeptide SEQ ID NO~
~ The term "collagen~blndlng proteins" i5 used herein:`.
;; : ~ to descrlbe~ pro~ei~s ~:having a domain capable of -;~

: : :

-- '"' - ;

. ` ., , ,; . , , !` : , . i ' ` ' , : `

WO94tll395 21 ~ 838 PCT/US93/11~04 9 ':
specifically binding to or interacting with collagen.
The term "gelatin-binding domain of fibronectin" is ~.
used herein ~o describe the amino acid sequence of mammalian fibronectin which is the site of interaction of -~
S mammalian fibronectin with gelatin. The ~gelatin" is used to define denatured collagen.
The term ~specific binding affinity for fibronectin~
is used herein to mean at least l% by weight binding of fibronectin to peptides under saturating peptide conditions. The symbol (~) indicates binding of 1 to 5%, (++) i~dicates from more than:5 to 50% binding and (+++) indicate3 more than 5Q% bindlng to fibronectin.
The one letter codes for amino acids and ~he :~.
corresp2nding:three letter codes which are used herein are as follows~
':
W--~rp . , - ~.
K=Lys Y=Tyr P-Pro D-~sp :
R-~xg F~Phe :
Q=Gln : E-Glu T-Thr .
G-Gly :~
~S-Ser ~ ~ :
A=Ala , `

,::
- , WO 94J1~395 P~/US93/~1104 ;`.; ~
. f 2i~83 lo DEq~hILED DE:SCRIPTION O~ E I~TION
The peptides according to the invention are deri~ed from the extra cellular matrix protein thrombospondin.
In particular, the peptides are deri~ed from one of the 5domains that mediate interaction of thrombospondin with the matrix adhesi~e protein fibronectin. The peptides of the pr ient invention are derived from the second type 1 repeat of thro~bospondLn. Thrombospondin is a modular adhesi~e glycoprotein that contains three domains that ha~e been: implicated in the ~a~ttachment of cells to thrombospondin. One s~gion of thrombospondin, consisting of a chymotryptic SO kDa peptide contains the three type 1 repeats of a~57 residue segment homologous to two ; malarial proteins (Lawler~J.~and Hynes, R~O~ 1986, J.
;15 Cell~ Biol. 103: 1635-16~). The amino acid sequence of the second type~ 1 repeat is incorporated herein by ~ re~erence thereto. ; ~
: ~ ~ :The peptides of this invention interact direct1y with the ge1at1n-binding domain~of fLbronectin. The ZO gelatln-blndLng domain;of~fibronectin is~necessary for the interaction of fibronectin with gelatin. ~ Specific and pref~rred sequences of the peptides of the~invention are set ~ar~h;1n Table l~and may be produced~by alny oflia arie~y of~art~known methods. Such methods of peptide ~ synthesis; include, for~example, solid phase peptide synthesis, genetic eng1neering te~hniques 1ncluding ~he clonLng of; DNA encod1ng the peptide in an expression .
- .;' ~:

WO94~11395 PCT/US93/11104 . . 21~83S3 ve~tor, and ~he direct isolation of the peptide from ~hrombospondin.
Table l also includes other peptides (FN Binding-) which while ha~iny homo~ogy to the p~ptides according to the in~entlon (FN Binding ~, ++, +~) do not include the essential His-Trp (H-W) sequence.
: .:
Table l Flbronectin blndlng to immobilized pep~ides. ;;

Peptide Seq. Id:No: Sequence FN Bindi~g 239 8 :SHWSPWSS
246 7 ~R~KQDGG~5HWSPWSS ++ . .
~56 6 GGWSH~SP~SS ~+
~5 25g 17 ~G&~SHASPWSS - ~
263 5 ~RF~QDGGWS~WSP ~+ .
266 ~7 ~ ~RF~QDGGASH~5P -~64 4 G&WSHWSP +
285 3 ~ : WSHWS
20 :~9~ ~ WSHWSP ~ +
300 .1 . GGWSHW ~+~
322 28 ~ QDGGWS
: 3l7 l5 DGW5PW
318 21 : GGWGPW
319 : 29 GTWSEW
:: 320 30 ~: GFWSEW -The peptides of the in~ention ~ind speci~ically to the gelatin-bindi~g domaln of f~ibronec~in. The se~uences responsible ~or ~flbronectln-blndlng o~ the present pep~tdes are contalned in a peptide pre~iously identified as a heparin-bindi~g peptide, however, Lhe optimal -.
: sequence for binding fibronectin has only weak heparin-: ~

:: :: :

W~ 94/113g5 PCT/US~3/11104 21~83~3 binding acti~i~y lU.S.P. application 07/801,812). ~or~ -particularly, a 17 amino acid peptide (peptide 246) (SEQ
ID NO: 7) comprising part of the second type I repea~ of ~hrombospondin ~KRFRQDGGWSHWSPW5S) exhibits s~rong .
affinity for fibronectin and is a potent inhibitor of fibronectin binding to gelatin and of fibxonectin- ~
mediated cell adhesion to a gelatin matrix. Binding of ~,:
~he pre~ent pep~ides to fibronectin does not require the `=`
amino terminal basic residues of peptide 246, since -~`
peptide 2~6 tGGWS ~ SPWSS) (SEQ ID NO: 6) which lac~s these resi~ues also binds fibronectin.
At least two tryptophan residues in the correct `
p~sition are required for binding. The central .
try~tophan residue of peptide 2 4 6 ~ SEQ ID NO: 18 ) :: -(residue 9) is required for binding since a sLmilar peptide ~25~ lack~ng this trypt~phan residue f~ils to . -bind (Table 1 ~nd Figure 1). Am~ng overlappin~ peptides derived from the 17 amino ~cid peptide 246 (SEQ ID NO: 7) the peptide ha-ring the sequence GlyGlyTrpSerHis'$rp (SEQ
ID NO~ has strongest binding activity (Figure 1 and .
. .- .
Table 1). Most pxeferred among the peptides of ~he `~
invention is th . hexapeptide ha~ing the sequence ;~
Glye1yTrpSerHisTrp (SEQ ID NO 1). I ; -The~peptldes of the present invention may contain .`
sequences deri~ed from the con~ecuti~Tely occurxing human thrombospondln amino acids. Prefer~bly, the ~`
consecutiYely occurrlng amino acid sequences are selec~ed , .
- .-W~94/1~395 21 PCT/US93tlllO4 ~ ~83,~3 ~ ~

from amino acids 39 4-42D and 4 24-45 O. Especially praferred are the consecutive amino acîds 410-420 and 4~4-~34.
The binding acti~ity of several peptides deri~ed : ~
5from the second Type 1 repeat of thrombospondin are shown ~:
in Figure l. At saturating peptide concentra~ions of 100 -.~
~g/ml, more~than 50% of added fibronectin is bound to the , :
immobilized peptides of this invention. ~he hexapeptide ~consisting of the peptide sequence GlyGlyTxpSerHisTpr (SEQ ID NO. l) :binds ~fibronectin be~er than larger ~.
peptides containing the same sequence or intact .; ~
:thrombospondin~, indicating that ~he 6 residue ~equence is .

: partially cryptic:in thrombospondin. It is no~ neces~ary that the peptides:of the in~entlon contain the entir~ ~

: hexapeptid~ sequence ~or effecti~e fibronecti~ binding `::

; howeYer, it i5 necessary that the pep~ides qhare some : seqùence homology wi~h:the hexapeptide. Two tryptophan ::

~ sidues and the His residue of: the hexapeptide are :
essential Ln the~peptides ~of the lnYention since even ZO~ con exvative substitution~ with similar amino acids renders the peptides~ Lnact~lve ~(Figure 2j. Preferably, he serine res~idue lS present but it is not essential. ~:
Optimal;bindlng:is obtained Wlth peptides~cdmpri ng ~he ~ : : : :: ~ . ~ .:
serlne resldue~(Figùre 2B). ConserYative substitution of :~
~; the: serine résldue:with~a~threonlne residue decreases ac l~ity~approxi:mately fl~e-~old and substitutlon of ~he ~.
serLne residue~wlth~an alanlne residue pxoducies an ac~i~e ~`

~ ~ .

W~4/11~9~ P~T~US93/11104 ~
21~4~383 I ~

peptide having a slightly higher inhibition constant than the threonine analog. The presence of two glycine residues st~ongly enhances acti~ity relative to a peptide analog ha~ing only one glycine residue (Figure 2B).
S Conservati~e substitution of both glycine residues wi~h alanine or of ~he first glycine residue with aspartic acid abolishes ac~ivity. Pxeferably, the pep~ide comprises two gly~ine residues, however, pep~ides lacking one or both glycine residues are wi~hin the scopa of the in~ention.
Accordingly, the peptides of this in~ention ha~e a sequence of at least 5 amino acids residues, which include at leas~ kwo tryptophan residues, at least one of which is present as the sequence HisTrp (XW) wherein said peptide has specific binding affinity for the gelatin-binding site of ibronectin. More preferred is a peptide with a~ least 5 amino acid residues including the sequence XaaHisTrp, wherein Xaa is an amino acid selected from serine, threonine and alanine and wherein the 5 amino acids has at leas~ two Trp residues. Even more pre~erred lS the~hexapeptide GlyGlyTrp~aaHi~Trp, wherein Xaa is serine, threonine ~r alanine.
In a particularly preferred embodimentl of the invention, the peptide according to the invention is a : 25 peptide ha~in~ a sequence independently selected from SEQ
ID NO:l, SEQ ID N0: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID
NO:~, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Most -' ' '.' ., '"~

~094/113~5 21~ PCT/US93/11104 preferable is the peptide SEQ ID N0: 1.
The peptides of the present in~ention retain proper conformation in solution for binding to fibronectin with specific binding affinity. Moreover, the peptides of the p~esent i~vention bind speci ic~lly to the gelatin-binding site of fibronec$in and preferably, at le~ast 5%
. ~bind~ng afflnity~ under saturatLng pending conditions.
The:present peptides are potent inhibitors of fibronectin . binding to ~gelatin and :of fibronec~in-mediated cell adhesion to a gelatin matrix.
~ Fibronectin is an~ im~ortant extracellular matrix :: ~ component that ~mediatas cell in~exac~ions and extracellular matrix assembly during noxmal development.
: It also participates in homeostasis and wound heali~g, .15. and peptides~that inhibit fibronectin intexactions with : : cells have been demons~rated to inhibit tumor metastasis in animal models~(~umphries,~ M.J. Olden, X and Yamad , K., (1986) Science 233, 467-470).
The present peptides: represent a new ~class~of ~ ~inhibitors~of fibronectln:~function. The peptldes of this in~ention~:bind~ specifically:to ihronectin :and inhiblt interactions of fibronectin with collagen but not with cell surf ace:integrin receptors f Gr fi~ronectin. As such,~hese p-ptldes;are~appllcable 1n regulatlny cell 25` ~ m trîx ~interactions~ inYolYed in tumorigenesis, :metasta:sis~, wound~ repair:~and~homeos~asis. For~example, the~ presert ~peptldes Lnhlblt fibronectln-dependent : ~ , W094/11395 PCT/US93/1l104 21q`~'383 ' -: `

adhesion of the human breast carcinoma cell line MDA-MB-435 to immobilized gelhtin. The inhibition of adhesion of th~se cancer cells by the peptides of the present in~ention is specific in that a homologous peptide lacking fibronectin binding activity fails to inhibit adhesion (sea Figur~s 6-A, 6-B and 7).
As noted aboYe, only par~ial se~uence homology wi~h the hexapep~ide GlyGlyT~pSerHisTrp (SEQ XD N0: 1) is n~eded to inhibit fibronec~in bi~ding to gelatin. The existence of homologous sequences comp ising active ~ite~
in ~arious proteins suggests that ~he acti~ities of other proteins containing domains homologous to the fibronectin gelatin=binding domain can he regulated with the peptides of the present in~ention. For example, such proteins as matrix metalloproteinase-2 and ma~rix metalloproteinase share homology with the fibranectin gelatin-binding domain andr ~herefoxe, may be rPgUlated by the peptides of this in~ention.
. There is a high degree of conser~ation of the : .~
fibrQnectin-binding hexapeptide GlyGlyTrpSerHisTrp (SEQ
ID ~0: 1~ Ln the thrombo~pondin gene family. The sequence is completely conserved in the second type I
repeats~of mouse and human ThbsI genes andl in Thbs 2 genes from mou5e and chic~en (Laherty, et al. 1992, J.
2~ Biol. Chem. 2~670:3274-3281). A search of the Swiss-Prot data base uslng the search se~uence GlyGlyTrpSerHisTrp ~SEQ ID N0: 1) yielded several o~her proteins with , ' , W094/~t395 21 ~ PCT/U593/111~4 similar sequences. (see Table II). Epstein-Barr virus ribonucleotide reductase contains the most homologous sequences, GlyGlyTrpPheHisTrp (SEQ ID N0: 18). The ~-subunit of the nicotinic ace~ylcholine receptor, which is highly conserved among ~ertebrate acetylcholine receptor sequences also shares sequence homology with ~he hexapeptide through the sequence GlyTrp~ys~isTrp (SEQ ID
~N0: l9j. A peptide of~acetylcholine receptor contai~ing the sequence Gly~rpLysHisTrp ~SEQ ID N0: 19) plays a role in acetylcholine~bindLng :to the~receptor. Furthermore . ano~her similar sequence, TrpSerXTrpSer~, has been : i~enti~ied as a poten~ial ~'binding" se~uence (Miyazaki, et al. lggl; Emb~ J. , 3191-3197; Yoshimura, et al. 1992, J. Biol. Chem. 267: 11619-11625~). The ligands that : 15 interact with thls site ha~e not been ident~fied, buk ~: : loss of recept~or processing: or activity following mutation of the~consensus sequence suggests that cellular factors involved: in protein~ folding, intracellular `: processing, or slgnal transduGtion are potential ligands.
; ~ :

~ ~ , WO94/1139S , PCT/US93~11104 . . ; . -2 i 4 ~ 3 8 3 18 . .:~:
TABLE II
Amino acid sequence homologies with the fibronectin~
binding sequenee from thrombospondin. .

Source Sequence human Thb~l ~RF~QDGGWSHWSPWSSC
mouse Thbsl R~F~QDGGWS~HSPWSSC
mous~ Thbs2 TRIRQNGGW5~WSF~5SC
chicken Th~s2 HRIRQDGG~SBWSPWSSC
E3V ribonucleo~ide reductase ~QS~Y~GG~S~WHD~AGC
chicken AchR WVN~D~G~NVYYACC ~ .
T. California ~hR WVN~DYRGW~VrYTCC
rat AchR WVI~EAR ~ ~SCC ;
mouse AchR iWVI ~ GWREWVFYSCC
bo~ine AchR ~ WVI~ESRGW~WVF~ACC`
,~
Se~uence identity with the thrombospondin sequence is ..
indicated by bold residues.
Campositions contalning a therapeutically effective ~-amoun~ of at least one pPptide of the present invention .~
~ ,.
are prepared by:any methods known in the art. For example, the compositions are~prepared by forming an admixture of at~ least one~peptide of the present nvention and a pharmaceutically acceptable carrier or excipient, such as for~example, s~eril~ saline, Ring rs solutionj Ringer~s lactate or 5~ dextrose. The peptides ............ ................................................................. ... ..
, :
may be mixed with a variety of carrier compou~ds depe~ding on the form of preparation desired for --: adminls~ration ; -In another~embodiment,~the peptides of the presen~
in~ention may~ be~ con]ugated with a suitable carrier ~ ,. .
polymex or protein. For exa~ple, chemical conjugation to ~
.
- .

WO~4/ll395 ~3383 PCT/US93/ll104 .

the peptide can be used to target specific cells by attachment to an antibody against a surface protein of the targe~ cell.
Various methods of administration of peptides axe known to those skilled in the art. Such methods of adminis~ration may include, but are not lLmited to, sux~ace application, oral and parenteral routes, injection into joints, subcutaneous injection via sustained release, intra~enous injection or other pharmaceutical methods of delivery.
Approp iate dosages of the peptides of ~he in~en~ion will depend upon the condition presented by the indi~idual subject. The skilled medical~worker will be able to determin~e appropriate dosages requlred to combat th~ physiological acti~ity.~ However, in general, ~mounts of from about 1 ~g to 100 ~g~kg body weigh~fday of the biologically active peptide should be useful.
The following examples are provided to more fully illus~rate the principles~and practl~ces of the in~ention.
20 ~ ~ The examples are~not intended ln any way to l~mit the sc~,pe of the lnYention.
~XANP~E l This example~illustrates the blnding of peptides of the invention to~fibronectin.
S ~ Fibronectin was isolated from pla~elet-depleted plasma as~described ~AIiyama, et al. 1985, J. Biol. Chem.
260:4492 450~0) and ~iodlnated using Iodogen tPierce ....

WO94/1139~ ~ PCT/US93/11104 8 ~

Chemical Co., Rockford, ILL) as described (Roberts, et al. 1987, J. Cell. Biol. 104:131 139~.
Peptides were synthesized corresponding to sequences .
of human thrombospondin deduced from a cDNA sequence for S human endothelial cell throm~ospondin (C Lawler and Hynes, 1986, J. Cell. Biol. 103~1635-1648). Peptides -;:
were synthesi~ed on a Biosearch ~odel 9600 peptide syn~hesizer usiny standard Merrifield solid phase ~:
synthesis protocols and t-Boc chemistry. Peptides were .
. .~.
analyzed and where nece~sary, further purified by reverse-phase HPLC chromatography. Tdentities of some of the peptides, including peptlde 246 (SEQ ID NO: 7), were :
: .
~erified by complet~ amino acid sequence analysis.
: Peptide solutlons were neutralized~by addition of dilute NaOH and stored in solution at -20C. ~-Direct~ binding of the fibronectin to immobilized peptides was carried out by ~irst absorbing the peptides on pol~ inyl: chloride mLcrotiter wells for 3 hours at .
: 25~C at the indicated concentrations. Unbound pep~ides :~ 20 were removed by washlng and~wells were incubated for 30 .,`
minut~s in ~ris-BSA (50 mN tris-HCQ, pH7-8, 110 mm NaCl, 1% ~5~) ~Slgma fatty acid and globulin-free, 0.1% NaN3).
The wells were washed and incubated ~with 30 ml df b . 5 g/ml ~25I-fibrone~tln~for 2 hours at 25C. The wells .-25 : wexe washed five~imes with ~ulbecco's phosphate buf~ered ~:
saline and cut from~the plate and bound radioacti~ity was :~ counted.

:~ ~ . '''':

S383 ~ , The peptides lis~ed in Table 1 were synthesized as described abo~e and absorbed to microtiter wells. Of the larger peptides tested from the type I repeats or ~he carboxyl terminal domain of thrombospondin, only peptide 246 (SEQ ID NO- 7) bound labelled fibronectin. The peptide GlyGIy~rpSerHisTrp (SEQ ID NO: 1) exhibited the strongest ~inding activity. This peptide se~uence is located in the second type I repeat of the human ThbsI
gene. Synthetic peptides 317 ~SEQ ID NO: 15) and 3l8 (SEQ ID NO~ 20), derived from the correspo~ding positions in the firs~ and third type I repeats of thrombospondin were inactive.
The results~ are shown in Table 1 and Figure 1.
Figure l sh~ws bindin~ of I-fibronectin to peptides 300 :15 ~ ) (Gly~lyTrpSerhlsTrp) (SEQ ID :NO: 1); 246 (o) : ~(hysArgPheLysGlnAspGlyGlyTrpSerXi~sTrpSe ProTrpSerSer) : ~( S E Q I D : N O : : :7 ) ;; 2 6~ 3 ( (LysArgPheLysGlnAspGlyGlyTrpSerHLsTrpserPro) SEQ ~ID NO: 5) (-)~ and 264 (GlyGlyTrpSerElsTroSerPro) Z0: ~ (S~EQ ID~NO:~4).
E$a~P~E 2;
This ~example lllustrates~ the ;specifLcity of~
inhibition :of fibronectln bindlng to thr~ os~ondin : peptide 3oo~(GlyGlyTrpserHlsTrp) by~synthetlc peptldes~
: 25~ Mlcrotlter~pl;ate~wel~ls were coated with~O~q/ml of peptide~GlyGlyTrpSerHisTrp~(SEQ ID NO: 1) as in Example 3ind- Dg ~ of ~ fibronectl- (0 5 ~g/ml) =o bound W094/1~395 PCT/US93/11104 2`~4g383 peptide was determined in the presence of increasing amoun~s of the peptides: Panel A: the peptides are GlyGlyTrpSerHisTrp (SEQ ID NO: 1) (^); GlyGlyTrpSerLysTrp (SEQ ID N0: 9) (~); GlyGlyTyrSerHisTrp ~SEQ ID N0: lO) (~) and GlyGlyTrpSer~isTyr (SEQ ID N0: ll) (); Panel B:
GlyGlyTrpSerHisTrp (SEQ ID N0~ ); GlyGl~TrpThrHisTrp (SEQ ID NO: 12) (- ~; GlyG1yTrpAla~isTrp (SEQ ID NO: 13) ~); Gly~rpSerHisTrp (SEQ ID N0: 14) (+);
AlaAlaTrpSerHisTrp (SEQ ID N0: 21) (); and AspGlyTrpSerHisTrp (SEQ ID N0: 22) (0).
The results are shown in Figure 2.
Binding of fibronectln to immobilized pep~ide G~yGlyTrpSerHisTrp (SEQ ID ~0: 1) was inhibited by soluble peptide 300 with 50% inhibition at 27 ~m. Three ~hexapeptides containing conservati~e single amino acid substitutions, ~l~GlyTrpSerLysTrp (SEQ ID N0: 9);
GlyGlyTyrSerHisTrp (SEQ ID N0: lD) and GlyGlyTrpSer~isTyr (SEQ ID N0: ll) were inactive. The corresponding hexapeptides from the flrst and third repeats of 20 ~ ~ thrombQspondln were also lnactive~.
E~AMP~E 3 : . `
: This example illustrates the inhibition of fibron~ctin : binding to thrombospondin peptide GlyGlyTrpSerHisTrp by solublefibronectin,thrombospondin ~: .
and syn~hetic::peptide GlyGlyTrpSerHisTrp.
: ~ ~Human platelet:thro~bospondin was purified according ~ to the method of Roberts, et al. 1985 J. Cell Biol.

- - .
~ .
;, . :

W~94/~1395 PCT/U~93/ll104 " .. ;. . 21~,3,83 , ', ~
, ; ,, 23 `
104:131-139. :~
Binding of l25I-fibro~ectin (0.5 ~g/ml) to microtiter pla~e wells coated with 50 ~gJml of peptide GlyGlyTrpSerHisTrp (SEQ ID NO: 1) was determined in the presence of synthetic~peptide GlyGlyTrpSerHisTrp (SEQ ID
NO: 1), fibronectin or:thrombospondin. The results are shown in Figure 3.
- . , : Unlabelled fibronectln and thrcmbospondin competed for binding~ of l25I-fi~ronectln to ~the peptide ~GlyGlyTrpSerHisTrp :(S:EQ ID NO~: l), but~several control proteins, i.e.: ovalbumin, transferrin, fetuin, goat:IgG
and murine laminin~did not (IC50>200 ~g~ml).
X~P~ 4 This example illustrates~that peptLdes of the 15~ ~pres~nt inventlon and whole thrombospondin bind to the g~latin-binding~domain of fibronectin.
Nicrotiter plates were~coated with ~50 ~g/ml of synthetic peptide GlyGlyTrpSerHisTrp (SEQ~ID NO: l) as described ln Example~ nding of l25I-fibronectin ~0.:5 0~ g/ml)~ to: the~bound:~peptide :was ~determined in the:~
presence~of~proteolytic~fragments of flbronectln,~which:
were~obtained ~from~Telios~ Pharmaceutlcals,~ Inc. San iego,~l C~.' The ~proteoly~i~c~fragments included linta~
. . ~ .
fibronectln;~ 30 ~kDa~ gel3tln-blndin~g doma~in; 33~ kDa~ ::

`;25~ recomblnant:cell;bl~ lng ~omaLn~; 2~8 kDa recombinant cel;I~

binding ~omaln:and transf~errln.~;The results are sho~m in Figure~4. ~

- , : : : , .

W~94/~13~ PCT/VS93/11104 ., ~ , . . .
2 1 ~ g 3 8 3 Of the proteolytic fragments tested, the gelatin-binding domain was the strongest inhibi~or of 125I-fibronec~in binding to immobilized peptide. The 33 kDa fragm~nt, which con$~ins the cell binding domain was S inhibitory. Howevex, ~he 28 kDa fragment which contains all but the N-terminal 5 kDa of the 33 kDa Iragment was inacti~e a~d a 40 kDa fragment with the same amino terminus as the 28 kDa fragment was approximately 10-fold le~s active. Since the sequ~nce of the 33 kDa recombinant fragment is contained in the 120 kDa fra~ment of fibronectin and the 120 ~Da fragment was only a weak inhibitor, the ac~i~ity expressed by the 33 kDa fra~m~nt is probably due to a cryptic site. T~e acti~ity of the 33 kDa fra~ment is less than that of the gelatin binding lS fragment.
E~MPLE 5 This example illus~rates the specificity of inhibition of ibronec~in binding to gelatin by the peptide GlyGlyTrpSerHisTrp.
Nells of a microtiter plate were incubated for 3 hours at 25C with 2~g~ml gelatin in Dulbecco's PBS. The wells were emptied and incubated with Dulbecco's PBS
contai~ing 1~ ~SA for 30 minutes. The welLs were emptied and in~ubated with 0.5 ~g/ml l25I~fibronectin in the presence of peptide GlyGlyTrpSerHisTrp (SEQ ID NO: 1) (-); GlyGl~TrpSerLysTrp ~SEQ ID NO- 9~ ( A );
GlyGlyTyrSerHlsTrp (SEQ ID NO: 10) (-)i or ..

WO94/1l3~5 PCT/US93/11Id?4 ~ 21~383 ` ~

GlyGlyTrpSerHisTyr ~SEQ ID NO: 11) (~) for 2 hours a~
25~C. The wells were washed 4 times with PBS, cut from ..
the plat~ and the bound radioactivity was coun~ed. The ^-:
results are shown in Fi~ure 5. ~.
~he inhibitlo~ by peptide GiyGlyTrpSerHisTrp (SEQ ID
NO: 1) was specific in that the relate~ peptides :
GlyG1yTrpSerLysTrp (SEQ ID NO- 9); GlyG1yTyrSerHisTrp ,-:
~SEQ ID NO: lO~ and GlyGlyTrpSerHisTyr (SEQ ID NO: 11), ~ ' with single amino :acid substitutions from the active :~
: sequence were inacti~e.
. EXa~P~iE 6 ;
: ,:
A ligand binding:assay as described in~Example S was performed exc~pt that~mLcrotlter plates were coated wi~h i~mobilized native type I collagen in place of g~latinO :,:
The peptide GlyGIyTrpSerHisTrp~ ~(SEQ ID NC: 1) : ~ `
inhibited fibronectin~binding to typ?e I col:lagen with an ~.
IC50=40~m. GlyGlyTrpA1a~isTrp (SEQ ID NO: 13) was a weak inhibitor and the peptldes TrpSerHisTrpSerPro (SEQ
ID NO: 2); :GlyGlyTrpSerLysTrp ~(SEQ ID NO: 9); ~ :
20~ ;~;:GlyGlyTyrSerHisTrp :(SEQ ID NO~:~lO); GlyGlyTrpSerHisTyr~
(SEQ ID: NO~ Al ~laTrpSerHisTrp~ (SEQ ID NO: 21~
AspG1yTrpSerHisTrp ~(5EQ ID :NO: 22); and i IGlyGlyTxpThr~isTrp~;(SEQ ID NO- 24) were inactive.~"~, ;E~A~PLE 7 25~ ; Thls example~ ~illustrates?~ the inhLbi~tion of fibronec~tin-dependent~cell adhesion to de~ature~ collagen WO 94/11395 PCT/US93/11104 ~
2i~383 26 .
by peptide GlyGlyTrpSerHisTrp (SEQ ID NO: 1). ..
Gelatin (l ~g/ml) in Dulbecco's PBS was coated on plastic discs in 24 well pla~es and incubated for 2 hours at 37C. After ~he supernatant was r~moved, the discs ~;
were trea~ed with l~ BSA-tris pH 7. 8 for 30 minutes at ambient temperature. The discs were washed twice with PBS and 0.4 ml of RPNl 1640 medium containing 0.1% BSA :~
and l ~g/ml fibronectin and 2, 20 or 200 ~g of the ~, ~"
inhibitory peptides were added to ~he wells. A
suspension of lO5 cells in 0.1 ml RPMI medium containing 0.1% BSA was added~ to the wells and incubated at 37C
wi~h 5% C2 fox 1 hour. The discs were washed to remoYe : ~ nonadherent cells and stained. Adhsrent cells were co~nted ~icroscopically. The results are shown in Figures 6 and 7.
In panel~A of Figure 6 human melanoma cell of cell line A2058 were added to the wells and in Panel A of Figure 7 N~A 435s breast carcinoma cells were added to . immobili~ed gelatin.: At the concentrations used, the cells weakly adhered to gelatin in the absence of added : . fibronectin. The s:timulated adhesion in ~he presence of -: fibronec~in was inhibited in a dose-dependent:manner ~y `;
the peptide ~GlyG1yTrpSerHLsTrp; (SEQ.ID NO:, 1).l The inhibitlon was specific in that ~he homologous peptides :~
25 ; ~ that lacked~ ~fibronectln binding acti~ity, :
GlyG1yTrpSerLysTrp (SEQ ID NO: 9) and TrpSerHisTrpSerPro (SEQ ID NO: 2), were inactive.

.

- .~

W~g4/11395 21 PCT/US93/11104 ~ ~83 ~7 Direct adhesion of A2058 cells (Figure 6s) or MDA ~;~
435s cells (Figure 7B) to immobilized fi~ronec~in was not inhibited by the peptide GlyGlyTrpSerHisTrp (SEQ ID NO:
1) or the control peptides, bu~ adhesion of the breast :~
carcinoma cells to fibronectin was inhibited by the peptide GlyAxgGlyAspSer ~SEQ I~ ~O: 25). Thus, interaction of fibronectin with integrin receptors on the breast carcinoma cells is not inhibited by the peptide GlyGlyTrpSerHisTrp (SEQ ID NO: 1). ;
~0 E$aMP~E 8 `~-This example illustrates the inhibition of :
. . .
fibronectin-mediated adhesion of breast carrinoma cells to nati~e type I collagen by peptide GlyGlyTrpSerHisTrp (SEQ ID NO: 1). : .
Discs were treated as~in Example 7 except that 1 ~g/ml of type I collagen in Dul~ecco~s P~S was coa~ed on ~~
; ~ .
the discs. Cell attachment of breast carcinoma cells (c~ll line MDA 435s) to immo~ilized t~pe I collagen was determined in the presence of fibronectin alone and in .....
the presence of peptide:GlyGlyTrpSerHisT~p (SEQ ID NO~
l); GlyGlyTrpSerLysTrp (SEQ ~ID NO: 9 ); TrpSerElisTrpSerPro (SEQ ID NO: 2) or GlyArgGly~spSer ~SEQ ID N0: 25). The results are shown in Figure 8.
Fibronectin-mediated:adhesion of breast carcinoma cells to native type 1 collagen was inhibited by peptides ~lyGl~ rpSerHIsTrp (SEQ ID NO: 1) and GlyArgGly~spSer (SEQ ID NO: 25) but not Glv~lyTrpSerLysTrp ~SEQ ID NQ: 9) . ~

WO94/1~39~ ~ ~ 48 3 8~ PCT/US93/11104 or TrpSerHisTrpSerPro (SEQ ID NO: 2). Direct adhesion tO
type 1 collagen was stronger th~n tO gelatin. The .
effec~s of the peptide GlyGlyTrpSerHisTrp (SEQ ID NO: l) were specific for fi~ronectin-stimulated adhesion, S however, this pep~ide does not inhibit direct binding of ~:~
cells to type 1 collagen.
E~AMP~E 9 ~his example illustrates inhibition of gelatinase ~y p~ptide GlyGlyTrpS~erHisTrp. Degradation of 1~5I-gelatin by gelatinase activl~y in condLtioned medium from bovine :~
; corneal endothelial cells was determined by release of radioacti~ity fram microtiter plate wells coated with the radiolabeled substrate and lncubated for 60 or 90 min ~:
wi~h conditioned :medium a~ti~ated using p~
. . .
hydroxymercuribenzoa:te. Acti~1~y ln the pre~ence of the indicated concentrations of:peptide GlyGlyTrpSerHisTrp (SEQ ID NO: l) is presented~ as~ a per~ent of co~trol acti~ity determined in the absence of peptid~ and corrected for background~release~ determined in the absence of mercurial acti~a~ion. Inhibi~ion was also determined by a~g~elatin zymogram ~of endathelial cell conditioned ~ medium:: :a,~ ~ntrol; b, 50 ~/ml `~
GlyGlyTrpSerHisTrp ~ (SEQ~ ID NO~ , c, 500 1 ~g~
GlyGlyTrpSerHLsTrp~(:SEQ :ID NO: l); d, 50 ~g/ml ; . .
&lyGlyTrpSerLysTrp (SEQ ID~ NO: 9); e, 500 ~gfml Gl~Gl~T ~ Ser~ysTrp; and : f, 500: ~g/ml peptide LysArgPheLysGlnAspGlyGlyTrpSerHisTrpSerProTrpSerSer(SEQ -:

.
:
.
- ::

WO 94/11395 2l~ PCI'/US93/11104 ID NO: 7 ) .

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;' ' ~ ~ ''.'' W094/1~3~5 2 1 ~ ~ 3 8 PCT/US~3/lll04 Raw Sequence~
S.~ _in~
(1) GENERAL INFORMATION:
(i) APPLICAN~:~HE GOVERNMENT OF THE UNITED STA~ES OF AMERICA, as represen~ed by ~he Secretary of Health and Human Services (ii) TITLE OF INVENTIO~:Peptide Inhibitors of Fibronection and Re~a~ed Collagen~Bindi~g Proteins (iii) NUNBER OF SEQUEN OE S:30 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRE55EE:Lowe, Price, LeBlanc & Becker (B~ S~REET:Suite 300, 99 Can~l C~nter Plaza (C) CITY:Alexandria (~) STATE:VixgLnia (E) COUNTRY:USA
(F) ZIp:223l4 : ~
(~) 0MPUTER READ~BLE FORM: .
(A) MEDIUM TYP:E-Floppy Disk (B) COMPUTER:IBM PC Compatible : (C) OPERATING SYSTEM:PC-DOS/MS-DOS
(D) SOFTN ~ E~:Word~Per~ect, ~ersion 5.1 ~vi) CURRENT APPLICATION DATA:
(A)~ APPLICATION NUMBER:
(B) FILIN~ DATE~
~ (C) ~L~SSIFIC~TION: -:; ~(vii) ATTORNEY/AGENT IN~ORMATION- .
. (A) NANE:Ro~ert L. Price (B) `REGIST ~ ION NUNBER:22,685 (C) REFERENCE/DOC~ET NUMBER:
! ( i'X j iTELECO~UNIC~TION INFORM~TION~
; : (A) TBLEPHONE:(:703)684-1111 (B) TELEFAX:(;703)~84-1124 :

~:: ( 2 j INFORKAT~ON FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS:~
(A) LENG~H:6 amlno acids -: :

' WO94/11395 ~ 38 PCT/US93/11104 (B) TYPE:amino acid (C) TOPOLOGY:linear (ii) MOhECULE TYPE:Peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO~
Gly Gly Trp Ser His Trp ~;-(3) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTHo 6 amino acids ~B) T~PE:amino~acids ~;
.. ~; -(C) TOPOLOGY:linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 2: ~:
Txp Ser His Trp Ser Prol 5 t2) INFOR~TION FO~ SEQ ID NO: 3: .`
: (i) SEQUENCE CXARAC~ERISTICS: .`
(A) LENGTH:5 amino acids ~`
~B) TYPE:amino acids .:~
(D) TOPOLOGY:linear (ii) MOLECULE TYPE:peptide . -:`~
(xi) SEQUENCE ~ES~RIPTION:SEQ ID NO: 3 Trp:Ser His Trp Ser .~
l 5 ;::`
i (2) INFORMATION FOR SEQEUNCE ID NO: 4O
(i) SEQUENCE CXARACTERISTICS:
(A) LENG~H:B~amino acids (B) TYPE:amino aclds~
. .
~ (D) TOPOLOGY:linear .:-''~

~
' ' - ~'`.''~

W094/1139~ PCT/US93/1~1~4 - 2 1 4 ~ 3 ~ 3 (ii~ MO~ECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 4:
Gly Gly Trp Ser His Trp Ser Pro ( 2 ? INFORMATION FOR SEQ ID NO: 5 (i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH:14 amlno acids (B) T~PE:amino acids (D) TOPOLOGY;linear (ii) MO~ECULE TYPE:pep~ide ::
(xi) SEQUENCE DESCRIPTIONoSEQ ID NO: 5: ~;
Lys Arg Phe Lys Gln:Asp Gly Gly Trp Ser His Trp Ser Pro ~-l 5: l0 ..
: (2) INFORM~TI~ON FOR~SEQ ID~NO:: 6:
( L ) SEQUENCE C~ARACTERISTICS:
( A ) LENGTH: 11 ~nino acids ( B ) T~!PE: a~nino acids ( D ) TOPOLOGY: linear ~ ~-:~ ( ii )MOLECTJLE TYPE:peptide : ( xi ) S~:QUENCE DESCRIPTION- SEQ ID NO ~ 6 ~
Gly Gly Trp Ser Hl~s Trp Ser Pro Trp Ser Ser , (2) INFOREATION FOR:SEQUEN~E ID NO. 7:
(ii) SEQUENCE CNARACTERISTICS~
(A) LE~GTH:17 amlno acids (B) TYPE:amino acids . ~ ~
;~ : (D) TOPOLOGY:linear ~ .
::
(ii) MOLECULE TYPE:peptide - ~ ' -WO94~11395 PCT/U~93/11104 .;~
_ 21~83 ~ ~

xi) SEQUENCE DESCRIPTION:SEQ ID NO: 7:
Lys Arg Phe Lys Gln Asp Gly Gly Trp Ser His Trp Ser Pro ; :
l 5 l0 Trp Ser Ser (2) INFORMATION FOR SEQUENCE ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LE~GTH:8 amino acids ~) m E:amino acids .
(D) TOPOLOGY:linear (ii) MOLECUI,E TYPE:peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 8: :.
, .....
Ser His Trp Ser Pro Trp Ser Ser .,:
(2) INFORMATION FOR SEQUENCE ID NO: 9: ~
,.,; .
(i) SEQUENCE CHAR~CTERISTICS:
~A) ~ENGT~:6 amino acids (B) m E:a~ino acids :
(D) TOPOLQGY:lineax -i (ii) ~OLECUL~ TYPE:peptide `~-~xi) SEQUENCE DESCRIPTION:SEQ ID NO: g:
ly Gly Trp Ser Lys Trp ~
~ :".
(2) INFO~MATION FOR SEQUENCE ID NO: l0:
:- (i)l SEQUENCE CHARACTERISTICS:
(A) LENGTH:6 amlno acids (B) m E:amino~acids : f D~ TOPOLOGY:linear .
~ii) MOLECULE TYPE:peptide '` ~;
'~

W~94~11395 P~T/US93~11104 .
2 1 4 8 3 ~: 3 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: l0:
Gly Gly Tyr Ser His Trp l 5 (2) INFO~MATION FOR SEQUENCE ID NO~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:6 amino acids (B) TYPE:amino acids ;
(D) TOPOLOGY:linear (ii) MOLECULE TYPE:peptide (xi) SEQUE~CE DESCRIPTION:SEQ ID NO~
Gly Gly Trp Ser His Tyr 5 ' ` ~ ;` ~'.
(2) INFORMATION FOR SEQU~NCE ID NO: 12: .
(i) 5EQU~NCE CHARACTERISTICS: ~:
:~A) LENGTH:6 amino acids ~ -(B) TYPE:amino acids ( D ) TOPOLOGY: 1 inear (il) MOLEC~E TYPE:peptide : ~ .
.~ (xi) SEQUE~CE DESCRTPTION:SEQ ID~NO: 12~:
: Gly Gly Trp Thr Hi~ Trp 1 5 ~ :
(2) INFORMATION FOR SEQUENCE ID Nn: l3:
(i) SEQUENCE C~ARACTERISTICS:
,.! i ` I (~A) LE~GTHa6 ~ino acids ~ ;
:(B) T~PE:amino acids - .
(D) TOPOLOGY:linear ~ ~;
(ii) MOLECULE TYPE peptide . ~

~.
,: '.'. ', : ~ ~

;: .

WOg4~1~395 PC~/US93/11104 _~ 21~3~3 I ~:

~ -(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 13: ;
ly Gly Trp ~la His Trp (2) INFORMATION FOR SEQUENCE ID NO: l4:
(i) SEQUENCE CHAR~CTERISTICS: ,~
(A) LENGTH: 5 amino acids (B) TYPE:amino acids ~.
(D) ~OPOLOGY:linear ;.;
(ii) MOLECULE TYPE:peptide txi~ SEQUENCE DESCRIP~ION:SEQ ID NO: 14:
Gly Trp Ser His Trp ~(2) INFORMATION FOR SEQUENCE ID NO: lSô
(i) SEQUENCE C~ARACTERISTICS~
~) LENGTH:6 amino acids '.
~B) TYPE:amino acids . :
(D) TOPOLOGY:linear (ii) MOLECULE TYPE:peptide ; :
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 15:
Asp Gly Trp Ser His Trp. ~:

(2) INFO~ATION FOR~SEQUENCE ID NO: 16: `;
(i) SEQUENCE C~ARACTERISTICS: ~ -(A) LENGTH:~5 amino ~cids ` i ;-: ~B) TYPE:amino acids .;
(D~ TOPOLOGY:11near tii~ MOLECULE TYPE:peptide ,:' W094/11395 2 1 4 8 ~ 8 3 PCT/US93/11104 :
36 :
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: l6:
Gly Arg Gly Asp Ser l 5 -:
(2) INFORMATION FOR SEQUENCE ID NO: l7:
~i) SEQUENCE CH~RACTERISTICS:
(A) LENG~H:lO amino acids ~
(B) TYPE:amino acids --(D) TOPOLOGY:linear (ii) MOLECUhE TYPE:peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 17:
Gly Trp Ser His Ala Ser Pro Trp Ser Ser `~
l 5 lO
(2) IN~ORMATION FOR SEQUENCE ID NO: 18: ~ .
(i) SEQUENCE C~AR~CTERISTICS:
(A) LENG~H:6 amino acids (B) TYPE:amino acids: -...
(D) TOPOLOGY:lLnea~ .
(ii) MOLECULE TYPE:pep~ide (xi): SEQUENCE DESCRIPTIONsSEQ ID NO: l8: ' Gly Gly Trp Phe~His Trp ~.
~.
~2) INFORMATION FOR SEQUENCE ID NO: Ig: ~
(1) SEQUENCE~CEARA~TERISTICS: ~ :
LENGTH: 5~mino acids .
(B) TYPE:amino aclds :(D) T~PQ10GY::linear: ~.
: ~( Ll j MOLECULE T~PE::peptide ~ ~

: ':

:

WO94/~139~ PCT/VS93/~1104 , ~\ 7 ~ ~8383 (xi) SEQUENCE DESCRIPTION:SEQ ID NO~ l9:
Gly Trp Lys His Trp l 5 ~2) INFORMATION FOR SEQUENCE ID NO: 20: .:.
(i) SEQUENCE CHAR~CTERISTICS: -~
(A) ~ENGTH:6 amino acids ``
(B) TYPE:amlno acids (D) TOP~LOGY:linear ~
(ii) MOLECULE TYPE:peptide ..
, .
(xi) SEQUENCE DESCR~PTION:SEQ ID NO: 20:
Gly Gly Trp Gly Pro Trp . . .
(2) INFORM~TION FOR SEQUENCE ID NO: 21 tij SEQUENCE CEAR~TERISTICS~
(~) LENGTH:6 amino acids (B) TYPE:amino acids ;
(D) TOPOLOGY:linear ::~
(ii) MOLECULE TYPE:peptide (XL ) SEQUENCE DESCRIPTION:SEQ ID NO: 2I~
:~ Ala Ala Trp Ser His Trp .~::
l 5 (2) INFORMATION FOR SEQUENCE ID NO: 22:
(i~ SEQUENCE CHARACTERISTI~S:
; i (A) LENGTH:6~amino acids (B) TYPE:~min~:aclds :(Dj TOPOLOGY::linear .
~ (ii)~MOLECULE TYPE:peptide :

~ : :

W~94/1139~ P~T/US93/11104 , 2'1~g383 (xi) SEQUE~CE DESCRIPTION:SEQ ID NO: 22:
Asp Gly Trp Ser His Trp l 5 (2) INFORMATION FOR SEQUENCE ID NO~ 23:
(i) SEQUENCE C ~ CTERISTICS:
(A) LENG~H:6~amino aciàs (B~ TYPE:amino acids (D) TOPOLOGY:linear (ii) MOLECU~E TYPE:peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:~23:
Trp Ser His Trp Ser Pro (2~) INFORNATION FOR SEQUENCE ID NO:: 24:
SEQUENCE CHARACTERISTICS:;
) LENGT~:6 amino acids (B) TYPE:~mino acids ) TOPOL~GY:linear :
tii:) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION:SEQ:ID NO: 24:
Gly GIy Trp Thr His Trp :~

~ (2)~ INFORXATION FOR SEQUENCE~ID~NO: 25 : (i) SEQUENCE CEARaCTERISTICS~
(A) ILENGTH~5 amino acids~
B) ~T~PE:~mino acids (D)~TopoLoGy~lLDear MOLECULE~TYPE:peptide ::~

~,. .

;::

WO94/113~5 ~ PCT/US93/11104 ; :

'- 8383 39 :
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 25: :~
Gly Arg Gly Ala Ser l 5 (2) INFORMATION FOR SEQUENCE ID NO: 26:
(i) SEQUEN~E CP~ACTERISTICS:
(A) LENGTH:14 amino acids (B) TYPE:amino acids ..
(D) TOPOLOGY:linear~
(ii) NGLECULE TYPE:peptide .
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 2~
Lys Arg Phe Lys Gln Asp Gly ~ly Ala Ser His ~la Ser Phe (2) INFORM~TION~FOR SEQUENCE ID NO:: 27:
(i) 5EQVENCE CP~RA~TERISTICS: -,, (A) LE~GTH:6 amLno acids (B) TYPE:amino acids (D): TOPOLOGY:lînear : .
; (ii) :MOLECULE TYPE`:peptide . ;-~(xi) SE~UENCE DESCRIPTION:SEQ ID NO: 27: .
Gln Asp Gly Gly Trp Ser. .`
l ~ 5 (2) INFOP~qATION FOR SEQUENCE~ID NO: 2 (i) SEQUENCE CPIRACTERISTICS~
` (A) ~ENGT~ 6 amino acids : :
- :
(B~ T~PE:amino acids (D) TOPOLOGY:linear (ii) ~MOLECULE TYPE:peptide .

:: .

: : ' .. :-.~

.

WO9~/11395 PCT/US93/11104 .. .. . .
21~383 ;
(Xi) SEQUENCE DESCRIPTION:SEQ ID NO: 28:
G1Y Thr TrP Ser G1U TrP

(2) INFORMATION FOR SEQUENCE ID NO: 29: -(i) SEQUENCE CHARACTERISTICS: ~-(A) LENGTH;6 aminO aCidS
I (B) TYPE:am1nO aCidS
(D) TOPOLOGY:1inear .
OLECULE TYPE:PePtide ~Xi) SEQUENCE DESCRIPTION:SEQ ID NO: 29:
G1Y Phe TrP Ser G1U TrP ;~

(2) INFORMATION ~OR SEQUENCE ID NO: 30:
(i) SEQUENCE CEARACTERISTIC5~
(A) LENGTH:7 aminO aCidS
~: ~(B)~ TYPE:amLn~:aCL
(D) TOPOLOGY:1inear ~ MOLECULE TYPE:PePtide ~ , (Xi)~ SE~UE~CE DESCRIPTION:SEQ ID NO 30: . .
Gly ~ Gly ~ Trp ~ Ser;~ IJYS S-r Trp , ~ ~ . ...

, . ~; .

Claims (20)

WHAT IS CLAIMED IS:

1. A peptide having a specific binding affinity for the gelatin-binding domain of fibronectin, said peptide comprising at least 5 amino acids and including the sequent HisTrp and at least one other tryptophan residue.

2. The peptide according to claim 1 wherein said peptide comprises the sequence XaaHisTrp, wherein Xaa is selected from the group of amino acids consisting of serine, threonine and alanine.

3. The peptide according to claim 1 wherein said peptide comprises from 5 to 30 amino acid residues.

4. The peptide according to claim 1 wherein said peptide comprises from 6 to 20 amino acids.

5. The peptide according to claim 1 further comprising an amino-terminal N-acetyl and a carboxyl-terminal amide.

6. A peptide having a specific binding affinity for the gelatin-binding domain of fibronectin, said peptide having up to thirty amino acids and comprising the sequence (A) Xa-Xb-His-Trp-Xc, wherein Xa is H or an amino acid sequence of from 1 to 27 amino acids, Xb is serine, alanine, tryptophan, or threonine and Xc is OH, NH2, or an amino acid sequence of from 1 to 27 amino acids and at least one of Xa and Xb includes a tryptophan residue located within three amino acid residues from Trp in sequence (A).

7. The peptide according to claim 6 wherein Xa is an amino acid sequence of from 1 to 27 amino acids derived from the consecutively occurring thrombospondin amino acid sequence including amino acid residues 394-420, Xb is serine, alanine or threonine and Xc is an amino acid sequence of from 1 to 27 amino acids derived from the consecutively occurring human thrombospondin amino acid sequence including amino acid residues 424-450.

8. A peptide according to claim 1, wherein said peptide has a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

9. A peptide according to claim 1, wherein said peptide has a sequence comprising GlyGlyTrpXaaHisTrp, wherein Xaa is selected from the group of amino acids consisting of serine, threonine and alanine.

10. The peptide according to claim 4 wherein Xaa is alanine.

11. The peptide according to claim 4 wherein Xaa is serine.

12. A pharmaceutical composition for binding fibronectin or other related collagen-binding proteins comprising an effective amount of at least one peptide according to claim 1 and a pharmaceutically acceptable excipient or carrier.

13. A pharmaceutical composition according to claim 12, wherein said effective amount of a peptide is a peptide having a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

14. A pharmaceutical composition according to claim 12, wherein the peptide is a peptide having a specific binding affinity for the gelatin-binding domain of fibronectin, said peptide having up to thirty amino acids and comprising the sequence (A) Xa-Xb-His-Trp-Xc, wherein Xa is H or an amino acid sequence from 1 to 27 amino acids, Xb is serine, alanine, tryptophan, or threonine and Xc is OH, NH2, or an amino acid sequence of from 1 to 27 amino acids and at least one of Xa and Xb includes a tryptophan residue located within three amino acid residues from Trp in sequence (A).

15. A pharmaceutical composition according to claim 14, wherein Xa is an amino acid sequence of from 1 to 27 amino acids derived from the consecutively occurring thrombospondin amino acid sequence including amino acid residues 394 420, Xb is serine, alanine or threonine and Xc is an amino acid sequence of from 1-27 amino acids derived from the consecutively occurring human thrombospondin amino acid sequence including amino acid residues 424-450.

16. A pharmaceutical composition for binding fibronectin or other related collagen-binding proteins comprising an effective amount of at least one peptide according to claim 4 and a pharmaceutically acceptable excipient or carrier.

17. A pharmaceutical composition according to claim 13, wherein said peptide is a peptide having a sequence according to SEQ ID NO:1.

18. A method for binding fibronectin or other related collagen-binding proteins in a patient in need of such treatment comprising administering to said patient an effective amount of a pharmaceutical composition according to claim 12.

19. A method for binding fibronectin, matrix metalloproteinase-2, matrix metalloproteinase-9 or other related collagen-binding proteins according to claim 18, wherein said peptide is a peptide having a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7 and SEQ ID NO:8.

20. A method for inhibiting enzymatic activity of collagenases and other proteases homologous to fibronectin in a patient in need of such treatment, which comprises administering to said patient an effective amount of at least one peptide contained in a pharmaceutically acceptable excipient or carrier, wherein said peptide is a peptide having a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.