U.S. patent application number 10/654578 was filed with the patent office on 2004-11-18 for compounds and methods for modulating desmosomal cadherin-mediated functions.
This patent application is currently assigned to Adherex Technologies, Inc.. Invention is credited to Blaschuk, Orest W., Gour, Barbara J., Symonds, James Matthew.
Application Number | 20040229811 10/654578 |
Document ID | / |
Family ID | 24136060 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229811 |
Kind Code |
A1 |
Blaschuk, Orest W. ; et
al. |
November 18, 2004 |
Compounds and methods for modulating desmosomal cadherin-mediated
functions
Abstract
Modulating agents for inhibiting or enhancing desmosomal
cadherin mediated cell adhesion are provided. The modulating agents
comprise one or more of: (a) a peptide sequence that is at least
50% identical to a desmosomal cadherin CAR sequence; (b) a
non-peptide mimetic of a desmosomal cadherin CAR sequence; (c) a
substance, such as an antibody or antigen-binding fragment thereof,
that specifically binds a desmosomal cadherin CAR sequence; and/or
(d) a polynucleotide encoding a polypeptide that comprises a
desmosomal cadherin CAR sequence or analogue thereof. Methods for
using such modulating agents for modulating desmosomal
cadherin-mediated cell adhesion in a variety of contexts are also
provided.
Inventors: |
Blaschuk, Orest W.;
(Westmount, CA) ; Symonds, James Matthew; (Durham,
NC) ; Gour, Barbara J.; (Kemptville, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Adherex Technologies, Inc.
Ottawa
CA
|
Family ID: |
24136060 |
Appl. No.: |
10/654578 |
Filed: |
September 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10654578 |
Sep 3, 2003 |
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09535852 |
Mar 27, 2000 |
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6638911 |
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09535852 |
Mar 27, 2000 |
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09187859 |
Nov 6, 1998 |
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6358920 |
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09187859 |
Nov 6, 1998 |
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09073040 |
May 5, 1998 |
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6472367 |
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Current U.S.
Class: |
514/19.3 ;
514/19.1; 514/21.1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 5/101 20130101; C07K 5/1008 20130101; C07K 5/0808 20130101;
C07K 14/705 20130101; C07K 7/06 20130101; C07K 2319/00 20130101;
Y10S 977/805 20130101 |
Class at
Publication: |
514/015 ;
514/016; 514/017; 514/018 |
International
Class: |
A61K 038/10; A61K
038/08; A61K 038/06 |
Claims
1. A modulating agent that: (a) comprises a desmosomal cadherin CAR
sequence; and (b) contains 3-16 amino acid residues linked by
peptide bonds.
2. (Canceled)
3. A modulating agent that: (a) comprises a desmosomal cadherin CAR
sequence having the formula:
Aaa-Phe-Baa-Ile/Leu/Val-Asp/Asn/Glu-Caa-Daa-- Ser/Thr/Asn-Gly (SEQ
ID NO:3) wherein Aaa, Baa, Caa and Daa are independently selected
amino acid residues; Ile/Leu/Val is an amino acid that is selected
from the group consisting of isoleucine, leucine and valine,
Asp/Asn/Glu is an amino acid that is selected from the group
consisting of aspartate, asparagine and glutamate; and Ser/Thr/Asn
is an amino acid that is selected from the group consisting of
serine, threonine or asparagine; and (b) contains no more than 50
consecutive amino acid residues present within the desmosomal
cadherin.
4. (Canceled)
5. A modulating agent that comprises a desmosomal cadherin CAR
sequence selected from the group consisting of NQK, NRN, NKD, EKD,
ERD, RAL, YAL, YAT, FAT and YAS, wherein the agent contains no more
than 50 consecutive amino acid residues present within a desmosomal
cadherin.
6. A modulating agent according to any one of claims 3 and 5,
wherein the agent is a peptide ranging in size from 3 to 50 amino
acid residues.
7. A modulating agent according to any one of claims 1, 3 and 5,
wherein the agent is a peptide ranging in size from 4 to 16 amino
acid residues.
8. A modulating agent according to any one of claims 1, 3 and 5,
wherein the CAR sequence is present within a cyclic peptide.
9. A modulating agent according to claim 8, wherein the cyclic
peptide has the formula: 4wherein W is a tripeptide selected from
the group consisting of NQK, NRN, NKD, EKD, ERD, RAL, YAL, YAT, FAT
and YAS; wherein X.sub.1, and X.sub.2 are optional, and if present,
are independently selected from the group consisting of amino acid
residues and combinations thereof in which the residues are linked
by peptide bonds, and wherein X.sub.1 and X.sub.2 independently
range in size from 0 to 10 residues, such that the sum of residues
contained within X.sub.1 and X.sub.2 ranges from 1 to 12; wherein
Y.sub.1 and Y.sub.2 are independently selected from the group
consisting of amino acid residues, and wherein a covalent bond is
formed between residues Y.sub.1 and Y.sub.2; and wherein Z.sub.1
and Z.sub.2 are optional, and if present, are independently
selected from the group consisting of amino acid residues and
combinations thereof in which the residues are linked by peptide
bonds.
10. A polynucleotide encoding a modulating agent according to any
one of claims 1, 3 and 5.
11. An expression vector comprising a polynucleotide according to
claim 10.
12. A host cell transformed or transfected with an expression
vector according to claim 11.
13. A modulating agent comprising an antibody or antigen-binding
fragment thereof that specifically binds to a desmosomal cadherin
CAR sequence and modulates a desmosomal cadherin-mediated function,
wherein the desmosomal cadherin CAR sequence has the formula:
Aaa-Phe-Baa-Ile/Leu/Val-Asp/Asn/Gl- u-Caa-Daa-Ser/Thr/Asn-Gly (SEQ
ID NO:3) wherein Aaa, Baa, Caa and Daa are independently selected
amino acid residues; Ile/Leu/Val is an amino acid that is selected
from the group consisting of isoleucine, leucine and valine,
Asp/Asn/Glu is an amino acid that is selected from the group
consisting of aspartate, asparagine and glutamate; and Ser/Thr/Asn
is an amino acid that is selected from the group consisting of
serine, threonine and asparagine; and wherein the modulating agent
inhibits or enhances a function mediated by the desmosomal
cadherin.
14. (Canceled)
15. A modulating agent according to any one of claims 1, 3 and 5,
wherein the agent comprises one or more desmoglein CAR sequences
selected from the group consisting NQK, NQKT (SEQ ID NO:63), NQKTG
(SEQ ID NO:64), INQK (SEQ ID NO:65), INQKT (SEQ ID NO:66), INQKTG
(SEQ ID NO:67), VINQK (SEQ ID NO:68), VINQKT (SEQ ID NO:69),
VINQKTG (SEQ ID NO:70), FVINQK (SEQ ID NO:71), FVINQKT (SEQ ID
NO:72), FVINQKTG (SEQ ID NO:73), IFVINQK (SEQ ID NO:74), IFVINQKT
(SEQ ID NO:75), IFVINQKTG (SEQ ID NO:76), NRN, NRNT (SEQ ID NO:77),
NRNTG (SEQ ID NO:78), INRN (SEQ ID NO:79), INRNT (SEQ ID NO:80),
INRNTG (SEQ ID NO:81), IINRN (SEQ ID NO:82), IINRNT (SEQ ID NO:83),
IINRNTG (SEQ ID NO:84), FIINRN (SEQ ID NO:85), FIINRNT (SEQ ID
NO:86), FIINRNTG (SEQ ID NO:87), MFIINRN (SEQ ID NO:88), MFIINRNT
(SEQ ID NO:89), MFIINRNTG (SEQ ID NO:90), NKD, NKDT (SEQ ID NO:91),
NKDTG (SEQ ID NO:92), LNKD (SEQ ID NO:93), LNKDT (SEQ ID NO:94),
LNKDTG (SEQ ID NO:95), YLNKD (SEQ ID NO:96), YLNKDT (SEQ ID NO:97),
YLNKDTG (SEQ ID NO:98), FYLNKD (SEQ ID NO:99), FYLNKDT (SEQ ID
NO:100), FYLNKDTG (SEQ ID NO:101), VFYLNKD (SEQ ID NO:102),
VFYLNKDT (SEQ ID NO:103), VFYLNKDTG (SEQ ID NO:104), RAL, RALN (SEQ
ID NO:151), RALNS (SEQ ID NO:152), RALNSM (SEQ ID NO:153), RALNSL
(SEQ ID NO:154), RALNSMG (SEQ ID NO:155), RALNSLG (SEQ ID NO:156),
CRAL (SEQ ID NO:157), CRALN (SEQ ID NO:158), CRALNS (SEQ ID
NO:159), CRALNSM (SEQ ID NO:160), CRALNSL (SEQ ID NO:161), CRALNSMG
(SEQ ID NO:162), CRALNSLG (SEQ ID NO:163), YCRAL (SEQ ID NO:164),
YCRALN (SEQ ID NO:165), YCRALNS (SEQ ID NO:166), YCRALNSM (SEQ ID
NO:167), YCRALNSL (SEQ ID NO:168), YCRALNSMG (SEQ ID NO:169),
YCRALNSLG (SEQ ID NO:170), IYRAL (SEQ ID NO:171), IYRALN (SEQ ID
NO:172), IYRALNS (SEQ ID NO:173), IYRALNSM (SEQ ID NO:174),
IYRALNSL (SEQ ID NO:175), IYRALNSMG (SEQ ID NO:176), IYRALNSLG (SEQ
ID NO:177), YAL, YALD (SEQ ID NO:180), YALDA (SEQ ID NO:181),
YALDAR (SEQ ID NO:182), YALDARG (SEQ ID NO:183), GYAL (SEQ ID
NO:184), GYALD (SEQ ID NO:185), GYALDA (SEQ ID NO:186), GYALDAR
(SEQ ID NO:187), GYALDARG (SEQ ID NO:188), TGYAL (SEQ ID NO:189),
TGYALD (SEQ ID NO:190), TGYALDA (SEQ ID NO:191), TGYALDAR (SEQ ID
NO:192), TGYALDARG (SEQ ID NO:193), LTGYAL (SEQ ID NO:194), LTGYALD
(SEQ ID NO:195), LTGYALDA (SEQ ID NO:196), LTGYALDAR (SEQ ID
NO:197), LTGYALDARG (SEQ ID NO:198), RALNA (SEQ ID NO:200), RALNAQ
(SEQ ID NO:201), RALNAL (SEQ ID NO:202), RALNAQG (SEQ. ID NO:203),
RALNALG (SEQ ID NO:204), CRALNA (SEQ ID NO:205), CRALNAQ (SEQ ID
NO:206), CRALNAL (SEQ ID NO:207), CRALNAQG (SEQ ID NO:208),
CRALNALG (SEQ ID NO:209), TCRAL (SEQ ID NO:210), TCRALN (SEQ ID
NO:211), TCRALNA (SEQ ID NO:212), TCRALNAQ (SEQ ID NO:213),
TCRALNAL (SEQ ID NO:214), TCRALNAQG (SEQ ID NO:215), TCRALNALG (SEQ
ID NO:216), ITCRAL (SEQ ID NO:217), ITCRALN (SEQ ID NO:218),
ITCRALNA (SEQ ID NO:219), ITCRALNAQ (SEQ ID NO:220), ITCRALNAL (SEQ
ID NO:221), ITCRALNAQG (SEQ ID NO:222) and ITCRALNALG (SEQ ID
NO:223).
16. A modulating agent according to claim 15, wherein the agent
comprises a linear peptide having the sequence
N-Ac-IFVINQKTG-NH.sub.2 (SEQ ID NO:105), N-Ac-MFIINRNTG-NH.sub.2
(SEQ ID NO:106), N-Ac-VFYLNKDTG-NH.sub.2 (SEQ ID NO:107),
N-Ac-IYRALNSMG-NH.sub.2 (SEQ ID NO:______), N-Ac-IYRALNSLG-NH.sub.2
(SEQ ID NO:______), N-Ac-LTGYALDARG-NH.sub.2 (SEQ ID NO:______),
N-Ac-ITCRALNAQG-NH.sub.2 (SEQ ID NO:______) and
N-Ac-ITCRALNALG-NH.sub.2 (SEQ ID NO:______).
17. A modulating agent according to claim 15, wherein a desmoglein
CAR sequence is present within a cyclic peptide.
18. A modulating agent according to claim 17, wherein the cyclic
peptide comprises a sequence selected from the group consisting of
CNQKC (SEQ ID NO:3066), CNQKTC (SEQ ID NO:3067), CNQKTGC (SEQ ID
NO:3068), CINQKC (SEQ ID NO:3069), CINQKTC (SEQ ID NO:3070),
CINQKTGC (SEQ ID NO:3071), CVINQKC (SEQ ID NO:3072), CVINQKTC (SEQ
ID NO:3073), CVINQKTGC (SEQ ID NO:3074), CFVINQKC (SEQ ID NO:3075),
CFVINQKTC (SEQ ID NO:3076), CFVINQKTGC (SEQ ID NO:3077), CIFVINQKC
(SEQ ID NO:3078), CIFVINQKTC (SEQ ID NO:3079), CIFVINQKTGC (SEQ ID
NO:3080), CNRNC (SEQ ID NO:3081), CNRNTC (SEQ ID NO:3082), CNRNTGC
(SEQ ID NO:3083), CINRNC (SEQ ID NO:3084), CINRNTC (SEQ ID
NO:3085), CINRNTGC (SEQ ID NO:3086), CIINRNC (SEQ ID NO:3087),
CIINRNTC (SEQ ID NO:3088), CIINRNTGC (SEQ ID NO:3089), CFIINRNC
(SEQ ID NO:3090), CFIINRNTC (SEQ ID NO:3091), CFIINRNTGC (SEQ ID
NO:3092), CMFIINRNC (SEQ ID NO:3093), CMFIINRNTC (SEQ ID NO:3094),
CMFIINRNTGC (SEQ ID NO:3095), CNKDC (SEQ ID NO:3096), CNKDTC (SEQ
ID NO:3097), CNKDTGC (SEQ ID NO:3098), CLNKDC (SEQ ID NO:3099),
CLNKDTC (SEQ ID NO:3100), CLNKDTGC (SEQ ID NO:3101), CYLNKDC (SEQ
ID NO:3102), CYLNKDTC (SEQ ID NO:3103), CYLNKDTGC (SEQ ID NO:3104),
CFYLNKDC (SEQ ID NO:3105), CFYLNKDTC (SEQ ID NO:3106), CFYLNKDTGC
(SEQ ID NO:3107), CVFYLNKDC (SEQ ID NO:3108), (CVFYLNKDTC (SEQ ID
NO:3109), CVFYLNKDTGC (SEQ ID NO:3110), ENQKK (SEQ ID NO:3111),
ENQKTK (SEQ ID NO:3112), ENQKTGK (SEQ ID NO:3113), EINQKK (SEQ ID
NO:3114), EINQKTK (SEQ ID NO:3115), EINQKTGK (SEQ ID NO:3116),
EVINQKK (SEQ ID NO:3117), EVINQKTK (SEQ ID NO:3118), EVINQKTGK (SEQ
ID NO:3119), EFVINQKK (SEQ ID NO:3120), EFVINQKTK (SEQ ID NO:3121),
EFVINQKTGK (SEQ ID NO:3122), EIFVINQKK (SEQ ID NO:3123), EIFVINQKTK
(SEQ ID NO:3124), EIFVINQKTGK (SEQ ID NO:3125), ENRNK (SEQ ID
NO:3126), ENRNTK (SEQ ID NO:3127), ENRNTGK (SEQ ID NO:3128), EINRNK
(SEQ ID NO:3129), EINRNTK (SEQ ID NO:3130), EINRNTGK (SEQ ID
NO:3131), EIINRNK (SEQ ID NO:3132), EIINRNTK (SEQ ID NO:3133),
EIINRNTGK (SEQ ID NO:3134), EFIINRNK (SEQ ID NO:3135), EFIINRNTK
(SEQ ID NO:3136), EFIINRNTGK (SEQ ID NO:3137), EMFIINRNK (SEQ ID
NO:3138), EMFIINRNTK (SEQ ID NO:3139), EMFIINRNTGK (SEQ ID
NO:3140), ENKDK (SEQ ID NO:3141), ENKDTK (SEQ ID NO:3142), ENKDTGK
(SEQ ID NO:3143), ELNKDK (SEQ ID NO:3144), ELNKDTK (SEQ ID
NO:3145), ELNKDTGK (SEQ ID NO:3146), EYLNKDK (SEQ ID NO:3147),
EYLNKDTK (SEQ ID NO:3148), EYLNKDTGK (SEQ ID NO:3149), EFYLNKDK
(SEQ ID NO:3150), EFYLNKDTK (SEQ ID NO:3151), EFYLNKDTGK (SEQ ID
NO:3152), EVFYLNKDK (SEQ ID NO:3153), EVFYLNKDTK (SEQ ID NO:3154),
EVFYLNKDTGK (SEQ ID NO:3155), KNQKD (SEQ ID NO:3156), KNQKTD (SEQ
ID NO:3157), KNQKTGD (SEQ ID NO:3158), KINQKD (SEQ ID NO:3159),
KINQKTD (SEQ ID NO:3160), KINQKTGD (SEQ ID NO:3161), KVINQKD (SEQ
ID NO:3162), KVINQKTD (SEQ ID NO:3163), KVINQKTGD (SEQ ID NO:3164),
KFVINQKD (SEQ ID NO:3165), KFVINQKTD (SEQ ID NO:3166), KFVINQKTGD
(SEQ ID NO:3167), KIFVINQKD (SEQ ID NO:3168), KIFVINQKTD (SEQ ID
NO:3169), KIFVINQKTGD (SEQ ID NO:3170), KNRND (SEQ ID NO:3171),
KNRNTD (SEQ ID NO:3172), KNRNTGD (SEQ ID NO:3173), KINRND (SEQ ID
NO:3174), KINRNTD (SEQ ID NO:3175), KINRNTGD (SEQ ID NO:3176),
KIINRND (SEQ ID NO:3177), KIINRNTD (SEQ ID NO:3178), KIINRNTGD (SEQ
ID NO:3179), KFIINRND (SEQ ID NO:3180), KFIINRNTD (SEQ ID NO:3181),
KFIINRNTGD (SEQ ID NO:3182), KMFIINRND (SEQ ID NO:3183), KMFIINRNTD
(SEQ ID NO:3184), KMFIINRNTGD (SEQ ID NO:3185), KNKDD (SEQ ID
NO:3186), KNKDTD (SEQ ID NO:3187), KNKDTGD (SEQ ID NO:3188), KLNKDD
(SEQ ID NO:3189), KLNKDTD (SEQ ID NO:3190) KLNKDTGD (SEQ ID
NO:3191), KYLNKDD (SEQ ID NO:3192), KYLNKDTD (SEQ ID NO:3193),
KYLNKDTGD (SEQ ID NO:3194), KFYLNKDD (SEQ ID NO:3195), KFYLNKDTD
(SEQ ID NO:3196), KFYLNKDTGD (SEQ ID NO:3197), KVFYLNKDD (SEQ ID
NO:3198), KVFYLNKDTD (SEQ ID NO:3199), KVFYLNKDTGD (SEQ ID
NO:3200), DNQKK (SEQ ID NO:3201), DNQKTK (SEQ ID NO:3202), DNQKTGK
(SEQ ID NO:3203), DINQKK (SEQ ID NO:3204), DINQKTK (SEQ ID
NO:3205), DINQKTGK (SEQ. ID NO:3206), DVINQKK (SEQ ID NO:3207),
DVINQKTK (SEQ ID NO:3208), DVINQKTGK (SEQ ID NO:3209), DFVINQKK
(SEQ ID NO:3210), DFVINQKTK (SEQ ID NO:3211), DFVINQKTGK (SEQ ID
NO:3212), DIFVINQKK (SEQ ID NO:3213), DIFVINQKTK (SEQ ID NO:3214),
DIFVINQKTGK (SEQ ID NO:3215), DNRNK (SEQ ID NO:3216), DNRNTK (SEQ
ID NO:3217), DNRNTGK (SEQ ID NO:3218), DINRNK (SEQ ID NO:3219),
DINRNTK (SEQ ID NO:3220), DINRNTGK (SEQ ID NO:3221), DIINRNK (SEQ
ID NO:3222), DIINRNTK (SEQ ID NO:3223), DIINRNTGK (SEQ ID NO:3224),
DFIINRNK (SEQ ID NO:3225), DFIINRNTK (SEQ ID NO:3226), DFIINRNTGK
(SEQ ID NO:3227), DMFIINRNK (SEQ ID NO:3228), DMFIINRNTK (SEQ ID
NO:3229), DMFIINRNTGK (SEQ ID NO:3230), DNKDK (SEQ ID NO:3231),
DNKDTK (SEQ ID NO:3232), DNKDTGK (SEQ ID NO:3233), DLNKDK (SEQ ID
NO:3234), DLNKDTK (SEQ ID NO:3235), DLNKDTGK (SEQ ID NO:3236),
DYLNKDK (SEQ ID NO:3237), DYLNKDTK (SEQ ID NO:3238), DYLNKDTGK (SEQ
ID NO:3239), DFYLNKDK (SEQ ID NO:3240), DFYLNKDTK (SEQ ID NO:3241),
DFYLNKDTGK (SEQ ID NO:3242), DVFYLNKDK (SEQ ID NO:3243), DVFYLNKDTK
(SEQ ID NO:3244), DVFYLNKDTGK (SEQ ID NO:3245), KKNQKE (SEQ ID
NO:3246), KNQKTE (SEQ ID NO:3247), KNQKTGE (SEQ ID NO:3248), KINQKE
(SEQ ID NO:3249), KINQKTE (SEQ ID NO:3250), KINQKTGE (SEQ ID
NO:3251), KVINQKE (SEQ ID NO:3252), KVINQKTE (SEQ ID NO:3253),
KVINQKTGE (SEQ ID NO:3254), KFVINQKE (SEQ ID NO:3255), KFVINQKTE
(SEQ ID NO:3256), KFVINQKTGE (SEQ ID NO:3257), KIFVINQKE (SEQ ID
NO:3258), KIFVINQKTE (SEQ ID NO:3259), KIFVINQKTGE (SEQ ID
NO:3260), KNRNE (SEQ ID NO:3261), KNRNTE (SEQ ID NO:3262), KNRNTGE
(SEQ ID NO:3263), KINRNE (SEQ ID NO:3264), KINRNTE (SEQ ID
NO:3265), KINRNTGE (SEQ ID NO:3266), KIINRNE (SEQ ID NO:3267),
KIINRNTE (SEQ ID NO:3268), KIINRNTGE (SEQ ID NO:3269), KFIINRNE
(SEQ ID NO:3270), KFIINRNTE (SEQ ID NO:3271), KFIINRNTGE (SEQ ID NO
3272), KMFIINRNE (SEQ ID NO:3273), KMFIINRNTE (SEQ ID NO:3274),
KMFIINRNTGE (SEQ ID NO:3275), KNKDE (SEQ ID NO:3276), KNKDTE (SEQ
ID NO:3277), KNKDTGE (SEQ ID NO:3278), KLNKDE (SEQ ID NO:3279),
KLNKDTE (SEQ ID NO:3280), KLNKDTGE (SEQ ID NO:3281), KYLNKDE (SEQ
ID NO:3282), KYLNKDTE (SEQ ID NO:3283), KYLNKDTGE (SEQ ID NO:3284),
KFYLNKDE (SEQ ID NO:3285), KFYLNKDTE (SEQ ID NO:3286), KFYLNKDTGE
(SEQ ID NO:3287), KVFYLNKDE (SEQ ID NO:3288), KVFYLNKDTE (SEQ ID
NO:3289), KVFYLNKDTGE (SEQ ID NO:3290), NQKTG (SEQ ID NO:3291),
INQKT (SEQ ID NO:3292), INQKTG (SEQ ID NO:3293), VINQK (SEQ ID
NO:3294), VINQKT (SEQ ID NO:3295), VINQKTG (SEQ ID NO:3296), FVINQK
(SEQ ID NO:3297), FVINQKT (SEQ ID NO:3298), FVINQKTG (SEQ ID
NO:3299), IFVINQK (SEQ ID NO:3300), IFVINQKT (SEQ. ID NO:3301),
IFVINQKTG (SEQ ID NO:3302), NRNTG (SEQ ID NO:3303), INRNT (SEQ ID
NO:3304), INRNTG (SEQ ID NO:3305), IINRN (SEQ ID NO:3306), IINRNT
(SEQ ID NO:3307), IINRNTG (SEQ ID NO:3308), FIINRN (SEQ ID
NO:3309), FIINRNT (SEQ ID NO:3310), FIINRNTG (SEQ ID NO:3311),
MFIINRN (SEQ ID NO:3312), MFIINRNT (SEQ ID NO:3313), MFIINRNTG (SEQ
ID NO:3314), NKDTG (SEQ ID NO:3315), LNKDT (SEQ ID NO:3316), LNKDTG
(SEQ ID NO:3317), YLNKD (SEQ ID NO:3318), YLNKDT (SEQ ID NO:3319),
YLNKDTG (SEQ ID NO:3320), FYLNKD (SEQ ID NO:3321), FYLNKDT (SEQ ID
NO:3322), FYLNKDTG (SEQ ID NO:3323), VFYLNKD (SEQ ID NO:3324),
VFYLNKDT (SEQ ID NO:3325) and VFYLNKDTG (SEQ ID NO:3326), CRALC,
CRALNC, CRALNSC, CRALNSMC, CRALNSLC, CRALNSMGC, CRALNSLGC, CCRALC,
CCRALNC, CCRALNSC, CCRALNSMC, CCRALNSLC, CCRALNSMGC, CCRALNSLGC,
CYCRALC, CYCRALNC, CYCRALNSC, CYCRALNSMC, CYCRALNSLC, CYCRALNSMGC,
CYCRALNSLGC, CIYRALC, CIYRALNC, CIYRALNSC, CIYRALNSMC, CIYRALNSLC,
CIYRALNSMGC, CIYRALNSLGC, ERALK, ERALNK, ERALNSK, ERALNSMK,
ERALNSLK, ERALNSMGK, ERALNSLGK, ECRALK, ECRALNK, ECRALNSK,
ECRALNSMK, ECRALNSLK, ECRALNSMGK, ECRALNSLGK, EYCRALK, EYCRALNK,
EYCRALNSK, EYCRALNSMK, EYCRALNSLK, EYCRALNSMGK, EYCRALNSLGK,
EIYRALK, EIYRALNK, EIYRALNSK, EIYRALNSMK, EIYRALNSLK, EIYRALNSMGK,
EIYRALNSLGK, KRALE, KRALNE, KRALNSE, KRALNSME, KRALNSLE, KRALNSMGE,
KRALNSLGE, KCRALE, KCRALNE, KCRALNSE, KCRALNSME, KCRALNSLE,
KCRALNSMGE, KCRALNSLGE, KYCRALE, KYCRALNE, KYCRALNSE, KYCRALNSME,
KYCRALNSLE, KYCRALNSMGE, KYCRALNSLGE, KIYRALE, KIYRALNE, KIYRALNSE,
KIYRALNSME, KIYRALNSLE, KIYRALNSMGE, KIYRALNSLGE, DRALK, DRALNK,
DRALNSK, DRALNSMK, DRALNSLK, DRALNSMGK, DRALNSLGK, DCRALK, DCRALNK,
DCRALNSK, DCRALNSMK, DCRALNSLK, DCRALNSMGK, DCRALNSLGK, DYCRALK,
DYCRALNK, DYCRALNSK, DYCRALNSMK, DYCRALNSLK, DYCRALNSMGK,
DYCRALNSLGK, DIYRALK, DIYRALNK, DIYRALNSK, DIYRALNSMK, DIYRALNSLK,
DIYRALNSMGK, DIYRALNSLGK, KRALD, KRALND, KRALNSD, KRALNSMD,
KRALNSLD, KRALNSMGD, KRALNSLGD, KCRALD, KCRALND, KCRALNSD,
KCRALNSMD, KCRALNSLD, KCRALNSMGD, KCRALNSLGD, KYCRALD, KYCRALND,
KYCRALNSD, KYCRALNSMD, KYCRALNSLD, KYCRALNSMGD, KYCRALNSLGD,
KIYRALD, KIYRALND, KIYRALNSD, KIYRALNSMD, KIYRALNSLD, KIYRALNSMGD,
KIYRALNSLGD, RALNS, RALNSM, RALNSL, RALNSMG, RALNSLG, CRALN,
CRALNS, CRALNSM, CRALNSL, CRALNSMG, CRALNSLG, YCRAL, YCRALN,
YCRALNS, YCRALNSM, YCRALNSL, YCRALNSMG, YCRALNSLG, IYRAL, IYRALN,
IYRALNS, IYRALNSM, IYRALNSL, IYRALNSMG, IYRALNSLG, CYALC, CYALDC,
CYALDAC, CYALDARC, CYALDARGC, CGYALC, CGYALDC, CGYALDAC, CGYALDARC,
CGYALDARGC, CTGYALC, CTGYALDC, CTGYALDAC, CTGYALDARC, CTGYALDARGC,
CLTGYALC, CLTGYALDC, CLTGYALDAC, CLTGYALDARC, CLTGYALDARGC, EYALK,
EYALDK, EYALDAK, EYALDARK, EYALDARGK, EGYALK, EGYALDK, EGYALDAK,
EGYALDARK, EGYALDARGK, ETGYALK, ETGYALDK, ETGYALDAK, ETGYALDARK,
ETGYALDARGK, ELTGYALK, ELTGYALDK, ELTGYALDAK, ELTGYALDARK,
ELTGYALDARGK, KYALE, KYALDE, KYALDAE, KYALDARE, KYALDARGE, KGYALE,
KGYALDE, KGYALDAE, KGYALDARE, KGYALDARGE, KTGYALE, KTGYALDE,
KTGYALDAE, KTGYALDARE, KTGYALDARGE, KLTGYALE, KLTGYALDE,
KLTGYALDAE, KLTGYALDARE, KLTGYALDARGE, DYALK, DYALDK, DYALDAK,
DYALDARK, DYALDARGK, DGYALK, DGYALDK, DGYALDAK, DGYALDARK,
DGYALDARGK, DTGYALK, DTGYALDK, DTGYALDAK, DTGYALDARK, DTGYALDARGK,
DLTGYALK, DLTGYALDK, DLTGYALDAK, DLTGYALDARK, DLTGYALDARGK, KYALD,
KYALDD, KYALDAD, KYALDARD, KYALDARGD, KGYALD, KGYALDD, KGYALDAD,
KGYALDARD, KGYALDARGD, KTGYALD, KTGYALDD, KTGYALDAD, KTGYALDARD,
KTGYALDARGD, KLTGYALD, KLTGYALDD, KLTGYALDAD, KLTGYALDARD,
KLTGYALDARGD, YALDA, YALDAR, YALDARG, GYALD, GYALDA, GYALDAR,
GYALDARG, TGYAL, TGYALD, TGYALDA, TGYALDAR, TGYALDARG, LTGYAL,
LTGYALD, LTGYALDA, LTGYALDAR, LTGYALDARG, CRALC, CRALNC, CRALNAC,
CRALNAQC, CRALNALC, CRALNAQGC, CRALNALGC, CCRALC, CCRALNC,
CCRALNAC, CCRALNAQC, CCRALNALC, CCRALNAQGC, CCRALNALGC, CTCRALC,
CTCRALNC, CTCRALNAC, CTCRALNAQC, CTCRALNALC, CTCRALNALGC,
CTCRALNALGC, CITCRALC, CITCRALNC, CITCRALNAC, CITCRALNAQC,
CITCRALNALC, CITCRALNAQGC, CITCRALNALGC, ERALK, ERALNK, ERALNAK,
ERALNAQK, ERALNALK, ERALNAQGK, ERALNALGK, ECRALK, ECRALNK,
ECRALNAK, ECRALNAQK, ECRALNALK, ECRALNAQGK, ECRALNALGK, ETCRALK,
ETCRALNK, ETCRALNAK, ETCRALNAQK, ETCRALNALK, ETCRALNAQGK,
ETCRALNALGK, EITCRALK, EITCRALNK, EITCRALNAK, EITCRALNAQK,
EITCRALNALK, EITCRALNAQGK, EITCRALNALGK, KRALE, KRALNE, KRALNAE,
KRALNAQE, KRALNALE, KRALNAQGE, KRALNALGE, KCRALE, KCRALNE,
KCRALNAE, KCRALNAQE, KCRALNALE, KCRALNAQGE, KCRALNALGE, KTCRALE,
KTCRALNE, KTCRALNAE, KTCRALNAQE, KTCRALNALE, KTCRALNAQGE,
KTCRALNALGE, KITCRALE, KITCRALNE, KITCRALNAE, KITCRALNAQE,
KITCRALNALE, KITCRALNAQGE, KITCRALNALGE, DRALK, DRALNK, DRALNAK,
DRALNAQK, DRALNALK, DRALNAQGK, DRALNALGK, DCRALK, DCRALNK,
DCRALNAK, DCRALNAQK, DCRALNALK, DCRALNAQGK, DCRALNALGK, DTCRALK,
DTCRALNK, DTCRALNAK, DTCRALNAQK, DTCRALNALK, DTCRALNAQGK,
DTCRALNALGK, DITCRALK, DITCRALNK, DITCRALNAK, DITCRALNAQK,
DITCRALNALK, DITCRALNAQGK, DITCRALNALGK, KRALD, KRALND, KRALNAD,
KRALNAQD, KRALNALD, KRALNAQGD, KRALNALGD, KCRALD, KCRALND,
KCRALNAD, KCRALNAQD, KCRALNALD, KCRALNAQGD, KCRALNALGD, KTCRALD,
KTCRALND, KTCRALNAD, KTCRALNAQD, KTCRALNALD, KTCRALNAQGD,
KTCRALNALGD, KITCRALD, KITCRALND, KITCRALNAD, KITCRALNAQD,
KITCRALNALD, KITCRALNAQGD, KITCRALNALGD, RALNA, RALNAQ, RALNAL,
RALNAQG, RALNALG, CRALN, CRALNA, CRALNAQ, CRALNAL, CRALNAQG,
CRALNALG, TCRAL, TCRALN, TCRALNA, TCRALNAQ, TCRALNAL, TCRALNAQG,
TCRALNALG, ITCRAL, ITCRALN, ITCRALNA, ITCRALNAQ, ITCRALNAL,
ITCRALNAQG and ITCRALNALG.
19. A modulating agent comprising an antibody or antigen-binding
fragment thereof that: (a) specifically binds to a desmoglein CAR
sequence selected from the group consisting of
N-Ac-IFVINQKTG-NH.sub.2 (SEQ ID NO:105), N-Ac-MFIINRNTG-NH.sub.2
(SEQ ID NO:106), N-Ac-VFYLNKDTG-NH.sub.2 (SEQ ID NO:107),
N-Ac-IYRALNSMG-NH.sub.2 (SEQ ID NO:______), N-Ac-IYRALNSLG-NH.sub.2
(SEQ ID NO:______), N-Ac-LTGYALDARG-NH.sub.2 (SEQ ID NO:______),
N-Ac-ITCRALNAQG-NH.sub.2 (SEQ ID NO:______) and
N-Ac-ITCRALNALG-NH.sub.2 (SEQ ID NO:______); and (b) modulates a
desmoglein-mediated function.
20. A modulating agent according to any one of claims 1, 3 and 5,
wherein the agent comprises one or more desmocollin CAR sequences
selected from the group consisting EKD, EKDT (SEQ ID NO:108), EKDTG
(SEQ ID NO:109), IEKD (SEQ ID NO:10), IEKDT (SEQ ID NO:111), IEKDTG
(SEQ ID NO:112), YIEKD (SEQ ID NO:113), YIEKDT (SEQ ID NO:114),
YIEKDTG (SEQ ID NO:115), FYIEKD (SEQ ID NO:116), FYIEKDT (SEQ ID
NO:117), FYIEKDTG (SEQ ID NO:118), LFYIEKD (SEQ ID NO:119),
LFYIEKDT (SEQ ID NO:120), LFYIEKDTG (SEQ ID NO:121), ERD, ERDT (SEQ
ID NO:122), ERDTG (SEQ ID NO:123), VERD (SEQ ID NO:124), VERDT (SEQ
ID NO:125), VERDTG (SEQ ID NO:126), YVERD (SEQ ID NO:127), YVERDT
(SEQ ID NO:128), YVERDTG (SEQ ID NO:129), FYVERD (SEQ ID NO:130),
FYVERDT (SEQ ID NO:131), FYVERDTG (SEQ ID NO:132), LFYVERD (SEQ ID
NO:133), LFYVERDT (SEQ ID NO:134), LFYVERDTG (SEQ ID NO:135), IERD
(SEQ ID NO:136), IERDT (SEQ ID NO:137), IERDTG (SEQ ID NO:138),
YIERD (SEQ ID NO:139), YIERDT (SEQ ID NO:140), YIERDTG (SEQ ID
NO:141), FYIERD (SEQ ID NO:142), FYIERDT (SEQ ID NO:143), FYIERDTG
(SEQ ID NO:144), LFYIERD (SEQ ID NO:145), LFYIERDT (SEQ ID NO:146)
and LFYIERDTG (SEQ ID NO:147), YAT, YATT (SEQ ID NO:226), YATTA
(SEQ ID NO:227), YATTAD (SEQ ID NO:228), YATTADG (SEQ ID NO:229),
GYAT (SEQ ID NO:230), GYATT (SEQ ID NO:231), GYATTA (SEQ ID
NO:232), GYATTAD (SEQ ID NO:233), GYATTADG (SEQ ID NO:234), AYAT
(SEQ ID NO:235), AYATT (SEQ ID NO:236), AYATTA (SEQ ID NO:237),
AYATTAD (SEQ ID NO:238), AYATTADG (SEQ ID NO:239), YGYAT (SEQ ID
NO:240), YGYATT (SEQ ID NO:241), YGYATTA (SEQ ID NO:242), YGYATTAD
(SEQ ID NO:243), YGYATTADG (SEQ ID NO:244), YAYAT (SEQ ID NO:245),
YAYATT (SEQ ID NO:246), YAYATTA (SEQ ID NO:247), YAYATTAD (SEQ ID
NO:248), YAYATTADG (SEQ ID NO:249), LYGYAT (SEQ ID NO:250), LYGYATT
(SEQ ID NO:251), LYGYATTA (SEQ ID NO:252), LYGYATTAD (SEQ ID
NO:253), LYGYATTADG (SEQ ID NO:254), LYAYAT (SEQ ID NO:255),
LYAYATT (SEQ ID NO:256), LYAYATTA (SEQ ID NO:257), LYAYATTAD (SEQ
ID NO:258), LYAYATTADG (SEQ ID NO:259), VYGYAT (SEQ ID NO:260),
VYGYATT (SEQ ID NO:261), VYGYATTA (SEQ ID NO:262), VYGYATTAD (SEQ
ID NO:263), VYGYATTADG (SEQ ID NO:264), VYAYAT (SEQ ID NO:265),
VYAYATT (SEQ ID NO:266), VYAYATTA (SEQ ID NO:267), VYAYATTAD (SEQ
ID NO:268), VYAYATTADG (SEQ ID NO:269), IYGYAT (SEQ ID NO:270),
IYGYATT (SEQ ID NO:271), IYGYATTA (SEQ ID NO:272), IYGYATTAD (SEQ
ID NO:273), IYGYATTADG (SEQ ID NO:274), IYAYAT (SEQ ID NO:275),
IYAYATT (SEQ ID NO:276), IYAYATTA (SEQ ID NO:277), IYAYATTAD (SEQ
ID NO:278) or IYAYATTADG (SEQ ID NO:279), FAT, FATT (SEQ ID
NO:286), FATTP (SEQ ID NO:287), FATTPD (SEQ ID NO:288), FATTPDG
(SEQ ID NO:289), AFAT (SEQ ID NO:290), AFATT (SEQ ID NO:291),
AFATTP (SEQ ID NO:292), AFATTPD (SEQ ID NO:293), AFATTPDG (SEQ ID
NO:294), IAFAT (SEQ ID NO:295), IAFATT (SEQ ID NO:296), IAFATTP
(SEQ ID NO:297), IAFATTPD (SEQ ID NO:298), IAFATTPDG (SEQ ID
NO:299), IIAFAT (SEQ ID NO:300), IIAFATT (SEQ ID NO:301), IIAFATTP
(SEQ ID NO:302), IIAFATTPD (SEQ ID NO:303), IIAFATTPDG (SEQ ID
NO:304), LIAFAT (SEQ ID NO:305), LIAFATT (SEQ ID NO:306), LIAFATTP
(SEQ ID NO:307), LIAFATTPD (SEQ ID NO:308), LIAFATTPDG (SEQ ID
NO:309), YAS, YAST (SEQ ID NO:312), YASTA (SEQ ID NO:313), YASTAD
(SEQ ID NO:314), YASTADG (SEQ ID NO:315), AYAS (SEQ ID NO:316),
AYAST (SEQ ID NO:317), AYASTA (SEQ ID NO:318), AYASTAD (SEQ ID
NO:319), AYASTADG (SEQ ID NO:320), IAYAS (SEQ ID NO:321), IAYAST
(SEQ ID NO:322), IAYASTA (SEQ ID NO:323), IAYASTAD (SEQ ID NO:324),
IAYASTADG (SEQ ID NO:325), LIAYAS (SEQ ID NO:326), LIAYAST (SEQ ID
NO:327), LIAYASTA (SEQ ID NO:328), LIAYASTAD (SEQ ID NO:329),
LIAYASTADG (SEQ ID NO:330).
21. A modulating agent according to claim 20, wherein the agent
comprises a linear peptide having the sequence
N-Ac-LFYIEKDTG-NH.sub.2 (SEQ ID NO:148), N-Ac-LFYVERDTG-NH.sub.2
(SEQ ID NO:149), N-Ac-LFYIERDTG-NH.sub.2 (SEQ ID NO:150),
N-Ac-LYGYATTADG-NH.sub.2 (SEQ ID NO:280), N-Ac-LYAYATTADG-NH.sub.2
(SEQ ID NO:281), N-Ac-VYGYATTADG-NH.sub.2 (SEQ ID NO:282),
N-Ac-VYAYATTADG-NH.sub.2 (SEQ ID NO:283), N-Ac-IYGYATTADG-NH.sub.2
(SEQ ID NO:284), N-Ac-IYAYATTADG-NH.sub.2 (SEQ ID NO:285),
IIAFATTPDG-NH.sub.2 (SEQ ID NO:310), N-Ac-LIAFATTPDG-NH.sub.2 (SEQ
ID NO:311) or N-Ac-LIAYASTADG-NH.sub.2 (SEQ ID NO:331).
22. A modulating agent according to claim 20, wherein a desmocollin
CAR sequence is present within a cyclic peptide.
23. A modulating agent according to claim 22, wherein the cyclic
peptide comprises a sequence selected from the group consisting of
CEKDC (SEQ ID NO:3327), CEKDTC (SEQ ID NO:3328), CEKDTGC (SEQ ID
NO:3329), CIEKDC (SEQ ID NO:3330), CIEKDTC (SEQ ID NO:3331),
CIEKDTGC (SEQ ID NO:3332), CYIEKDC (SEQ ID NO:3333), CYIEKDTC (SEQ
ID NO:3334), CYIEKDTGC (SEQ ID NO:3335), CFYIEKDC (SEQ ID NO:3336),
CFYIEKDTC (SEQ ID NO:3337), CFYIEKDTGC (SEQ ID NO:3338), CLFYIEKDC
(SEQ ID NO:3339), CLFYIEKDTC (SEQ ID NO:3340), CLFYIEKDTGC (SEQ ID
NO:3341), CERDC (SEQ ID NO:3342), CERDTC (SEQ ID NO:3343), CERDTGC
(SEQ ID NO:3344), CVERDC (SEQ ID NO:3345), CVERDTC (SEQ ID
NO:3346), CVERDTGC (SEQ ID NO:3347), CYVERDC (SEQ ID NO:3348),
CYVERDTC (SEQ ID NO:3349), CYVERDTGC (SEQ ID NO:3350), CFYVERDC
(SEQ ID NO:3351), CFYVERDTC (SEQ ID NO:3352), CFYVERDTGC (SEQ ID
NO:3353), CLFYVERDC (SEQ ID NO:3354), CLFYVERDTC (SEQ ID NO:3355),
CLFYVERDTGC (SEQ ID NO:3356), CIERDC (SEQ ID NO:3357), CIERDTC (SEQ
ID NO:3358), CIERDTGC (SEQ ID NO:3359), CYIERDC (SEQ ID NO:3360),
CYIERDTC (SEQ ID NO:3361), CYIERDTGC (SEQ ID NO:3362), CFYIERDC
(SEQ ID NO:3363), CFYIERDTC (SEQ ID NO:3364), CFYIERDTGC (SEQ ID
NO:3365), CLFYIERDC (SEQ. ID NO:3366), CLFYIERDTC (SEQ ID NO:3367),
CLFYIERDTGC (SEQ ID NO:3368), EEKDK (SEQ ID NO:3369), EEKDTK (SEQ
ID NO:3370), EEKDTGK (SEQ ID NO:3371), EIEKDK (SEQ ID NO:3372),
EIEKDTK (SEQ ID NO:3373), EIEKDTGK (SEQ ID NO:3374), EYIEKDK (SEQ
ID NO:3375), EYIEKDTK (SEQ ID NO:3376), EYIEKDTGK (SEQ ID NO:3377),
EFYIEKDK (SEQ ID NO:3378), EFYIEKDTK (SEQ ID NO:3379), EFYIEKDTGK
(SEQ ID NO:3380), ELFYIEKDK (SEQ ID NO:3381), ELFYIEKDTK (SEQ ID
NO:3382), ELFYIEKDTGK (SEQ ID NO:3383), EERDK (SEQ ID NO:3384),
EERDTK (SEQ ID NO:3385), EERDTGK (SEQ ID NO:3386), EVERDK (SEQ ID
NO:3387), EVERDTK (SEQ ID NO:3388), EVERDTGK (SEQ ID NO:3389),
EYVERDK (SEQ ID NO:3390), YVERDTK (SEQ ID NO:3391), EYVERDTGK (SEQ
ID NO:3392), EFYVERDK (SEQ ID NO:3393), EFYVERDTK (SEQ ID NO:3394),
EFYVERDTGK (SEQ ID NO:3395), ELFYVERDK (SEQ ID NO:3396), ELFYVERDTK
(SEQ ID NO:3397), ELFYVERDTGK (SEQ ID NO:3398), EIERDK (SEQ ID
NO:3399), EIERDTK (SEQ ID NO:3400), EIERDTGK (SEQ ID NO:3401),
EYIERDK (SEQ ID NO:3402), EYIERDTK (SEQ ID NO:3403), EYIERDTGK (SEQ
ID NO:3404), EFYIERDK (SEQ ID NO:3405), EFYIERDTK (SEQ ID NO:3406),
EFYIERDTGK (SEQ ID NO:3407), ELFYIERDK (SEQ ID NO:3408), ELFYIERDTK
(SEQ ID NO:3409), ELFYIERDTGK (SEQ ID NO:3410), KEKDD (SEQ ID
NO:3411), KEKDTD (SEQ ID NO:3412), KEKDTGD (SEQ ID NO:3413), KIEKDD
(SEQ ID NO:3414), KIEKDTD (SEQ ID NO:3415), KIEKDTGD (SEQ ID
NO:3416), KYIEKDD (SEQ ID NO:3417), KYIEKDTD (SEQ ID NO:3418),
KYIEKDTGD (SEQ ID NO:3419), KFYIEKDD (SEQ ID NO:3420), KFYIEKDTD
(SEQ ID NO:3421), KFYIEKDTGD (SEQ ID NO:3422), KLFYIEKDD (SEQ ID
NO:3423), KLFYIEKDTD (SEQ ID NO:3424), KLFYIEKDTGD (SEQ ID
NO:3425), KERDD (SEQ ID NO:3426), KERDTD (SEQ ID NO:3427), KERDTGD
(SEQ ID NO:3428), KVERDD (SEQ ID NO:3429), KVERDTD (SEQ ID
NO:3430), KVERDTGD (SEQ ID NO:3431), KYVERDD (SEQ ID NO:3432),
KYVERDTD (SEQ ID NO:3433), KYVERDTGE (SEQ ID NO:3434), KFYVERDD
(SEQ ID NO:3435), KFYVERDTD (SEQ ID NO:3436), KFYVERDTGD (SEQ ID
NO:3437), KLFYVERDD (SEQ ID NO:3438), KLFYVERDTD (SEQ ID NO:3439),
KLFYVERDTGD (SEQ ID NO:3440), KIERDD (SEQ ID NO:3441), KIERDTD (SEQ
ID NO:3442), KIERDTGD (SEQ ID NO:3443), KYIERD (SEQ ID NO:3444),
KYIERDTD (SEQ ID NO:3445), KYIERDTGD (SEQ ID NO:3446), KFYIERDD
(SEQ ID NO:3447), KFYIERDTD (SEQ ID NO:3448), KFYIERDTGD (SEQ ID
NO:3449), KLFYIERDD (SEQ ID NO:3450), KLFYIERDTD (SEQ ID NO:3451),
KLFYIERDTGD (SEQ ID NO:3452), DEKDK (SEQ ID NO:3453), DEKDTK (SEQ
ID NO:3454), DEKDTGK (SEQ ID NO:3455), DIEKDK (SEQ ID NO:3456),
DIEKDTK (SEQ ID NO:3457), DIEKDTGK (SEQ ID NO:3458), DYIEKDK (SEQ
ID NO:3459), DYIEKDTK (SEQ ID NO:3460), DYIEKDTGK (SEQ ID NO:3461),
DFYIEKDK (SEQ ID NO:3462), DFYIEKDTK (SEQ ID NO:3463), DFYIEKDTGK
(SEQ ID NO:3464), DLFYIEKDK (SEQ ID NO:3465), DLFYIEKDTK (SEQ ID
NO:3466), DLFYIEKDTGK (SEQ ID NO:3467), DERDK (SEQ ID NO:3468),
DERDTK (SEQ ID NO:3469), DERDTGK (SEQ ID NO:3470), DVERDK (SEQ ID
NO:3471), DVERDTK (SEQ ID NO:3472), DVERDTGK (SEQ ID NO:3473),
DYVERDK (SEQ ID NO:3474), DYVERDTK (SEQ ID NO:3475), DYVERDTGK (SEQ
ID NO:3476), DFYVERDK (SEQ ID NO:3477), DFYVERDTK (SEQ ID NO:3478),
DFYVERDTGK (SEQ ID NO:3479), DLFYVERDK (SEQ ID NO:3480), DLFYVERDTK
(SEQ ID NO:3481), DLFYVERDTGK (SEQ ID NO:3482), DIERDK (SEQ ID
NO:3483), DIERDTK (SEQ ID NO:3484), DIERDTGK (SEQ ID NO:3485),
DYIERDK (SEQ ID NO:3486), DYIERDTK (SEQ ID NO:3487), DYIERDTGK (SEQ
ID NO:3488), DFYIERDK (SEQ ID NO:3489), DFYIERDTK (SEQ ID NO:3490),
DFYIERDTGK (SEQ ID NO:3491), DLFYIERDK (SEQ ID NO:3492), DLFYIERDTK
(SEQ ID NO:3493), DLFYIERDTGK (SEQ ID NO:3494), KEKDE (SEQ ID
NO:3495), KEKDTE (SEQ ID NO:3496), KEKDTGE (SEQ ID NO:3497), KIEKDE
(SEQ ID NO:3498), KIEKDTE (SEQ ID NO:3499), KIEKDTGE (SEQ ID
NO:3500), KYIEKDE (SEQ ID NO:3501), KYIEKDTE (SEQ ID NO:3502),
KYIEKDTGE (SEQ ID NO:3503), KFYIEKDE (SEQ ID NO:3504), KFYIEKDTE
(SEQ ID NO:3505), KFYIEKDTGE (SEQ ID NO:3506), KLFYIEKDE (SEQ ID
NO:3507), KLFYIEKDTE (SEQ ID NO:3508), KLFYIEKDTGE (SEQ ID
NO:3509), KERDE (SEQ ID NO:3510), KERDTE (SEQ ID NO:3511), KERDTGE
(SEQ ID NO:3512), KVERDE (SEQ ID NO:3513), KVERDTE (SEQ ID
NO:3514), KVERDTGE (SEQ ID NO:3515), KYVERDE (SEQ ID NO:3516),
KYVERDTE (SEQ ID NO:3517), KYVERDTGE (SEQ ID NO:3518), KFYVERDE
(SEQ ID NO:3519), KFYVERDTE (SEQ ID NO:3520), KFYVERDTGE (SEQ ID
NO:3521), KLFYVERDE (SEQ ID NO:3522), KLFYVERDTE (SEQ ID NO:3523)
KLFYVERDTGE (SEQ ID NO:3524), KIERDE (SEQ ID NO:3525) KIERDTE (SEQ
ID NO:3526), KIERDTGE (SEQ ID NO:3527), KYIERDE (SEQ ID NO:3528),
KYIERDTE (SEQ ID NO:3529), KYIERDTGE (SEQ ID NO:3530), KFYIERDE
(SEQ ID NO:3531), KFYIERDTE (SEQ ID NO:3532), KFYIERDTGE (SEQ ID
NO:3533), KLFYIERDE (SEQ ID NO:3534), KLFYIERDTE (SEQ ID NO:3535),
KLFYIERDTGE (SEQ ID NO:3536), EKDTG (SEQ ID NO:3537), IEKDT (SEQ ID
NO:3538), IEKDTG (SEQ ID NO:3539), YIEKD (SEQ ID NO:3540), YIEKDT
(SEQ ID NO:3541), YIEKDTG (SEQ ID NO:3542), FYIEKD (SEQ ID
NO:3543), FYIEKDT (SEQ ID NO:3544), FYIEKDTG (SEQ ID NO:3545),
LFYIEKD (SEQ ID NO:3546), LFYIEKDT (SEQ ID NO:3547), LFYIEKDTG (SEQ
ID NO:3548), ERDTG (SEQ ID NO:3549), VERDT (SEQ ID NO:3550), VERDTG
(SEQ ID NO:3551), YVERD (SEQ ID NO:3552), YVERDT (SEQ ID NO:3553),
YVERDTG (SEQ ID NO:3554), FYVERD (SEQ ID NO:3555), FYVERDT (SEQ ID
NO:3556), FYVERDTG (SEQ ID NO:3557), LFYVERD (SEQ ID NO:3558),
LFYVERDT (SEQ ID NO:3559), LFYVERDTG (SEQ ID NO:3560), IERDT (SEQ
ID NO:3561), IERDTG (SEQ ID NO:3562), YIERD (SEQ ID NO:3563),
YIERDT (SEQ ID NO:3564), YIERDTG (SEQ ID NO:3565), FYIERD (SEQ ID
NO:3566), FYIERDT (SEQ ID NO:3567), FYIERDTG (SEQ ID NO:3568),
LFYIERD (SEQ ID NO:3569), LFYIERDT (SEQ ID NO:3570), LFYIERDTG (SEQ
ID NO:3571), CYATC, CYATTC, CYATTAC, CYATTADC, CYATTADGC, CGYATC,
CGYATTC, CGYATTAC, CGYATTADC, CGYATTADGC, CAYATC, CAYATTC,
CAYATTAC, CAYATTADC, CAYATTADGC, CYGYATC, CYGYATTC, CYGYATTAC,
CYGYATTADC, CYGYATTADGC, CYAYATC, CYAYATTC, CYAYATTAC, CYAYATTADC,
CYAYATTADGC, CLYGYATC, CLYGYATTC, CLYGYATTAC, CLYGYATTADC,
CLYGYATTADGC, CLYAYATC, CLYAYATTC, CLYAYATTAC, CLYAYATTADC,
CLYAYATTADGC, CVYGYATC, CVYGYATTC, CVYGYATTAC, CVYGYATTADC,
CVYGYATTADGC, CVYAYATC, CVYAYATTC, CVYAYATTAC, CVYAYATTADC,
CVYAYATTADGC, CIYGYATC, CIYGYATTC, CIYGYATTAC, CIYGYATTADC,
CIYGYATTADGC, CIYAYATC, CIYAYATTC, CIYAYATTAC, CIYAYATTADC,
CIYAYATTADGC, EYATK, EYATTK, EYATTAK, EYATTADK, EYATTADGK, EGYATK,
EGYATTK, EGYATTAK, EGYATTADK, EGYATTADGK, EAYATK, EAYATTK,
EAYATTAK, EAYATTADK, EAYATTADGK, EYGYATK, EYGYATTK, EYGYATTAK,
EYGYATTADK, EYGYATTADGK, EYAYATK, EYAYATTK, EYAYATTAK, EYAYATTADK,
EYAYATTADGK, ELYGYATK, ELYGYATTK, ELYGYATTAK, ELYGYATTADK,
ELYGYATTADGK, ELYAYATK, ELYAYATTK, ELYAYATTAK, ELYAYATTADK,
ELYAYATTADGK, EVYGYATK, EVYGYATTK, EVYGYATTAK, EVYAYATTADK,
EVYGYATTADGK, EVYAYATK, EVYAYATTK, EVYAYATTAK, EVYAYATTADK,
EVYAYATTADGK, EIYGYATK, EIYGYATTK, EIYGYATTAK, EIYGYATTADK,
EIYGYATTADGK, EIYAYATK, EIYAYATTK, EIYAYATTAK, EIYAYATTADK,
EIYAYATTADGK, KYATE, KYATTE, KYATTAE, KYATTADE, KYATTADGE, KGYATE,
KGYATTE, KYATTAE, KGYATTADE, KGYATTADGE, KAYATE, KAYATTE, KAYATTAE,
KAYATTADE, KAYATTADGE, KYGYATE, KYGYATTE, KYGYATTAE, KYGYATTADE,
KYGYATTADGE, KYAYATE, KYAYATTE, KYAYATTAE, KYAYATTADE, KYAYATTADGE,
KLYGYATE, KLYGYATTE, KLYGYATTAE, KLYGYATTADE, KLYGYATTADGE,
KLYAYATE, KLYAYATTE, KLYAYATTAE, KLYAYATTADE, KLYAYATTADGE,
KVYGYATE, KVYGYATTE, KVYGYATTAE, KVYGYATTADE, KVYGYATTADGE,
KVYAYATE, KVYAYATTE, KVYAYATTAE, KVYAYATTADE, KVYAYATTADGE,
KIYGYATE, KIYGYATTE, KIYGYATTAE, KIYGYATTADE, KIYGYATTADGE,
KIYAYATE, KIYAYATTE, KIYAYATTAE, KIYAYATTADE. KIYAYATTADGE, DYATK,
DYATTK, DYATTAK, DYATTADK, DYATTADGK, DGYATK, DGYATTK, DGYATTAK,
DGYATTADK, DGYATTADGK, DAYATK, DAYATTK DAYATTAK, DAYATTADK,
DAYATTADGK, DYGYATK, DYGYATTK, DYGYATTAK, DYGYATTADK, DYGYATTADGK,
DYAYATK, DYAYATTK, DYAYATTAK, DYAYATTADK, DYAYATTADGK, DLYGYATK,
DLYGYATTK, DLYGYATTAK, DLYGYATTADK, DLYGYATTADGK, DLYAYATK,
DLYAYATTK, DLYAYATTAK, DLYAYATTADK, DLYAYATTADGK, DVYGYATK,
DVYGYATTK, DVYGYATTAK, DVYGYATTADK, DVYGYATTADGK, DVYAYATK,
DVYAYATTK, DVYAYATTAK, DVYAYATTADK, DVYAYATTADGK, DIYGYATK,
DIYGYATTK, DIYGYATTAK, DIYGYATTADK, DIYGYATTADGK, DIYAYATK,
DIYAYATTK, DIYAYATTAK, DIYAYATTADK, DIYAYATTADGK, KYATD, KYATTD,
KYATTAD, KYATTADD, KYATTADGD, KGYATD, KGYATTD, KGYATTAD, KGYATTADD,
KGYATTADGD, KAYATD, KAYATTD, KAYATTAD, KAYATTADD, KAYATTADGD,
KYGYATD, KYGYATTD, KYGYATTAD, KYGYATTADD, KYGYATTADGD, KYAYATD,
KYAYATTD, KYAYATTAD, KYAYATTADD, KYAYATTADGD, KLYGYATD, KLYGYATTD,
KLYGYATTAD, KLYGYATTADD, KLYGYATTADGD, KLYAYATD, KLYAYATTD,
KLYAYATTAD, KLYAYATTADD, KLYAYATTADGD, KVYGYATD, KVYGYATTD,
KVYGYATTAD, KVYGYATTADD, KVYGYATTADGD, KVYAYATD, KVYAYATTD,
KVYAYATTAD, KVYAYATTADD, KVYAYATTADGD, KIYGYATD, KYGYATTD,
KIYGYATTAD, KIYGYATTADD, KIYGYATTADGD, KIYAYATD, KIYAYATTD,
KIYAYATTAD, KIYAYATTADD, KIYAYATTADGD, YATTA, YATTAD, YATTADG,
GYATT, GYATTA, GYATTAD, GYATTADG, AYATT, AYATTA, AYATTAD, AYATTADG,
YGYAT, YGYATT, YGYATTA, YGYATTAD, YGYATTADG, YAYAT, YAYATT,
YAYATTA, YAYATTAD, YAYATTADG, LYGYAT, LYGYATT, LYGYATTA, LYGYATTAD,
LYGYATTADG, LYAYAT, LYAYATT, LYAYATTA, LYAYATTAD, LYAYATTADG,
VYGYAT, VYGYATT, VYGYATTA, VYGYATTAD, VYGYATTADG, VYAYAT, VYAYATT,
VYAYATTA, VYAYATTAD, VYAYATTADG, IYGYAT, IYGYATT, IYGYATTA,
IYGYATTAD, IYGYATTADG, IYAYAT, IYAYATT, IYAYATTA, IYAYATTAD,
IYAYATTADG, CFATC, CFATTC, CFATTPC, CFATTPDC, CFATTPDGC, CAFATC,
CAFATTC, CAFATTPC, CAFATTPDC, CAFATTPDGC, CIAFATC, CIAFATTC,
CIAFATTPC, CIAFATTPDC, CIAFATTPDGC, CIIAFATC, CIIAFATTC,
CIIAFATTPC, CIIAFATTPDC, CIIAFATTPDGC, CLIAFATC, CLIAFATTC,
CLIAFATTPC, CLIAFATTPDC, CLIAFATTPDGC, EFATK, EFATTK, EFATTPK,
EFATTPDK, EFATTPDGK, EAFATK, EAFATTK, EAFATTPK, EAFATTPDK,
EAFATTPDGK, EIAFATK, EIAFATTK, EIAFATTPK, EIAFATTPDK, EIAFATTPDGK,
EIIAFATK, EIIAFATTK, EIIAFATTPK, EIIAFATTPDK, EIIAFATTPDGK,
ELIAFATK, ELIAFATTK, ELIAFATTPK, ELIAFATTPDK, ELIAFATTPDGK, KFATE,
KFATTE, KFATTPE, KFATTPDE, KFATTPDGE, KAFATE, KAFATTE, KAFATTPE,
KAFATTPDE, KAFATTPDGE, KIAFATE, KIAFATTE, KIAFATTPE, KIAFATTPDE,
KIAFATTPDGE, KIIAFATE, KIIAFATTE, KIIAFATTPE, KIIAFATTPDE,
KIIAFATTPDGE, KLIAFATE, KLIAFATTE, KLIAFATTPE, KLIAFATTPDE,
KLIAFATTPDGE, DFATK, DFATTK, DFATTPK, DFATTPDK, DFATTPDGK, DAFATK,
DAFATTK, DAFATTPK, DAFATTPDK, DAFATTPDGK, DIAFATK, DIAFATTK,
DIAFATTPK, DIAFATTPDK, DIAFATTPDGK, DIIAFATK, DIIAFATTK,
DIIAFATTPK, DIIAFATTPDK, DIIAFATTPDGK, DLIAFATK, DLIAFATTK,
DLIAFATTPK, DLIAFATTPDK, DLIAFATTPDGK, KFATD, KFATTD, KFATTPD,
KFATTPDD, KFATTPDGD, KAFATD, KAFATTD, KAFATTPD, KAFATTPDD,
KAFATTPDGD, KIAFATD, KIAFATTD, KIAFATTPD, KIAFATTPDD, KIAFATTPDGD,
KIIAFATD, KIIAFATTD, KIIAFATTPD, KIIAFATTPDD, KIIAFATTPDGD,
KLIAFATD, KLIAFATTD, KLIAFATTPD, KLIAFATTPDD, KLIAFATTPDGD, FATTP,
FATTPD, FATTPDG, AFATT, AFATTP, AFATTPD, AFATTPDG, IAFAT, IAFATT,
IAFATTP, IAFATTPD, IAFATTPDG, IIAFAT, IIAFATT, IIAFATTP, IIAFATTPD,
IIAFATTPDG, LIAFAT, LIAFATT, LIAFATTP, LIAFATTPD, LIAFATTPDG,
CYASC, CYASTC, CYASTAC, CYASTADC, CYASTADGC, CAYASC, CAYASTC,
CAYASTAC, CAYASTADC, CAYASTADGC, CIAYASC, CIAYASTC, CIAYASTAC,
CIAYASTADC, CIAYASTADGC, CLIAYASC, CLIAYASTC, CLIAYASTAC,
CLIAYASTADC, CLIAYASTADGC, EYASK, EYASTK, EYASTAK, EYASTADK,
EYASTADGK, EAYASK, EAYASTK, EAYASTAK, EAYASTADK, EAYASTADGK,
EIAYASK, EIAYASTK, EIAYASTAK, EIAYASTADK, EIAYASTADGK, ELIAYASK,
ELIAYASTK, ELIAYASTAK, ELIAYASTADK, ELIAYASTADGK, KYASE, KYASTE,
KYASTAE, KYASTADE, KYASTADGE, KAYASE, KAYASTE, KAYASTAE, KAYASTADE,
KAYASTADGE, KIAYASE, KIAYASTE, KIAYASTAE, KIAYASTADE, KIAYASTADGE,
KLIAYASE, KLIAYASTE, KLIAYASTAE, KLIAYASTADE, KLIAYASTADGE, DYASK,
DYASTK, DYASTAK, DYASTADK, DYASTADGK, DAYASK, DAYASTK, DAYASTAK,
DAYASTADK, DAYASTADGK, DIAYASK, DIAYASTK, DIAYASTAK, DIAYASTADK,
DIAYASTADGK, DLIAYASK, DLIAYASTK, DLIAYASTAK, DLIAYASTADK,
DLIAYASTADGK, KYASD, KYASTD, KYASTAD, KYASTADD, KYASTADGD, KAYASD,
KAYASTD, KAYASTAD, KAYASTADD, KAYASTADGD, KIAYASD, KIAYASTD,
KIAYASTAD, KIAYASTADD, KIAYASTADGD, KLIAYASD, KLIAYASTD,
KLIAYASTAD, KLIAYASTADD, KLIAYASTADGD, YASTA, YASTAD, YASTADG,
AYAST, AYASTA, AYASTAD, AYASTADG, IAYAS, IAYAST, IAYASTA, IAYASTAD,
IAYASTADG, LIAYAS, LIAYAST, LIAYASTA, LIAYASTAD and LIAYASTADG.
24. A modulating agent comprising an antibody or antigen-binding
fragment thereof that: (a) specifically binds to a desmocollin CAR
sequence selected from the group consisting of LFYIEKDTG (SEQ ID
NO:545), LFYVERDTG (SEQ ID NO:559) and LFYIERDTG (SEQ ID NO:571),
N-Ac-LYGYATTADG-NH.sub.2 (SEQ ID NO:______),
N-Ac-LYAYATTADG-NH.sub.2 (SEQ ID NO:______),
N-Ac-VYGYATTADG-NH.sub.2 (SEQ ID NO:______),
N-Ac-VYAYATTADG-NH.sub.2 (SEQ ID NO:______),
N-Ac-IYGYATTADG-NH.sub.2 (SEQ ID NO:______),
N-Ac-IYAYATTADG-NH.sub.2 (SEQ ID NO:______), IIAFATTPDG-NH.sub.2
(SEQ ID NO:______), N-Ac-LIAFATTPDG-NH.sub.2 (SEQ ID NO:______) and
N-Ac-LIAYASTADG-NH.sub.2 (SEQ ID NO:______); and (b) modulates a
desmocollin-mediated function.
25-31. (Canceled)
32. A pharmaceutical composition comprising a modulating agent
according to any one of claims 1, 3 and 5 or 13, in combination
with a physiologically acceptable carrier.
33-39. (Canceled)
40. A method for modulating cell adhesion, comprising contacting a
desmosomal cadherin-expressing cell with a modulating agent
according to any one of claims 1, 3 and 5 or 13, and thereby
modulating cell adhesion.
41. A method according to claim 40, wherein the cell is selected
from the group consisting of epithelial cells, endothelial cells
and tumor cells.
42. A method according to claim 40, wherein the modulating agent is
present within a pharmaceutical composition, in combination with a
physiologically acceptable carrier.
43. A method according to claim 40, wherein cell adhesion is
inhibited.
44. A method according to claim 40, wherein cell adhesion is
enhanced.
45-70. (Canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/535,852, filed Mar. 27, 2000, now allowed;
which is a continuation-in-part of U.S. patent application Ser. No.
09/187,859, filed Nov. 6, 1998, and issued as U.S. Pat. No.
6,358,920; which is a continuation-in-part of U.S. patent
application Ser. No. 09/073,040, filed May 5, 1998, and issued as
U.S. Pat. No. 6,472,367, all of which applications are incorporated
by reference herein in their entireties.
STATEMENT REGARDING SEQUENCE LISTING SUBMITTED ON CD-ROM
[0002] The Sequence Listing associated with this application is
provided on CD-ROM in lieu of a paper copy, and is hereby
incorporated by reference into the specification. Three CD-ROMs are
provided, containing identical copies of the sequence listing:
CD-ROM No. 1 is labeled COPY 1, contains the file 407c10.app.txt
which is 0.51 MB and created on Sep. 3, 2003; CD-ROM No. 2 is
labeled COPY 2, contains the file 407c10.app.txt which is 0.51 MB
and created on Sep. 3, 2003; CD-ROM No. 3 is labeled CRF, contains
the file 407c10.app.txt which is 0.51 MB and created on Sep. 3,
2003.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to methods for
modulating desmosomal cadherin-mediated functions, and more
particularly to the use of modulating agents derived from
desmocollin and desmoglein cell adhesion recognition sequences for
inhibiting or enhancing functions mediated by such desmosomal
cadherins.
[0005] 2. Description of the Related Art
[0006] Cadherins are a rapidly expanding superfamily of
calcium-dependent cell adhesion molecules (CAMs) (for review, see
Munro et al., In: Cell Adhesion and Invasion in Cancer Metastasis,
P. Brodt, ed., pp. 17-34, RG Landes Co., Austin Tex., 1996). All
cadherins appear to be membrane glycoproteins that generally
promote cell adhesion through homophilic interactions (a cadherin
on the surface of one cell binds to an identical cadherin on the
surface of another cell), although cadherins also appear to be
capable of forming heterotypic complexes with one another under
certain circumstances and with lower affinity.
[0007] There are many different types of cadherins. The most
extensively studied group of cadherins is known as the classical,
or type I, cadherins. Classical cadherins have been shown to
regulate epithelial, endothelial, neural and cancer cell adhesion,
with different cadherins expressed on different cell types. All
classical cadherins have a similar structure. As illustrated in
FIG. 1A, classical cadherins are composed of five extracellular
domains (EC1-EC5), a single hydrophobic domain (TM) that
transverses the plasma membrane (PM), and two cytoplasmic domains
(CP1 and CP2). The calcium binding motifs DXNDN (SEQ ID NO:1), DXD
and LDRE (SEQ ID NO:2) are interspersed throughout the
extracellular domains, and each 110 amino acid region that contains
such motifs is considered a cadherin repeat. The first
extracellular domain (EC1) contains the cell adhesion recognition
(CAR) sequence, HAV (His-Ala-Val), along with flanking sequences on
either side of the CAR sequence that play a role in conferring
specificity. Synthetic peptides containing the HAV sequence and
antibodies directed against such peptides have been shown to
inhibit classical cadherin-dependent processes (Munro et al.,
supra; Blaschuk et al., J. Mol. Biol. 211:679-82,1990; Blaschuk et
al., Develop. Biol. 139:227-29, 1990; Alexander et al., J. Cell.
Physiol. 156:610-18, 1993).
[0008] Cadherins that contain calcium binding motifs within
extracellular domain cadherin repeats, but do not contain an HAV
CAR sequence, are considered to be nonclassical cadherins
(illustrated in FIGS. 1B to 1AA). To date, nine groups of
nonclassical cadherins have been identified (types II-X). These
cadherins are also membrane glycoproteins. Type II, or atypical,
cadherins include OB-cadherin (cadherin-11; see Getsios et al.,
Developmental Dynamics 211:238-247,1998; Simonneau et al., Cell
Adhesion and Communication 3:115-130,1995; Okazaki et al., J.
Biological Chemistry 269:12092-12098,1994), cadherin-5
(VE-cadherin; see Navarro et al., J. Cell Biology 140:1475-1484,
1998), cadherin-6 (K-cadherin; see Shimoyama et al., Cancer
Research 55:2206-2211, 1995; Shimazui et al., Cancer Research
56:3234-3237,1996; Inoue et al., Developmental Dynamics
211:338-351,1998; Getsios et al., Developmental Dynamics
211:238-247, 1998), cadherin-7 (see Nakagawa et al., Development
121:1321-1332, 1995), cadherin-8 (see Suzuki et al., Cell
Regulation 2:261-270, 1991), cadherin-12 (Br-cadherin; see Tanihara
et al., Cell Adhesion and Communication 2:15-26,1994), cadherin-14
(see Shibata et al., J. Biological Chemistry 272:5236-5240, 1997),
cadherin-15 (M-cadherin; see Shimoyama et al., J. Biological
Chemistry 273:10011-10018,1998), and PB-cadherin (see Sugimoto et
al., J. Biological Chemistry 271:11548-11556, 1996). For a general
review of atypical cadherins, see Redies and Takeichi,
Developmental Biology 180:413-423, 1996 and Suzuki et al., Cell
Regulation 2:261-270,1991.
[0009] Types III-X include LI-cadherin (type III; see Bemdorff et
al., J. Cell Biology 125:1353-1369, 1994), T-cadherin (type IV; see
Ranscht, U.S. Pat. No. 5,585,351; Tkachuk et al., FEBS Lett.
421:208-212, 1998; Ranscht et al., Neuron 7:391-402, 1991;
Sacristan et al., J. Neuroscience Research 34:664-680, 1993; Vestal
and Ranscht, J. Cell Biology 119:451-461,1992; Fredette and
Ranscht, J. Neuroscience 14:7331-7346,1994; Ranscht and
Bronner-Fraser, Development 111:15-22, 1991), protocadherins (type
V; e.g., protocadherins 42, 43 and 68; see Sano et al., EMBO J.
12:2249-2256, 1993; GenBank Accession Number AF029343),
desmocollins (type VI; e.g., desmocollins 1, 2, 3 and 4; see King
et al., Genomics 18:185-194,1993; Parker et al., J. Biol. Chem.
266:10438-10445,1991; King et al., J. Invest Dermatol. 105:314-321,
1995; Kawamura et al., J. Biol. Chem. 269:26295-26302, 1994),
desmogleins (type VII; e.g., desmogleins 1 and 2; see Wheeler et
al., Proc. Natl. Acad. Sci. USA 88:4796-4800; Koch et al., Eur. J.
Cell. Biol. 55:200-208,1991), and cadherin-related neuronal
receptors (type X; see Kohmura et al., Neuron 20:1137-1151,
1998).
[0010] Most studies of nonclassical cadherins have focused on
atypical or type 11 cadherins. The structure of such cadherins is
similar to that of the type I cadherins, but they do not contain
the CAR sequence, HAV (FIG. 1B). Atypical cadherins appear to
mediate a wide variety of functions. Additional variation is seen
in the structures of types III-X cadherins. Although less studied,
such cadherins also appear to play a role in diverse functions.
Desmosomal cadherins, for example, are present within desmosomes,
the intercellular junctions that provide adhesion and membrane
anchors for the intermediate filament cytoskeleton. These cadherins
(e.g., desmogleins and desmocollins) play a role in desmosomal
adhesion, which is critically important for normal tissue
construction and epidermis structure. Abnormal desmosomes appear in
certain types of carcinomas and other skin disorders (see Chidgey,
Histol. Histopathol. 12:1159-1168, 1997).
[0011] Notwithstanding these recent advances, nonclassical cadherin
function remains poorly understood at the biological and molecular
levels. Accordingly, there is a need in the art for identifying
sequences involved in modulating nonclassical cadherin-dependent
functions, such as cell adhesion mediated by desmosomal cadherins,
and for the development of methods employing such sequences to
inhibit processes such as cell adhesion. The present invention
fulfills these needs and further provides other related
advantages.
BRIEF SUMMARY OF THE INVENTION
[0012] Briefly stated, this invention provides compositions and
methods for modulating desmosomal cadherin-mediated functions, such
as cell adhesion. Within certain aspects, modulating agents are
provided. Such agents are capable of modulating (i.e., inhibiting
or enhancing) one or more functions mediated by the desmosomal
cadherins desmoglein (dsg) or desmocollin (dsc). A modulating agent
may comprise at least one of: (a) a native dsc or dsg CAR sequence;
(b) an analogue of such a CAR sequence that is capable of
modulating dsc- or dsg-mediated cell adhesion; (c) a non-peptide
peptidomimetic of a dsg or dsg CAR sequence that is capable of
modulating dsc- or dsg-mediated cell adhesion; (d) an antibody, or
antigen-binding fragment thereof, that specifically binds a dsc or
dsg CAR sequence; and/or (e) a polynucleotide encoding a native dsc
or dsg CAR sequence or analogue thereof that is capable of
modulating dsc- or dsg-mediated cell adhesion. Certain preferred
modulating agents comprise a desmoglein-1, desmoglein-2,
desmoglein-3, desmocollin-1, desmocollin-2, desmocollin-3 or
desmocollin-4 CAR sequence, or an analogue of any of the foregoing
CAR sequences. An analogue is generally a peptide sequence that is
at least 50% identical to a dsg or dsc CAR sequence, and (i)
detectably inhibits a function that is modulated by the dsg or dsc;
or (ii) detectably enhances adhesion of cells that express the dsg
or dsc. Modulating agents preferably contain no more than 85, and
preferably no more than 50, consecutive amino acid residues present
within the dsg or dsc. Certain modulating agents comprise a dsg or
dsc CAR sequence and are 3-16 amino acid residues in length. Within
certain specific embodiments, a modulating agent as described above
is a peptide ranging in size from 3 to 50, preferably from 4 to 16,
amino acid residues.
[0013] For certain modulating agents, the dsg or dsc CAR sequence
has the formula:
Aaa-Phe-Baa-Ile/Leu/Val-Asp/Asn/Glu-Caa-Daa-Ser/Thr/Asn-Gly (SEQ ID
NO:3)
[0014] wherein Aaa, Baa, Caa and Daa are independently selected
amino acid residues; Ile/Leu/Val is an amino acid that is selected
from the group consisting of isoleucine, leucine and valine,
Asp/Asn/Glu is an amino acid that is selected from the group
consisting of aspartate, asparagine and glutamate; and Ser/Thr/Asn
is an amino acid that is selected from the group consisting of
serine, threonine or asparagine. For other modulating agents as
described above, the dsg or dsc CAR sequence consists of at least
three consecutive amino acid residues, and preferably at least five
consecutive amino acid residues, of a region of the dsg or dsc
having the formula recited above.
[0015] Within certain specific embodiments, a modulating agent as
provided herein comprises: (a) one or more desmoglein CAR sequences
selected from the group consisting of NQK, NQKT (SEQ ID NO:63),
NQKTG (SEQ ID NO:64), INQK (SEQ ID NO:65), INQKT (SEQ ID NO:66),
INQKTG (SEQ ID NO:67), VINQK (SEQ ID NO:68), VINQKT (SEQ ID NO:69),
VINQKTG (SEQ ID NO:70), FVINQK (SEQ ID NO:71), FVINQKT (SEQ ID
NO:72), FVINQKTG (SEQ ID NO:73), IFVINQK (SEQ ID NO:74), IFVINQKT
(SEQ ID NO:75), IFVINQKTG (SEQ ID NO:76), NRN, NRNT (SEQ ID NO:77),
NRNTG (SEQ ID NO:78), INRN (SEQ ID NO:79), INRNT (SEQ ID NO:80),
INRNTG (SEQ ID NO:81), IINRN (SEQ ID NO:82), IINRNT (SEQ ID NO:83),
IINRNTG (SEQ ID NO:84), FIINRN (SEQ ID NO:85), FIINRNT (SEQ ID
NO:86), FIINRNTG (SEQ ID NO:87), MFIINRN (SEQ ID NO:88), MFIINRNT
(SEQ ID NO:89), MFIINRNTG (SEQ ID NO:90), NKD, NKDT (SEQ ID NO:91),
NKDTG (SEQ ID NO:92), LNKD (SEQ ID NO:93), LNKDT (SEQ ID NO:94),
LNKDTG (SEQ ID NO:95), YLNKD (SEQ ID NO:96), YLNKDT (SEQ ID NO:97),
YLNKDTG (SEQ ID NO:98), FYLNKD (SEQ ID NO:99), FYLNKDT (SEQ ID
NO:100), FYLNKDTG (SEQ ID NO:101), VFYLNKD (SEQ ID NO:102),
VFYLNKDT (SEQ ID NO:103) and VFYLNKDTG (SEQ ID NO:104); or (b) an
analogue of any of the foregoing sequences that differs in one or
more substitutions, deletions, additions and/or insertions such
that that ability of the analogue to modulate a desmoglein-mediated
function is not substantially diminished. For example, the agent
may comprise a linear peptide having the sequence
N-Ac-IFVINQKTG-NH.sub.2 (SEQ ID NO:105), N-Ac-MFIINRNTG-NH.sub.2
(SEQ ID NO:106) or N-Ac-VFYLNKDTG-NH.sub.2 (SEQ ID NO:107). The
desmoglein CAR sequence may, but need not, be present within a
cyclic peptide.
[0016] Within other specific embodiments, a modulating agent as
provided herein may comprise: (a) one or more desmocollin CAR
sequences selected from the group consisting of EKD, EKDT (SEQ ID
NO:108), EKDTG (SEQ ID NO:109), IEKD (SEQ ID NO:110), IEKDT (SEQ ID
NO:111), IEKDTG (SEQ ID NO:112), YIEKD (SEQ ID NO:113), YIEKDT (SEQ
ID NO:114), YIEKDTG (SEQ ID NO:115), FYIEKD (SEQ ID NO:116),
FYIEKDT (SEQ ID NO:117), FYIEKDTG (SEQ ID NO:118), LFYIEKD (SEQ ID
NO:119), LFYIEKDT (SEQ ID NO:120), LFYIEKDTG (SEQ ID NO:121), ERD,
ERDT (SEQ ID NO:122), ERDTG (SEQ ID NO:123), VERD (SEQ ID NO:124),
VERDT (SEQ ID NO:125), VERDTG (SEQ ID NO:126), YVERD (SEQ ID
NO:127), YVERDT (SEQ ID NO:128), YVERDTG (SEQ ID NO:129), FYVERD
(SEQ ID NO:130), FYVERDT (SEQ ID NO:131), FYVERDTG (SEQ ID NO:132),
LFYVERD (SEQ ID NO:133), LFYVERDT (SEQ ID NO:134), LFYVERDTG (SEQ
ID NO:135), IERD (SEQ ID NO:136), IERDT (SEQ ID NO:137), IERDTG
(SEQ ID NO:138), YIERD (SEQ ID NO:139), YIERDT (SEQ ID NO:140),
YIERDTG (SEQ ID NO:141), FYIERD (SEQ ID NO:142), FYIERDT (SEQ ID
NO:143), FYIERDTG (SEQ ID NO:144), LFYIERD (SEQ ID NO:145),
LFYIERDT (SEQ ID NO:146) and LFYIERDTG (SEQ ID NO:147); or (b) an
analogue of any of the foregoing sequences that differs in one or
more substitutions, deletions, additions and/or insertions such
that that ability of the analogue to modulate a
desmocollin-mediated function is not substantially diminished. For
example, the agent may comprise a linear peptide having the
sequence N-Ac-LFYIEKDTG-NH.sub.2 (SEQ ID NO:148),
N-Ac-LFYVERDTG-NH.sub.2 (SEQ ID NO:149) or N-Ac-LFYIERDTG-NH.sub.2
(SEQ ID NO:150). The desmocollin CAR sequence may, but need not, be
present within a cyclic peptide.
[0017] Within other specific embodiments, a modulating agent as
provided herein may comprise: (a) one or more Desmoglein-1 CAR
sequences selected from the group consisting of: RAL, RALN (SEQ ID
NO:151), RALNS (SEQ ID NO:152, RALNSM (SEQ ID NO:153), RALNSL (SEQ
ID NO:154), RALNSMG (SEQ ID NO:155), RALNSLG (SEQ ID NO:156), CRAL
(SEQ ID NO:157), CRALN (SEQ ID NO:158), CRALNS (SEQ ID NO:159),
CRALNSM (SEQ ID NO:160), CRALNSL (SEQ ID NO:161), CRALNSMG (SEQ ID
NO:162), CRALNSLG (SEQ ID NO:163), YCRAL (SEQ ID NO:164), YCRALN
(SEQ ID NO:165), YCRALNS (SEQ ID NO:166), YCRALNSM (SEQ ID NO:167),
YCRALNSL (SEQ ID NO:168), YCRALNSMG (SEQ ID NO:169), YCRALNSLG (SEQ
ID NO:170), IYRAL (SEQ ID NO:171), IYRALN (SEQ ID NO:172), IYRALNS
(SEQ ID NO:173), IYRALNSM (SEQ ID NO:174), IYRALNSL (SEQ ID
NO:175), IYRALNSMG (SEQ ID NO:176) or IYRALNSLG (SEQ ID NO:177); or
(b) an analogue of any of the foregoing sequences that differs in
one or more substitutions, deletions, additions and/or insertions
such that that ability of the analogue to modulate a
desmoglein-mediated function is not substantially diminished.
Linear peptides having such sequences may be modified at the N-
and/or C-termini, as in the peptides N-Ac-IYRALNSMG-NH.sub.2 (SEQ
ID NO:178) and N-Ac-IYRALNSLG-NH.sub.2 (SEQ ID NO:179).
[0018] Within other specific embodiments, a modulating agent as
provided herein may comprise: (a) one or more Desmoglein-2 CAR
sequences selected from the group consisting of: YAL, YALD (SEQ ID
NO:180), YALDA (SEQ ID NO:181), YALDAR (SEQ ID NO:182), YALDARG
(SEQ ID NO:183), GYAL (SEQ ID NO:184), GYALD (SEQ ID NO:185),
GYALDA (SEQ ID NO:186), GYALDAR (SEQ ID NO:187), GYALDARG (SEQ ID
NO:188), TGYAL (SEQ ID NO:189), TGYALD (SEQ ID NO:190), TGYALDA
(SEQ ID NO:191), TGYALDAR (SEQ ID NO:192), TGYALDARG (SEQ ID
NO:193), LTGYAL (SEQ ID NO:194), LTGYALD (SEQ ID NO:195), LTGYALDA
(SEQ ID NO:196), LTGYALDAR (SEQ ID NO:197) or LTGYALDARG (SEQ ID
NO:198); or (b) an analogue of any of the foregoing sequences that
differs in one or more substitutions, deletions, additions and/or
insertions such that that ability of the analogue to modulate a
desmoglein-mediated function is not substantially diminished.
Linear peptides having such sequences may be modified at the N-
and/or C-termini, as in the peptides N-Ac-LTGYALDARG-NH.sub.2 (SEQ
ID NO:199).
[0019] Within other specific embodiments, a modulating agent as
provided herein may comprise: (a) one or more Desmoglein-3 CAR
sequences selected from the group consisting of: RAL, RALN (SEQ ID
NO:151), RALNA (SEQ ID NO:200), RALNAQ (SEQ ID NO:201), RALNAL (SEQ
ID NO:202), RALNAQG (SEQ ID NO:203), RALNALG (SEQ ID NO:204), CRAL
(SEQ ID NO:157), CRALN (SEQ ID NO:158), CRALNA (SEQ ID NO:205),
CRALNAQ (SEQ ID NO:206), CRALNAL (SEQ ID NO:207), CRALNAQG (SEQ ID
NO:208), CRALNALG (SEQ ID NO:209), TCRAL (SEQ ID NO:210), TCRALN
(SEQ ID NO:211), TCRALNA (SEQ ID NO:212), TCRALNAQ (SEQ ID NO:213),
TCRALNAL (SEQ ID NO:214), TCRALNAQG (SEQ ID NO:215), TCRALNALG (SEQ
ID NO:216), ITCRAL (SEQ ID NO:217), ITCRALN (SEQ ID NO:218),
ITCRALNA (SEQ ID NO:219), ITCRALNAQ (SEQ ID NO:220), ITCRALNAL (SEQ
ID NO:221), ITCRALNAQG (SEQ ID NO:222), ITCRALNALG (SEQ ID NO:223);
or (b) an analogue of any of the foregoing sequences that differs
in one or more substitutions, deletions, additions and/or
insertions such that that ability of the analogue to modulate a
desmoglein-mediated function is not substantially diminished.
Linear peptides having such sequences may be modified at the N-
and/or C-termini, as in the peptides N-Ac-ITCRALNAQG-NH.sub.2 (SEQ
ID NO:224) and N-Ac-ITCRALNALG-NH.sub.2 (SEQ ID NO:225).
[0020] Within other specific embodiments, a modulating agent as
provided herein may comprise: (a) one or more Desmocollin-1 CAR
sequences selected from the group consisting of: YAT, YATT (SEQ ID
NO:226), YATTA (SEQ ID NO:227), YATTAD (SEQ ID NO:228), YATTADG
(SEQ ID NO:229), GYAT (SEQ ID NO:230), GYATT (SEQ ID NO:231),
GYATTA (SEQ ID NO:232), GYATTAD (SEQ ID NO:233), GYATTADG (SEQ ID
NO:234), AYAT (SEQ ID NO:235), AYATT (SEQ ID NO:236), AYATTA (SEQ
ID NO:237), AYATTAD (SEQ ID NO:238), AYATTADG (SEQ ID NO:239),
YGYAT (SEQ ID NO:240), YGYATT (SEQ ID NO:241), YGYATTA (SEQ ID
NO:242), YGYATTAD (SEQ ID NO:243), YGYATTADG (SEQ ID NO:244), YAYAT
(SEQ ID NO:245), YAYATT (SEQ ID NO:246), YAYATTA (SEQ ID NO:247),
YAYATTAD (SEQ ID NO:248), YAYATTADG (SEQ ID NO:249), LYGYAT (SEQ ID
NO:250), LYGYATT (SEQ ID NO:251), LYGYATTA (SEQ ID NO:252),
LYGYATTAD (SEQ ID NO:253), LYGYATTADG (SEQ ID NO:254), LYAYAT (SEQ
ID NO:255), LYAYATT (SEQ ID NO:256), LYAYATTA (SEQ ID NO:257),
LYAYATTAD (SEQ ID NO:258), LYAYATTADG (SEQ ID NO:259), VYGYAT (SEQ
ID NO:260), VYGYATT (SEQ ID NO:261), VYGYATTA (SEQ ID NO:262),
VYGYATTAD (SEQ ID NO:263), VYGYATTADG (SEQ ID NO:264), VYAYAT (SEQ
ID NO:265), VYAYATT (SEQ ID NO:266), VYAYATTA (SEQ ID NO:267),
VYAYATTAD (SEQ ID NO:268), VYAYATTADG (SEQ ID NO:269), IYGYAT (SEQ
ID NO:270), IYGYATT (SEQ ID NO:271), IYGYATTA (SEQ ID NO:272),
IYGYATTAD (SEQ ID NO:273), IYGYATTADG (SEQ ID NO:274), IYAYAT (SEQ
ID NO:275), IYAYATT (SEQ ID NO:276), IYAYATTA (SEQ ID NO:277),
IYAYATTAD (SEQ ID NO:278) or IYAYATTADG (SEQ ID NO:279); or (b) an
analogue of any of the foregoing sequences that differs in one or
more substitutions, deletions, additions and/or insertions such
that that ability of the analogue to modulate a
desmocollin-mediated function is not substantially diminished.
Linear peptides having such sequences may be modified at the N-
and/or C-termini, as in the peptides N-Ac-LYGYATTADG-NH.sub.2 (SEQ
ID NO:280) N-Ac-LYAYATTADG-NH.sub.2 (SEQ ID NO:281)
N-Ac-VYGYATTADG-NH.sub.2 (SEQ ID NO:282) N-Ac-VYAYATTADG-NH.sub.2
(SEQ ID NO:283) N-Ac-IYGYATTADG-NH.sub.2 (SEQ ID NO:284) and
N-Ac-IYAYATTADG-NH.sub.2 (SEQ ID NO:285).
[0021] Within other specific embodiments, a modulating agent as
provided herein may comprise: (a) one or more Desmocollin-2 CAR
sequences selected from the group consisting of: FAT, FATT (SEQ ID
NO:286), FATTP (SEQ ID NO:287), FATTPD (SEQ ID NO:288), FATTPDG
(SEQ ID NO:289), AFAT (SEQ ID NO:290), AFATT (SEQ ID NO:291),
AFATTP (SEQ ID NO:292), AFATTPD (SEQ ID NO:293), AFATTPDG (SEQ ID
NO:294), IAFAT (SEQ ID NO:295), IAFATT (SEQ ID NO:296), IAFATTP
(SEQ ID NO:297), IAFATTPD (SEQ ID NO:298), IAFATTPDG (SEQ ID
NO:299), IIAFAT (SEQ ID NO:300), IIAFATT (SEQ ID NO:301), IIAFATTP
(SEQ ID NO:302), IIAFATTPD (SEQ ID NO:303), IIAFATTPDG (SEQ ID
NO:304), LIAFAT (SEQ ID NO:305), LIAFATT (SEQ ID NO:306), LIAFATTP
(SEQ ID NO:307), LIAFATTPD (SEQ ID NO:308), LIAFATTPDG (SEQ ID
NO:309); or (b) an analogue of any of the foregoing sequences that
differs in one or more substitutions, deletions, additions and/or
insertions such that that ability of the analogue to modulate a
desmocollin-mediated function is not substantially diminished.
Linear peptides having such sequences may be modified at the N-
and/or C-termini, as in the peptides N-Ac-IIAFATTPDG-NH.sub.2 (SEQ
ID NO:310) and N-Ac-LIAFATTPDG-NH.sub.2 (SEQ ID NO:311).
[0022] Within other specific embodiments, a modulating agent as
provided herein may comprise: (a) one or more desmocollin-3 or
desmocollin-4 CAR sequences selected from the group consisting of:
YAS, YAST (SEQ ID NO:312), YASTA (SEQ ID NO:313), YASTAD (SEQ ID
NO:314), YASTADG (SEQ ID NO:315), AYAS (SEQ ID NO:316), AYAST (SEQ
ID NO:317), AYASTA (SEQ ID NO:318), AYASTAD (SEQ ID NO:319),
AYASTADG (SEQ ID NO:320), IAYAS (SEQ ID NO:321), IAYAST (SEQ ID
NO:322), IAYASTA (SEQ ID NO:323), IAYASTAD (SEQ ID NO:324),
IAYASTADG (SEQ ID NO:325), LIAYAS (SEQ ID NO:326), LIAYAST (SEQ ID
NO:327), LIAYASTA (SEQ ID NO:328), LIAYASTAD (SEQ ID NO:329),
LIAYASTADG (SEQ ID NO:330); or (b) an analogue of any of the
foregoing sequences that differs in one or more substitutions,
deletions, additions and/or insertions such that that ability of
the analogue to modulate a desmocollin-mediated function is not
substantially diminished. Linear peptides having such sequences may
be modified at the N- and/or C-termini, as in the peptides
N-Ac-LIAYASTADG-NH.sub.2 (SEQ ID NO:331).
[0023] Within other embodiments, a modulating agent comprises a dsc
or dsg CAR sequence that is present within a cyclic peptide. Such
cyclic peptides may have the formula: 1
[0024] wherein W is a tripeptide selected from the group consisting
of NQK, NRN, NKD, EKD, ERD, RAL, YAL, YAT, FAT, and YAS; wherein
X.sub.1, and X.sub.2 are optional, and if present, are
independently selected from the group consisting of amino acid
residues and combinations thereof in which the residues are linked
by peptide bonds, and wherein X.sub.1 and X.sub.2 independently
range in size from 0 to 10 residues, such that the sum of residues
contained within X.sub.1 and X.sub.2 ranges from 1 to 12; wherein
Y.sub.1 and Y.sub.2 are independently selected from the group
consisting of amino acid residues, and wherein a covalent bond is
formed between residues Y.sub.1 and Y.sub.2; and wherein Z.sub.1
and Z.sub.2 are optional, and if present, are independently
selected from the group consisting of amino acid residues and
combinations thereof in which the residues are linked by peptide
bonds.
[0025] Within other aspects of the present invention,
polynucleotides encoding a modulating agent as described above are
provided, along with expression vectors comprising such a
polynucleotide and host cells transformed or transfected with such
an expression vector.
[0026] The present invention further provides modulating agents
that comprise an antibody or antigen-binding fragment thereof that
specifically binds to a dsg or dsc CAR sequence as described above
and modulates a desmosomal cadherin-mediated function.
[0027] Within further aspects, the present invention provides
modulating agents comprising a non-peptide mimetic of any one of
the desmosomal cadherin CAR sequences provided above.
[0028] In certain embodiments, a modulating agent may be linked to
a drug, a detectable marker, a targeting agent and/or a support
material. Modulating agents may also, or alternatively, comprise
one or more of: (a) a cell adhesion recognition sequence other than
a dsc or dsg CAR sequence; and/or (b) an antibody or
antigen-binding fragment thereof that specifically binds to a CAR
sequence other than a dsc or dsg CAR sequence. For example, such an
adhesion molecule may be a classical cadherin, nonclassical
cadherin, integrin, occludin, claudin, N-CAM, fibronectin, laminin
or other extracellular matrix protein.
[0029] Within further aspects, pharmaceutical compositions are
provided, comprising a modulating agent as described above, in
combination with a pharmaceutically acceptable carrier. Such
compositions may additionally comprise a drug and/or one or more
of: (a) a peptide comprising a cell adhesion recognition sequence
other than a dsc or dsg CAR sequence; and/or (b) an antibody or
antigen-binding fragment thereof that specifically binds to a CAR
sequence other than a dsc or dsg CAR sequence.
[0030] The present invention further provides, within other
aspects, methods for modulating one or more desmosomal
cadherin-mediated functions. Such methods generally comprise
contacting a dsg- or dsc-expressing cell with a modulating agent as
described above. Suitable cells include, but are not limited to,
epithelial cells, endothelial cells and tumor cells. Within such
methods, the modulating agent may, but need not, be present within
a pharmaceutical composition as recited above.
[0031] The present invention further provides, within other
aspects, methods for modulating cell adhesion, comprising
contacting a dsc- and/or dsg-expressing cell with a modulating
agent or a pharmaceutical composition as described above. Such
modulating agents and compositions may inhibit or enhance cell
adhesion.
[0032] Within other aspects, the present invention provides methods
for inhibiting adhesion of dsc- and/or dsg-expressing cells in a
mammal, comprising administering to a mammal a modulating agent as
described above, wherein the modulating agent inhibits dsc- and/or
dsg-mediated cell adhesion.
[0033] Within further aspects, the present invention provides
methods for enhancing the delivery of a drug through the skin of a
mammal, comprising contacting epithelial cells of a mammal with a
drug and a modulating agent as described above, wherein the step of
contacting is performed under conditions and for a time sufficient
to allow passage of the drug across the epithelial cells, and
wherein the modulating agent inhibits dsg- or dsc-mediated cell
adhesion. Such modulating agents may pass into the blood stream of
the mammal. Within certain embodiments, the modulating agent is
linked to the drug. The step of contacting may, but need not, be
performed via a skin patch comprising the modulating agent and the
drug, and such skin patches are further provided herein.
[0034] Methods are further provided for facilitating blood sampling
in a mammal, comprising contacting epithelial cells of a mammal
with a modulating agent as described above, wherein the modulating
agent inhibits dsg- or dsc-mediated cell adhesion, and wherein the
step of contacting is performed under conditions and for a time
sufficient to allow passage of one or more blood components across
the epithelial cells. The step of contacting may be performed via a
skin patch comprising the modulating agent, and (optionally) a
reagent for detecting a blood component of interest, and such kits
are specifically provided herein. Within certain embodiments, the
epithelial cells are skin cells or are gum cells.
[0035] Within further aspects, methods are provided for enhancing
the delivery of a drug to a tumor in a mammal, comprising
administering to a mammal a modulating agent as described above,
wherein the modulating agent inhibits dsg- or dsc-mediated cell
adhesion. Suitable tumors include, but are not limited to, bladder
tumors, ovarian tumors, breast tumors, stomach tumors and kidney
tumors, and the modulating agent may be administered locally to the
tumor or may be administered systemically.
[0036] Within other aspects, the present invention provides methods
for treating cancer in a mammal, comprising administering to a
mammal a modulating agent as described above, wherein the
modulating agent inhibits dsg- or dsc-mediated cell adhesion. The
mammal may be afflicted with a cancer such as a carcinoma, leukemia
or melanoma, and the modulating agent may be administered to a
tumor or systemically.
[0037] The present invention further provides, within other
aspects, methods for inducing apoptosis in a desmosomal
cadherin-expressing cell, comprising contacting a desmosomal
cadherin-expressing cell with a modulating agent as described
above, wherein the modulating agent inhibits desmosomal
cadherin-mediated cell adhesion.
[0038] Within other aspects, the present invention provides methods
for enhancing adhesion of desmosomal cadherin-expressing cells,
comprising contacting dsg- or dsc-expressing cells with a
modulating agent as described above, wherein the modulating agent
enhances dsg- or dsc-mediated cell adhesion, wherein the step of
contacting is performed under conditions and for a time sufficient
to detectably enhance adhesion of the cells. Within certain
embodiments, modulating agents for use within such methods are
linked to a support molecule or a solid support.
[0039] Within related aspects, the present invention provides
methods for facilitating wound healing and/or reducing scar tissue
in a mammal, comprising contacting a wound in a mammal with a
modulating agent as described above, wherein the modulating agent
enhances desmosomal cadherin-mediated cell adhesion. Within certain
embodiments, modulating agents for use within such methods are
linked to a support molecules or a solid support.
[0040] Methods are also provided, within other aspects, for
enhancing adhesion of foreign tissue implanted within a mammal,
comprising contacting a site of implantation of foreign tissue in a
mammal with a modulating agent as described above, wherein the
modulating agent enhances dsg- or dsc-mediated cell adhesion. Such
foreign tissue may be a skin graft or organ implant. Within certain
embodiments, modulating agents for use within such methods are
linked to a support molecules or a solid support.
[0041] Within further aspects, methods are provided for treating an
autoimmune blistering disorder in a mammal, comprising
administering to a mammal a modulating agent as described above,
wherein the modulating agent enhances desmosomal cadherin-mediated
cell adhesion. Within certain embodiments, such an agent may be
administered topically to a blister. Modulating agents for use
within such methods may be linked to a support molecule or a solid
support.
[0042] The present invention further provides methods for detecting
the presence of desmosomal cadherin-expressing cells in a sample,
comprising: (a) contacting a sample with an antibody or
antigen-binding fragment thereof that binds to a dsg or dsc CAR
sequence as described above under conditions and for a time
sufficient to allow formation of an antibody-cadherin complex; and
(b) detecting the level of antibody-cadherin complex, and therefrom
detecting the presence of desmosomal cadherin-expressing cells in a
sample. The antibody may be linked to a support material or a
detectable marker such as a fluorescent marker. In certain
embodiments, the step of detecting is performed using fluorescence
activated cell sorting.
[0043] Kits for detecting the presence of dsg- or dsc-expressing
cells in a sample are also provided. Such kits may comprise: (a) an
antibody or antigen-binding fragment thereof that specifically
binds to a desmosomal cadherin CAR sequence; and (b) a detection
reagent.
[0044] Within other aspects, the present invention provides methods
for identifying a compound capable of modulating a desmosomal
cadherin-mediated function, comprising: (a) contacting an antibody
or antigen-binding fragment thereof that specifically binds to a
dsg or dsc CAR sequence as described above with a test compound;
and (b) detecting the level of antibody or fragment that binds to
the test compound, and therefrom identifying a compound capable of
modulating desmosomal cadherin-mediated cell adhesion.
[0045] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings. All references disclosed herein are hereby
incorporated by reference in their entirety as if each was
incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIGS. 1A-1M are diagrams depicting the structure of
classical (FIG. 1A) and nonclassical cadherins (FIGS. 1B to 1AA).
The extracellular domains are designated EC1-EC5 for most
cadherins; EC1-EC7 for LI-cadherin and EC1-EC6 for protocadherins
and cnr. The hydrophobic domain that transverses the plasma
membrane (PM) is represented by TM, and the varying number of
cytoplasmic domains are represented by CP. The calcium binding
motifs for classical cadherins are shown in FIG. 1A by DXNDN (SEQ
ID NO:1), DXD, LDRE (SEQ ID NO:2), DXE, and DVNE (SEQ ID NO:341),
and the calcium binding motifs for other cadherins are also
indicated above the extracellular domains. Below the extracellular
domains, the nine amino acid CAR sequences are shown.
[0047] FIG. 2 provides the amino acid sequences of representative
mammalian nonclassical cadherin extracellular domains, as indicated
(SEQ ID NOs:4-43). Calcium binding motifs are shown in bold, and
representative CAR sequences are shown in bold and underlined.
[0048] FIG. 3 provides the amino acid sequences of representative
mammalian desmosomal cadherin EC1 domains, as indicated. Amino
acids are represented by their IUPAC codes and - represents a gap.
The desmosomal cadherin CAR sequence is indicated in bold.
[0049] FIG. 4 depicts the structures of representative cyclic
peptide modulating agents.
DETAILED DESCRIPTION OF THE INVENTION
[0050] As noted above, the present invention provides methods for
modulating desmosomal cadherin-mediated functions, such as cell
adhesion. The present invention is based, in part, upon the
identification of previously unknown cell adhesion recognition
(CAR) sequences present in desmosomal cadherins. A modulating agent
may comprise one or more such desmosomal cadherin CAR sequences (or
analogues or mimetics thereof), with or without one or more
additional cell adhesion molecule CAR sequences, as described
below. Peptide CAR sequences may be present within a linear or
cyclic peptide. Alternatively, or in addition, a modulating agent
may comprise a polynucleotide encoding a polypeptide comprising one
or more desmosomal cadherin CAR sequences and/or a modulating agent
may comprise a substance (such as an antibody or antigen-binding
fragment thereof) that specifically binds to a desmosomal cadherin
CAR sequence.
[0051] In general, to modulate a desmosomal cadherin-mediated
function, a cell that expresses a desmosomal cadherin is contacted
with a modulating agent either in vivo or in vitro. Within certain
aspects, the methods provided herein inhibit a desmosomal
cadherin-mediated function. Such methods include, for example,
methods for treating diseases or other conditions characterized by
undesirable cell adhesion or for facilitating drug delivery to a
specific tissue or tumor. Certain methods may inhibit cell adhesion
(e.g., cancer cell adhesion). Alternatively, a modulating agent
may, such as when linked to a matrix or to another modulating agent
via a linker, be used to enhance a desmosomal cadherin-mediated
function, such as cell adhesion. Such conjugates may be used, for
example, to facilitate wound healing or the adhesion of
implants.
[0052] Modulating Agents
[0053] The term "modulating agent," as used herein, refers to a
molecule comprising at least one of the following components:
[0054] (a) a linear or cyclic peptide sequence that is at least 50%
identical to a desmosomal cadherin CAR sequence (i.e., a desmosomal
cadherin CAR sequence or an analogue thereof that retains at least
50% sequence identity);
[0055] (b) a mimetic (e.g., peptidomimetic or small molecule mimic)
of a desmosomal cadherin CAR sequence;
[0056] (c) a substance, such as an antibody or antigen-binding
fragment thereof, that specifically binds a desmosomal cadherin CAR
sequence; and/or
[0057] (d) a polynucleotide encoding a polypeptide that comprises a
desmosomal cadherin CAR sequence or analogue thereof.
[0058] A modulating agent may consist entirely of one or more of
the above elements, or may additionally comprise further peptide
and/or non-peptide regions.
[0059] Additional peptide regions may be derived from a desmosomal
cadherin (preferably an extracellular domain that comprises a CAR
sequence) and/or may be heterologous. Within certain preferred
embodiments, a modulating agent contains no more than 85
consecutive amino acid residues, and preferably no more than 50
consecutive amino acid residues, present within a desmosomal
cadherin.
[0060] A modulating agent is further capable of modulating a
function mediated by a desmosomal cadherin. Such activity may
generally be assessed using, for example, representative assays
provided herein. Certain modulating agents inhibit an interaction
between desmosomal cadherin molecules and/or between a desmosomal
cadherin and a different adhesion molecule. For functions (e.g.,
cell adhesion) that are inhibited by a full length desmosomal
cadherin, such a modulating agent may inhibit the function with an
activity that is not substantially diminished relative to the full
length desmosomal cadherin (i.e., the modulating agent inhibits the
function at least as well as soluble cadherin, when contacted with
cells that express the cadherin). For example, a modulating agent
may be as effective as soluble desmosomal cadherin in preventing
and/or disrupting adhesion of desmosomal cadherin-expressing cells.
Alternatively, to enhance adhesion of desmosomal
cadherin-expressing cells, a modulating agent may comprise an
antibody or antigen-binding fragment thereof and/or multiple
peptides or mimetics linked to a support material. Such modulating
agents may function as a biological glue to bind desmosomal
cadherin-expressing cells, and should result in a detectable
enhancement of cell adhesion (preferably an enhancement that is at
least as great as that observed for immobilized desmosomal cadherin
or antibody directed against the cadherin).
[0061] The term "nonclassical cadherin," as used herein, refers to
a polypeptide that contains characteristic cadherin repeats, but
does not contain an HAV CAR sequence. As used herein, a "cadherin
repeat" refers to an amino acid sequence that is approximately 110
amino acid residues in length (generally 100 to 120 residues,
preferably 105 to 115 residues), comprises an extracellular domain,
and contains three calcium binding motifs (DXD, XDXE and DXXDX; SEQ
ID NOS:332 and 333 respectively) in the same order and in
approximately the same position (see, e.g., FIG. 2). The presence
of an extracellular domain may generally be determined using well
known techniques, such as the presence of one or more of: a
hydrophilic sequence, a region that is recognized by an antibody, a
region that is cleaved by trypsin and/or a potential glycosylation
site with the glycosylation motif Asn-X-Ser/Thr. The second calcium
binding motif commonly has the sequence LDRE (SEQ ID NO:2),
although variants of this sequence with conservative substitutions
are also observed, including MDRE (SEQ ID NO:334), LDFE (SEQ ID
NO:335), LDYE (SEQ ID NO:336), IDRE (SEQ ID NO:337), VDRE (SEQ ID
NO:338) and IDFE (SEQ ID NO:339). Within most cadherin repeats, the
third calcium binding motif has the sequence
[L,I,V]-X-[L,I,V]-X-D-X-N-D-[N,H]-X-P (SEQ ID NO:340), wherein
residues indicated in brackets may be any one of the recited
residues. A preferred third calcium binding motif has the sequence
DXNDN (SEQ ID NO:1), although one or both of the D residues may be
replaced by an E. Homology among cadherin repeats is generally at
least 20%, preferably at least 30%, as determined by the ALIGN
algorithm (Myers and Miller, CABIOS 4:11-17, 1988). Most cadherins
comprise at least five cadherin repeats, along with a hydrophobic
domain that transverses the plasma membrane and, optionally, one or
more cytoplasmic domains, as shown in FIGS. 1B-1AA. Occasionally,
however, a cadherin may substitute an extracellular domain that
contains fewer than three calcium binding motifs for one or more of
the cadherin repeats. For example, as shown in FIG. 2, the second
extracellular domain of LI-cadherin comprises only the first
calcium binding motif (DXD). As noted above, atypical, or type 11,
cadherins include cadherin-5 (VE-cadherin), cadherin-6
(K-cadherin), cadherin-7, cadherin-8, cadherin-11 (OB-cadherin),
cadherin-12, cadherin-14, cadherin-15 and PB-cadherin. Types III-X
include LI-cadherin, T-cadherin, protocadherins (e.g.,
protocadherins 42, 43 and 68), desmocollins (e.g., desmocollins 1,
2, 3 and 4), desmogleins (e.g., desmogleins 1 and 2), and
cadherin-related neuronal receptors.
[0062] The term "desmosomal cadherin" refers to a nonclassical
cadherin that is present within the intercellular junction known as
the desmosome. Desmosomal cadherins include desmogleins and
desmocollins (see e.g., King et al., Genomics 18:185-194,1993;
Parker et al., J. Biol. Chem. 266:10438-10445, 1991; King et al.,
J. Invest. Dermatol. 105:314-321,1995; Kawamura et al., J. Biol.
Chem. 269:26295-26302,1994; Wheeler et al., Proc. Natl. Acad. Sci.
USA 88:4796-4800; and Koch et al., Eur. J. Cell. Biol. 55:200-208,
1991). Desmogleins and desmocollins are expressed by cells that
possess desmosomes, such as epithelial cells, cardiac muscle cells
and meningeal cells. These cadherins are involved in intercellular
adhesion of such cells, and may function in a heterotypic manner,
whereby a desmocollin isoform and a desmoglein isoform are both
required for adhesion. Desmogleins and desmocollins are involved in
a number of autoimmune blistering disorders, such as pemphigus
vulgaris, pemphigus foliaceus and intercellular IgA dermatosis, and
have been shown to have reduced expression in some human
carcinomas. The sequences of various extracellular domains of known
desmosomal cadherins are shown in FIGS. 2 and 3 (SEQ ID NOs:4-43
and SEQ ID NOs:44-57).
[0063] A desmosomal cadherin CAR sequence, as used herein, is an
amino acid sequence that is present within in a naturally occurring
desmosomal cadherin and that is capable of detectably modulating a
desmosomal cadherin-mediated function, such as cell adhesion, as
described herein. In other words, contacting a desmosomal
cadherin-expressing cell with a peptide comprising a CAR sequence
results in a detectable change in a desmosomal cadherin-mediated
function using at least one of the representative assays provided
herein. CAR sequences are generally recognized in vivo by a
desmosomal cadherin or other adhesion molecule (i.e., a molecule
that mediates cell adhesion via a receptor on the cell surface),
and are necessary for maximal heterophilic and/or homophilic
interaction. CAR sequences may be of any length, but generally
comprise at least three amino acid residues, preferably 4-16 amino
acid residues, and more preferably 5-9 amino acid residues. A
peptide modulating agent may comprise any number of amino acid
residues, but preferred agents comprise 3-50 residues, preferably
4-16 residues, and more preferably 6-15 residues.
[0064] It has been found, within the context of the present
invention, that certain nonclassical cadherin CAR sequences share
the consensus sequence:
Aaa-Phe-Baa-Ile/Leu/Val-Asp/Asn/Glu-Caa-Daa-Ser/Thr/Asn-Gly (SEQ ID
NO:3)
[0065] Within the consensus sequence, Aaa, Baa, Caa and Daa
indicate independently selected amino acid residues; "Ile/Leu/Val"
indicates an amino acid that is isoleucine, leucine or valine;
"Asp/Asn/Glu" indicates an amino acid that is aspartic acid,
asparagine or glutamic acid; and "Ser/Thr/Asn" indicates an amino
acid that is serine, threonine or asparagine. Representative
desmosomal cadherin CAR sequences, as well as other nonclassical
cadherin CAR sequences, are provided within Table I. CAR sequences
specifically provided herein further include portions of such
representative CAR sequences, as well as longer polypeptides that
comprise at least a portion of such sequences. Additional
nonclassical cadherin CAR sequences may be identified based on
sequence homology to the nonclassical cadherin CAR sequences
provided herein, and based on the ability of a peptide comprising
such a sequence to modulate a nonclassical cadherin-mediated
function within a representative assay described herein. Within
certain embodiments, a modulating agent comprises at least three
consecutive residues, preferably at least five consecutive residues
and more preferably at least seven consecutive residues, of a
desmosomal cadherin CAR sequence that satisfies the above consensus
sequence. Similarly, if a CAR sequence of a different nonclassical
cadherin is used in combination with a desmosomal cadherin CAR
sequence, such CAR sequence(s) should comprise at least three
consecutive residues, preferably at least five consecutive residues
and more preferably at least seven consecutive residues, of a
nonclassical cadherin CAR sequence that satisfies the above
consensus sequence.
1TABLE I Representative Nonclassical Cadherin CAR Sequences
Cadherin CAR Sequence Human OB-cadherin EC1 FFVIEEYTG (SEQ ID
NO:367) Human OB-cadherin EC1 IFVIDDKSG (SEQ ID NO:368) Human
OB-cadherin EC2 YFSVEAQTG (SEQ ID NO:369) Human cadherin-5 EC1
VFRVDAETG (SEQ ID NO:370) Human cadherin-6 EC1 FFLLEEYTG (SEQ ID
NO:371) Human cadherin-6 EC1 LFIINENTG (SEQ ID NO:372) Human
cadherin-6 EC2 YFSVESETG (SEQ ID NO:373) Human cadherin-6 EC4
IFNIDSGNG (SEQ ID NO:374) Chicken cadherin-7 EC1 IFIIDENTG (SEQ ID
NO:375) Chicken cadherin-7 EC2 YFSVEPKTG (SEQ ID NO:376) Chicken
cadherin-7 EC4 YFNIDANSG (SEQ ID NO:377) Human cadherin-8 EC1
MFVLEEFSG (SEQ ID NO:378) Human cadherin-8 EC1 IFQINDVTG (SEQ ID
NO:379) Human cadherin-12 EC1 VFTIDETTG (SEQ ID NO:380) Human
cadherin-12 EC2 YFSIDPKTG (SEQ ID NO:381) Human cadherin-14 EC1
IFIIDDTTG (SEQ ID NO:382) Human cadherin-14 EC2 YFSVDPKTG (SEQ ID
NO:383) Human cadherin-14 EC4 FFNIDANTG (SEQ ID NO:384) Human
cadherin-15 EC1 VFSIDKFTG (SEQ ID NO:385) Human cadherin-15 EC2
LFSIDELTG (SEQ ID NO:386) Human T-cadherin EC1 IFRINENTG (SEQ ID
NO:387) Rat PB-cadherin EC1 FFVVEEYTG (SEQ ID NO:388) Rat
PB-cadherin EC1 IFLIDELTG (SEQ ID NO:389) Rat PB-cadherin EC2
HFTVDPKTG (SEQ ID NO:390) Rat PB-cadherin EC4 IFDIDADTG (SEQ ID
NO:391) Human LI-cadherin EC2 YFQINNKTG (SEQ ID NO:392) Human
protocadherin 43 LFALDLVTG (SEQ ID NO:393) EC3 Human protocadherin
43 YFTINRDNG (SEQ ID NO:394) EC5 Human protocadherin 68 LFSIDPKTG
(SEQ ID NO:395) EC3 Human protocadherin 68 LFEIDPSSG (SEQ ID
NO:396) EC6 Human desmoglein1 EC1 IFVINQKTG (SEQ ID NO:397) Human
desmoglein1 EC2 MFIINRNTG (SEQ ID NO:398) Human desmoglein2 EC2
VFYLNKDTG (SEQ ID NO:399) Human desmocollin 1 LFYIEKDTG (SEQ ID
NO:400) EC1 Human desmocollin 2 LFYVERDTG (SEQ ID NO:401) EC1 Human
desmocollin 3/4 LFYIERDTG (SEQ ID NO:402) EC1 Mouse Cnr1 EC3
KFHIDPVSG (SEQ ID NO:403) Mouse Cnr2 EC3 QFSIDADTG (SEQ ID NO:404)
Mouse Cnr3 EC3 TFHIDSVSG (SEQ ID NO:405) Mouse Cnr5 EC3 AFNIDSNSG
(SEQ ID NO:406) Mouse Cnr6 EC3 KFTIDSSSG (SEQ ID NO:407) Mouse Cnr7
EC3 LFTLDEKNG (SEQ ID NO:408) Mouse Cnr8 EC3 KFLINEKTG (SEQ ID
NO:409) CONSENSUS xFxidxxtG (SEQ ID NO:3) v n s l e n
[0066] Nonclassical cadherin CAR sequences are generally physically
located within the cadherin molecule in or near the binding site of
an adhesion molecule (i.e., within 10 amino acids, and preferably
within 5 amino acids). The location of a binding site may generally
be determined using well known techniques, such as evaluating the
ability of a portion of the nonclassical cadherin to bind to the
same nonclassical cadherin or to another adhesion molecule. Any
standard binding assay may be employed for such an evaluation.
Recognition of a CAR sequence by the nonclassical cadherin or other
adhesion molecule results in a measurable effect on an adhesion
molecule function, such as cell adhesion. Peptides comprising a CAR
sequence generally inhibit such a function unless linked, as
described herein, to form an enhancer of adhesion molecule
function.
[0067] Certain preferred desmosomal cadherin CAR sequences comprise
3-9 amino acid residues of a desmoglein or desmocollin sequence
provided in Table I. For example, a CAR sequence may comprise 3, 4
or 5 residues of a 9 amino acid sequence in Table I. Within certain
embodiments, a CAR sequence may include at least residues 5-7 of a
sequence in Table I. A desmoglein CAR sequence may comprise, for
example, one or more of the sequences: NQK, NQKT (SEQ ID NO:63),
NQKTG (SEQ ID NO:64), INQK (SEQ ID NO:65), INQKT (SEQ ID NO:66),
INQKTG (SEQ ID NO:67), VINQK (SEQ ID NO:68), VINQKT (SEQ ID NO:69),
VINQKTG (SEQ ID NO:70), FVINQK (SEQ ID NO:71), FVINQKT (SEQ ID
NO:72), FVINQKTG (SEQ ID NO:73), IFVINQK (SEQ ID NO:74), IFVINQKT
(SEQ ID NO:75), IFVINQKTG (SEQ ID NO:76), NRN, NRNT (SEQ ID NO:77),
NRNTG (SEQ ID NO:78), INRN (SEQ ID NO:79), INRNT (SEQ ID NO:80),
INRNTG (SEQ ID NO:81), IINRN (SEQ ID NO:82), IINRNT (SEQ ID NO:83),
IINRNTG (SEQ ID NO:84), FIINRN (SEQ ID NO:85), FIINRNT (SEQ ID
NO:86), FIINRNTG (SEQ ID NO:87), MFIINRN (SEQ ID NO:88), MFIINRNT
(SEQ ID NO:89), MFIINRNTG (SEQ ID NO:90), NKD, NKDT (SEQ ID NO:91),
NKDTG (SEQ ID NO:92), LNKD (SEQ ID NO:93), LNKDT (SEQ ID NO:94),
LNKDTG (SEQ ID NO:95), YLNKD (SEQ ID NO:96), YLNKDT (SEQ ID NO:97),
YLNKDTG (SEQ ID NO:98), FYLNKD (SEQ ID NO:99), FYLNKDT (SEQ ID
NO:100), FYLNKDTG (SEQ ID NO:101), VFYLNKD (SEQ ID NO:102),
VFYLNKDT (SEQ ID NO:103) and VFYLNKDTG (SEQ ID NO:104). Linear
peptides having such sequences may be modified at the N- and/or
C-termini, as in the peptides N-Ac-IFVINQKTG-NH.sub.2 (SEQ ID
NO:105), N-Ac-MFIINRNTG-NH.sub.2 (SEQ ID NO:106) and
N-Ac-VFYLNKDTG-NH.sub.2 (SEQ ID NO:107).
[0068] A desmocollin CAR sequence may comprise, for example, one or
more of the sequences EKD, EKDT (SEQ ID NO:108), EKDTG (SEQ ID
NO:109), IEKD (SEQ ID NO:110), IEKDT (SEQ ID NO:111), IEKDTG (SEQ
ID NO:112), YIEKD (SEQ ID NO:113), YIEKDT (SEQ ID NO:114), YIEKDTG
(SEQ ID NO:115), FYIEKD (SEQ ID NO:116), FYIEKDT (SEQ ID NO:117),
FYIEKDTG (SEQ ID NO:118), LFYIEKD (SEQ ID NO:119), LFYIEKDT (SEQ ID
NO:120), LFYIEKDTG (SEQ ID NO:121), ERD, ERDT (SEQ ID NO:122),
ERDTG (SEQ ID NO:123), VERD (SEQ ID NO:124), VERDT (SEQ ID NO:125),
VERDTG (SEQ ID NO:126), YVERD (SEQ ID NO:127), YVERDT (SEQ ID
NO:128), YVERDTG (SEQ ID NO:129), FYVERD (SEQ ID NO:130), FYVERDT
(SEQ ID NO:131), FYVERDTG (SEQ ID NO:132), LFYVERD (SEQ ID NO:133),
LFYVERDT (SEQ ID NO:134), LFYVERDTG (SEQ ID NO:135), IERD (SEQ ID
NO:136), IERDT (SEQ ID NO:137), IERDTG (SEQ ID NO:138), YIERD (SEQ
ID NO:139), YIERDT (SEQ ID NO:140), YIERDTG (SEQ ID NO:141), FYIERD
(SEQ ID NO:142), FYIERDT (SEQ ID NO:143), FYIERDTG (SEQ ID NO:144),
LFYIERD (SEQ ID NO:145), LFYIERDT (SEQ ID NO:146) and LFYIERDTG
(SEQ ID NO:147). Linear peptides having such sequences may be
modified at the N- and/or C-termini, as in the peptides
N-Ac-LFYIEKDTG-NH.sub.2 (SEQ ID NO:148), N-Ac-LFYVERDTG-NH.sub.2
(SEQ ID NO:149) and N-Ac-LFYIERDTG-NH.sub.2 (SEQ ID NO:150).
[0069] Further desmosomal cadherin CAR sequences are derived from
the EC1 domains recited in FIG. 3. A desmoglein-1 CAR sequence may
comprise, for example, one or more of the sequences RAL, RALN (SEQ
ID NO:151), RALNS (SEQ ID NO:152), RALNSM (SEQ ID NO:153), RALNSL
(SEQ ID NO:154), RALNSMG (SEQ ID NO:155), RALNSLG (SEQ ID NO:156),
CRAL (SEQ ID NO:157), CRALN (SEQ ID NO:158), CRALNS (SEQ ID
NO:159), CRALNSM (SEQ ID NO:160), CRALNSL (SEQ ID NO:161), CRALNSMG
(SEQ ID NO:162), CRALNSLG (SEQ ID NO:163), YCRAL (SEQ ID NO:164),
YCRALN (SEQ ID NO:165), YCRALNS (SEQ ID NO:166), YCRALNSM (SEQ ID
NO:167), YCRALNSL (SEQ ID NO:168), YCRALNSMG (SEQ ID NO:169),
YCRALNSLG (SEQ ID NO:170), IYRAL (SEQ ID NO:171), IYRALN (SEQ ID
NO:172), IYRALNS (SEQ ID NO:173), IYRALNSM (SEQ ID NO:174),
IYRALNSL (SEQ ID NO:175), IYRALNSMG (SEQ ID NO:176) and IYRALNSLG
(SEQ ID NO:177). Linear peptides having such sequences may be
modified at the N- and/or C-termini, as in the peptides
N-Ac-IYRALNSMG-NH.sub.2 (SEQ ID NO:178) and N-Ac-IYRALNSLG-NH.sub.2
(SEQ ID NO:179).
[0070] A desmoglein-2 CAR sequence may comprise, for example, one
or more of the sequences YAL, YALD (SEQ ID NO:180), YALDA (SEQ ID
NO:181), YALDAR (SEQ ID NO:182), YALDARG (SEQ ID NO:183), GYAL (SEQ
ID NO:184), GYALD (SEQ ID NO:185), GYALDA (SEQ ID NO:186), GYALDAR
(SEQ ID NO:187), GYALDARG (SEQ ID NO:188), TGYAL (SEQ ID NO:189),
TGYALD (SEQ ID NO:190), TGYALDA (SEQ ID NO:191), TGYALDAR (SEQ ID
NO:192), TGYALDARG (SEQ ID NO:193), LTGYAL (SEQ ID NO:194), LTGYALD
(SEQ ID NO:195), LTGYALDA (SEQ ID NO:196), LTGYALDAR (SEQ ID
NO:197), LTGYALDARG (SEQ ID NO:198). Linear peptides having such
sequences may be modified at the N- and/or C-termini, as in the
peptides N-Ac-LTGYALDARG-NH.sub.2 (SEQ ID NO:199).
[0071] A desmoglein-3 CAR sequence may comprise, for example, one
or more of the sequences RAL, RALN (SEQ ID NO:151), RALNA (SEQ ID
NO:200), RALNAQ (SEQ ID NO:201), RALNAL (SEQ ID NO:202), RALNAQG
(SEQ ID NO:203), RALNALG (SEQ ID NO:204), CRAL (SEQ ID NO:157),
CRALN (SEQ ID NO:158), CRALNA (SEQ ID NO:205), CRALNAQ (SEQ ID
NO:206), CRALNAL (SEQ ID NO:207), CRALNAQG (SEQ ID NO:208),
CRALNALG (SEQ ID NO:209), TCRAL (SEQ ID NO:210), TCRALN (SEQ ID
NO:211), TCRALNA (SEQ ID NO:212), TCRALNAQ (SEQ ID NO:213),
TCRALNAL (SEQ ID NO:214), TCRALNAQG (SEQ ID NO:215), TCRALNALG (SEQ
ID NO:216), ITCRAL (SEQ ID NO:217), ITCRALN (SEQ ID NO:218),
ITCRALNA (SEQ ID NO:219), ITCRALNAQ (SEQ ID NO:220), ITCRALNAL (SEQ
ID NO:221), ITCRALNAQG (SEQ ID NO:222), ITCRALNALG (SEQ ID NO:223).
Linear peptides having such sequences may be modified at the N-
and/or C-termini, as in the peptides N-Ac-ITCRALNAQG-NH.sub.2 (SEQ
ID NO:224) and N-Ac-ITCRALNALG-NH.sub.2 (SEQ ID NO:225).
[0072] A desmocollin-1 CAR sequence may comprise, for example, one
or more of the sequences YAT, YATT (SEQ ID NO:226), YATTA (SEQ ID
NO:227), YATTAD (SEQ ID NO:228), YATTADG (SEQ ID NO:229), GYAT (SEQ
ID NO:230), GYATT (SEQ ID NO:231), GYATTA (SEQ ID NO:232), GYATTAD
(SEQ ID NO:233), GYATTADG (SEQ ID NO:234), AYAT (SEQ ID NO:235),
AYATT (SEQ ID NO:236), AYATTA (SEQ ID NO:237), AYATTAD (SEQ ID
NO:238), AYATTADG (SEQ ID NO:239), YGYAT (SEQ ID NO:240), YGYATT
(SEQ ID NO:241), YGYATTA (SEQ ID NO:242), YGYATTAD (SEQ ID NO:243),
YGYATTADG (SEQ ID NO:244), YAYAT (SEQ ID NO:245), YAYATT (SEQ ID
NO:246), YAYATTA (SEQ ID NO:247), YAYATTAD (SEQ ID NO:248),
YAYATTADG (SEQ ID NO:249), LYGYAT (SEQ ID NO:250), LYGYATT (SEQ ID
NO:251), LYGYATTA (SEQ ID NO:252), LYGYATTAD (SEQ ID NO:253),
LYGYATTADG (SEQ ID NO:254), LYAYAT (SEQ ID NO:255), LYAYATT (SEQ ID
NO:256), LYAYATTA (SEQ ID NO:257), LYAYATTAD (SEQ ID NO:258),
LYAYATTADG (SEQ ID NO:259), VYGYAT (SEQ ID NO:260), VYGYATT (SEQ ID
NO:261), VYGYATTA (SEQ ID NO:262), VYGYATTAD (SEQ ID NO:263),
VYGYATTADG (SEQ ID NO:264), VYAYAT (SEQ ID NO:265), VYAYATT (SEQ ID
NO:266), VYAYATTA (SEQ ID NO:267), VYAYATTAD (SEQ ID NO:268),
VYAYATTADG (SEQ ID NO:269), IYGYAT (SEQ ID NO:270), IYGYATT (SEQ ID
NO:271), IYGYATTA (SEQ ID NO:272), IYGYATTAD (SEQ ID NO:273),
IYGYATTADG (SEQ ID NO:274), IYAYAT (SEQ ID NO:275), IYAYATT (SEQ ID
NO:276), IYAYATTA (SEQ ID NO:277), IYAYATTAD (SEQ ID NO:278) and
IYAYATTADG (SEQ ID NO:279). Linear peptides having such sequences
may be modified at the N- and/or C-termini, as in the peptides
N-Ac-LYGYATTADG-NH.sub.2 (SEQ ID NO:280) N-Ac-LYAYATTADG-NH.sub.2
(SEQ ID NO:281) N-Ac-VYGYATTADG-NH.sub.2 (SEQ ID NO:282)
N-Ac-VYAYATTADG-NH.sub.2 (SEQ ID NO:283) N-Ac-IYGYATTADG-NH.sub.2
(SEQ ID NO:284) and N-Ac-IYAYATTADG-NH.sub.2 (SEQ ID NO:285).
[0073] A desmocollin-2 CAR sequence may comprise, for example, one
or more of the sequences FAT, FATT (SEQ ID NO:286), FATTP (SEQ ID
NO:287), FATTPD (SEQ ID NO:288), FATTPDG (SEQ ID NO:289), AFAT (SEQ
ID NO:290), AFATT (SEQ ID NO:291), AFATTP (SEQ ID NO:292), AFATTPD
(SEQ ID NO:293), AFATTPDG (SEQ ID NO:294), IAFAT (SEQ ID NO:295),
IAFATT (SEQ ID NO:296), IAFATTP (SEQ ID NO:297), IAFATTPD (SEQ ID
NO:298), IAFATTPDG (SEQ ID NO:299), IIAFAT (SEQ ID NO:300), IIAFATT
(SEQ ID NO:301), IIAFATTP (SEQ ID NO:302), IIAFATTPD (SEQ ID
NO:303), IIAFATTPDG (SEQ ID NO:304), LIAFAT (SEQ ID NO:305),
LIAFATT (SEQ ID NO:306), LIAFATTP (SEQ ID NO:307), LIAFATTPD (SEQ
ID NO:308), LIAFATTPDG (SEQ ID NO:309). Linear peptides having such
sequences may be modified at the N- and/or C-termini, as in the
peptides N-Ac-IIAFATTPDG-NH.sub.2 (SEQ ID NO:310) and
N-Ac-LIAFATTPDG-NH.sub.2 (SEQ ID NO:311).
[0074] A desmocollin-3 or desmocollin-4 CAR sequence may comprise,
for example, one or more of the sequences YAS, YAST (SEQ ID
NO:312), YASTA (SEQ ID NO:313), YASTAD (SEQ ID NO:314), YASTADG
(SEQ ID NO:315), AYAS (SEQ ID NO:316), AYAST (SEQ ID NO:317),
AYASTA (SEQ ID NO:318), AYASTAD (SEQ ID NO:319), AYASTADG (SEQ ID
NO:320), IAYAS (SEQ ID NO:321), IAYAST (SEQ ID NO:322), IAYASTA
(SEQ ID NO:323), IAYASTAD (SEQ ID NO:324), IAYASTADG (SEQ ID
NO:325), LIAYAS (SEQ ID NO:326), LIAYAST (SEQ ID NO:327), LIAYASTA
(SEQ ID NO:328), LIAYASTAD (SEQ ID NO:329), LIAYASTADG (SEQ ID
NO:330). Linear peptides having such sequences may be modified at
the N- and/or C-termini, as in the peptides
N-Ac-LIAYASTADG-NH.sub.2 (SEQ ID NO:331).
[0075] Those of ordinary skill in the art will recognize that
similar peptide sequences may be designed to modulate a function
mediated by other cadherins, following identification of a CAR
sequence as described herein.
[0076] It will be apparent that certain of the peptide sequences
provided above may modulate a function mediated by multiple
nonclassical cadherins. In general, peptides comprising a greater
number of consecutive residues derived from a desmosomal cadherin
have a greater specificity for that cadherin. In addition, further
flanking sequences may be included to enhance specificity. Such
flanking sequences may be identified based on the sequences
provided in FIGS. 2 and 3, or based on published sequences. To
achieve specificity (i.e., modulation of a desmosomal cadherin
function that is enhanced relative to the modulation of a function
mediated by a different cadherin), the addition of 2 to 5 flanking
residues (preferably at least one residue on either side of the CAR
sequence) is generally sufficient.
[0077] As noted above, modulating agents provided herein may
comprise an analogue or mimetic of a desmosomal cadherin CAR
sequence. An analogue generally retains at least 50% identity to a
native desmosomal cadherin CAR sequence, and modulates a desmosomal
cadherin-mediated function as described herein. Such analogues
preferably contain at least three consecutive residues of, and more
preferably at least five consecutive residues of, a desmosomal
cadherin CAR sequence. An analogue may contain any of a variety of
amino acid substitutions, additions, deletions and/or modifications
(e.g., side chain modifications). Preferred amino acid
substitutions are conservative. A "conservative substitution" is
one in which an amino acid is substituted for another amino acid
that has similar properties, such that one skilled in the art of
peptide chemistry would expect the secondary structure and
hydropathic nature of the polypeptide to be substantially
unchanged. Amino acid substitutions may generally be made on the
basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity and/or the amphipathic nature of the
residues. For example, negatively charged amino acids include
aspartic acid and glutamic acid; positively charged amino acids
include lysine and arginine; and amino acids with uncharged polar
head groups having similar hydrophilicity values include leucine,
isoleucine and valine; glycine and alanine; asparagine and
glutamine; and serine, threonine, phenylalanine and tyrosine. Other
groups of amino acids that may represent conservative changes
include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys,
ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his;
and (5) phe, tyr, trp, his. The critical determining feature of a
desmosomal cadherin CAR sequence analogue is the ability to
modulate a desmosomal cadherin-mediated function, which may be
evaluated using the representative assays provided herein.
[0078] A mimetic is a non-peptidyl compound that is
conformationally similar to a desmosomal cadherin CAR sequence,
such that it modulates a desmosomal cadherin-mediated function as
described below. Such mimetics may be designed based on techniques
that evaluate the three dimensional structure of the peptide. For
example, Nuclear Magnetic Resonance spectroscopy (NMR) and
computational techniques may be used to determine the conformation
of a desmosomal cadherin CAR sequence. NMR is widely used for
structural analyses of both peptidyl and non-peptidyl compounds.
Nuclear Overhauser Enhancements (NOE's), coupling constants and
chemical shifts depend on the conformation of a compound. NOE data
provides the interproton distance between protons through space and
can be used to calculate the lowest energy conformation for the
desmosomal cadherin CAR sequence. This information can then be used
to design mimetics of the preferred conformation. Linear peptides
in solution exist in many conformations. By using conformational
restriction techniques it is possible to fix the peptide in the
active conformation. Conformational restriction can be achieved by
i) introduction of an alkyl group such as a methyl which sterically
restricts free bond rotation; ii) introduction of unsaturation
which fixes the relative positions of the terminal and geminal
substituents; and/or iii) cyclization, which fixes the relative
positions of the sidechains. Mimetics may be synthesized where one
or more of the amide linkages has been replaced by isosteres,
substituents or groups which have the same size or volume such as
--CH.sub.2NH--, --CSNH--, --CH.sub.2S--, --CH.dbd.CH--,
--CH.sub.2CH.sub.2--, --CONMe-- and others. These backbone amide
linkages can also be part of a ring structure (e.g., lactam).
Mimetics may be designed where one or more of the side chain
functionalities of the desmosomal cadherin CAR sequence are
replaced by groups that do not necessarily have the same size or
volume, but have similar chemical and/or physical properties which
produce similar biological responses. Other mimetics may be small
molecule mimics, which may be readily identified from small
molecule libraries, based on the three-dimensional structure of the
CAR sequence. It should be understood that, within embodiments
described below, an analogue or mimetic may be substituted for a
desmosomal cadherin CAR sequence.
[0079] Modulating agents, or peptide portions thereof, may be
linear or cyclic peptides. The term "cyclic peptide," as used
herein, refers to a peptide or salt thereof that comprises (1) an
intramolecular covalent bond between two non-adjacent residues and
(2) at least one desmosomal cadherin CAR sequence or an analogue
thereof. The intramolecular bond may be a backbone to backbone,
side-chain to backbone or side-chain to side-chain bond (i.e.,
terminal functional groups of a linear peptide and/or side chain
functional groups of a terminal or interior residue may be linked
to achieve cyclization). Preferred intramolecular bonds include,
but are not limited to, disulfide, amide and thioether bonds. One
or more of any of the above desmosomal cadherin CAR sequences, or
an analogue or mimetic thereof, may be incorporated into a cyclic
peptide, with or without one or more other adhesion molecule
binding sites. Additional adhesion molecule binding sites are
described in greater detail below.
[0080] The size of a cyclic peptide ring generally ranges from 5 to
about 15 residues, preferably from 5 to 10 residues. Additional
residue(s) may be present on the N-terminal and/or C-terminal side
of a desmosomal cadherin CAR sequence, and may be derived from
sequences that flank a desmosomal cadherin CAR sequence, with or
without amino acid substitutions and/or other modifications.
Alternatively, additional residues present on one or both sides of
the CAR sequence(s) may be unrelated to an endogenous sequence
(e.g., residues that facilitate cyclization, purification or other
manipulation and/or residues having a targeting or other
function).
[0081] Within certain embodiments, a modulating agent may comprise
a cyclic peptide that contains a desmosomal cadherin CAR sequence
as provided in Table I (or a portion of such a CAR sequence).
Certain cyclic peptides have the formula: 2
[0082] Within this formula, W is a tripeptide selected from the
group consisting of NQK, NRN, NKD, EKD, ERD, RAL, YAL, YAT, FAT,
and YAS; X.sub.1, and X.sub.2 are optional, and if present, are
independently selected from the group consisting of amino acid
residues and combinations thereof in which the residues are linked
by peptide bonds, and wherein X.sub.1 and X.sub.2 independently
range in size from 0 to 10 residues, such that the sum of residues
contained within X.sub.1 and X.sub.2 ranges from 1 to 12; Y.sub.1
and Y.sub.2 are independently selected from the group consisting of
amino acid residues, and wherein a covalent bond is formed between
residues Y.sub.1 and Y.sub.2; and Z.sub.1 and Z.sub.2 are optional,
and if present, are independently selected from the group
consisting of amino acid residues and combinations thereof in which
the residues are linked by peptide bonds.
[0083] Cyclic peptides may comprise any of the above CAR
sequence(s). Such cyclic peptides may be used as modulating agents
without modification, or may be incorporated into a modulating
agent.
[0084] Representative cyclic peptides comprising a desmoglein CAR
sequence include: CNQKC (SEQ ID NO:410), CNQKTC (SEQ ID NO:411),
CNQKTGC (SEQ ID NO:412), CINQKC (SEQ ID NO:413), CINQKTC (SEQ ID
NO:414), CINQKTGC (SEQ ID NO:415), CVINQKC (SEQ ID NO:416),
CVINQKTC (SEQ ID NO:417), CVINQKTGC (SEQ ID NO:418), CFVINQKC (SEQ
ID NO:419), CFVINQKTC (SEQ ID NO:420), CFVINQKTGC (SEQ ID NO:421),
CIFVINQKC (SEQ ID NO:422), CIFVINQKTC (SEQ ID NO:423), CIFVINQKTGC
(SEQ ID NO:424), CNRNC (SEQ ID NO:425), CNRNTC (SEQ ID NO:426),
CNRNTGC (SEQ ID NO:427), CINRNC (SEQ ID NO:428), CINRNTC (SEQ ID
NO:429), CINRNTGC (SEQ ID NO:430), CIINRNC (SEQ ID NO:431),
CIINRNTC (SEQ ID NO:432), CIINRNTGC (SEQ ID NO:433), CFIINRNC (SEQ
ID NO:434), CFIINRNTC (SEQ ID NO:435), CFIINRNTGC (SEQ ID NO:436),
CMFIINRNC (SEQ ID NO:437), CMFIINRNTC (SEQ ID NO:438), CMFIINRNTGC
(SEQ ID NO:439), CNKDC (SEQ ID NO:440), CNKDTC (SEQ ID NO:441),
CNKDTGC (SEQ ID NO:442), CLNKDC (SEQ ID NO:443), CLNKDTC (SEQ ID
NO:444), CLNKDTGC (SEQ ID NO:445), CYLNKDC (SEQ ID NO:446),
CYLNKDTC (SEQ ID NO:447), CYLNKDTGC (SEQ ID NO:448), CFYLNKDC (SEQ
ID NO:449), CFYLNKDTC (SEQ ID NO:450), CFYLNKDTGC (SEQ ID NO:451),
CVFYLNKDC (SEQ ID NO:452), CVFYLNKDTC (SEQ ID NO:453), CVFYLNKDTGC
(SEQ ID NO:454), ENQKK (SEQ ID NO:455), ENQKTK (SEQ ID NO:456),
ENQKTGK (SEQ ID NO:457), EINQKK (SEQ ID NO:458), EINQKTK (SEQ ID
NO:459), EINQKTGK (SEQ ID NO:460), EVINQKK (SEQ ID NO:461),
EVINQKTK (SEQ ID NO:462), EVINQKTGK (SEQ ID NO:463), EFVINQKK (SEQ
ID NO:464), EFVINQKTK (SEQ ID NO:465), EFVINQKTGK (SEQ ID NO:466),
EIFVINQKK (SEQ ID NO:467), EIFVINQKTK (SEQ ID NO:468), EIFVINQKTGK
(SEQ ID NO:469), ENRNK (SEQ ID NO:470), ENRNTK (SEQ ID NO:471),
ENRNTGK (SEQ ID NO:472), EINRNK (SEQ ID NO:473), EINRNTK (SEQ ID
NO:474), EINRNTGK (SEQ ID NO:475), EIINRNK (SEQ ID NO:476),
EIINRNTK (SEQ ID NO:477), EIINRNTGK (SEQ ID NO:478), EFIINRNK (SEQ
ID NO:479), EFIINRNTK (SEQ ID NO:480), EFIINRNTGK (SEQ ID NO:481),
EMFIINRNK (SEQ ID NO:482), EMFIINRNTK (SEQ ID NO:483), EMFIINRNTGK
(SEQ ID NO:484), ENKDK (SEQ ID NO:485), ENKDTK (SEQ ID NO:486),
ENKDTGK (SEQ ID NO:487), ELNKDK (SEQ ID NO:488), ELNKDTK (SEQ ID
NO:489), ELNKDTGK (SEQ ID NO:490), EYLNKDK (SEQ ID NO:491),
EYLNKDTK (SEQ ID NO:492), EYLNKDTGK (SEQ ID NO:493), EFYLNKDK (SEQ
ID NO:494), EFYLNKDTK (SEQ ID NO:495), EFYLNKDTGK (SEQ ID NO:496),
EVFYLNKDK (SEQ ID NO:497), EVFYLNKDTK (SEQ ID NO:498), EVFYLNKDTGK
(SEQ ID NO:499), KNQKD (SEQ ID NO:500), KNQKTD (SEQ ID NO:501),
KNQKTGD (SEQ ID NO:502), KINQKD (SEQ ID NO:503), KINQKTD (SEQ ID
NO:504), KINQKTGD (SEQ ID NO:505), KVINQKD (SEQ ID NO:506),
KVINQKTD (SEQ ID NO:507), KVINQKTGD (SEQ ID NO:508), KFVINQKD (SEQ
ID NO:509), KFVINQKTD (SEQ ID NO:510), KFVINQKTGD (SEQ ID NO:511),
KIFVINQKD (SEQ ID NO:512), KIFVINQKTD (SEQ ID NO:513), KIFVINQKTGD
(SEQ ID NO:514), KNRND (SEQ ID NO:515), KNRNTD (SEQ ID NO:516),
KNRNTGD (SEQ ID NO:517), KINRND (SEQ ID NO:518), KINRNTD (SEQ ID
NO:519), KINRNTGD (SEQ ID NO:520), KIINRND (SEQ ID NO:521),
KIINRNTD (SEQ ID NO:522), KIINRNTGD (SEQ ID NO:523), KFIINRND (SEQ
ID NO:524), KFIINRNTD (SEQ ID NO:525), KFIINRNTGD (SEQ ID NO:526),
KMFIINRND (SEQ ID NO:527), KMFIINRNTD (SEQ ID NO:528), KMFIINRNTGD
(SEQ ID NO:529), KNKDD (SEQ ID NO:530), KNKDTD (SEQ ID NO:531),
KNKDTGD (SEQ ID NO:532), KLNKDD (SEQ ID NO:533), KLNKDTD (SEQ ID
NO:534) KLNKDTGD (SEQ ID NO:535), KYLNKDD (SEQ ID NO:536), KYLNKDTD
(SEQ ID NO:537), KYLNKDTGD (SEQ ID NO:538), KFYLNKDD (SEQ ID
NO:539), KFYLNKDTD (SEQ ID NO:540), KFYLNKDTGD (SEQ ID NO:541),
KVFYLNKDD (SEQ ID NO:542), KVFYLNKDTD (SEQ ID NO:543), KVFYLNKDTGD
(SEQ ID NO:544), DNQKK (SEQ ID NO:545), DNQKTK (SEQ ID NO:546),
DNQKTGK (SEQ ID NO:547), DINQKK (SEQ ID NO:548), DINQKTK (SEQ ID
NO:549), DINQKTGK (SEQ ID NO:550), DVINQKK (SEQ ID NO:551),
DVINQKTK (SEQ ID NO:552), DVINQKTGK (SEQ ID NO:553), DFVINQKK (SEQ
ID NO:554), DFVINQKTK (SEQ ID NO:555), DFVINQKTGK (SEQ ID NO:556),
DIFVINQKK (SEQ ID NO:557), DIFVINQKTK (SEQ ID NO:558), DIFVINQKTGK
(SEQ ID NO:559), DNRNK (SEQ ID NO:560), DNRNTK (SEQ ID NO:561),
DNRNTGK (SEQ ID NO:562), DINRNK (SEQ ID NO:563), DINRNTK (SEQ ID
NO:564), DINRNTGK (SEQ ID NO:565), DIINRNK (SEQ ID NO:566),
DIINRNTK (SEQ ID NO:567), DIINRNTGK (SEQ ID NO:568), DFIINRNK (SEQ
ID NO:569), DFIINRNTK (SEQ ID NO:570), DFIINRNTGK (SEQ ID NO:571),
DMFIINRNK (SEQ ID NO:572), DMFIINRNTK (SEQ ID NO:573), DMFIINRNTGK
(SEQ ID NO:574), DNKDK (SEQ ID NO:575), DNKDTK (SEQ ID NO:576),
DNKDTGK (SEQ ID NO:577), DLNKDK (SEQ ID NO:578), DLNKDTK (SEQ ID
NO:579), DLNKDTGK (SEQ ID NO:580), DYLNKDK (SEQ ID NO:581),
DYLNKDTK (SEQ ID NO:582), DYLNKDTGK (SEQ ID NO:583), DFYLNKDK (SEQ
ID NO:584), DFYLNKDTK (SEQ ID NO:585), DFYLNKDTGK (SEQ ID NO:586),
DVFYLNKDK (SEQ ID NO:587), DVFYLNKDTK (SEQ ID NO:588), DVFYLNKDTGK
(SEQ ID NO:589), KKNQKE (SEQ ID NO:590), KNQKTE (SEQ ID NO:591),
KNQKTGE (SEQ ID NO:592), KINQKE (SEQ ID NO:593), KINQKTE (SEQ ID
NO:594), KINQKTGE (SEQ ID NO:595), KVINQKE (SEQ ID NO:596),
KVINQKTE (SEQ ID NO:597), KVINQKTGE (SEQ ID NO:598), KFVINQKE (SEQ
ID NO:599), KFVINQKTE (SEQ ID NO:600), KFVINQKTGE (SEQ ID NO:601),
KIFVINQKE (SEQ ID NO:602), KIFVINQKTE (SEQ ID NO:603), KIFVINQKTGE
(SEQ ID NO:604), KNRNE (SEQ ID NO:605), KNRNTE (SEQ ID NO:606),
KNRNTGE (SEQ ID NO:607), KINRNE (SEQ ID NO:608), KINRNTE (SEQ ID
NO:609), KINRNTGE (SEQ ID NO:610), KIINRNE (SEQ ID NO:611),
KIINRNTE (SEQ ID NO:612), KIINRNTGE (SEQ ID NO:613), KFIINRNE (SEQ
ID NO:614), KFIINRNTE (SEQ ID NO:615), KFIINRNTGE (SEQ ID NO:616),
KMFIINRNE (SEQ ID NO:617), KMFIINRNTE (SEQ ID NO:618), KMFIINRNTGE
(SEQ ID NO:619), KNKDE (SEQ ID NO:620), KNKDTE (SEQ ID NO:621),
KNKDTGE (SEQ ID NO:622), KLNKDE (SEQ ID NO:623), KLNKDTE (SEQ ID
NO:624), KLNKDTGE (SEQ ID NO:625), KYLNKDE (SEQ ID NO:626),
KYLNKDTE (SEQ ID NO:627), KYLNKDTGE (SEQ ID NO:628), KFYLNKDE (SEQ
ID NO:629), KFYLNKDTE (SEQ ID NO:630), KFYLNKDTGE (SEQ ID NO:631),
KVFYLNKDE (SEQ ID NO:632), KVFYLNKDTE (SEQ ID NO:633), KVFYLNKDTGE
(SEQ ID NO:634), NQKTG (SEQ ID NO:635), INQKT (SEQ ID NO:636),
INQKTG (SEQ ID NO:637), VINQK (SEQ ID NO:638), VINQKT (SEQ ID
NO:639), VINQKTG (SEQ ID NO:640), FVINQK (SEQ ID NO:641), FVINQKT
(SEQ ID NO:642), FVINQKTG (SEQ ID NO:643), IFVINQK (SEQ ID NO:644),
IFVINQKT (SEQ ID NO:645), IFVINQKTG (SEQ ID NO:646), NRNTG (SEQ ID
NO:647), INRNT (SEQ ID NO:648), INRNTG (SEQ ID NO:649), IINRN (SEQ
ID NO:650), IINRNT (SEQ ID NO:651), IINRNTG (SEQ ID NO:652), FIINRN
(SEQ ID NO:653), FIINRNT (SEQ ID NO:654), FIINRNTG (SEQ ID NO:655),
MFIINRN (SEQ ID NO:656), MFIINRNT (SEQ ID NO:657), MFIINRNTG (SEQ
ID NO:658), NKDTG (SEQ ID NO:659), LNKDT (SEQ ID NO:660), LNKDTG
(SEQ ID NO:661), YLNKD (SEQ ID NO:662), YLNKDT (SEQ ID NO:663),
YLNKDTG (SEQ ID NO:664), FYLNKD (SEQ ID NO:665), FYLNKDT (SEQ ID
NO:666), FYLNKDTG (SEQ ID NO:667), VFYLNKD (SEQ ID NO:668),
VFYLNKDT (SEQ ID NO:669) and VFYLNKDTG (SEQ ID NO:670).
[0085] Representative cyclic peptides comprising a desmocollin CAR
sequence include: CEKDC (SEQ ID NO:671), CEKDTC (SEQ ID NO:672),
CEKDTGC (SEQ ID NO:673), CIEKDC (SEQ ID NO:674), CIEKDTC (SEQ ID
NO:675), CIEKDTGC (SEQ ID NO:676), CYIEKDC (SEQ ID NO:677),
CYIEKDTC (SEQ ID NO:678), CYIEKDTGC (SEQ ID NO:679), CFYIEKDC (SEQ
ID NO:680), CFYIEKDTC (SEQ ID NO:681), CFYIEKDTGC (SEQ ID NO:682),
CLFYIEKDC (SEQ ID NO:683), CLFYIEKDTC (SEQ ID NO:684), CLFYIEKDTGC
(SEQ ID NO:685), CERDC (SEQ ID NO:686), CERDTC (SEQ ID NO:687),
CERDTGC (SEQ ID NO:688), CVERDC (SEQ ID NO:689), CVERDTC (SEQ ID
NO:690), CVERDTGC (SEQ ID NO:691), CYVERDC (SEQ ID NO:692),
CYVERDTC (SEQ ID NO:693), CYVERDTGC (SEQ ID NO:694), CFYVERDC (SEQ
ID NO:695), CFYVERDTC (SEQ ID NO:696), CFYVERDTGC (SEQ ID NO:697),
CLFYVERDC (SEQ ID NO:698), CLFYVERDTC (SEQ ID NO:699), CLFYVERDTGC
(SEQ ID NO:700), CIERDC (SEQ ID NO:701), CIERDTC (SEQ ID NO:702),
CIERDTGC (SEQ ID NO:703), CYIERDC (SEQ ID NO:704), CYIERDTC (SEQ ID
NO:705), CYIERDTGC (SEQ ID NO:706), CFYIERDC (SEQ ID NO:707),
CFYIERDTC (SEQ ID NO:708), CFYIERDTGC (SEQ ID NO:709), CLFYIERDC
(SEQ ID NO:710), CLFYIERDTC (SEQ ID NO:711), CLFYIERDTGC (SEQ ID
NO:712), EEKDK (SEQ ID NO:713), EEKDTK (SEQ ID NO:714), EEKDTGK
(SEQ ID NO:715), EIEKDK (SEQ ID NO:716), EIEKDTK (SEQ ID NO:717),
EIEKDTGK (SEQ ID NO:718), EYIEKDK (SEQ ID NO:719), EYIEKDTK (SEQ ID
NO:720), EYIEKDTGK (SEQ ID NO:721), EFYIEKDK (SEQ ID NO:722),
EFYIEKDTK (SEQ ID NO:723), EFYIEKDTGK (SEQ ID NO:724), ELFYIEKDK
(SEQ ID NO:725), ELFYIEKDTK (SEQ ID NO:726), ELFYIEKDTGK (SEQ ID
NO:727), EERDK (SEQ ID NO:728), EERDTK (SEQ ID NO:729), EERDTGK
(SEQ ID NO:730), EVERDK (SEQ ID NO:731), EVERDTK (SEQ ID NO:732),
EVERDTGK (SEQ ID NO:733), EYVERDK (SEQ ID NO:734), YVERDTK (SEQ ID
NO:735), EYVERDTGK (SEQ ID NO:736), EFYVERDK (SEQ ID NO:737),
EFYVERDTK (SEQ ID NO:738), EFYVERDTGK (SEQ ID NO:739), ELFYVERDK
(SEQ ID NO:740), ELFYVERDTK (SEQ ID NO:741), ELFYVERDTGK (SEQ ID
NO:742), EIERDK (SEQ ID NO:743), EIERDTK (SEQ ID NO:744), EIERDTGK
(SEQ ID NO:745), EYIERDK (SEQ ID NO:746), EYIERDTK (SEQ ID NO:747),
EYIERDTGK (SEQ ID NO:748), EFYIERDK (SEQ ID NO:749), EFYIERDTK (SEQ
ID NO:750), EFYIERDTGK (SEQ ID NO:751), ELFYIERDK (SEQ ID NO:752),
ELFYIERDTK (SEQ ID NO:753), ELFYIERDTGK (SEQ ID NO:754), KEKDD (SEQ
ID NO:755), KEKDTD (SEQ ID NO:756), KEKDTGD (SEQ ID NO:757), KIEKDD
(SEQ ID NO:758), KIEKDTD (SEQ ID NO:759), KIEKDTGD (SEQ ID NO:760),
KYIEKDD (SEQ ID NO:761), KYIEKDTD (SEQ ID NO:762), KYIEKDTGD (SEQ
ID NO:763), KFYIEKDD (SEQ ID NO:764), KFYIEKDTD (SEQ ID NO:765),
KFYIEKDTGD (SEQ ID NO:766), KLFYIEKDD (SEQ ID NO:767), KLFYIEKDTD
(SEQ ID NO:768), KLFYIEKDTGD (SEQ ID NO:769), KERDD (SEQ ID
NO:770), KERDTD (SEQ ID NO:771), KERDTGD (SEQ ID NO:772), KVERDD
(SEQ ID NO:773), KVERDTD (SEQ ID NO:774), KVERDTGD (SEQ ID NO:775),
KYVERDD (SEQ ID NO:776), KYVERDTD (SEQ ID NO:777), KYVERDTGD (SEQ
ID NO:778), KFYVERDD (SEQ ID NO:779), KFYVERDTD (SEQ ID NO:780),
KFYVERDTGD (SEQ ID NO:781), KLFYVERDD (SEQ ID NO:782), KLFYVERDTD
(SEQ ID NO:783), KLFYVERDTGD (SEQ ID NO:784), KIERDD (SEQ ID
NO:785), KIERDTD (SEQ ID NO:786), KIERDTGD (SEQ ID NO:787), KYIERD
(SEQ ID NO:788), KYIERDTD (SEQ ID NO:789), KYIERDTGD (SEQ ID
NO:790), KFYIERDD (SEQ ID NO:791), KFYIERDTD (SEQ ID NO:792),
KFYIERDTGD (SEQ ID NO:793), KLFYIERDD (SEQ ID NO:794), KLFYIERDTD
(SEQ ID NO:795), KLFYIERDTGD (SEQ ID NO:796), DEKDK (SEQ ID
NO:797), DEKDTK (SEQ ID NO:798), DEKDTGK (SEQ ID NO:799), DIEKDK
(SEQ ID NO:800), DIEKDTK (SEQ ID NO:801), DIEKDTGK (SEQ ID NO:802),
DYIEKDK (SEQ ID NO:803), DYIEKDTK (SEQ ID NO:804), DYIEKDTGK (SEQ
ID NO:805), DFYIEKDK (SEQ ID NO:806), DFYIEKDTK (SEQ ID NO:807),
DFYIEKDTGK (SEQ ID NO:808), DLFYIEKDK (SEQ ID NO:809), DLFYIEKDTK
(SEQ ID NO:810), DLFYIEKDTGK (SEQ ID NO:811), DERDK (SEQ ID
NO:812), DERDTK (SEQ ID NO:813), DERDTGK (SEQ ID NO:814), DVERDK
(SEQ ID NO:815), DVERDTK (SEQ ID NO:816), DVERDTGK (SEQ ID NO:817),
DYVERDK (SEQ ID NO:818), DYVERDTK (SEQ ID NO:819), DYVERDTGK (SEQ
ID NO:820), DFYVERDK (SEQ ID NO:821), DFYVERDTK (SEQ ID NO:822),
DFYVERDTGK (SEQ ID NO:823), DLFYVERDK (SEQ ID NO:824), DLFYVERDTK
(SEQ ID NO:825), DLFYVERDTGK (SEQ ID NO:826), DIERDK (SEQ ID
NO:827), DIERDTK (SEQ ID NO:828), DIERDTGK (SEQ ID NO:829), DYIERDK
(SEQ ID NO:830), DYIERDTK (SEQ ID NO:831), DYIERDTGK (SEQ ID
NO:832), DFYIERDK (SEQ ID NO:833), DFYIERDTK (SEQ ID NO:834),
DFYIERDTGK (SEQ ID NO:835), DLFYIERDK (SEQ ID NO:836), DLFYIERDTK
(SEQ ID NO:837), DLFYIERDTGK (SEQ ID NO:838), KEKDE (SEQ ID
NO:839), KEKDTE (SEQ ID NO:840), KEKDTGE (SEQ ID NO:841), KIEKDE
(SEQ ID NO:842), KIEKDTE (SEQ ID NO:843), KIEKDTGE (SEQ ID NO:844),
KYIEKDE (SEQ ID NO:845), KYIEKDTE (SEQ ID NO:846), KYIEKDTGE (SEQ
ID NO:847), KFYIEKDE (SEQ ID NO:848), KFYIEKDTE (SEQ ID NO:849),
KFYIEKDTGE (SEQ ID NO:850), KLFYIEKDE (SEQ ID NO:851), KLFYIEKDTE
(SEQ ID NO:852), KLFYIEKDTGE (SEQ ID NO:853), KERDE (SEQ ID
NO:854), KERDTE (SEQ ID NO:855), KERDTGE (SEQ ID NO:856), KVERDE
(SEQ ID NO:857), KVERDTE (SEQ ID NO:858), KVERDTGE (SEQ ID NO:859),
KYVERDE (SEQ ID NO:860), KYVERDTE (SEQ ID NO:861), KYVERDTGE (SEQ
ID NO:862), KFYVERDE (SEQ ID NO:863), KFYVERDTE (SEQ ID NO:864),
KFYVERDTGE (SEQ ID NO:865), KLFYVERDE (SEQ ID NO:866), KLFYVERDTE
(SEQ ID NO:867), KLFYVERDTGE (SEQ ID NO:868), KIERDE (SEQ ID
NO:869), KIERDTE (SEQ ID NO:870), KIERDTGE (SEQ ID NO:871), KYIERDE
(SEQ ID NO:872), KYIERDTE (SEQ ID NO:873), KYIERDTGE (SEQ ID
NO:874), KFYIERDE (SEQ ID NO:875), KFYIERDTE (SEQ ID NO:876),
KFYIERDTGE (SEQ ID NO:877), KLFYIERDE (SEQ ID NO:878), KLFYIERDTE
(SEQ ID NO:879), KLFYIERDTGE (SEQ ID NO:880), EKDTG (SEQ ID
NO:881), IEKDT (SEQ ID NO:882), IEKDTG (SEQ ID NO:883), YIEKD (SEQ
ID NO:884), YIEKDT (SEQ ID NO:885), YIEKDTG (SEQ ID NO:886), FYIEKD
(SEQ ID NO:887), FYIEKDT (SEQ ID NO:888), FYIEKDTG (SEQ ID NO:889),
LFYIEKD (SEQ ID NO:890), LFYIEKDT (SEQ ID NO:891), LFYIEKDTG (SEQ
ID NO:892), ERDTG (SEQ ID NO:893), VERDT (SEQ ID NO:894), VERDTG
(SEQ ID NO:895), YVERD (SEQ ID NO:896), YVERDT (SEQ ID NO:897),
YVERDTG (SEQ ID NO:898), FYVERD (SEQ ID NO:899), FYVERDT (SEQ ID
NO:900), FYVERDTG (SEQ ID NO:901), LFYVERD (SEQ ID NO:902),
LFYVERDT (SEQ ID NO:903), LFYVERDTG (SEQ ID NO:904), IERDT (SEQ ID
NO:905), IERDTG (SEQ ID NO:906), YIERD (SEQ ID NO:907), YIERDT (SEQ
ID NO:908), YIERDTG (SEQ ID NO:909), FYIERD (SEQ ID NO:910),
FYIERDT (SEQ ID NO:911), FYIERDTG (SEQ ID NO:912), LFYIERD (SEQ ID
NO:913), LFYIERDT (SEQ ID NO:914) and LFYIERDTG (SEQ ID
NO:915).
[0086] Representative cyclic peptides comprising a desmoglein-1 CAR
sequence include: CRALC (SEQ ID NO:916), CRALNC (SEQ ID NO:917),
CRALNSC (SEQ ID NO:918), CRALNSMC (SEQ ID NO:919), CRALNSLC (SEQ ID
NO:920), CRALNSMGC (SEQ ID NO:921), CRALNSLGC (SEQ ID NO:922),
CCRALC (SEQ ID NO:923), CCRALNC (SEQ ID NO:924), CCRALNSC (SEQ ID
NO:925), CCRALNSMC (SEQ ID NO:926), CCRALNSLC (SEQ ID NO:927),
CCRALNSMGC (SEQ ID NO:928), CCRALNSLGC (SEQ ID NO:929), CYCRALC
(SEQ ID NO:930), CYCRALNC (SEQ ID NO:931), CYCRALNSC (SEQ ID
NO:932), CYCRALNSMC (SEQ ID NO:933), CYCRALNSLC (SEQ ID NO:934),
CYCRALNSMGC (SEQ ID NO:935), CYCRALNSLGC (SEQ ID NO:936), CIYRALC
(SEQ ID NO:937), CIYRALNC (SEQ ID NO:938), CIYRALNSC (SEQ ID
NO:939), CIYRALNSMC (SEQ ID NO:940), CIYRALNSLC (SEQ ID NO:941),
CIYRALNSMGC (SEQ ID NO:942), CIYRALNSLGC (SEQ ID NO:943), ERALK
(SEQ ID NO:944), ERALNK (SEQ ID NO:945), ERALNSK (SEQ ID NO:946),
ERALNSMK (SEQ ID NO:947), ERALNSLK (SEQ ID NO:948), ERALNSMGK (SEQ
ID NO:949), ERALNSLGK (SEQ ID NO:950), ECRALK (SEQ ID NO:951),
ECRALNK (SEQ ID NO:952), ECRALNSK (SEQ ID NO:953), ECRALNSMK (SEQ
ID NO:954), ECRALNSLK (SEQ ID NO:955), ECRALNSMGK (SEQ ID NO:956),
ECRALNSLGK (SEQ ID NO:957), EYCRALK (SEQ ID NO:958), EYCRALNK (SEQ
ID NO:959), EYCRALNSK (SEQ ID NO:960), EYCRALNSMK (SEQ ID NO:961),
EYCRALNSLK (SEQ ID NO:962), EYCRALNSMGK (SEQ ID NO:963),
EYCRALNSLGK (SEQ ID NO:964), EIYRALK (SEQ ID NO:965), EIYRALNK (SEQ
ID NO:966), EIYRALNSK (SEQ ID NO:967), EIYRALNSMK (SEQ ID NO:968),
EIYRALNSLK (SEQ ID NO:969), EIYRALNSMGK (SEQ ID NO:970),
EIYRALNSLGK (SEQ ID NO:971), KRALE (SEQ ID NO:972), KRALNE (SEQ ID
NO:973), KRALNSE (SEQ ID NO:974), KRALNSME (SEQ ID NO:975),
KRALNSLE (SEQ ID NO:976), KRALNSMGE (SEQ ID NO:977), KRALNSLGE (SEQ
ID NO:978), KCRALE (SEQ ID NO:979), KCRALNE (SEQ ID NO:980),
KCRALNSE (SEQ ID NO:981), KCRALNSME (SEQ ID NO:982), KCRALNSLE (SEQ
ID NO:983), KCRALNSMGE (SEQ ID NO:984), KCRALNSLGE (SEQ ID NO:985),
KYCRALE (SEQ ID NO:986), KYCRALNE (SEQ ID NO:987), KYCRALNSE (SEQ
ID NO:988), KYCRALNSME (SEQ ID NO:989), KYCRALNSLE (SEQ ID NO:990),
KYCRALNSMGE (SEQ ID NO:991), KYCRALNSLGE (SEQ ID NO:992), KIYRALE
(SEQ ID NO:993), KIYRALNE (SEQ ID NO:994), KIYRALNSE (SEQ ID
NO:995), KIYRALNSME (SEQ ID NO:996), KIYRALNSLE (SEQ ID NO:997),
KIYRALNSMGE (SEQ ID NO:998), KIYRALNSLGE (SEQ ID NO:999), DRALK
(SEQ ID NO:1000), DRALNK (SEQ ID NO:1001), DRALNSK (SEQ ID
NO:1002), DRALNSMK (SEQ ID NO:1003), DRALNSLK (SEQ ID NO:1004),
DRALNSMGK (SEQ ID NO:1005), DRALNSLGK (SEQ ID NO:1006), DCRALK (SEQ
ID NO:1007), DCRALNK (SEQ ID NO:1008), DCRALNSK (SEQ ID NO:1009),
DCRALNSMK (SEQ ID NO:1010), DCRALNSLK (SEQ ID NO:1011), DCRALNSMGK
(SEQ ID NO:1012), DCRALNSLGK (SEQ ID NO:1013), DYCRALK (SEQ ID
NO:1014), DYCRALNK (SEQ ID NO:1015), DYCRALNSK (SEQ ID NO:1016),
DYCRALNSMK (SEQ ID NO:1017), DYCRALNSLK (SEQ ID NO:1018),
DYCRALNSMGK (SEQ ID NO:1019), DYCRALNSLGK (SEQ ID NO:1020), DIYRALK
(SEQ ID NO:1021), DIYRALNK (SEQ ID NO:1022), DIYRALNSK (SEQ ID
NO:1023), DIYRALNSMK (SEQ ID NO:1024), DIYRALNSLK (SEQ ID NO:1025),
DIYRALNSMGK (SEQ ID NO:1026), DIYRALNSLGK (SEQ ID NO:1027), KRALD
(SEQ ID NO:1028), KRALND (SEQ ID NO:1029), KRALNSD (SEQ ID
NO:1030), KRALNSMD (SEQ ID NO:1031), KRALNSLD (SEQ ID NO:1032),
KRALNSMGD (SEQ ID NO:1033), KRALNSLGD (SEQ ID NO:1034), KCRALD (SEQ
ID NO:1035), KCRALND (SEQ ID NO:1036), KCRALNSD (SEQ ID NO:1037),
KCRALNSMD (SEQ ID NO:1038), KCRALNSLD (SEQ ID NO:1039), KCRALNSMGD
(SEQ ID NO:1040), KCRALNSLGD (SEQ ID NO:1041), KYCRALD (SEQ ID
NO:1042), KYCRALND (SEQ ID NO:1043), KYCRALNSD (SEQ ID NO:1044),
KYCRALNSMD (SEQ ID NO:1045), KYCRALNSLD (SEQ ID NO:1046),
KYCRALNSMGD (SEQ ID NO:1047), KYCRALNSLGD (SEQ ID NO:1048), KIYRALD
(SEQ ID NO:1049), KIYRALND (SEQ ID NO:1050), KIYRALNSD (SEQ ID
NO:1051), KIYRALNSMD (SEQ ID NO:1052), KIYRALNSLD (SEQ ID NO:1053),
KIYRALNSMGD (SEQ ID NO:1054), KIYRALNSLGD (SEQ ID NO:1055), RALNS
(SEQ ID NO:1056), RALNSM (SEQ ID NO:1057), RALNSL (SEQ ID NO:1058),
RALNSMG (SEQ ID NO:1059), RALNSLG (SEQ ID NO:1060), CRALN (SEQ ID
NO:1061), CRALNS (SEQ ID NO:1062), CRALNSM (SEQ ID NO:1063),
CRALNSL (SEQ ID NO:1064), CRALNSMG (SEQ ID NO:1065), CRALNSLG (SEQ
ID NO:1066), YCRAL (SEQ ID NO:1067), YCRALN (SEQ ID NO:1068),
YCRALNS (SEQ ID NO:1069), YCRALNSM (SEQ ID NO:1070), YCRALNSL (SEQ
ID NO:1071), YCRALNSMG (SEQ ID NO:1072), YCRALNSLG (SEQ ID
NO:1073), IYRAL (SEQ ID NO:1074), IYRALN (SEQ ID NO:1075), IYRALNS
(SEQ ID NO:1076), IYRALNSM (SEQ ID NO:1077), IYRALNSL (SEQ ID
NO:1078), IYRALNSMG (SEQ ID NO:1079) and IYRALNSLG (SEQ ID
NO:1080).
[0087] Representative cyclic peptides comprising a desmoglein-2 CAR
sequence include: CYALC (SEQ ID NO:1081), CYALDC (SEQ ID NO:1082),
CYALDAC (SEQ ID NO:1083), CYALDARC (SEQ ID NO:1084), CYALDARGC (SEQ
ID NO:1085), CGYALC (SEQ ID NO:1086), CGYALDC (SEQ ID NO:1087),
CGYALDAC (SEQ ID NO:1088), CGYALDARC (SEQ ID NO:1089), CGYALDARGC
(SEQ ID NO:1090), CTGYALC (SEQ ID NO:1091), CTGYALDC (SEQ ID
NO:1092), CTGYALDAC (SEQ ID NO:1093), CTGYALDARC (SEQ ID NO:1094),
CTGYALDARGC (SEQ ID NO:1095), CLTGYALC (SEQ ID NO:1096), CLTGYALDC
(SEQ ID NO:1097), CLTGYALDAC (SEQ ID NO:1098), CLTGYALDARC (SEQ ID
NO:1099), CLTGYALDARGC (SEQ ID NO:1100), EYALK (SEQ ID NO:1101),
EYALDK (SEQ ID NO:1102), EYALDAK (SEQ ID NO:1103), EYALDARK (SEQ ID
NO:1104), EYALDARGK (SEQ ID NO:1105), EGYALK (SEQ ID NO:1106),
EGYALDK (SEQ ID NO:1107), EGYALDAK (SEQ ID NO:1108), EGYALDARK (SEQ
ID NO:1109), EGYALDARGK (SEQ ID NO:1110), ETGYALK (SEQ ID NO:1111),
ETGYALDK (SEQ ID NO:1112), ETGYALDAK (SEQ ID NO:1113), ETGYALDARK
(SEQ ID NO:1114), ETGYALDARGK (SEQ ID NO:1115), ELTGYALK (SEQ ID
NO:1116), ELTGYALDK (SEQ ID NO:1117), ELTGYALDAK (SEQ ID NO:1118),
ELTGYALDARK (SEQ ID NO:1119), ELTGYALDARGK (SEQ ID NO:1120), KYALE
(SEQ ID NO:1121), KYALDE (SEQ ID NO:1122), KYALDAE (SEQ ID
NO:1123), KYALDARE (SEQ ID NO:1124), KYALDARGE (SEQ ID NO:1125),
KGYALE (SEQ ID NO:1126), KGYALDE (SEQ ID NO:1127), KGYALDAE (SEQ ID
NO:1128), KGYALDARE (SEQ ID NO:1129), KGYALDARGE (SEQ ID NO:1130),
KTGYALE (SEQ ID NO:1131), KTGYALDE (SEQ ID NO:1132), KTGYALDAE (SEQ
ID NO:1133), KTGYALDARE (SEQ ID NO:1134), KTGYALDARGE (SEQ ID
NO:1135), KLTGYALE (SEQ ID NO:1136), KLTGYALDE (SEQ ID NO:1137),
KLTGYALDAE (SEQ ID NO:1138), KLTGYALDARE (SEQ ID NO:1139),
KLTGYALDARGE (SEQ ID NO:1140), DYALK (SEQ ID NO:1141), DYALDK (SEQ
ID NO:1142), DYALDAK (SEQ ID NO:1143), DYALDARK (SEQ ID NO:1144),
DYALDARGK (SEQ ID NO:1145), DGYALK (SEQ ID NO:1146), DGYALDK (SEQ
ID NO:1147), DGYALDAK (SEQ ID NO:1148), DGYALDARK (SEQ ID NO:1149),
DGYALDARGK (SEQ ID NO:1150), DTGYALK (SEQ ID NO:1151), DTGYALDK
(SEQ ID NO:1152), DTGYALDAK (SEQ ID NO:1153), DTGYALDARK (SEQ ID
NO:1154), DTGYALDARGK (SEQ ID NO:1155), DLTGYALK (SEQ ID NO:1156),
DLTGYALDK (SEQ ID NO:1157), DLTGYALDAK (SEQ ID NO:1158),
DLTGYALDARK (SEQ ID NO:1159), DLTGYALDARGK (SEQ ID NO:1160), KYALD
(SEQ ID NO:1161), KYALDD (SEQ ID NO:1162), KYALDAD (SEQ ID
NO:1163), KYALDARD (SEQ ID NO:1164), KYALDARGD (SEQ ID NO:1165),
KGYALD (SEQ ID NO:1166), KGYALDD (SEQ ID NO:1167), KGYALDAD (SEQ ID
NO:1168), KGYALDARD (SEQ ID NO:1169), KGYALDARGD (SEQ ID NO:1170),
KTGYALD (SEQ ID NO:1171), KTGYALDD (SEQ ID NO:1172), KTGYALDAD (SEQ
ID NO:1173), KTGYALDARD (SEQ ID NO:1174), KTGYALDARGD (SEQ ID
NO:1175), KLTGYALD (SEQ ID NO:1176), KLTGYALDD (SEQ ID NO:1177),
KLTGYALDAD (SEQ ID NO:1178), KLTGYALDARD (SEQ ID NO:1179),
KLTGYALDARGD (SEQ ID NO:1180), YALDA (SEQ ID NO:1181), YALDAR (SEQ
ID NO:1182), YALDARG (SEQ ID NO:1183), GYALD (SEQ ID NO:1184),
GYALDA (SEQ ID NO:1185), GYALDAR (SEQ ID NO:1186), GYALDARG (SEQ ID
NO:1187), TGYAL (SEQ ID NO:1188), TGYALD (SEQ ID NO:1189), TGYALDA
(SEQ ID NO:1190), TGYALDAR (SEQ ID NO:1191), TGYALDARG (SEQ ID
NO:1192), LTGYAL (SEQ ID NO:1193), LTGYALD (SEQ ID NO:1194),
LTGYALDA (SEQ ID NO:1195), LTGYALDAR (SEQ ID NO:1196) and
LTGYALDARG (SEQ ID NO:1197).
[0088] Representative cyclic peptides comprising a desmoglein-3 CAR
sequence include: CRALC (SEQ ID NO:916), CRALNC (SEQ ID NO:917),
CRALNAC (SEQ ID NO:1198), CRALNAQC (SEQ ID NO:1199), CRALNALC (SEQ
ID NO:1200), CRALNAQGC (SEQ ID NO:1201), CRALNALGC (SEQ ID
NO:1202), CCRALC (SEQ ID NO:923), CCRALNC (SEQ ID NO:924), CCRALNAC
(SEQ ID NO:1203), CCRALNAQC (SEQ ID NO:1204), CCRALNALC (SEQ ID
NO:1205), CCRALNAQGC (SEQ ID NO:1206), CCRALNALGC (SEQ ID NO:1207),
CTCRALC (SEQ ID NO:1208), CTCRALNC (SEQ ID NO:1209), CTCRALNAC (SEQ
ID NO:1210), CTCRALNAQC (SEQ ID NO:1211), CTCRALNALC (SEQ ID
NO:1212), CTCRALNAQGC (SEQ ID NO:1213), CTCRALNALGC (SEQ ID
NO:1214), CITCRALC (SEQ ID NO:1215), CITCRALNC (SEQ ID NO:1216),
CITCRALNAC (SEQ ID NO:1217), CITCRALNAQC (SEQ ID NO:1218),
CITCRALNALC (SEQ ID NO:1219), CITCRALNAQGC (SEQ ID NO:1220),
CITCRALNALGC (SEQ ID NO:1221), ERALK (SEQ ID NO:944), ERALNK (SEQ
ID NO:945), ERALNAK (SEQ ID NO:1222), ERALNAQK (SEQ ID NO:1223),
ERALNALK (SEQ ID NO:1224), ERALNAQGK (SEQ ID NO:1225), ERALNALGK
(SEQ ID NO:1226), ECRALK (SEQ ID NO:951), ECRALNK (SEQ ID NO:952),
ECRALNAK (SEQ ID NO:1227), ECRALNAQK (SEQ ID NO:1228), ECRALNALK
(SEQ ID NO:1229), ECRALNAQGK (SEQ ID NO:1230), ECRALNALGK (SEQ ID
NO:1231), ETCRALK (SEQ ID NO:1232), ETCRALNK (SEQ ID NO:1233),
ETCRALNAK (SEQ ID NO:1234), ETCRALNAQK (SEQ ID NO:1235), ETCRALNALK
(SEQ ID NO:1236), ETCRALNAQGK (SEQ ID NO:1237), ETCRALNALGK (SEQ ID
NO:1238), EITCRALK (SEQ ID NO:1239), EITCRALNK (SEQ ID NO:1240),
EITCRALNAK (SEQ ID NO:1241), EITCRALNAQK (SEQ ID NO:1242),
EITCRALNALK (SEQ ID NO:1243), EITCRALNAQGK (SEQ ID NO:1244),
EITCRALNALGK (SEQ ID NO:1245), KRALE (SEQ ID NO:972), KRALNE (SEQ
ID NO:973), KRALNAE (SEQ ID NO:1246), KRALNAQE (SEQ ID NO:1247),
KRALNALE (SEQ ID NO:1248), KRALNAQGE (SEQ ID NO:1249), KRALNALGE
(SEQ ID NO:1250), KCRALE (SEQ ID NO:979), KCRALNE (SEQ ID NO:980),
KCRALNAE (SEQ ID NO:1251), KCRALNAQE (SEQ ID NO:1252), KCRALNALE
(SEQ ID NO:1253), KCRALNAQGE (SEQ ID NO:1254), KCRALNALGE (SEQ ID
NO:1255), KTCRALE (SEQ ID NO:1256), KTCRALNE (SEQ ID NO:1257),
KTCRALNAE (SEQ ID NO:1258), KTCRALNAQE (SEQ ID NO:1259), KTCRALNALE
(SEQ ID NO:1260), KTCRALNAQGE (SEQ ID NO:1261), KTCRALNALGE (SEQ ID
NO:1262), KITCRALE (SEQ ID NO:1263), KITCRALNE (SEQ ID NO:1264),
KITCRALNAE (SEQ ID NO:1265), KITCRALNAQE (SEQ ID NO:1266),
KITCRALNALE (SEQ ID NO:1267), KITCRALNAQGE (SEQ ID NO:1268),
KITCRALNALGE (SEQ ID NO:1269), DRALK (SEQ ID NO:1000), DRALNK (SEQ
ID NO:1001), DRALNAK (SEQ ID NO:1270), DRALNAQK (SEQ ID NO:1271),
DRALNALK (SEQ ID NO:1272), DRALNAQGK (SEQ ID NO:1273), DRALNALGK
(SEQ ID NO:1274), DCRALK (SEQ ID NO:1007), DCRALNK (SEQ ID
NO:1008), DCRALNAK (SEQ ID NO:1275), DCRALNAQK (SEQ ID NO:1276),
DCRALNALK (SEQ ID NO:1277), DCRALNAQGK (SEQ ID NO:1278), DCRALNALGK
(SEQ ID NO:1279), DTCRALK (SEQ ID NO:1280), DTCRALNK (SEQ ID
NO:1281), DTCRALNAK (SEQ ID NO:1282), DTCRALNAQK (SEQ ID NO:1283),
DTCRALNALK (SEQ ID NO:1284), DTCRALNAQGK (SEQ ID NO:1285),
DTCRALNALGK (SEQ ID NO:1286), DITCRALK (SEQ ID NO:1287), DITCRALNK
(SEQ ID NO:1288), DITCRALNAK (SEQ ID NO:1289), DITCRALNAQK (SEQ ID
NO:1290), DITCRALNALK (SEQ ID NO:1291), DITCRALNAQGK (SEQ ID
NO:1292), DITCRALNALGK (SEQ ID NO:1293), KRALD (SEQ ID NO:1028),
KRALND (SEQ ID NO:1029), KRALNAD (SEQ ID NO:1294), KRALNAQD (SEQ ID
NO:1295), KRALNALD (SEQ ID NO:1296), KRALNAQGD (SEQ ID NO:1297),
KRALNALGD (SEQ ID NO:1298), KCRALD (SEQ ID NO:1034), KCRALND (SEQ
ID NO:1035), KCRALNAD (SEQ ID NO:1299), KCRALNAQD (SEQ ID NO:1300),
KCRALNALD (SEQ ID NO:1301), KCRALNAQGD (SEQ ID NO:1302), KCRALNALGD
(SEQ ID NO:1303), KTCRALD (SEQ ID NO:1304), KTCRALND (SEQ ID
NO:1305), KTCRALNAD (SEQ ID NO:1306), KTCRALNAQD (SEQ ID NO:1307),
KTCRALNALD (SEQ ID NO:1308), KTCRALNAQGD (SEQ ID NO:1309),
KTCRALNALGD (SEQ ID NO:1310), KITCRALD (SEQ ID NO:1311), KITCRALND
(SEQ ID NO:1312), KITCRALNAD (SEQ ID NO:1313), KITCRALNAQD (SEQ ID
NO:1314), KITCRALNALD (SEQ ID NO:1315), KITCRALNAQGD (SEQ ID
NO:1316), KITCRALNALGD (SEQ ID NO:1317), RALNA (SEQ ID NO:1318),
RALNAQ (SEQ ID NO:1319), RALNAL (SEQ ID NO:1320), RALNAQG (SEQ ID
NO:1321), RALNALG (SEQ ID NO:1322), CRALN (SEQ ID NO:1323), CRALNA
(SEQ ID NO:1324), CRALNAQ (SEQ ID NO:1325), CRALNAL (SEQ ID
NO:1326), CRALNAQG (SEQ ID NO:1327), CRALNALG (SEQ ID NO:1328),
TCRAL (SEQ ID NO:1329), TCRALN (SEQ ID NO:1330), TCRALNA (SEQ ID
NO:1331), TCRALNAQ (SEQ ID NO:1332), TCRALNAL (SEQ ID NO:1333),
TCRALNAQG (SEQ ID NO:1334), TCRALNALG (SEQ ID NO:1335), ITCRAL (SEQ
ID NO:1336), ITCRALN (SEQ ID NO:1337), ITCRALNA (SEQ ID NO:1338),
ITCRALNAQ (SEQ ID NO:1339), ITCRALNAL (SEQ ID NO:1340), ITCRALNAQG
(SEQ ID NO:1341) and ITCRALNALG (SEQ ID NO:1342).
[0089] Representative cyclic peptides comprising a desmocollin-1
CAR sequence include: CYATC, CYATTC, CYATTAC, CYATTADC, CYATTADGC,
CGYATC, CGYATTC, CGYATTAC, CGYATTADC, CGYATTADGC, CAYATC, CAYATTC,
CAYATTAC, CAYATTADC, CAYATTADGC, CYGYATC, CYGYATTC, CYGYATTAC,
CYGYATTADC, CYGYATTADGC, CYAYATC, CYAYATTC, CYAYATTAC, CYAYATTADC,
CYAYATTADGC, CLYGYATC, CLYGYATTC, CLYGYATTAC, CLYGYATTADC,
CLYGYATTADGC, CLYAYATC, CLYAYATTC, CLYAYATTAC, CLYAYATTADC,
CLYAYATTADGC, CVYGYATC, CVYGYATTC, CVYGYATTAC, CVYGYATTADC,
CVYGYATTADGC, CVYAYATC, CVYAYATTC, CVYAYATTAC, CVYAYATTADC,
CVYAYATTADGC, CIYGYATC, CIYGYATTC, CIYGYATTAC, CIYGYATTADC,
CIYGYATTADGC, CIYAYATC, CIYAYATTC, CIYAYATTAC, CIYAYATTADC,
CIYAYATTADGC, EYATK, EYATTK, EYATTAK, EYATTADK, EYATTADGK, EGYATK,
EGYATTK, EGYATTAK, EGYATTADK, EGYATTADGK, EAYATK, EAYATTK,
EAYATTAK, EAYATTADK, EAYATTADGK, EYGYATK, EYGYATTK, EYGYATTAK,
EYGYATTADK, EYGYATTADGK, EYAYATK, EYAYATTK, EYAYATTAK, EYAYATTADK,
EYAYATTADGK, ELYGYATK, ELYGYATTK, ELYGYATTAK, ELYGYATTADK,
ELYGYATTADGK, ELYAYATK, ELYAYATTK, ELYAYATTAK, ELYAYATTADK,
ELYAYATTADGK, EVYGYATK, EVYGYATTK, EVYGYATTAK, EVYGYATTADK,
EVYGYATTADGK, EVYAYATK, EVYAYATTK, EVYAYATTAK, EVYAYATTADK,
EVYAYATTADGK, EIYGYATK, EIYGYATTK, EIYGYATTAK, EIYGYATTADK,
EIYGYATTADGK, EIYAYATK, EIYAYATTK, EIYAYATTAK, EIYAYATTADK,
EIYAYATTADGK, KYATE, KYATTE, KYATTAE, KYATTADE, KYATTADGE, KGYATE,
KGYATTE, KGYATTAE, KGYATTADE, KGYATTADGE, KAYATE, KAYATTE,
KAYATTAE, KAYATTADE, KAYATTADGE, KYGYATE, KYGYATTE, KYGYATTAE,
KYGYATTADE, KYGYATTADGE, KYAYATE, KYAYATTE, KYAYATTAE, KYAYATTADE,
KYAYATTADGE, KLYGYATE, KLYGYATTE, KLYGYATTAE, KLYGYATTADE,
KLYGYATTADGE, KLYAYATE, KLYAYATTE, KLYAYATTAE, KLYAYATTADE,
KLYAYATTADGE, KVYGYATE, KVYGYATTE, KVYGYATTAE, KVYGYATTADE,
KVYGYATTADGE, KVYAYATE, KVYAYATTE, KVYAYATTAE, KVYAYATTADE,
KVYAYATTADGE, KIYGYATE, KIYGYATTE, KIYGYATTAE, KIYGYATTADE,
KIYGYATTADGE, KIYAYATEi, KIYAYATTE, KIYAYATTAE, KIYAYATTADE,
KIYAYATTADGE, DYATK, DYATTK, DYATTAK, DYATTADK, DYATTADGK, DGYATK,
DGYATTK, DGYATTAK, DGYATTADK, DGYATTADGK, DAYATK, DAYATTK,
DAYATTAK, DAYATTADK, DAYATTADGK, DYGYATK, DYGYATTK, DYGYATTAK,
DYGYATTADK, DYGYATTADGK, DYAYATK, DYAYATTK, DYAYATTAK, DYAYATTADK,
DYAYATTADGK, DLYGYATK, DLYGYATTK, DLYGYATTAK, DLYGYATTADK,
DLYGYATTADGK, DLYAYATK, DLYAYATTK, DLYAYATTAK, DLYAYATTADK,
DLYAYATTADGK, DVYGYATK, DVYGYATTK, DVYGYATTAK, DVYGYATTADK,
DVYGYATTADGK, DVYAYATK, DVYAYATTK, DVYAYATTAK, DVYAYATTADK,
DVYAYATTADGK, DIYGYATK, DIYGYATTK, DIYGYATTAK, DIYGYATTADK,
DIYGYATTADGK, DIYAYATK, DIYAYATTK, DIYAYATTAK, DIYAYATTADK,
DIYAYATTADGK, KYATD, KYATTD, KYATTAD, KYATTADD, KYATTADGD, KGYATD,
KGYATTD, KGYATTAD, KGYATTADD, KGYATTADGD, KAYATD, KAYATTD,
KAYATTAD, KAYATTADD, KAYATTADGD, KYGYATD, KYGYATTD, KYGYATTAD,
KYGYATTADD, KYGYATTADGD, KYAYATD, KYAYATTD, KYAYATTAD, KYAYATTADD,
KYAYATTADGD, KLYGYATD, KLYGYATTD, KLYGYATTAD, KLYGYATTADD,
KLYGYATTADGD, KLYAYATD, KLYAYATTD, KLYAYATTAD, KLYAYATTADD,
KLYAYATTADGD, KVYGYATD, KVYGYATTD, KVYGYATTAD, KVYGYATTADD,
KVYGYATTADGD, KVYAYATD, KVYAYATTD, KVYAYATTAD, KVYAYATTADD,
KVYAYATTADGD, KIYGYATD, KIYGYATTD, KIYGYATTAD, KIYGYATTADD,
KIYGYATTADGD, KIYAYATD, KIYAYATTD, KIYAYATTAD, KIYAYATTADD,
KIYAYATTADGD, YATTA, YATTAD, YATTADG, GYATT, GYATTA, GYATTAD,
GYATTADG, AYATT, AYATTA, AYATTAD, AYATTADG, YGYAT, YGYATT, YGYATTA,
YGYATTAD, YGYATTADG, YAYAT, YAYATT, YAYATTA, YAYATTAD, YAYATTADG,
LYGYAT, LYGYATT, LYGYATTA, LYGYATTAD, LYGYATTADG, LYAYAT, LYAYATT,
LYAYATTA, LYAYATTAD, LYAYATTADG, VYGYAT, VYGYATT, VYGYATTA,
VYGYATTAD, VYGYATTADG, VYAYAT, VYAYATT, VYAYATTA, VYAYATTAD,
VYAYATTADG, IYGYAT, IYGYATT, IYGYATTA, IYGYATTAD, IYGYATTADG,
IYAYAT, IYAYATT, IYAYATTA, IYAYATTAD and IYAYATTADG.
[0090] Representative cyclic peptides comprising a desmocollin-2
CAR sequence include: CFATC, CFATTC, CFATTPC, CFATTPDC, CFATTPDGC,
CAFATC, CAFATTC, CAFATTPC, CAFATTPDC, CAFATTPDGC, CIAFATC,
CIAFATTC, CIAFATTPC, CIAFATTPDC, CIAFATTPDGC, CIIAFATC, CIIAFATTC,
CIIAFATTPC, CIIAFATTPDC, CIIAFATTPDGC, CLIAFATC, CLIAFATTC,
CLIAFATTPC, CLIAFATTPDC, CLIAFATTPDGC, EFATK, EFATTK, EFATTPK,
EFATTPDK, EFATTPDGK, EAFATK, EAFATTK, EAFATTPK, EAFATTPDK,
EAFATTPDGK, EIAFATK, EIAFATTK, EIAFATTPK, EIAFATTPDK, EIAFATTPDGK,
EIIAFATK, EIIAFATTK, EIIAFATTPK, EIIAFATTPDK, EIIAFATTPDGK,
ELIAFATK, ELIAFATTK, ELIAFATTPK, ELIAFATTPDK, ELIAFATTPDGK, KFATE,
KFATTE, KFATTPE, KFATTPDE, KFATTPDGEi, KAFATE, KAFATTE, KAFATTPE,
KAFATTPDE, KAFATTPDGE, KIAFATE, KIAFATTE, KIAFATTPE, KIAFATTPDE,
KIAFATTPDGE, KIIAFATE, KIIAFATTE, KIIAFATTPE, KIIAFATTPDE,
KIIAFATTPDGE, KLIAFATE, KLIAFATTE, KLIAFATTPE, KLIAFATTPDE,
KLIAFATTPDGE, DFATK, DFATTK, DFATTPK, DFATTPDK, DFATTPDGK, DAFATK,
DAFATTK, DAFATTPK, DAFATTPDK, DAFATTPDGK, DIAFATK, DIAFATTK,
DIAFATTPK, DIAFATTPDK, DIAFATTPDGK, DIIAFATK, DIIAFATTK,
DIIAFATTPK, DIIAFATTPDK, DIIAFATTPDGK, DLIAFATK, DLIAFATTK,
DLIAFATTPK, DLIAFATTPDK, DLIAFATTPDGK, KFATD, KFATTD, KFATTPD,
KFATTPDD, KFATTPDGD, KAFATD, KAFATTD, KAFATTPD, KAFATTPDD,
KAFATTPDGD, KIAFATD, KIAFATTD, KIAFATTPD, KIAFATTPDD, KIAFATTPDGD,
KIIAFATD, KIIAFATTD, KIIAFATTPD, KIIAFATTPDD, KIIAFATTPDGD,
KLIAFATD, KLIAFATTD, KLIAFATTPD, KLIAFATTPDD, KLIAFATTPDGD, FATTP,
FATTPD, FATTPDG, AFATT, AFATTP, AFATTPD, AFATTPDG, IAFAT, IAFATT,
IAFATTP, IAFATTPD, IAFATTPDG, IIAFAT, IIAFATT, IIAFATTP, IIAFATTPD,
IIAFATTPDG, LIAFAT, LIAFATT, LIAFATTP, LIAFATTPD, LIAFATTPDG.
[0091] Representative cyclic peptides comprising a desmocollin-3 or
desmocollin-4 CAR sequence include: CYASC, CYASTC, CYASTAC,
CYASTADC, CYASTADGC, CAYASC, CAYASTC, CAYASTAC, CAYASTADC,
CAYASTADGC, CIAYASC, CIAYASTC, CIAYASTAC, CIAYASTADC, CIAYASTADGC,
CLIAYASC, CLIAYASTC, CLIAYASTAC, CLIAYASTADC, CLIAYASTADGC, EYASK,
EYASTK, EYASTAK, EYASTADK, EYASTADGK, EAYASK, EAYASTK, EAYASTAK,
EAYASTADK, EAYASTADGK, EIAYASK, EIAYASTK, EIAYASTAK, EIAYASTADK,
EIAYASTADGK, ELIAYASK, ELIAYASTK, ELIAYASTAK, ELIAYASTADK,
ELIAYASTADGK, KYASE, KYASTE, KYASTAE, KYASTADE, KYASTADGE, KAYASE,
KAYASTE, KAYASTAE, KAYASTADE, KAYASTADGE, KIAYASE, KIAYASTE,
KIAYASTAE, KIAYASTADE, KIAYASTADGE, KLIAYASE, KLIAYASTE,
KLIAYASTAE, KLIAYASTADE, KLIAYASTADGE, DYASK, DYASTK, DYASTAK,
DYASTADK, DYASTADGK, DAYASK, DAYASTK, DAYASTAK, DAYASTADK,
DAYASTADGK, DIAYASK, DIAYASTK, DIAYASTAK, DIAYASTADK, DIAYASTADGK,
DLIAYASK, DLIAYASTK, DLIAYASTAK, DLIAYASTADK, DLIAYASTADGK, KYASD,
KYASTD, KYASTAD, KYASTADD, KYASTADGD, KAYASD, KAYASTD, KAYASTAD,
KAYASTADD, KAYASTADGD, KIAYASD, KIAYASTD, KIAYASTAD, KIAYASTADD,
KIAYASTADGD, KLIAYASD, KLIAYASTD, KLIAYASTAD, KLIAYASTADD,
KLIAYASTADGD, YASTA, YASTAD, YASTADG, AYAST, AYASTA, AYASTAD,
AYASTADG, IAYAS, IAYAST, IAYASTA, IAYASTAD, IAYASTADG, LIAYAS,
LIAYAST, LIAYASTA, LIAYASTAD and LIAYASTADG.
[0092] As noted above, certain preferred modulating agents comprise
a peptide (containing a desmosomal cadherin CAR sequence or an
analogue thereof) in which at least one terminal amino acid residue
is modified (e.g., the N-terminal amino group is modified by, for
example, acetylation or alkoxybenzylation and/or an amide or ester
is formed at the C-terminus). It has been found, within the context
of the present invention, that the addition of at least one such
group to a linear or cyclic peptide modulating agent may improve
the ability of the agent to modulate a desmosomal cadherin-mediated
function. Certain preferred modulating agents contain modifications
at the N- and C-terminal residues, such as N-terminal acetylation
and/or C-terminal amidation.
[0093] The present invention further contemplates desmosomal
cadherin CAR sequences from other organisms. Such CAR sequences may
be identified based upon similarity to the sequences provided
herein, and the ability to modulate a desmosomal cadherin-mediated
function such as may be confirmed as described herein.
[0094] Within certain embodiments, as discussed below, cyclic
peptides that contain small CAR sequences (e.g., three residues
without significant flanking sequences) are preferred for
modulating desmosomal cadherin-mediated functions. Such peptides
may contain an N-acetyl group and a C-amide group. Small cyclic
peptides may generally be used to specifically modulate adhesion of
cancer and/or other cell types by topical administration or by
systemic administration, with or without linking a targeting agent
to the peptide, as discussed below.
[0095] Within embodiments in which inhibition of a desmosomal
cadherin-interaction is desired, a modulating agent may contain one
desmosomal cadherin CAR sequence, or multiple CAR sequences that
are adjacent to one another (i.e., without intervening sequences)
or in close proximity (i.e., separated by peptide and/or
non-peptide linkers to give a distance between the desmosomal CAR
sequences that ranges from about 0.1 to 400 nm). A linker may be
any molecule (including peptide and/or non-peptide sequences) that
does not contain a CAR sequence and that can be covalently linked
to at least two peptide sequences. Using a linker, CAR
sequence-containing peptides and other peptide or protein sequences
may be joined end-to-end (i.e., the linker may be covalently
attached to the carboxyl or amino group of each peptide sequence),
and/or via side chains. One linker that can be used for such
purposes is (H.sub.2N(CH.sub.2).sub.nCO.sub.2H), or derivatives
thereof, where n ranges from 1 to 4. Other linkers that may be used
will be apparent to those of ordinary skill in the art. Peptide and
non-peptide linkers may generally be incorporated into a modulating
agent using any appropriate method known in the art.
[0096] Within embodiments in which enhancement of cell adhesion
mediated by a desmosomal cadherin is desired, a modulating agent
may contain multiple desmosomal cadherin CAR sequences, or
antibodies that specifically bind to such sequences, joined by
linkers as described above. For enhancers of cadherin function, the
linker distance should generally be 400-10,000 nm. One linker that
can be used for such purposes is
(H.sub.2N(CH.sub.2).sub.nCO.sub.2H).sub.m, or derivatives thereof,
where n ranges from 1 to 10 and m ranges from 1 to 4000. For
example, if glycine (H.sub.2NCH.sub.2CO.sub.2H) or a multimer
thereof is used as a linker, each glycine unit corresponds to a
linking distance of 2.45 angstroms, or 0.245 nm, as determined by
calculation of its lowest energy conformation when linked to other
amino acids using molecular modeling techniques. Similarly,
aminopropanoic acid corresponds to a linking distance of 3.73
angstroms, aminobutanoic acid to 4.96 angstroms, aminopentanoic
acid to 6.30 angstroms and amino hexanoic acid to 6.12 angstroms.
Enhancement of cell adhesion may also be achieved by attachment of
multiple modulating agents to a support material, as discussed
further below.
[0097] Certain preferred modulating agents may comprise a
desmoglein CAR sequence in combination with a desmocollin CAR
sequence. When the CAR sequences are joined by linkers as described
above, such agents may be used, for example, to promote cell
adhesion in a variety of contexts. By way of example, such an agent
could comprise the dsg-2 CAR sequence YAL in combination with the
dsc-2 CAR sequence FAT.
[0098] A modulating agent as described herein may additionally
comprise one or more CAR sequences for one or more different
adhesion molecules (including, but not limited to, nonclassical
cadherins and other CAMs) and/or one or more substances, such as
antibodies or fragments thereof, that bind to such sequences.
Linkers may, but need not, be used to separate such CAR sequence(s)
and/or antibody sequence(s) from the CAR sequence(s) and/or each
other. Such modulating agents may generally be used within methods
in which it is desirable to simultaneously disrupt a function
mediated by multiple adhesion molecules. As used herein, an
"adhesion molecule" is any molecule that mediates cell adhesion via
a receptor on a cell's surface. Adhesion molecules include cell
adhesion proteins (e.g., other members of the cadherin gene
superfamily, such as N-cadherin and E-cadherin); integrins;
extracellular matrix proteins such as laminin, fibronectin,
collagens, vitronectin, entactin and tenascin; and members of the
immunoglobulin supergene family, such as N-CAM. Preferred CAR
sequences for inclusion within a modulating agent include the
classical cadherin CAR sequence His-Ala-Val (HAV); Arg-Gly-Asp
(RGD), which is bound by integrins (see Cardarelli et al., J. Biol.
Chem. 267:23159-64, 1992); Tyr-Ile-Gly-Ser-Arg (YIGSR; (SEQ ID
NO:______), which is bound by .alpha.6.beta.1 integrin; KYSFNYDGSE
(SEQ ID NO:______), which is bound by N-CAM; the N-CAM heparin
sulfate-binding site IWKHKGRDVILKKDVRF (SEQ ID NO:______); the
putative claudin CAR sequence IYSY (SEQ ID NO:______) and/or the
occludin CAR sequence LYHY (SEQ ID NO:______).
[0099] Other preferred CAR sequences for inclusion within a
modulating agent include OB-cadherin CAR sequences such as DDK,
IDDK (SEQ ID NO:______) DDKS (SEQ ID NO:______ VIDDK (SEQ ID
NO:______), IDDKS (SEQ ID NO:______), VIDDKS (SEQ ID NO:______),
DDKSG (SEQ ID NO:______), IDDKSG (SEQ ID NO:______), VIDDKSG (SEQ
ID NO:______), FVIDDK (SEQ ID NO:______), FVIDDKS (SEQ ID
NO:______), FVIDDKSG (SEQ ID NO:______), IFVIDDK (SEQ ID
NO:______), IFVIDDKS (SEQ ID NO:______), IFVIDDKSG (SEQ ID
NO:______), EEY, IEEY (SEQ ID NO:______), EEYT (SEQ ID NO:______),
VIEEY (SEQ ID NO:______), IEEYT (SEQ ID NO:______), VIEEYT (SEQ ID
NO:______), EEYTG (SEQ ID NO:______), IEEYTG (SEQ ID NO:______),
VIEEYTG (SEQ ID NO:______), FVIEEY (SEQ ID NO:______), FVIEEYT (SEQ
ID NO:______), FVIEEYTG (SEQ ID NO:______), FFVIEEY (SEQ ID
NO:______), FFVIEEYT (SEQ ID NO:______), FFVIEEYTG (SEQ ID
NO:______), EAQ, VEAQ (SEQ ID NO:______), EAQT (SEQ ID NO:______),
SVEAQ (SEQ ID NO:______), VEAQT (SEQ ID NO:______), SVEAQT (SEQ ID
NO:______), EAQTG (SEQ ID NO:______), VEAQTG (SEQ ID NO:______),
SVEAQTG (SEQ ID NO:______), FSVEAQ (SEQ ID NO:______), FSVEAQT (SEQ
ID NO:______), FSVEAQTG (SEQ ID NO:______), YFSVEAQ (SEQ ID
NO:______), YFSVEAQT (SEQ ID NO:______) and YFSVEAQTG (SEQ ID
NO:______).
[0100] Still further CAR sequences that may be included in a
modulating agent are cadherin-5 CAR sequences such as DAE, VDAE
(SEQ ID NO:______), DAET (SEQ ID NO:______), RVDAE (SEQ ID
NO:______), VDAET (SEQ ID NO:______), RVDAET (SEQ ID NO:______),
DAETG (SEQ ID NO:______), VDAETG (SEQ ID NO:______), RVDAETG (SEQ
ID NO:______), FRVDAE (SEQ ID NO:______), FRVDAET (SEQ ID
NO:______), FRVDAETG (SEQ ID NO:______), VFRVDAE (SEQ ID
NO:______), VFRVDAET (SEQ ID NO:______) and VFRVDAETG (SEQ ID
NO:______).
[0101] Using linkers, such modulating agents may form linear or
branched structures. Within one embodiment, modulating agents
having a branched structure comprise four different CAR sequences,
such as IFVIDDKSG (SEQ ID NO:______), RGD, YIGSR (SEQ ID NO:______)
and HAV. Bi-functional modulating agents that comprise a desmosomal
cadherin CAR sequence joined via a linker to a classical cadherin
CAR sequence are also preferred for certain embodiments. As noted
above, linkers preferably produce a distance between CAR sequences
ranging from 0.1 to 10,000 nm, more preferably ranging from 0.1-400
nm. A separation distance between recognition sites may generally
be determined according to the desired function of the modulating
agent.
[0102] The total number of CAR sequences (including the desmosomal
cadherin CAR sequence, with or without other CAR sequences derived
from one or more different adhesion molecules) present within a
modulating agent may range from 1 to a large number, such as 100,
preferably from 1 to 10, and more preferably from 1 to 5. Peptide
modulating agents comprising multiple CAR sequences typically
contain from 6 to about 1000 amino acid residues, preferably from 6
to 50 residues. When non-peptide linkers are employed, each CAR
sequence of the modulating agent is present within a peptide that
generally ranges in size from 3 to 50 residues in length,
preferably from 4 to 25 residues, and more preferably from 5 to 15
residues.
[0103] As noted above, modulating agents may be polypeptides or
salts thereof, containing only amino acid residues linked by
peptide bonds, or may contain non-peptide regions, such as linkers.
Peptide regions of a modulating agent may comprise residues of
L-amino acids, D-amino acids, or any combination thereof. Amino
acids may be from natural or non-natural sources, provided that at
least one amino group and at least one carboxyl group are present
in the molecule; .alpha.- and .beta.-amino acids are generally
preferred. The 20 L-amino acids commonly found in proteins are
identified herein by the conventional three-letter or one-letter
abbreviations, and the corresponding D-amino acids are designated
by a lower case one letter symbol.
[0104] A modulating agent may also contain rare amino acids (such
as 4-hydroxyproline or hydroxylysine), organic acids or amides
and/or derivatives of common amino acids, such as amino acids
having the C-terminal carboxylate esterified (e.g., benzyl, methyl
or ethyl ester) or amidated and/or having modifications of the
N-terminal amino group (e.g., acetylation or alkoxycarbonylation),
with or without any of a wide variety of side-chain modifications
and/or substitutions (e.g., methylation, benzylation, t-butylation,
tosylation, alkoxycarbonylation, and the like). Preferred
derivatives include amino acids having a C-terminal amide group.
Residues other than common amino acids that may be present with a
modulating agent include, but are not limited to,
2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric
acid, .alpha.-aminoadipic acid, m-aminomethylbenzoic acid and
.alpha.,.beta.-diaminopropionic acid.
[0105] Peptide modulating agents (and peptide portions of
modulating agents) as described herein may be synthesized by
methods well known in the art, including chemical synthesis and
recombinant DNA methods. For modulating agents up to about 50
residues in length, chemical synthesis may be performed using
solution or solid phase peptide synthesis techniques, in which a
peptide linkage occurs through the direct condensation of the
.alpha.-amino group of one amino acid with the .alpha.-carboxy
group of the other amino acid with the elimination of a water
molecule. Peptide bond synthesis by direct condensation, as
formulated above, requires suppression of the reactive character of
the amino group of the first and of the carboxyl group of the
second amino acid. The masking substituents must permit their ready
removal, without inducing breakdown of the labile peptide
molecule.
[0106] In solution phase synthesis, a wide variety of coupling
methods and protecting groups may be used (see Gross and
Meienhofer, eds., "The Peptides: Analysis, Synthesis, Biology,"
Vol. 1-4 (Academic Press, 1979); Bodansky and Bodansky, "The
Practice of Peptide Synthesis," 2d ed. (Springer Verlag, 1994)). In
addition, intermediate purification and linear scale up are
possible. Those of ordinary skill in the art will appreciate that
solution synthesis requires consideration of main chain and side
chain protecting groups and activation method. In addition, careful
segment selection is necessary to minimize racemization during
segment condensation. Solubility considerations are also a
factor.
[0107] Solid phase peptide synthesis uses an insoluble polymer for
support during organic synthesis. The polymer-supported peptide
chain permits the use of simple washing and filtration steps
instead of laborious purifications at intermediate steps.
Solid-phase peptide synthesis may generally be performed according
to the method of Merrifield et al., J. Am. Chem. Soc. 85:2149,
1963, which involves assembling a linear peptide chain on a resin
support using protected amino acids. Solid phase peptide synthesis
typically utilizes either the Boc or Fmoc strategy. The Boc
strategy uses a 1% cross-linked polystyrene resin. The standard
protecting group for .alpha.-amino functions is the
tert-butyloxycarbonyl (Boc) group. This group can be removed with
dilute solutions of strong acids such as 25% trifluoroacetic acid
(TFA). The next Boc-amino acid is typically coupled to the amino
acyl resin using dicyclohexylcarbodiimide (DCC). Following
completion of the assembly, the peptide-resin is treated with
anhydrous HF to cleave the benzyl ester link and liberate the free
peptide. Side-chain functional groups are usually blocked during
synthesis by benzyl-derived blocking groups, which are also cleaved
by HF. The free peptide is then extracted from the resin with a
suitable solvent, purified and characterized. Newly synthesized
peptides can be purified, for example, by gel filtration, HPLC,
partition chromatography and/or ion-exchange chromatography, and
may be characterized by, for example, mass spectrometry or amino
acid sequence analysis. In the Boc strategy, C-terminal amidated
peptides can be obtained using benzhydrylamine or
methylbenzhydrylamine resins, which yield peptide amides directly
upon cleavage with HF.
[0108] In the procedures discussed above, the selectivity of the
side-chain blocking groups and of the peptide-resin link depends
upon the differences in the rate of acidolytic cleavage. Orthoganol
systems have been introduced in which the side-chain blocking
groups and the peptide-resin link are completely stable to the
reagent used to remove the .alpha.-protecting group at each step of
the synthesis. The most common of these methods involves the
9-fluorenylmethyloxycarbonyl (Fmoc) approach. Within this method,
the side-chain protecting groups and the peptide-resin link are
completely stable to the secondary amines used for cleaving the
N-.alpha.-Fmoc group. The side-chain protection and the
peptide-resin link are cleaved by mild acidolysis. The repeated
contact with base makes the Merrifield resin unsuitable for Fmoc
chemistry, and p-alkoxybenzyl esters linked to the resin are
generally used. Deprotection and cleavage are generally
accomplished using TFA.
[0109] Those of ordinary skill in the art will recognize that, in
solid phase synthesis, deprotection and coupling reactions must go
to completion and the side-chain blocking groups must be stable
throughout the entire synthesis. In addition, solid phase synthesis
is generally most suitable when peptides are to be made on a small
scale.
[0110] Acetylation of the N-terminus can be accomplished by
reacting the final peptide with acetic anhydride before cleavage
from the resin. C-amidation is accomplished using an appropriate
resin such as methylbenzhydrylamine resin using the Boc technology.
Following synthesis of a linear peptide, with or without
N-acetylation and/or C-amidation, cyclization may be achieved if
desired by any of a variety of techniques well known in the art.
Within one embodiment, a bond may be generated between reactive
amino acid side chains. For example, a disulfide bridge may be
formed from a linear peptide comprising two thiol-containing
residues by oxidizing the peptide using any of a variety of
methods. Within one such method, air oxidation of thiols can
generate disulfide linkages over a period of several days using
either basic or neutral aqueous media. The peptide is used in high
dilution to minimize aggregation and intermolecular side reactions.
This method suffers from the disadvantage of being slow but has the
advantage of only producing H.sub.2O as a side product.
Alternatively, strong oxidizing agents such as 12 and
K.sub.3Fe(CN).sub.6 can be used to form disulfide linkages. Those
of ordinary skill in the art will recognize that care must be taken
not to oxidize the sensitive side chains of Met, Tyr, Trp or His.
Cyclic peptides produced by this method require purification using
standard techniques, but this oxidation is applicable at acid pHs.
Oxidizing agents also allow concurrent deprotection/oxidation of
suitable S-protected linear precursors to avoid premature,
nonspecific oxidation of free cysteine.
[0111] DMSO, unlike I.sub.2 and K.sub.3Fe(CN).sub.6, is a mild
oxidizing agent which does not cause oxidative side reactions of
the nucleophilic amino acids mentioned above. DMSO is miscible with
H.sub.2O at all concentrations, and oxidations can be performed at
acidic to neutral pHs with harmless byproducts.
Methyltrichlorosilane-diphenylsulfoxide may alternatively be used
as an oxidizing agent, for concurrent deprotection/oxidation of
S-Acm, S-Tacm or S-t-Bu of cysteine without affecting other
nucleophilic amino acids. There are no polymeric products resulting
from intermolecular disulfide bond formation. Suitable
thiol-containing residues for use in such oxidation methods
include, but are not limited to, cysteine, .beta.,.beta.-dimethyl
cysteine (penicillamine or Pen), .beta.,.beta.-tetramethylene
cysteine (Tmc), .beta.,.beta.-pentamethylene cysteine (Pmc),
.beta.-mercaptopropionic acid (Mpr),
.beta.,.beta.-pentamethylene-.beta.-mercaptopropionic acid (Pmp),
2-mercaptobenzene, 2-mercaptoaniline and 2-mercaptoproline.
Peptides containing such residues are illustrated by the following
representative formulas, in which the desmosomal cadherin is a
desmoglein, the underlined portion is cyclized, N-acetyl groups are
indicated by N-Ac and C-terminal amide groups are represented by
--NH.sub.2:
2 i) N-Ac-Cys-Asn-Gln-Lys-Cys-NH.sub.2 (SEQ ID NO:_) ii)
N-Ac-Cys-Ile-Asn-Gln-Lys-Thr-Gly-Cys-NH.sub.2 (SEQ ID NO:_) iii)
N-Ac-Cys-Ile-Asn-Gln-Lys-Cys-NH.sub.2 (SEQ ID NO:_) iv)
N-Ac-Cys-Asn-Gln-Lys-Thr-Cys-NH.sub.2 (SEQ ID NO:_) v)
N-Ac-Cys-Ile-Asn-Gln-Lys-Thr-Cys-NH.sub.2 (SEQ ID NO:_) vi)
N-Ac-Cys-Asn-Gln-Lys-Thr-Cys-OH (SEQ ID NO:_) vii)
H-Cys-Ile-Asn-Gln-Lys-Thr-Cys-NH.sub.2 (SEQ ID NO:_) viii)
N-Ac-Cys-Asn-Gln-Lys-Pen-NH.sub.2 (SEQ ID NO:_) ix)
N-Ac-Cys-Phe-Val-Ile-Asn-Gln-Lys-Thr-Gly-Cys-NH.sub.2 (SEQ ID NO:_)
x) N-Ac-Cys-Ile-Phe-Val-Ile-Asn-Gln-Lys-Thr-Gly-Cys-NH.sub.2 (SEQ
ID NO:_) xi) N-Ac-Ile-Tmc-Val-Ile-Asn-Gln-Lys-Thr-Cy-
s-Glu-NH.sub.2 (SEQ ID NO:_) xii) N-Ac-Ile-Pmc-Val-Ile-Asn-
-Gln-Lys-Thr-Gly-Cys-NH.sub.2 (SEQ ID NO:_) xiii)
Mpr-Val-Ile-Asn-Gln-Lys-Thr-Gly-Cys-NH.sub.2 (SEQ ID NO:_) xiv)
Pmp-Val-Ile-Asn-Gln-Lys-Thr-Gly-Cys-NH.sub.2 (SEQ ID NO:_)
[0112] It will be readily apparent to those of ordinary skill in
the art that, within each of these representative formulas, any of
the above thiol-containing residues may be employed in place of one
or both of the thiol-containing residues recited. Similar formulas
comprising different nonclassical cadherin CAR sequences may be
generated by those of ordinary skill in the art, based on the CAR
sequences provided herein.
[0113] Within another embodiment, cyclization may be achieved by
amide bond formation. For example, a peptide bond may be formed
between terminal functional groups (i.e., the amino and carboxy
termini of a linear peptide prior to cyclization). One such cyclic
peptide comprising a desmoglein CAR sequence is INQKTG (SEQ ID
NO:______) with or without an N-terminal acetyl group and/or a
C-terminal amide. Within another such embodiment, the linear
peptide comprises a D-amino acid (e.g., NQKtS; SEQ ID NO:______).
Alternatively, cyclization may be accomplished by linking one
terminus and a residue side chain or using two side chains, as in
KNQKD (SEQ ID NO:______) or KINQKTGD (SEQ ID NO:______), with or
without an N-terminal acetyl group and/or a C-terminal amide.
Residues capable of forming a lactam bond include lysine, ornithine
(Orn), .alpha.-amino adipic acid, m-aminomethylbenzoic acid,
.alpha.,.beta.-diaminopropionic acid, glutamate or aspartate.
[0114] Methods for forming amide bonds are well known in the art
and are based on well established principles of chemical
reactivity. Within one such method, carbodiimide-mediated lactam
formation can be accomplished by reaction of the carboxylic acid
with DCC, DIC, EDAC or DCCl, resulting in the formation of an
O-acylurea that can be reacted immediately with the free amino
group to complete the cyclization. The formation of the inactive
N-acylurea, resulting from O.fwdarw.N migration, can be
circumvented by converting the O-acylurea to an active ester by
reaction with an N-hydroxy compound such as 1-hydroxybenzotriazole,
1-hydroxysuccinimide, 1-hydroxynorbornene carboxamide or ethyl
2-hydroximino-2-cyanoacetate. In addition to minimizing O.fwdarw.N
migration, these additives also serve as catalysts during
cyclization and assist in lowering racemization. Alternatively,
cyclization can be performed using the azide method, in which a
reactive azide intermediate is generated from an alkyl ester via a
hydrazide. Hydrazinolysis of the terminal ester necessitates the
use of a t-butyl group for the protection of side chain carboxyl
functions in the acylating component. This limitation can be
overcome by using diphenylphosphoryl acid (DPPA), which furnishes
an azide directly upon reaction with a carboxyl group. The slow
reactivity of azides and the formation of isocyanates by their
disproportionation restrict the usefulness of this method. The
mixed anhydride method of lactam formation is widely used because
of the facile removal of reaction by-products. The anhydride is
formed upon reaction of the carboxylate anion with an alkyl
chloroformate or pivaloyl chloride. The attack of the amino
component is then guided to the carbonyl carbon of the acylating
component by the electron donating effect of the alkoxy group or by
the steric bulk of the pivaloyl chloride t-butyl group, which
obstructs attack on the wrong carbonyl group. Mixed anhydrides with
phosphoric acid derivatives have also been successfully used.
Alternatively, cyclization can be accomplished using activated
esters. The presence of electron withdrawing substituents on the
alkoxy carbon of esters increases their susceptibility to
aminolysis. The high reactivity of esters of p-nitrophenol,
N-hydroxy compounds and polyhalogenated phenols has made these
"active esters" useful in the synthesis of amide bonds. The last
few years have witnessed the development of
benzotriazolyloxytris-(dimethylamino)phosphonium
hexafluorophosphonate (BOP) and its congeners as advantageous
coupling reagents. Their performance is generally superior to that
of the well established carbodiimide amide bond formation
reactions.
[0115] Within a further embodiment, a thioether linkage may be
formed between the side chain of a thiol-containing residue and an
appropriately derivatized .alpha.-amino acid. By way of example, a
lysine side chain can be coupled to bromoacetic acid through the
carbodiimide coupling method (DCC, EDAC) and then reacted with the
side chain of any of the thiol containing residues mentioned above
to form a thioether linkage. In order to form dithioethers, any two
thiol containing side-chains can be reacted with dibromoethane and
diisopropylamine in DMF. Examples of thiol-containing linkages are
shown below: 3
[0116] Cyclization may also be achieved using
.delta..sub.1,.delta..sub.1-- Ditryptophan (e.g.,
Ac-Trp-Asn-Gln-Lys-Trp-OMe) (SEQ ID NO:______).
[0117] The structures and formulas recited herein are provided
solely for the purpose of illustration, and are not intended to
limit the scope of the cyclic peptides described herein.
[0118] For longer modulating agents, recombinant methods are
preferred for synthesis. Within such methods, all or part of a
modulating agent can be synthesized in living cells, using any of a
variety of expression vectors known to those of ordinary skill in
the art to be appropriate for the particular host cell. Suitable
host cells may include bacteria, yeast cells, mammalian cells,
insect cells, plant cells, algae and other animal cells (e.g.,
hybridoma, CHO, myeloma). The DNA sequences expressed in this
manner may encode portions of a desmosomal cadherin or other
adhesion molecule, or may encode a peptide comprising a desmosomal
cadherin analogue or an antibody fragment that specifically binds
to a desmosomal cadherin CAR sequence. Such DNA sequences may be
prepared based on known cDNA or genomic sequences, or from
sequences isolated by screening an appropriate library with probes
designed based on the sequences of known desmosomal cadherins. Such
screens may generally be performed as described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratories, Cold Spring Harbor, N.Y., 1989 (and references cited
therein). Polymerase chain reaction (PCR) may also be employed,
using oligonucleotide primers in methods well known in the art, to
isolate nucleic acid molecules encoding all or a portion of an
endogenous adhesion molecule. To generate a nucleic acid molecule
encoding a desired modulating agent, an endogenous cadherin
sequence may be modified using well known techniques. For example,
portions encoding one or more CAR sequences may be joined, with or
without separation by nucleic acid regions encoding linkers, as
discussed above. Alternatively, portions of the desired nucleic
acid sequences may be synthesized using well known techniques, and
then ligated together to form a sequence encoding the modulating
agent.
[0119] As noted above, polynucleotides may also function as
modulating agents. In general, such polynucleotides should be
formulated to permit expression of a polypeptide modulating agent
following administration to a mammal. Such formulations are
particularly useful for therapeutic purposes, as described below.
Those of ordinary skill in the art will appreciate that there are
many ways to achieve expression of a polynucleotide within a
mammal, and any suitable method may be employed. For example, a
polynucleotide may be incorporated into a viral vector such as, but
not limited to, adenovirus, adeno-associated virus, retrovirus, or
vaccinia or other pox virus (e.g., avian pox virus). Techniques for
incorporating DNA into such vectors are well known to those of
ordinary skill in the art. A retroviral vector may additionally
transfer or incorporate a gene for a selectable marker (to aid in
the identification or selection of transfected cells) and/or a
targeting moiety, such as a gene that encodes a ligand for a
receptor on a specific target cell, to render the vector target
specific. Targeting may also be accomplished using an antibody, by
methods known to those of ordinary skill in the art. Other
formulations for polynucleotides for therapeutic purposes include
colloidal dispersion systems, such as macromolecule complexes,
nanocapsules, microspheres, beads, and lipid-based systems
including oil-in-water emulsions, micelles, mixed micelles, and
liposomes. A preferred colloidal system for use as a delivery
vehicle in vitro and in vivo is a liposome (i.e., an artificial
membrane vesicle). The preparation and use of such systems is well
known in the art.
[0120] As noted above, a modulating agent may additionally, or
alternatively, comprise a substance such as an antibody or
antigen-binding fragment thereof, that specifically binds to a
desmosomal cadherin CAR sequence. As used herein, a substance is
said to "specifically bind" to a desmosomal cadherin CAR sequence
(with or without flanking amino acids) if it reacts at a detectable
level with a peptide containing that sequence, and does not react
detectably with peptides containing a different CAR sequence or a
sequence in which the order of amino acid residues in the cadherin
CAR sequence and/or flanking sequence is altered. Such antibody
binding properties may generally be assessed using an ELISA, which
may be readily performed by those of ordinary skill in the art and
is described, for example, by Newton et al., Develop. Dynamics
197:1-13, 1993.
[0121] Polyclonal and monoclonal antibodies may be raised against a
desmosomal cadherin CAR sequence using conventional techniques.
See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1988. In one such technique, an immunogen
comprising the CAR sequence is initially injected into any of a
wide variety of mammals (e.g., mice, rats, rabbits, sheep or
goats). The smaller immunogens (i.e., less than about 20 amino
acids) should be joined to a carrier protein, such as bovine serum
albumin or keyhole limpet hemocyanin. Following one or more
injections, the animals are bled periodically. Polyclonal
antibodies specific for the CAR sequence may then be purified from
such antisera by, for example, affinity chromatography using the
modulating agent or antigenic portion thereof coupled to a suitable
solid support.
[0122] Monoclonal antibodies specific for a desmosomal cadherin
sequence may be prepared, for example, using the technique of
Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and
improvements thereto. Briefly, these methods involve the
preparation of immortal cell lines capable of producing antibodies
having the desired specificity from spleen cells obtained from an
animal immunized as described above. The spleen cells are
immortalized by, for example, fusion with a myeloma cell fusion
partner, preferably one that is syngeneic with the immunized
animal. Single colonies are selected and their culture supernatants
tested for binding activity against the modulating agent or
antigenic portion thereof. Hybridomas having high reactivity and
specificity are preferred.
[0123] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies, with or without the use of various
techniques known in the art to enhance the yield. Contaminants may
be removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation and extraction.
Antibodies having the desired activity may generally be identified
using immunofluorescence analyses of tissue sections, cell or other
samples where the target cadherin is localized.
[0124] Within certain embodiments, the use of antigen-binding
fragments of antibodies may be preferred. Such fragments include
Fab fragments, which may be prepared using standard techniques.
Briefly, immunoglobulins may be purified from rabbit serum by
affinity chromatography on Protein A bead columns (Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988; see especially page 309) and digested by papain to yield Fab
and Fc fragments. The Fab and Fc fragments may be separated by
affinity chromatography on protein A bead columns (Harlow and Lane,
1988, pages 628-29).
[0125] Evaluation of Modulating Agent Activity
[0126] Modulating agents as described above are capable of
modulating one or more desmosomal cadherin-mediated functions. An
initial screen for such activity may be performed by evaluating the
ability of a modulating agent to bind to a desmosomal cadherin
using any binding assay known to those of ordinary skill in the
art. For example, a Pharmacia Biosensor machine may be used, as
discussed in Jonsson et al., Biotechniques 11:520-27, 1991. For
example, a modulating agent may comprise a CAR sequence that binds
to a desmosomal cadherin. A specific example of a technology that
measures the interaction of peptides with molecules can be found in
Williams et al., J. Biol. Chem. 272, 22349-22354, 1997.
Alternatively, real-time BIA (Biomolecular Interaction Analysis)
uses the optical phenomenon surface plasmon resonance to monitor
biomolecular interactions. The detection depends upon changes in
the mass concentration of macromolecules at the biospecific
interface, which in turn depends upon the immobilization of test
molecule or peptide (referred to as the ligand) to the surface of a
Biosensor chip, followed by binding of the interacting molecule
(referred to as the analyte) to the ligand. Binding to the chip is
measured in real-time in arbitrary units of resonance (RU).
[0127] By way of example, surface plasmon resonance experiments may
be carried out using a BIAcore X.TM. Biosensor (Pharmacia Ltd.,
BIAcore, Uppsala, Sweden). Parallel flow cells of CM 5 sensor chips
may be derivatized, using the amine coupling method, with
streptavidin (200 .mu.g/ml) in 10 mM Sodium Acetate, pH 4.0,
according to the manufacturer's protocol. Approximately 2100-2600
resonance units (RU) of ligand may be immobilized, corresponding to
a concentration of about 2.1-2.6 ng/mm.sup.2. The chips may then
coated be with desmosomal cadherin derivatized to biotin. Any
non-specifically bound protein is removed.
[0128] To determine binding, test analytes (e.g., peptides
containing a desmosomal cadherin CAR sequence) may be placed in
running buffer and passed simultaneously over test and control flow
cells. After a period of free buffer flow, any analyte remaining
bound to the surface may be removed with, for example, a pulse of
0.1% SDS bringing the signal back to baseline. Specific binding to
the derivatized sensor chips may be determined automatically by the
system by subtraction of test from control flow cell responses. In
general, a modulating agent binds to a desmosomal cadherin at a
detectable level within such as assay. The level of binding is
preferably at least that observed for the full length desmosomal
cadherin under similar conditions.
[0129] The ability to modulate a desmosomal cadherin-mediated
function may be evaluated using any of a variety of in vitro assays
designed to measure the effect of the peptide on a response that is
generally mediated by the desmosomal cadherin. As noted above,
modulating agents may be capable of enhancing or inhibiting a
desmosomal cadherin-mediated function.
[0130] Certain desmosomal cadherins are associated with adhesion of
particular cell types (e.g., epithelial cells). The ability of an
agent to modulate cell adhesion may generally be evaluated in vitro
by assaying the effect on adhesion between appropriate cells. In
general, a modulating agent is an inhibitor of cell adhesion if
contact of the test cells with the modulating agent results in a
discernible disruption of cell adhesion. Modulating agents that
enhance cell adhesion (e.g., agents comprising multiple desmosomal
cadherin CAR sequences and/or desmosomal cadherin CAR sequences
linked to a support material) are considered to be modulators of
cell adhesion if they are capable of promoting cell adhesion, as
judged by plating assays to assess cell adhesion to a modulating
agent attached to a support material, such as tissue culture
plastic.
[0131] Within certain cell adhesion assays, the addition of a
modulating agent to cells that express a desmosomal cadherin
results in disruption of cell adhesion. A "desmosomal
cadherin-expressing cell," as used herein, may be any type of cell
that expresses a desmosomal cadherin at a detectable level, using
standard techniques such as immunocytochemical protocols (e.g.,
Blaschuk and Farookhi, Dev. Biol. 136:564-567, 1989). For example,
such cells may be plated under standard conditions that, in the
absence of modulating agent, permit cell adhesion. In the presence
of modulating agent (e.g., 1 mg/mL), disruption of cell adhesion
may be determined visually within 24 hours, by observing retraction
of the cells from one another and the substratum.
[0132] Suitable cells for use within such assays may be any of a
variety of cells that express the desmosomal cadherin of interest.
Certain cells express one or more cadherins endogenously. In
general, MDCK cells or keratinocytes may be used to evaluate
desmocollin- or desmoglein-mediated cell adhesion. It will be
apparent that other cells may also be used within such assays,
provided that the cells express the desmosomal cadherin of
interest.
[0133] Alternatively, cells that do not naturally express a
cadherin may be used within such assays. Such cells may be stably
transfected with a polynucleotide (e.g., a cDNA) encoding one or
more cadherins of interest, such that the cadherin or cadherins are
expressed on the surface of the cell. For example, as noted above,
both a desmoglein and a desmocollin may be required for optimal
cell adhesion, and such assays may be performed using cells
transformed with polynucleotides encoding both of these desmosomal
cadherins. Expression of the cadherin(s) may be confirmed by
assessing adhesion of the transfected cells, in conjunction with
immunocytochemical techniques using antibodies directed against the
cadherin of interest. The stably transfected cells that aggregate,
as judged by light microscopy, following transfection express
sufficient levels of the desmosomal cadherin(s). Preferred cells
for use in such assays include L cells, which do not detectably
adhere and do not express any cadherin (Nagafuchi et al., Nature
329:341-343, 1987). Following transfection of L cells with a cDNA
encoding one ore more cadherins, aggregation is observed.
Modulating agents that detectably inhibit such aggregation may be
used to modulate functions mediated by the cadherin. Such assays
have been used for numerous nonclassical cadherins, including
OB-cadherin (Okazaki et al., J. Biol. Chem. 269:12092-98,1994),
cadherin-5 (Breier et al., Blood 87:630-641, 1996), cadherin-6
(Mbalaviele et al., J. Cell. Biol. 141:1467-1476,1998), cadherin-8
(Kido et al., Genomics 48:186-194, 1998), cadherin-15 (Shimoyama et
al., J. Biol. Chem. 273:10011-10018,1998), PB-cadherin (Sugimoto et
al., J. Biol. Chem. 271:11548-11556, 1996), LI-cadherin (Kreft et
al., J. Cell. Biol. 136:1109-1121, 1997), protocadherin 42 and 43
(Sano et al., EMBO J. 12:2249-2256,1993) and desmosomal cadherins
(Marcozzi et al., J. Cell. Sci. 111:495-509, 1998; Tselepis et al.,
Proc. Natl. Acad. Sci. USA 95:8064-8069, 1998). It will be apparent
to those of ordinary skill in the art that assays may be performed
in a similar manner for other cadherins.
[0134] Transfection of cells for use in cell adhesion assays may be
performed using standard techniques and published cadherin
sequences. For example, sequences of desmosomal cadherins may be
found within references cited herein and in the GenBank database.
GenBank accession numbers for certain desmosomal cadherins include:
X56654 (human desmoglein 1); Z26317 and S64273 (human desmoglein
2); X72925 (human desmocollin 1); X56807 (human desmocollin 2);
X83929 (human desmocollin 3); and D17427 (human desmocollin 4).
[0135] By way of example, an assay for evaluating a modulating
agent for the ability to inhibit a desmosomal cadherin mediated
function may evaluate the effect of a modulating agent on the
electrical resistance across a monolayer of cells. For example,
Madin Darby canine kidney (MDCK) cells can be exposed to the
modulating agent dissolved in medium (e.g., at a final
concentration of 0.5 mg/ml for a period of 24 hours). The effect on
electrical resistance can be measured using standard techniques.
This assay evaluates the effect of a modulating agent on tight
junction formation in epithelial cells. In general, the presence of
500 .mu.g/mL modulating agent should result in a statistically
significant decrease in electrical resistance after 24 hours.
[0136] Other assays involve visual evaluation of cell adhesion,
using standard methods. In general, a modulating agent that is
derived from a particular desmosomal cadherin CAR sequence (i.e.,
comprises such a CAR sequence, or an analog or mimetic thereof, or
an antibody that specifically recognizes such a CAR sequence) and
that detectably modulates adhesion of cells that express the same
desmosomal cadherin is considered to modulate a function mediated
by the desmosomal cadherin.
[0137] Other assays may be used to assess the effect of a
modulating agent on specific desmosomal cadherin-mediated
functions. For example, modulating agents that inhibit interactions
of desmogleins and desmocollins may enhance skin permeability. This
ability may be assessed by evaluating, for example, the effect of a
modulating agent on permeability of adherent epithelial and/or
endothelial cell layers (e.g., human skin). Such skin may be
derived from a natural source or may be synthetic. Human abdominal
skin for use in such assays may generally be obtained from humans
at autopsy within 24 hours of death. Briefly, a modulating agent
(e.g., 500 .mu.g/ml) and a test marker (e.g., the fluorescent
markers Oregon Green.TM. and Rhodamine Green.TM. Dextran) may be
dissolved in a sterile buffer (e.g., phosphate buffer, pH 7.2), and
the ability of the marker to penetrate through the skin and into a
receptor fluid (e.g., phosphate buffer) may be measured using a
Franz Cell apparatus (Franz, Curr. Prob. Dermatol. 7:58-68, 1978;
Franz, J. Invest. Dermatol. 64:190-195, 1975). The penetration of
the markers through the skin may be assessed at, for example, 6,
12, 24, 36, and 48 hours after the start of the experiment. In
general, a modulating agent that enhances the permeability of human
skin results in a statistically significant increase in the amount
of marker in the receptor compartment after 6-48 hours in the
presence of 500 .mu.g/mL modulating agent.
[0138] Modulating Agent Modification and Formulations
[0139] A modulating agent as described herein may, but need not, be
linked to one or more additional molecules. In particular, as
discussed below, it may be beneficial for certain applications to
link multiple modulating agents (which may, but need not, be
identical) to a support material, such as a single molecule (e.g.,
keyhole limpet hemocyanin) or a solid support, such as a polymeric
matrix (which may be formulated as a membrane or microstructure,
such as an ultra thin film), a container surface (e.g., the surface
of a tissue culture plate or the interior surface of a bioreactor),
or a bead or other particle, which may be prepared from a variety
of materials including glass, plastic or ceramics. For certain
applications, biodegradable support materials are preferred, such
as cellulose and derivatives thereof, collagen, spider silk or any
of a variety of polyesters (e.g., those derived from hydroxy acids
and/or lactones) or sutures (see U.S. Pat. No. 5,245,012). Within
certain embodiments, modulating agents and molecules comprising
other CAR sequence(s) (e.g., an HAV or RGD sequence, or an
OB-cadherin or cadherin-5 CAR sequence as described above) may be
attached to a support such as a polymeric matrix, preferably in an
alternating pattern.
[0140] Suitable methods for linking a modulating agent to a support
material will depend upon the composition of the support and the
intended use, and will be readily apparent to those of ordinary
skill in the art. Attachment may generally be achieved through
noncovalent association, such as adsorption or affinity or,
preferably, via covalent attachment (which may be a direct linkage
between a modulating agent and functional groups on the support, or
may be a linkage by way of a cross-linking agent). Attachment of a
modulating agent by adsorption may be achieved by contact, in a
suitable buffer, with a solid support for a suitable amount of
time. The contact time varies with temperature, but is generally
between about 5 seconds and 1 day, and typically between about 10
seconds and 1 hour.
[0141] Covalent attachment of a modulating agent to a molecule or
solid support may generally be achieved by first reacting the
support material with a bifunctional reagent that will also react
with a functional group, such as a hydroxyl or amino group, on the
modulating agent. For example, a modulating agent may be bound to
an appropriate polymeric support or coating using benzoquinone, by
condensation of an aldehyde group on the support with an amine and
an active hydrogen on the modulating agent or by condensation of an
amino group on the support with a carboxylic acid on the modulating
agent. A preferred method of generating a linkage is via amino
groups using glutaraldehyde. A modulating agent may be linked to
cellulose via ester linkages. Similarly, amide linkages may be
suitable for linkage to other molecules such as keyhole limpet
hemocyanin or other support materials. Multiple modulating agents
and/or molecules comprising other CAR sequences may be attached,
for example, by random coupling, in which equimolar amounts of such
molecules are mixed with a matrix support and allowed to couple at
random.
[0142] Although modulating agents as described herein may
preferentially bind to specific tissues or cells, and thus may be
sufficient to target a desired site in vivo, it may be beneficial
for certain applications to include an additional targeting agent.
Accordingly, a targeting agent may also, or alternatively, be
linked to a modulating agent to facilitate targeting to one or more
specific tissues. As used herein, a "targeting agent," may be any
substance (such as a compound or cell) that, when linked to a
modulating agent enhances the transport of the modulating agent to
a target tissue, thereby increasing the local concentration of the
modulating agent. Targeting agents include antibodies or fragments
thereof, receptors, ligands and other molecules that bind to cells
of, or in the vicinity of, the target tissue. Known targeting
agents include serum hormones, antibodies against cell surface
antigens, lectins, adhesion molecules, tumor cell surface binding
ligands, steroids, cholesterol, lymphokines, fibrinolytic enzymes
and those drugs and proteins that bind to a desired target site.
Among the many monoclonal antibodies that may serve as targeting
agents are anti-TAC, or other interleukin-2 receptor antibodies;
9.2.27 and NR-ML-05, reactive with the 250 kilodalton human
melanoma-associated proteoglycan; and NR-LU-10, reactive with a
pancarcinoma glycoprotein. An antibody targeting agent may be an
intact (whole) molecule, a fragment thereof, or a functional
equivalent thereof. Examples of antibody fragments are
F(ab').sub.2, -Fab', Fab and F[v] fragments, which may be produced
by conventional methods or by genetic or protein engineering.
Linkage is generally covalent and may be achieved by, for example,
direct condensation or other reactions, or by way of bi- or
multi-functional linkers.
[0143] For certain embodiments, it may be beneficial to also, or
alternatively, link a drug to a modulating agent. As used herein,
the term "drug" refers to any bioactive agent intended for
administration to a mammal to prevent or treat a disease or other
undesirable condition. Drugs include hormones, growth factors,
proteins, peptides and other compounds. The use of certain specific
drugs within the context of the present invention is discussed
below.
[0144] Modulating agents as described herein may be present within
a pharmaceutical composition. A pharmaceutical composition
comprises one or more modulating agents in combination with one or
more pharmaceutically or physiologically acceptable carriers,
diluents or excipients. Such compositions may comprise buffers
(e.g., neutral buffered saline or phosphate buffered saline),
carbohydrates (e.g., glucose, mannose, sucrose or dextrans),
mannitol, proteins, polypeptides or amino acids such as glycine,
antioxidants, chelating agents such as EDTA or glutathione,
adjuvants (e.g., aluminum hydroxide) and/or preservatives. Within
yet other embodiments, compositions of the present invention may be
formulated as a lyophilizate. One or more modulating agents (alone
or in combination with a targeting agent and/or drug) may, but need
not, be encapsulated within liposomes using well known technology.
Compositions of the present invention may be formulated for any
appropriate manner of administration, including for example,
topical, oral, nasal, intravenous, intracranial, intraperitoneal,
subcutaneous, or intramuscular administration.
[0145] For certain embodiments, as discussed below, a
pharmaceutical composition may further comprise a modulator of cell
adhesion that is mediated by one or more molecules other than the
particular desmosomal cadherin. Such modulators may generally be
prepared as described above, using one or more CAR sequences and/or
antibodies thereto. Such compositions are particularly useful for
situations in which it is desirable to inhibit cell adhesion
mediated by multiple cell adhesion molecules, such as other members
of the cadherin gene superfamily such as the classical cadherins
(e.g., N-cadherin and E-cadherin); nonclassical cadherins,
integrins; occludin; claudins; N-CAM and/or extracellular matrix
proteins such as laminin, fibronectin, collagens, vitronectin,
entactin and tenascin.
[0146] A pharmaceutical composition may also, or alternatively,
contain one or more drugs, which may be linked to a modulating
agent or may be free within the composition. Virtually any drug may
be administered in combination with a modulating agent as described
herein, for a variety of purposes as described below. Examples of
types of drugs that may be administered with a modulating agent
include analgesics, anesthetics, antianginals, antifungals,
antibiotics, anticancer drugs (e.g., taxol or mitomycin C),
antiinflammatories (e.g., ibuprofen and indomethacin),
anthelmintics, antidepressants, antidotes, antiemetics,
antihistamines, antihypertensives, antimalarials, antimicrotubule
agents (e.g., colchicine or vinca alkaloids), antimigraine agents,
antimicrobials, antiphsychotics, antipyretics, antiseptics,
anti-signaling agents (e.g., protein kinase C inhibitors or
inhibitors of intracellular calcium mobilization), antiarthritics,
antithrombin agents, antituberculotics, antitussives, antivirals,
appetite suppressants, cardioactive drugs, chemical dependency
drugs, cathartics, chemotherapeutic agents, coronary, cerebral or
peripheral vasodilators, contraceptive agents, depressants,
diuretics, expectorants, growth factors, hormonal agents,
hypnotics, immunosuppression agents, narcotic antagonists,
parasympathomimetics, sedatives, stimulants, sympathomimetics,
toxins (e.g., cholera toxin), tranquilizers and urinary
antiinfectives.
[0147] For imaging purposes, any of a variety of diagnostic agents
may be incorporated into a pharmaceutical composition, either
linked to a modulating agent or free within the composition.
Diagnostic agents include any substance administered to illuminate
a physiological function within a patient, while leaving other
physiological functions generally unaffected. Diagnostic agents
include metals, radioactive isotopes and radioopaque agents (e.g.,
gallium, technetium, indium, strontium, iodine, barium, bromine and
phosphorus-containing compounds), radiolucent agents, contrast
agents, dyes (e.g., fluorescent dyes and chromophores) and enzymes
that catalyze a colorimetric or fluorometric reaction. In general,
such agents may be attached using a variety of techniques as
described above, and may be present in any orientation.
[0148] The compositions described herein may be administered as
part of a sustained release formulation (i.e., a formulation such
as a capsule or sponge that effects a slow release of modulating
agent following administration). Such formulations may generally be
prepared using well known technology and administered by, for
example, oral, rectal or subcutaneous implantation, or by
implantation at the desired target site. Sustained-release
formulations may contain a modulating agent dispersed in a carrier
matrix and/or contained within a reservoir surrounded by a rate
controlling membrane (see, e.g., European Patent Application
710,491 A). Carriers for use within such formulations are
biocompatible, and may also be biodegradable; preferably the
formulation provides a relatively constant level of modulating
agent release. The amount of modulating agent contained within a
sustained release formulation depends upon the site of
implantation, the rate and expected duration of release and the
nature of the condition to be treated or prevented.
[0149] Pharmaceutical compositions of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). Appropriate dosages and a suitable duration and
frequency of administration will be determined by such factors as
the condition of the patient, the type and severity of the
patient's disease and the method of administration. In general, an
appropriate dosage and treatment regimen provides the modulating
agent(s) in an amount sufficient to provide therapeutic and/or
prophylactic benefit. Within particularly preferred embodiments of
the invention, a modulating agent or pharmaceutical composition as
described herein may be administered at a dosage ranging from 0.001
to 50 mg/kg body weight, preferably from 0.1 to 20 mg/kg, on a
regimen of single or multiple daily doses. For topical
administration, a cream typically comprises an amount of modulating
agent ranging from 0.00001% to 1%, preferably 0.0001% to 0.002%.
Fluid compositions typically contain about 10 ng/ml to 5 mg/ml,
preferably from about 10 .mu.g to 2 mg/mL modulating agent.
Appropriate dosages may generally be determined using experimental
models and/or clinical trials. In general, the use of the minimum
dosage that is sufficient to provide effective therapy is
preferred. Patients may generally be monitored for therapeutic
effectiveness using assays suitable for the condition being treated
or prevented, which will be familiar to those of ordinary skill in
the art.
[0150] Modulating Agent Methods of Use
[0151] In general, the modulating agents and compositions described
herein may be used for modulating a function, such as cell
adhesion, of desmosomal cadherin-expressing cells. Such modulation
may be performed in vitro and/or in vivo, preferably in a mammal
such as a human, using any method that contacts the desmosomal
cadherin-expressing cell with the modulating agent. As noted above,
modulating agents for purposes that involve the disruption of
desmosomal cadherin-mediated cell adhesion may comprise a
desmosomal cadherin CAR sequence, multiple desmosomal cadherin CAR
sequences in close proximity and/or a substance (such as an
antibody or an antigen-binding fragment thereof) that recognizes a
desmosomal cadherin CAR sequence. When it is desirable to also
disrupt cell adhesion mediated by other adhesion molecules, a
modulating agent may additionally comprise one or more CAR
sequences bound by such adhesion molecules (and/or antibodies or
fragments thereof that bind such sequences), preferably separated
from each other and from the desmosomal cadherin CAR sequence by
linkers. As noted above, such linkers may or may not comprise one
or more amino acids. For enhancing cell adhesion, a modulating
agent may contain multiple desmosomal cadherin CAR sequences
derived from one or more desmosomal cadherins or antibodies (or
fragments), preferably separated by linkers, and/or may be linked
to a single molecule or to a support material as described above.
When it is desirable to also enhance cell adhesion mediated by
other adhesion molecules, a modulating agent may additionally
comprise one or more CAR sequences bound by such adhesion molecules
(and/or antibodies or fragments thereof that bind such sequences),
preferably separated from each other and from the desmosomal
cadherin CAR sequence by linkers.
[0152] Certain methods involving the disruption of cell adhesion as
described herein have an advantage over prior techniques in that
they block tumor cell adhesion. As described in greater detail
below, modulating agents as described herein may also be used to
disrupt or enhance cell adhesion in a variety of other contexts.
Within each of the methods described herein, one or more modulating
agents may generally be administered alone, or within a
pharmaceutical composition. In each specific method described
herein, as noted above, a targeting agent may be employed to
increase the local concentration of modulating agent at the target
site.
[0153] Within one aspect, methods are provided in which cell
adhesion is diminished. In one such aspect, the present invention
provides methods for reducing unwanted cellular adhesion in a
mammal by administering a modulating agent as described herein.
Unwanted cellular adhesion can occur, for example, between tumor
cells, between tumor cells and normal cells or between normal cells
as a result of surgery, injury, chemotherapy, disease, inflammation
or other condition jeopardizing cell viability or function. In one
particularly preferred embodiment, a modulating agent is further
capable of disrupting cell adhesion mediated by multiple adhesion
molecules. Such an agent may comprise, in addition to one or more
desmosomal cadherin CAR sequences, CAR sequences such as the
classical cadherin CAR sequence HAV sequence, an RGD sequence,
which is bound by integrins, an OB-cadherin or cadherin-5 CAR
sequence as described above, the occludin CAR sequence LYHY (SEQ ID
NO:______); and/or the claudin CAR sequence IYSY (SEQ ID
NO:______), preferably separated from the desmosomal cadherin CAR
sequence via a linker. Alternatively, separate modulators of cell
adhesion mediated by other adhesion molecules may be administered
in conjunction with the modulating agent(s), either within the same
pharmaceutical composition or separately.
[0154] Topical administration of the modulating agent(s) is
generally preferred, but other means may also be employed.
Preferably, a fluid composition for topical administration
(comprising, for example, physiological saline) comprises an amount
of modulating agent as described above, and more preferably from 10
.mu.g/mL to 1 mg/mL. Creams may generally be formulated as
described above. Topical administration in the surgical field may
be given once at the end of surgery by irrigation of the wound or
as an intermittent or continuous irrigation with the use of
surgical drains in the post-operative period or by the use of
drains specifically inserted in an area of inflammation, injury or
disease in cases where surgery does not need to be performed.
Alternatively, parenteral or transcutaneous administration may be
used to achieve similar results.
[0155] Certain modulating agents as provided herein may be used to
facilitate transdermal drug delivery. Transdermal delivery of drugs
is a convenient and non-invasive method that can be used to
maintain relatively constant blood levels of a drug. In general, to
facilitate drug delivery via the skin, it is necessary to perturb
adhesion between the epithelial cells (keratinocytes) and the
endothelial cells of the microvasculature. Using currently
available techniques, only small, uncharged molecules may be
delivered across skin in vivo. The methods described herein are not
subject to the same degree of limitation. Accordingly, a wide
variety of drugs may be transported across the epithelial and
endothelial cell layers of skin, for systemic or topical
administration. Such drugs may be delivered to melanomas or may
enter the blood stream of the mammal for delivery to other sites
within the body.
[0156] To enhance the delivery of a drug through the skin, a
modulating agent as described herein and a drug are contacted with
the skin surface. Multifunctional modulating agents comprising
multiple nonclassical cadherin CAR sequences (e.g., a desmosomal
cadherin CAR sequence in combination with a CAR sequence derived
from OB-cadherin or cadherin-5) may also be used. Such modulating
agents may also, or alternatively, comprise the classical cadherin
CAR sequence HAV, the fibronectin CAR sequence RGD, which is
recognized by integrins, and/or the occludin CAR sequence LYHY (SEQ
ID NO:______). Alternatively, a separate modulator of cell adhesion
may be administered in conjunction with the modulating agent(s),
either within the same pharmaceutical composition or
separately.
[0157] Contact may be achieved by direct application of the
modulating agent, generally within a composition formulated as a
cream or gel, or using any of a variety of skin contact devices for
transdermal application (such as those described in European Patent
Application No. 566,816 A; U.S. Pat. No. 5,613,958; U.S. Pat. No.
5,505,956). A skin patch provides a convenient method of
administration (particularly for slow-release formulations). Such
patches may contain a reservoir of modulating agent and drug
separated from the skin by a membrane through which the drug
diffuses. Within other patch designs, the modulating agent and drug
may be dissolved or suspended in a polymer or adhesive matrix that
is then placed in direct contact with the patient's skin. The
modulating agent and drug may then diffuse from the matrix into the
skin. Modulating agent(s) and drug(s) may be contained within the
same composition or skin patch, or may be separately administered,
although administration at the same time and site is preferred. In
general, the amount of modulating agent administered via the skin
varies with the nature of the condition to be treated or prevented,
but may vary as described above. Such levels may be achieved by
appropriate adjustments to the device used, or by applying a cream
formulated as described above. Transfer of the drug across the skin
and to the target tissue may be predicted based on in vitro studies
using, for example, a Franz cell apparatus, and evaluated in vivo
by appropriate means that will be apparent to those of ordinary
skill in the art. As an example, monitoring of the serum level of
the administered drug over time provides an easy measure of the
drug transfer across the skin.
[0158] Transdermal drug delivery as described herein is
particularly useful in situations in which a constant rate of drug
delivery is desired, to avoid fluctuating blood levels of a drug.
For example, morphine is an analgesic commonly used immediately
following surgery. When given intermittently in a parenteral form
(intramuscular, intravenous), the patient usually feels sleepy
during the first hour, is well during the next 2 hours and is in
pain during the last hour because the blood level goes up quickly
after the injection and goes down below the desirable level before
the 4 hour interval prescribed for re-injection is reached.
Transdermal administration as described herein permits the
maintenance of constant levels for long periods of time (e.g.,
days), which allows adequate pain control and mental alertness at
the same time. Insulin provides another such example. Many diabetic
patients need to maintain a constant baseline level of insulin
which is different from their needs at the time of meals. The
baseline level may be maintained using transdermal administration
of insulin, as described herein. Antibiotics may also be
administered at a constant rate, maintaining adequate bactericidal
blood levels, while avoiding the high levels that are often
responsible for the toxicity (e.g., levels of gentamycin that are
too high typically result in renal toxicity).
[0159] Drug delivery by the methods of the present invention also
provides a more convenient method of drug administration. For
example, it is often particularly difficult to administer
parenteral drugs to newborns and infants because of the difficulty
associated with finding veins of acceptable caliber to catheterize.
However, newborns and infants often have a relatively large skin
surface as compared to adults. Transdermal drug delivery permits
easier management of such patients and allows certain types of care
that can presently be given only in hospitals to be given at home.
Other patients who typically have similar difficulties with venous
catheterization are patients undergoing chemotherapy or patients on
dialysis. In addition, for patients undergoing prolonged therapy,
transdermal administration as described herein is more convenient
than parenteral administration.
[0160] Transdermal administration as described herein also allows
the gastrointestinal tract to be bypassed in situations where
parenteral uses would not be practical. For example, there is a
growing need for methods suitable for administration of therapeutic
small peptides and proteins, which are typically digested within
the gastrointestinal tract. The methods described herein permit
administration of such compounds and allow easy administration over
long periods of time. Patients who have problems with absorption
through their gastrointestinal tract because of prolonged ileus or
specific gastrointestinal diseases limiting drug absorption may
also benefit from drugs formulated for transdermal application as
described herein.
[0161] Further, there are many clinical situations where it is
difficult to maintain compliance. For example, patients with mental
problems (e.g., patients with Alzheimer's disease or psychosis) are
easier to manage if a constant delivery rate of drug is provided
without having to rely on their ability to take their medication at
specific times of the day. Also patients who simply forget to take
their drugs as prescribed are less likely to do so if they merely
have to put on a skin patch periodically (e.g., every 3 days).
Patients with diseases that are without symptoms, like patients
with hypertension, are especially at risk of forgetting to take
their medication as prescribed.
[0162] For patients taking multiple drugs, devices for transdermal
application such as skin patches may be formulated with
combinations of drugs that are frequently used together. For
example, many heart failure patients are given digoxin in
combination with furosemide. The combination of both drugs into a
single skin patch facilitates administration, reduces the risk of
errors (taking the correct pills at the appropriate time is often
confusing to older people), reduces the psychological strain of
taking "so many pills," reduces skipped dosage because of irregular
activities and improves compliance.
[0163] The methods described herein are particularly applicable to
humans, but also have a variety of veterinary uses, such as the
administration of growth factors or hormones (e.g., for fertility
control) to an animal.
[0164] As noted above, a wide variety of drugs may be administered
according to the methods provided herein. Some examples of drug
categories that may be administered transdermally include
anti-inflammatory drugs (e.g., in arthritis and in other condition)
such as all NSAID, indomethacin, prednisone, etc.; analgesics
(especially when oral absorption is not possible, such as after
surgery, and when parenteral administration is not convenient or
desirable), including morphine, codeine, Demerol, acetaminophen and
combinations of these (e.g., codeine plus acetaminophen);
antibiotics such as Vancomycin (which is not absorbed by the GI
tract and is frequently given intravenously) or a combination of
INH and Rifampicin (e.g., for tuberculosis); anticoagulants such as
heparin (which is not well absorbed by the GI tract and is
generally given parenterally, resulting in fluctuation in the blood
levels with an increased risk of bleeding at high levels and risks
of inefficacy at lower levels) and Warfarin (which is absorbed by
the GI tract but cannot be administered immediately after abdominal
surgery because of the normal ileus following the procedure);
antidepressants (e.g., in situations where compliance is an issue
as in Alzheimer's disease or when maintaining stable blood levels
results in a significant reduction of anti-cholinergic side effects
and better tolerance by patients), such as amitriptylin, imipramin,
prozac, etc.; antihypertensive drugs (e.g., to improve compliance
and reduce side effects associated with fluctuating blood levels),
such as diuretics and beta-blockers (which can be administered by
the same patch; e.g., furosemide and propanolol); antipsychotics
(e.g., to facilitate compliance and make it easier for care giver
and family members to make sure that the drug is received), such as
haloperidol and chlorpromazine; and anxiolytics or sedatives (e.g.,
to avoid the reduction of alertness related to high blood levels
after oral administration and allow a continual benefit throughout
the day by maintaining therapeutic levels constant).
[0165] Numerous other drugs may be administered as described
herein, including naturally occurring and synthetic hormones,
growth factors, proteins and peptides. For example, insulin and
human growth hormone, growth factors like erythropoietin,
interleukins and inteferons may be delivered via the skin.
[0166] Kits for administering a drug via the skin of a mammal are
also provided within the present invention. Such kits generally
comprise a device for transdermal application (e.g., a skin patch)
in combination with, or impregnated with, one or more modulating
agents. A drug may additionally be included within such kits.
[0167] Within a related aspect, modulating agents as described
herein may be used to increase the permeability of endothelial and
epithelial cell layers, thereby facilitating sampling of the blood
compartment by passive diffusion. Such methods permit the detection
and/or measurement of the levels of specific molecules circulating
in the blood. In general, to sample the blood compartment, it is
necessary to perturb adhesion between the epithelial cells
(keratinocytes) and the endothelial cells of the microvasculature.
Using currently available techniques, only small, uncharged
molecules may be detected across skin in vivo. The methods
described herein are not subject to the same degree of limitation.
Accordingly, a wide variety of blood components may be sampled
across epithelial and endothelial cell layers. Such sampling may be
achieved across any such cell layers, including skin and gums.
[0168] For example, application of one or more modulating agents to
the skin, via a skin patch as described herein, permits the patch
to function like a sponge to accumulate a small quantity of fluid
containing a representative sample of the serum. The patch is then
removed after a specified amount of time and analyzed by suitable
techniques for the compound of interest (e.g., a medication,
hormone, growth factor, metabolite or marker). Alternatively, a
patch may be impregnated with reagents to permit a color change if
a specific substance (e.g., an enzyme) is detected. Substances that
can be detected in this manner include, but are not limited to,
illegal drugs such as cocaine, HIV enzymes, glucose and PSA. This
technology is of particular benefit for home testing kits.
[0169] To facilitate sampling of blood in a patient, a modulating
agent as described above for enhancing drug delivery is contacted
with the skin surface. Modulating agent(s) and reagents for
assaying blood components may, but need not, be contained within
the same composition or skin patch. In general, the amount of
modulating agent administered via the skin may vary as described
above. Such levels may be achieved by appropriate adjustments to
the device used, or by applying a cream formulated as described
above. Transfer of the blood component across the skin may be
predicted based on in vitro studies using, for example, a Franz
cell apparatus, and evaluated in vivo by appropriate means that
will be apparent to those of ordinary skill in the art.
[0170] Kits for sampling blood component via, for example, the skin
or gums of a mammal, are also provided within the present
invention. Such kits generally comprise a device for transdermal
application (i.e., skin patch) in combination with, or impregnated
with, one or more modulating agents. A reagent for detection of a
blood component may additionally be included within such kits.
[0171] Within a further aspect, methods are provided for enhancing
delivery of a drug to a tumor in a mammal, comprising administering
a modulating agent in combination with a drug to a tumor-bearing
mammal. Such a modulating agent may comprise only a desmosomal
cadherin CAR sequence, or may further comprise one or more other
nonclassical or classical cadherin CAR sequences, such as a CAR
sequence derived from OB-cadherin, cadherin-5, cadherin-6,
E-cadherin and/or N-cadherin (e.g., HAV, SHAVSS (SEQ ID NO:______),
AHAVDI (SEQ ID NO:______) or a analogue of such a sequence).
Bi-functional modulating agents that comprise the desmosomal
cadherin CAR sequence with either flanking E-cadherin-specific
sequences or flanking N-cadherin-specific sequences joined via a
linker to the desmosomal cadherin CAR sequence are also preferred.
Preferably, the peptide portion(s) of a modulating agent comprises
6-16 amino acids, since longer peptides are difficult to dissolve
in aqueous solution and are more likely to be degraded by
peptidases.
[0172] In one particularly preferred embodiment, a modulating agent
is capable of disrupting cell adhesion mediated by multiple
adhesion molecules. For example, a single branched modulating agent
(or multiple agents linked to a single molecule or support
material) may disrupt adhesion mediated by a desmosomal cadherin,
as well as E-cadherin, N-cadherin, occludin, claudin and integrin
mediated cell adhesion. Such agents serve as multifunctional
disrupters of cell adhesion. Alternatively, a separate modulator
may be administered in conjunction with the modulating agent(s),
either within the same pharmaceutical composition or separately.
Preferred antibody modulating agents include Fab fragments directed
against a nonclassical or classical cadherin CAR sequence, as
described above. A Fab fragment may be incorporated into a
modulating agent or may be present within a separate modulator that
is administered concurrently.
[0173] Preferably, the modulating agent and the drug are formulated
within the same composition or drug delivery device prior to
administration. In general, a modulating agent may enhance drug
delivery to any tumor (e.g., breast tumor, stomach tumor, ovarian
tumor or kidney tumor), and the method of administration may be
chosen based on the type of target tumor. For example, injection or
topical administration as described above may be preferred for
melanomas and other accessible tumors (e.g., metastases from
primary ovarian tumors may be treated by flushing the peritoneal
cavity with the composition). Other tumors (e.g., breast tumors)
may be treated by injection of the modulating agent and the drug
(such as mitomycin C) into the site of the tumor. In other
instances, the composition may be administered systemically, and
targeted to the tumor using any of a variety of specific targeting
agents. Suitable drugs may be identified by those of ordinary skill
in the art based upon the type of cancer to be treated (e.g., taxol
for breast cancer). In general, the amount of modulating agent
administered varies with the method of administration and the
nature of the tumor, within the typical ranges provided above,
preferably ranging from about 1 .mu.g/mL to about 2 mg/mL, and more
preferably from about 10 .mu.g/mL to 1 mg/mL. Transfer of the drug
to the target tumor may be evaluated by appropriate means that will
be apparent to those of ordinary skill in the art. Drugs may also
be labeled (e.g., using radionuclides) to permit direct observation
of transfer to the target tumor using standard imaging
techniques.
[0174] Within a related aspect, the present invention provides
methods for treating cancer in a mammal. Such a modulating agent
may comprise only a desmosomal cadherin CAR sequence, or may
further comprise one or more other nonclassical or classical
cadherin CAR sequences, such as CAR sequences derived from
OB-cadherin, cadherin-5, cadherin-6, E-cadherin and/or N-cadherin.
CAR sequences involved in integrin-mediated cell adhesion (e.g.,
RGD) may also be used. For example, such a modulating agent may
further comprise a sequence such as HAV, SHAVSS (SEQ ID NO:______),
AHAVDI (SEQ ID NO:______), RGD, YIGSR (SEQ ID NO:______), or a
derivative of such a sequence. Preferably, the peptide portion(s)
of such modulating agents comprise 6-16 amino acids. Preferred
antibody modulating agents include Fab fragments directed against a
nonclassical or classical cadherin CAR sequence. The Fab fragments
may be either incorporated into a modulating agent or may be
present within a separate modulator that is administered
concurrently.
[0175] A modulating agent may be administered alone (e.g., via the
skin) or within a pharmaceutical composition. For melanomas and
certain other accessible tumors, injection or topical
administration as described above may be preferred. Other tumors
(e.g., bladder tumors, bronchial tumors or tracheal tumors) may be
treated by injection of the modulating agent into the cavity. In
other instances, the composition may be administered systemically,
and targeted to the tumor using any of a variety of specific
targeting agents, as described above. Preferably, the tumor is a
breast tumor, stomach tumor or kidney tumor. In general, the amount
of modulating agent administered varies depending upon the method
of administration and the nature of the cancer, but may vary within
the ranges identified above. The effectiveness of the cancer
treatment may be evaluated using well known clinical observations,
such as monitoring the level of serum tumor markers (e.g., CEA or
PSA).
[0176] The addition of a targeting agent as described above may be
beneficial, particularly when the administration is systemic.
Suitable modes of administration and dosages depend upon the
condition to be prevented or treated but, in general,
administration by injection is appropriate. Dosages may vary as
described above. The effectiveness of the inhibition may be
evaluated grossly by assessing the inability of the tumors to
maintain their growth and microscopically by observing an absence
of nerves at the periphery of the tumor.
[0177] In yet another related aspect, the present invention
provides methods for inducing apoptosis in a desmosomal
cadherin-expressing cell. Such a modulating agent may comprise only
a desmosomal cadherin CAR sequence, or may further comprise one or
more other nonclassical or classical cadherin CAR sequences, such
as HAV, SHAVSS (SEQ ID NO:______), AHAVDI (SEQ ID NO:______), RGD,
YIGSR (SEQ ID NO:______) or an analogue of such a sequence.
Preferably, the peptide portion(s) of such modulating agents
comprise 6-16 amino acids. Preferred antibody modulating agents
include Fab fragments directed against a nonclassical or classical
cadherin CAR sequence. The Fab fragments may be either incorporated
into a modulating agent or within a separate modulator that is
administered concurrently. Administration may be topical, via
injection or by other means, and the addition of a targeting agent
may be beneficial, particularly when the administration is
systemic. Suitable modes of administration and dosages depend upon
the location and nature of the cells for which induction of
apoptosis is desired but, in general, dosages may vary as described
above. A biopsy may be performed to evaluate the level of induction
of apoptosis.
[0178] In certain other aspects, the present invention provides
methods for enhancing adhesion of desmosomal cadherin-expressing
cells. Within certain embodiments, a modulating agent may be linked
to a solid support, resulting in a matrix that comprises multiple
modulating agents. Within one such embodiment, the support is a
polymeric matrix to which modulating agents and molecules
comprising other CAR sequence(s) are attached (e.g., modulating
agents and molecules comprising either HAV or RGD sequences may be
attached to the same matrix, preferably in an alternating pattern).
Such matrices may be used in contexts in which it is desirable to
enhance adhesion mediated by multiple cell adhesion molecules.
Alternatively, the modulating agent itself may comprise multiple
desmosomal cadherin CAR sequences or antibodies (or fragments
thereof), separated by linkers as described above. Either way, the
modulating agent(s) function as a "biological glue" to bind
multiple desmosomal cadherin-expressing cells within a variety of
contexts.
[0179] Within one such aspect, modulating agents comprising the
desmosomal cadherin CAR sequence and/or multiple modulating agents
linked to a single molecule or support material may be used to
facilitate wound healing and/or reduce scar tissue in a mammal.
Such a modulating agent may comprise one desmosomal cadherin CAR
sequence, or may comprise multiple such sequences (e.g., a
desmoglein CAR sequence and a desmocollin CAR sequence).
Optionally, a modulating agent may further comprise one or more
other nonclassical cadherin CAR sequences, such as CAR sequences
derived from OB-cadherin or cadherin-5 and/or one or more classical
cadherin CAR sequences, including HAV, SHAVSS (SEQ ID NO:______),
AHAVDI (SEQ ID NO:______), or an analogue of such a sequence.
Preferred antibody modulating agents include Fab fragments directed
against either the nonclassical cadherin or E-cadherin CAR
sequences. Modulating agents that are linked to a biocompatible and
biodegradable matrix such as cellulose or collagen are particularly
preferred. For use within such methods, a modulating agent should
have a free amino or hydroxyl group. The modulating agents are
generally administered topically to the wound, where they may
facilitate closure of the wound and may augment, or even replace,
stitches. Similarly, administration of matrix-linked modulating
agents may facilitate cell adhesion in skin grafting and prosthetic
implants, and may prolong the duration and usefulness of collagen
injection. In general, the amount of matrix-linked modulating agent
administered to a wound, graft or implant site varies with the
severity of the wound and/or the nature of the wound, graft, or
implant, but may vary as discussed above. Multi-functional
modulating agents comprising a desmosomal cadherin sequence, a
classical cadherin CAR sequence (HAV), and the CAR sequence bound
by certain integrins (RGD) may also be used as potent stimulators
of wound healing and/or to reduce scar tissue. Alternatively, one
or more separate modulators of classical cadherin- or
integrin-mediated cell adhesion may be administered in conjunction
with the modulating agent(s), either within the same pharmaceutical
composition or separately.
[0180] Within further aspects, modulating agents may be used for
the treatment of autoimmune blistering disorders, such as pemphigus
vulgaris, pemphigus foliaceus and intercellular IgA dermatosis.
These disorders are pathological conditions in which a patient
begins to develop antibodies against one or more desmosomal
cadherins. In consequence, adhesion of skin cells begins to fail,
resulting in various kinds of blistering. The use of modulating
agents to enhance desmosomal cadherin-mediated cell adhesion may
alleviate symptoms of such disorders. Optionally, such methods
would be performed in conjunction with a method for eliminating the
anti-cadherin antibodies. Modulating agents for use in such
therapies may comprise one desmosomal cadherin CAR sequence or,
preferably, comprise a desmoglein CAR sequence and a desmocollin
CAR sequence. Optionally, a modulating agent further comprises one
or more other nonclassical cadherin CAR sequences, such as CAR
sequences derived from OB-cadherin or cadherin-5 and/or one or more
classical cadherin CAR sequences, including HAV, SHAVSS (SEQ ID
NO:______), AHAVDI (SEQ ID NO:______), or an analogue of such a
sequence. Preferred antibody modulating agents include Fab
fragments directed against either the nonclassical cadherin or
E-cadherin CAR sequences. Modulating agents that are linked to a
biocompatible and biodegradable matrix such as cellulose or
collagen are particularly preferred. For use within such methods, a
modulating agent should have a free amino or hydroxyl group. The
modulating agents are generally administered topically to the site
of blisters. In general, the amount of matrix-linked modulating
agent administered to a blister site varies with the severity of
the condition, and generally ranges as discussed above.
Multi-functional modulating agents comprising one or more
desmosomal cadherin sequences, a classical cadherin CAR sequence
(HAV), and the CAR sequence bound by certain integrins (RGD) may
also be used. Alternatively, one or more separate modulators of
classical cadherin- or integrin-mediated cell adhesion may be
administered in conjunction with the modulating agent(s), either
within the same pharmaceutical composition or separately.
[0181] Within another aspect, one or more modulating agents may be
linked to the interior surface of a tissue culture plate or other
cell culture support, such as for use in a bioreactor. Such linkage
may be performed by any suitable technique, as described above.
Modulating agents linked in this fashion may generally be used to
immobilize cadherin-expressing cells. For example, dishes or plates
coated with one or more modulating agents may be used to immobilize
cadherin-expressing cells within a variety of assays and screens.
Within bioreactors (i.e., systems for large scale production of
cells or organoids), modulating agents may generally be used to
improve cell attachment and stabilize cell growth. Modulating
agents may also be used within bioreactors to support the formation
and function of highly differentiated organoids derived, for
example, from dispersed populations of fetal mammalian cells.
Bioreactors containing biomatrices of modulating agent(s) may also
be used to facilitate the production of specific proteins.
[0182] Modulating agents as described herein may be used within a
variety of bioreactor configurations. In general, a bioreactor is
designed with an interior surface area sufficient to support large
numbers of adherent cells. This surface area can be provided using
membranes, tubes, microtiter wells, columns, hollow fibers, roller
bottles, plates, dishes, beads or a combination thereof. A
bioreactor may be compartmentalized. The support material within a
bioreactor may be any suitable material known in the art;
preferably, the support material does not dissolve or swell in
water. Preferred support materials include, but are not limited to,
synthetic polymers such as acrylics, vinyls, polyethylene,
polypropylene, polytetrafluoroethylene, nylons, polyurethanes,
polyamides, polysulfones and poly(ethylene terephthalate);
ceramics; glass and silica.
[0183] Other aspects of the present invention provide methods that
employ antibodies raised against a desmosomal CAR sequence for
diagnostic and assay purposes. Assays typically involve using an
antibody to detect the presence or absence of a desmosomal cadherin
(free or on the surface of a cell), or proteolytic fragments
containing one or more EC domains in a suitable biological sample,
such as tumor or normal tissue biopsies, blood, lymph node, serum
or urine samples, or other tissue, homogenate, or extract thereof
obtained from a patient.
[0184] There are a variety of assay formats known to those of
ordinary skill in the art for using an antibody to detect a target
molecule in a sample. See, e.g., Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For
example, the assay may be performed in a Western blot format,
wherein a protein preparation from the biological sample is
submitted to gel electrophoresis, transferred to a suitable
membrane and allowed to react with the antibody. The presence of
the antibody on the membrane may then be detected using a suitable
detection reagent, as described below.
[0185] In another embodiment, the assay involves the use of
antibody immobilized on a solid support to bind to the target
cadherin, or a proteolytic fragment containing an extracellular
domain and encompassing a CAR sequence, and remove it from the
remainder of the sample. The bound cadherin may then be detected
using a second antibody or reagent that contains a reporter group.
Alternatively, a competitive assay may be utilized, in which a
cadherin is labeled with a reporter group and allowed to bind to
the immobilized antibody after incubation of the antibody with the
sample. The extent to which components of the sample inhibit the
binding of the labeled cadherin to the antibody is indicative of
the reactivity of the sample with the immobilized antibody, and as
a result, indicative of the level of the cadherin in the
sample.
[0186] The solid support may be any material known to those of
ordinary skill in the art to which the antibody may be attached,
such as a test well in a microtiter plate, a nitrocellulose filter
or another suitable membrane. Alternatively, the support may be a
bead or disc, such as glass, fiberglass, latex or a plastic such as
polystyrene or polyvinylchloride. The antibody may be immobilized
on the solid support using a variety of techniques known to those
in the art, which are amply described in the patent and scientific
literature.
[0187] In certain embodiments, the assay for detection of a
desmosomal cadherin in a sample is a two-antibody sandwich assay.
This assay may be performed by first contacting an antibody that
has been immobilized on a solid support, commonly the well of a
microtiter plate, with the biological sample, such that the
desmosomal cadherin within the sample is allowed to bind to the
immobilized antibody (a 30 minute incubation time at room
temperature is generally sufficient). Unbound sample is then
removed from the immobilized cadherin-antibody complexes and a
second antibody (containing a reporter group such as an enzyme,
dye, radionuclide, luminescent group, fluorescent group or biotin)
capable of binding to a different site on the cadherin is added.
The amount of second antibody that remains bound to the solid
support is then determined using a method appropriate for the
specific reporter group. The method employed for detecting the
reporter group depends upon the nature of the reporter group. For
radioactive groups, scintillation counting or autoradiographic
methods are generally appropriate. Spectroscopic methods may be
used to detect dyes, luminescent groups and fluorescent groups.
Biotin may be detected using avidin, coupled to a different
reporter group (commonly a radioactive or fluorescent group or an
enzyme). Enzyme reporter groups may generally be detected by the
addition of substrate (generally for a specific period of time),
followed by spectroscopic or other analysis of the reaction
products. Standards and standard additions may be used to determine
the level of cadherin in a sample, using well known techniques.
[0188] Such immunoassays may be used, for example, in the diagnosis
and monitoring of conditions associated with abnormal desmosomal
cadherin expression. As noted above, such conditions include
autoimmune blistering disorders, such as pemphigus vulgaris,
pemphigus foliaceus and intercellular IgA dermatosis. In general,
the presence or absence of such a condition in a patient may be
determined by (a) contacting a biological sample obtained from a
patient with an antibody or antigen-binding fragment thereof; (b)
detecting in the sample a level or pattern of polypeptide that
binds to the antibody or fragment thereof; and (c) comparing the
level or pattern of polypeptide with a predetermined cut-off value
or a normal pattern. In general, these diseases are associated with
an autoimmune response against desmosomal cadherins, which causes
the internalization of these cadherins. Such internalization may be
visualized as patchy cytoplasmic granules in stained biopsy
sections of lesional and perilesional keratinocytes (see Iwatsuki
et al., Br. J. Dermatol. 140:35-43). These granules are not
detected in normal patients.
[0189] Immunoassays may further be used to differentiate between
benign skin lesions, such as keratoacanthoma, and squamous cell
carcinoma. The location of desmoglein staining, in particular, may
be used to differentiate between a keratoacanthoma and a carcinoma
(see Krunic et al., Acta Derm. Venereol. 76:394-398, 1996). In
general, extensive pericellular staining indicates a
keratoacanthoma. Focal pericellular staining indicates a
keratoacanthoma or a well-differentiated squamous cell carcinoma.
No staining or juxtanuclear staining indicates a poorly
differentiated squamous cell carcinoma. Thus, a reduction of
pericellular staining (as determined visually or quantitatively, as
a reduction of 50%) is indicative of squamous cell carcinoma.
[0190] The present invention also provides kits for use in such
immunoassays. Such kits generally comprise one or more antibodies,
as described above. In addition, one or more additional
compartments or containers of a kit generally enclose elements,
such as reagents, buffers and/or wash solutions, to be used in the
immunoassay.
[0191] Within further aspects, modulating agents or antibodies (or
fragments thereof) may be used to facilitate cell identification
and sorting in vitro or imaging in vivo, permitting the selection
of cells expressing the desmosomal cadherin (or different
desmosomal cadherin levels). Preferably, the modulating agent(s) or
antibodies for use in such methods are linked to a detectable
marker. Suitable markers are well known in the art and include
radionuclides, luminescent groups, fluorescent groups, enzymes,
dyes, constant immunoglobulin domains and biotin. Within one
preferred embodiment, a modulating agent linked to a fluorescent
marker, such as fluorescein, is contacted with the cells, which are
then analyzed by fluorescence activated cell sorting (FACS).
[0192] Antibodies or fragments thereof may also be used within
screens of combinatorial or other nonpeptide-based libraries to
identify other compounds capable of modulating desmosomal
cadherin-mediated cell adhesion. Such screens may generally be
performed using an ELISA or other method well known to those of
ordinary skill in the art that detect compounds with a shape and
structure similar to that of the modulating agent. In general, such
screens may involve contacting an expression library producing test
compounds with an antibody, and detecting the level of antibody
bound to the candidate compounds. Compounds for which the antibody
has a higher affinity may be further characterized as described
herein, to evaluate the ability to modulate OB-cadherin-mediated
cell adhesion.
[0193] The following Example is offered by way of illustration and
not by way of limitation.
EXAMPLE
Preparation of Representative Modulating Agents
[0194] This Example illustrates the solid phase synthesis of
representative peptide modulating agents.
[0195] The peptides were synthesized on a 431A Applied Biosystems
peptide synthesizer using p-Hydroxymethylphenoxymethyl polystyrene
(HMP) resin and standard Fmoc chemistry. After synthesis and
deprotection, the peptides were de-salted on a Sephadex G-10 column
and lyophilized. The peptides were analyzed for purity by
analytical HPLC, and in each case a single peak was observed.
Peptides were made as stock solutions at 10 to 25 mg/mL in
dimethylsulfoxide (DMSO) or water and stored at -20.degree. C.
before use.
[0196] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 0
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