Us8841255 PDF

USOO8841255B2
(12) United States Patent (10) Patent No.: US 8,841,255 B2

Chilkoti (45) Date of Patent: *Sep. 23, 2014
(54) THERAPEUTICAGENTS COMPRISING (56) References Cited
FUSIONS OF VASOACTIVE INTESTINAL
PEPTIDE AND ELASTIC PEPTIDES U.S. PATENT DOCUMENTS
4,132,746 A 1/1979 Urry et al.
(75) Inventor: Ashutosh Chilkoti, Durham, NC (US) 4,187,852 A 2/1980 Urry et al.
4,474,851 A * 10/1984 Urry ............................. 428,373
(73) Assignee: Duke University, Durham, NC (US) 4,500,700 A 2/1985 Urry
4,589,882 A 5/1986 Urry
4,749,647 A 6, 1988 Thomas et al.
(*) Notice: Subject to any disclaimer, the term of this 4,752,638 A 6, 1988 Nowinski et al.
patent is extended or adjusted under 35 4,783,523 A 1 1/1988 Urry et al.
4,870,055 A 9/1989 Urry et al.
U.S.C. 154(b) by 0 days. 4,898,926 A 2/1990 Urry
5,147,855 A 9, 1992 Gozes et al.
This patent is Subject to a terminal dis 5,235,041 A 8/1993 Cappello et al.
claimer. 5,236,904 A 8/1993 Gerstenberg et al.
5,243,038 A 9, 1993 Ferrarietal.
(21) Appl. No.: 12/852.365 5,288,514 A 2f1994 Ellman
5,445,934 A 8, 1995 Fodor et al.
5.447,912 A 9/1995 Gerstenberg et al.
(22) Filed: Aug. 6, 2010 5,496,712 A 3/1996 Cappello et al.
5,514,581 A 5/1996 Ferrarietal.
(65) Prior Publication Data 5,519,004 A 5/1996 Urry
5,520,672 A 5/1996 Urry
US 2011 FO123487 A1 May 26, 2011 5,527,610 A 6/1996 Urry
5,545,617 A 8, 1996 Dartt et al.
5,624,711 A 4/1997 Sundberg et al.
5,641,648 A 6/1997 Ferrarietal.
Related U.S. Application Data 5,681,816 A 10, 1997 Korman
5,702.717 A 12/1997 Cha et al.
(63) Continuation-in-part of application No. 12/493.912, 5,747,646 A 5/1998 Hakimi et al.
filed on Jun. 29, 2009, now Pat. No. 8,178,495, which (Continued)
is a continuation-in-part of application No.
12/158,190, filed aS application No. FOREIGN PATENT DOCUMENTS
PCT/US2006/048572 on Dec. 20, 2006, now Pat. No.
8,334,257. EP O449592 B1 11, 1994
WO WO 86,06492 A1 11, 1986
(60) Provisional application No. 61/076,221, filed on Jun. (Continued)
27, 2008, provisional application No. 60/751,896,
filed on Dec. 20, 2005. OTHER PUBLICATIONS
Bidwell et al., “Application of thermally responsive polypeptides
(51) Int. Cl. directed against c-Myc transcriptional function for cancer therapy.”
A6 IK38/22 (2006.01)
A6 IK38/39 (2006.01) Mol. Cancer Ther. 4(7): 1076-1085 (2005).
C07K I4/745 (2006.01) (Continued)
C07K 4/78 (2006.01)
C07K I4/52 (2006.01)
A6 IK38/48 (2006.01) Primary Examiner — Christine J Saoud
A6 IK 47/48 (2006.01) Assistant Examiner — Jon M Lockard
C07K I4/7 (2006.01) (74) Attorney, Agent, or Firm — Cooley LLP
C07K I4/62 (2006.01)
C07K I4/605 (2006.01) (57) ABSTRACT
A6 IK 45/06 (2006.01)
A61 K38/00 (2006.01) The present invention provides therapeutic agents and com
(52) U.S. Cl. positions comprising elastic peptides and therapeutic pro
CPC ......... A61K 47/48246 (2013.01); C07K 14/745 teins. Such peptides exhibit a flexible, extended conforma
(2013.01); C07K 14/78 (2013.01); C07K tion. In some embodiments, the therapeutic protein is a
2319/31 (2013.01); C07K 14/52 (2013.01); GLP-1 receptor agonist (e.g., GLP-1, exendin), insulin, or
A61K 38/4846 (2013.01); A61 K38/00 Factor VII/VIIa, including functional analogs. The present
(2013.01); A61K38/2278 (2013.01); A61 K invention further provides encoding polynucleotides, as well
47/48292 (2013.01); C07K 14/71 (2013.01); as methods of making and using the therapeutic agents. The
C07K 14/62 (2013.01); C07K 14/605 therapeutic agents have improvements in relation to their use
(2013.01); A61K 45/06 (2013.01) as therapeutics, including, interalia, one or more of half-life,
USPC .......... 514/13.1: 514/21.2:530/350; 530/353 clearance and/or persistence in the body, Solubility, and bio
availability.
(58) Field of Classification Search
None
See application file for complete search history. 14 Claims, 32 Drawing Sheets
US 8,841,255 B2
Page 2
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US 8,841,255 B2
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Nilsson, J. et al., "Affinity Fusion Strategies for Detection, Purifica Transition Depends on Mean Residue Hydrophobicity.” J. Am.
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Onyüksel et al., “Human VIP-O: A long-acting, biocompatible and
biodegradable peptide nanomedicine for essential hypertension.” Elastic Structure Due to an Inverse Temperature Transition and Elas
Peptides 27:2271-2275 (2006). ticity Due to Internal Chain Dynamics,” Journal of Protein Chemis
Onyüksel et al., “A Novel Formulation of VIP in Sterically Stabilized try, vol. 7, No. 1, pp. 1-34 (1988).
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Sejourne, et al., “Development of a Novel Bioactive Formulation of J. Phys. Chem. B., vol. 101, No. 51, pp. 11007-11028, (1997).
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Pharmaceutical Research, vol. 14, No. 3, 1997, pp. 362-365. * cited by examiner
U.S. Patent Sep. 23, 2014 Sheet 1 of 32 US 8,841,255 B2
FIGURE 1
Xbal
BgII, Nde
* - ...
Y - T7 prom
A. ELP1-90 Nhe
, , Ban.H.
lact .* . Sac
EcoRI
flori iN
& Dralil
Kan A PS
ap Y----------------- na
Sria
Clai
pET24d-ELP1-90
FIGURE 2A
pET24d-Ex-4 ELP1-90
FIGURE 2B
1. aattaatacg acticactata ggggaattgt gag.cggataa caattcc.cct

ttaattatgc tagtegatat coccittaa.ca citc.gc.ctatt gtta agggga
>> . . . T7 pron. . . . >>
Xbal Nde
--- - - - - - - - ce - - -
P- - - - - - - -
51 ctagaaataa ttttgtttaa, ctittaagaag gagatataca tatgcatggc
gatctittatt aaaacaaatt gaaattettc. citctatatgt attacgitac.cg
13 nor m wom now we m nor
at g
> Erw C
X w. X w. X w. X w. X W X rar as a ras a rear a rap ar are area arra aro 2- - - - - - - - - - - or memorrow or one or on- or a
101 gaaggcacct ttaccagoga totgagcaaa cagatggaag aagaag.cggit

citt.ccgtgga aatggtc.gct agacitcgttt gtc.tacctitc ttctt.cgc.ca
an au as a bar a ra wa W M -w Wa aw w x -w as Sco w w w - or on was were w are ree re or or or w w or or or or or or or or or or or or
> . . . . . . . . . . . . . . . . . . . . Ex4 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
e g t t is c s k q in e e is a
Asc
mr -
>3 - - - - - - - -a or or or more
151 go.gc.ctgttt attgaatggc togaaaaacgg cggc.ccgagc agcggcgc.gc.
cgcggacaaa taacttaccg acttitttgcc gcc.gggctcg togcc.gc.gcg
95 k- w w w w w - - -
> . . . . . . . . . . . . . . . . . . . . Ex4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . >
if i e w k is S s g a
XhoI
-- ----
was M M M W- >
201 cgc.cgc.cgag cctogagggc atgggtgggc cggg.cgtggg togttccgggC

gcgg.cggctc. ggagctc.ccg tacccacccg gCC.cgcaccc acaaggc.ccg.
m --- - - --- v--- - - -m-, m-mm mm mm - umu m m PA
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . >EP-90
p p p S e g it g g p g v g v. p. g
251 gtgggtgttc cgggtggcgg togtgc.cgggc goaggtgttc ctggtgtagg (SEQ ID NO. 23)
cacccacaag gCCcaccgcc acacggc.ccg cgt.ccacaag gaccacatcc
> . . . . . . . . . . . . . . . . . . . . . . . . . ELP1-90. . . . . . . . . . . . . . . . . . . . >
w g v g v. p. g. a g v p g. v. (SEQ ID NO. 24)
P1 : SEQ ID NO. : 35
P2: SEQ ID NO. : 36
P3: SEQ ID NO. : 37
P4: SEQ ID NO. : 38
P5: SEC ID NO. : 39
P6: SEC ID NO. : 40
FIGURE 3A
Node
7- or -- - - - - - - - - - or no or w w we were we w w w w a rare err - - - - - - - - - - - - - - - - - -

1 tatggaaaac citgitatttcc aacatggcga aggcacctitt accagogatc
accttittg gaCatalaagg ttgtaccgct tcc.gtggaaa togg togctag
Kao as as a co - - - - - - - - - - - - - - - - - - - or r an or more on on to we wers - - - - - - - - 8
’’. . . . . . . . . . . . . . . . . . Ex4 ELPi"90 . . . . . . . . . . . . . . . . . . . . >
e y if g h g e g t if t is di
>> . . . . . . Teaw. . . . . . . >>
>
51 togagcaaaca gatggaagaa gaag.cggtgc gcctgtttat togaatggctg
acticgtttgt citaccttctt ctitc.gc.cacg cggacaaata acttaccgac
> . . . . . . . . . . . . . . . . . . . Ex- 4 EJ.P. - 90' . . . . . . . . . . . . . . . . . . . .
s k q t e e e a F i e w i.
Asce
I m o oa o or W.
101 aaaaacgg.cg gcc.cgag cag cqgcgc.gc.cg cc.gc.cgagcc (SEQ ID NO. : 25)

tttittgcc.gc cggg citcgt.c gcc.gc.gcggc gg.cggct.cgg agct
Kw we to a man w w w w w w or mm w w w w w w w mm or w w w w w m - or w. -- 4.
P . . . . . . . . . . . . . . Ex- 4 EP-90 . . . . . . . . . . . . . . >.
k a g g S S 2 S (SEC ID NO. : 26)
P4: SEQ ID NO. : 38

P7: SEQ ID NO. : 41
P8: SEQ ID NO. : 42
FIGURE 3B
Nde
P9 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
1 tatggatato coaacgaccg aaaacctgta titt.ccaa.cat gg.cgaaggca
acctatag ggttgctggc titttggacat aaaggttgta cc.gctt.ccgt.
K- - - - - - - - a new we own to a low was or a more lar se -- - - - - - - - - - - - - - - - - r ran or
?? . . . . . . . . . . . . . . . . . . Ex" 4 ELP. "90 . . . . . . . . . . . . . . . . . . . .
d i. p t t e n . y f q h g e g
>> . . . . . . . . . . . Linker lev . . . . . . . . . . . >>
r run wr rous r r ro r arror a run to a r-e M Pas >
51 cctttaccag cqatctgage aaacagatgg aagaagaagc ggtgcgc.ctg

ggaaatgg to gottagacitcg tttgtc.tacc ttcttctitcg ccacgcggac
- w w w w w W. W. O
? . . . . . . . . . . . . . . . . . . . Ex" 4 EB1. "90 . . . . . . . . . . . . . . . . . . . . . d

it f it s d l s k g it e a a v r
101 tttattgaat ggctgaaaaa cgg.cggc.ccg agcagogg.cg cgc.cgcc.gc.c

aaataactta ccgactittitt gcc.gc.cgggc togtogcc.gc gcgg.cgg.cgg
ag worm orw on m. m. m. w w w w w w w w w w w W w w w W. W. W. www.
. . . . . . . . . . . . . . . . . . . Ex- 4 EP "r 9C . . . . . . . . . . . . . . . . . . . . . >

if i e w k ia S is g a go
151 gagcc (SEQ ID NO. : 27)

citcggagct
m a PA
> . . ss Ex- 4 E. SO
S (SEQ ID NO. : 28)
P4 : SEQ ID NO. : 38
P9: SEQ ID NO. : 43
PO : SEQ ID NO. : 44
FIGURE 4A
NodeI.
P- - - - - - - - - - - - - - - - - - - - - - - - no e - - - - - - - - - - - - - - - - - - - - - - - - -
1 tatgaaaaag atttggctgg cgctggctgg tttagttitta gcgtttagcg
acttitt to taaaccgacc gcgaccgacc aaatcaaaat cgcaaatcgc
51 catcgg.cgca toggcgaaggc acctttacca gcgatctgag caaacagatg

gtagcc.gcgt. accgctitc.cg tggaaatggit cgctag actc gtttgttctac
ow n w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w P2
> . DsbA>>>> . . . . . . . . . . . . . . Ex-4 EE.P1-90 . . . . . . . . . . . . . . . . . >

a s a h g e g t if it s d . s k g a
101 gaagaagaag cggtgcgc.ct gtttattgaa toggctgaaaa acgg.cgg.ccc

cittcttctitc gocacgcgga caaataactt accgactittt togcc.gc.cggg
<-- - - -
. . . . . . . . . . . . . . . . . . . Ex- 4 E.P. - 90 . . . . . . . . . . . . . . . . . . . . . X
e e e a w f i. e. w k. I g g
151 gag cagcggc gogcc.gc.cgc cgagcc (SEQ ID NO. : 29)

citcgt.cgc.cg cgcggcggcg gctcggagct
- - - - - - - - - M M M - - - - - - - - - - - - - M. M. W. M. M. P4
. . . . . . Ex- 4 ELP1-wr 90 . . . . . . . >>

p s is g a p p p s (SEQ D NO. : 30)
P4: SEQ ID NO. : 38

P1.1 : SEQ ID NO. : 45
P12: SEQ ID NO. : 46
FIGURE 4B
p $1t 7 prom . . . Y.
DsbA ^,
Sy \
y A ac Ex- 4 ELP1-90 y \ Nhe
f f \ \ BamHI
| V \ -i Saci.
EcoRI
FspI -1 i A |
FBXi y
f1 or a -
w A DraIII
\ Kan PSI
N /
> s -
-
Sap ------ Pvt.
; Sra.
Xrnal
Cla
NI
pET24d-DsbA-Ex-4 ELP1-90
FIGURE SA
Xibal 47
Nde 86
Kpni 119
ECOR 170
Bgi 201
XhoI2O3
Mu 6043 D
BSt E 5861 GLP-1 (7-37)
ELP1-90
Bgll 1570
Nhe 1589
ppBO868 EcoR 1626
6784 bps
Bgll 4980
Dra 2041
Pvt 2743
FIGURE 6A
Xibal 47
Noel 86
Kpnl 119
Node 7419
BS36 7357
No. 7084
Xibal 7045
Bgll 6979
ELP1-120
Mu 6257
p
PB1022 Bgll 2005
Nhe 2024
BSt E 6O75 8145bps EcoR 2061
Dfa 2492
Pvt 3 194
FIGURE 7A
Bgll 12
PyL (530
Whe 895
CMV pro Bgll 1100
Amp
signal prepropetide Xibal 1434
FW-EP1-90
ppBO788
8086 bps Kpni 2081
Not 2233
Dra 484 3.32.

FIGURE 7B (5 of 6)
y
. . . . . . . . . . . . . . . . . . . . . . . Šiši ~ SS &
2851 gCaggcgtCC
Y
2901 Cagg agttcCtgga gtcggagtgc

s SSS
- - - - s - - - - - -
2.951
3051
3101 ccggagttgg agtgcc.cgga gttggagtcc caggaggcgg agtcCCCgga

S. S.
Siss SS . . . . . . . . . . . . . . . . . .
St. X
SR S S S S S SS
31.51 gCaggcgt.cc ccggagg.cgg agtgccgggg gtgggagttc. CC9g.cgtggg

SRSS SSS as SS
agttc.ccgga gg.cggagtgC CCggCgCagg agttcCtgga

. . . . . . . . . as a 1 1- SFS 8& . . . . . . . . . . . . . . . . . . . . .
SS
S
3251
3301 gCaggcgtcc ccggaggcgg agtgc.cgggc gtgggagttc ccggcgtggg

Y- W & s
w w x X w w x x . . . WY w w w w w w w w SYSSS & S.S.S. - SSR w
S. S. S. SS S. S. SS Ya S w & S 's
- SY
. . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . . . . . . 3 3 & 8 & & K & 4 8 w w sv&
3351 agttcc.cgga ggcggagtgc ccggcgcagg agttcCtgga gtoggagtgC

v - - - - - - - - - - - - - - a V a Swiss
as' S, SS
Six-S-S -- Sy
& - - - - - - - - - - - - - - - . . . . *
& S S S v & N
S.
3401 tggagtoc Caggaggcgg agtcCocgga

y
s S. & SS S &
FIGURE 7B (6 of 6)
3451 gCaggCgtCC CCggaggcgg agtgCCgggC gtgggagttc. CCggCgtggg

Y.
* & & & 4. & & 8 s w w . . . . . Sy. SS-SSS
. . . . S. Sa. a SSS W. W. s
cggcgcag
Y
3551 CCggagttgg agtgcCCgga gttggag to C Caggaggcgg

^ - - - - - - - - - - - - - - - - - - SSS S.
Xa.
an an a new
Xo
- -- - --
36O1 gccgggc toggcCttgat gactcgagtC
SSS. - SS . . . . . . . . . . . . . . . NY
3651 tagagggc.cc gtttaaacco gotgatcago citcgactgtg ccttctagtt (SEQ ID NO: 57)

FIGURE 8A
Ndle
as a as a rm - m r or
P5 w w w or m wo- w w w or or or - or - w w w mm
51 citagaaataa titttgtttaa ctittaagaag gagatataca tatgtttgttg
Instil...in Ew 90 >> . . . . . . )
if y
3 chain >> . . . )
- - - -->
101 alaccalacace tgtgcggcto acacctggtg gaagctcitct acctagtgtg
>... . ... ... a as a Insul in E.I.P. - 90 . . . . . . . . . . . . . . . . . . . Y.
rt c h c g s v e a y v
> . . . . . .. . . . w v v s 4 x w w P v w chain . . . . . . . . . . . . . . . . . . . . . . . .
151 cggggaacga. ggottcttct acacacccaa gaccc.gc.cgg gaggcagagg

w w w w & w Insul in ELP1 - 90. . . . . . w w x X w R - A & X
g f f y t t a
> . . . . . . . . . . . . . . B chain . . . . . . . . . . . . . >
C chain >> . . . . . . .
acctgcaggt. ggggcaggtg gag citggg.cg gggg.cCCtgg tycaggcago

> . . . . .. . . . . - - a a - Insulin ELP1" 90 . . . . . . . . . . . . . . . . . . . Y.
d q v g g v e g g a g S
. . . . . . . . .. - a - a C chain . . . . . . . . . . . . . . . . . . . . . . .
otgcagocct tgg occtgga gggg toccitg cagaag.cgtg goattgttgga.

> . . . . . . . . . . w w w w w & a m insii in ELF1 - 9C . . . . . . . . . . . . . . . . . . . )
I. C. p. a e g is q k r g i v
> . . . . . . . . . . . . . C chain . . . . . . . . . . . . . *
>>A chair. . . )
x or w w w www mar or Wr m or
30 acaatgctgt. accagoatct gctc.ccticta cca.gctggag aactactgca

cctic ttgatgacgt.
>. . . . . . . . . . a a a Inst... in E.P. - 90 . . . . . . . . . . . . . . . . . . . >
e q C. c. t is i. c is . y q e y c
- - - - - - - - - - w - - - - A Chair. . . . . . . . . . . . . . . . . . . . . . . . .
it is i. c is . y q i. e. in y C.
351. acctCgaggg Catgggtggg CCgggCgtgg gttgttcCggg C9tgggtgtt

tggagctc.cc gtaccc.
- -- 6
P . . . . ... . .. a Insul in ELP1 - 90 . . . . . . . . . . . . . . . . . . . P

in e g E. g g g g W g 9 g v g v.
>> A chain
401 cCgggtgg.cg gtgttgc.cggg cgcaggtgtt cotggtgtag gttgttgc.cggg (SEQ ID NO:31)

P . . . . . . . . . . a a as a insul in E.I.P." 90 . . . . . . . . . . . . . . . . . . . >
g w p g a g w p g v g w p (SEQ ID NO:32)
P5: SEQ ID NO. : A 7
16: SEQ ID NO 8
FIGURE 8B
MLI
s" Xbal
Ndel
t BcII
BstEII
| | | Xhol ar "w
Apal -17- per

EcoRy, ',y
w w Ns
B chairbas.
HincII,
HpaI, 7 A - (AE \ N\
y A lac Insulin ELP1-90 y \
FspI, l v i , NheI
Bar-I
FspAI \ fi ori 4 f EcoRI
\ k. Sac
\ A. DraIII
N N
Kan . M
/ psi
^ -
SapI ------,t '. PWu
Xmal
SmaI
Cla
Nr.
pET24d Insulin-ELP1-90
U.S. Patent Sep. 23, 2014 Sheet 25 Of 32 US 8,841,255 B2
FIGURE 9
FIGURE 10
Protein:
is Alyosin
Phosphorylase B
BSA
Glutarie dehydrogenise
Alcoholdehydrogenase
Carbonic anhydrase
viyoglobi-Red
protiin
Stitchi
FIGURE 11
Activation of FX by FVila or FVila-ELP
COC nM
4-P Fit: y = (A - D)/(1 + (x/C)^B) + D: A B C D R^2

C Plotil 1 (FW-1-90: Concentration vs Meanwalue) -0.967 0.596 4.28 31.1 0.998
Ploti:2 (FVila: Concentration vs Meanwalue) -0.698 0.496 1758+11 2.77e--06 O997
Curve Fit Option - Fixed Weight Value

FGURE 2
FVia-EP has a long PK in Rats

000
-- fa-EP
-- FVia
0.
was
cool
S.
5
&
s
:
&
s
s:
e
is 20 25 SO 3S
Time after administration (h)
FVia c. 45-60 in
FWa-P: 690 min
U.S. Patent Sep. 23, 2014 Sheet 29 Of 32 US 8,841,255 B2
FIGURE 13
High In vitro Activity of GLP1-ELP and Exendin-4-ELP Fusion

Proteins
150
t
100
E
-
50
1e-4 OOO1 0.01 O. 1 1O 1OO
Peptide or protein nM
A GLP-1-ELP
Exendin peptide
Exendin-ELP
FIGURE 14
PharmaCOkinetics of GLP1-ELP in Rats
10
10
O s EC s 13 2. {
tinae after administration hours)
T12 IV= 12.9 hours

T1/2 SO= 8.6 hours
FIGURE 5
Long Haif life of GP-1 EP in Rabbits
s
ss
s
area
re
ring
8.
s
ge
s
S.
s
2. i 8 3. 20 4. 60 8
tie after administration (rs)
12 We 2.920 hours
2 SOR:8.624 hours
FGURE 6
Sustained Glycemic Control of GP-EP in diabetic Mice
400
S 5 2 2S 3.
time after administration (hours
US 8,841,255 B2
1. 2
THERAPEUTICAGENTS COMPRISING sequences of the elastin protein (elastin-like-peptide or ELP).
FUSIONS OF VASOACTIVE INTESTINAL Such elastic peptide components provide certain therapeutic
PEPTIDE AND ELASTIC PEPTIDES advantages to the therapeutic agent, such as comparatively
better stability, solubility, bioavailability, half-life, persis
PRIORITY tence, and/or biological action of the therapeutic proteina
ceous component. Such properties may be determined, for
This application is a continuation-in-part of U.S. applica example, with respect to the therapeutic components
tion Ser. No. 12/493,912, filed Jun. 29, 2009, which claims unfused or unconjugated counterpart. In some embodiments,
priority to U.S. Provisional Application No. 61/076,221, filed the elastic peptide is an ELP that undergoes a reversible
Jun. 27, 2008, each of which is hereby incorporated by ref 10 inverse phase transition, which may impart additional practi
erence in its entirety. This application is also a continuation cal and/or therapeutic advantages. The invention further pro
in-part of U.S. application Ser. No. 12/158,190, which is a vides polynucleotides encoding the therapeutic agents of the
U.S. national stage of PCT/US06/048572, filed Dec. 20, invention, as well as methods of treatment or prophylaxis for
2006, which claims priority to U.S. Provisional Application certain biological conditions.
No. 60/751,896, filed Dec. 20, 2005, each of which is hereby 15 In a first aspect, the invention provides a therapeutic agent
incorporated by reference in its entirety. comprising an elastic peptide component and a therapeutic
proteinaceous component, as well as pharmaceutical compo
GOVERNMENT SUPPORT sitions containing the same for delivery to a subject or patient
in need. The therapeutic component may be selected from
This invention was made with Government support under active portions of the therapeutic proteins described herein,
grant number EB00188 and GM-061232 from National Insti including those listed in Table 1, or functional analogs
tutes of Health. The US Government has certain rights to this thereof. In certain embodiments, the therapeutic component
invention. is a GLP-1 receptor agonist, such as GLP-1, exendin-4, or a
functional analog thereof. Such therapeutic components are
DESCRIPTION OF THE TEXT FILE SUBMITTED 25 generally effective for, among other things, increasing insulin
ELECTRONICALLY secretion from the pancreas in a glucose-dependent manner.
In other embodiments, the therapeutic component is an insu
The contents of the text file submitted electronically here lin or functional analog thereof, which is generally effective
with are incorporated herein by reference in their entirety: A for promoting glucose uptake from the blood and storage
computer readable format copy of the Sequence Listing (file 30 within cells. In still other embodiments, the therapeutic com
name: PHAS 021 00US SubSeqList ST25.txt, date ponent is a Factor VII/VIIa or functional analog thereof,
recorded: Sep. 1, 2010, file size 50 kb). which is generally effective for promoting coagulation by
activation of Factor X or Factor IX.
BACKGROUND OF THE INVENTION The elastic peptide and therapeutic components may be
35 covalently coupled by various means, including chemical
Therapeutic proteins or peptides in their native state or coupling (e.g., conjugation) and recombinant fusion technol
when recombinantly produced can be labile molecules exhib ogy. In addition, the number of elastic peptide or therapeutic
iting, inter alia, short periods of serum stability, serum half components per molecule, and their respective positions
life (i.e., circulatory half-life), or limited persistence in the within the molecule, may vary as needed. The therapeutic
body. Such molecules can also be extremely labile when 40 agent may further include one or more spacer or linker moi
formulated, such as when formulated in aqueous solutions. eties, which in addition to providing the desired functional
In some instances, polyethylene glycol (PEG) conjugated independence of the elastic peptide and therapeutic compo
to a proteinaceous molecule results in a longer-acting, Sus nents, may optionally provide for additional functionalities,
tained activity of the molecule. PEG attachment, however, Such as a protease-sensitive feature to allow for proteolytic
can often substantially reduce or even destroy the protein's 45 release or activation of the therapeutic component. The thera
therapeutic activity. Therapeutic proteins and/or peptides peutic agent may further include one or more targeting com
have also been stabilized by fusion to certain proteins that are ponents such as, for example, a peptide or protein to target the
capable of extending serum half-life. For example, in some therapeutic agent to a particular cell type, e.g., a cancer cell,
instances, therapeutic proteins fused to albumin, transferrin, or to a particular organ.
and antibody fragments exhibit extended serum half-life 50 In a second aspect, the invention provides polynucleotides,
when compared to the therapeutic protein in the unfused state. Such polynucleotides comprising a nucleotide sequence
See U.S. Pat. No. 7,238,667 (particularly with respect to encoding a therapeutic agent of the invention. For example,
albumin conjugates), U.S. Pat. No. 7,176,278 (particularly the nucleotide sequence encodes an elastic peptide fusion
with respect to transferrin conjugates), and U.S. Pat. No. with a functional portion of at least one therapeutic protein
5,766,883, which are each hereby incorporated by reference 55 described herein, including those listed in Table 1 (or func
in their entireties. tional analog thereof). In certain embodiments, the therapeu
There remains a need in the art for more stable, longer tic component is a GLP-1 receptoragonist (including GLP-1
acting, and/or effective proteinaceous molecules. and exendin-4), insulin, Factor VII/VIIa, or functional analog
thereof. Such polynucleotides may further comprise addi
SUMMARY OF THE INVENTION 60 tional control element(s) operably linked to the nucleotide
sequence. Such as promoter elements and/or other transcrip
The present invention provides therapeutic agents com tion or expression-related signals. The polynucleotide may be
prising an elastic peptide component and a therapeutic pro inserted into various vectors, which may be useful for pro
teinaceous component. The elastic peptide component may duction of the therapeutic agent in host cells, including, for
form a spiral conformation, and/or may have an extended 65 example, bacterial and eukaryotic host cells.
structure relative to an alpha helix. The elastic peptide com In a third aspect, the invention provides a method for treat
ponent may be structurally related to, or derived from, ing or preventing a disease, disorder, or condition in a Subject,
US 8,841,255 B2
3 4
Such as in a mammalian patient, including a human patient. FIGS. 7A-B display a Factor VII-ELP1-90 fusion. FIG.7A
The method comprises administering an effective amount of depicts pPB0788, which encodes Factor VII-ELP1-90. FIG.
the therapeutic agent of the invention (or pharmaceutical 7B depicts the nucleotide and amino acid sequence of the
composition containing the same) to a Subject or patient in encoded fusion protein (SEQ ID NOS: 57 and 58, respec
need thereof. For example, the patient may be in need of an tively).
agent having a biological activity or preferred indication FIGS. 8A-B display an insulin-4/ELP fusion. FIG. 8A
listed herein (e.g., in Table 1). In certain embodiments depicts the nucleotide and amino acid sequence of an insulin
employing a GLP-1 receptor agonist/elastic peptide com (B, C, and A chains) having the ELP component cloned in
pound or employing an insulin/elastic peptide compound, the frame (SEQID NOS: 31 and 32). Primer sequences are indi
invention provides a method for treating one or more disor 10 cated (SEQ ID NOS: 47 and 48). FIG. 8B depicts plasmid
ders including type 1 or type 2 diabetes, hyperglycemia, and pET24d Insulin-ELP1-90 expressing the insulin/ELP fusion
impaired glucose tolerance. In certain other embodiments of FIG. 8A.
employing Factor VII/VIIa?elastic peptide compound, the FIG. 9 is a Western blot for FVII-ELP1-90 from transient
invention provides a method for treating one or more disor transfection of Freestyle HEK293, detected with mouse anti
ders including hemophilia, post-Surgical bleeding, anticoagu 15 human FVII monoclonal antibody. Lanes are: (1) culture
lation-induced bleeding, thrombocytopenia, factor VII defi media; (2) FVII ELP1-90 after purification by phase transi
ciency, factor XI deficiency, and intracranial hemorrhage. tion; and FVII control.
Various other aspects, features and embodiments of the FIG.10 is an SDS-PAGE showing recombinant production
invention will be more fully apparent from the following of an Exendin-4/ELP4-60 fusion. Lanes are: (M) Protein
disclosure and appended claims. markers: (1) Exendin-4 ELP4-60 from total lysate; (2) Exen
din-4 ELP4-60 from insoluble lysate; (3) Exendin-4 ELP4-60
BRIEF DESCRIPTION OF THE FIGURES from soluble lysate; (4) Exendin-4 ELP4-60 from 1st transi
tion (equal volume); (5) Exendin-4 ELP4-60 from 2nd tran
FIG. 1 depicts plasmid plT24d-ELP1-90, encoding an sition (concentrated); (6) Exendin-4 ELP4-60 from 3rd tran
elastin-like-peptide (ELP) component with a 10 unit VPGXG 25 sition (concentrated).
(SEQID NO:3) repeat motif, where guest positionX is V. G. FIG. 11 shows the activation of Factor X by FactorVIIa
and A in the ratio of 5:3:2. This motif is repeated eight times ELP1-90, and by Factor VIIa as a comparison. As shown,
with a final C-terminal 10-unit repeat where X is V. G, A, and FactorVIIa-ELP retains full activity.
W in the ratio 4:3:2:1. This ELP component is represented FIG. 12 shows that Factor VIIa-ELP1-90 has a long PK
generally as (VPGXG). 30 when administered by i.v. in rats. FactorVIIa has a T of
FIGS. 2A-B display an exendin-4/ELP fusion. FIG. 2A about 690 min. as compared to about 45-60 min. for Factor
depicts plasmid pET24d-Ex-4 ELP1-90 encoding an ELP VIIa.
component with VPGXG (SEQID NO:3) repeat motif (as in FIG. 13 shows the high in vitro activity of GLP1-ELP and
FIG. 1) cloned in frame with an N-terminal exendin-4 com Exendin-4-ELP, when compared to the activity of Exendin
ponent. FIG. 2B depicts the nucleotide and amino acid 35 peptide.
sequence of the exendin-4/ELP fusion (SEQID NOS: 23 and FIG. 14 shows that GLP1-ELP has a T of about 12.9
24). Primer sequences are indicated (SEQID NOS: 35-40). hours when administered by i.V. to rats, and a T of about 8.6
FIGS. 3A-B display an exendin-4 construct having an hours when administered subcutaneously (SQ).
N-terminal Tev cleavage site. FIG. 3A depicts the nucleotide FIG. 15 shows that GLP-1 ELP has a long half-life in
and amino acid sequence of an exendin-4 construct having an 40 rabbits of about 20 hours when administeredi.V., and about 24
N-terminal TeV (Tobacco Etch Virus cysteine protease) cleav hours when administered sub-cutaneously.
age site (SEQ ID NOS: 25 and 26). Primer sequences are FIG.16 shows sustained glycemic control in diabetic mice
indicated (SEQID NOS:38, 41, 42). FIG.3B also depicts the with GLP-1-ELP.
nucleotide and amino acid sequence of an exendin-4 con
struct having an N-terminal TeV cleavage site, but with an 45 DETAILED DESCRIPTION OF THE INVENTION
additional sequence N-terminal to the TeV cleavage site to
provide a better target for the protease (SEQID NOS: 27 and The present invention provides therapeutic agents com
28). Primer sequences are indicated (SEQ ID NOS: 38, prising an elastic peptide component and a therapeutic com
43.44). ponent. The therapeutic component may be selected from
FIGS. 4A-B display an exendin-4/ELP fusion with a DsbA 50 Table 1 (e.g., selected from a Therapeutic Protein, or func
leader sequence. FIG. 4A depicts the nucleotide and amino tional portion or functional analog thereof, listed in Table 1),
acid sequence of an exendin-4/ELP fusion as in FIGS. 1-3, but or described herein. In certain embodiments, the therapeutic
with a DsbA leader sequence to direct secretion into the component is a GLP-1 receptor agonist, Such as GLP-1 or
periplasmic space (SEQ ID NOS: 29 and 30). Primer exendin-4, or may be insulin, Factor VII/VIIa, or functional
sequences are indicated (SEQID NOS:38, 45, 46). FIG. 4B 55 analog thereof. The elastic peptide component exhibits a flex
depicts plasmid pET24d-DsbA-Ex-4 ELP1-90 encoding the ibility and freedom of movement that results from its second
fusion of FIG. 4A. ary structure characteristics, and overall or Substantial lack of
FIGS.5A-B display a GLP-1 (A8G,7-37)/ELP1-90 fusion. a rigid tertiary structure. The elastic peptide components may
FIG.5A depicts pPB0868, which encodes GLP-1 (A8G,7-37) contain structural units related to, or derived from, sequences
ELP1-90. FIG. 5B depicts the nucleotide and amino acid 60 of the elastin protein. The elastic peptide provides certain
sequence of the encoded fusion protein (SEQID NOS: 53 and therapeutic advantages, such as comparatively better persis
54, respectively). tence, stability, solubility, bioavailability, half-life, and/or
FIGS.6A-B display a GLP-1(A8G,7-37)ELP1-120 fusion. biological action of the therapeutic component. Such proper
FIG. 6A depicts pPB1022, which encodes GLP-1 (A8G,7-37) ties may be determined with respect to, for example, an
ELP1-120. FIG. 6B depicts the nucleotide and amino acid 65 unfused or unconjugated counterpart of the therapeutic com
sequence of the encoded fusion protein (SEQID NOS:55 and ponent. The invention further provides polynucleotides
56, respectively). encoding the therapeutic agents of the invention, as well as
US 8,841,255 B2
5 6
methods of treatment or prophylaxis for certain biological example, the elastic peptide-coupled therapeutic component
conditions, including the preferred indications listed in Table may be enhanced in, e.g., its bioavailability, bio-unavailabil
1, and including diabetes (e.g., Type I and Type II), hyperg ity, therapeutically effective dose, biological action, formu
lycemia, bleeding, hemophilia, and hemorrhage, among oth lation compatibility, resistance to proteolysis or other degra
CS. dative modality, solubility, half-life or other measure of
For ease of reference in the ensuing discussion, set out persistence in the body Subsequent to administration, rate of
below are definitions of some terms appearing in the discus clearance from the body Subsequent to administration, etc.
Sion. Such enhancement may be determined, for example, in rela
As used herein, the term “therapeutic agent” or “therapeu tion to a corresponding unconjugated or unfused counterpart
tic component” refers to an agent or component capable of 10 therapeutic (e.g., determined relative to native GLP-1, exen
inducing a biological effect in vivo and/or in vitro. The bio din, insulin, or Factor VII/VIIa, or a therapeutic protein
logical effect may be useful for treating and/or preventing a described herein).
condition, disorder, or disease in a subject or patient. In some embodiments, the therapeutic agent of the inven
As used herein, the term “coupled' means that the specified tion circulates or exists in the body in a soluble form, and
components are either directly covalently bonded to one 15 escapes filtration by the kidney thereby persisting in the body
another (e.g., via chemical conjugation or recombinant fusion in an active form. In some embodiments, the therapeutic
technology), or indirectly covalently joined to one another agents of the invention have a molecular weight of less than
(e.g., via chemical conjugation or recombinant fusion tech the generally recognized cut-off for filtration through the
nology) through an intervening moiety or moieties, such as a kidney, such as less than about 60 kD. or in some embodi
bridge, spacer, or linker. ments less than about 55, 50, 45, 40, 30, or 20kDa, and persist
As used herein, “half-life' (which generally refers to in in the body by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,
vivo half-life or circulatory half-life) is the period of time that 20-fold, or 100-fold or longer than an uncoupled (e.g.,
is required for a 50% diminution of bioactivity of the active unfused or unconjugated) therapeutic counterpart.
agent to occur. Such term is to be contrasted with “persis The number of elastic peptide and/or therapeutic compo
tence,” which is the overall temporal duration of the active 25 nents per molecule, and their respective positions within the
agent in the body, and “rate of clearance' as being a dynami molecule, may vary among embodiments of the invention.
cally changing variable that may or may not be correlative For example, in embodiments where the agent is a recombi
with the numerical values of half-life and persistence. nant fusion, at least one elastic peptide component may be
The term “functional analog refers to a protein that is an placed at one or both of the N-terminus and the C-terminus.
active analog (e.g., either chemical or protein analog), deriva 30 Where the elastic peptide component is at both the N-termi
tive, fragment, truncation isoform or the like of a native nus and C-terminus of the fusion, the elastic peptide compo
protein. For example, the functional analog may be a func nents will flank the therapeutic component. Alternatively, the
tional analog of a therapeutic protein listed in Table 1, or may therapeutic component may be positioned at either or both of
be a functional analog of a GLP-1 receptor agonist (e.g., the N-terminus and C-terminus. Where the therapeutic com
GLP-1, exendin), insulin, or Factor VII/VIIa. A polypeptide is 35 ponent is at both the N-terminus and C-terminus, the thera
active when it retains some or all of the biological activity of peutic component will flank the elastic peptide component. In
the corresponding native polypeptide, as determined in vivo a further embodiment, different therapeutic components are
or in one or more indicative in vitro assays. Exemplary activ positioned at the N-terminus and C-terminus of the molecule.
ity assays for certain therapeutic proteins, which are determi As discussed in detail herein, in certain embodiments, such
native of activity, are listed Table 1. Further, such biological 40 therapeutic component(s) may be released by proteolysis of a
activities and assays for GLP-1 receptoragonists, insulin, and spacer moiety separating the elastic peptide and therapeutic
Factor VII/VIIa, which are determinative of whether a given components. In certain embodiments, the therapeutic com
molecule is a “functional analog, are described in detail ponent may be inactive in the fused State, and becoming active
elsewhere herein. upon proteolytic release from the elastic peptide
As used herein, the term “native as used in reference to an 45 component(s). Alternatively, the therapeutic component
amino acid sequence, indicates that the amino acid sequence remains active in the fused State, making proteolytic process
is found in a naturally-occurring protein. ing of the therapeutic agent unnecessary for biological activ
As used herein, the term “spacer” refers to any moiety, ity.
peptide or other chemical entity, that may be interposed When prepared as recombinant fusions, the therapeutic
between the elastic peptide component and the therapeutic 50 agent can be prepared by known recombinant expression
component. For example, the spacer may be a divalent group techniques. For example, to recombinantly produce the thera
that is covalently bonded at one terminus to the elastic peptide peutic agent, a nucleic acid sequence encoding the chimeric
component, and covalently bonded at the other terminus to gene is operatively linked to a Suitable promoter sequence
the therapeutic component. The therapeutic agents may there Such that the nucleic acid sequence encoding Such fusion
fore be open to the inclusion of additional chemical structure 55 protein will be transcribed and/or translated into the desired
that does not preclude the efficacy of the agent for its intended fusion protein in the host cells. Preferred promoters are those
purpose. The spacer may, for example, be a protease-sensitive useful for expression in E. coli, such as the T7 promoter. Any
spacer moiety that is provided to control the pharmacokinet commonly used expression system may be used, including
ics of the agent, or the spacer may be a protease-resistant eukaryotic or prokaryotic systems. Specific examples include
moiety. 60 yeast (e.g., Saccharomyces spp., Pichia spp.), baculovirus,
The therapeutic component and the elastic peptide compo mammalian, and bacterial systems, such as E. coli, and Cau
nent may be coupled with one another in any Suitable covalent lobacter:
manner, including chemical coupling and recombinant tech The various aspects and embodiments of the invention are
nology, Such that the therapeutic agent is efficacious for its described in greater detail in the following sections.
intended purpose, and Such that the presence of the elastic 65 Elastic Peptide Component
peptide component enhances the therapeutic component in The therapeutic agent of the invention may comprise one or
Some functional, therapeutic or physiological aspect. For more elastic peptide components. The elastic peptide compo
US 8,841,255 B2
7 8
nents may comprise or consist of structural peptide units or gen bonding to other parts of the molecule or to other mol
sequences that are related to, or derived from, the elastin ecules. Otherwise a greater energy barrier to the libration
protein (e.g., elastin-like-peptides, or ELPs). Elastic peptides exists and motion will be restricted. Since non-hydrogen
are useful for improving the properties of therapeutic pro bonded segments having freedom of motion exist in the 3-spi
teins, such as those described herein (e.g., listed in Table 1), 5 ral between the points of hydrogen bonding for the B-turns,
including GLP-1 receptor agonists (e.g., GLP-1 or exendin these segments may be said to be librationally Suspended.
4), insulin, and Factor VII/VIIa in one or more of bioavail Librationally suspended segments therefore are a structural
ability, therapeutically effective dose, biological action, for feature that exists in certain elastic peptides because of the
mulation compatibility, resistance to proteolysis, Solubility, repeating 3-turns with relative infrequent hydrogen bonding.
half-life or other measure of persistence in the body subse- 10 Librationally suspended segments resulting from the B-spiral
quent to administration, and/or rate of clearance from the structure are thought to give rise to elasticity, as will be further
body.
The elastic peptide component may be constructed from discussed.
structural units of from three to about twenty amino acids, or Another factor leading to the high librational freedom of
in some embodiments, from four to ten amino acids, Such as 15 Such molecules is the absence of significant polar interactions
five or six amino acids. The length of the individual structural between the amino acid residues, either intrachain or inter
units, may vary or may be uniform. In certain embodiments, chain, other than a hydrogen bond within the B-turn. The
the elastic peptide component is constructed of a polytetra-, amino acid residues present are mostly hydrophobic or gly
polypenta-, polyhexa-, polyhepta-, polyocta, and polynon cine and accordingly do not exert significant forces on one
apeptide motif of repeating structural units. Exemplary struc- 20 another through space. If a significant number of charged or
tural units include units defined by SEQID NOS: 1-12 (be polar groups were present, electrostatic interactions might
low), which may be employed as repeating structural units, limit librational freedom and restrict the number of available
including tandem-repeating units, or may be employed in states in the relaxed (non-extended) form of the molecules.
Some combination, to create a peptide component effective Polar and charged amino acid residues are not strictly prohib
for improving the properties of the therapeutic component. 25 ited, however, if their presence does not destroy the elasticity
Thus, the elastic peptide component may comprise or consist of the elastic peptide component as a whole. For example, an
occasional serine residue is present in naturally occurring
essentially of structural unit(s) selected from SEQID NOS: tropoelastin
1-12, as defined below. without destroying elasticity. Accordingly,
The elastic peptide component, comprising such structural hydrophobic amino acid residues and glycines are preferred
units, may be of varying sizes. For example, the elastic pep- 30 in forming elastomeric polypeptides of the present type
tide component may comprise or consist essentially of from although other amino acids may be present to a some extent.
about 10 to about 500 structural units, or in certain embodi Although not intending to be boundby theory, the elasticity
ments about 15 to about 150 structural units, or in certain of polypeptides of the B-turn structure may be caused by
embodiments from about 20 to about 100 structural units, or thermodynamic drive toward greater entropy. The relaxed
from about 50 to about 90 structural units, including one or a 35 state of the B-spiral has a large degree of librational freedom
combination of units defined by SEQID NOS: 1-12. Thus, the and thus the atoms of the peptide chain can exist in a large
elastic peptide component may have a length of from about 50 number of positions. When the molecules are stretched, the
to about 2000 amino acid residues, or from about 100 to about degree of freedom is reduced, particularly for librational
600 amino acid residues, or from about 200 to about 500 motions, and when the tension is released, a thermodynamic
amino acid residues, or from about 200 to about 400 amino 40 driving force toward higher entropy results in reformation of
acid residues. the contracted B-spiral.
Elastic polymers (e.g., bioelastic polymers) are known and Other specific bioelastic polymers that can be used to carry
described in, for example, U.S. Pat. No. 5,520,672 to Urry et out the present invention are described in U.S. Pat. Nos.
al. In general, elastic peptides comprise elastomeric units of 4,132,746, 4,187,852, 4,500,700, 4,589,882, and 4,870,055,
bioelastic pentapeptides, tetrapeptides, and/or nonapeptides 45 each of which are hereby incorporated by reference. Still
(e.g., elastin-like peptides). Thus, in Some embodiments the other examples of bioelastic polymers are set forth in U.S.
elastomeric unit is a pentapeptide, in other embodiments the Pat. No. 6,699,294, U.S. Pat. No. 6,753,311, and U.S. Pat. No.
6,063,061, which are also incorporated by reference in their
elastomeric unit is a tetrapeptide, and in still other embodi entirety.
ments the elastomeric unit is a nonapeptide. Bioelastic poly
mers that may be used to carry out the present invention are 50 In some embodiments, the (3-turn may have the following
set forth in U.S. Pat. No. 4,474,851, which is hereby incor structure, in the formation of a B-spiral:
porated by reference in its entirety.
As disclosed in U.S. Pat. No. 4,474,851, elastomeric pep
tides may have a sequence of regularly appearing B-turns,
forming an overall spiral conformation (e.g., a B-spiral, which 55
is a series of regularly repeating 3-turns). The spiral structures
are more open than the more common C.-helix. As a result, the O =o
atoms in the peptide backbone have a high freedom of move O Rs O R4 NH
ment (e.g., as compared to the freedom of movement for an | | H H
C.-helix). This is particularly true of librational motions 60 C-C-N-HC-C-N-C-CH
H iii. H |
involving peptide moieties. A libration is a torsional oscilla O R3
tion involving simultaneous rotational motions of the two
single bonds on each side of a librating moiety. The moiety
involved in a libration may be a single peptide bond or several wherein R1-R5 represent side chains of amino acid residues
peptide residues. For adequate freedom of motion to exist, it 65 1-5, and m is 0 when the repeating unit is a tetrapeptide or 1
is important, however, that the carbonyl oxygen and the when the repeating unit is a pentapeptide. Nonapeptide
amino hydrogen of the peptide bond not be involved in hydro repeating units generally consist of sequential tetra- and pen
US 8,841,255 B2
10
tapeptides. The amino acid residues may be hydrophobic inverse transition cycling procedures, e.g., utilizing the tem
amino acid residues, such as those independently selected perature-dependent solubility of the therapeutic agent, or salt
from alanine, Valine, leucine, isoleucine, proline, phenylala addition to the medium. Successive inverse phase transition
nine, tryptophan, and methionine. In many cases, the first cycles can be used to obtain a high degree of purity. In addi
amino acid residue of the repeating unit is a residue of valine, tion to temperature and ionic strength, other environmental
leucine, isoleucine or phenylalanine; the second amino acid variables useful for modulating the inverse transition of the
residue is a residue of proline; the third amino acid residue is therapeutic agents include pH, the addition of inorganic and
a residue of glycine; and the fourth amino acid residue is organic Solutes and solvents, side-chain ionization or chemi
glycine or a hydrophobic residue such as tryptophan, pheny cal modification, and pressure.
lalanine or tyrosine. 10 In certain embodiments, the ELP component does not
In some embodiments, the elastic peptide component, or in undergo a reversible inverse phase transition, or does not
Some cases the therapeutic agent, has a size of less than about undergo Such a transition at a biologically relevant Tt, and
65 kDa, or less than about 60 kDa, or less than about 55 kDa, thus the improvements in the biological and/or physiological
or less than about 50 kDa, or less than about 40 kDa, or less properties of the molecule (as described elsewhere herein),
than about 30 or 25 kDa. Three major blood proteins, Human 15 may be entirely or Substantially independent of any phase
Serum Albumin (HSA), Transferrin (Tf) and IgG, or the Fc transition properties. Nevertheless, such phase transition
portion of IgGs in their glycosylated form, have been properties may impart additional practical advantages, for
exploited to extend the half-lives of proteins and peptides for example, in relation to the recovery and purification of Such
improved therapeutic use. These molecules are 585, 679 and molecules.
480 amino acids in length giving molecular weights of about In certain embodiments, the ELP component(s) may be
66, 77, and -75kDa (including glycosylations), respectively. formed of structural units, including but not limited to:
They are each globular and relatively compact. The halflife of (a) the tetrapeptide Val-Pro-Gly-Gly, or VPGG (SEQ ID
these molecules is determined by a number of factors, includ NO: 1);
ing charge distribution, rescue of molecules by the neonatal (b) the tetrapeptide Ile-Pro-Gly-Gly, or IPGG (SEQ ID
Fc receptor (FcRn) (HSA and Fc) or cycling of Tfthrough the 25 NO:2):
Tf receptor (TfR), and their size which prevents filtering (c) the pentapeptide Val-Pro-Gly-X-Gly (SEQ ID NO:3),
through the kidney glomerulus. HSA is slightly below the or VPGXG, where X is any natural or non-natural amino
generally regarded cut-off for filtration through the kidney acid residue, and where X optionally varies among poly
(~70 kDa) but its charge distribution helps prevent this. It meric or oligomeric repeats;
would be anticipated that, in order to achieve half-life exten 30 (d) the pentapeptide Ala-Val-Gly-Val-Pro, or AVGVP
sion of the same order as that achieved with HSA, Tfand Fc. (SEQID NO: 4):
a protein of at least this molecular weight range would be (e) the pentapeptide Ile-Pro-Gly-X-Gly, or IPGXG (SEQ
required or desirable, i.e. having over 550 amino acids and ID NO. 5), where X is any natural or non-natural amino
being over 65 kDa. However, an elastic peptide with a small acid residue, and where X optionally varies among poly
number of amino acids relative to HSA, Tfand Fc (e.g., in the 35 meric or oligomeric repeats;
range of about 300 to 400) and around 30 to 40 kDa may have (e) the pentapeptide Ile-Pro-Gly-Val-Gly, or IPGVG (SEQ
a half life that matches and/or exceeds that of HSA, Tf, and ID NO: 6):
Fc. (f) the pentapeptide Leu-Pro-Gly-X-Gly, or LPGXG (SEQ
Thus, in some embodiments, the elastic peptide component ID NO: 7), where X is any natural or non-natural amino
may have an extended, relatively unstructured (e.g., no defini 40 acid residue, and where X optionally varies among poly
tive tertiary structure due to rotational and/or librational free meric or oligomeric repeats;
dom of the peptide backbone) and non-globular form, and (g) the pentapeptide Leu-Pro-Gly-Val-Gly, or LPGVG
thus such molecules may have a large expanded structure in (SEQID NO: 8):
comparison to HSA, Tf and Fc, so as to escape kidney filtra (h) the hexapeptide Val-Ala-Pro-Gly-Val-Gly, O
tion. In Such embodiments, the therapeutic agents of the 45 VAPGVG (SEQID NO: 9);
invention have a molecular weight of less than the generally (I) the octapeptide Gly-Val-Gly-Val-Pro-Gly-Val-Gly, or
recognized cut-off for filtration through the kidney, Such as GVGVPGVG (SEQID NO: 10);
less than about 60 kD, or in some embodiments less than (J) the nonapeptide Val-Pro-Gly-Phe-Gly-Val-Gly-Ala
about 55, 50, 45, 40, 30, or 25kDa, and persist in the body by Gly, or VPGFGVGAG (SEQID NO: 11); and
at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 50 (K) the nonapeptides Val-Pro-Gly-Val-Gly-Val-Pro-Gly
100-fold longer than an uncoupled (e.g., unfused or uncon Gly, or VPGVGVPGG (SEQ ID NO:12).
jugated) therapeutic counterpart. Such structural units defined by SEQID NOS:1-12 may form
In certain embodiments, the elastic peptide component is structural repeat units, or may be used in combination to form
an ELP that undergoes a reversible inverse phase transition. an ELP component in accordance with the invention. In some
ELP components are structurally disordered and highly 55 embodiments, the ELP component is formed entirely (or
soluble in water below a transition temperature (Tt), but almost entirely) of one or a combination of (e.g., 2, 3 or 4)
exhibit a sharp (2-3°C. range) disorder-to-order phase tran structural units selected from SEQ ID NOS: 1-12. In other
sition when the temperature is raised above the Tt, leading to embodiments, at least 75%, or at least 80%, or at least 90% of
desolvation and aggregation of the ELP components. For the ELP component is formed from one or a combination of
example, the ELP forms insoluble polymers, when reaching 60 structural units selected from SEQID NOS: 1-12, and which
sufficient size, which can be readily removed and isolated may be present as repeating units.
from Solution by centrifugation. Such phase transition is In certain embodiments, the ELP component(s) contain
reversible, and isolated insoluble ELPs can be completely repeat units, including tandem repeating units, of the pen
resolubilized in buffer solution when the temperature is tapeptide Val-Pro-Gly-X-Gly (SEQID NO:3), where X is as
returned below the Tt of the ELPs. Thus, the therapeutic 65 defined above, and where the percentage of Val-Pro-Gly-X-
agents of the invention can, in some embodiments, be sepa Gly (SEQID NO:3) pentapeptide units taken with respect to
rated from other contaminating proteins to high purity using the entire ELP component (which may comprise structural
US 8,841,255 B2
11 12
units other than VPGXG (SEQ ID NO:3)) is greater than one or more amino acids also do not eliminate or Substantially
about 75%, or greater than about 85%, or greater than about affect the phase transition properties where ELP components
95% of the ELP component. The ELP component may con are employed (relative to the deletion of such one or more
tain motifs having a 5 to 15-unit repeat (e.g. about 10-unit amino acids).
repeat) of the pentapeptide of SEQID NO:3, with the guest For ELP sequences, in each repeat, X is independently
residue X varying among at least 2 or at least 3 of the units. selected. The structure of the resulting ELP components may
The guest residues may be independently selected, such as be described using the notation ELPk[X,Y-n), where k des
from the amino acids V.I., L, A, G, and W (and may be selected ignates a particular ELP repeat unit, the bracketed capital
So as to retaina desired inverse phase transition property). The letters are single letter amino acid codes and their correspond
repeat motif itself may be repeated, for example, from about 10 ing Subscripts designate the relative ratio of each guest resi
5 to about 12 times, such as about 8 to 10 times, to create an due X in the structural units (where applicable), and n
exemplary ELP component. The ELP component as describes the total length of the ELP in number of the struc
described in this paragraph may of course be constructed tural repeats. For example, ELP1VAG-10 designates an
from any one of the structural units defined by SEQID NOS: ELP component containing 10 repeating units of the pen
1-12, or a combination thereof. 15 tapeptide VPGXG (SEQ ID NO:3), where X is valine, ala
In some embodiments, the ELP component may include a nine, and glycine at a relative ratio of 5:2:3: ELP1 KVF-4
3-turn structure. Exemplary peptide sequences Suitable for designates an ELP component containing 4 repeating units of
creating a B-turn structure are described in International the pentapeptide VPGXG (SEQID NO:3), where X is lysine,
Patent Application PCT/US96/05186, which is hereby incor valine, and phenylalanine at a relative ratio of 1:2:1; ELP1
porated by reference in its entirety. For example, the fourth KV-7F-9 designates a polypeptide containing 9 repeating
residue (X) in the elastin pentapeptide sequence, VPGXG units of the pentapeptide VPGXG (SEQID NO:3), where X is
(SEQ ID NO:3), can be altered without eliminating the for lysine, Valine, and phenylalanine at a relative ratio of 1:7:1;
mation of a B-turn. Alternatively, the ELP component may ELP1 V-5 designates a polypeptide containing 5 repeating
lack a B-turn, or otherwise have a different conformation units of the pentapeptide VPGXG (SEQID NO:3), where X is
and/or folding character. 25 exclusively valine; ELP1 V-20 designates a polypeptide
In certain embodiments, the ELP components include containing 20 repeating units of the pentapeptide VPGXG
polymeric or oligomeric repeats of the pentapeptide VPGXG (SEQ ID NO:3), where X is exclusively valine; ELP2 (5)
(SEQID NO:3), where the guest residue X is any amino acid. designates a polypeptide containing 5 repeating units of the
X may be a naturally occurring or non-naturally occurring pentapeptide AVGVP (SEQ ID NO:4); ELP3 V-5) desig
amino acid. In some embodiments, X is selected from ala 30 nates a polypeptide containing 5 repeating units of the pen
nine, arginine, asparagine, aspartic acid, cysteine, glutamic tapeptide IPGXG (SEQ ID NO:5), where X is exclusively
acid, glutamine, glycine, histidine, isoleucine, leucine, valine; ELP4 V-5 designates a polypeptide containing 5
lysine, methionine, phenylalanine, serine, threonine, tryp repeating units of the pentapeptide LPGXG (SEQID NO:7),
tophan, tyrosine and Valine. In some embodiments, X is a where X is exclusively valine. Such ELP components as
natural amino acid other than proline or cysteine. 35 described in this paragraph may be used in connection with
The guest residue X (e.g., with respect to SEQID NO:3, or the present invention to increase the therapeutic properties of
other ELP structural unit) may be a non-classical (non-ge the therapeutic component.
netically encoded) amino acid. Examples of non-classical Further, the Tt is a function of the hydrophobicity of the
amino acids include: D-isomers of the common amino acids, guest residue. Thus, by varying the identity of the guest resi
2,4-diaminobutyric acid, C.-amino isobutyric acid, A-ami 40 due(s) and their mole fraction(s), ELPs can be synthesized
nobutyric acid, Abu, 2-amino butyric acid, Y-Abu, e-Ahk. that exhibit an inverse transition over a 0-100° C. range. Thus,
6-amino hexanoic acid, Aib, 2-amino isobutyric acid, the Tt at a given ELP length may be decreased by incorporat
3-amino propionic acid, ornithine, norleucine, norvaline, ing a larger fraction of hydrophobic guest residues in the ELP
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic sequence. Examples of Suitable hydrophobic guest residues
acid, t-butylglycine, t-butylalanine, phenylglycine, cyclo 45 include valine, leucine, isoleucine, phenyalanine, tryptophan
hexylalanine, B-alanine, fluoro-amino acids, designer amino and methionine. Tyrosine, which is moderately hydrophobic,
acids such as B-methylamino acids, CC-methylamino acids, may also be used. Conversely, the Tt may be increased by
NC.-methylamino acids, and amino acid analogs in general. incorporating residues, such as those selected from the group
Selection of X is independent in each ELP structural unit consisting of glutamic acid, cysteine, lysine, aspartate, ala
(e.g., for each structural unit defined herein having a guest 50 nine, asparagine, serine, threonine, glycine, arginine, and
residueX). For example, X may be independently selected for glutamine; preferably selected from alanine, serine, threonine
each structural unit as an amino acid having a positively and glutamic acid.
charged side chain, an amino acid having a negatively The ELP component in some embodiments is selected or
charged side chain, or an amino acid having a neutral side designed to provide a Tt ranging from about 10 to about 80°
chain, including in Some embodiments, a hydrophobic side 55 C., such as from about 35 to about 60°C., or from about 38 to
chain. about 45° C. In some embodiments, the Tt is greater than
In still other embodiments, the ELP component(s) may about 40° C. or greater than about 42° C., or greater than
include polymeric or oligomeric repeats of the pentapeptides about 45° C., or greater than about 50° C. The transition
VPGXG (SEQID NO:3), IPGXG(SEQID NO:5) or LPGXG temperature, in some embodiments, is above the body tem
(SEQ ID NO:7), or a combination thereof, where X is as 60 perature of the subject or patient (e.g., >37° C.) thereby
defined above. remaining soluble in vivo, or in other embodiments, the Tt is
In each embodiment, the structural units, or in Some cases below the body temperature (e.g., <37°C.) to provide alter
polymeric or oligomeric repeats, of the elastic peptide native advantages, such as in vivo formation of a drug depot
sequences may be separated by one or more amino acid for Sustained release of the therapeutic agent.
residues that do not eliminate the overall effect of the mol 65 The Tt of the ELP component can be modified by varying
ecule, that is, in imparting certain improvements to the thera ELP chain length, as the Tt generally increases with decreas
peutic component as described. In certain embodiments. Such ing MW. For polypeptides having a molecular weightD100,
US 8,841,255 B2
13 14
000, the hydrophobicity scale developed by Urry et al. (PCT/ Vancomycin; desferrioxamine (DFO); parathyroid hormone,
US96/05186, which is hereby incorporated by reference in its an immunogen or antigen, an antibody such as a monoclonal
entirety) is preferred for predicting the approximate Tt of a antibody.
specific ELP sequence. However, in some embodiments, ELP Where the therapeutic component is an antibody or anti
component length can be kept relatively small, while main body sequence, the antibody may be of any isotype, including
taining a target Tt, by incorporating a larger fraction of hydro IgG, IgM, IgA, Ig), and IgE. Where the antibody is IgG, the
phobic guest residues (e.g., amino acid residues having subtype may be IgG1, IgG2, IgG3, or IgG4. The antibody
hydrophobic side chains) in the ELP sequence. For polypep sequence may be humanized or chimeric. The term “anti
tides having a molecular weight<100,000, the Tt may be body' as used herein includes antibody fragments or seg
predicted or determined by the following quadratic function: 10 ments that retain the capability of binding to a target antigen,
Tt-M+MX+MX where X is the MW of the fusion pro for example, Fab, F(ab')2, and FV fragments, and the corre
sponding fragments obtained from antibodies other than IgG.
tein, and M-116.21; M=-1.7499; M=0.010349. Examples of therapeutic antibodies include but are not lim
While the Tt of the ELP component, and therefore of the ited to herceptin, rituxan, campath, gemtuzumab, herceptin,
ELP component coupled to a therapeutic component, is 15 panorex, rituximab, beXXar, edrecolomab, alemtuzumab,
affected by the identity and hydrophobicity of the guest resi mylotrag, IMC-C225, Smartin 195, and mitomomab.
due, X, additional properties of the molecule may also be The therapeutic component may also be a therapeutic com
affected. Such properties include, but are not limited to solu ponent listed in Table 1 (e.g., full length or functional portions
bility, bioavailability, persistence, and half-life of the mol or functional analogs thereof), as well as GLP-1 receptor
ecule. agonists such as GLP-1 or exendin-4, insulin, or Factor VII/
As described in PCT/US2007/077767 (published as WO VIIa, and functional analogs thereof. The structure and activ
2008/030968), which is hereby incorporated by reference in ity of Such therapeutic components are described in detail
its entirety, the ELP-coupled therapeutic component can below. In some forms of the therapeutic agent, the coupling of
retain the therapeutic component's biological activity. Addi the therapeutic component to the elastic peptide is effected by
tionally, ELPs themselves can exhibit long half-lives. There 25 direct covalent bonding or indirect (through appropriate
fore, ELP components in accordance with the present inven spacer groups) bonding (as described elsewhere herein). Fur
tion substantially increase (e.g. by greater than 10%. 20%, ther, the therapeutic component(s) and the elastic peptide
30%, 50%, 100%, 200%, 500% or more, in specific embodi component(s) can be structurally arranged in any Suitable
ments) the half-life of the therapeutic component when con manner involving Such direct or indirect covalent bonding,
30 relative to one another.
jugated thereto. Such half-life (or in some embodiments per Glucagon-Like Peptide (GLP)-1 Receptor Agonists
sistence or rate of clearance) is determined in comparison to In certain embodiments of the invention, the therapeutic
the half-life of the free (unconjugated or unfused) form of the agent comprises an ELP component fused or conjugated to a
therapeutic component. Furthermore, ELPs may target high GLP-1 receptor agonist, Such as GLP-1, exendin-4, or func
blood content organs, when administered in vivo, and thus, 35 tional analogs thereof.
can partition in the body, to provide a predetermined desired Human GLP-1 is a 37 amino acid residue peptide originat
corporeal distribution among various organs or regions of the ing from preproglucagon which is synthesized in the L-cells
body, or a desired selectivity or targeting of a therapeutic in the distal ileum, in the pancreas, and in the brain. Process
agent. In Sum, the therapeutic agents contemplated by the ing of preproglucagon to give GLP-1 (7-36)amide, GLP-1
invention are administered or generated in vivo as active 40 (7-37) and GLP-2 occurs mainly in the L-cells. A simple
compositions having extended half-lives (e.g., circulatory system is used to describe fragments and analogs of this
half-life), among other potential benefits described herein. peptide. For example, Gly-GLP-1 (7-37) designates a frag
The invention thus provides various agents for therapeutic ment of GLP-1 formally derived from GLP-1 by deleting the
(in vivo) application, where the therapeutic component is amino acid residues Nos. 1 to 6 and Substituting the naturally
biologically active. Such therapeutic components include, 45 occurring amino acid residue in position 8 (Ala) by Gly.
without limitation, growth hormone (GH) particularly human Similarly, Lys (N-tetradecanoyl)-GLP-1 (7-37) designates
and bovine growth hormone, growth hormone-releasing hor GLP-1 (7-37) wherein the e-amino group of the Lys residue in
mones; interferon including C-. B-, or Y-interferons, etc., inter position 34 has been tetradecanoylated. Where reference in
leukin-1, interleukin-II; erythropoietin including C- and this text is made to C-terminally extended GLP-1 analogues,
B-erythropoietin (EPO), granulocyte colony stimulating fac 50 the amino acid residue in position 38 is Arg unless otherwise
tor (GCSF), granulocyte macrophage colony stimulating fac indicated, the optional amino acid residue in position 39 is
tor (GM-CSF), anti-angiogenic proteins (e.g., angiostatin, also Arg unless otherwise indicated and the optional amino
endostatin) PACAP polypeptide (pituitary adenylate cyclase acid residue in position 40 is Asp unless otherwise indicated.
activating polypeptide), vasoactive intestinal peptide (VIP), Also, ifa C-terminally extended analogue extends to position
thyrotrophin releasing hormone (TRH), corticotropin releas 55 41, 42, 43, 44 or 45, the amino acid sequence of this extension
ing hormone (CRH), vasopressin, arginine vasopressin is as in the corresponding sequence in human preproglucagon
(AVP), angiotensin, calcitonin, atrial naturetic factor, Soma unless otherwise indicated.
to statin, adrenocorticotropin, gonadotropin releasing hor The parent peptide of GLP-1, proglucagon (PG), has sev
mone, oxytocin, insulin, Somatotropin, plasminogen tissue eral cleavage sites that produce various peptide products
activator, coagulation factors including coagulation factors 60 dependent on the tissue of origin including glucagon (PG32
VIII and IX, glucosylceramidase, SargramoStim, lenograstin, 62) and GLP-17-36NH. (PG|72-107) in the pancreas, and
filgrastin, dornase-C, molgramostim, PEG-L-asparaginase, GLP-17-37 (PG|78-108) and GLP-17-36NH. (PG 78
PEG-adenosine deaminase, hirudin, eptacog-C. (human blood 107) in the L cells of the intestine where GLP-17-36NH
coagulation factor VIIa) nerve growth factors, transforming (78-107 PG) is the major product. The GLP-1 component in
growth factor, epidermal growth factor, basic fibroblast 65 accordance with the invention may be any biologically active
growth factor, VEGF; heparin including low molecular product or derivative of proglocagon, or functional analog
weight heparin, calcitonin; antigens; monoclonal antibodies; thereof, including: GLP-1 (1-35), GLP-1(1-36), GLP-1
US 8,841,255 B2
15 16
(1-36)amide, GLP-1 (1-37), GLP-1 (1-38), GLP-1 (1-39), which is hereby incorporated by reference in its entirety.
GLP-1 (1-40), GLP-1 (1-41), GLP-1 (7-35), GLP-1 (7-36), Other fragments and modified sequences of GLP-1 are known
GLP-1 (7-36)amide, GLP-1 (7-37), GLP-1 (7-38), GLP-1 in the art (U.S. Pat. No. 5.614492; U.S. Pat. No. 5,545,618:
(7-39), GLP-1 (7-40) and GLP-1 (7-41), or a analog of the European Patent Application, Publication No. EP 0658568
foregoing. Generally, the GLP-1 component in Some embodi 5 A1: WO93/25579, which are hereby incorporated by refer
ments may be expressed as GLP-1 (A-B), where A is an ence in their entireties). Such fragments and modified
integer from 1 to 7 and B is an integer from 38 to 45, option sequences may be used in connection with the present inven
ally with one or more amino acid Substitutions as defined tion, as well as those described below.
below. Certain structural and functional analogs of GLP-1 have
As an overview, after processing in the intestinal L-cells, 10 been isolated from the venom of the Gila monster lizards
GLP-1 is released into the circulation, most notably in (Heloderma suspectum and Heloderma horridum) and have
response to a meal. The plasma concentration of GLP-1 rises shown clinical utility. Such molecules find use in accordance
from a fasting level of approximately 15 pmol/L to a peak with the present invention. In particular, exendin-4 is a 39
postprandial level of 40 pmol/L. For a given rise in plasma amino acid residue peptide isolated from the venom of Helo
glucose concentration, the increase in plasma insulin is 15 derma suspectum and shares approximately 52% homology
approximately threefold greater when glucose is adminis with human GLP-1. Exendin-4 is a potent GLP-1 receptor
tered orally compared with intravenously (Kreymann et al., agonist that stimulates insulin release, thereby lowering
1987, Lancet 2(8571): 1300-4). This alimentary enhance blood glucose levels. Exendin-4 has the following amino acid
ment of insulin release, known as the incretin effect, is pri sequence: HGEGTFTSDLSKQMEEEAVRLFEWLKNG
marily humoral and GLP-1 is now thought to be the most GPSSGAPPPS (SEQ ID NO: 14). A synthetic version of
potent physiological incretin in humans. GLP-1 mediates exendin-4 known as exenatide (marketed as Byetta R) has
insulin production via binding to the GLP-1 receptor, known been approved for the treatment of Type-2 Diabetes.
to be expressed in pancreatic B cells. In addition to the insuli Although exenatide is structurally analogous to native GLP
notropic effect, GLP-1 Suppresses glucagon Secretion, delays 1, it has a longer half-life after injection.
gastric emptying (Wettergen et al., 1993, Dig Dis Sci 38: 25 While exenatide has the ability to lower blood glucose
665-73) and may enhance peripheral glucose disposal levels on its own, it can also be combined with other medica
(D’Alessio et al., 1994, J. Clin Invest 93: 2293-6). tions such as metformin, a thioZolidinedione, a Sulfonylureas,
A combination of actions gives GLP-1 unique therapeutic and/or insulin to improve glucose control. Exenatide is
advantages over other agents currently used to treat non administered by injection Subcutaneously twice per day using
insulin-dependent diabetes mellitus (NIDDM). First, a single 30 a pre-filled pen device. Typical human responses to exenatide
Subcutaneous dose of GLP-1 can completely normalize post include improvements in the initial rapid release of endog
prandial glucose levels in patients with NIDDM (Gutniak et enous insulin, an increase in B-cell growth and replication,
al., 1994, Diabetes Care 17: 1039-44). This effect may be Suppression of pancreatic glucagon release, delayed gastric
mediated both by increased insulin release and by a reduction emptying, and reduced appetite-all of which function to
in glucagon Secretion. Second, intravenous infusion of 35 lower blood glucose. Unlike Sulfonylureas and meglitinides,
GLP-1 can delay postprandial gastric emptying in patients exenatide increases insulin synthesis and secretion in the
with NIDDM (Williams et al., 1996, J. Clin Endo Metab 81: presence of glucose only, thus lessening the risk of hypogly
327-32). Third, unlike sulphonylureas, the insulinotropic cemia. Despite the therapeutic utility of exenatide, it has
action of GLP-1 is dependent on plasma glucose concentra certain undesirable traits, including the requirement of twice
tion (Holz et al., 1993, Nature 361:362-5). Thus, the loss of 40 daily injections, gastrointestinal side effects, and similar to
GLP-1-mediated insulin release at low plasma glucose con native GLP-1, a relatively short half-life (i.e. approximately 2
centration protects against severe hypoglycemia. hr).
When given to healthy subjects, GLP-1 potently influences Various functional analogs of GLP-1 and exendin-4 are
glycemic levels as well as insulin and glucagon concentra known, and which find use in accordance with the invention.
tions (Orskov, 1992, Diabetologia 35:701-11), effects which 45 These include liraglutide (Novo Nordisk, WO98/008871),
are glucose dependent (Weir et al., 1989, Diabetes 38: 338 R1583/taspoglutide (Roche, WO00/034331), CJC-1 131
342). Moreover, it is also effective in patients with diabetes (Conjuchem, WO00/069911), ZP-10/AVE0010 (Zealand
(Gutniak, M., 1992, N. Engl. Med 226: 1316-22), normaliz Pharma, Sanofi-Aventis, WO01/004156), and LY548806 (Eli
ing blood glucose levels in type 2 diabetic Subjects and Lilly, WO03/018516).
improving glycemic control in type 1 patients (Nauck et al., 50 Liraglutide, also known as NN2211, is a GLP-1 receptor
1993, Diabetologia 36: 741-4, Creutzfeldt et al., 1996, Dia agonistanalog that has been designed for once-daily injection
betes Care 19:580-6). (Harder et al., 2004, Diabetes Care 27: 1915-21). Liraglutide
GLP-1 is, however, metabolically unstable, having a has been tested in patients with type-2 diabetes in a number of
plasma half-life (t) of only 1-2 minutes in vivo. Moreover, studies and has been shown to be effective over a variety of
exogenously administered GLP-1 is also rapidly degraded 55 durations. In one study, treatment with liraglutide improved
(Deacon et al., 1995, Diabetes 44: 1126–31). This metabolic glycemic control, improved f-cell function, and reduced
instability has limited the therapeutic potential of native GLP endogenous glucose release in patients with type-2 diabetes
1. after one week of treatment (Degn et al., 2004, Diabetes 53:
GLP-17-36NH has the following amino acid sequence: 1187–94). In a similar study, eight weeks of 0.6-mgliraglutide
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID 60 therapy significantly improved glycemic control without
NO: 13), which may be employed as the GLP-1 component in increasing weight in Subjects with type 2 diabetes compared
accordance with the invention. Alternatively, the GLP-1 com with those on placebo (Harder et al., 2004, Diabetes Care 27:
ponent may contain glycine (G) at the second position, giv 1915-21).
ing, for example, the sequence HGEGTFTSDVS Thus, in certain embodiments, the GLP-1 receptoragonist
SYLEGOAAKEFIAWLVKGR (SEQ ID NO: 17). The 65 in accordance with the invention is as described in WO987
GLP-1 component may be a biologically active fragment of 008871, which is hereby incorporated by reference in its
GLP-1, for example, as disclosed in US 2007/0041951, entirety. The GLP-1 receptor agonist may have at least one
US 8,841,255 B2
17 18
lipophilic Substituent, in addition to one, two, or more amino Calif.). At the conclusion of the study, the percentages of
acid substitutions with respect to native GLP-1. For example, patients with Hb A1c-7% ranged from 47-69% for once daily
the lipophilic Substituent may be an acyl group selected from dosing compared to 32% for placebo. In addition, AVE0010
CH(CH2)CO , wherein n is an integer from 4 to 38, such treated patients showed dose-dependent reductions in weight
as an integer from 4 to 24. The lipophilic Substituent may be and post-prandial plasma glucose.
an acyl group of a straight-chain or branched alkyl or fatty Thus, in certain embodiments, the GLP-1 receptoragonist
acid (for example, as described in WO98/008871, which is as described in WO01/004156, which is hereby incorpo
description is hereby incorporated by reference). rated by reference in its entirety. For example, the GLP-1
In certain embodiments, the GLP-1 component is Arg receptor agonist may have the sequence:
GLP-1 (7-37), Arg-GLP-1 (7-37), Lys-GLP-1 (7-37), 10
Arg-Lys-GLP-I (7-37), Arg-Lys-GLP-I (7-38),
Arg: Lys-GLP-1 (7-39), Arg-Lys-GLP-1 (7-40), (SEQ ID NO: 18)
Arg'Lys-GLP-1 (7-37), Arg'Lys-GLP-1 (7-37), HGEGTFTSDLSKOMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2
(e.g. AVEO010).
Arg-Lys-GLP-1 (7-39), Arg'Lys-GLP-1 (7-40),
Arg-Lys-GLP-I (7-39), Arg-Lys-GLP-1 (7- 15 LY548.806 is a GLP-1 derivative designed to be resistant to
40), GlyArg-GLP-1 (7-37); GlyArg-GLP-1 (7-37): proteolysis by dipeptidase-peptidyl IV (DPP-IV) (Jackson et
GlyLys-GLP-1 (7-37); GlyArg-Lys-GLP-1 (7-37), al., Abstract No. 562, Jun. 10-14, 2005, 65th American Dia
GlyArg-Lys-GLP-1 (7-39), Gly-Arg-Lys-GLP-1 betes Association Meeting, San Francisco, Calif.). In an ani
(7-40), GlyArg-Lys-GLP-1 (7-37), GlyArg-Lys mal model of hyperglycemia, LY548.806 has been shown to
GLP-1(7-37), GlyArg-Lys-GLP-1 (7-39); produce a significant lowering of blood glucose levels during
GlyArg“Lys-GLP-1 (7-40), GlyArg-Lys--GLP-1 the hyperglycemic phase (Saha et al., 2006, J. Pharm. Exp.
(7-39) and GlyArg-Lys-GLP-1 (7-40), each option Ther. 316: 1159-64). Moreover, LY548.806 was shown to
ally having a lipophilic substituent. For example, the GLP-1 produce a significant increase in insulin levels consistent with
receptor agonist may have the sequence/structure its known mechanism of action, namely stimulation of insulin
Arg'Lys-(N-e-(y-GlucN-C-hexadecanoyl)))-GLP-I(7- 25 release in the presence of hyperglycemia.
37). Thus, in certain embodiments, the GLP-1 receptoragonist
Taspoglutide, also known as R1583 or BIM 51077, is a is as described in WO03/018516, which is hereby incorpo
GLP-1 receptor agonist that has been shown to improve gly rated by reference in its entirety. In some embodiments, the
cemic control and lower body weight in subjects with type 2 therapeutic agents of the present invention comprise GLP-1
diabetes mellitus treated with metformin (Abstract No. 30 analogs wherein the backbone for Such analogs or fragments
A-1604, Jun. 7, 2008, 68th American Diabetes Association contains an amino acid other than alanine at position 8 (posi
Meeting, San Francisco, Calif.). tion 8 analogs). The backbone may also include L-histidine,
Thus, in certain embodiments, the GLP-1 receptoragonist D-histidine, or modified forms of histidine such as desamino
is as described in WO00/034331, which is hereby incorpo histidine, 2-amino-histidine, B-hydroxy-histidine, homohis
rated by reference in its entirety. In certain exemplary 35 tidine, C-fluoromethyl-histidine, or C.-methyl-histidine at
embodiments, the GLP-1 receptor agonist has the sequence position 7. In some embodiments, these position 8 analogs
AibhGLP-1 (7-36)NH. (e.g. taspoglutide), wherein Aib may contain one or more additional changes at positions 12,
is alpha-aminoisobutyric acid. 16, 18, 19, 20, 22, 25, 27, 30, 33, and 37 compared to the
CJC-1 131 is a GLP-1 analog that consists of a DPP-IV corresponding amino acid of native GLP-1. In other embodi
resistant form of GLP-1 joined to a reactive chemical linker 40 ments, these position 8 analogs may contain one or more
group that allows GLP-1 to form a covalent and irreversible additional changes at positions 16, 18, 22, 25 and 33 com
bond with serum albumin following Subcutaneous injection pared to the corresponding amino acid of native GLP-1. In
(Kim et al., 2003, Diabetes 52: 751-9). In a 12-week, ran certain exemplary embodiments, the GLP-1 receptor agonist
domized, double-blind, placebo-controlled multicenter has the sequence: HVEGTFTSDVSSYLEEQAAKEFIAW
study, CJC-1131 and metformin treatment was effective in 45 LIKGRG-OH (SEQ ID NO: 19) (e.g. LY548.806).
reducing fasting blood glucose levels in type 2 diabetes Thus, the present invention provides therapeutic agents
patients (Ratner et al., Abstract No. 10-OR, Jun. 10-14, 2005, comprising an elastic peptide (e.g., an ELP) and a GLP-1
65th American Diabetes Association Meeting, San Francisco, receptor agonist. For example, in certain embodiments, the
Calif.). GLP-1 receptoragonist is GLP-1 (SEQID NO:13, 17, or 59)
Thus, in certain embodiments, the GLP-1 receptoragonist 50 or a functional analog thereof. In other embodiments, the
is as described in WO00/069911, which is hereby incorpo GLP-1 receptor agonist is exendin-4 (SEQ ID NO:14) or a
rated by reference in its entirety. In some embodiments, the functional analog thereof. Such functional analogs of GLP-1
GLP-1 receptor agonist is modified with a reactive group or exendin-4 include functional fragments truncated at the
which reacts with amino groups, hydroxyl groups or thiol C-terminus by from 1 to 10 amino acids, including by 1, 2, 3,
groups on blood components to form a stable covalent bond. 55 or up to about 5 amino acids (with respect to SEQID NOS:
In certain embodiments, the GLP-1 receptor agonist is modi 13, 14, 17, or 59). Such functional analogs may contain from
fied with a reactive group selected from the group consisting 1 to 10 amino acid insertions, deletions, and/or substitutions
of Succinimidyl and maleimido groups. In certain exemplary (collectively) with respect to the native sequence (e.g., SEQ
embodiments, the GLP-1 receptor agonist has the sequence? ID NOS 13, 14, and 59), and in each case retaining the activity
structure: D-Ala Lys-(2-(2-(2-maleimidopropionamido 60 of the peptide. For example, the functional analog of GLP-1
(ethoxy)ethoxy)acetamide))-GLP-1 (7-37) (e.g. CJC-1131). or exendin-4 may have from 1 to about 3, 4, or 5 insertions,
AVE0010, also known as ZP-10, is a GLP-1 receptorago deletions and/or substitutions (collectively) with respect to
nist that may be employed in connection with the invention. SEQID NOS: 13, 59 and 14, and in each case retaining the
In a recent double-blind study, patients treated with once activity of the peptide. Such activity may be confirmed or
daily dosing of AVE0010 demonstrated significant reductions 65 assayed using any available assay, including those described
in HbA1c levels (Ratner et al., Abstract No. 433-P, 68th herein. In these or other embodiments, the GLP-1 receptor
American Diabetes Association Meeting, San Francisco, agonist component has at least about 50%, 75%, 80%, 85%,
US 8,841,255 B2
19 20
90%, or 95% identity with the native sequence (SEQID NOS: GLP-1 receptor agonist/elastic peptide compound can be
13, 59, and 14). The determination of sequence identity introduced into a cell. Such as an immortalized B-cell, and the
between two sequences (e.g., between a native sequence and resulting cell can be contacted with glucose. If the cell pro
a functional analog) can be accomplished using any align duces insulin in response to the glucose, then the modified
ment tool, including Tatusova et al., Blast 2 sequences—a new 5 GLP-1 is generally considered biologically active in vivo
tool for comparing protein and nucleotide sequences, FEMS (Fehmann et al., 1992, Endocrinology 130: 159-166). An
Microbial Lett. 174:247-250 (1999). Such functional analogs exemplary assay is described in greater detail herein.
may further comprise additional chemical modifications, The ability of an GLP-1 receptor agonist/elastic peptide
Such as those described in this section and/or others known in compound to enhance B-cell proliferation, inhibit B-cell apo
the art. 10 ptosis, and regulate islet growth may also be measured using
In certain embodiments, the GLP1-ELP fusion has a known assays. Pancreatic B-cell proliferation may be
sequence exemplified herein as SEQ ID NOS: 54 and 56. assessed by H-tymidine or BrdU incorporation assays (See
When processed, the mature form of such fusion protein will e.g. Buteau et al., 2003, Diabetes 52: 124-32), wherein pan
begin with the His 7 of GLP. creatic B-cells such as INS(832/13) cells are contacted with a
In another aspect, the present invention provides methods 15 GLP-1 receptor agonist/elastic peptide compound and ana
for the treatment or prevention of type 2 diabetes, impaired lyzed for increases in H-thymidine or BrdU incorporation.
glucose tolerance, type 1 diabetes, hyperglycemia, obesity, The antiapoptotic activity of a GLP-1 receptoragonist/elastic
binge eating, bulimia, hypertension, syndrome X, dyslipi peptide compound can be measured in cultured insulin-se
demia, cognitive disorders, atherosclerosis, non-fatty liver creting cells and/or in animal models where diabetes occurs
disease, myocardial infarction, coronary heart disease and as a consequence of an excessive rate of beta-cell apoptosis
other cardiovascular disorders. The method comprises (See e.g. Bulotta et al., 2004, Cell Biochem Biophy's 40(3
administering the therapeutic agent comprising the elastin suppl): 65-78).
like peptide (ELP) and the GLP-1 receptor agonist (as In addition to GLP-1, other peptides of this family, such as
described above) to a patient in need of such treatment. In those derived from processing of the pro-glucagon gene, Such
these or other embodiments, the present invention provides 25 as GLP-2, GIP, and oxyntomodulin, could be conjugated or
methods for decreasing food intake, decreasing B-cell apop fused to the elastic peptide component (as described herein)
tosis, increasing B-cell function and B-cell mass, and/or for to enhance the therapeutic potential.
restoring glucose sensitivity to B-cells. Generally, the patient Insulin
may be a human or non-human animal patient (e.g., dog, cat, In other embodiments, the present invention provides a
cow, or horse). Preferably, the patient is human. 30 therapeutic agent comprising an elastin peptide component
The treatment with a ELP/GLP-1 receptor agonist com coupled to insulin (e.g., via fusion or conjugation). Insulin
pound according to the present invention may also be com injections, e.g. of human insulin, can be used to treat diabetes.
bined with one or more pharmacologically active Substances, The insulin-making cells of the body are called B-cells, and
e.g. selected from antidiabetic agents, antiobesity agents, they are found in the pancreas gland. These cells clump
appetite regulating agents, antihypertensive agents, agents for 35 together to form the “islets of Langerhans', named for the
the treatment and/or prevention of complications resulting German medical student who described them.
from or associated with diabetes and agents for the treatment The synthesis of insulin begins at the translation of the
and/or prevention of complications and disorders resulting insulin gene, which resides on chromosome 11. During trans
from or associated with obesity. In the present context, the lation, two introns are spliced out of the mRNA product,
expression “antidiabetic agent' includes compounds for the 40 which encodes a protein of 110 amino acids in length. This
treatment and/or prophylaxis of insulin resistance and dis primary translation product is called preproinsulin and is
eases wherein insulin resistance is the pathophysiological inactive. It contains a signal peptide of 24 amino acids in
mechanism. length, which is required for the protein to cross the cell
The ability of a GLP-1 or exendin-4 analog, oran GLP-1 membrane.
receptoragonist/elastic peptide compound, to bind the GLP-1 45 Once the preproinsulin reaches the endoplasmic reticulum,
receptor may be determined by standard methods, for a protease cleaves off the signal peptide to create proinsulin.
example, by receptor-binding activity Screening procedures Proinsulin consists of three domains: an amino-terminal B
which involve providing appropriate cells that express the chain, a carboxyl-terminal Achain, and a connecting peptide
GLP-1 receptor on their surface, for example, insulinoma cell in the middle known as the C-peptide. Insulin is composed of
lines such as RINmSF cells or INS-1 cells. In addition to 50 two chains of amino acids named chain A (21 amino acids—
measuring specific binding of tracer to membrane using GIVEQCCASVCSLYQLENYCN) (SEQ ID NO: 15) and
radioimmunoassay methods, cAMP activity or glucose chain B (30 amino acids FVNQHLCG
dependent insulin production can also be measured. In one SHLVEALYLVCGERGFFYTPKA) (SEQ ID NO: 16) that
method, a polynucleotide encoding the GLP-1 receptor is are linked together by two disulfide bridges. There is a 3rd
employed to transfect cells to thereby express the GLP-1 55 disulfide bridge within the A chain that links the 6th and 11th
receptor protein. Thus, these methods may be employed for residues of the A chain together. In most species, the length
testing or confirming whether a suspected GLP-1 receptor and amino acid compositions of chains A and B are similar,
agonist is active. An exemplary assay is described in greater and the positions of the three disulfide bonds are highly con
detail herein. served. For this reason, pig insulin can replace deficient
In addition, known methods can be used to measure or 60 human insulin levels in diabetes patients. Today, porcine insu
predict the level of biologically activity of a GLP-1 receptor lin has largely been replaced by the mass production of
agonist or GLP-1 receptoragonist/elastic peptide in vivo (See human proinsulin by bacteria (recombinant insulin).
e.g. Siegel, et al., 1999, Regul Pept 79(2-3):93-102). In par Insulin molecules have a tendency to form dimers in Solu
ticular, GLP-1 receptor agonists or GLP-1 receptor agonist/ tion, and in the presence of Zinc ions, insulin dimers associate
elastic peptide compounds can be assessed for their ability to 65 into hexamers. Whereas monomers of insulin readily diffuse
induce the production of insulin in vivo using a variety of through the blood and have a rapid effect, hexamers diffuse
known assays for measuring GLP-1 activity. For example, a slowly and have a delayed onset of action. In the design of
US 8,841,255 B2
21 22
recombinant insulin, the structure of insulin can be modified Substitution of asparagine at position 3 by lysine and lysine at
in a way that reduces the tendency of the insulin molecule to position 29 by glutamine of human insulin B chain. Insulin
form dimers and hexamers but that does not interrupt binding glulisine has more rapid onset of action and shorter duration
to the insulin receptor. In this way, a range of preparations are of action compared to regular human insulin.
made, varying from short acting to long acting. In another embodiment, the insulin may contain an A and/
Within the endoplasmic reticulum, proinsulin is exposed to or B chain of glargine (also known as Lantu, Sanofi-Aventis).
several specific peptidases that remove the C-peptide and Insulin glargine differs from human insulin in that the amino
generate the mature and active form of insulin. In the Golgi acid asparagine at position 21 of the A chain is replaced by
apparatus, insulin and free C-peptide are packaged into secre glycine and two arginines are added to the C-terminus of the
tory granules, which accumulate in the cytoplasm of the 10 B-chain. Compared with bedtime neutral protamine Hage
B-cells. Exocytosis of the granules is triggered by the entry of dorn (NPH) insulin (an intermediate acting insulin), insulin
glucose into the beta cells. The secretion of insulin has abroad glargine is associated with less nocturnal hypoglycemia in
impact on metabolism. patients with type 2 diabetes.
There are two phases of insulin release in response to a rise In yet another embodiment, the insulin may contain an A
in glucose. The first is an immediate release of insulin. This is 15 and/or B chain from detemir (also known as Levemir, Novo
attributable to the release of preformed insulin, which is Nordisk). Insulin detemir is a soluble (at neutral pH) long
stored in secretory granules. After a short delay, there is a acting insulin analog, in which the amino acid threonine at
second, more prolonged release of newly synthesized insulin. B30 is removed and a 14-carbon, myristoyl fatty acid is
Once released, insulin is active for a only a brief time acetylated to the epsilon-amino group of LysE29. After sub
before it is degraded by enzymes. Insulinase found in the liver cutaneous injection, detemir dissociates, thereby exposing
and kidneys breaks down insulin circulating in the plasma, the free fatty acid which enables reversible binding to albu
and as a result, insulin has a half-life of only about 6 minutes. min molecules. So at Steady state, the concentration of free
This short duration of action results in rapid changes in the unbound insulin is greatly reduced resulting in stable plasma
circulating levels of insulin. glucose levels.
Insulin analogs have been developed with improved thera 25 In some embodiments, the insulin may be a single-chain
peutic properties (Owens et al., 2001, Lancet 358: 739-46; insulin analog (SIA) (e.g. as described in U.S. Pat. No. 6,630,
Vajo et al., 2001, Endocr Rev 22: 706-17), and such analogs 438 and WO08/019,368, which are hereby incorporated by
may be employed in connection with the present invention. reference in their entirety). Single-chain insulin analogs
Various strategies, including elongation of the COOH-termi encompass a group of structurally-related proteins wherein
nal end of the insulin B-chain and engineering of fatty acid 30 the A and B chains are covalently linked by a polypeptide
acylated insulins with substantial affinity for albumin are linker. The polypeptide linker connects the C-terminus of the
used to generate longer-acting insulin analogs. However, in B chain to the N-terminus of the A chain. The linker may be
Vivo treatments with available longer-acting insulin com of any length so long as the linker provides the structural
pounds still result in a high frequency of hypo- and hyperg conformation necessary for the SIA to have a glucose uptake
lycemic excursions and modest reduction in Hb A. Accord 35 and insulin receptor binding effect. In some embodiments,
ingly, development of a truly long-acting and stable human the linker is about 5-18 amino acids in length. In other
insulin analog still remains an important task. embodiments, the linker is about 9-15 amino acids in length.
Functional analogs of insulin that may be employed in In certain embodiments, the linker is about 12 amino acids
accordance with the invention include rapid acting analogs long. In certain exemplary embodiments, the linker has the
Such as lispro, aspart and glulisine, which are absorbed rap 40 sequence KDDNPNLPRLVR (SEQ ID NO. 20) or
idly (<30 minutes) after Subcutaneous injection, peak at one GAGSSSRRAPQT (SEQID NO. 21). However, it should be
hour, and have a relatively short duration of action (3 to 4 understood that many variations of this sequence are possible
hours). In addition, two long acting insulin analogs have been such as in the length (both addition and deletion) and substi
developed: glargine and detemir, and which may be employed tutions of amino acids without Substantially compromising
in connection with the invention. The long acting insulin 45 the effectiveness of the produced SIA in glucose uptake and
analogs have an onset of action of approximately two hours insulin receptor binding activities. For example, several dif
and reach a plateau of biological action at 4 to 6 hours, and ferent amino acid residues may be added or removed from
may last up to 24 hours. either end without substantially decreasing the activity of the
Thus, in one embodiment, the insulin component may con produced SIA.
tain the A and/or B chain of lispro (also known as Humalog, 50 An exemplary single-chain insulin analog currently in
Eli Lilly). Insulin lispro differs from human insulin by the clinical development is albulin (Duttaroy et al., 2005, Diabe
substitution of proline with lysine at position 28 and the tes 54: 251-8). Albulin can be produced in yeast or in mam
substitution of lysine with proline at position 29 of the insulin malian cells. It consists of the BandA chain of human insulin
B chain. Although these modifications do not alter receptor (100% identity to native human insulin) linked together by a
binding, they help to block the formation of insulin dimers 55 dodecapeptide linker and fused to the NH terminals of the
and hexamers, allowing for larger amounts of active mono native human serum albumin. For expression and purification
meric insulin to be available for postprandial injections. of albulin, Duttaroy et al. constructed a synthetic gene con
In another embodiment, the insulin may contain an A and/ struct encoding a single-chain insulin containing the B- and
or B chain of aspart (also known as Novolog, Novo Nordisk). A-chain of mature human insulin linked together by a dode
Insulin aspart is designed with the single replacement of the 60 capeptide linker using four overlapping primers and PCR
amino acid proline by aspartic acid at position 28 of the amplification. The resulting PCR product was ligated in
human insulin B chain. This modification helps block the frame between the signal peptide of human serum albumin
formation for insulin hexamers, creating a faster acting insu (HSA) and the NH terminus of mature HSA, contained
lin. within a pSAC35 vector for expression in yeast. In accor
In yet another embodiment, the insulin may contain an A 65 dance with the present invention, the HSA component of
and/or B chain of glulisine (also known as Apidra, Sanofi abulin may be replaced with an ELP component as described
Aventis). Insulin glulisine is a short acting analog created by herein.
US 8,841,255 B2
23 24
Thus, in one aspect, the present invention provides thera be measured in any number of cells types, for example, H4Ile
peutic agents comprising an elastic peptide and an insulin or hepatoma cells. In this assay, pretreatment with a biologically
functional analog thereof. For example, in certain embodi active analog will generally result in a dose-dependent inhi
ments, the insulin is a mammalian insulin, Such as human bition of the amount of glucose released.
insulin or porcine insulin. In accordance with the invention, Factor VII (VIIa)
the elastic peptide component may be coupled (e.g., via In certain embodiments, the invention provides therapeutic
recombinant fusion or chemical conjugation) to the insulin A agents comprising an elastic peptide component coupled
chain, or B chain, or both. The insulin may comprise each of (e.g., via fusion or conjugation) to a Factor VII/VIIa. Coagul
chains A, B, and C (SEQID NOS: 51 and 52), or may contain lation is the biological process of blood clot formation involv
a processed form, containing only chains A and B. In some 10
ing many different serine proteases as well as their essential
embodiments, chains A and B are connected by a short link cofactors and inhibitors. It is initiated by exposure of Factor
ing peptide, to create a single chain insulin. The insulin may
be a functional analog of human insulin, including functional VII (FVII) and Factor VIIa (FVIIa) to its membrane bound
fragments truncated at the N-terminus and/or C-terminus (of cofactor, tissue factor (TF), resulting in production of Factor
either or both of chains A and B) by from 1 to 10 amino acids, 15 Xa (FXa) and more FVIIa. The process is propagated upon
including by 1, 2, 3, or about 5 amino acids. Functional production of Factor IXa (FIXa) and additional FXa that,
analogs may contain from 1 to 10 amino acid insertions, upon binding with their respective cofactors FVIIIa and FVa,
deletions, and/or substitutions (collectively) with respect to form platelet bound complexes, ultimately resulting in the
the native sequence (e.g., SEQ ID NOS 15 and 16), and in formation of thrombinanda fibrin clot. Thrombin also serves
each case retaining the activity of the peptide. For example, to further amplify coagulation by activation of cofactors such
functional analogs may have 1, 2, 3, 4, or 5 amino acid as FV and FVII and Zymogens such as Factor XI. Moreover,
insertions, deletions, and/or substitutions (collectively) with thrombin activates platelets leading to platelet aggregation,
respect to the native sequence (which may contain chains A which is necessary for the formation of a hemostatic plug.
and B, or chains A, B, and C). Such activity may be confirmed Factor VII circulates in the blood in a zymogen form, and
or assayed using any available assay, including those 25 is converted to its active form, Factor VIIa, by either factor
described herein. In these or other embodiments, the insulin IXa, factor Xa, factor XIIa, or thrombin by minor proteolysis.
component has at least about 75%, 80%, 85%, 90%. 95%, or Factor VIIa is a two-chain, 50 kilodalton (kDa) plasma serine
98% identity with each of the native sequences for chains A protease. The active form of the enzyme comprises a heavy
and B (SEQ ID NOS:15 and 16). The determination of chain (254 amino acid residues) containing a catalytic
sequence identity between two sequences (e.g., between a 30 domain and a light chain (152 residues) containing 2 epider
native sequence and a functional analog) can be accom mal growth factor (EGF)-like domains. The mature factor
plished using any alignment tool, including Tatusova et al., VII/VIIa that circulates in plasma is composed of 406 amino
Blast 2 sequences—a new tool for comparing protein and acid residues (SEQID NO: 33). The light and heavy chains
nucleotide sequences, FEMS Microbiol Lett. 174:247-250 are held together by a disulfide bond.
(1999). The insulin component may contain additional 35 As noted above, Factor VIIa is generated by proteolysis of
chemical modifications known in the art. a single peptide bond from its single chain Zymogen, Factor
In another aspect, the present invention provides methods VII, which is present at approximately 0.5 ug/ml in plasma.
for the treatment or prevention of diabetes, including type I The conversion of Zymogen Factor VII into the activated
and II diabetes. The method comprises administering an two-chain molecule occurs by cleavage of an internal peptide
effective amount of the therapeutic agent comprising an elas 40 bond. In human Factor VII, the cleavage site is at Arg152
tic peptide (e.g., ELP) component and an insulin (or func Ile153 (Hagen et al., 1986, PNAS USA 83: 24 12-6).
tional analog thereof) component to a patient in need thereof. “Factor VII/VIIa as used in this application means a prod
Generally, the patient may be a human or non-human animal uct consisting of either the unactivated form (factor VII) or the
(e.g., dog, cat, cow, or horse) patient. Preferably, the patient is activated form (factor VIIa) or mixtures thereof. "Factor VII/
human. 45 VIIa within the above definition includes proteins that have
To characterize the in vitro binding properties of an insulin an amino acid sequence of native human factor VII/VIIa. It
analog or an elastic peptide-containing insulin analog, com also includes proteins with a slightly modified amino acid
petition binding assays may be performed in various cell lines sequence, for instance, a modified N-terminal end including
that express the insulin receptor (Jehle et al., 1996, Diabeto N-terminal amino acid deletions or additions so long as those
logia 39: 421-432). For example, competition binding assays 50 proteins substantially retain the activity of factor VIIa. “Fac
using CHO cells overexpressing the human insulin receptor tor VII' within the above definition also includes natural
may be employed. Insulin can also bind to the IGF-1 receptor allelic variations that may exist and occur from one individual
with a lower affinity than the insulin receptor. To determine to another. Also, degree and location of glycosylation or other
the binding affinity of an ELP-containing insulin analog, a post-translation modifications may vary depending on the
competition binding assay can be performed using 'I-la 55 chosen host cells and the nature of the host cellular environ
beled IGF-1 in L6 cells. ment.
The activities of insulin include stimulation of peripheral In the presence of calcium ions, Factor VIIa binds with
glucose disposal and inhibition of hepatic glucose produc high affinity to TF. TF is a 263 amino acid residue glycopro
tion. The ability of an elastic peptide-containing insulin ana tein composed of a 219 residue extracellular domain, a single
log to mediate these biological activities can be assayed in 60 transmembrane domain, and a short cytoplasmic domain
vitro using known methodologies. For example, the effect of (Morrissey et al., 1987, Cell 50: 129-35). The TF extracellular
an elastic peptide-containing analog on glucose uptake in domain is composed of two fibronectin type III domains of
3T3-L1 adipocytes can be measured and compared with that about 105 amino acids each. The binding of FVIIa is mediated
of insulin. Pretreatment of the cells with a biologically active entirely by the TF extracellular domain (Muller et al., 1994,
analog will generally produce a dose-dependent increase in 65 Biochem. 33:10864-70). Residues in the area of amino acids
2-deoxyglucose uptake. The ability of an elastic peptide 16-26 and 129-147 contribute to the binding of FVIIa as well
containing insulin analog to regulate glucose production may as the coagulant function of the molecule. Residues Lys20.
US 8,841,255 B2
25 26
Trp45, Asp58, Tyr94, and Phel40 make a large contribution frequent injections associated with currently available FVIIa
(1 kcal/mol) to the free energy (AG) of binding to FVIIa. therapy and the potential for obtaining more optimal thera
TF is expressed constitutively on cells separated from peutic FVIIa levels with concomitant enhanced therapeutic
plasma by the vascular endothelium. Its expression on endot effect, there is a clear need for improved FVII or FVIIa-like
helial cells and monocytes is induced by exposure to inflam molecules with a longer half-life in vivo.
matory cytokines or bacterial lipopolysaccharides (Drake et Recombinant human coagulation factor VIIa (rEVIIa,
al., 1989, J. Cell Biol. 109:389). Upon tissue injury, the NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has
exposed extracellular domain of TF forms a high affinity, proven to be efficacious for the treatment of bleeding episodes
calcium dependent complex with FVII. Once bound to TF, in hemophilia patients with inhibitors. A small fraction of
FVII can be activated by peptide bond cleavage to yield serine 10 patients may be refractory to rRVIIa treatment and could
protease FVIIa. The enzyme that catalyzes this step in vivo potentially benefit from genetically modified FVIIa mol
has not been elucidated, but in vitro FXa, thrombin, TF:FVIIa ecules with increased potencies. To this end, FVIIa analogs
and FIXa can catalyze this cleavage. FVIIa has only weak with increased intrinsic activity have been investigated that
activity upon its physiological substrates FX and FIX exhibit superior hemostatic profiles in vitro (see e.g. WO02/
whereas the TF:FVIIa complex rapidly activates FX and FIX. 15 077218 or WO05/074975, which are hereby incorporated by
The TF:FVIIa complex constitutes the primary initiator of reference in their entirety, and Tranholm et al., 2003, Blood
the extrinsic pathway of blood coagulation. The complex 102(10): 3615-20, which is also incorporated by reference).
initiates the extrinsic pathway by activation of FX to Factor These analogs may also be used as more efficacious hemo
Xa (FXa), FIX to Factor IXa (FIXa), and additional FVII to static agents in other indications where efficacy of rEVIIa has
FVIIa. The action of TF:FVIIa leads ultimately to the con been observed, including in thrombocytopenia and trauma.
version of prothrombin to thrombin, which carries out many Thus, in some embodiments, the Factor VIIa analog that
biological functions. Among the most important activities of may be used in accordance with the invention is as described
thrombin is the conversion of fibrinogen to fibrin, which in WO02/077218 or WO05/074975. For example, the FVIIa
polymerizes to form a clot. The TF:FVIIa complex also par analog may have a glutamine Substituted for methionine at
ticipates as a secondary factor in extending the physiological 25 position 298 (i.e. M298Q-FVIIa). In certain exemplary
effects of the contact activation system. embodiments, the FVIIa analog contains two additional
The initiation and Subsequent regulation of coagulation is mutations, valine at position 158 replaced by aspartic acid
complex, since maintenance of hemostasis is crucial for Sur and glutamic acid at position 296 replaced by valine (i.e.
vival. There is an exquisite balance between hemostasis (nor V158D/E296V/M298Q-FVIIa). Additionally or alterna
mal clot formation and dissolution) and thrombosis (patho 30 tively, the Factor VIIa analog may have an alanine residue
genic clot formation). Serious clinical conditions involving substitution for lysine at position 337 (i.e. V158D/E296V/
aberrations in coagulation include deep vein thrombosis, M298Q/K337A-FVIIa). In still other embodiments, the Fac
myocardial infarction, pulmonary embolism, stroke and dis tor VIIa analog has a substitution or insertion selected from
seminated intravascular coagulation (in sepsis). There are Q250C; P406C; and 407C, wherein a cysteine has also been
also many bleeding coagulopathies where there is insufficient 35 introduced in the C-terminal sequence (see, e.g. U.S. Pat. No.
clot formation. These include hemophilia A (FVIII defi 7,235,638, which is hereby incorporated by reference in its
ciency) or hemophilia B (FIX deficiency), where procoagul entirety). The Factor VIIa analog may further comprise a
lant therapy is required. The challenge in this therapeutic area substitution or insertion at one or more of positions 247, 260,
is to operate in the narrow window between too much and too 393, 396, and/or 405.
little coagulation. 40 In these or other embodiments, the Factor VIIa analog
The use of exogenous FVIIaaSatherapeutic agent has been comprises a Substitution relative to the sequence of native
shown to induce hemostasis in patients with hemophilia A Factor VIIa selected from: (a) a substitution of Lys157 with an
and B (Hedner, 2001, Seminars Hematol. 38 (suppl. 12): amino acid selected from the group consisting of Gly, Val,
43-7: Hedner, 2004, Seminars Hematol. 41 (suppl. 1): 35-9). Ser. Thr, Asp, and Glu; (b) a substitution of Lys337 with an
It also has been used to treat bleeding in patients with liver 45 amino acid selected from the group consisting of Ala, Gly,
disease, anticoagulation-induced bleeding, Surgery, thromb Val, Ser. Thr, Gln, Asp, and Glu; (c) a substitution of Asp334
ocytopenia, thrombasthenia, Bemard-Soulier syndrome, Von with any amino acid other than Ala or ASn; and (d) a Substi
Willebrand disease, and other bleeding disorders (See e.g. tution of Ser336 with any amino acid other than Ala or Cys
Roberts et al., 2004, Blood 104:3858-64). (see e.g. U.S. Pat. No. 7,176,288, which is hereby incorpo
Commercial preparations of human recombinant FVIIa are 50 rated by reference in its entirety). Additionally or alterna
Soldas NovoSevenTM. NovoSevenTM is indicated for the treat tively, the Factor VIIa analog comprises a substitution of the
ment of bleeding episodes in hemophilia A or B patients and Leu at position 305 of Factor VII with an amino acid residue
is the only recombinant FVIIa effective for bleeding episodes selected from the group consisting of Val, Ile, Met, Phe, Trp,
currently available. A circulating recombinant FVIIa half-life Pro, Gly, Ser, Thr, Cys, Tyr, Asn. Glu, Lys, Arg, His, Asp and
of 2.3 hours was reported in “Summary Basis for Approval for 55 Gln (see e.g. U.S. Pat. No. 6,905,683, which is hereby incor
NovoSevenTM FDA reference number 96-0597. Moreover, porated by reference in its entirety).
the half-life of recombinant FVIIa is shorter in pediatric Thus, in one aspect, the present invention provides thera
patients (~1.3 hours), Suggesting that higher doses of recom peutic agents comprising an elastic peptide, e.g., an elastin
binant FVIIa may be required in this population (Roberts et like peptide (ELP) and a Factor VII/VIIa, or functional analog
al., 2004, Blood 104: 3858-64). Accordingly, relatively high 60 thereof. For example, in certain embodiments, the Factor
doses and frequent administration are necessary to reach and VII/VIIa is human Factor VII/VIIa (e.g., SEQ ID NO: 33).
Sustain the desired therapeutic or prophylactic effect. As a The Factor VII/VIIa may be a functional analog of human
consequence, adequate dose regulation is difficult to obtain Factor VII/VIIa, including functional fragments truncated at
and the need of frequent intravenous administrations imposes the N-terminus and/or C-terminus by from 1 to 10 amino
restrictions on the patient's way of living. 65 acids, including by 1, 2, 3, or about 5 amino acids. Functional
A molecule with a longer circulation half-life would analogs may contain from 1 to 10 amino acid insertions,
decrease the number of necessary administrations. Given the deletions, and/or substitutions (collectively) with respect to
US 8,841,255 B2
27 28
the native sequence (e.g., SEQID NO: 33), and in each case tide-containing Factor VIIa analog diluted to varying concen
retaining the activity of the peptide. For example, such ana trations directly into FVII deficient plasma. In a coagulom
logs may have from 1 to about 5 amino acid insertions, dele eter, one part plasmata FVIIa analog can be mixed with 2
tions, and/or substitutions (collectively) with respect to the parts InnovinTM (Dade, Miami, Fla.) prothrombin time
native full length sequence, or with respect to one or both of 5 reagent (recombinant human tissue factor with phospholipids
the heavy and light chains. Such activity may be confirmed or and CaCl2). Clot formation is detected optically and time to
assayed using any available assay, including those described clotting measured. Clotting time (seconds) is compared to the
herein. In these or other embodiments, the Factor VII/VIIa mean clotting time of FVII-deficient plasma alone and plotted
component has at least about 75%, 80%, 85%, 90%. 95%, or as the fractional clotting time versus FVIIa analog concen
98% identity with the native sequence (SEQID NO:33). The 10 tration.
determination of sequence identity between two sequences Therapeutic Proteins
(e.g., between a native sequence and a functional analog) can The present invention further provides therapeutic agents
be accomplished using any alignment tool, including comprising an elastic peptide component and at least one
Tatusova et al., Blast 2 sequences—a new tool for comparing therapeutic protein selected from Table 1. The elastic peptide
protein and nucleotide sequences, FEMS Microbiol Lett. 174: 15 (e.g., ELP) component and therapeutic protein may be
247-250 (1999). coupled by recombinant fusion or chemical conjugation as
In exemplary embodiments, the FactorVII-ELP fusion has described herein. Such therapeutic proteins are listed in Table
the amino acid sequence of SEQID NO:58. SEQID NO:58 1 by protein name and GeneSeq Accession No. The amino
further comprises a TEV protease cleavage site between the acid sequence of each Therapeutic Protein, which is known in
FactorVII and ELP sequences, which may be beneficial for the art, is hereby incorporated by reference for each Thera
removing the ELP sequence post expression where desired. peutic Protein listed in Table 1. Such therapeutic proteins are
However, in accordance with the invention, the tev sequence further described in US patent or PCT publications that are
may be entirely removed, or replaced with another linking also listed in Table 1, and such US patent and PCT publica
sequence as disclosed herein. tions are hereby incorporated by reference, especially with
In another aspect, the present invention provides methods 25 respect to the structure of Such therapeutic proteins and
for the treatment or prevention of bleeding-related disorders. described functional analogs.
The method comprises administering an effective amount of Table 1 further describes the biological activity of each
the therapeutic agent comprising an elastic peptide and a listed Therapeutic Protein, as well as an exemplary assay for
Factor VII/VIIa or functional analog thereof to a patient in determining the activity of functional analogs or agents of the
need. In certain embodiments, the bleeding-related disorder 30 invention (e.g., fusion with an elastic peptide component).
is one or more of hemophilia (A or B), post-Surgical bleeding, Generally, functional analogs of therapeutic proteins listed in
anticoagulation-induced bleeding, thrombocytopenia, Factor Table 1 may include functional fragments truncated at the
VII deficiency, Factor XI deficiency, bleeding in patients with N-terminus and/or C-terminus by from 1 to 10 amino acids,
liver disease, thrombasthenia, Bemard-Soulier syndrome, including by 1, 2, 3, 4 or about 5 amino acids. Functional
von Willebrand disease, and intracranial hemorrhage. Gener 35 analogs may contain from 1 to 10 amino acid insertions,
ally, the patient is a human or non-human animal (e.g., dog, deletions, and/or substitutions (collectively) with respect to
cat, cow, or horse) patient. Preferably, the patient is human. the base sequence (e.g., as listed in Table 1), and in each case
To characterize the in vitro binding properties of a sus retaining the full or partial biological activity (as listed in
pected Factor VII/VIIa analog, or an elastic peptide-contain Table 1) of the therapeutic protein. For example, functional
ing Factor VIIa analog, TF binding assays can be performed 40 analogs may have 1, 2, 3, 4, or 5 amino acid insertions,
as described previously (See, e.g., Chaing et al., 1994, Blood deletions, and/or substitutions (collectively) with respect to
83 (12): 3524–35). Briefly, recombinant human TF can be the base sequence. Such activity may be confirmed or assayed
coated onto Immulon II plates in carbonate antigen buffer using any available assay, including those described in the
overnight at 4°C. BSA is also coated onto the plates for use Table. In these or other embodiments, the therapeutic protein
as a control. Elastic peptide-containing Factor VIIa analogs 45 has at least about 75%, 80%, 85%, 90%, 95%, or 98% identity
may be added at various concentrations in TBS-T buffer. with the corresponding base sequence. The molecules may
After several washes, monospecific polyclonal rabbit anti further comprise additional chemical modifications known
human FVIIa sera is added and incubated for approximately for each in the art.
an hour at room temperature. Next, goat anti-rabbit IgG con In some embodiments, the therapeutic protein (e.g., as
jugated to alkaline phosphatase is added, followed by the 50 selected from Table 1) has a size of less than about 25kDa, or
alkaline phosphatase substrate PNPP, which is used for detec less than about 10 kDa, or less than about 5 kDa, and the
tion. After subtraction of background, the absorbance at 405 corresponding therapeutic agent of the invention (e.g., com
nm is taken to be directly proportional to the degree of Factor prising the ELP component) has a molecular weight of less
VIIa binding to the immobilized TF. These values can then be than about 60 kDa, 55 kDa, 50 kDa, or 40 kDa.
compared to control plasma containing Factor VIIa. 55 Table 1 further lists preferred indications for each thera
The clotting ability of a Factor VII/VIIa analog oran elastic peutic protein, for which the corresponding therapeutic agent
peptide-containing Factor VIIa analog can be measured in finds use. Such as in a method for treatment or prevention
human FVII deficient plasma. In this assay, the elastic pep related to Such indication.
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(12) United States Patent (10) Patent No.: US 8,841,255 B2

(56) References Cited 2009,0270317 A1 10, 2009 Chikoti

1. aattaatacg acticactata ggggaattgt gag.cggataa caattcc.cct

101 gaaggcacct ttaccagoga totgagcaaa cagatggaag aagaag.cggit

201 cgc.cgc.cgag cctogagggc atgggtgggc cggg.cgtggg togttccgggC

7- or -- - - - - - - - - - or no or w w we were we w w w w a rare err - - - - - - - - - - - - - - - - - -

101 aaaaacgg.cg gcc.cgag cag cqgcgc.gc.cg cc.gc.cgagcc (SEQ ID NO. : 25)

P4: SEQ ID NO. : 38

51 cctttaccag cqatctgage aaacagatgg aagaagaagc ggtgcgc.ctg

? . . . . . . . . . . . . . . . . . . . Ex" 4 EB1. "90 . . . . . . . . . . . . . . . . . . . . . d

101 tttattgaat ggctgaaaaa cgg.cggc.ccg agcagogg.cg cgc.cgcc.gc.c

. . . . . . . . . . . . . . . . . . . Ex- 4 EP "r 9C . . . . . . . . . . . . . . . . . . . . . >

151 gagcc (SEQ ID NO. : 27)

51 catcgg.cgca toggcgaaggc acctttacca gcgatctgag caaacagatg

> . DsbA>>>> . . . . . . . . . . . . . . Ex-4 EE.P1-90 . . . . . . . . . . . . . . . . . >

101 gaagaagaag cggtgcgc.ct gtttattgaa toggctgaaaa acgg.cgg.ccc

151 gag cagcggc gogcc.gc.cgc cgagcc (SEQ ID NO. : 29)

. . . . . . Ex- 4 ELP1-wr 90 . . . . . . . >>

P4: SEQ ID NO. : 38

Dra 484 3.32.

2901 Cagg agttcCtgga gtcggagtgc

3101 ccggagttgg agtgcc.cgga gttggagtcc caggaggcgg agtcCCCgga

31.51 gCaggcgt.cc ccggagg.cgg agtgccgggg gtgggagttc. CC9g.cgtggg

agttc.ccgga gg.cggagtgC CCggCgCagg agttcCtgga

3301 gCaggcgtcc ccggaggcgg agtgc.cgggc gtgggagttc ccggcgtggg

3351 agttcc.cgga ggcggagtgc ccggcgcagg agttcCtgga gtoggagtgC

3401 tggagtoc Caggaggcgg agtcCocgga

3451 gCaggCgtCC CCggaggcgg agtgCCgggC gtgggagttc. CCggCgtggg

3551 CCggagttgg agtgcCCgga gttggag to C Caggaggcgg

3651 tagagggc.cc gtttaaacco gotgatcago citcgactgtg ccttctagtt (SEQ ID NO: 57)

151 cggggaacga. ggottcttct acacacccaa gaccc.gc.cgg gaggcagagg

acctgcaggt. ggggcaggtg gag citggg.cg gggg.cCCtgg tycaggcago

otgcagocct tgg occtgga gggg toccitg cagaag.cgtg goattgttgga.

30 acaatgctgt. accagoatct gctc.ccticta cca.gctggag aactactgca

351. acctCgaggg Catgggtggg CCgggCgtgg gttgttcCggg C9tgggtgtt

P . . . . ... . .. a Insul in ELP1 - 90 . . . . . . . . . . . . . . . . . . . P

401 cCgggtgg.cg gtgttgc.cggg cgcaggtgtt cotggtgtag gttgttgc.cggg (SEQ ID NO:31)

Apal -17- per

Activation of FX by FVila or FVila-ELP

4-P Fit: y = (A - D)/(1 + (x/C)^B) + D: A B C D R^2

Curve Fit Option - Fixed Weight Value

FVia-EP has a long PK in Rats

High In vitro Activity of GLP1-ELP and Exendin-4-ELP Fusion

1e-4 OOO1 0.01 O. 1 1O 1OO

PharmaCOkinetics of GLP1-ELP in Rats

tinae after administration hours)

T12 IV= 12.9 hours

Long Haif life of GP-1 EP in Rabbits

Sustained Glycemic Control of GP-EP in diabetic Mice

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