Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 9513~003 1 ~ S' 't
21 93752
MODIFIED INSUL~ E GROWTE[ FACI~RS
FiPI~I of the I
This invention relates to the ' ~ of ~/ly~Li~kD~ and more ~Li~.ul~uly to
the "~ of insulin-like growth factors and to methods of making and using such
modified yulyy~lJLhh~
r ~ ~ of ~hP ~vP~
The insulin gene family, comprised of insulin, relaxin, insulin-like growth factors
1 ^Dnd 2, and possibly the beta subunit of 7S nerve growth factor, represents a group of
structurally related puly~ Lid~D whose biological functions have diverged as reported in
10 Dull, et al., ~ 310:777-781 (1984).
T " I;IU; growth factors I and 2 aGF-l and IGF-2) are about 7-8 kilodalton
proteins that are structurally related to each other and to insulin. IGF-1 and IGF-2 s_are
about 70% amino acid identity with each other and about 30% amino acid identity with
insulin. IGF-I and IGF-2 are believed to have related tertiary structures as reported in PCT
Application Publication No. WO 90100569, published on January 25, 1990. The structural
similarity between IGF-I and IGF-2 permits both to bind to IGF receptors. Two IGF
receptors are known to exist. IGF-I and IGF-2 bind to the IGF type I receptor, while
msulin binds with less affinity to this receptor. The type I receptor l r '' ~ binds
IGF-I and is believed to transduce the mitogenic effects of IGF-I and IGF-2. IGF-2 binds
20 to the type I receptor with a 10-fold lower affinity than IGF-I. The second or type ~ IGF
receptor, r " ~y binds IGF-2. Receptor binding is beheved to be necessary for the
biolodcal activities of IGF-I and IGF-2.
IGF-I and IGF-2 are mitogenic for a large number of cell types, including
fibroblasts, I " ~U~D, endothelialcellsandosteoblasts (bone-formingcells). IGF-I and
25 IGF-2 also stimulate .~ of many cell types, e.g., synthesis and secretion of
collagens by, ' ' IGF-I and IGF-2 exert their mitogenic and cell ;~
effects by binding to the specific IGF cell surface receptors. IGF-I also has been shown
to inhibit protein catabolism in vivo, stimulate glucose uptake by cells and to promote
survival of isolated neurons in culture. These properties have led to IGF-I being tested as
a therapeutic agent for a variety of disease indications as reported in Froesch et al.,
WO95/32003 2 1 ~ 0 7 5 2 F~~ S ~ ~c ~
;n r..~ ry and ~ nl 254-260 ~aay/June 1990) and Cotterill, Clinical
r.~ Il" ~ vy 37~ 16 (1992). In addition lO specif~c cell surface receptors, there exist
at least six distinct IGF binding proteins aGFBP-l through IGFBP-6) that circulate
throughout the body. These proteins bind IGF-I and IGF-2 with high affinity. The birlding
S of IGF-1 and IGF-2 to binding proteins reduces the action of these IGFs on cells by
preventing their interaction with cell surface IGF receptors. IGF bmding proteins,
IGFBP-3, also function to prolong the circulating half-lives of IGF-1 and IGF-2
in the blood stream. In the absence of IGF binding proteins, the half-life of IGF-1 in blood
is less than 10 minutes. In contrast, when IGF-1 is bound to IGFBP-3, its half-life in blood
is lengthened to about 8 hours. The circulating half-life of IGF-1 bound to the other
smaller binding proteins is about 30 minutes as reported in Davis et al., I~Lf
rn.l,.. .;...~l,~vy~ 123 469-475 (1989); Guler et al., Acta r-~- ' ' 121:753-758(1989); and T~ l ' et al., J. of r ~.- i l..~. 123:461-468 (1989). When IGF is
bound to bindimg proteims, it is unable to bind to the IGF receptors and is therefore, no
longer active in the body. Decreased affu ity to binding proteins allows more of the IGF
to be active im the body. Situations when this decreased affnity to brnding proteins may
be useful include, for exarnple, cachexia, . u,;~, and peripheral i..,.,... ' -
r the therapeutic utility of IGF can be modifled by the presence or
absence of these IGF binding proteins, vhich may potentiate or inhibit the beneficial effects
of MF. The levels of certain IGF binding proteins can vary greatly, depending upon the
disease state. For example, IGFBP-1 levels are very high in diabetes patients, whereas they
are nearly ~ ' ' '- in normal patients as reported in Brismar et al., J. of
rn.l.,..; ~ 11:599-602 (1988); Suil~i et al., J. of t'l-
rn~ y and Metabolism, 66:266-273 (1988); and Unterman et al., Biochem.
. Biophys. Res. Cornm., 163:882-887 (1989). IGFBP-3 levels are reduced in severely ill
patients such as those tbat have undergone major surgery as repo~ted in Davies et al., L
r~ , 130 469-473 (1991); Davenport et al., J. t'lin r ~ MP~q~, 75:590-
595 (1992). The reduced levels of IGFBP-3, and consequent shorter circulating half-life of
IGF-l, may contribute to the cachexia (weight loss) seen in these patients.
Insuhn-like growth factor 1 aGF-l), also known as " C, has long been
studied for its role in the growth of various tissues. Its role as a useful therapeutic agent
for several disease conditions has been suggested. Ci" ~ 'y reduced levels of IGF-1
-
Wo s5/32003 2 ~ ~ 0 7 5 2 ~ 5.c
were found in 23 patients with varying extent and severity of burns as reported in Moller
et al., Bums, 17(4):279-281 (1991). A marked rise in serum type m ,,.~ G' '', a marker
of bone formation, occurred after one week of ~ ' ' ' " of '~, produced
IGF-1 to patients with dwarfism otherwise non-responsive to growth hommone as reported
in Laron et al., '1 ' T'-'- ' ' , 35: 145-150 (1991). The effects of the infusion
of IGF-I in a child with Laron Dwarfism is described in Walker et al., Th,- New T~.n~
Tm~ of M~irinA 324(21):1483-1488 (1991). Increased weight g~un, nitrogen retention
and muscle protein synthesis following treatment of diabetic rats with IGF-I or IGF-I
having a deletion of the first three amino acids ordinarily found in IGF-I (referred to as
"(desl-3)IGF-l") were .~ ' in Tomas et al., r ~ J.. 276:547-554 (1991).
Growth restoration in msulin-deficient diabetic rats by ' of .- .1~,
produced human IGF-I is reported in Scheiwiller et al., ~hlL, 323:169 (1986). IGF-I
and (desl-3)IGF-I enhance growth in rats after gut resection, as reported in Lemmey et al.,
J. Physiol.. 260 (F-' ' Metab. 23) E213-E219 (1991). A . ' of
platelet-derived growth factor and insulin-like growth factors, including IGF-I, enhanced
"'G~ ''1;"" in beagle dogs as reported by Lynch et al., J. ~
16:545-548 (1989). The synergistic effects of platelet-derived growth &ctor and IGF-1 in
wound healing are reported in Lynch et al., P~oc. I~Tqt7 A~ l Sci.. 84:7696-7700 (1987).
'rhe effects of IGF-I and growth hormone on 1 ~ " I bone growth in vitro are set forth
in Scheven and Hamilton, .A~`tq rn ~ 1"~ (cove~h~ 124:602-607 (1991). In vivo
actions of IGF-I on bone formation and resorption in rats are shown in Spencer et al.,
~, 12:21-26 (1991). The use of IGF-1 and IGF-2 for enhancing the survival of non-
mitotic, cholinergic neuronal cells in a mammal is described rn U.S. Patent 5,093,317 to
Lewis et aT. In addition, PCT Application Publication No. WO 92111865 published on July
23, 1992, describes the use of IGF-I for the treatment of caTdiac disorders.
Various ' ~ to the naturally occurring or wild-type IGF-I have been
described. For example, the naturally occurring variant of IGF-I missing from the first
three N-terminal amino acids, (desl-3)IGF-I, was discovered in cerebral spinal fluid and
- in colostrum as reported in Sara, et al., Proc.l~Tqtl Ar-q~l .Cri 83:4904~907 (1986) and
Francis et al., Rin.~Amirql Jmlr~ql 251:95-103 (1988). In vitro studies have shown that
this variant is equipotent to IGF-I in binding to IGF cell surface receptorS and in
stimulating cell X,, Thus, the fTrst three amino acids of IGF-1 appear to be
Wo gs/32003 2 1 9 0 7 5 2 r~.,u~: c'oc. 10 ~
I for the binding of IGF-I to its specific cell surface receptors. (Desl-3)IGF-Iwas found to have greatly reduced affinity (100-fold less) for certain IGF binding proteins,
specifically IGFBP-I and IGFBP-2, as reported in Forbes et al., Ri~h~ m Biovhys. Res.
Comm., 157:196-202 (1988); and Carlsson-Skwirut et al., r Bi~-h~ys. A~
1101:192-197 (1989). The binding of IGFBP-3 to (desl-3)IGF-I also is affected, being
reduced by two to three fold. Animal stndies have shown that (desl-3)IGF-I, when given
by continual ' infusion, is more potent than IGF-I m stimulating a mlmber of
anabolic functions, such as growth, reported in Cascieli et al., J. E ' ' ~y, 123:373-
381 (1988) and Gillespie et al., J. ~ Iu~ -y, 127:401-405 (1990). The enhanced
properties of (desl-3)1GF-1 are believed to result from its reduced affinity for IGF binding
proteins, reported in Carlsson-Skwirut et al., ~- - Bi~-h!ys. A~ 1101:192-197
(1989). The reduced affuuty of (desl-3)IGF-I for IGF binding proteiAs results in (desl-
3)IGF-I having a shorter circulating half-life than wild type IGF-I as reported in Cascieri
et al., J. rA~ py. 123:373-381 (1988). Therefore, (desl-3)IGF-I must be
- ' l by continual infusion or by multiple daily injections in order to effoct its
enhanced potency.
PCT Application Publication No. WO 89105822 publishod on June 29, 1989,
describes other ,~ of IGF-I. This application describes substituting the third
amino acid from the N-terminal end of the naturally occurring IGF-I with glycime,
glut~mime, leucine, arginine or Iysine to form IGF-I muteins. This reference however doos
not teach replacing the third amino acid with cysteine.
The potential therapeutic usefulness of IGF-I and (desl-3) IGF-I is limited by their
short circulating alf-lives to situations when the proteins can be r ' by continual
infusion or by multiple daily injections to achieve thoir maximum therapoutic potontial. As
am example, Woodall et al., T~ M~-h Res., 23: 581-584 (1991), reports that the same
total dose of IG~-I r four times daily by ' injection was far superior
in stimulating growth im mutant lit/lit mice (growth hormone deficient mice) than was the
same total dose ~ ' ' once daily.
There are many cases in wmch it would be preferable to administer IGF-1 in a
single onc~a-day injection or in a single injection given once every several days. For
injeciable drugs, patient . , " is expected to be higher for drugs that can be
r- ~ once a day rather than several times a day. In order for IGF-I to be
~ woss/320n3 2 l 9 3 7 52 r~llu ~r~
lly effective when given once a day or once every few days, methods must be
developeo to increase its circulating half-life.
Increasing the molecular weight of a protein by covalently bonding am iner~ polymer
chain such as ~ .h~k ..., glycol (PEG) to the protein is known to increase the circulating
5 half-life of the proteirl in the body. See, for example, Davis et al, F~ 1 polymp~
Polymeric r~ - and F' '~ for p~inmPrlir~ll Use. p. 441-451 (1980).
However, since multiple PEG molecules can bind to each protein molecule, and because
there are typically a large number of sites on each protein molecule suitable for bindirlg to
several PEG molecules using known methods, there has been little success in attaching PEG
to yield l- O reaction products. See Goodson et al, Biotechnologv, 8:343 (1990),and U.S. Patent 4,904,584. This lack of site attachment specificity can give rise to a
number of problems, including loss of activity of the protein.
Thus, a need exists for prolonging the circulating half life of IGF without
, its usefulness as a therapeutic agent. The present invention satisfies this need
15 by increasmg the molecular weight of the IGF. This is . ' ' ' by providing muteins
of IGF suitable for thiol-specific attachment of PEG to IGF and PEG conjugates formed
from such muteins.
Q of the InvP ~inn
The mvention relates to various modifled forms of IGF. One type of modified IGF,20 referred to as mutems, is produced by replacmg cysteine residues for specific amino acids
in the wild type molecule, or by inse~ting cysteine residues adjacent to or bet~veen amino
acids in the wild type molecule. Cysteine residues which are not involved in
disulfide bonds are considered to be "free". Cysteine residues can be substituted or inserted
in regions of the IGF molecule that are exposed on the protein's surface. For example, the
25 non-native cysteime can be inserted or substituted within the first or last twenty amino acids
of wild-type IGF-I. This non-native cysteine is expected to be free and to serve as the
attachment site for the pol,~,LI~ , glycol (PEG) molecules to IGF, resulting in PEGylated
molecules. In some cases, however, during refolding of the mutem or during reduction of
the mutein before reaction with PEG, the non-native cysteine can become mvolved in a
30 disulfide bond and thereby free a native cysteine for PEGylation. Attachment of the PEG
wo gsl32003 2 1 9 0 7 5 2 F~l/u..: cr r A ~
molecule to a mutein creates a further modified for n of IGF, or IGF-PEG conjugate of
defmed structure, where the PEG is attached to the IGF at a free cysteine residue.
Thus, the present invention is directed to a PO~ glycol (PEG) conjugate
comprising PEG and a mutein of IGF, and IJ~ti~,ul~ly IGF-1, where the PEG is attached
to the mutein at a non-native cysteine. PEG can be attached to the free cysteine through
a thiol-specific activating group including, for example, maleimide, sulfhydryl, thiol,
triflate, tresylate, aziridine, exirane and S-pyridyl. A suitable PEG can have a molecular
weight of 5 kDa, 8.5 kDa, 10 kDa or 20 kDa. The PEG conjugate of tùe present invention
can also contain a second protein to form a dumbbell. Methods of making the PEG
conjugates are also provided.
Moreover, PEG is known to increase the circulating half-life of a protein in thebody. Thus, IGF can be ~' ' to patients less frequently with equal or better
.,rr~L;~ than in the past.
The present invention is further directed to muteins of IGF l.~u Liuul~iy those having
IS a non-native cysteime in the N-terrninal or C-terrninal region of the mutein. The muteins
can be produced by I ` methods.
Also provided in the present invention are I ' ' l~ comprising
the IGF-PEG conjugate and methods of using the IGF-PEG conjugate to treat a patient
havimg or potentially having an IGF associated condition.
~ P.~r.lrfinn of fhP Tnv,onri~n
The present invention is directed to modified forms of insulin-like growth factors
aGF) that provide beneficial proper~ies not exhibited by wild-type IGF. The modified
forms of IGF include muteins of these growth factors, containing at least one free cysteime.
Conjugates containing the IGF muteins attached to pol~.LhJh,..~, glycol (PEG) are also
25 considered modified forms of IGF.
Terms used throughout the ~ and claims are defined as follows:
The term "IGF" refers to any pol~id~ that binds to the IGF type I Receptor,
including, for ex~unple, IGF-l, IGF-2, (desl-3)1GF-1, (R3)1GF-1 which is a mutein having
a non-native arginine at residue number 3, and insulin. This hormone family is described
in Blundell and Humbel, ~a~L, 287:781-787 (1980). Due to this common receptor
w0 95/32003 2 1 9 ~ 7 5 2 r~"u ~r ^
binding, the teachings of the present invention which are described with respect to IGF-1
are mtended to encompass IGF-2, des(1-3) IGF-l, (R3)IGF-1, and insulin also.
The term "wild type IGF" refers to the, "~ ' or naturally occurring IGF. This
term is used , ' ~ ' 'y with "IGF," "naturally occurring IGF," or "native IGF." The
S term "wild type IGF" also refers to native IGF to which a methionine residue has been
added at the N-termimus.
The term "wild type IGF-1" refers to the I ' ~ ' or naturally-occurring 70
amino acid form of IGF-1. This term is used ' , "y with "IGF-1," "naturally
occurring IGF-1," and "native IGF-1." The term "wild type IGF-1" also refers to native
IGF-1 to which a methionine residue has been added at the N:
The term "~.u.. ~ " refers tO that which is not found im the native molecule.
The term "IGF-PEG conjugate" refers to an IGF molecule attached to a IJUI~
glycol molecule. This is also referred to as a "Peg conjugaten.
The term "N-terminal region" refers to ~ , the first twenty ammo acids
IS from the N-termimus of IGF or an IGF mutein, and up to twelve amimo acids preceding the
frst amino acid of the N-terminus of IGF.
The term "N; " refers to the frst amimo acid at the N-ter~ninal region im the
sequence of wild-type IGF, for example, glycine in IGF-1.
The term ~C-terminal region" refers to ~I,UI~ / the last twenty amino acids
20 from the C-termimus of IGF or an IGF mutein and up to twelve amino acids following the
last amino acid of the C-termimus of IGF.
The term "C-termimus" refers to the last amimo acid at the C-termmal region in the
sequence of wild-type IGF, for example, alanine m IGF-I.
The term "mutein" refers to a modified form of IGF, which has been modified to
25 contain a non-native cysteine.
The term "retain biological activity" refers to having at least 10% of the mitogenic
activity of wild type . ' IGF as measnred by the relative amount of 3H-thymidine
, ~ into UMR106 rat . cells, in the absence of IGF binding protein-1,
usimg the assay described herein. The muteins and the conjugates of the present invention
30 retain biûlogical activity.
The term "activating group" refers to a site on the PEG molecule which attaches to
the muteim.
woss/32003 2! 90~52 ~ 5~ l^
,
The term "I' lly acceptable carrier" refers to a ylly..;~lu~ y
' ', aqueous or non-aqueous solvent.
The term "free cysteine" refers to any cysteine residue not involved in an
i l.- . -l. '-. disulfidebond.
The term "IGF associated condition" refers to an existing or potential adverse
Ly ~;ulù~ l condition which results from an over-production or ' , ' of IGF,
IGF binding protem or IGF receptor, , ~,, or inadequate binding of IGF to binding
proteins or receptors and any disease in which IGF: ' aLeviates disease
symptoms. An IGF associated condition also refers to a condition in ~hich - '
of IGF to a normal patient has a desired effect.
The term "patient" refers to any human or animal in need of treatment for an IGFassociated condition.
The IGF muteins oFthe present rnvention are produced by modifying wild-type IGF,particularly at the N-terminal or C-terminal region of the protein. Such . ~.~;ri. ~ can
be l or additions of at least one cysteine residue. An IGF mutein can be
produced by replacing a specific amimo acid with a cysteine, such as, for example,
~, one of the frrst or last four amino acids of IGF-1 with a cysteine residue. The
amino acid sequence of wild type IGF-1 starting from the N-terminal end is: G P E T L
CGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTG
IVDECCFRSCDLRRLEMYCAPLKPAKSA~SEQIDNO. 1).
Other ' include, for example, adding at least one cysteine residue in
Front of the first or after the last amino acid of IGF. Fûr example, a cysteine residue can
be inserted in front of and adjacent to the ftrst amino acid of IGF. For muteins produced
by E. cûli, the non-native cysteine can appear between Met and the frrst amino acid of IGF.
A free cysteine residue can also appear in a group of about twelve or less amino acids
inserted before the first or after the last amimo acid of IGF to form a longer IGF mutein.
In IJ~u~uLuly useful " the non-native cysteine residues are located in
regiûns of the IGF-I molecule exposed to the protein's surface. The N-terminal region, for
example, is involved in the binding of the IGF to binding proteins, but is not involved in
binding of IGF to cell surface IGF receptors. ~
An IGF-I mutein of the present invention is also referred to as "a cysteine mutein
of IGF-I." The non-native cysteine residue can act as the attachment site for covalent
wo 95/32003 2 ~ 9 ~ 7 5 2 r~ r - 10
linkage of the activating group on the pvl.~ l.jh..~ glycol. Altemavively, the non-native
cysteine can become involved in disulfide bonding thereby freeing a native cysteine residue
for thiol-specific attachmem to PEG. The newly created molecule comprising the cysteme
mutem of IGF with the PEG attached is referred to as a "PEG conjugate of IGF~.
S The IGF muteins of the present invention can be prcpared by methods well known
to one skilled in the art. Such metbods include mutagenic techniques for ~ ~; ormsertion of an ammo acid residue, as described, for example, in U.S. Patent 4,518,584,
l ' herein by reference. The mutems produced by mutagenic techniques can then
be expressed as ' products according to procedures known to those skilled in the10 art. The muteins can ~llt~ V~,ly be synthesized by Cu_llliv~ methods known in the
art. The IGF muteims can also be prepared according to the methods atld techniques
described in the examples set forth below.
The present invention also provides IGF-PEG conjugates and methods of making
such conjugates by attaching the IGF muteins to pol~,lhjk,l.c glycol to mcrease the
circulating half-life of the molecule in the body as well as decrease its afftnity to IGF
bmding prvteins.
In the present invention long chain polymer units of pvl~.hgl~llC glycol (PEG) are
bonded to the mutein via a covalent attachment to the sulfhydryl group of a free cysteine
residue on the IGF mutem. Various PEG polymers with different molecular weights, 5.0
kDa (PEG~ ), 8.5 kDa (PEGUoo), 10 kDa (PEGIoooo), amd 20 kDa (PEG200o~) can be used
to make the IGF-PEG conjugates. In order to obtain selectivity of reaction and
~" reaction mixtures, it is useful to use ' ' ' polymer units that will
react specifically with sulfhydryl or tniol grvups. The functional or reactive group attached
to the long chain pvl~ glycol polymer is the activatmg grvup to which the IGF
mutein attaches at a free cysteme site. Useful activating groups include, for example,
maleimide, sulfhydryl, thiol, triflate, tresylate, aziridme, exirane, or 5-pyridyl.
In another I t, PVIJ~hjh~IIC glycol (PEG) polymers containing two
activating grvups can be used to create "dumbbell" type molecules containing two IGF
- muteins attached to one molecule of PEG at each end of the PEG molecule. For example,
30 PEG 1.;1 ' (a pUl~, ~k,llc glycol polymer containing a maleimide activating group
on each end of the PEG molecule) can be used to create these "dumbbell" type molecules.
These dumbbell molecules can also comprise a single IGF mutein covalently attached via
wos~/32003 21 9 07 52 r~ ,5,.~
PEG to a second protein or peptide of different structure. The second prooein or peptide
can be, for example, a growth factor such as platelet-derived growth factor, or fibroblast
growth factor.
One skilled in the art can readily deoermine the appropriate pH, of
S protein, and ratio of protein:PEG necessary to produce a useful yield of either mono-
pegylated IGF- I aGF-PEG), or dumbbell IGF- I aGF-PEG-IGF, IGF-PEG-PDGF, or IGF-PEG-FGF) using ~;u..~v...~u.~l methods known to one skilled in the alt for making these
, . . . .
The invention present also includes l ' l ~ . The IGF muteins
1û and PEG conjugates can be in a 1' ~ 'ly-acceptable catrier to form the
~,h ".. . ~..';. Al , ~' of the present invention. The term ~l' "S~
acceptable carrier" as used herein means a non-toxic, generaUy inert vehicle for the active
ingredient, wmch does not adversely affect the ingredient or the patient to whom the
is _' ' Suitable vehicles or catriers can be found in standard
~' 'texts,forexample,inr 's~' 'Sri~n~ 16thed.,Mack
Publishing Co., Easton, PA (1980), i . ' herein by reference. Such carriers
include, for example, aqueous solutions such as L- ' buffers, phosphate buffers,Ringer's solution and ,uh~;vlv~ saline. In addition, the carrier can contitin other
l' '~S,-acceptable excipients for modifying or v the pII, osmolarity,
viscosity, clarity, color, sterility, stability, rate of ~' ' or odor of the r~
The l' ', , can be prepared by methods krlown in the art,
imcluding, by way of am example, the simple mixing of reagents. Those skilled in the art
will know that the choice of the ,ul~ ' carrier and the appropriate preparation of
the, l depend on the intended use and mode of
In one i ' ' t, it is envisioned that the carrier and the IGF muoeirl or conjugate
constitutes a ,u~ ;vlvo;~lly- , ' ' ~lv....1~., ' ' The primary solvent in
such a carrier can be either aqueous or non-aqueous in nature. In addition, the carrier can
contilin ULh~ -acceptable excipients for modifyimg or ,, the pH,
osmolarity, viscosity, clarity, color, sterility, stability, rate of ~" ' or odor of the
' ' Simil3rly, the catrier can contain still other I ' ' g l'~-acceptable
excipients for modifying or l" the stability, raoe of ' ' release, or
absorption of the IGF mutein or conjugate. Such excipients are those substances usually
. .
woss/32003 2 1 93752 1~ s~
and customarily employed to formulate dosages for parenteral ~ ;.... in either ulut
dose or multi-dose form.
Once the, ' I, . has been r 1~ it can be stored im sterile
vials as a solution, suspension, gel, emulsion, soLid, or dehydrated or IyophiLized powder.
5 Such 1( 1 may be stored either in a ready to use form or requiring
'y prior to r' ' The preferred storage of such r ~ is at
at least as low as 4rc and preferably at -70C. It is also preferred that such
containing the IGF mutem or conjugate are stored and ' ~ at or near
J~h~lo~h~l pH. It is presently believed that ' im a r~ at a high pH
(i.e. greater thfm 8) or at a low pH (i.e. Iess than 5) is l ' ' '
The manner of ' v the r ~ ' contaming the IGF mutem or
conjugate for systemic delivery can be via ' , ~ 0~15~ oral,
intranasal, or vaginal or rectal - r r " y . Preferably the manner of: ' of the
COntSdilliiîg the IGF muteins or conjugates for local delivery is via
;.. l. - i;. .. l~., ' l, orimstiLlationorinhalationstotherespiratorytract. Inaddition
it may be desirable to administer the IGF muteins or conjugates to specified portions of the
alimentary can~ either by oral - ' of the IGF muteims or conjugates in an
or device
For oral: ' the the IGF muteins or conjugates are r ~ ~ The
, ' ' IGF muteins or conjugates may be formulated with or without
cceptable carriers . ~!~ used in the . , " _ of solid dosage
forms. Preferably, the capsule is designed so that the active portion of the r ~ ' is
released at that poimt m the gastro-intestinal tract when ~ ,/ is maximized and
pre-systemic .l. v,~ ;.... is minimized. Additional excipients may be included to facilitate
absorption of the IGF muteins or conjugates. Diluents, flavorings, low melting point
waxes, vegetable oils, lubricants, suspending agents, tablet ~" v v agents, and
bimders may also be employed.
Regardless of the manner of r ' ' ' ' " , the specific dose is calculated accordmg
to the, . body weight of the patient. Other factors in !' ' ' ' ~ the appropriate
dosage can imclude the disease or condition to be treated or prevented, route of: ' and the age, sex and medical condition of the pateint. In certain
' ' the dosage and -' is designed to create a preselected
woss/32003 21 q07 52 r~ c--10 ~
range of the IGF muteins or conjugates in the patient's blood stream. It is
believed that the of circulating .,. . ~ of the IGF muteins or conjugates
of less than 0.01 ng per ml of plasma may not be an effective ~ , while the
prolonged of circulating levels in excess of 100 ~g per ml may have
S undesirable side effects. Further refmement of the ~ .nc necessary to determine the
appropriate dosage for treatment involving each of the above mentioned r '- isroutinely made by those of ordinary skiU in the art and is ~ithin the ambit of tasks routinely
performed by them without undue, l , especiaUy in light of the dosage
' ~ and assays disclosed herein. These dosages may be ascertamed through use
10 of the established assays for .' ~ l,, dosages utilized in ; with appropriatedù~_ ...,~u...._ data.
It should be noted that the IGF mutein and conjugate l ' ' ~
described herem may be used for veterinary as weU as humam d~ ;- .-- - and that the term
"patient" should not be construed in a limiting manner. In the case of veterinary
"1~ 'A, the dosage ranges should be the same as specified above.
The l' ~ f the present iAvention can be used to treat a
patient having or potentiaUy having an IGF associated condition. Some of these conditions
can include, for example, dwarfism, diabetes, periodontal disease and U~tW,UU.J~;~. The
': , . of the present invention can also be used to treat a condition
in which ' of IGF to a normal patient has a desired effect; for example, using
IGF-I to enh~mce growth of a patient ûf normal shture.
The foUowing examples are intended to illustrate the present imvention and are not
intended to be limiting.
EXAMPL~ I
A. C of the IGF-I ge~e
The IGF-1 gene was assembled in two shges. InitiaUy, the DNA sequence encoding
IGF-1 was joined to DNA sequences encoding the secretory leader sequence of the E. coli
OMP A protein (ompAL). This gene fusion was rnnctr~ t~ in order to determine whether
IGF-I could be efficiently secreted from E. coli. A second construct, in which IGF-1 is
expressed as an int~.ll ' protein in E. coli, was created by deleting DNA sequences
12
wo9sl32003 2 1 ~0752 F~"~,~ s~6 1~
encoding the OmpA leader sequence and replacing them with DNA sequences that allow
inrr~rf~ r expression of IGF-l.
B. r - of tbe OmpAL-IGF-I gene fusion
The four synthetic oligonucleotides labeled OmpAlU:
5'GATCCGATCGTGGAGGATGATTAAATGAAAAAGACAGCTATCGCGATCGCA3'
(SEQ ID NO. 2), OmpA2U: 5'GTGGCACTGGCTGGTTTCGCTACCGTA
GCGCAGGCCGCTCCGTGGCAGTGC3' ~SEQ ID NO. 3), OmpAlL: 5'CAGTGC
CACTGCGATCGCGATAG~ ATTTAATCATCCTCCACGATCG3 ' (SEQ
ID NO. 4) and OmpA2L: 5'GCACTGCCACGGAGCGGCCTGCGCTAC
GGTAGCGAAACCAGC3' (SEQ ID NO. 5), were annealed pairvise (lU + lL amd 2U
+ 2L) and the pairs ligated together. All four of these ~ , ' ' were synthesizedusing DNA ~ purchased from Applied Biosystems (Models 391 and 380A). The
ligation mixture was then digested with the restriction enzyme Haem. The resulting
BamE~/EIaem restriction fragment coding for a ~ l start signal and the first 21
amino acids of the ompA signal sequence was purified. This DNA fragment was mixed
with BamElI + PstI-digested PUC18 DNA (. ~;~I r available from Boehringer
Marmhein r -~ 's, T " . ', IN) and the two synthetic 'i~ ~ ' [IGF-I
(1-14) U + L] 5'CCGGTCCGGAGACTCTGTGCGGCGCAGAACTGGTTGAC
GCTCTGCA3' (SEQ ID NO. 6) and 5'GAGCGTCAACCAGTTCTGCGCCGC
ACAGAGTCTCCGGACCGG3' (SEQ ID NO. 7) were ligated together. The ligation
mixture was used to tr.msform ~QIi strain ~M109 (. ~Iy available from New
England Biolabs, Beverly, MA) and individual colonies isolated. These plasmids
(OmpALlGF-lpUC18) have a i ' ' start signal followed by DNA sequences
encoding the OmpA signal sequence amd the first 14 amino acids of IGF-I.
An M13 phage containing DNA sequences encoding amino acids 15 through 70 of
IGF-I was created by ligating together the two ~ ,' y pairs of ~-"~, ' '
(tGFIU + IL and IGF2U + 2L) 5'GTTCGTATGCGGCGACCGTGGCTTC
TACTTCAACAAACCGACTGGCTACGGTTCCAGCTCTCGTCGTGCACCGCAG
ACTGGTATC3' (SEQ ID NO. 8) and ~ ~lC(JluAACGATACCAGTCTGCGGTGC
ACGACGAGAGCTGGAACCGTAGCCAGTCG~ l l l ~ l l ~AAGTAGAAGCCACG
GTCGCCGCATACGAACTGCA3' (SEQ ID NO. 9) and cloning the DNA fragment into
13
woss/320o3 21 90752 P~llu~ ~c 10
Pstl + Hindm-digested M13 mplg DNA (commercially available from New England
Biolabs, Beverly, MA). Double-stranded DNA was purified from a phage clone and the
PstI/Hindm fragment encoding ammo acids 15-70 of the IGF-1 protein were isolated. This
DNA fragment was ligated together with Pstl + Hindm-digested plasmid OrnpALlGF-
lpUC18 DNA and used to transform E. coli strain 3M107 (. l~y available from
GIBCOBRL, t~ h ..~l....v, MD). TheBamHI/HindmfragmentcontainingthelGF-I gene
fused to the OmpAL sequence was isolated and cloned into the BamHI + Eindm generated
site of plasmid pT3~-2 (described in PCT Application pubhication WO 91/08285 pub~ished
on June 13, 1991). The completed plasmid containing the ompA~IGF-I gene fusion is
called pT3XI-2 010C(TC3)ompALlGF-1.
C. I' of the ?' - ~1 IGF-l gene
The BamHI/Hindm fragment contaming the OmpA~IGF-I gene fusion described
above was purified from plasmid pT3XI-2010C(TC3)ompALlGF-I and digested with Hinf~.
The all 200 bp Hinff/Hindm DNA fragment was mixed with the almealed,
, . y synthetic -'iv ' ' (MetIGFlU + lL) 5'GATCCGATCGT
GGAGGATGATTAAATGGCCG~l~CGGAG3' (SEQ ID NO. 10) and 5'AGT
CTCCGGACCGGCCATTTAATCATCCTCCACGATCG3' (SEQ ID NO. I l) arid ligated
with BamHI + Hindm-digested plasmid pT3XI2 DNA, and used to transform E. coli
JM107. The completed plasmid construct is called 010C(TC3)IGF-lpT3XI-2 and contains
an extra alanine residue in between the initiator methionine and the beginning of the IGF-1
sequence. The BamHI/Hindm fragment containing the mutant IGF-1 gene was isolated and
ligated into the BamE~ + Hindm generated site of plasmid pT5T (described in Nature,
Vol. 343, No. 6256, pp. 341-346, 1990). The ligation mixture was used to trarlsform E.
coli BL21/DE3 described in US Patent 4,952,496 and the resulting individual colorlies were
isolated. This construct was named 010C(TC3)IGF-lpT5T.
The extra alanine codon was removed by in vitro v In vitro
was performed usmg a Muta-Gene kit purchased from Bio-Rad T ' ' (R' ' 1,
CA). The v procedure followed was essentially that described in the i -~. . I ;., -
that accompany the l~it. Plasmid 010C(TC3)IGF-lpT3XI-2 was digested with BamHI +Hindm and the 200 bp DNA fragment contair~ing the mutant IGF-1 gene was purified and
cloned into the BamE~ and Hindm sites of plasmid M13 mpl9.
wo gs/32003 2 1 9 0 7 5 2 l'~ u~,S C :^
Uracll-containing single-str~mded template DNA was pre~oared follu . . illg ,UI~J.~""LiOII
of the phage in E. coli strain CJ236 (supplieA with Muta-Gene Kit purchased from Bio-Rad
T ' - ,Richmûnd,CA). The,-li, ' ' usedfor , hadthesequence:
5'GATGATTAAATGGGTCCGGAGACT3' (SEQII) NO. 12). The ~ reaction
5 producf was used to transform E. coli strain JM109 and individual plaques picked.
Double-stranded replicative form DNA from imdividualphages was isolated, digested
with BamHI + Hindm and the 200 bp fragment containing the IGF-1 gene purified. The
purified DNA was cloned into the BamHI + Hindm generated site of plasmid pTST and
used to transform E. coli strain BI~1/DE3. One bacterial cûlony with the correct plasmid
was named 010(TC3)mutlGF-lpT5T. Several isolates were sequenced, and all were
correct.
EXAMPLE 2
C of IGF-1 Muteins
Several muteins of IGF-1 were cn"cfn~rf~A Three of the muteins replaced each of
the first three amino acids of IGF-I with a cysteine residue. These muteins are referred
to as Cl, C2, and C3, I~i.,U~i~.,ly. A fûurth muteim introduced a cysteine residue between
the N-terminal methiûnine residue and the first amino acid of IGF-I. This mutein is
referred to as -lC.
The -lC, C1, C2 and C3 muteins of IGF-1 were made using the 1,~ chain
reaction (PCR) technique as described below. The starting plasmid used for the Ie, was 010(TC3)mutIGF-lpT5T, which is described in Example 1. This plasmid
contains DNA sequences encûding an irlitiator methionine followed by the sequence of the
natural humam IGF-1 protein. Mut mt IGF-1 DNA sequences were amplified frvm tms gene
using a 5' ~ iv ' ' that contained the desired mutation and a 3'
~i~, ' ' of correct sequence. The 5' ~ ' were designed
sû that they , ' the first methionine of the gene as part of am Nde I restriction
enzyme cleavage site (CATATG). Each ~ ~ 'i,, ' ' contained the desired
mutation followed by lS to 21 nucleotides that were a perfffl match to the IGF-1 gene
sequences im plasmid 010(TC3)mutlGF-lpTST. The 3' ~'iv ' ' was 25 nucleotides
long and was designed to encode the last 6 codons of IGF-1 amd to cûntain the Hind m site
that follows the stop codon.
wo 95/32003 2 l 9 0 7 5 2 r~l~L~ ~ , ~
In addition, a wild type clone was made in the same malmer using the Ndel site of
pTST (described above) as the first I ' This wild type clone is designated 85p-11.
The, 'i~, ' ' used to construct the -1 C, C1, C2 and C3 muteins and the 85p-11 wild
type clone are set forth in Table 1.
16
WO 9S/32003 2 1 9 0 7 5 2
-
r
. _ ~
,
'
J
-' J
X ~
V ~ O .
~, m E~ 3
. m P~ m m ~
x
O ~ ~
a
w0 95/32003 2 19 0 7 5 2 r "u.
Polymerase chain reaction (PCR) was performed in 100ul reactions containing 20
mM Tris pH 8.8, 10 mM KCI, 6 mM (NH4)2S04, 1.5 mM MgC12, 0.1% Triton X-100
using 20 pmole of each oligo and ~ 'y Ing of plasmid 010(TC3)mutIGF-lpT5T
as template DNA. 0.5ul (1.25 units) of Pfu pol~ (Stratagene, San Diego, CA) was
5 added after the first .l ~ ,.. step with the tubes held at 65C. The reactions were
oYerlayed with 2 drops of mineral oil at that time. The reactions were cycled 30 times for
I mim. at 95C, I min. at 65C, and I min. at 72C in an Ericomp Twinblock'Y thermal
cycler (Ericomp, San Diego, CA). After the last cycle the reactions were held at 720C for
10 minutes.
After PCR, 80ul of the reactions were phenol extracted one time then ethanol
l ' The r ~ DNA was . ' ' in 80ul of TE buffer (10 mM Tris-
HCI pH 8.0, I nlM EDTA) and 20ul was digested with Nde I and Hind m and
~,I~L.I I ' ' on a 1.5 % agarose gel. The amplified DNA bands that ran at
210bp were eluted using NA45 paper (Schleicher and Schuell, Keene, NH)
15 according to the r 'S ' ' '' The eluted DNA was ~ in 20ul of
TE buffer and 2ul was ligated to gel-purifled Nde I and Hind m digested plasmid pTST in
a volume of 20ul. Plasmid pT5T is described in Example 1. The ligation reactions were
used to transform ~.~QU stram BL21/DE3 and colonies selected on LB agar plates
containing 50ug/ml of ampicillm. Milu plasmid DNA preps were made from several
20 colonies from the j r " plates. The DNAs were digested with Eco RV and Hmd
m to determine which j r ' contained IGF-I DNA inserts. Plasmids containing
IGF-I DNA inserts were sequenced to verify that the inserts were correctly mutated (the
entire IGF gene was sequenced for each mutein).
P~ - ' ' y growth studies were performed by growing a - ~ ; V~; i r
25 for each mutein in Luria Broth + 12ug/ml t~ a~ " to an ~, OD60o of about
1Ø Isopropyl-beta-D-i'-~ y ' aPTG) was added to I mM to induce
expression of T7 ~,ol~ and the subsequent ~ ;.... and translation of the IGF
muteins. ~A~ '!~ 0.1 OD unit of cells were Iysed in SDS sample buffer by boilmg
for two mimutes and ~ .' ' on a 16% P~IJ~ SDS gel. The gel was0 stained with Coomassie blue. IGF-1 protein bands of the expected size, which is
/ 7-8 kDa, were observed in the lanes loaded with induced cells for each
mutein as well as for the wild-type control.
18
1~ wo 95/32003 2 19 0 7 5 2 r~l,u~ c -- ^
Muteins in which cysteine was substituted for the amino acids at residues 11, 12,
15, 16, 55, 64, 65, 67 and 69 of the wild type IGF-l were also prepared using PCR. The
PCR template was either the full-length plasmid from clone 85p-11 or the NdeI-Hindm
fragment from clone 85p-1 I containing the wild type IGF-I gene.
S For muteims Cll, C12, C15 and C16, 5' ~" ~ ' ' were synthesized that
' the furst methionine as part of the NdeI site (CATATG) and each contained
the mutation desired followed by 21-24 nucleotides that were a perfect match to clone 85p-
11. These, 'i, ' ' are set forth im Table 2. For the 3' end of the gene, a 25-mer
~ aGF(262p)25) that nnatched the last 6 codons of IGF-I plus the Hindm site
followmg the stop codon was used. IGF(262p)25 is set forth in Table 1.
19
W09V3~003 21~0~2 r~ o
A
~`
.
~'
. _
00
ô~ 6
~ ~ o~ ~ .
t
8 : c~
S '~
" ~
V'~
wo 95t32003 2 1 9 0 7 5 2 r~ l,u~s loG~ ^
For muteins CSS, C64, C65, C67 and C69, 3' oli~,. ' ' that I ~ the
Hindm site (AAGCTT), the stop codon (TAA) and containing the mutation desired
followed by 21-27 nucleotides that were a perfect match to clone 85p-11 were synthesized.
For the S' end of the gene, a 28-mer, li, ' ' (IGF(85p)28) that matched the first
5 . 7 codons of IGF-I plus the initiation codon for methionine ill~,ul~ ' into an NdeI site
was used. These ~ ' ~ ' ' are set forth in Table 3.
W~9~32003 21 90752 r ~u~ ~ ~
~",
~ . `'
~ , .
. .
& ~ ~
YY
.
u
~ . ~ u ~
y ~ ~ ~J l ~ -
U ~ ~ ~
,
22
~ o
~ WO 95/32003 2 1 9 7 5 2 ~ ~, ., ~ ~ c
Polymerase chain reaction (PCR) was performed in 100ul reactions containing 20
mM Tris p~ 8.8, 10 mM KCI, 6 mM (NH4)2SO4, 1.5 mM MgC12, 0.1% Triton X-100
usmg 20 pmole of each oligo, rr ' ' ~1 lng of template DNA, 200uM each of dATP,
dCTP, dGTP, TTP, 20pmole of each oligo primer, and lul (2.5Tl) of Pfu polymeraseS (Stratagene, San Diego, CA). The reactions vere cycled 30 times for I mirl. at 95C, 1
min. at 65C and I min. at 72C in a GeneAmp PCR System 9600 (Perkin Elmer Cetus).
After the last cycle the reactions were held at 72C for 10 min.
After PCR, the reaction mixtures were purified by passing through ~'' S1 100
columns (Clontech Lab. Inc., Palo Alto, CA). Purified PCR fragments were digested with
NdeI and lIindm and the--210bp bands eluted from 1.5% agarose gel using NA45 paper
(Schleicher and SchueD, Keene, NEI) according to the r ~S ~ The
double cut and gel-purif~ed DNA fragments were ligated to similarly cut and gel purifled
pTST plasmid at 15C for 18 hrs. The resulting ligation mixtures were used to transform
E. Coli strain DHSa and plated on LB + ampiciDin (SOuglml) plates. Plasmid DNAs were
prepared from several colonies using the Qiagen plasmid kit and the entire IGF-I gene
sequenced with the Taq DyeDeoxy Terminator Cycle Sequencmg Kit (Applied
Biu~y ) A correct construct was selected for each mutein (clones Cl 1-1, C12-3, C15-
1, C16-4, C55-1, C64-1, C65-1, C67-2, C69-1) and j r ~ mto E. Coli strain
BL21/DE3 for expression.
r y expression studies of the muteins were performed by growing two
t, j r ' for each mutem in LB + t~LI,.~,' (12uglml) to an
lj r OD600 0f about 0.6-0.8. IPTG was added to a final . of ImM and
the ceDs aDowed to grow for an additional 2 hrs. The IPTG induces the expression of T7
polymerase and the subsequent and translation o~ the IGF muteins.
~, '~, 0.1 OD unit of the cells (both uninduced and IPIG-induced) were Iysed in
SDS sample buffer containing B , ' ~ ' and ~1~, , ' ' on 15 % SDS-PAGE.
The gel was stained by Coomassie blue and the IPTG-inducible IGF mutein bands of the
expected size were observed in lanes loaded with induced cells for each mutein.
23
w095/32003 21 907 52 P~
EXAMPLE 3
r Refolding, and I'; ~ of MuteiLfs
Although the following is written for the C3 mutein, the satne procedure applies to
other muteins l , ' ' by the instant invention. The only difference is in the star~utg
S cells used.
E coli cells expressing the IGF-1 C3 mutein were suspended in Buffer A (50 rnM
Tris, pH 7.5, 20 mM NaCI and l r~tM " ' ' ' (DTI~ at a . of ~fO ml
Buffer A to 10 g cell paste, and disrupted at 1800 psi using a French pressure ceil (SIM
Inc., Urbana IL). The suspension was centrifuged at 20,000 X g for 30 min,0 and a1iquots of the pellet and supernatant analyzed by SDS-PAGE. A major band
to the IGF-1 C3 mutein was present in the pellet, but not the C~ f~ f
The pellet was suspended in Buffer A at a . of 40 ml Buffer A to 10 g cell
paste, and re-centrifuged at 20,000 X g for 30 ~un. Tilis wash procedure was repeafed 2
times. The final pellet containing the IGF-I C3 mutein was suspended in 6 M guanidine,
50 rn~I Tris, pE 7.5, 6 mM DTT at a: of 25 ml to 10 g cells using a ground
glass I ~ . The suspension was incubated at room i r ' for 15 r un. The
'._;1 protein was removed by ~ ;r..L,.-:;.... at 20,000 X g for 30 mirt. The funal
of the C3 mutein was 1.0 mg/ml. SDS-PAGE analysis of the pellet and
~ ~ following the procedure of Example 2 showed that IGF-I C3 mutein was
20 present in the supernatant only.
The denatured and reduced IGF-I mutein was subjected to the following three-steprefolding procedure:
1) Oxidized ~ , the mixed-disulfide producing reagent (GSSG), was
added to the supernatant to a fmal of 25 rnM, and incubated
at room i , for 15 min.
2) The solution was then diluted 10 fold graduaUy with 50 mM TAs, pH 9.7,
amd l' ~ and cysteine were added to funal
of lrnM and S rltM, I~,~l;.~ly. Filtal . of
protein was 100ug/ml.
3) The refolding mixture was incubated overnight at 40C, and then centrifitged at 20,000 x g for 15 min. SDS-PAGE analysis of the pellet and
24
~ Wo gs/32003 2 ~ 9 0 7 5 2 PCTIUS95/06540
showed that the supematant was composed of relatively I O IGF-I
C3 muteim.
Aliquots (50ul) of the supernatant were diluted to 200ul with Buffer B (0.05%
TFA), injected onto a reverse phase column (RP~, I x 250mm, Synchrom, Lafayette, IN),
amd eluted with 80 % ~ 1~, 0.042 % TFA (Buffer C) using a linear gradient (increase
of 2% Buffer C/min) at a flow rate of 0.25 ml/min.
A smgle major peak ~ , refolded IGF-I C3 mutein eluted at 26.5 mm.
Refolded IGF-1 C2 mutein eluted at 26.0 min. The retention times of refolded IGF-1 C3
and IGF-I C2 muteins shifted to 32.2 min and 31.7 min, I~~ iv~l~, after beimg
completely reduced and denatured in 5 M guanidine, 50 mM Tris p:EI 7.5, 100 mM DTT.
These results imdicate that both the C3 and C2 muteins refold to a smgle major species
under the conditions described. N-terminal sequence analysis of IGF-I C3 mutein eluting
at 26.6 min gave the sequence: M G P C T L C (SEQ ID NO. 29) con}lrming that a
cysteine residue has been substituted for glutamic acid im the 3 position of the N-terminal
sequence of natural human IGF-I . An extra methionine residue is present at the N-terminus
of the ' proteim expressed by E coli. N-terminal sequence analysis of IGF-I C2
muteim eluting at 26.0 min gave the sequence: G C E T L C (SEQ ID NO. 30) conflrming
that a cysteime residue has been substituted for proline in the 2 position of the N-temlinal
sequence of natural human IGF-l.
The refold . containing the C55, C64, C65, C67 or C69 muteins were
analyzed by diluting 50 ul aliquots of the . to 200ul with Buffer B (0.05 % TFA),
injecting the diluted , onto a reverse phase column (RP-4, I x 250mm,
Synchrom, Lafayette, IN), and eluting with 100% ~1 ', 0.042% TFA (Buffer D)
usimg a linear gradient (increase of 2% Buffer D/min) at a flow rate of 0.25 ml/mm. Two
major distinct ,.~ I peaks (1:1 ratio of each), Pld & Pkll, eluted at 20.5 and 21.5
min, l~D~ . This pattem closely resembles the pattem observed for wild type
("WT") IGF-I (See, Meng, et al., J. Cl i Vol. 443, pp. 183-192 (1988)),
specifically ~ ' herein by reference. The earlier eluting peak observed in the WT
refold has been shown to be an isomeric form of IGF-I with S-S ~ C6-C47, C48-
C52, and C18-C61; whereas the later eluting peak is correctly refolded with S-S
,, C6-C48, C47-C52, C18-C61 (See, Raschdorf et al., Ri~m~Ai~
WO95/32003 21 90752 r~ c~
F ~ r S~ u~ uvy~ Vol. 16, pp.3-8 (1988), specifically I ' herein
by reference).
An ~,~ ' peak of varying size elutulg at 21.5 - 23.0 min is also present in
the refûld , SDS-PAGE analysis of this material shows that it contains
5 misfolded monomer and multimer forms of IGF-I. RP4 analysis of the refold
contaming the C11, C12, C15 or C16 muteins showed the presence of several peaks eluting
from 2û.5 - 21.5 min, as well as signific~mt (50 to 75 % of the total) amounts of apparently
mis-folded material elutmg at 21.5 - 23.0 min. The retention time of the refolded muteins
shifted to 27 min after being completely reduced and denatured im 5 M guanidine, 50 n~M
Tris pH 7.5, 100 mM DTT. Tbis ~' that the refolded forms "collapse" to a
single ~uul~ tiJc after reduction of disulfides. The RP4 peaks, therefore, represent
distinct forms of IGF muteins with different disulflde bonds.
l~XA~E 4
Isolation of Refolded IGF-I Mutein
lS Refold mixtures (435 ml) prepared from 20g of E. coli paste containing either the
refolded C3 or C2 mutein of Example 3 were . ' to lOOml, acidified to pH 5.5
with 5M HCI, dialyzed agamst 20 mM sodium acetate, pH 5.5, and loaded onto an S-Sepharose (PharmacialLKB, Piscataway, N" column (1.6 X 15 cm) previously ~
with the sodium acetate buffer described above. The bound protein was eluted with a 300
20 ml linear gradient from 0 to 0.5M NaCI. Three ml fractions were collected. A smgle
major protem peak eluted at 0.2-0.3M NaCI. Aliquots (100 ul and 25 ul) of each peak
were analyzed separately by reverse phase (RP-4 1 X 250 mm) and gel filtration
-" ' ~ A ',Y (Superdex 75 3.2 X 300 mm, Pharmacia/LKB, Piscataway, NJ). Gel
filtration effectively separates monomers from dimeric and multimeric forms of IGF present
25 m the refold . Fractions contaming ~ , monomeric (determined
from gel filtration and RP-4 analysis) C3 mutein were pooled, ~ ' to 2.0 mg
protein/ml, and 2.0-5.0 ml aliquots were loaded onto a Sephacryl S-100 (Pharmacia/LKB,
Piscataway, N" gel filtration column (2.6 X 100 cm) previously ur, "' ' with 20 mM
sodium acetate, pH 5.5, 250 mM NaCI. The proteim was eluted at 2.0 ml/min, and aliquots
30 (lOul) of each fraction were analyzed by RP-4 reverse phase ' . ~,, ' y and SDS-
PAGE followmg the procedure of Example 2. Fractions containing a sirlgle refolded
26
,
~ wO 9sl32003 2 1 9 0 7 5 2 . ~
species of IGF-1 C3 or IGF-1 C2 mutein monomer of 95 % or more purity were pooled and
r ' to 250 ug/ml. This material was assayed for bioactivity and reacted with an
8.5 kDa p~ glycol as described below.
For the Cll, C12, C15, C16, C55, C64, C65, C67 and C69 muteins, the refold
. was dialyzed imto 20mM Tris, pH 7.4,: l ' 10-lSX and loaded onto
a Superdex 75 gel filtration column previously r~ ' ' with the same buffer. The
monomers were then pooled and loaded onto an RP-4 column (RP-4, 2.1 x 250mm,
Synchrom), and eluted with 100% ~ nifnl~, 0.042% TFA ~3uffer B) using a lmear
gradient (increase of 2 % Bufer B/min) at a flow rate of 1.0 ml/min. The C12, C55, C64,
C65, C67 & C69 muteins refolded into 2 distinct RP-4 monomer peaks (1:1 ratio of each,
PkI & Pk~) closely resembling the pattern observed for WT-IGF-I (See Meng at al., cited
above). The C12 & C15 monomer fractions from Superdex 75 also contained significant
amounts of apparently mis-folded material eluting after PII (21.5 - 23 0 min). The Cll,
C15 and C16 monomer fractions contained multiple (3-6) peaks when analyzed by RP-4.
The monomer peaks were coDected separately and assayed for bioactivity and subjected to
mass analysis (see below).
Mass analysis of the monomer peaks was performed using an API m obtained from
Sciex, Toronto, Canada. Mass analysis was performed on both the refolded (disulfide
bouD Dba) ~I-d ledu d b d bll bled IlWllOmeD. ~ odowiD Inbse~ we~e obbDed:
27
WO95132003 2 1 90752 r~ 5~
TABLE 4
.
~ ~5
Ql pl 7ns wn6 wn3 ~Dsn U~
Cll p2 wn- usn
S C12 pl mn n~s n~s U7~ ~03
n2 p2 n~s U7~
ns pl 77~6 n77 n7s Wl u~n
ns p2 nu ~33
cls p3 n7s W7
0 a6pl 7736 ns7 nss U l ~z
n6 p2 ns6 0~2
csspl 7n~ n~s n~7 U33 L32
c5s p2 n~7 U32
C6 pl ml ng2 nu L7~ n7s
5 C ~2 n~ n7~
a~s pl 7757 nn n7~ W2 ~59
(Ys p n7s n62
C67pl m3 W3~ W32 ~ 117
C67p W31 ~119
20 aispl nn3 Wl~ wls ~m2 ~mn
c6s p2 W15 ~ml
28
~ W095/32003 21 ~0752 r ~ c- ^
These masses of the reduc~ed muteins match, within VA~ erwr, the expected
masses for a pol~lidv with the indicated cysteine mutation. The masses of the refolded
monomers match the expected mass of ~oly~,~liJv~ having 3 ' ' disulfide bonds
and a single mixed disulfide of either cysteine - glutathione (cyS-s-s-r' ~.) or5 cysteine - cysteine (Cys-S-S-Cys). The mixed disulfides form during refoldimg and remain
intact because there is no other cysteine residue present in the molecule available to form
an ' ' disuhf~de.
A scale up ~, .. ;1;. ~;.... for the C69 mutein was also perfornled. The refold mixture
fwm 8 gm of washed imclusion bodies (WIBS) was . ' ~ 10X to 400 ml, dialyzed
against 20mM sodium acetate, pH 5.5. 200 ul aliquots was loaded onto am Sephacryl S-100
(Pharmacia/LKB, Piscataway, N~) gel filtration column (10 X 80 cm) previously
~ . ' with 20 mM sodium acetate, pH 5.5 and 250 mM NaCI. The fractions were
eluted at 25 ml per minute, and aliquots (50 ul) of each fraction were analyzed by
SDS/PAGE. Fractions contanting monomers were pooled.
To separate the two isomeric forms of C69, 200 ml of the S-100 monomer pool was
diluted with 800 ml of 1.1 M ammonium sulfate-20mM sodium acetate, pH 5.5 (Buffer A)
and loaded onto an Octyl Sephawse (Pharmacia/IXB, Piscataway, NJ) column (2.5 X 20
cm) previously ~, ' ' ' with Buffer A. The bound proteim was eluted with a 750 ml
linear gradient from Buffer A to 50% Buffer B (50% ethamol-20 mM sodium acetate, pH
5.5). 12 ml fractions were collected. Two major protein peaks eluted at 25% and 32% of
Buffer B. Aliquots (50 ul) of each pealA were analyzed by reverse phase (RP-4, I X 250
mm). Fractions containing ~ , correctly refolded (determined from RP-4
analysis) C69, eluting at--32-38 ~ Buffer B were pooled. Reverse phase analysis showed
the correctly refolded C69 pool was 95 % or more 1 - . ~L,. A ~ This material was assayed
for bioactivity (see below).
EXAMPLE S
Pegylation of Mtlteins
The C3, C2 and C69 muteins were covalently joined to an 8.5 kDa ~ul~vLlljlv,.v
glycol (8.5 kDa PEG) or an 20 kDa pV~ ..rlv.lv glycol (20 kDa PEG) having a maleimide
30 activatimg group in a two step process:
29
wo 95/32003 2 1 9 0 7 5 2 P~ . C : ~
1) The purifled IGF-l muteins were partially reduced with DTT in a 15 ml reaction
mixture containing 2.3 mg (296 nmoles) IGF-1 muteim, 170 ug DTT (1110 nmoles) im 14
mM sodium acetate, 33 mM sodium phosphate, pH 7Ø The final of protem
was 10 ug/ml, and the molar ratio of DTT:proteim was 3.75:1. For reaction with the C69
S mutein, 91ug DTT (592 nmoles) was used and the molar ratio of DTT:protein was 2:1.
The reaction mixture was incubated at room i . ci for 3 hours (5 hours for reaction
with the C69 mutein) and terminated by the addition of 1.0 ml of lM sodium acetate, pH
5.5. The reaction mixture was dialyzed at 4C overnight against 20 mM sodium acetate
pH5.5.
2) The par~ially reduced IGF-I mutein was reacted with either the 8.5 kDa PEG orthe 20 kDa PEG in a 20 ml reaction mixture containing 2.3 mg (296 nmoles) of proteim,
9.985 mg (1174 mmoles) 8.5 kDa PEG im 15 mM sodium acetate, 26 mM sooium
phosphate, pH 7Ø The final: of proteim was 112 mg/l. The molar ratio of
8.5 kDa PEG:protein was 4:1; for reaction with the C69 mutein the molar ratio of 20kDa
PEG:protein was 4:1. The reaction mixture was incubated at room ~ for 3
hours, and terminated by placing at 40C or freezing at -20C. SDS-PAGE analysis of the
reaction mixture following the procedure of Example 2 showed that ~ , 50% of
the partially reduced PEGylated C2 and C3 mutein was conver~ed to a mono-PEGylated
species. The C3 and C2 20kDa-PEG conjugates migrated at a relative molecular weight
of a~ 'S, 60kDa on SDS PAGE; The C3 and C2 8.5kDa-Peg conjugates migrated
at a relative molecular weight of about 23 kDa on SDS PAGE. A~l, 'S, 20 % of thepartially reduced PEGylated C69 mutein was converted to a mono-PEGylated speciesmigrating at a relative molecular weight of 67 kDa on SDS PAGE.
Wild type IGF-l subjected to the same partial reduction conditions and PEGylation
procedures did not become PEGylated.
EXAI~LE 6
n - of Pegyla~d Muteins
The pegylated C2 or C3 mutein reaction mixtures (containing ~ S~ 100-200
mg protein) were dialyzed extensively at 4C agamst 5 mM CitliC acid, pH 2.6. The
pegylated mutein was separated from the unPEGylated muteim usmg an S-Sepharose
(Pharmacia/LKB, Piscataway, NJ) cation exchange column (2.5 X 25 cm) previously
wo s~/32oo3 2 1 9 0 7 5 2 ~ 0
eq.li1ih~rA with 5 mM citric acid buffer, pH 2.6. The bound protein was eluted with a
2000 ml linear Oradient from 0 to 1 M NaCl. 25 ml fractions were collected. Pegylated
C2 or C3 muteins eluted at 0.25-0.4M NaCI and the h~ oy' ' protein eluted at 0.8-0.9
M NaCI.
S Fractions containing the pegylated C2 or C3 muteins were pooled, . ' and
rulLh~ I y .i~cl by Sephacryl S-200 gel filtration ' . O , ' .~ . 15 ml of the .fractions containing ~.yyl, ~, 20 mg of total protein was loaded onto a Sephacryl S200
(PharmacialLKB, Piscataway, NJ) column (2.6 X 100 cm) previously , ' ' with 20
mM sodium acetate, pH 5.5 containing 250 mM NaCI. The protem was eluted at 2.0 ml
per min. The bulk of the material eluted with an apparent MW of 200 kDa.
The C69-PEG was separated from the " ,.~ ' C69 muteim by Q-Sepharose
anion exchange ~ L. , . y . 100 ml of the reaction mixture contaming 11 mg of total
protein was loaded onto a Q-Sepharose anion exchange column column (Pharmacia/LE~B,
Piscataway, NJ) column a.6 X 100 cm) previously , "' ' With 20mM Tris, pH 9.0
(buffer A). The bound protein was eluted with a linear gradient of 20mM tris Ph 9.0, IM
NaCI (buffer B) at 5.0 mUmm. 10 ml fraction were collected. The C69-PEG eluted at
50mM NaCI, well separated from the unreacted monomer which eluted at 100mM NaCI.C69-PEG was pooled, ' to 13 ml amd loaded onto an S-200 gel filtration column
a.6 x 100cm) previously , with 20mM sodium acetate, ph 5.5, 250 mM NaCI.
BA~E 7
Bioassay of Pegylaled Muteins
r human metIGF-I (rIGF-l) (13achem California, Torrance, CA), various
I, ,,.~' ' and PEGylated muteins were tested for their relative mitogenic activity and
affinity for .~ ' msulin-lLI~e growth factor binding protem I ("IGF-BPI"), whichis described in PCT Application publication WO 89/09792, published on October 19, 1989.
A. Relative Mitogenic Activity
The relative mitogenic (growth s~ ' , ` activities of the C3 and C2 muteins and
pegylated C3 and C2 muteins were compared to that of wild type IGF-I by measuring the
relative amount of ~H ~ . ihl~,v~ ' ' into rat U~'~,V~olw~d cells when varying
amounts of the proteins were present under serum free conditions. The rat o t .,- --
31
wo gs/32003 2 1 9 0 7 5 2 r~l~u~ s.~ - ~
cells (the UMR106 cell line; obtained from American Type Culture Collection, Accession
No. CRL-1661, Rockville, Maryland) were plated at 5-6 X 104 cells in 0.5 ml of Ham's
F12 Media (M~ ' ' Herndon, VA) containing 7% fetal bovime serum, 100 U/ml
penicillin and 100 ~g/ml ~ r' y~ ' and 2 mM L-glutamime per well im 48-well tissue
S culture ptates (Costar, Cambridge, MA). After incubating for 72 hours at 37C when the
cells were confluent, the cells were washed twice with phosphate buffered saline (PBS) md
prc- ' ` ' in serum-free Ham's F12 medium containmg 100 U/ml penicillin amd 100
mg/ml ~ tu...y~ amd 2 mM L-glutamime for 24 hours. After the pre 0.5 ml
of F12 serum-free medium containing serial dilutions (1.0 - 1,000 ng/ml) of dGF-1, C3
10 and C2 muteins, and pegylated C3 amd C2 muteims were incubated separately for an
additional 20-24 hours. Each well was then pulse labeled with 0.5 uCi of 3II ~
(NEN Research Products, DuPont Co., Boston, MA) for 4 hours, then washed with cold
PBS three times and . ' 3H-thymidine was , . ` ` ' with cold 7 %
l.;~,t.lu~ , acid a.T. Baker Inc., Phillipsburg, Nl). After rinsing with 95% ethanol,
15 cells were solubilized with 0.3 M NaOH and aliquots removed for ~ ;counting.
3H-thymidine was quantitated by liquid ' counting. All assays were performed
im triplicate.
The C3 and C2 muteims and pegylated C3 and C2 muteins were found to stimulate
the same maximal level of 3EI lt.~ " . into DNA as ' IGF-1.
The potencies of the C3 and C2 muteins and the pegylated C3 and C2 muteins were about
3 to 4 fold lower than ' IGF-I. The ED50 (dose required for half maximal
activity) of IGF-1 was 5-10 ng/ml compared with 30-40 ng/ml for ~ . ~,y-
C3 amd C2 muteims, and the pegylated C3 amd C2 muteins.
These CA~ ` ' that the mitogenic activity of IGF-1 has been
substantiaUy retained by the U amd C2 mutems and the pegylated U and C2 muteins. All
four molecules capable of simulating cells to divide, as measured by 3H-thymidine
into DNA. All four molecules are capable of stunulating cells to divide to
the same maximal extent.
Using the assay described above, the relative mitogenic activity of of the E~P-4 peaks
(described above) of the C11, 12, C15, C16, CSS, C64, C65, C67 and C69 IGF muteins
and of C69-PEG was also ,~ ' The results of the latter assay is set forth in Table
32
~ W09s/32003 21qO752 P~ Sc
TABLE 5
APPPROXn~IATE ED~o OF IGF-I MUT~ I~S ~ C69-PEG (NG/ ML)
PEAK I PEAIC II PEAK m PEAK IV
(20.5 min) (21.5 min) (22.0 min) (22.5 min)
Cl 1 - 200 ~ 200 ~ 200 ND
C12 50 - 55 6 - 8 ND ND
SC15 ~ 150 ~70 ~5 ~300
C16 ~20 ~8 ND ND
CSS ~40 5 - 7 ~40 ND
C64 ~ 90 ~ 22 ND ND
C65 ~ 45 ~ 45 ND ND
10 C67 ~25 5 - 6 ND ND
C69 .20 - 25 5 - 6 ND ND
C69-PEG 5-6
~WT IGF-I 20 4-6
WT IGF-I ED~o ~4-6 ng/ml
ND - NOT DETERMINED
33
wo9~/32003 2 1 90752 1~ S~
The mitogenic activity of Peaks ~ of the C12, C16, C55, C67 and C69 mutein
monomers was not ~ -- ly different from the mitogenic activity of colrectly refolded
WT rIGF-I (Peak II). The ED~o of wild type rIGF-I was 4-6 ng/ml compared with 5-8
ng/ml for these unPEGylated mutein monomers. Table 5 shows that Peaks I of the various
S muteins had lower (5-30 fold) bioactivity than correctly refolded WT rIGF-I si~ilar to the
bioactivity of WT rIGF-I peak I. The C69-PEG conjugate, synthesized from peak II of
C69, had the same bioactivity as C69 peak II and correctly refolded WT rIGF-I.
B. Relative AffiDiq for IGF-BP1
The relative affinities of the C3, C2 and C69 muteins and the PEGylated foTms ofthose mutems for IGF binding protein-l (IGF-BP1) were compared to that of the wild type
IGF-I by measunng the ability of IGF-BPI to inhibit the mitogenic activities of the protems
on rat ~- cells. The rat U~IR106 ceDs were plated at 5-6 X 104
cells in 0.5 ml of Ham's F12 containing 7% fetal bovine serum, 100 U/ml peniciDin and
100 mg/ml , y~ih~ and 2 mM ~glutamine per well m 48-well tissue culture plates.
After mcubating for 72 hours at 3rC when the cells became confluent, the cells were
washed twice with PBS and prc ' ' m serum-free Ham's F12 medium containing 100U/ml penicillin, 100 mg/ml ,tl~ , amd 2 mM ~glutamme for 24 hours. After the
- 0.5 ml of F12 serum-free medium containing either 50 ng/ml or 200ng/ml
of rIGF-I, C3 or C2 mutein, or pegylated C3 or C2 mutein were incubated separately with
varying amounts of IGF-BPI (100 nglml - I X 104 ng/ml) for an additional 20-24 hours.
Each well was then pulse labeled with 0.5 uCi of 3H-thymidine (NEN Research Products,
Du Pont Co., Boston, MA) for 4 hours, then washed with cold PBS three times and
~, ' 3H-thymidine was ~ , ' with cold 7% i ' acid a.T. Baker
Inc., r ~ NJ). 3H-thymidine was quamtitated by liquid ~ -- counting. AU
assays were performed in triplicate.
The results of this experiment indicated that the affu~ities of the,, ~' ' C3
mutein and the pegylated C3 mutein for IGFBPI were greatly reduced. At a molar ratio
of 20:1 (IGFBPl:rIGF-I), the mitogenic activity of rIGF-I (50 ng/ml) was reduced 80%;
however, the mitogenic activities of the same . of the ~ ' ' C3 mutein
and pegylated C3 mutein were reduced 35% and 0%, I~ ~liv.l.~. Similarly, when 200
ng/ml of the proteins were incubated with a 20 fold molar excess of IGF-BPI, 70% of the
34
~ W095/32003 2 19~752 rV.,IJ~J~06~10
mitogenic activity of rIGF-I was inhibited, whereas none of the mitogenic activity of the
pegylaoed C3 mutein was inhibited. The affmities of of both the ~ ' C2 muoein
and the pegylated C2 mutein were identical to that of wild type IGF-1. The affinity of both
the I, v.r' ' C69 and C69-PEG for IGF-BP1 were not v ~ different from that
of WT rIGF-I
These data indicate that the pegylated C3 muoem has vreatly reduced affmity for
IGFBP1 when compared to IGF-1. Thus the mitogenic activity of the pegylaoed C3 muoein
wi~l not be inhibited by IGF binding prooeins under conditions where the mitogenic activity
of IGF-1 will be inhibited. However, the affinity of pegylated C2 and C69 muoeins for
IGFBP1 is the same as the affinity of wild type IGF-1. Thus the mitogenic activity of
pegylaoed C2 and C69 muoeins will be inhibioed by IGF binding proteins under the same
conditions where the mitogenic activity of IGF-1 will be inhibioed.
E~fA~LE 8
Animal Tests
Animal studies were performed to compare the pl-~-.. ,ov; properties of the
muoeins and PEG conjugates of the present invention to the l' '~ properties
of wild type IGF-1.
Animals
Male Spravue Dawley rats with pituitary glands surgicaUy removed
(h~l~vlJhr.,~Lu.. ~vd or Hypox rats) and weighing 110-121 grams were o~tained from
Charles River Co. The rats were maintained in cages with lighting controlled over a 12
h-lighV12 h-dark cycle.
The animals had continuous access to waoer and food. Five animals were housed per
cage. The weights of the rats were monitored daily and only rats with weight gains of less
than 2 grams per week during the 2-3 weeks after arrival were considered to be succesfully
~J~,ul~h~ l and used for the ~ r
B. M~:thods
In r, I, animals (10 Hypox rats per group) were mjecoed every third day
(ETD) ' 'y (sc) with ~T rIGF-I (160 mg, 320mg), 1 . v.v ' C2 (320mg),
Woss/32003 21 901752 r~ 'o~
I . ,,y' ' C3 (320mg), pegylated C2 O(C2-PEG, 320mg) or Pegylated C3 (C3-
pF.G,r~r,n ) dissolved in 0.2 ml of binding buffer (0.1 M HEPES-0.05 M NaH2PO4). A
separate group of 10 animals received 0.2ml vehicle. Injections were given between 0700
hours and 0800 hours and body weights were recorded daily between 1600 h and 1?00 h.
5 rI'he weights of rats on the day after the last imjection were taken as the fmal weight.
F, ' ~, animals (9 Hypox rats per group) were imjected every third day sub-
'S, with WT rIGF-I (320mg, single injection daily, S~; 320mg ETD; 640mg
ErID), or C3-PEG (320mg ETD, 640mg ErD, 960mg ETD).dissolved in 0.2 ml of binding
buffer (0.1 M HEPES-0.05 M NaH2PO~). A separate group of 9 animals received 0.2ml
vehicle. Injections were given between 0700 h and 0800 h amd body weight were recorded
daily between 1600 h and 1700 h. r~he weights of rats on the day after the last injection
were taken as the fmal weight.
In F, m, animals (10 Hypox rats) were injected every third day sub-
lS, with C3-PEG (160mg lV, 320mg ETD), dissolved in 0.2 ml vehicle (0.1 M
HEPES-0.05 M NaH2PO~). A separate group of 10 animals received 0.2ml vehicle.
Injections were given betwoen 0700 h and 0800 h amd body weight were recorded daily
betwoen 1600-1700 h. The weights of rats on the day after the last injection were taken as
the fmal weight.
At the end of F, I & II, rats were r, ' .~ ' ' with CO2 and weighed. In
20 F . m, the tibia were removed and the epiphyseal width measured.
C. R~sults
r ~ Rats treated with sc injections with either 160mg or 320mg of WT
IGF-I ErD showed no significant weight gain compared with animals injected with vehicle
(Table 6). Similarly, animals injected ErID with 320 ng un-PEGylated C2 IGF-I or un-
25 PEGylated C3 mutein did not show signif~cant weight gain. Animals injected ErID with320 mg C2-PEG and C3-PEG gained 4.42 i:0-74 g and 5.45 i 0.98 g, ~ .,ly,
which was ! ,, .r. '',Sl greater than the weight gain of animals injected with wild type IGF-
I (p < 0.01) . The weight gain of animals injected with PEGylated C2 or PEGylated C3 was
,, ~ 'S greater than the weight gain of animals injected with the un-PEGylated C2 or
30 C3 mutein (p~0.05). The PEGylated proteins clearly showed effficacy; however, the
36
~ WO 95J32003 2 1 ~ 0 7 5 2 P~ C ~o
identical dose of WT IGF-I showed no efficacy. S~ ;ly, the addition of PEG
improves the biological potency of the molecule.
TABA~ 6
THE EFl~ECT OF IGF~~ [~ANS (U~AA;~YA~ATAFD ~ PEGYI~ATAED) ON TA~E
GA~OWTHA OF AA YA~A ~Ai~DA~ 1 ~J~IZED ~ATS
? ''lT.TJrT~T.T~ A1)0SE AL~ 2UA~NI- Y MEAN WT GA~I P VALUE
ug/day (g) vs
Vehicle ETD -1.28 + 0.95
WT IGF-I 160 ETAT) 0.23 + 0.87
WT IGF-I 320 AT~D 0.59 + 0.79
10 C2 320 ETD -0.52 + 0.67
C2-PEG 320 ETD 4.42 + 0.74 WT 320 0.01
C2 320 0.01
C3 320 <0.05
C3 320 ~D I .75 i 0.90
C3-PEG 320 ETAr) 5.45 + 0.98 WT 320 <0.01
C2 320 <0.05
C3 320 < .05
These results ' that the PEGylated muteins exhibit enhanced
~ over WT IGF-I and the un-PEGylated IGF muteins.
r lATA A~ats treated with sc injections of WT IGF-I 320mg SATD~ 320mg ETD
and 640mg ETAr) gained 4.02g + 0.46g, 0.81g + 0.81g and 1.41g + 0.52g, ~Dy~Li~vl~
(Table 7). However, animals given 160mg, 320mg, 640mg 960mg of C3-PEG ETAI) gained
5.22g + 0.46g, 5.50g + 0.52g, 8.69g + 0.67g, and 10.43g + 0.77g, ~ (Table
20 6). All doses of C3-PEG ETD stimulated ~ more weight gain than both WT
w09~32003 21 907 52 r~ m~5,~
IGF-I doses given ETD. Animals injected with either 640mg or 960mg of C3-PEG ETDgained ~ , more weight than those dven 320mg WT IGF-I SID. C3-PEG doses
of 160mg and 320mg ETD stimulated greater weight gam than 320mg of WT IGF-I SID;however, these differences did not reach statistical
5 TABLE 7
1~ EFFECI OF C3-PEGylated IGF-I ON TF~ GR(~WTH OF
~Y~Ul'~D~l~D RATS
MOLE~ i Dasr. ~u~r MliAN r ~ALUE
14ddqr ~rr ~N ns
Vdsicle Ell~ -0.77 i
0.38
0Silll IaF-I 320 SID 4.02 + sNT 320 ETD0.01
0,46 w~r 640 r.TD o.ol
Wl IaF-I 320 ElD 0.81 i
0.81
WT IGF-I 640 EID 1.41 i
0.52
c3-PrSG 160 EID 5.22 i W8 320 ETD 0.01
0.46 Wl 640 EID 0.01
C3-PEa 320 El D 5 50 i Wl' 320 Ell~ 0.01
0.52 s~ 640 ETD 0.01
5C3 PEa 640 Era 8.69 + Wl 320 SID <0.01
0.67 C3PK~s 160 ETD C0.01
C3PECs 320 El D ~0.01
c3-Pr,a 960 r~D 10.43 + Wl 320SID <0.01
0.77 c3Pra 160 ElD ~0.01
C3PEa320ElD C0.01
F ' ~ ~ ' that C3-PEG r' ' ' ' SC ETD exhibits gS.~ater
potency than WT IGF-I: ' ' sc ETD. All doses of C3-PEG stimulated greater
mean weight gain tham animals given 320mg WT IGF-I SID. The e~hanced
20 1 ' ~ of C3-PEG make it more potent than WT IGF-I in the animal model
described.
38
wo 9513z003 2 1 9 0 7 5 2 ~ 5.'06'l^
r~ - m: Rats treated with sc injections of C3-PEG 160 mg an~ 320 mg
gained 8.3g + 0.7g and 9.0g + 0.6g, ~ (Table 4). Vehicle gained 4.2g + 0.3g.
The weight gained induced by U-PEG was statistically greater than anunals given vehicle.
Similarly, the tibial epiphyseal widths of rats receiving C3-PEG were statistically greater
than rats receiving vehicle (Table 8).
.
TABLE 8
'1~ EE7FECT OF C3-PEGylafed IGE7-l ON T~ GROWT~ Ol~
~lYl ~rr~ ~l~ RATS (WElGElT GAIN)
MOLECUI~E WSE ~ N Wl' GAIN MEAN T1131A WIDTH P VALUE
y (d (~ ) V8
0 Ve~iclo ETD 4.2 + 03 0.136 + 0.0~4
C3-PEG 160 ETD 8.3 + 0.46 0.159 + o oo~ Vebicle
~0.01
C3-PEG 320 El'D 9.0 + 0.~ 0.151 _ 0.004 Vebicle
<0.01
39
WO 95132003 , ., ~ ,C l f~
21 90752
TABIE 9
TEIE l~FFECT OF C3-PEGylat~d IGF-I ON T~IE GROWT~ OF
H Yl J~ 1 J~II~D RATS ~IIBIA ls~ I .. ~ ~AL W~)
DOSE l,~U~ MEAN TIBIAL P VAllllE
t~ ~n.~ ugld-y WIIYIH (
S Vchicle ErD 0.136 i 0.008
C3-PEG 160 ETD 0.180 ~ 0.01 Vchicle <0.01
C3-PE1~ 320 ETD 0.167iO.06 Vehicle <0.01
F, m ~ that C3-PEG stimulates not only weight gain, but also
bone growth in EIYPOX rats. This indicates that C3-PEG may be a useful r
10 for the induction of bone for~nation.
Although this invention has been described with respect to specific ' ' , it
is not intended to be limited thereto. Various ' which will be apparent to thoseskilled in the art are deemed to fall within the spirit and scope of the present invention.
