Note: Descriptions are shown in the official language in which they were submitted.
wV 93/121~2 2 ~ 2 ~ ~ 7 ~ Pcr/US92/lO~9
CYTOK~ES VVl~I AN UNPAIRED CYSIE~E I~ESIDUE
AND CONlIJGATES T~IE:REOF
S F~eld of the In~en~ion
The invention relates to cytoldnes which are site-specifically muta~d to have
one unpair~ cysteine residue located apart from the recept~r-binding site, and
conjugates ~ereof, in particu1ar, conjugates where a lip~philic group is conjugated to
the unpaired cysteine residue, and to such conjugates of cytoldnes which have ~
10 unpairedl cysteine residue located apart ~rom the ref~eptor-binding ~ite in ~e native
form.
Background oî Inqention
Cytokines act on cells to regulate cell grow~h, maturation or cellular ac~vi~r,
by eith~r stimula~ng or inhibiting growt}) or maturation or activity of neighbonng,
15 fimc~onally rela~ed cell ~s. Cytokines are syn~hesiz~d and s~creted by
lymphocytes~ macr~phages, fibroblasts, neuronal cells? or o~er cell types.
The in vilro studies on cytokines indica~e that they are generally very po~ent
factors which a~t at very low concentrations. Each cytol~ne can have many different
effects ~leiotropici~ , ~d act loca:lly on seYeral functionally related cell t~pes. For
20 e~cample, a~ the site of tissue repair, or inflammation, or of immune response9 a
cytoldne is generally involved in a loop of regulatory processes. The cyt~kine
regula~es not only the mah~ ion and activity of the t~rget cells, but the production
of ~e cytol~ne itself is also regulated by certai~ actors. :E~or example, in the
microenvironment of a site into which an immunogen is administered, the
25 ma~ophages are ac~ated and induced to secrete interleu~n-l ("IL-lN). IL-l
Wo 93/12142 ~ ;7 2 PCI/US92/1~889
~ 2
a~tivates eh~ n~ighboring an~gen-specific T cells. These ac~vated T cells then secrete
interleukin~ IL-2n) which acts upon the T cells in an autocnne fashion and the
ac~vat~d T cells also secre~ interferon (nIFN-yn3 which further activates the
macrophages and B cell growth factors, which in tu~ ivate the neighboring
S alltigell-~fic B cells.
The l~ ~unction of cytokines is to be con~rasted with hormones of ~e
endocmle system. A hormone is synthesized by a lo~alized tissue or organ and
~ by ~e blood circulation to ~ct on a tissue ~r organ systemically, or one whichis located in a dif~e~ent part of the body.
Cytol~nes generally are small proteins, and are made in small quantities. They
e generally not detectable in the circulation eYen wi~ very sensi~ve
immunochen~ical assa~ys. Cytokines have very high affinity ~r their receptors on ~
target cells, and ne~d only Wnd t~ a small portion of the re~tors on the surface of
the target cells t~ trigger the subsequent bi~logical even~s.
: ~ 15 Many cytoldnes have beerl identifi&d, and the genes of many cytoldnes have
been cloned and expressed in various systems. R~combinant cytoldnes have been
prod~, purifi~d, and used for v:anous in vitro ~nd in YiVo therapeutic purposes.Ce*ain of ~ese r~combinant:cytokines have been approved by the United States Food
and Drug Administration or the regulatory agencies of o~er countries for various: : 20 therapeu~c pu~ses. For example, erythropoie~n ("EPO") is approved for increasing
red blood eell counts in patients r~ceiving kidn~y dialysis; granulocyte colony
stimulation factor ('GCSF') and glanulocy~ macrcpha~e colony s~mulabon factor
~' .
;
WO 93/12142 2 1 2 ~ 6 7 2 PCr/US~2/1088g
(nGMCSFn) are approved for boosting granulocy~e counts in cancer patients receiving
chemothe~apy or imdiation; IL-2 is approved for ~nhancing immuni~y in pa~dents with
certain types of cancer; a-interferon (~IPN-cYn) is approved for patients ~h certain
viral infections (such as hepati~s C) ~r with certain types of cancer.
Cy~okin~s w}tich target cells in the blood t,ircul~on or bone marrow, such as
EPO and the af~rementioned colony s~mul~n factors ~categoriz~d as hemopoietic
grow~h factors)? are safer and more efficacious (or at least have been easier tod~elop) than ~ose which target cells in the lymph nodes or o~er ti~sues. Numerous
in vivo human clinical studies have indica~ at many cytoldnes7 such as IL-2, IL-l,
tumor necrosis factor ~"~n), IFN-a, and IFN-y, are extremely toxic when
administered systemically. In addi~on, due in part to their small siæ, cyto~nes have
very short phanna~ological half li~es and are cleared wry rapidly from ~e
; circul~tion.
,:
The c~kines: ~hich do not target cells in the blood or circula~on seem to be
toxic when sys~,mieally administered because th~y cannQt act on target cells over
short distances or locally, or:in a micro*nvironment in tissues, as ~hey would in viv~.
Systemic administr~on results in ~e cyto ine diffusing throughout the cir~ulation and
~: ~ els~wh~re, ~ather~ ~an localizing it t~ the target site.
Several cytol~nes have been or are being investigated for ~ ng diseases
affecting local or régional areas. Epidennal growth factor ("EGF"?, fibroblast growth
factor ("FGF"), insulin-like growth ~actor (nIGF"), and platelet~e~ived growth factor
("PDGFn) bave been studied in clinical trials for accelerating healing uf wounds
;~ :
wo 93/12142 2 1 2 ~ 6 7 2 PCr/US92/1088s
resulting from accidents or surg~ry. Some of these cytokines are also being studied
for ~eating stomach ulcer.
It seems dlat if the delivery of cytoldnes and therapeutic enzymes could be
localized to maintain sufficient concentration for an appropriate p~ at the target
S site, they could be more therapeutically effective. Also, because ~e toxic eff~ts
ciated un~h systemic administration of cytolcines would be eliminate~l, high doses
of cyto~ne could be administered to the diseas~d tissue site. Some poten~al
applica~ons of lo~ cytoldne ~ministration would include trea~ng solid tumors
witlh TNF, IFN-a, IFN- y, or IL-2. An injured part of the spine could possibly be
,, ~,
trea~ed wi~ nerve growth factor (nNGFn) or ciliary nerve ~ophic factor ("CNTF").: ; : Where the infec~on by microorganisms is lo~alized to ~fect only some tissues, such
as only the e~ternal portions of the mouth, nose, or eyes, or only the external genital
- organs, or only the toes, or only part of the skin, the adminîstra~on IFN-a, IFN
IFN- y, or IL 2 to these tissues may be effective. Af~er a surgical procedure, the local
15~ administra~on~of:PDGF, BGF, FGF, or l(iF to the areas o the injured tissues may
pr~m~te healing. The inflamma~on of the nasal lining caused by an allergic reaction
or by infec~on may also be trea1ed with EGF, FGF, or IGF~ in ~mbination with anti-
~: :
allergy medication or antiblotics. Injured gums caused, for example, by ~rthodontic
surgery may b~ Ueated with E G F or IG F, E G F or IGP could also be used to trea~
eye traunna caused by ophthalmic surgery or injury. ~dness could be ~ea~d by
: s~bcu~aneDus b~topi~al adn~nisbra~on of E G F or IG F to the af~ec~3d areas, in order
~ D~pro m ote haur~growth.;
::: : :
WO 93/12142 2 1 2 4 1~7 2 - PCr/U~92/10889
Fatty acylation of cytokines potentially offers a means to localize the cytokine,
thereby preventing ~he toxîcity associated with systemic administ~ation. ~ization
of ~e ~ 3cinc ~or a sufficient p~riod also keeps it in an area where it can be
~e~apeuticaUy ef~ec~e. Pro~eins which are artificially acylated by fatty acids have
S been shown to have increased affini~r for ~e cellNlar plasma membrane. Por
example, ncin A chain (which is a to~n) after modification by ~atty acid conjugation9
has greatly increased non-sp&cific cyt~ icity on vari~us cell types. KabanoY, A.V.
et el. Biomed. Sci. 1:33 (199~). Among the cytokines, TNF has been fatty acylated
in orde~ ~o enhan~e its ability to incorporate into liposomes. Utsumi, T. et al. Cancer
1~ Res. 51:3362 (1991). Iheliposomes w~re ~o be administered in~ravenously into
patients to deliver ehe TNF to cell~ of ~e re~culoendoth~lial system. The fatty æyl
group w~s ~onjugat~d by eo~ g ~he ~ amino group of the ly~ine resîdues, or the a-
~0 gTOUp of the first amino acid residue of ~he TNF polype~de chain, with ~e
N-hydroxysuccinimide es~er of the fat~r acid. Since they are 6 lysine residues per
15 ~ polypeptide ch~un, the final products varied in terms of number of fatty groups
per molecule and the ~cular lysine residues ~ which the fatty acids were
con~ugated.
It i~ hlown th~t some na~ve proteins are post~anslationalLly modifi~d in vz~o
by ~he covalen~ conjugahon of fatty acyl groups. The linkages through which fatty
20 acids are attached to proteins can be divided into two general categories. Wilcox, C.
A. and Olson, E. N. Bioc~emistry 26:1029 (1987). The ~Irst category Qf acylated
p~teins contain fatty acid linked to ~e polypeptide by an ~ester or thiol (SH) ester
- W093~12142 212 46~ ~ ` PCr/U592~1088g
. 6 ~ -
bond. These acylated proteins are localized to cell membranes and are acylated
prim~r~ly by saturated palmitic acid, a 16~bon residue fatty acid.
The s~cond catego~y of acylated proteisls contaill fatty acids linked to the
po~yp~tide by an amide bond. These proteins a~e acylated with m~s~c acid, a 14-
carbon sah~ d fatty acid, which is linked to an ~o ~terminal glycine residue. Incon~ to proteins acylated with palmitate, my~ista~e contai~g proteins have been
~hown to be b~ 3luble and memb~ane-bound.
Among tlle cytol~nes, an a~yl~ted native palmi~ 1 has ~en discovered.
This isoform of IL-1 is membrane-bound on the su~face of monocytes and
io macs~phages. Bakouche, O. et al. J. Immunol. 147:2164 ~1991). The O ester and
S~ster linkages ~ the fat~r acyl group are carried out enzymatically in ~he cis-Golgi
appara~us ~d in the transitional ~lements of ~he end~plasmic re~iculum, where
enzymes ~e¢og~ c,~o,rtain structural features of ~e proteins which are to be acyla~d.
Only a small frac~os~ of the total IL-l is acyla~ed, and only one OI' mally OH group~
associa~ with serine residues, or only one of many SH groups associated with
cysteine residues9 is ~onjugated wi~h the p~lmityl group.
The difficulty with fa~ty acyla~on by the present chen~ical synthetic methods
as has been p~r~ormed with ~icin A chain and TNF, therefore, lies in ensuring th~t
the cytokine~are in ~act acyla~ed and form a homogeneolls conjugated population.Another dif~culty is ensuring that the cytokine binding site does not become fatty
æylated, as ~is could inhibit or pr~vent its bindLng and/or its biological activity.
wo 9~121~2 2 1 2 ~ 6 ~ ~ PCr/US92/10889
Sumnaary of the In~ention
The invention includes cytol~nes which are site-specifically mutated to c~ntain
one unpaired cysteine residue located apart from the rece~tor-binding site. Morespecifically, a cyt~ldne molecule is site-specifically mutate~ by recombinan~ DNA
methods so as to yield an unpaired cysteine residue at a site apart ~rom the receptor
b~ding site. This can be achi~ved, for e~ple, by subs~tu~ng an w~ed cysteine
residue for another amino aci~ ~esidue, such as serine, a~ a site apart from ~e
~?r b~nding site. The cytokines that contain an unpaired cysteine residue loca~
apart from t~e receptor-binding sites accessible. ~or conjug~tion with a ~roper~r-
modifying group, whe~er the un~ed residue is native ~r in~r~uced by site-~cific
mutation, are refe~r~ to as AUC cytokines? (AUC denotes "allosteric unpaired
cysteine"),
The LnventioKI further includes site-speci~c lipophiliza~on of such AUC
cytoldnes. Llpophilizati~o increases the li~ophilicity (af~ for lipid), which aids
the resulting product in attaching to csll membranes or m localization to a target site,
and thereby reduces its clearan~ rate. The lip~philiæd cytolcine caIl attach to the
cells at ~e site of administration, which slows the rate of diffusion into ~e vascular
~ ~ ~ or into the mucous fluid and hence~retains the product at the ~arget site fo~
longer dur~tion. The biologi~al ac~vity and binding prop~r~es of the lipophiliæd, 20 cytol~ne, however, are substan~ally ~e same as the native form.
e prefe~red embcdiment of the inYention has a cytolcine lipophilized by
~valently conjugating it t~ one fatty a~yl group. The preferred fatty acyl groups m~y
wo 93/12l42 21~ 4 ~ 7 2 pcr/uss2/1o889
- 8
range from ~18 carbon atoms in length.
The invention further includes AUC cytoldnes cQnjuga~ to an antibody or
anothe~ ~ype of binding molecule, horseradish pero~idase, aLkaline phosphatase,
fluo~escein substances, or biotin. Such conjugates are useful as reagents for cellular
5 and cytokine rece~tor assays. AUC cyt~kines, such as IL-2~ lI,~, and EGF, may
also be conjugated with cytoto~cins, such as Acin A chain or ps~udomonus e~coto%in,
for targedng tumor cells expressing high densities of the respective receptors.
l~e ~llvendon further includes pharmaceutical compositions of lipophilized
cyto~es, or the other cytoldne conjugates described above. The phann~ceutical
,. ~
10 composition may include suitable adjuvant~, diluents and solvents.
Detailed Descfip~ion of the Invention
A. CytQk~nç~i~ç fo~ Li~ophiliz~tiQn ~d Rel~vant Con~ ns Ther~
:
As noted above, a number of native (non-modified) cyto~dnes have been
inves~ga~ed or are being developed fo~ trea~ng localized disease sites. These same
c~ldnes ~inclu~ing E~GF, ~:GF, IGF, PDGF~ , IFN-~B, IPN- y, IL-2 IL-l,
NGF, and CNT~) ~ be lipophilized with the techniques of the invention to make
them suitable ~or such trea~ment. Ce~tain enzymes, such as DNAase, which can be
en by inhalation and is po~entially useful in dissolving DNA in alve~ mucus in
~: :
patien~ with cystic fibrosis, and superoxide dismutase, which can be administe~ed to
~, 20 the inflarned ioints of patients with os~oarthritis, can also be lipophilized with the
teehniqucs of the mven~on to make it suitable for sueh treatment. All of these
cytokines are relatively small proteins, and the human genes expressing them have
.. '
WO 93/12142 21 2 4 6 7 2 Pcr~uss2/lo889
been cloned, characteriæd, and expressed in host cells. The~efore, recombinant
~rtokirles can be readily produced in quaI-ti~ies suitable for site-specific mutation and
lipophilization is feasible.
The site-specific mutation of the cytal~ne, which pe~ts conjuga~on of ~e
lipophilic substance apart from the binding site, can be accomplished in a number of
ways, including by reoombinant techniques. The lipophilic substance can be
~onjugated to th~ cytol~ne by a number of techniques, including a chemical reaction
be~en the lipophilic substance and the cytokine. The lipophilic group itself, and ~e
final lipophilized product, should be subs~ally resistant to enzyme cleavage. The
lipophilized p~oduet should have substantially the same receptor-binding activity and
; : biological a~tivity as the native ~orm.
The lip~philic sub~tance ~ be a number of agents, including fat~f acid
groups, and lipophilic and uncharged pe~'ddes. The lip~philic group should ~e
~ .
nanimmunogemc, nonantigenic, and not so large as to affect biological ac~vity orreceptor binding. Furth~r, as noted above, ~ather than a lipophilic substance1 the
cytobne can be con~ugated to other chemical modifier groups, including an antibody
: : or ano~er t~pe of ~binding:mol~cule, horseradish peroxidase, alkaline phosphatase,
fluDrcscem subs~ances, biotin, or a cytotoxin molecule.
A prefcrred li~philized product is conjugated to a fatty acyl group. The
~, 20 length of the fatty a~yl group determines the lipophilicity of the final product, and the
philicity of the cyt~ldne itself de~ennines ~e equilibAum/distribution of it between
: . :
~e cellular plasma membrane and the ext~ellular space. One can select ~rom fa~ty~ ~ .
,
~:
:
WO 93/12142 2 1 2 4 6 7 2 P~r/USs2/108~9
acids with a wide range of chain lengths (including, 6, 8, 10, 12, 14, 16, and 18
carbons) to selec~ the fatty acyl group which provides optimal phannaeol~netic
proper~es of the fina~ product for ~he targeted application.
Generally, greater lipophilicity is pr~vided by a longer f~ty acyl grol~p. The
5 gre~ter ~ philidty renders a final produ~t which will be distributed in a more limited
area, and will be less diffusive, and will rema~n longer at the si~e of admi~istra~on.
A cytol~ne conjugated with a 16 or 18 carbon acyl group will attach well to the
cellular plasma membrane and h~ve limi~ed dli~fusibili~y. However, gr~ter
lip~philici~r is not necessarily m~re desirable1 because some solubility and diffusibili~
10 are requir~ to achieve maximum rece~tor binding and biological effect. The fatq~
yl groups providing ~ptimal pharmacoldne~cs f~r a ~ypical cytokine are 8-14
ca~on long, sah-ra~, and unbranched.
B. ~ L~
A ~kine is suitable for localized as ~3p~sed to systernic adminis~ation,
~: 15 bas~d on the:~esults ~or the in v~vo human clinical studies, if it has one or mor~ of the
following properties:
~ e substance is seriously to%ic when administered i.v. or by a gene~alized
systemic route;
:~ ~ (2) the substance has~a rapid clealance xate, i.e., a s~rum half life of less than 3
20 hc~urs;
(3) If ~e subs~ is.systemical~y administered, the therapeutic e~fect on the targeted
~: ~ disease i~ marginal;
wO 93/12142 - ~ 1 2 4 ~ ~ 2 PC~USg2/10~9
11
(4) The a~fec~d site which is to be targeted by the substance is appropriately locali~d
such that the delivery of the substance by a local, nonsystemic administ~atioll is
~easible; e.g., a nonmetasta~c solid tumor or an eye infection is appropriat~ly
localized, while erythro~iesis in the bone marrow9 although anatomically and
S histologically lo~li~d, is not approp~ately localized.
Lipop~tion of a cytokine should provide a product wi~ a high~r affinity
for the cellular plasma memb~ane than the native cytokine, ther~by proYiding a
lipophiliæd product which will remain at the target site longer ~an the na~ve product.
~he lipc~ph~ product should stay attached to the cells for periods of time and not
io rapidly dif~use into ~e capillaIies to be carri~ away in the blood str~n. Similarly,
when a lipophilized p~oduct is administered t~ a mucosal su~ace, it should be a~le to
attach to the ~ells and a~oid being rapidly washed away by the mucous fluids.
C. Dis~ases uit~121e for l[reatme~wi~ hilized (:ytoldnes and Meth~
~ÇQf
A number of 1iseases and conditions can be treate~l with local administration
of lip~philized cyt~kines. If a di~ condition is appropnately localized and the
affeeted tissue site is accessible for drug delivery, the local administration of a
lipophilized cytokine is desirable, as relatively high doses can be administered without
the ~oxicity ass~iated with systemie administration. The mode of administration
20 should be such ~hat it ~elivers the drug t~ all of ~he a~cted tissue. "lHigh density"
ir~jec~ion, whe~o small volumes of the therapeutic solution are injected into a large
number of sites per unit volume of tissue, is the preferred mode of administration.
:For ~ample, inj~c~ons of lO ~1 of therapeu~c ss)lution in~o 10 sites ~ l ml of solid
WO 93/121~2 2 1 2 4 6 7 2 Pcr/US92/~ 89
12
~ssue would be a ~ypical protocol for administration.
Solid tumors are one e~ample of a condition suitable ~or treatment with a
lip~philized cytokine. One would directly in~eet lipophilized IFN-a, IlF~ y, TNF, IL-
1 ~r IL-2 into the solid tumor site. This treatment would be especially attractive when
the tumor has not gone into metastasis, or has only limited metastasis. This direct
i~jection can also ~ minister~d in conjunction with surgery, in order to provide an
even more direct ~ss to the tumor site. Such injec~on near the e~cision site also
ensures the optimal imn une response to ~id in des~oying the residual tumor cells.
In a di~ condition, in which the af~cted tissue is SUperflCial and ~rery thin ore~posed, such as an infected cornea, nasal lining, superficial wounds, or bald s~dn,
~ical ~pplication of a lipoplhilized cyt~ne may be effective.
Lesions or ~enital warts can be trea~ed wi~h direct injection of li~philized
IFN-a, IFN-,B, ~r DFN-~y. Again, there is dir~t access to the affected site wi~h direct
inj~ction. Spinal injunes ~ ~e treated with dir~t injection sf lipophized NGF orCNTF. Lipophiliz~d superoxide dismut~e may be administered to the inflamed joints
of patients with os~nbs.
In~ec~ions affecting the lungs, such as an inlluenza or bacterial, viral, or
parasitic pneumonia, can be treated with inhalation of lipophilized IFN-a, IPN-,B7 s:llr
I~;N- y. In cystic fibrosis7 lipophilized DNase may be talsen inhala~ion. DNase can
~, 20 dissolve ~NA in the alveolar mucus, thus reducing the viscosity of ~he fluid. A
preferred inhalation device is a metered dose inhaler, which ensures administra~ion of
a measured dosage.
Wo 93/12142 2 ~ PCI /US92/10889
13
L~calizecl viral, bacterial or yeast infections of the vagina, rectum, mou~,
thro~t, nasal linings9 eyes~ andlor ears can be treated with applieation of lipophilized
IFN-a, IP~-~B, or IFN~y to the mucosal surface9 preferably with a dropper or
spraying de~/ice. Such t~pical ~pplication of lipophilized EGF, FGF, or IGF can also
5 be e~fective in promoting healing of wounds or surgic~il ~cisions or lesions.
To ~eat baldness, or to ~enew or promote hair grow~, lipophilized EC~, FGF
or IGF can be applied by int~adennal or subcutaneous inj~ction, or by topical
applicadon.
D. ~ and ~o~uuea~ ~5y~Q}dne~
10As noted above, several methods can be employed to conjugate a fatty ac~rl
group to a cytoldne. Fat~ a~yl group~ whieh are modified t~ contain active linl~g
:~ g~oups include N-hydroxysuecinimide esters of fatty acid, and can be prepared
accorditlg to ~e m~hod of Lapidot, Y. et al. J. Lip~ Res. 8:142 (1976). Some of
~:: these active esters are als~ available ~rom commercial sources, such as Sigma
15 Chemi~al Co. (St. ~ouis, MO) and Matreya, Inc. ~Pleasant Gap, PA,).
The coupling of the N-hydroxysuccinimide ester to ~e fatty acid to ~e
cytol~ne can be performed according to the techniques describ~d in Utsumi, T. et al.
Cancer Res. ~51:3362 (l~l). This:paper also descnbes how to ~dtrate to determine
: : the number of f~t~r acyl group9: ~r molecule on the modified protein. As noted
: 20 above, it is ~efe red that only one fatty acyl g~oup be eonjugated to each cytoldne
- ~molecule. The pref~ed stoichiome~y can be ~chie ~ed by con~olling the ratio o~
reactants during the o~upling reaction. The lipohili~ed cytokines which have one fatty
;
W~ 93/12142 2 1 2 4fi 7 2 PCr/US92/10889
.,,
acyl group per molecule Gm be purified by a rou~ne procedure.
If the fatty acyl groups are conjuga~ed via the ~-amino groups of lysine
residues on the cytokine, one cannot ensure the coupling of one fatty ~cyl group to the
~-amino group of a particular lysine residue on ~e cytokine. ThereiFore, the final
S lipophili~d cytoldne will be heterogenic in terms of the number of fatty acyl groups
pe~ cytokine moleeule and the location of ~e fatty acyl groups. This type of
conjugation c~tes the possibility that some of the modified cytoldne molecules will
lo~ recs~tor-binding activity, because the fatty acyl groups may couple to ~ino
g~oups at or near the cytokine receptor binding sites. Further~ this type of
~,~
hete~ogeneous conjugation increases the possibility of the fatty acyl grou~modified
sites b~minlg immunogenic. Therefore, the preferred method of conjuga~on is to
.
first modify the cytolcine in a site-~cific manner so that the lipophilic group will
bind to the site-~ecific modi~lca~on rather ~n at other locations on ~e cytol~nemolecule.
: ~ ~ 15 Por c~strucdng the lipophilized ~ytokines of the invention, the cytokine genes
are site-specifically mutated by recombinant DNA methods so that the mutated
cytolcille has an unpai~ed cysteine residue far enough away from (allosteric to) the
r~ tor binding sites to pre~ent it from interfering in rece~tor binding or biological
tivi.~. ~
Generally, na~ive cyto~dne molecules are single chain polyp~ides and contain
; ~ even Dumbers of cysteine residues. These cysteine residues form disulfide bonds
between pairs of cys eme residues. The specific pairing of ~he cysteine residue~ is
: .
wo 93/l2142 212 ~ 6 7 2 Pcr/uss2/lo889
detenTIined by the 3-dimensional folding of the polype~tide chain, whieh is de~rmined
by the sequence of the polypeptide. The disulfide bonds are not usually exposed on
the surface of the proteLrl molecule, and their function is ~o hold the pro~ein in ~ rigid
s~ueture to withstand the relatively harsh and variable conditions which e~;ist out:side
the cyt~plasm. Secreted proteills, such as cytol~ines, have disulfide bonds, whe~eas
proteins which remain in the cyto~lasm or on ~e inner surface of the plasma
membrane do not have disulfide bonds.
A cysteine residue can be introduced onto the cyt~kine at a particular site to
provide a docking site for fatty acyl ~group conjugation. The c~teria for choosing
where to place this residue are ~at:
(1) the subs~dtu~on should not affect the 3~imensional folding of the cytoldlle
molecule;
O the residue should be away from the rece~tor binding site; and
(3) Ihe residue should be on the surface of the p~otein mol~cule and accessible ~or
fa~y acyla~ion.
Generally, a se ine residue which is in ~r near a pep~ide str~tch that is highlyhydrophilic is most suited ~or ~eplacement with a cysteine residue. Cyst~ine andsenne residues are structurally highly homologous. ~e close proximity to or the
location in a hydrophilic peptide s~eteh will ensure that the residue will be Oll the
~0 surface of the protein mole~ule, so as ~ be available for chemical conjugation after
substitu~on. Other residues whieh can be subs~ituted are those which ar~ polar or
charged, sucb a~ asparagine, glutamine, ty~osine, histidine, lysille, arginine, aspartate,
wo 93/12l42 21~ 4 fi 7 2 - pcr/us9~/1o889
16
,.
and glut~nate9 provided they are in ~r near a peptide sbretcll that is hydrophilic.
Since the X-ray crystallographic 3~imensional structure of most cytol~ne
molecules has not b~en detennined, it is not possible to predict whether an amino acid
residue is or is not in or near ~e rece~tor-binding site of a particular cytoldne. The
5 procedure for ma~ng this determination and identifying a suitable residue ~or
subs~ ion ~volves systen~ically dete~ng whe~er ~e subs~tution of par~cular
re~idues by cystehle, ~ollowed by lipophilizadon, affects the rece~tor binding or the
biologic~l ac~y of the substitut~xl pr~uct.
A ste~by-step procedure t~ obtain an AUC cytokirle follows.
(i) ~
The first ste~ is ~o determine the amino acid sequence. Por most c~ldlles,
: ~ the sequences are available from the literature, and sequencing is not necessa~y. For
others, sequencing can be perfonned by mlcleo~de sequ~ncing of ~e cDNA clones
of the mRN~ of the c~kines. The deduced amino acid sequerlces can ~ confirmed
15 by N-t:enninal amino acid sequence analysis and from a molecular weight
det~rmination of the cy~oldne proteins.
(ii) ~yd~hili~zly~is
The next step is to analyze the hydr~philicity of ~he cytokine polypeptide.
Seve~al software prograTns that plot the hydrophilicity ~or hydropa~y~ in qua~titati:ve
20 indices in ~elation to the line~r amino acid sequence are available and can be used.
One of such compu~er progr~n is developed by Hopp, T.P. and Wood, K.R., and
des~ribed in A~ol. ~unol. 20:483 ~1983). Micr~Geme sequenæ analysis package
WO 93/1~142 2 ~ 2 4 ~7 2 Pcr/llS92/10889
17
distributed by Beckman Inst~Nm~nts, Inc. Palo, Alto, CA. provides a software
program for p~rforming hydrophiliei~h~r plots.
(iii) Ident~fyin~ ~ndi~r~sidues
The next ste~ is to identify the hydrophilic regions in the polyp~tide chain and
5 ~ identify the best-suites~ residues in or near hydrophilic stretches f~r l~e substi~u~on
with ~e cys~eine residue. Ihe preferred residue for subslitution is a serine residue.
. Howev~r, if a se~ine residue is not available or not sui~able, a histidine, tyrosine,
glutamate, aspar~ate, lysine, histidine, as~aragine, or glut~ne residue could be an
adequate alt~mative choiee. One generates a number of mutant cons~ucts (as
io many as een) each having only one subs~dtution per mutant cons~uct. Eventually,
us~g the procedures ~escribed furtha below, ~he mutant constructs are screened to
determine which haLYe a ~ubstitution allosteric to the bindillg site.
(iV) ~
~he next s~ is to synthesiæe the native and mutant gene~. Polyme~ase chairl
reac~o n (PCR) can ~ used to construct the natiYe cytokine gene. One would use
ohgonutcl~otidc primers tha~ co~respond ~o ~he 59 and 3' end of ehe ml~A of the
cyto~e, and that contain prope~ cloning s~quences. One would start with ~he ~A
pr~ ation from ~ctivated lymphocytes, leukocytes, fibroblasts, or o~her cell lines
producing ~he par~cular cy~kines, ~rom which the cDNA are to be cl~ned. The
20 doned cDNA, after s~quencing confirm~tion, is insened into a plasn~id, such as
pUCl9, for subsequent ~roc~dures. One rou~ne laboratory pr~edure for site-direeted
mutagenesis is to start with the syn~esis of oligonucl~otide pnmers of abollt 25
!
WO 93/12142 PCI/US92/10X89
fi ~ '~
18
nucleotides which ~ontain the triplet condoll of a cysteine residue in place of the triplet
condon of the serine (or other) residue which is to be re~laced. These primers with
the inshlled mutations pe~t ~e synthesis of ~ull leng~ DNA genes wi~ the site-
directed mutations. A ~onvenient method was developed by Kunkel, T.J~.., Proc.
S Natl. ~dac. Sci. U.S.A., 82:488 (1985~. A ste~by-st~ pr~toc~l wi~ the reagents is
descIibed by Kunkel, T.A. in Current Prot~cols is ~olccular Biology, Supp. 6 ~
8.2.1, Eds. Ausubel, F.M. et al, Wiley Intersciences (1990). A PCR method for
in~oducing point muta~ons in cloned DNA is als~ rou~nely used by many molecular
biology lab~rato~ies. A ste~by-ste~ procedure i~ ,deseribed by Cormaclc, B. ~urrent
1û Proto~ols in Molecular Biology, Supp. 15 ~ 8.5.1 ~ds. Ausubel, F.M. et al, Wiley
Intersciences (1991).
A preferred method to construct the en~re ~amily of native genes and mutant
cons~cts is t~ synthesize ~he complete genes wi~ a DNA syn~esiær. Over}apping
oligorlucl~des of 11;0 80 nucleo~des from the positive and negati~re stands which a~e
complemen~ am~g the adjacent oligonu~leo~des at their 3' ends can be syn~esiæd
with one of the commercial DNA synthesizers, such as one ~om Applied Biosystems,
Inc. Th~ oligonucleo~des pr~vide both the templates and primer~ ~mutually primed
syn~hesis) to generate ~e desi~ed sequence in one-sillgle step. After etongation is
p~formed witlh T7 DNA polymerase, the segments are linlced by a ligase. The
o~igonucleotide~ at the two ends of the genes are properly designed to include
restric~i~n enzyme si~es, so that the synthes~d genes can be inser~ed into the prop~r
c~pression vector. The reagents to be p~parul and the stepwise procedure is
Wo 93/12142 - pcr/us92/1o889
21'~4fi72
19
described by Moore, D.D., Current Proto~ols in A~olecularBiology, Supp. 6 ~ 8.2.8,
Eds. Ausubel, F.M. et al, Wiley Intersciences (1990). This method is a~trac~ive
becauæ it easily allows const~uc~on of the large number of site~irected mutations
ne~ded to make the various mutant constructs, and be~ause ~ytoldne genes in general
S are relativi~ small, shorter than 600 nucleotides. All of the oligonucleotides, except
~e one with the specific mutation, may be shared for the individual constructs.
Complete synthesized interferon genes have been madc. See Edge, M.D. et al.
In~e~eron 7, ~. Gresser" I pp. 2-46 (Acadenaic Press, London, 1986).
(v) ~pression
,~, ~,.
The ne~t s~ is to express the wild type and the mutated sets of cDNA in a
eukary~c or prolsryotic expression system and produce the na~ive cytoldne and tlhc
mutant cytoldnes, and then to purify the cytoldnes to produce sufficient amounts of
ea~h. For most cyt~:\ldnes which contain disulfide bonds or are glycosylated, the
cuhryotic ex~ressio n system is prefe~ cause it assures ~iisul~lde bond formation
lS and :the proper folding of the polypeptide chains. Among the various eukaryotic
e~pression systems, a pre~erred system ~or the expression of the foreign cyto~negenes u~es the expression vector derived from nuclear polyhedron protein gene of the
insect vws baculovirus ~Aa~tographa (:~alifomi~a). The virus can be grown in the;~ inses~t cells. Sp70dopte~a frugiper~ ce1ls (sf9 ce~ls3. Luckow~ V.A. and Summers,
M.D., Vîrolo~ 167:31 (19~9). This expre~siQn system has been packaged into a
- convenient Idt (designa:ted "M~c Bac Baculovirus E~press Systemn) and is sold by
Invitrogell (San Diego, CA.).
,, ,
W~ 93/1;!142 2 1 2 ~ 6 7 2 Pcr/uss2/~08~9
For cytokines which do not contain a disulfide bond, such as IFN- y, and for
which a earbohydrate moiety either do~ not exist or is not involved in the binding to
the cytokine rece~tor, the e~cpression may be carried ~ut in prokaryotie cells. When
an E. coli expression system is used, the e~pressed cytol~ne proteins need to be
S solubilize~, reduced to unfold the polypep'dde chain, and allow~d to renature to fonn
~e most favor~le 3~imensi~nal structure. A pref~ed system is the FLAG
~iosystem Icit, offered by In~em~onal Biotechnologies of Kodak (New Haven7 CT.).
This system also contains the ~eagents for the detec~on and purifieatiotl of the non-
fu~ p~oteill~ Monoclonal arldbodies for most cytoldnes ha~e been dev~loped and are
10 commerGially available. These monoclonal antibodies can be used to affinity pu~
t~e r~tive cyt~es.
(vi) ~Qniu~ation
~e next ste~ is to conjugate the purified na~ve and mutant cytokines untll
~: : fatty acyl groups, prefe~ably o~ about 8-14 carborls in length. An example of a
~; 15 preferred chemical reac~on suitable to accomplish ~his conjugation using a 1~carbon
fatty ~yl group, which is adopted from the method ~f Carlsson et al. Bi~ch~m. J.
:~ 173:723 (1978), is as foll~ws:.
In Reaction I, 1-bromodecane (~m Aldrich Chem. Co., Milwaukee9
Wisconsin) is reacted with 2~ 2'~ipyridy1 disulfide (Aldrich) to form pyridy1 decane
20 disulfide. In ReactioJ 2, *is product is reacted with AUC cytokine to ~orm ~e
decanoyl pro~ein.
wo 93/12142 212 4 fi 7 ~ pcr/us92/lo8Bs
21
Before performing the ~njuga~on reaction, howev~r, the first step is to c~
a f~ee SH gr~up on ~e cyto~ne. The ~r&c SH group ~ the unpair~d cyst~ine resic~ue
of a cytoldne, ~Nhich o~ten is c~upled to other sul~ydryl grou~containing metabolites
during biosynthesis, is first ~educed by mild reducing conditions ~ free it ~rom such
meeabolites. The mild reducing conditions, however, d~ not reduce ~e disulfide
bonds buried inside ~e m~lecular ba~kbone of the cy~ldne. AfteY reduetion, the
reducing agent is rem6~ved ~y gel filtra~on or ion exchange chrotn~t~graphy. The~eated cytokine is then reac~d with the f~y acyl groups, which haYe ~n previously
IS modifi~d with an ac~ve group.
The native cytokine likely will not conjugate with the fatty acyl groups, as ~hen~ve cytoldne usually does not have ~y accessible~ unp~red ysteille residues.
Howe~er, ~or those n~tive c~e~ whi~h ~ have accessible unpaired cysteine
~esidues, th~y will al~ be conjuga~d ~D ~atty acyl groups by ~he procedure described
above. lllereafter, they ~ be analyzed ~or r~eptor bindinglbi~logical a~tivity as
describe~ imm~diately bel~w, to de~e whe~her they are AUC cytol~nes. If this
a~lysi~ reve~ls that they are rlot AUC cytclfines, then the unpair~d cysteine residue
wo 93/12142 Pcr/us92/lo889
212~6~2
- 22
may be replaced by a senne residue ~to ensure that it does not conjugat:e with the ~atty
acyl group), and another residue at another l~cation can be replaced with a cysteine
residue~ This substi~tion of a seAne for a cysteine will not affect the receptorbinding or biological ac~vity. The conjugation reac~on will only create fatty
S acyladon of such cytoldnes at ~e one unpaired cysteine residue, and not elsewhe~e,
(vii) 1~12i~inglbiolo~i~1 ~tivity
The last st~ is to analyze and compare ~e rece~tor-binding and biological
activit~r of the na~ve cytokine and the ~atty acyl-conjugated mutant cytoldne. Those
mu~ant cytol~ne molecules with pardcular cysteine residue ~ubstit~tions which have
,~.,,
10 substasl~ially the same rece~tor-binding and biological activity as the native cytokines
are ref~rred ~o as AUC cytol~nes.
2. ~a~pl~: The ~Da~tion of AU~-IFN-a2
The st~ps described above in Section D.(i)-(vii) can be used, for exam~le, to
, ~ .
produ~ the A~C cy~ol~ne IFN-a2. IFN-a2 is a member of ~e family of interferon
c yto~nes, and Table I below summariæs the p~rtinent key pr~ es of this family,
::: and ~more particularly includes information about the cysteine residues ~hereof.
ere are 19 forms of :IFN-a, one form of IFN-~, and one form of IFN-~y.
All but~two of ~e IFN-CY have 4 cysteine residues, forming 2 disulfide ~onds, between
residue Nos. I an~ ~ (or 98 or lOO) and Setween Nos. 29 and 39. Only I~ al and
20 IFN-aD h~ve an unpaired ~ysteine residue ~No. 86). IFN-,B has 1 disulfide bond
n eysteine residue:Nos. 31 and 141) and one unpaired cysteine ~esidue (No.
:: ~
17). IPN-~yhas no cysteine residue.
:: ,
WO 93~121422 1 2 ~ 6 7 2 PCI/US92/1~889
23
TABLE 1. CYSTEINE RESIDUES OF HIJMAN INTERFERONS
IFN# Total # Cysteine S-S Bonds Free
l~esidues Residues Cysteine
IFN-a6 166 4 Cl-C99, C29~C39 0
IFN-a~S 166 ~ Cl-C~9, C29-C39 0
IFN-a2 165 4 Cl-C98, C29-C39 0
IFN-c~l 1~ 5 4~ 9~, C29-(~39 C~6
IFN-atD 166 5 Cl-C99, (~29-C39 C86
IFN-~Hl 166 4 Cl-C99~ C29-C39 0
2~
IF~-tY8 166 4 Cl-C99, C29-~39 0
E:N-aB 166 4 Cl-C100, (::29-C39 0
IPN-a4b 166 4 Cl-C99, C29-C39 C)
IFN-aC 1~6 4 Cl-C99, C29-C39 0
IPN-aL l66 4 Cl-~99, C29-C39 0
IFN-~Jl l66 4 Cl-C99, C29-(:39
IP~-~2 l~6 4 ~l-C~g, C29-C39 3~
IFN-lxI l66 4 Cl-C99, C29-C39 0
~FN-aF l~6 4 Cl-C99~ C29-C39 0
IFN-aWA 166 4 Cl-C99~ C29-C39 0
IPN-cYCix-l l66 4 Cl-C99, C29-C39 0
CY76 l6~i 4 Cl-C99, iC2g-(~3g
w~ 93/12142 - P~r/US92/10889
67~
24
IF~-cr88 166 ~ Cl-C99, C29 C39 0
IFN-,B 166 3 C31-C141 C17
IFN- y 143 0 0 0
F~r plepanng an AUC cyt~kine, ~e pro~ure described above in Section
~: D-(V to (vii) where an unpairc;l cysteine residue is introduced into the cytol~ne should
be employ~d for most of the IFN, but not for IFN-~1, IFN-aD, and IFN-~B. E~ecause
10 ~N-a1, IFN-aD, and IFN-,~ all have an unpaired cysteine residue, ~e procedure
~: described in S~c~on D.~V1) (where an unpaired cysteine residue is substituted by a
serine and then a cysteine is substituted elsewhere) should instead be followed.
The fact that amino acid residue No. 86 is a serine or t~r~sine residue in all
IFN-a exce~t IFN-al and IFN-aD suggests that the cysteine residue No. 86 in IFN-
15 al and IFN-aD is not crucial~. Thus, for IFN-al and IFN-aD, one would first try
direct conjugahon of a fatty acyl group to Cys 86 ac~rding to the t~hnique~ of
Seetion D.(vi), and then use the methods ~f Section D.(vii) to determine if the
,
conJugate remains receptor-binding and biologic~l activities. If this substitution is not
: : :
easible or if the analysis reveals that the cytol~ne is not an AUC cytokine, one could
20 replace Cys 86 with~ a~ senne ~esidue and proceed to prepare an AUC-IFN-al and
IFN-~D acco:ding to the methods of Section D.(vi).
Similar.approaches can be us~d to prepare ~he AUC fo~m of I~;N-~B. It is noted
that if the native ~unpaired cysteine residue is replaced by a serine residue or an~ther
residue, and an ~unpaired cysteine residue is in~oduced replaeing another residue in
: ` :
~::
.
- Wo 93/12142 2 -I 2 ~ 6 7 2 Pcr/US92tlO~89
~ 25
a different location, the mutant cytokine will have two site-sp~cific mutations, but only
one site swted f~r conjugation with a lipophilic substance.
As Table 1 indicates, IFN-a2 has four cysteine re~idue~ forming two disulfide
bonds. The nucleotide ~equence of the cDNA gene and the deduc~d amino acid
S sequence ar~ known and published. U~g a hydrophicili~ analysis program pro~ided
by l~ o(3enie, which adopts the pnnciples of Hopp, T.P. and Wood, K.R. Mol.
Inununol. ~0:483 (1983), a hydrophicility plot is made (n~t shown). The plot
indicates ~egions or ~de segments of relatively }ligh hydrophicili~. Using ~e
itena diseussed ~e in Sec~on D., the amino acid r~sidues ~elected f~r site-
dirccted mutagenesis (i.e., for subs~hltion with cysteine residues) are: serine No. 11,
: arginine No. 22, lydne No. 31, glutamic acid No. 42, glutamic acid No. 51~ e
No. 72, glut~nic a~id No. 113, se~ine No. 115, lysine No. 133, serine No.160, and
S~ne No. 163 (c~ating eleven mutant constn;lcts in total).
:: :
The prefened method for prq~ing the na~ve I~N~a2 gene and the 11 mutuan~
:~ 15 gene cons~ucts is with the oligonucleotide syn~esis method as discussed aboYe in
Section D.(iv). ~ ~he prefQred conjugation method is described in Section D.(~i).
The~ ste~by-step procedure described above generally, and for IPN-~Y2
:
: specifically, :c~n be f~ollowed for prepa~ing other AUC ~ytokines. As analyzed above,
most native cysoldnes have e~en numbers of cysteine ~esidues and do not con~a~n an
i ~ 2 0 u~ d cysteine residue. For example, human EGF poly~e~tîde is 53 amino acid
:
;: ~esidue in leng~ c~ontaining 6 cysteine ~esidues (3 disulfide bonds); human basi~ FGF
tide is 155 amino acid residue in length containing 4 cysteine ~esidues ~2
,
wo 93~12142 pcr/us92/loB89
2124672 26
disulfide bonds), human IGF polype~tide is 70 amino acid residue in leng~ containing
6 cysteine residues (3 disu1fide bonds); human TNF-a polypeptide is 157 amino acid
residue in length containing 2 cysteine residues (l disulfide bond)9 and human ~-NGF
polype~tide is 188 amino acid residue in length con~aining 6 cysteine residues (3
S disulfide bonds). ~elatively few cy~okines have odd numbers of cysteine residues and
~us an unp~ d cysteine residue. One is human IL-2 polype~tide, which is 153
. ~G acid residue in length containing 3 cysteinle Fesidues (l disulfide bond). The
u~paL~ed cystehle residue No. 125 can be subs~tuted to a non-cysteine residue without
losing IL-2's re~tor-bir.ding and biological ac~vity. E.P.A. application l36489
10 Thus~ this cysteiJIe residue (No. 125) provides a very likely site ~or conjugation.
Dia~n~i~ ~s~y
The present invention focuses main1y on the fatty aeyl derivatives of AUC
cytokines for local or mucosal therapeutic administ~ation. How~ver, these uses are
1~ not the only ~herapeutic applications of AUC cytoldnes. Cyto~nes, such as IL-2, IL-
4, and EGF have been explored as binding molecules and vehicles for cytotoxins, such
as pseudomonus exotoxin,to tumor cells expressing high densities of the receptoFs of
thG respective cytokines. The coupling of cytoto~;ns to the nadve cyto~nes by the
conv~n~ional m~hods will yield heterogeneous conjugates. The eoupling of cytotoxins
20 to AUC cytokines via the unpa~red cysteine residue will yield impr~ved conjugates ~or
the targe~ng of cytotoxins to cells.
Therapeul~e applications are not the only applications of AUC eytolcin~s.
Several applica~ons of AUC cytokines for providing useful reagents for research and
WO 93/12142 - pcr~us92/1o889
2127~672
diagnosis may also be developed. In studying cytokine fune~on, it is important to
quantitate ~e le~els of the cytoldne in culture or in body fluuds, the e~pression levels
o~ the cyt~kine re~tors ~n tar~et cells, and/or ~e l~vels of rece~tor~xpressing cells
in a tissue. In certain diseases or cl)nditions, the levels of ~ese bi~logieal palameters
S may change. MonitDrillg ~ese levels, therefore, can offer a means to determine or
monitor disease status.
For such ~uantita~ive assays, which include immunochemical and cell-binding
ass~ysl cytoldnes conjuga~ with indi~tor substances (such as an~bodies, h~rseradish
pe~oxid;ase, aLkaline phosphatase, fluoresceirl substances, or bio~n, for use in
10 conJunction with avidin~upled indicato~s) are useful reagents. In the conventional
methods ~or prelwing these conjugates, ~he ~oupling is made through the ~-amino
groups of ~e Iysine res~dues. However, as elucidated above, the c~njuga~s formed
in ~is manner will b~ hetero~eneous in terms of the numbers and the loca~ons of the
conjugated groups. Also, the activity and rece~to~ binding pr~perties of some of these
~5 conjugated cytokine molecules may be affected by ~e coupling of the modifier group
to a Iysine residue w~lich is in or elose to the binding site. Conjugation of these
modifier groups via the sîngle wlpaired cysteine ~esidue of an AUC cytokine, using
~ ~ ~ the me~hods described a~ve, can minimize or elimi12ate these problems and thus
`: provide improved reagents for research and diagnosis. Cytoldne~ are better than
20 ~eeeptor-specific antibodies for studying the rece~tors, because cy~kines usually have
high~ affinities than antibodie~ for their r~sp~tive receptors. Cytokines are smaller
in sizç than antibodies and thus are especially sui~able for staining tissue sections,
' .
wo 93/12142 212 4 6 7 2 pcr/us92/lo8B9
28
where ~ssue penetra~on and diffusion of the reagents is importarlt.
I~ should be understood that the terms, expressions and examples herein are
e~emplary only and not limiting, and those skilled in the art will recognizel or be able
to asc~rtain using no more ~ outine experimenta'don, many equivalents ~o the
S s~ecific embodimerlts of ~e irlvention described herein. All such equivalents are
intended to be encompassed by the following claims.
