Note: Descriptions are shown in the official language in which they were submitted.
CA 02214029 1997-08-27
WO 96/34109 PCTrUS96/05035
~1 .
SINGLE-VIAL FORMULATIONS OF DNAILIPID COMPLEXES
FIELD OF THE INVENTION
The invention relates to - _' .;.,l fG,I ' i of plasmid DNAlcationic lipid - . ' - for human
r clinical use, and related I .
BACK6ROUND OF THE INVENTION
The use of plasmid DNAlcationic lipid - .' 9 to transfer genes in vivo for the Ll~al..._..l of
human diseases, including ' :y and ca, . .a;,cular disorders, is; ' r~ d1 in active human clinical trials.
The R ' I DNA Advisory Committee (RACJ of the National Institutes of Health (NIH) approved
Nabel et al. to conduct a human clinical protocol using lipid mediated transfer of an _ encoding gene
into tumors for i ;' .1 of re r~ ~ Nabel et al., Proc. Natl. Acad. Sci. USA 90:11307 (1993); see
Nabel et al., Human Gene TheraPv 3:399 (1992); see also Nabel et al., Human Gene TheranY 5:57 (1994);
PCT Patent Al.' -: - WO# 94129469. The gene encoding a foreign major ' - . l' '~y complex
protein, HLA-B7, was ;..l..~ ~ into HLA-B7-negative patients with advanced ' by injection of
DN~lli,- - - .' using a DC-ChollDOPE cationic lipid mixture. Nabel et al., Proc. Natl. Acad. Sci.
USA, supra. Six - ' ;Idi- were ~ . ' ' without ~ . ' - in five HLA-B7-negative patients with
stage IV ' (~, one patient received a second -' ;,~ ) Plasmid DNA was detected by
p~ a~e chain reaction within biopsies of treated tumor nodules 3-7 days after injection but was not found
in the serum at any time. F. - ' ~ I HLA-B7 protein was ~l ~.dll,d by -' ~ y in tumor
biopsy tissue in all five patients, and immune, , to HLA-B7 and àul Rg tumors could be detected.
No ? ' ~ ' to DNA were detected in any patient. One patient d ~,dl~d ,~!J,. of injected nodules
on two ', ' 1l~ : . which was e . ' by ll_ . at distant sites. These studies
;' alud the r. ' ' ~y, safety, and Ol~l.."_..li~, potential of 1i, ' ' : ' gene transfer in humans. See
also Stewart et al., Human Gene TheraDy, 3:267 (1992) (safety and : y of gene transferin vivo with
DNAllipid ~ '; Nabel et al., Human Gene Therapv, 3:649 (1992) (lack of L i ~y and gonadal
1 - ~ by gene transfer in vivo with DNAllipid c~ .' s!; San et al., Human Gene TheraPv, 4:781
(1993) (safety and nl lu~dl.ity of new cationic lipid mixture, DMRIEIDOPE, for human gene therapy).
As a result of the Nabel et al. study, Vical ' ~ alud proposed a ' L~..lel clinical trial using
an improved cationic lipid mixture, DMRIEIDOPE. Vog ' _ et al., Human Gene Therapv 5:1357 (1994);
Hersh et al., Human Gene TherapY 5:1371 (1994); and Rubin et al., Human Gene TheraPY 5:1385 (1994).
The Food and Drug A~' Id6uil (FDA) has aui- i~d these clinical p.ul
The FDA also recently allowed Vical 'c r aL d to conduct a clinical protocol using lipid mediated
transfer of a cytokine encoding gene into tumors for ll~allll~lll of lil y. See Example 8. In ~,._ '
studies, the i..l, injection of a plasmid DNA ~AIJI ' vector ~ ~ ~ _ the human ;..1~l; ' 2 (IL-2)
gene reduced the incidence of tumor f. Illai and slowed tumor growth. By local e~ of ~,y~l ' s
at the site of the tumor, it is _.. ; ~ that lower levels of cytokines will be required for efficacy as
L . ud to systemic - ' , and that these levels will be ~ur~ low to avoid r ~ ~ ~ _ toxicity
CA 02214029 1997-08-27
W O96/34109 PCT~US96/05035
-2-
in the patient. The findings suggest that i..ll--' liO.. of IL-2 into a tumor, by il",all...,or injection of plasmid
DNA t:A~llU ' vectors, can stimulate an, : response. In the proposed trial, this approach will be
applied to human patients with solid ~ I tumors, using a plasmid DNA that encodes the human IL-2
protein and a DMRIEIDOPE cationic lipid mixture.
The w; ' , ~ad utility of a pul~llULIt:ui ' '!i~ ~ ' approach has been eai ' ' ' ~ from studies showing
cationic lipid de, ' - delivery of DNA (Felgner et al., Proc. Natl Acad. Sci. U.S.A., 84:7413 (1987); Felgner,
P.L., Adv. Druq Delivery Rev., 5:167 (1990); Felgner et al., Nature, 337:387 (1989); Brigham et al., Am. J.
Respir. Cell. Mol. Biol., 1:95 (1989); Muller et al., DNA Cell Biol., 9:221 (1990); Burger et al., Proc. Natl.
Acad. Sci. U.S.A., 89:2145 (1992)), mRNA (Weiss et al" J. Virol., 63:5310 (1989); Malone et al., Proc. Natl.
Acad. Sci. U.S.A., 86:6077 (1989)), and antisense oligomers (Chiang et al., J. Biol. Chem., 266:18162
(1991); Bennett et al., Mol. Phdll~dLoL~ 41:1023 (1992)) into living cells. Since the initial published
di , i- in 1987, several reagents have become u , 'l~ available (Behr et al., Proc. Natl. Acad. Sci.
U.S.A., 86:6982 (1989); Rose et al., e i ' ~ 10:520 (1991); Leventis et al., Biochim BioPhys. Acta.,
1023:124 (1990)), and additional cationic lipid reagents have been described reporting ad~ relative
to the u;dl products (Farhood et al., Biochim BioPhYs. Acta., 1111:239 (1992); Gao et al., Biochem
BioPhYs. Res. Commum., 179:280 (1991); Legendre et al., FhallllacL..IiLal Res., 9:1235 (1992); Zhou et al.,
Biochim. BioPhvs. Acta., 1065:8 (1991); r; - ~ . e et al., Biochim. Biophvs. Acta., 986:33 (1989)). The
broad 3~ of this approach has been further e: '' ' ' in ,~I~ ' ~ ' in vivo studies showing cationic
gene delivery to talh~L~HL~d blood vessels (Nabel et al., Proc. Natl. Acad. Sci. U.S.A.,
89:5157 (1992); Lim et al., Circulation, 83:2007 (1991); Yao et al., Proc. Natl. Acad. Sci. U.S.A., 88:8101
(1991); Nabel et al., Science, 249:1285 (1990)), lung epithelial cells (Stribling et al., Proc. Natl. Acad. Sci.
U.S.A., 89:11277 (1992); Brigham et al., Am. J. Med. Sci., 298:278 (1989); Yoshimura et al., Nucleic Acids
~" 20:3283 (1992)), brain tissue (Jiao et al., Exp. Neurol., 115:400 (1992); Ono et al., Neurosci. Lett.,
117:259 (1990)), Xenopus embryos (U ~ et al., Int. J. Dev. Biol., 35:481 (1991); Holt et al., Neuron,
4:203 (1990)), and the syâtemic ~ ' (Zhu et al., Science, 261:209 (1993); Philip et al., J. Biol. Chem.,
268:16087 (1993)).
Yet, l~" ' ~ of plasmid DNA for i' , purposes requires that a "ha, ~c 11~ aL-.opi ' '
vehicle be found in which the DNA can be taken from the ~-.llJHIl9 site to the clinical site with a
viable interim shelf-life. Although buffers c i ~ ~ " tris-(h~d~uA~ ' (Tris),
usually ~ - ~ ~, chelating agents, are r N, used to handle plaâmid DNA in the research ' ' dllJIy,
these buffers are ' i,~d for P~IILIIl~l~l use in the clinic. In their absence, a vehicle must be identified
that provides for efficient handling of the plasmid DNA in the, 'a.,lL,i ~, setting, preserves the chemical
and biological integrity of the plasmid DNA during shipping and storing, and allows efficient delivery of the
DNA to the desired tissue target by the preferred route of ~ .dliun.
The inclusion of cationic lipids in a r., ' liu.. with plasmid DNA illll.d~ ad ' ' ~ y
into the choice of a suitable vehicle for phall ~~ ' use. In this case, the solubility and stability of the
CA 02214029 1997-08-27
W O96/34109 PCTrUS96/05035
-3-
individual o~pc- ly charged c r ' and the - , ' they form must be ~vvv -~ ' in a single
medium. If ~lulai and ,ult~ dliun of the plasmid DNAlcationic lipid r ,' - is s~ ' Ld to be
essential for the rl : v of the product, it is , dli~r, that the vehicle chosen for 1 ' lllaceuliL~dl use
not interfere with this dLi-
The stability of the product can be enhanced by frozen storage, which imposes ~ lli.. i s
on the choice of vehicle for mixtures of plasmid DNA and cationic lipid. In this instance, the stability of the
r ,' ~ formed between plasmid DNA and lipid must be rJ~ ' Ld in addition to that of the individual
S r- IS. Sp61,ir- -~ly~ the fG, ' - must be designed so as to preserve the solubility and integrity of
the plasmid DNAllipid -- ,' -- over the course of storage at the frozen ~ , dlL.~.
The Nabel et al. initial work in human patients P ~ :Idl~d a 1l, ~ ~",enl for administration of
a ~t ' of plasmid DNA with cationic lipids for ~ce 'ul Lld"~reLliu" of tumor cells. SuPra. Indeed,
many in vivo gene therapy c,,' ~ - have shown a 1~, 1,...~..l for _~ Idli~a of a mixture of DNA
and lipid for efficacy. In these ,u,ui S7~. the gene therapy dose has been provided to the clinical site in a
multiple vial r 'ig, - because the plasmid DNAlcationic lipid ,' s are not stable. In this
: '~ di- n, the plasmid DNA in vehicle is present in one vial. The lipid mixture is present in a another,
separate vial either as a dried film or in solution. When the lipid mixture is provided in dried form, still
another vial c - v the diluent for ~ J~di . of the lipid is provided to the clinic.
With this s 'iv of product, the clinic staff must prepare the patient dose at the site by
r ~Ull ~ ~ several , : ' dilution and addition steps. First, the clinic staff must obtain the DNA in
vehicle. Second, they must obtain the lipid. Third, if it is a dried film, which event is likely because of
stability issues, they must rehydrate the lipid with a ~u, ': buffer by vortexing until h-
Fourth, they must transfer the r. : ~ lipid into the DNA solution and mix. Finally, fifth, they must
the amount to the patient. Optionally, the clinic staff may prepare dilutions of DNAllipid
C~for the r~ : . of eec~ doses.
Although this type of s ';., dliOII is accl:,ui '' for Phase I clinical trials, it is - ' . - to
'dl,lUI~, difficult to manage in s '~ Illily with 1. ' ~ Y v ' ' s, costly to ship and store, and
-,' d for clinic staff and ,JhJa;";d", to use. ' pr lallll~ there is no control or _ a""e of
c: y in, ~, dliUn of the final product, which is the dose 1~ ~ I,d to the patient. These
~- liO-Is clearly Pl ~dle the need for a plasmid DNAlcationic lipid ~ Jl r, ~ lr for human
clinical use.
Ar- . ~'~, it is an object of the invention to provide a . "' .;~1 rur ' - in which DNA and
lipid are combined in a ,' ",ac~ aLG~"; '' vehicle.
It is another object to provide a . ~ "' viol f".l ' - that retains the potency of the DNA and lipid.
It is a further object to provide a single vial lu,l ': that enjoys stability over a time course of
storage.
It is still another object to provide a single vial ~. I ' that is universal in 3~,' ~-
CA 02214029 1997-08-27
W O96/34109 PCTrUS96/05035
-4-
lt is yet another object to provide a single-vial ft" ' that is safe as shown by suitable safety
studies.
It is an additional object to provide a single-vial fo" ' i 1 that is easy for a clinic staff to prepare
and a physician to - '
These and other objects of the invention will be apparent to the ordinary artisan upon 't dliu
of the a,uel,;ri~ai as a whole.
SUMMARY OF THE INVENTION
The invention provides a process for making a - v .;al fu,l ' i of poly,' i'~l1i, '
in a 1' 11~ a, p~ '' vehicle for human clinical use in vivo or ex vivo ~.UIII~UUa;llu the
steps of: (a) sterilizing a lipid solution; (b) sterilizing a r~H~ ~ liJe solution; (c) ' _ the sterilized
p~l~,luoluul;de solution of step (b) with the sterilized lipid solution of step (a), in dilute form, at an ionic
strength that is lower than isotonicity, to form p~1~, ' i ' '!i, ' c ,' and (d) adjusting the
p~'~ ' li'e.'li, ' c .' - of step (c) to near ;aui ' '~y.
The invention further provides a process for making a _' .;JI rG~ ' t;"" of plasmid DNAlcationic
lipid c . ' for human clinical use . i the steps of: (a) autoclave sterilizing a cationic lipid solution
at high c di' , (b) diluting the sterilized cationic lipid solution of step (a); (c) filter sterilizing a plasmid
DNA solution; (d) adding the sterilized plasmid DNA solution of step (c) to the diluted sterilized cationic lipid
solution of step (b) at an ionic strength that is lower than ;aui ' '~ to form DNAllipid .t .' ;, and (e)
adjusting the DNAllipid c~ .' ~ of step (d) to near ;aui ' 'ty.
The invsntion also provides a process for making a single-vial ru" j'~r of plasmid DNAlcationic
lipid -- . ' ~ for human clinical use c . i g the steps of: (a) autoclave sterilizing a cationic lipid solution
ata Cll r l~dliunsurri~ highto ' ~ preventlipid~dddl;u~duringtheautoclaveùl~," liu~,
(b) diluting the sterilized cationic lipid solution of step (a) to a degree sufficient to ~ b~ prevent lipid
e_ ._ during step (d) below; (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid
DNA solution of step (c) to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that
is lower than ;aui ' 'ty to form DNAllipid c ,' and (e) adjusting the DNAllipid r ,' of step (d)
to near iaui ' '~y.
The invention moreover provides a process for making a _' . ' ~r I ': of plasmidDNAlcationic lipid - , ' for human clinical use - , i _ the steps of: (a) autoclave sterilizing a cationic
lipid solution having a toll~ " . in the range of from about 0.5 to about 5.0 M; (b) diluting the sterilized
cationic lipid solution of step (a) with a diluent to achieve a CUIII,I,..Ildi' in the range of from about 0.01
to about 1.0 M; (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution
of step (c), having a ~ lldLiun in the range of from about 0.05 to about 10 mglmL, to the diluted
sterilized cationic lipid solution of step (b) at an ionic strength that is lower than iaul ' '~y to form DNAllipid
- ,' and (e) adjusting the DNAllipid c ,' of step (d) to near ;au~ ' "y.
= =
CA 02214029 1997-08-27
WO96134109 PCTIUS96/05035
-5-
The invention ?''~ y provides a process for making a single-vial f~", ' - of plasmid
DNAlcationic lipid cl , ' ~ for human clinical use c , v the steps of: (a) ~ ~ sterilizing a cationic
lipid solution of DMRIEIDOPE, having a molar ratio in the range of from about 90:10 to about 10:90, and
having a rs lldlion in the range of from about 2 to about 10 mg DMRlElmL; (b) diluting the sterilized
cationic lipid solution of step (a) with a diluent to achieve a CUllbclllldi - of < about 2 mg DMRlElmL; (c)
filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c), having a
concb.,l, of < about 10 mg plasmid DNAlmL, to the diluted sterilized cationic lipid solution of step (b)
at an ionic strength that is lower than ;aût ~ 'y to form DNAllipid - ' s at a mass ratio of from about
50:1 to about 1:10 DNA to DMRIE; and (e) adjusting the DNAllipid c , ' s of step (d) to near ;so
with sodium chloride.
The invention Pb,ll,c" c provides a process for making a _'~ .;al fl Illu6liun of plasmid
DNAlcationic lipid - ' for human clinical use l ,;.;.., the steps of: (a) . ~obla~,c sterilizing a cationic
lipid solution of DMRIEIDOPE, having a molar ratio of about 50:50, and having a con~b..l,di of about 8
mg DMRlElmL; (b) diluting the sterilized cationic lipid solution of step (a) with a diluent to achieve a
cs 'IdtiU~ of < about 1 mg DMRlElmL; (c) filter sterilizing a plasmid DNA solution; (d) adding the
sterilized plasmid DNA solution of step (c), having a cûnc~..lldliùn of < about 5 mg plasmid DNAlmL, to the
diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than ;aù~ ~ 'y to form
DNAllipid cl , ' at a mass ratio of about 5:1 DNA to DMRIE; and (e) adjusting the DNAllipid complexes
of step (d) to near ph~ ' ' salinity.
The: ~.,.. i . as well, provides a process for making a '~ d flJllllbdai of plasmid
DNAlcationic lipid ' in about 0.9% sodium chloride with about 1% glycerol and about 0.01 YO Vitamin
E for human clinical use r ia;..g the steps of: (a) autoclave sterilizing a cationic lipid solution of
DMRIEIDOPE, having a molar ratio of about 50:50 molar ratio, and having a r ', " of about 8 mg
DMRlElmL; (b) diluting the sterilized cationic lipid solution of step (a) with a diluent to achieve a c~ ~,dliun
of < about 1 mg D'' ,'EI L, and with glycerol and Vitamin E; (c) filter sterilizing a plasmid DNA solution;
(d) adding the sterilized plasmid DNA solution of step (c), having a r - di' of < about 5 mg plasmid
DNAlmL, to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than
iaui ' ~y to form DNAllipid ~ at a mass ratio of about 5:1 DNA to DMRIE; and (e) adjusting the
DNAllipid cl ,' of step (d) to about 0.9% sodium chloride, wherein the final CU~bL..II_ of glycerol
is about 1% and of Vitamin E is about 0.01%.
According to other e ' ' . the invention provides - 'l viol fo~ r of plasmid
DNAlcationic lipid ' for human clinical use prepared by any of the above, ucesses.
In another ' - " 1, the invention provides a _' .;al rb~ of plasmid DNAlcationic lipid
L ,' for human clinical use - i _ a cationic lipid s and a plasmid DNA c~ 1,
wherein the plasmid DNA ~r L and the cationic lipid r , ' are combined at an ionic strength that
is lower than ;aui ' '~y to form the plasmid DNAlcationic lipid c , ' s, and further c , _ a nearly
CA 02214029 1997-08-27
W O96/34109 PCT~US96/05035
-6
isotonic aqueous medium, optionally LIJ~ no buffering agent except the plasmid DNA itself. The single-
vial fl liuil may be stable in frozen, IGrliy_~dlGd~ room IGIII,UGI G, or body IG---uG-dlL.G form. The
single-vial D . It, may be stable in frozen, IGrliyGldl~d, or room tGIll~Jeldi G form for at least about 8
weeks. The single-vial rl Illl~lai ~ may further comprise about 1% glycerol and about 0.01% Vitamin E in
nearly ,JI-, ~ ' v ' saline.
In still another: l 1, the invention provides a ~ ' .;.,l rul ' for human clinical use
c" i;.;..J plasmid DNAlcationic lipid ~ ' having storage stability in frozen, IGDi~GldlGd, or room
di G form for at least about 8 weeks.
in yet another ' ' l, the invention provides a . "' .;"l ru, j-~r for human clinical use
10 , ia;l,g plasmid DNAlcationic lipid ~ ~, ' retaining in vitro ~1 ~GI.liU~ efficacy of freshly prepared
plasmid DNAlcationic lipid - ,' s for at least about 8 weeks.
DETAILED DESCRIPTION OF THE INVENTION
The objects of the invention are achieved by col.ll~" ' ' ~ of a plasmid DNA solution with
a cationic lipid solution, ~J~GrGI ' 1~ in dilute form, at an ionic strength that is lower than ;au ~ t~" to obtain
15 plasmid DNAlcationic lipid ~ ' s, which, when admixed with solutes to generate iaui ~ ~" provide
_' .;al fG. ' :- suitable for human clinical use.
We have d;~-,..._.Gd that the order in which individual plasmid DNA and cationic lipid
are mixed to produce plasmid DNAlcationic lipid c ,' - is ~, i I to the final results, as is the ionic
strength at the time of initial complex ru.l :- Optimal in vitro 11_ CLLi- GrR~iGll~y and ,UI~.G of
20 lipid ~ dlion have been obtained by slow addition of a plasmid DNA solution to a cationic lipid solution
at an ionic strength that is lower than i~u; ~ ty. In this scheme, we have optimized in vitro 11 C~.,liu.~
Gfri ~ y and r.~.G..liui) of lipid ag~ - ~ even further by using the cationic lipid ~ , in a dilute
form upon its - ' ~ : - with the plasmid DNA c pc : to form the DNAllipid ' ~r
We have also found that alG.i' ~- of lipid mixtures by filtration c ~ ~ s their integrity. In
25 contrast, we have ': ~ ' that our lipid mixtures can survive alG-' i by autoclave IrGdl.. ~.. l.
Maximum in vitro lld~aOG~i Grri ~ y and ~.u.. of lipid d~.allai have been produced by autoclave
alG.i' i In this scenario,in vitro ll~..;.r. efficiency is further ~ ~ ' and lipid ' _ adai- further
.~..L~d by ~ - - - ~ the lipid mixtures at higher c~ during autoclave alG. ' ~-
Preferred single-vial r. . ' of the invention have dCCuld "~', been obtained by preparing
30 cationic lipid mixtures c ~ ~ _ a cationic lipid cs~ and a neutral lipid ~ t, having a molar
ratio in the range of from about 90:10 to about 10:90, ~ulGOGl.lbly about 50:50. A cationic lipid solution is
prepared from the cationic lipid mixture by hydrating a dried lipid film with a suitable diluent",.GOGI.Jbl~ using
water, to obtain a solution in highly cûnc~.llldluJ form. The cationic lipid solution is cc..i~idGIGd to be highly
~ : dlcd by having a ~r ll ~- in the range of from about 0.5 to about 5.0 M. This highly
35 c dlGd cationic lipid solution is s-~l-s~ subjected to al~li' ~- - by standard autoclave IIGdi 1,
e.~., 30 minutes at 121~C, in the usual way.
CA 02214029 1997-08-27
WO96/34109 PCTrUS96/05035
-7-
Al~ aL._I~, a cationic lipid solution can be prepared and sterilized by filtration or i"_ ~D or
other d,u,ulI, ial~ means at any desired cc c ~ :lai the higher c l~aiiar, of cationic lipid solution being
ad~, i g for protecting the material during sterilization by aulûL.ia~,~, llealllll:lll.
- Upon obtaining a highly c llal~d cationic lipid solution, one p.~r~l.. hly dilutes the solution,
,u~r~,_"y using sterilized water I~L, sterile WFI). Other suitable diluents, such as salines, can be
lud for water, and are selected on the basis of having low ionic strength, that is, lower than
i~u; ~y. The cationic lipid solution is diluted to achieve a c : a ~ in the range of from about 0.01
to about 1.0 M. Ori 'I~, at this point, suitable r. Iu.y agents may be illllu~ d, as di~ d below,
pa, li.,~da;l~ to facilitate the , of DNA and lipid during the cc ' liun step (~).
Plasmid DNA, which is p'~ ~ ' 9. ' in quality, is I . ' ' obtained. The plasmid DNA,
in aqueous solution, such as water (~k, sterile WFI) or other a,, u,ulial~ diluent, such as saline (~Ek,
, by ' -' saline), is sterilized. Sterilization is ~,~r~ by filtration, for instance, through a 0.2 ~m
filter. It is preferred that the diluent have an ionic strength that is no higher than ;au ~ y.
The sterilized plasmid DNA solution and diluted sterilized cationic lipid solution are usually brought
to room I di ~ prior to - ' ' The DNA solution may be used at crz l,_ - within a range
that extends from about 0.05 to about 10 mglmL. The solutions are then ' d, ~ rel "~, by c , " d
addition of the plasmid DNA solution to the cationic lipid solution at low ionic strength with mixing to form
DNAllipid ~ . ' - By low ionic strength is meant lower than ;~ui ~y. C7 mixing of the lipid
solution and plasmid DNA solution as they are combined is preferred, with a further short period of mixing
after the c ' i is complete. The mixing may be achieved by l,.,.i _ manual agitation (shaking),
__L ~ ~ mixing or stirring, or other suitable means.
DNA and lipid are combined to produce - ' - at a mass ratio that is optimal for Ll fc:
~rr- ~y, as evaluated, for example, by in vitro 11_ fu: assays.
The L , ' are b~ adjusted with a tonicifier to ; ,ui ~y for, ' y ' _
e' .~ For example, adequate sterile sodium chloride stock may be added to give a final
Jrc 1_ of about 0.9% NaCI (j~, ,' y ' g ' saline). The final ru. '; ~. can next be a-qpt- 'Iy
filled into sterile ', ,-~_ lud vials, and can then be stored frozen at about 10~C to -70~C,, ~r~
about -20~C.
At the clinic site, the DNAllipid vials are c( .~ thawed and typically mixed (e.a., by vortexing
or manual agitation, i_, shaking). They can be ~ ~ at room i alL.~, and are -' ~d
2d~, ~_ '~ within 24 hours of thawing. Each vial may deliver DNA in unit dosage form, or, all~luali.
may cu.. ,lilule a multidose container.
For use by the, ' ~ ~ . the ' .;..l ru~ ' i - are provided in cc l,_: up to about 10
mg DNAlmL, ,u,~ru. "~ up to about 0.5 mg DNAlmL. As will be r~ c _ ~ ' by those in the field, an effective
amount of DNA will vary with many factors, including the condition being treated, the chala~ liL.s of the
CA 022l4029 l997-08-27
W O96/34109 PCT~US96/05035
-8-
patient, and other factors. Typical doses will contain from about 5 mg to about 10 ~L~g DNA, although wide
va.idliu,,a from this range are possible while yet achieving useful results.
Other preferred single-vial iOI ' liùns of the invention include pll~ v 'l~ aGc~"i '' ~ . ~
as called for by accepted ,' ~~ac~li"dlpractice. These may ~ buffers, a: ' ~ amino acids,
- ' 'i~la, starches, sugars, ' ' " a, rdCi la, , ~' _ agents, t( ';cla, wetting agents, etc.
It has been found that the single-vial rO, provided herein maintain pl~y ' l ' pH without the use
of buffering agents except the plasmid DNA itself While additional buffering agents may be omitted, they
may dll~.-.dli.~ly be added as occasion provides
Preferred r. . F - are suitable for ~ that is pa,~"ld~dl, that is, by any means other
than oral. Pa.~ -dl - .dliul~ includes ; : s, such as i l-u~.-uv~ llddll~,idl~ i--ll
. ' - ' _, ;~1- ' I l i--ll . ilUIledl~ iull_' ~~, and i-~ lalilial '; ' 5, infusions, and by inhalation.
Injections include -' : dOUn through " '~y,i _ and catheters.
In pdli' ' 1'~ preferred - b~" Is, the invention provides v~ l r-,. ~ having final
c~ dlions of 0.9% sodium chloride l~"Jh~ ' ' saline) for iau~ y, 1% glycerol as an e,-,lairi~,
(and a ".yl, ot~ , and 0.01% Vitamin E as a IJ~:a~vdlN~ (and an ' ~).
CATIONIC LIPIDS
Cationic lipid reagents that are in use today for DNA 1. '~6UII are r~ d as lipid vesicles
or li, - - cu,.i _ cationic or positively charged lipids in r ' ' " with other lipids. The rl ':
may be prepared from a mixture of positively charged lipids, _ ';..,l~ charged lipids, neutral lipids, and
20 ' ' ul or a similar sterol.
The positively charged lipid can be one of the cationic lipids, such as DMRIE, described in U.S.
Patent No. 5,Z64,618, or one of the cationic lipids DOTMA, DOTAP, or ~ ' - thereof, or a -
of these. DMRIE is 1,2d 11ialylUAy,Ulu,uyl-3-dimethylh~dlU~y~lh11- ' bromide, and is preferred
See Felgner et al., J. Biol. Chem. ~f;9-7!;50(19941. DMRIE can be Sylli' - ' according to Example 1.
Neutral and r _ ';.al~ charged lipids can be any of the natural or synthetic I ' s, b 'i, ~ ' or mono-,
di-, or llial,ylylyi,~i.ula. The natural ~h- ,'-1i, '- may be derived from animal and plant sources, such as
'y ~ V ~ 'yl~: ~ e, i, _ ~ ~ P , i ~la~li or r ~ ~
Synthetic l'-~,' 1i, ' may be those having identical fatty acid groups, including, but not limited to,
dimyristoylphospha~tidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,
" uy\p~rS,' il~;'-' - and the r l~_r ~'V synthetic rh- .' i 1~ ! and
,' ,' lidylgl~ .v!s. The neutral lipid can be ,' ,' ~' '~' e, Cdll" ~i~', rhc~ di' ' ' e,
mono-, di- or IOd~.yl~ ll ' or ~-- ' v thereof, such as " ' ~1, ' , ' i 1~1~i' -' (DOPE), which
is preferred. DOPE can be ~ ul-dsed from Avanti Polar Lipids (Alabaster, Ala). The ll~ydli.~l~ charged lipid
can be .' ,' 1y!91~elul, ,'- .' " acid or a similar rh~ .' li~'' analog. Other additives such as
35 .' ' : ul,yl~ ' fatty acids,, ' v li, 's, p-- v' " 5, 9 v' ' neobee, - orany other
CA 02214029 1997-08-27
W O96/34109 = PCTrUS96/0503
9.
natural or synthetic , ' er' ' ~ can also be used in liposome ru",.u6liun~, as is c~r,. ..i 'Iy known for
the ".,, . - of li, s
In a L pC "' for preparing cationic 'i, -~ s, the cationic lipid can be present at a I Jr lldi
- of between about 0.1 mole % and 100 mole Y~, Lr~rb~l3r'1~ 5 to 100 mole %, and most, ~rb~ bl~ between
20 and 100 mole % The neutral lipid can be present in a CUnC~:Illldliua of between about 0 and 99.9 mole
- %, ~.~rb~ , O to 95 mole %, and most ~JIbrbl_~y 0 to 80 mole %. In order to produce lipid vesicles or
'i, having a net positive charge, the quantity of the positively charged component must exceed that
of the r,bsdl;.~ly charged ~ , Jr 1. The "?li._l~ charged lipid can be present at between about O to
49 mole % and, brbl~.bly O to 40 mole %. Ch ' ul or a similar sterol can be present at 0 to 80 mole
%, and, brb~ O to 50 mole %.
Lipid ~ r having at least one amphipathic lipid can ;, i3. ~ assemble to form
'i,-s Lipid reagents having a cationic lipid species can be prepared as cationic 'i,-- s The
- pr I lipids can be dissolved in a solvent such as r' ' Ulu~lll. The mixture can be _., dlbd to dryness
as a film on the inner surface of a glass vial. On ~ in an aqueous solvent, the --n,' pc ' Iipid
-' ' will assemble ih M,s into 'i, . See Example 2.
The 'i, - - can be analyzed for potency by in vitro lla--;~lL - assays. In these assays, plasmid
DNA and cationic lipid - ,' - are formed by mixing of the two sb,ualdlul~ diluted r r ' The
mixture is then added to cells in culture and 1- Ct:b assessed according to the I .re' b of Example
3.
PLASMID DNAs
The plasmid DNA required for 'i, ' mc~ tPd gene transfer has been widely and routinely prepared
in the ' ' dlulr~ for many years. S ' ~.ok, Fritsch, and Maniatis, Ml' ' Clonin~: a LaLûldl Y Manual,
2nd ed., Cold Spring Harbor LaLu,dluly Press, New York, 1989.
These plasmids aLcul J -~ can be selected from among p-.' ~UIib and: ' yUIib vectors, pBR322-
and pUC based vectors, and their deli.dli._~, etc. They can utilize any of various origins of ,., " for
instance, prokaryotic origins of ~I,' i . such as pMB1 and ColE1, and ' ~rul;b origins of ~I,' .
such as those r " ~".. " in yeast, fungi, insect, and ' cells (~, SW0 ori). They can
GIdle any of . genetic elements to facilitate cloning and bA~UI ' n, such as ir' r '' genes,
~G~ Lbl~ leaderpeptide , er,,introns,l~ ' i Pl " ~,Kozaksey- -- ~s.P~'lraJ_.. ~Iai signals, 1~ i,ui l~ , 5' UTRs, 3' UTRs, etc. The selection of vectors, origins,
and genetic elements will vary based on ", and is well within the skill of workers in this art.
Genes encoding any of diverse ~lll al proteins (or peptides, p~ s. "1~cn, ui
.' r Ui ' S, amidated proteins, etc.) can be inserted into the plasmids for delivery into cells. These
genes may rs : genomic DNA, cDNA, synthetic DNA, Fe'~ ~ _liJb~ r~ -liJd, etc.
Transfer of mRNA, antisense oligomers, and triple helix agents are also expressly r .' ~ as falling
within the scope of the present invention. These ., -e~ may be obtained using chemical synthesis or
CA 02214029 1997-08-27
W O96t34109 PCTrUS96/05035
-10
gene . ' i t-~ , They can be inserted into plasmids, and the plasmids ' , ll~ lJJùbLd
into host cells for p~pal, :iun.
Host cells can be selected from among prokaryotes and t~ dl yUII:S, including bacterial, yeast, fungi,
insect and ' cells. Preferred hosts are bacteria, such as E. coli. Any suitable strain of E. coli is
-or j : ~
Plùpagdi - of plasmid DNA~ i ~, hosts can be carried out using known IJIu~,eaa~:5. Such
p .~ ~ may utilize b~lola, ' ~ ,lu~a, lu.l..~ ,.a, etc., according to batch fe~....ldl fed batch
f~ll IdOul1, c..,.; ~ culture, Type 1, Il, and lll ~" Ldliull, aseptic ~l Idliun, uu~laOI i
f~" ~ n, protected ~., : . etc. Fitting the c ' (~, medium, i dllJI~:, pH, hours,
agitation, aeration, etc.) for ~.(n~ _ to the Lb._ : :CS is empirical and well within the skill of those
in the art.
r".i~i ~r of plasmid DNA to ~ ,acL..li-,al grade quality may proceed using well 6ai '' '
p,. Allel"dl;..,ly, ~Jlucesads for ,~ iun of I ' ,,,aceuli~.dlgrade plasmid DNA disclosed in Example
8 are preferred. A~ for reducing RNA ~ ~ di- in cell Iysates using didi _~
earth materials also according to Example 8 are also preferred in purifying plasmid DNA to phz.",ac"~.li"al
grade aldndd.da.
REGULATORY PRQCFSSEC
Gene therapy requires approvals from several different " ': y agencies in the United States,
including the Food and Drug A~' ~.dliu.l (FDA). The FDA oversees and regulates inter alia that medical
drugs are safe and L~R"d";uua and how they are '~Li ud (~L., GMP). Similar approvals are required
by most foreign countries. The . _'l .; ' fo" ' i of the invention comply with these r,_ ' y
r. .
APPLICATION
In one i,, ' : DMRIEIDOPE cationic lipid mixtures are prepared at from about 90:10 to about
10:90 molar ratio,, e~ at about 50:50 molar ratio. A cationic lipid solution is prepared from the
cationic lipid mixture by hydrating a dried lipid film with a suitable diluent, I e~el hl~ with water, at ~
about 2 mg DMRlElmL, I ~, hl~ at about 2 to about 10 mg DMRlElmL, most ~ at about 8 mg
DMRlElmL. This highly L lalud cationic lipid solution is ~ subjected to sl~,i' i - by
standard autoclave ll~,i 1, e.q., 30 minutes at 121 ~C, in the usual way.
Upon obtaining an ' ~d highly ~ lldlLd DMRIEIDOPE lipid solution, one then dilutes the
solution, I Lre~, ' 1y with sterilized water (e.~.. sterile WFI), to < about 2 mg DMRlElmL, p,~ to <
about 1 mg DMRlElmL, most I ~ to < 0.2 mg G'' I I ' Other suitable diluents, such as salines,
can be ': ' for water, and are selected on the basis of having low ionic strength, that is, lower than
iaui ~ ~y. Optionally, at this point, suitable 1~, ' y agents may be h,l,.' d, zd~dlll _ 'y by
addition into the diluent, for , l d into the cationic lipid solution. For example,; ' fiLla
may be bllllld~ -d,, ~ glycerol, in amounts to provide a final r~ - ~.dliUil in the ' .;il
CA 02214029 1997-08-27
WO96134109 PCTrUS96/05035
1 1 --
~u" ': - of from about 0.5 to about 5% glycerol,, ~re~ ~ 'y 1% glycerol. A ' ' ~ ,, t~e,va~ s may
also be added at this time,, ~ Vitamin E, in amounts to give a final c~ - ai' in the ' '~ .; '
f....~da ~ of from about 0.005 to about 0.05% Vitamin E,, ~r~lably about 0.01% Vitamin E.
- Plasmid DNA that has been purified to ,' UlaLL.. ~ ur~l quality is ~ ' ' obtained. A
process for the ~ iun of ,uI,a,,,,àLL~LiLdl yrade plasmid DNA is provided in Example 4. The plasmid DNA,
- in aqueous solution, such as water (~L, sterile WFI) or other ., r~r iale diluent, such as saline (~,
,' ~ ' -' saline), is sterilized. Sl~, ' li_a is pl~,ably by filtration, for instance, through a 0.2 ~m
' _ filter. It is preferred that the diluent have an ionic strength that is no higher than ;sui~ ~ ~y.
The sterilized plasmid DNA solution and diluted sterilized DMRIEIDOPE lipid solution are usually
broughttoroomI~ IdlUI~priorto c ' - TheDNAmaybeusedat c ll nrwithinarange
that extends from c dliui,sthat are quite high, for example, 10 mglmL, to r - llai - that are quite
low, for example, 0.02 mglml. The solutions are then s ' I, I ~f~, ''y, by LOIllll'' ' addition of the
plasmid DNA solution to the DMRIEIDOPE solution with mixing to form DNAllipid ~ , ' at low ionic
strength. Low ionic strength means lower than ;au' ~ ~y.
DNA and lipid are combined to produce -( , ' s at a mass ratio of from about 50:1 to about 1:10
DNA to DMRIE,, Lr~.dbly at a mass ratio of from about 10:1 to about 1:5 DNA to DMRIE, most, ~, ' 't
at a mass ratio of about 5:1 DNA to DMRIE.
S ' , 'y, the; , ' are adjusted with solutes to iaui ~ y for, ' ~. ' ' )y' ' ~ , d lr
In pal i' ' Iy preferred bJ~ the invention provides _' .;al f~ ,uuldi having final
con~,~.. lld;' of 0.9% sodium chloride, 1% glycerol, and 0.01% Vitamin E.
UNIVERSALITY
S _' . ' fo, ' - of the invention have been ', 'l~ tested with several different plasmid
DNAs, for exampie, plasmids c -i _ reporter genes, for instance, luL;r~laae and 1~ gà6Llua;llase, and
plasmids Op~ldli..,ly encoding rJr't~,_,.lid~s suitable for human gene therapy, ~, HLA-B7 and human IL-2.
See Examples 5-10. A~'' 'I~, we have applied the present invention to numerous diverse cationic lipid
species and cationic lipid mixtures, for example, DMRIE, DMRIEIDOPE, DOSPA, DOSPAIDOPE, HP-DORIE, HP-
DORIEIDOPE, T-MU-DMRIE, T-MU-DMRIEIDOPE, T-MC-DMRIE, T-MC-DMRIEIDOPE, ~-Ser-DMRlE, ~-Ser-
DMRlElDOPE"~AE-DMRlE"t~AE-DMRlElDOPE, Arabinose-TU-T-DMRlE,Arabinose-TU-rDMRlElDOPE, C~ t~se-
TU-T-DMRIE, ~ rtose-TU-T-DMRlElDOPE, Glucose-TU-T-DMRlE, and Glucose-TU-T-DMRlElDOPE. These
results ' :~dle the ._.~ My of the present invention.
SAFETY
s- _' .; ' R ll~ulai of the invention - " DNAIDMRIE-DOPE at a mass ratio of 5:1 DNA
to cationic lipid in 0.9% sodium chloride with 1% glycerol and 0.01% Vitamin E ' ,aled no signs of
toxicity in acute .J._ - toxicity studies in mice, repeat dose safety studies in mice, and repeat dose
safety studies in cj~ 1~ monkeys.
STABILITY
CA 02214029 1997-08-27
W O96/34109 PCTrUS9610503
-12-
The above single-vial ru~luuldliul1s were found to be stable. A,,c~ dlt:d testing e.;' ~d full
stability over a time course of 8 weeks at 20~C"~Oiy~ldi , and room l~ e.dl.J.t:. This testing showed
full stability over a time course of one month at body i , dlU~. These studies d~ Idl~d the utility
of the ~ , L of the ru~llluldliull. Example 11.
HUMAN GENE THERAPY
The single-vial ru~ ~ lians of the invention are suitable for human clinical use in vivo ~Example 12-
13) or ex vivo (~ U.S. Patent No. 5,399,346 to Anderson et al. for "Gene Therapy").
Particular aspects of the invention may be more readily u-,.R:,~luod by reference to the following
,' which are intended to exemplify the invention, without limiting its scope to the particular
10 ~ ,''i~d P-bc'
EXAMPLE 1
PREPARATION OF 1.2-DIMYRISTYLOXYPROPYL-
3-DIMETHYL-HYDROXYETHYL AMMONIUM BROMIDE (DMRIE)
DMRIE was ;~y~LIl~ a;~ed using minor ~--GdiR-.di c of the, .r ' t: ~ ~r~ r d for the synthesis of
15 DOTMA (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987)). Thus, 3 :" II-y' 1,2-~.. , " '
was r ' d with myristyl mesylate - ,' ~ ~, basic catalysis to generate the c~ r " ~, diether.
S- ' , to ~ . I..yl . ' , iR~ai of this lipophilic amine, q alli~di' was effected by ll~d~
with 2 ! ' :' ~' at elevated; , L:l. The ' . : " , ' ~'ly purified product exhibited IR, TLC,
and elemental analyses ~ : with those predicted for the desired hyJIu~.y "~b M salt.
EXAMPLE 2
CATIONIC LIPOSOME PREPARATION
DMRIE was ~ylllh6a;~t:d accordiny to Example 1. DOPE was ,uululla;.~.l from Avanti Polar Lipids
(Alabaster, Ala). Cationic ~i, ~ were prepared by mixing a ~' ' urullll solution of the lipids in a sterile
glass flask. The solvent was removed by e. . , under reduced pressure to produce a dried lipid film.
25 Vials were placed under vacuum overnight to remove any solvent traces. The lipid mixture was hydrated by
addition of sterile water for injection.
EXAMPLE 3
IAI VITF~O TRAN; ttL; l lrJN PROTOCOLS
Plasmid DNAlcationic lipid ,' were prepared by mixing an aliquot of an pth,. ' ;ida
30 solution with an aliquot of a liposome solution at room i , di 1:. Different ratios of positively charged
- to F l~llull~ui ' ~ can be used to suit the need. The methods are a "fi~_: of those
described in Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987), and Felgner and Holm, Focus 11(2)
Spring, 1989.
Tl f~.; were carried out in 96-well plates, as follows:
35 (1) The wells of a 96-well ulil~. plate were seeded with 20,000 to 40,000 cells per well;
CA 022l4029 l997-08-27
W O96/34109 PCTrUS96/OSO35
-13-
(2) S; ' .;JI r., ~ - of plasmid DNAlcationic lipid ~ ' s were prepared in sterile vials
or tubes;
(3) Dilutions of the ' . ' fl,., ' liuns prepared~as in (2) were carried out by serial
d6UI~ into cell culture medium (with or without fetal calf serum) ~ :, ' l~d in 3~ ~ ~r ial~ volumes
in 96-well plates;
- (4) The cell culture medium covering the cells that had been seeded as in (1) and grown to
rll e was removed by ~, dliu.\,
(5) The cells were washed with an -'' ' volume of cell culture medium without fetal calf
serum, and the wash medium was removed by e, di- 1,
(6) A volume of the diluted plasmid DNAlcationic lipid complexes was added to the washed cells
in a well of the ulil~, plate; the volume 1, '~ d usually consisted of 80 to 100 ,uL;
(7) Adequate 50% fetal calf serum was added to each well to bring the cùnL~ dliull of serum
in the wells to 10%;
(8) The plates were b. ' at 37~C (5% CO2). At 12 and 24 hours post llall.rL~.; . an
aliquot of 10% serum in Opti-MEM~ reduced-serum media (GIBCO BRL Life T~ - ' ' y, MD)
was added to each well;
(9) At the end of the ' (usually 48 or 72 hours), the medium covering the cells or a whole
cell Iysate was assayed for ~AIJII ~ activity.
Where~-gd6,,i'- wasthereportergene,the-, I was ~d 1l i :,i 'Iy,using
-' ~ r~11 red~-5dla~(CPRG) as a substrate, reading the plates with a olil~ reader at
405 nm.
EXAMPLE 4
PROCESS FOR THE PURIFICATION OF
PHARMACEUTICAL-GRADE PLASMID DNA
Cell Lysis. A cell paste was ll r ~ in 6 mL per gram wet bacterial weight of cold
Solution 1(61 mM glucose + 25 mM Tris buffer pH 8.0 + 10 mM EDTA at 5~C) with stirring at room
alL.~. To this solution 12 mL per gram wet bacterial weight Solution 11(0.2 N NaOH + 1% SDS)
was added and mixed end-over-end until ~ ~9 This was ' ~ ' on wet ice for 3~ 10
minutes. To the Iysed cell solution, 9 mL per gram wet bacterial weight of cold Solution lll (3.0 M
30 p~rl acetate pH 5.0 at 5~C) was added, mixed end-over-end until a white r;
appeared, and ' - on wet ice for .".~"l ~ '~ 5 minutes.
Filtration. The cell debris was removed from the Iysate by filtration, LL~llliruy-: or s- '
The . llat was collected and clarified by adding 3" ~~ ILI~ 25 911 Celite~' :' iearth and
filtering through a ~, ~rer "~ pltl,oalLd) filter ' ~ (Whatman # 1, 113 or ~ ' :' arranged in a
table top Buchner funnel. All~lua~ , the cell debris was removed from the Iysate by direct Celite@3 aided
filtration. In this case, 3~, 1 ~ ILI~ 90 911 Celite'~' diai L earth was added directly to the Iysis
CA 02214029 1997-08-27
W O96/34109 PCTrUS96/05035
-14-
solution and mixed by swirling until ~1 - The Iysate was then filtered through a ~ r
,u,~cGal~d) filter ' a"a ~hài # 1, 113 or ~ ' t: arranged in a table top Buchner funnel.
DNA P~u, i rul~lh~l~"d glycol ~PEG, e.a., PEG 8000) was added to the filtrate to 5 ~5%
~wlv), plus NaCI to 0.3-1.5 M. The PEG s--~, was stirred I rr~aLly overnight at 2 8~C. The DNA
1 ~.,;,u;ldle was collected by adding ~ 25 gll of Celite~ did~ rDu ~ earth to the PEG S_-r - -
and filtering through a ~,u.er~.~,51~ ,u~r~,Odl~d) filter ' arranged in a table top Buchner funnel. The
DNA ,ulu~,;r; dl~ was captured in the Celite'~ cake and recovered by s--r ' ~, the cake in TE buffer ~0.01
M Tris base pH 8.0 + 0.001 M EDTA).
RNA, Protein and I i~ , v'~aaLLhdride Removal. ~ acetate was added to the TE buffer to
2.5 M and stirred for ~,, uAi~l~alal~ 30 minutes at 2-8~C. The , which still c ~~
die earth, was filtered through a ~JIrr~ ' Iy ~ oal~d) filter ' a..a arranged in a table top
Buchner funnel. The DNA filtrate was then optionally clarified by alJh ~ I filtration.
Final DNA P", A final DNA ~.", Iiu-- was prlrul ' with 0.6 volumes of cold~ , ~p, ' for a minimum of 2 hours at -20~C. The ,u., , I~d DNA was ~,~..lriruudd in a Sorvall table
top LL.. llirL_ for 30 minutes at Z000 x 9 or , .' I. The DNA pellets were Ir, ~ hd in column
buffer prior to gel filtration ' I i _ ~,' ,.
6el Filtration Chl~ lu~. . ' r. A rhalllld~;a S 1000 tandem size exclusion column, DNA exclusion
limit of 20,000 bp, ~Phdl ~ Pia-,dl~.. d~, NJ) was poured. The S 1000 matrix was an inert and highly
stable matrix that was prepared by co. ' ll~ cross linking allyl dextran with N,N' Illt:lh~, ' - ~; '
The column was poured in two rhdlu._~;a XK261100 columns ~PI.a.. aL;d, ria~,al~ ay, NJ) with a final bed
height of 80 85 cm ~7 ~y8orln) resulting in a total column volume of ,, uJd~dl~ly 900 mL and a total
length of d~,plu,.i...dlul~ 160 cm. The columns were 'i~;' 'l~ pressure packed in one direction, reversed
and c -: ' in series for:, '' di- and ~ , - The column was e, "' dl~d in column buffer and
run at an 3p~ u~JI;aL~ flow rate. Cleared Iysate plasmid DNA was filtered through a 0.2 ~m filter and loaded
onto the column. Column operation and rldbi ti were i l_d with a Pha.llld.dd FPLC ~Phd~l
ria-,dlL. a1, NJ). Fractions ~a,r u)dllldlal~ 0.5 5% of column volume) were collected over the product
elution zone and analyzed by 0.8% agarose gel ~IU~llUr~ ra;a. Arr.u,u.iale fractions were pooled and
, ., tud with 2 volumes of cold ethanol. This column purified DNA was stored frozen at 20~C until
needed for ~.L~Jdldliùn of bulk plasmid DNA.
Bulk Plasmid DNA r,l,,- dliUII. The ethanol r.r~ l, column purified DNA was spun at
maximum speed in a Sorvall table top C~.~tli6 ~ for 30 minutes at 4-10~C or ~ ' : The pellets were
air-dried and pooled. The pooled pellets were ,. , ' ' in vehicle, for example, sterile WFI or l.h~ ' '
saline. The DNA was then filtered through a 0.2 ,um filter into a pyrogen free container. Samples were
optionally taken for quality control testing and the ,. ' stored frozen at -10~C to -70~C" r,
at-20~C.
CA 02214029 1997-08-27
W O96/34109 PCTrUS96/05035
-15-
EXAMPLE 5
HLA-B71DMRIE DOPE COMPLEXES
HLA-B7 plasmid DNAIDMRIE-DOPE lipid c ,' . intended for use in human gene therapy,
- dl'~dlLdi77 vitro potency as - ~' ~ial rull ' l;uns of the invention. See Nabel et al., Human Gene
Therapv 3:399 (1992); Nabel et al., Proc. Natl. Acad. Sci. USA 90:11307 (1993); PCT Patent A~," i
WO# 94129469; Nabel et al., Human Gene TheraPy 5:57 (1994); Vogelzang et al., Human Gene TheraPy
5:1357 (1994); Hersh et al., Human Gene Therapv 5:1371 (1994); and Rubin et al., Human Gene Therapv
5:1385 (1994).
An HLA B7 encoding plasmid was c~ ~.u.,lcd of about 5000 bp in size. It derived from a pBR322-
based plasmid c a bacterial origin of r~ It encoded the heavy (human HLA B7 cDNA) and
light (~ r~-2 ,_' ' ' cDNA) chains of a Class 1 MHC antigen '~ ~, lLd HLA-B7. These two
proteins were ,A~ ;,aed on a bi cistronic mRNA. Cukalyuli,, cell t:A~ ;Un of this mRNA was :'~, ' l
on a Rous Sarcoma Virus (RSV) promoter sequence derived from the 3' Long Terminal Repeat (LTR).
'Y" was also J, ' on a llan~ ,liua ~ 'ya ~l ~lalion signal sequence derived from
the bovine growth hormone gene. CAIJ.U of the heavy chain was regulated by the 5' c~p d,
protein ll. ' liul. start site. EA~.~ of the light chain was regulated by a Cap ' d~ Tr
Enhancer (CITE) sequence derived from the '- ,'-' ,.,caldili, Virus. The plasmid also encoded a
k.. ,u;.. r~ .a~e gene derived from Tn903.
CA 02214029 1997-08-27
W O96/34109 PCTrUS96/05035
-16
EXAMPLE 6
PHARMACEUTICAL-GRADE PURIFIED HLA-B7 PLASMID DNA
The HLA-B7 encoding plasmid was purified to pha~ r~ ' grade - ' da as del~ ' by the
criteria given in Table 1 below.
TABLE 1: HLA-B7 PLASMID DNA QUALITY CONTROL CRITERIA
TEST SPEGlrlCATlON METHOD
Size Identity A~, uAilllal~a. Agarose Gel [I~"l",' l~a;a
4900 bp
Realli.. liun Sites A r u~dlll~l~s ~ ' Agarose Gel [~ r~ a;a
XhollXbal- 3500 & 1400 bp,
BgllllXhol - 2100, 1700 8
1000 bp
Circular Plasmid DNA > 95YO of visualized nucleic Agarose Gel [I~,llupllO,~a;a
acid
A2601A280 Ratio 1.75 to 2.00 UV Absu,'-
E coli DNA < O.01 ,ugl~g plasmid DNA Southern Slot Blot
RNA N .- ' ' on gel Agarose Gel Elc.,lll,,Jhult:a;a
Protein L~ BCA C~' :liL Assay
Pyl~ t~, Not pyrogenic at 5 ,uglKg Rabbit Pyrogen Assay
rabbit body weight
'- ' i < 0.1 EUI~g plasmid DNA Limulus A~ ' lcyle Lysate
(LAL) Assay
Sterility No growth through 14 days Fluid T' ~ Assay
Potency 50 200% of reference In Vitro Tl~llal~l,liulll
Fl ~SLC~.e
General Safety Test Passes per 21 C.F.R. 610.11
EXAMPLE 7
IN VITRO POTENCY OF HLA-B71DMRIE-DOPE COMPLEXES
Tr_ f~. err ; was dLt~, ~' by HLA-B7 gene LA~ aa;un in SW480 cells, a human colon
P 'I -~ . cell line, ATCC # 228-CCL, or UM449 cells, a human ' ~ cell line, Alfred Chang,
University of Michigan, following in vitro 1, 'u,,; using HLA-B7 plasmid DNAIDMRIE-DOPE lipid ~
formed by the :hc~ ' ~.1 of the invention in 0.9% sodium chloride with 1% glycerol and 0.01% Vitamin
E.
CA 02214029 1997-08-27
W O96/34109 PCTrUS96/05035
-17-
From 200,00 to 400,000 UM449 cells were seeded per well into a 6-well plate the day before
Il. CL_ ' Cells were a >75% confluent -~ . prior to ll .~,:..liun. The cells were llàll~r~lad
with 10,1lg plasmid DNA in the presence of 2 ~9 DMRIE (Sylllll~a;~t:d in house) and 2 ~9 DOPE '~, _ "has.,d
- from Avanti Polar Lipids, Alabaster, Ala). The cells were ' -l~d at 37~C, 5% CO2 Ih.~ ,.' l. Reduced
serum media, e.q., Opti-MEM~' reduced-serum media (GIBCO BRL Life T~' ~", s, G ' ~b~J~u~ MD),
_ . r~ with fetal calf serum, was added to the cells 1-4 hours and 24 hours post-l,. 'C.,liull. Cells
were l~a~r,~l~d 48 hours post-l~ ~L_
HLA-B7 ., on the cell surface was measured by labelling with anti-HLA-B7 mouse antibody,
followed by a rluul ~aCu.\l se -- ' y antibody (anti mouse IgG - ' ' antibody R ph~..L.~. y 11ll bl ~ ~ "_ )
I r' daC~.. I staining of the cells was analyzed by flow cy~ lly. A two-fold increase in mean
1' ~ ? intensity was observed for ll_ h~l~d cells in contrast to negative controls (non-lla"al~LI~d
cells or cells ll C~Ll~d with an irrelevant gene). Potency was e~u;. ' I to that of freshly prepared
plasmid DNAlcationic lipid - ,'
EXAMPLE 8
IL-21DMRIE-DOPE COMPLEXES
IL-2 plasmid DNAIDMRIE-DOPE lipid r ,' , intended for use in human gene therapy,~alLdin vitro potency as - ' ~;àO rul ' of the invention.
A plasmid encoding IL-2 was cû.l~llu~ d of about 5000 bp in size. It derived from a pUC-based
plasmid cu"i ~, a bacterial origin of r ,' i It encoded an IL-2 fusion protein. The protein was
- ~., d by cloning a portion encoding a short segment of the 5' :lallalal~d region and the first six
amino acids of the leader peptide of the rat insulin 11 gene 5' of the human IL-2 coding sequence minus the
first two amino acids of its leader peptide. This fusion protein was placed under the ~ ' ~ulil,
Il i~, ' control of the l,yt _ ' .;-ua (CMV) " l~ early 1 r ~ This
sequence D " l~d ~ ,., of a r , ~ ~ mRNA c i ~, a 5' ..llàualal~d sequence from the CMV
": early 1 gene, including the 800+ bp intron, the IL-2 fusion protein coding , and a 3'
....~6l~d sequence derived from the bovine growth hormone gene having ll iuUua
.du..11~ -signal , r~ Theplasmidalsoencodeda' ~.,;"r~;;,i genederived
from Tn903.
CA 02214029 1997-08-27
W O96/34109 PCT~US96/05035
-18-
EXAMPLE 9
PHARMACEUTICAL-GRADE PURIFIED IL-2 PLASMID DNA
The IL-2 encoding plasmid was purified to ~' ludG~u~ .adeslalldallla as d~ d by the
criteria given in Table 2 below.
TABLE 2: IL-2 PLASMID DNA QUALITY CONTROL CRITERIA
TEST S~EOlrlCATlON METHOD
Size Identity Ap~.. uAilllal~s Agarose Gel [IL~ll.r' ~a;a
4900 bp
R6alli Sites A~, UAi"lal~a ~." ": ' Agarose Gel EIL~II p~ ~a;a
EcoRI - 3000 & 1900 bp,
Ncol - 3700 & 1200 bp
Circular Plasmid DNA > 95% of visualized nucleic Agarose Gel [IL~I-upl,u,t~,;a
acid
A2601A280 Ratio 1.75 to 2.00 UV Al3so,L
E coli DNA < 0.01 ~91~9 plasmid DNA Southern Slot Blot
RNA r:~ . on gel Agarose Gel [IL~,III, ' ~a;a
Protein < 0.016,ugllJg plasmid DNA Protein Slot Blot
Residual Ethanol < 500 ppm Gas Chlul,.ai ,'1
P~ y Not pyrogenic at 5 llglKg Rabbit Pyrogen Assay
rabbit body weight
r- ' Iu,;,~ < 0.1 EUI~g plasmid DNA Limulus l,r- bc yl~ Lysate
(LAL) Asâay
Sterility No growth through 14 days USP Direct Transfer
Potency 50-200% of reference In Vitro Trallru~,L;ù
ELISA
General Safety Test Passes USP General Safety Test
EXAMPLE 10
//1/ VIT~O POTENCY OF IL-21DMRIE-DOPE COMPLEXES
Tlallalu: t~R ~ was 1~:, ' by IL-2 gene, .s ~r in B16F0 cells, a mouse
cell line, ATCC # CRL 6322, following in vitro llàllar~_ using IL-2 plasmid DNAIDMRIE-DOPE lipid
, ' formed by the ... '3d('~,y of the invention in 0.9% sodium chloride with 1% glycerol and 0.01%
Vitamin E.
From 200,00 to 400,000 B16F0 cells were seeded per well into a 6-well plate the day before
l-_ CL_ ' - Cells were a >75% confluent ' 1~. prior to 1, Cu~.liun. The cells were llallar~cled
with 2.5 ,ug plasmid DNA in the presence of 0.5 ,ug DMRIE (s~lllh6a;LLIl in house) and 0.5 ,ug DOPE
CA 02214029 1997-08-27
W O96/34109 PCTrUS96/05035
-19-
("...,,hased from Avanti Polar Lipids, Alabaster, Ala). The cells were ;Il~.ubdlud at 37~C, 5% CO2 ' ~
A reduced serum medium, e.n., Opti-MEM'~ d~,bed scrum media lGlBCO BRL Life T~ ' ~, s, G -' ' y,
MD), , ' l~d with fetal calf serum, was added to the cells at - : of l~a";.r~ r and 24
~ hours post llall~r~"i Cell ~u~u~lllaldul was hal~c~l~d 48 to 80 hours post llan~r~
IL 2 e~,u~l in the cell 5l~ Illaldlll was measured by an enzyme amplified ~e"~;li.ity
~ IM ~5 ELlSA, Medgenix r ~ -: 5, Fleurus, Belgium). Potency was ~, . ' I to that of
freshly prepared plasmid DNAlcationic lipid complexes.
EXAMPLE 11
STABILITY OF IL 21DMRIE DOPE COMPLEXES
The stability of IL 2 plasmid DNAIDMRIE DOPE lipid c , ' formed by the ' ~ of the
invention and stored in 0.9% sodium chloride with 1% glycerol and 0.01% Vitamin E was E.' lod at ~20~C,
2~C, 25~C, and 37~C. An ~ .a, of DNAIDMRIE-DOPE - ' in 0.9% sodium chloride
with 1% glycerol and no Vitamin E was evaluated at the 37~C storage I , di ~. The stability of the
materials in the study was analyzed by a number of methods, including 96 well l~ar,~r~ assay. The
results of the study showed that IL-21DMRIE DOPE ~( , ' - and free DNA in 0.9% sodium chloride with
1% glycerol and 0.01% Vitamin E retained full stability over 57 days of storage at -20~C and 2~C. At 25~C
IL 21DMRIE-DOPE - ' ~ retained apparent full activity over 57 days of storage; free DNA showed a half
Iife of 500 days for c .~.. from circular to linear form at this Ir"".~,dl...~. At 37~C, IL 21DMRIE-DOPE
c ' s showed a half-life of 34 days; free DNA was r .~ d from circular to linear form with a 290
day half life at this l~ lai ~. In - , in the absence of Vitamin E, IL 21DMRIE-DOPE c
showed a half-life of 12 days at 37~C; and free DNA was - ..,. I~d from circular to linear form with a 108
day half-life at this ~ Idlu.~. Therefore, the presence of Vitamin E at the level of 0.01% in the subject
bc " I provided 2.7 to 3 times greater stability to the - of the vial.
EXAMPLE 12
PREPARATION OF IL 21DMRIE DOPE COMPLEXES
A. LIPID FORMULATION and AUTOCLAVE STERILIZATION.
1. PI~lJaldl of the DMRIEIDOPE Bulk Solution.
Using an _ ~yli~,dl balance, the batch amount of DMRIE Br was weighed out.
Working in a ~~..lilaled laminar flow hood, the DMRIE Br was placed in a clean 50 mL round bottom
flask.
Using a 5 mL glass pipet, five (5) mL of ' ' uru~lll was added to the round bottom flask from
above. The flask was swirled to dissolve the DMRIE Br.
Using a SMI pipet, the batch amount of DOPE was added.
The neck of the round bottom flask was rinsed with about five (5) mL of r'' uru~lll and the
contents swirled gently for at least one minute to mix. Any solution adhering to the neck after swirling was
rinsed into the flask with '" ' r'' uR..Ill.
CA 02214029 1997-08-27
W O96/34109 PCT~US96/05035
-20-
Using a rotary ~........................... dpUld~UI~ the ' ' U~u,l~ was removed from the solution from above. The flask
was kept on the rotary e~, di ~ untii ce~ u~ was no longer visible in the ~
A dea;.,cd was IhGI~__"y wiped down with alcohol and placed in a ~..Li6led laminar flow hood.
The round bottom flask from above was placed in the d~ Ca~ . The desi,,cdlu, was cc : ' to a
vacuum pump, the dl ~ e~d~ualed~ and the round bottom flask ~ d under vacuum for at least
12 hours.
After at least 12 hours exposure to vacuum, the deJ;I~cdi was isolated from the vacuum pump.
Working in the ~,..li6l~,d laminar flow hood, the ~4,: ~ al~ was c ' to a nitrogen gas source,
the gas turned on, and the vacuum released. When the d - lù~ was filled with nitrogen yas, the gas
source was removed.
The round bottom flask was removed from the ~le-~ lu,. Using a sterile ', - ' ' pipet, sterile
water for injection was added to the flask. The flask was capped, the liquid swirled, and vortexed for at
least 5 minutes or until luh~l' di ~r was achieved.
2. i'~"a,dliun of DMRIEIDOPE bulk liPid mixture vials for autoclave Irt:dlult:llL.
The solution from above was lldll~ d in --0.5 mL aliquots to clean 2-mL Type 1 glass vials.
Each vial was capped with a clean teflon-coated gray butyl stopper, and the cap secured with an aluminum
crimp.
The filled vials from above were l,ui ' .~d using a standard liquid cycle on the ~ ul,l~ (no less
than 121~C for 30 minutes). If i...ll~cl~d vials were not used ~ ' 1y, they were stored at 2 to 8~C.
The ' .~d vials were not held at room ~ , u for more than 6 hours.
B. PLASMID DNA FORMULATION.
1. Sterile filtration of IL-2 Plasmid DNA Standard Bulk Solution.
Working in a ~~..li6lGd laminar flow hood, the plasmid DNA standard bulk solution was filtered
through a 0.2,um sterile filtration unit. The filtrate was collected in a sterile d, ~- '' tube. The tube
was stored at 15 to 30~C.
C. PLASMID DNAIDMRIE-DOPE LIPID MIXTURE FORMULATION AND FILL.
1. P~UIJdldi of the DMRIEIDOPE lipid solution.
Working in a ~..li6l~d laminar flow hood with all materials at room l~ JLIdlul~ (15 to 30~C),
sterile water for injection was Lld~ d into a sterile li, - ' 250-mL bottle.
An dU~,.I,iale amount of stock glycerin and an a,, ., idle amount of stock Vitamin E was added
to the bottle from above and swirled to mix.
An au,u~, idle amount of autoclave sterilized DMRIEIDOPE bulk lipid mixture from above was added
to the solution from above and swirled to mix.
An aPP~r iale amount of the filtered IL-2 plasmid DNA solution from above was added to the
DMRIEIDOPE lipid solution from above. The bottle was capped and the mixture was swirled ~ by
hand for at least one minute.
.
~ ~ =
CA 02214029 1997-08-27
WO 96/34109 PCTrUS96/05035
-21-
An ., up,idle amount of stock (5%) sodium chloride injection was added to the IL-2 plasmid
DNAIDMRIE-DOPE lipid complex solution from above. The bottle was capped and the mixture was swirled
~,;grn ly by hand for at least 1 minute.
2. FilllFinish.
Working in a ~,.lilaltd laminar flow hood, 1.2 mL of the IL2 plasmid DNAIDMRIE-DOPE lipid
complex from above was ar~p~ filled into prepared 2 mL type I glass vials using a Drummond Pipet-Aid
and sterile ', Q~'' 2 mL pipets.
The vials were sealed with prepared 13 mm teflon-coated gray butyl stoppers and 13 mm tab-top
aluminum caps. All vial caps were crimped with a hand crimper.
EXAMPLE 13
IL-21DMRIE DOPE COMPLEXES IN
PHASE I HUMAN CLINICAL TRIALS
Direct ;..I~di injection of plasmid DNA ~:AIJII ~ vectors provides a method for the i"ll~d : Jr
of IL 2 genes into the tumor. In the Phase I study proposed in this protocol, the sponsor tests for safety
and dose a, i of the direct gene transfer approach for delivering the IL-2 gene directly into solid
tumors and 1~, hr r'~ I ~ ~ of the IL-2 gene is - 'il ' The IL-2 produced should elicit an
~ ' _ antitumor response which in turn may lead to a systemic ' _ -' ~' of other
tumor cells. Dose 1l r 1! are ~ 1~6lud with specific immune ", ~r. -
S,r~ '; '1~, the phase I protocol is designed:
1) to minimize the risks to the patient;
2) to derive the maximum i"r. . regarding the , . of ,~ ~l ' I genes in vivo;
3) to maximize potential benefit to the patient; and
4) to gain new ' . 'L '9 regarding gene delivery to tissues in vivo and the immune response
to tumors.
The . b; : .~s of the clinical plan include:
1) To confirm the in vivo eAIl~U ~ of the IL-2 gene in the tumor cells.
2) To r!: , the safety and toxicity of direct _' ' injections of b~ tda;~ld amounts
of a DNAllipid mixture, IL-21DMRIE-DOPE, into patients with advanced 'ig y.
3) To d~l~l ~ the biological activity and, ' --' : of ll~dll_l with IL-21DMRIE-
DOPE.
4) To chalaclLIi~: the clinical response to escdldi " doses of the study drug by assessing
the size of the injected tumor and of other tumor masses that may be present.
The product is L d of plasmid DNA coding for IL-2, ~ d in an injection vehicle with
the cationic lipid mixture DlAh~ OPt. When ~,-' d into the target tumor tissue, the lipid ~
ll_ h ~ of cells with the plasmid. On ll~l : of the plasmid into cells, the ru ' : gene is
eA~.,eased. The lipid mixture is a ~ ' i of two ~r ' DOPE (CAS name: 1,2-dioleoyl-sn-glycero-
CA 02214029 1997-08-27
W O96/34109 PCTrUS96/05035
-22-
3,'-;p~v ' ' ~ a) and DMRIE ~yllLh~ ud as DMRlE-Br (CAS name: I+l-)-N-(2-h~-huxytlhYI)-N,N-
dimethyl-2,3-L;~ ' jlo~y)-1-~ r bromide) which are assumed to be rapidly : bc'
The, '~ ~ ~'li, ~ ' mixture and injection vehicle are produced in aLGull' with the "It: ' - ' ' "y of
the invention. The plasmid DNA is R..lllalal~d with DMRIEIDOPE lipid mixture in the injection vehicle which
s~ : 1% glycerol and 0.01% Vitamin E in normal aqueous saline (O.9% sodium chloride in sterile water
for injection). The dosage form is delivered by injection into solid tumor tissue.
The c~ - lld" of plasmid DNA and DMRIEIDOPE in each dose package is spec!fied in the
following table:
TABLE OF DOSE PACKAGES
Dose Package Plasmid DNA Mass dried Volume sterile
v llaliun (mglmL) DMRIEIDOPE (mglmL) Injection vehicle (mL)
lO,ug 0.01 0.0041.2
30 ~ug 0.03 0.0121.2
100 ~9 0.10 0.041.2
30~,ug 0.30 0.121.2
This is a Phase I ~ ?1 study in which up to 25 patients are enrolled for injections directly into
tumor nodulés with a lipid-~G~I ' Ird IL-2 plasmid complex. Solid tumors ( ' ' ~ bony tumors), and
,: of 1il ' renal cell Call a, and hepatic -l~ s of advanced cc~:~.ldl
ca",;"ull,a, and l~,. . ' are the tumor types to be evaluated.
Eligible patients have a primary tumor nodule injected several times at specified intervals with a
specified dose of the study drug (see below). There are four groups with 5 patients each treated at the
..,;bEd dose (10, 30, 100 or 300 ~g), with a group of 5 patients retreated at the maximum tolerated
dose (MTD), or at 300 ~g if the MTD is not reached. The highest dose that does not yield Grade 3 or
higher toxicities is ' ~d the MTD. All toxicities are graded according to the World Health Olya~ ai
(WHO) Re ~ :- for Grading of Acute and Sub-Acute Toxic Effects.
CA 022l4029 1997-08-27
W O96/3~109 PCT~US96/0~03
-23-
TABLE OF SPECI~ltu DOSES
Dosage No. Of Dose PerTotal No. Of Days
GroupPatientsTreatment Tl~ai Between
Per Group Per Patient Tl~a~ s
1 5 10~9 6 7
Il 5 30~9 6 7
lll 5 100 ~9 6 7
IV 5 300 ~9 6 7
V 5 MTD 6 7
The study drug is P' : ~d and toxicities are ~d. Tumor lesions are selected for
ll~d~ if they are acc~ '' to lldi -' . by direct needle injection. These Ill~la~lali.,
lesions are located at any ' ' site such as skin, nodes, lung, liver, soft tissues etc. Bony tumors are
excluded. The amount of study drug material injected into each tumor is based on the algorithm outlined
below. The p,t~..,ib~d dose (10, 30, 100 or 300~19)iS thawed and diluted with injection vehicle to the
3" ~, ial~ volume. If s y, the study drug is injected with the aid of ~l. .' or CAT scan
~; ' of the - Id~lda;~. Prior to injection, following ,' of the needle, gentle , d ' is
applied to the syringe to ensure that no material is injected ;.,IIu. '~. After injection of the drug and
with the needle still in place, the dead space is flushed with 0.25-0.50 mL of sterile normal saline (0.9%
sodium chloride in sterile water for injection).
Tumor Diameter (cm) Volume of Injection (cc)
1.0-1.5 1.0
1.6-2.0 2.0
Z.1-3.0 3.0
3.1X 4.0
Vital signs are measured every 15 minutes at the start of, during, and after the injection for at least
2 hours or until the patient is stable. If the systolic blood pressure drops below 80 mm Hg, the injection
isl~ y and the patient is closely ad and treated a~ r ial~ until blood pressure
is r s
Patients are closely ~d for toxicity for 34 hours post injection, then 24 hours and 7 days
after the first and second : For injections 3-6, patients are ~d for 3-4 hours post injection
then 7 days post injection as long as they have i~""cd no toxicity during the 4 and 24 hour st ,~ v
periods following injections 1 and 2.
CA 022l4029 l997-08-27
W O96/34109 PCT~US96/05035
-24-
TABLE OF SCHEDULE FOR POST-INJECTION MONITORING
Treatment # 34 Hrs 24 Hrs 7 Days 14 Days
X X X ---
2 X X X ---
3 X -- X ---
4 X --- X ---
X --- X ---
6 X --- X X
Before each L , : injection, patients are evaluated for toxicities from the prior injection and
injected with the next dose only if no Grade 3 or higher toxicity occurs. A tumor sizing is done at each
dl..,..o(al injection of the nodule. If the tumor shrinks to a point where it can no longer be injected,
c~ doses are ~ ' ~ ~ ~d into another tumor nodule if any are present.
After the 6th injection, patient follow-up includes e.~' i with tumor sizings at weeks 8 and
16. After the week 16 visit, patients are evaluated a minimum of every 4 months.If a patient r,A~JLli stable disease or a partial response (see below) at 4-8 weeks after the last
injection of their initial course, helshe may receive an additional course of ll~alll-r,--l identical to the first
course of 1~ ; or the next higher dose. The patient must, however, continue to meet the entry criteria.
Adverse events are ~- Gd, and patients are removed from the study if ullacL~J '' toxicity
(Grade lll or IV) develops.
Classical, ' ~c~' ., ' studies of drug ' ~,iL . half time" : ' ' . and excretion are not
entirely relevant to in vivo gene injection and , . ~ However, the fate of the plasmid and detection
of the gene product (IL-2) are relevant to the ' ..', ~ of this agent. In addition, immune a-.li.ai is
25 i , lalll. Therefore, as part of the ~,.. a.,t of the efficacy of this study, - '.11 gene transfer and
e"~,.t..~;u.. is evaluated by molecular and i ' ' analyses. The following pal_ : ~ are measured
to evaluate the tumor Ir c~ and ~AIJII ~ of IL-2: 1) the presence of DNA from the IL-2 gene is
assessed by PCR - , ' C;~ai of cells obtained by biopsy of the treated site after the injection of the study
drug, 2)i '~~ ' ~ ' staining of tumor biopsy samples is used to assess ~ ", response and
30 soluble IL-2 eJ~Ir~;u~, 3) serum IL-2 levels are measured pre-llr,ai I and 2 times post the start of
therapy, however, the detection of serum IL-2 levels is not liL;~Jal~d due to IL-2 ~ : ' ' ~" 4) PCR analysis
uf pr,li~Jllr,,al blood samples is used to test for the presence of plasmid DNA after the start of llr,allll.:llL and
c , _ ~,d to pre-therapy, but detection of the gene in r~-~;.' dl blood samples is not . i , d, 5) the
cellular immune response is evaluated by l.._a~....i ~, baseline and pOst-llr,allll_..llL-2 induced a..li., of
35 PBMC by Ih,~ ' - uptake assay and NKI LAK response in ~ ,' dl blood pre-therapy and post-therapy,
and 6) an attempt is made to excise tumor tissue from another site prior to ll, l for di;~ ~ .
CA 02214029 1997-08-27
WO 96/34109 PCT~US96105035
-25-
y, cryo; t;~lvai and to evaluate r i ~ al blood 1~, ' yl~ ' -' reactions to
the tumor before and after 1,.
As an -'' ' part of the ~. ' i of the efficacy~of this study, the clinical response is
J measured. Standard oncologic criteria are applied to d~l~l whether or not a patient responds to the
study drug. All tumor - ~ la are recorded in ~ : a and cr ~ the longest diameter and
the r ~ ~ ~ diameter at the widest portion of the tumor. The tumor response d~ri Iisted below
are used to compare current total tumor size to pre-lu I total tumor size.
There is a complete tumor response upon ' ., a"Le of all clinical evidence of active tumor for
a minimum of four weeks, and the patients is free of all 51~ . i of cancer.
There is a partial tumor response upon fifty percent (50%) or greater decrease in the sum of the
products of all ' ~ a of ~ '' lesions. These " ' : - in tumor size must endure for a minimum
of four weeks. No ' increase in the size of any lesion or ~, a"..e of new lesions may occur.
The a~ , iàle ' _ ~_ tests used to d~ ..,al,dl~ the response must be repeated four weeks after initial
~b~ v: - in order to d~ I this duration.
There is stable disease upon less than 50% decrease in the sum of the products of all ' : a
of - ' ' lesions, or an increase in the tumor mass less than 25% in the absence of the du.. ' r
of new lesions.
There is r ~_ I ~ disease upon tumor, ~v bi.S;U~ as defined if one or more of the following
criteria are met: 1 ) 3~ ~ ~ . e of any new ~t 's).2) increase in tumor size of 225% in the sum of the
products of all d;a",dlL,a of I ' ' lesions, 3) _ " I clinical 'l iUld'- that cannot be dllH'
to ll~ai ~ or other medical c~ " and is assumed to be related to increased tumor burden, and 4)
., _ of tumor related a~ , deemed clinically: ' : by physician.
The principles of informed consent described in Food and Drug A ' ~,di' (FDA) R v ~ 21
C.F.R. Part 50 are followed.
The ~" ., idl~ approvals are obtained from the relevant h,~lilui ' Review Board (IRB), the
l;~r ' ~ DNA Advisory Cc ~ (RAC) of the National Institutes of Health (NIH), and the Food and
Drug A~ (FDA).
While particular bc ' : of the invention have been described in detail, it will be apparent to
those skilled in the art that these - bc ' Ia are e , ' y rather than limiting, and the true scope of the
invention is that defined within the attached claims.
