Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~27~i0~
- 2 - HOE/B 018
The invention relates to a process for the prepar~
ation of a therapeutic agent from tumor cells for the
therapy of tumorous diseases, and ~o a therapeutic agent
of this type.
S It ;s known, for example from "Mechanisms of Tumor
}mmuni~yf" &ree ~t al.~ e~s., John W;~ey and Sons, ~.Y.,
197~, pa~e 156, that attempts have already ~een made to
treat tumorous diseases by inoculat;on with tumor ce~ls
~h;ch have been mod;f;ed by freez;ng and thaw;ng, freeze-
dry;ng, pressure or homogenization Subcellular fractions
or cell extracts have also been used for th;s purposeO
However, as yet no vacc;ne aga;nst a tumorous d;sease has
been d;sclosed.
We have foundO surprisingly,-that cells, which have
been freeze-dr;ed or treated ~ith an aldehyde, from human
tumors or from cell aggregates obta;ned from the ~atter,
wh;ch carry ant;gens wh;ch are bound by ~he monoclonal ant;-
bod;es descr;bed ;n ou~ co-pending Cana~ian Patent A~plication Serial
Number 460,725, filed August 10, 1984, can be used as a therapeutic agent for
t~e treatment of t~orous diseases~
Thus the invention relates to a therapeutic agent ~or
the ereatment of a tumorous d;sease, contain;ng human cells
~hich have been dr;ed or stab;l;zed by a chemical trea~Ment
and ~hich carry ant;gens ~h;ch are bound by the monoclonal
antibod;es described in the above-ident.ified Canadiar~ Patent application.
One advantage of a therapeut;c agent of th;s type
compared w;th the use.of unmod;f;ed cells ;s that unmod;-
f;ed cells are not stable, so that they have to be pre-
pared fre~h each time Moreoever, for thi~ reason, they
cannot be standard;zed
The invent;on furthermore relates to a process for
the preparat;on of a therapeutic agent for the treatment
of a tumorous d;sease, which comprises the drying or sta-
bilizat;on, by a chemical treatment, and the process;ng
to a therapeutic agent, neuram;nidase being added where
appropr;ate, of human tu~or cells which carry antigens
wh;ch are bound by the monoclonal ant;bod;es described in
-- 3
the above-identified Canadian Patent Application.
The invention also relates to a process for the
preparation of a therapeutic agent for the treatment of a
tumorous disease, which comprises the isolation, by means of
a monoclonal antibody described in the above-identified
Canadian Patent Application, of an antigen from human cells
and its processing to a therapeutic agent, neuraminidase
being added where appropriate.
The possibility of potentiating an immune response
by neuraminidase is disclosed in German Offenlegungsschrift
2,620,649.
A therapeutic agent is to be understood to be an
agent which may be suitable both as a prophylactic and for
the treatment of a manifest disease.
The monoclonal antibodies described in co-pending
Canadian patent application serial number 46~,725 are those
which recognize an antigen (Agl) having a molecular weight of
approximately 33 KD + 3, or an an-tigen (Ag2) having a mol-
ecular weight of approximately 134 KD + 3, or an antigen
(Ag3) having a molecular weight of approximately 80 KD + 3,
or an antigen (Ag4) having a molecular weight of approx-
imately 55 KD + 3, or an antigen (Ag5) having a molecular
weight of approxima-tely 60 KD + 3, or an antigen (Ag6) having
a molecular weight of approximately 54 KD + 3, or an antigen
(Ag7) having a molecular weight of approximately 260 KD + 3,
or an antigen (Ag8) having a non-determinable molecular
weight, or an antigen (Ag9) having a non-determinable molec-
ular weight, or an antigen (AglO) having a molecular weight
of approximately 143 KD + 3 ancl 119 KD + 3, or an antigen
(Agll) having a molecular weight of approximately 178 KD + 3,
or an antigen (Agl2) having a non-determinable molecular
weight, or an antigen (Agl3) having a molecular weight of
approximately 34 KD + 3, or an antigen (Agl4) having a molec-
ular weight of approximately 195 KD ~ 3, or an antigen (Agl5)
having a molecular weight of approximately 44 KD + 7, or an
antigen (Agl6) having a molecular weight of approximately 43
~2~
- 3a -
KD + 3, or an antigen (Agl7) having a molecular weight of
approximately 130 KD -~ 3.
The antigens 4, 8, 9 and 10 are mycoplasma antigens
which are associated with the cell lines described in the
table.
The immunogens used to induce these antibodies are
human cell lines cultured in vitro, cell extracts or extracts
of human tissues. Permanent human cell lines, in particular
the CaLu-l, Chago, Oat 75, PaTu II and Bewo cell lines, are
preferred. It is also possible to use Agl-Agl7 to induce
Abl-Abl7.
Mammals, preferably mice, are immunized intra-
peritoneally with 1 x 106-108 cells J but preferably 107
cells, of a cell line of this type (days 0-120, but pre-
ferably on days 0 and 7) and, after 1-150 days, but prefer-
ably on day 11, the spleen cells from such animals are fused
with the X63 Ag 8653 cell line (The Journal of I~munology
173, 4, ~548-1550, 1979) (Nature 256, 495-497~ 1975).
The hybridomas resulting after 3 weeks are tested
for antibodies of the desired specificity. In this case, a
panel of 30 cell lines cultured in vitro, human peripheral
blood cells and human bone marrow were tested for reactivity
with the antibodies by means of indirect immunofluorescence
(Behring Inst. Mitt. 59, 64-70, 1976) and cell-sorter
analysis (Acta Cytol. 19, 374-377, 1975), (Proc. Natl. Acad.
Sci. USA 77/8, 4914-4917, 1980).
Surprisingly, among the hybridoma supernatants which
were tested, there were some which contained antibodies with
the interesting specificities described above and in Table I.
3~ ~.277~0~
Table 1
Reactivity o~ A~ 1-17 with cells in vitro
Cells tested 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1b 17
Lun~ tumor
celL lines
5 CaLu-1 + + ~ + ~
E-14 ~ - - + ~ ~ t -~ ~ ~ t
109/4 ~ +
~ 549 ~ + +
Oat 75 + + - ~ - + ~ + -
SHP-77 + * ~ + ~ + 1
Chago -~ t -~ +
Bro-Ca-Hoff + + -
Pancreas tun-
or cell l;nes
15 Pa Tu II ~ + - ~ +
Pa Tu III
MIA-Pa-Ca 2 ~ + - - -
Panc-1 * ~ - ~ +
Gynecologi-
caL tu~nor
cell Lincs
Bew~ * l I
~eLa + + * -
Pa-1 ~ ~ ~ +
- 3_
Cells tested 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
_, . ~ . .. .. _ .
Melanoma cell
l;nes
Mel-ULF + ~ + t - + - +
S Mel-RPMI ~ I ~ * - ~ - +
llel-51-2 +
Mel-21-C-48 + +
Unrelatc~
tumor cell
10 lines
~R-75-1 ~ * - -
MCF-7
Mamr,la-Ca~12 ~ - - * ~ ~ -
Colon-Ca-hx
15 Colon-lli + + - ~
HT~-10~0 ~ ~ _
Leiomyo- +
sarcoma
Hyper- _
20 nephroma TUW
IMR 32
Raj;
L)a~di
173~
6666/1
Immunocytoma
~27~6~
~ 3d -
Cells tested 1 2 3 4 5 6 7 ~ 9 10 11 1Z 13 14 15 16 17
Normal lymph-
o;d cel~s
Lymphocytes ~ ~ ~ ~ +
S Monocytes ~ * ~
Granulocytes ~ +
rythrocytes
Rone m a r r o w _+ + - - +~
l~ormal hur,lan
10 f;broblasts
LL-29 t
1~u-F i-Br ~2
Hu-Fi-Br 43 + ~ ~ - - - - - - - -
Hu-Fi-Br 47 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
~5 ~lu-F;-Br 10
Hu F;-Mel-1
H u- F; -l~l a ~l -13
Animal cells
~le~ - + ~ - -
Grcyhound
R~t fibro- - - - - - - - - - - - ~ -
~ asts
Mouse f;br o~
2 ~ b l a 9 1: S
~ 3e -
+ = sign;ficant positive reaction ;n an ;ndirect
;mmuno,luorescence assay, ;n radioimmunoassay and
;n cytofluorometr;c analys;s.
~ = ueakly positive reaction with part of the popula-
t;on tested
- ~ no s;gnif;cant reaction
The cells ~hich can be used ~ith1n the scope of the
~nvent1On are obtained from tumors by kno~n cell culture
processes~ Cells ~hich have been obtained from such cul-
tures by mechanical or enzymatlc means and have~ where
appropriate, been inactivated ~ith mitomycin C, are dried,
preferabl~ freeze~dr;ed~ or treated ~;th an agent kno~n to
those sk;lled in the art as a stabil1z1ng agent for organ;c
tissue, preferably a monoaldehyde or d1aldehyde having I
lS to 6 carbon a~oms.
The agents which are particularly suitable for
chemical stabiliza~1On and fixation include, in part~cular,
bifunct1Onal compounds~ that is to say those ~hich con-
tain t~o groups which ca~ react w1th functional groups
on the biological materiaL - in other words car. "cross-
link" ;t. Examples of these are dialdehydes, in particular
aliphatic dialdehydes having 2-8 carbon atoms. Ho~ever,
monoalkanals hav~ng 1-4 carbon atoms, such as formaldehyde,
~hich can undergo bifunctional reactions, as ~ell as bi-
functional imino esters, such as suberim;date, isocyanates
or lsothiocyanates, are also suitable for this purpose.
It is also possible ~or so-ralled tanning agents
such as, for example, tann1c acld and its derivatives, or
chro~1um ~alts, to be used as agents ~h1ch can stab1~1ze
biolog1cal mater1al. Sulfosalicylic ac1d is also su1t-
able.
~776~
-- 4 --
In general, the cells are ;n the form of cell aggre-
gates or single cells. $t ;s also poss;ble to use cell
fragments or antigens isolated from the tu~or cells. Ant;-
gens of th;s type can be a~tained from tumorous tissue
S from pat;ents, as uell as from human tu~crs ~h~eh grow ;n
immunef;cient an;mals, and can be used.
Defined ant;gens are obta;ned from the tumor cells
or fragments thereof using the monoclonal antibod;es des~
rr;bed in co-pending Canadi~n Pat~en~ Application Serial Nwnber
460,725.
Examples of ant;gens of this type are CEA tcarc;no-
embyron;c antlgen; J.exp. Med. (1965) 122, 467) NCA ~nor,-
speclfic crossreacting an~;gen; J. ~mmun. (1973) III" 1926)
~hich can be isolated from the tumor cells by ~mmunoadsorp-
t1On chromatography. For th;s purpose, the monoclonalantlbod;es described ;n Table I of German Offenlegungs-
schr;ft 30416,774 are covalently bound as pur;fied prs-
teins, to CNBr-activated sepharos~ 4~O and the antigens
~CEA, NCA) recognised by these monoclonal antib3dies, are
;solated from DE-TA colon carclnoma cell extract . A suit-
able process ;s descr;bed in the Pharmacia book "Aff;n;ty
Chromatography~ Principles and Methods"~ 12-t8 ~1979),
sum~arized on page 15.
An ant;gen of th;s type obta;ned uC;ng monoclonal
ant;bod;es can be used as a therapeut;c agent, for example
as act;ve compound ;n vacc;nes aga;nst a disease wh;ch
1s caused by the tumor cells from ~h;ch the antigen ~as
obtained.
qual;ty control of a mater;al ~hich is to be used
as a vaccine is carr;ed ou~ by, for çxample~ typing w;th
monoclonal antibodies, or by ~ractionation of the total
cellular proteins using SDS-polyacrylamide gel electro~
phoresis or 1s~electric focusing ~1st dimension) combined
~ith SDS-polyacrylam;de gel electrophores;s (2nd d~men-
sion3 followed by sta;ning of the gel ~s;lver sta;n).
The antigenic mater;al is preferably adm~nisteredir,tradermally, preferably by the checkerboard vacc;nation
method ~Cancer Immunol. and Immunother. 6,47-58 t1979), ;n
rart;cular page 4~) in comb;nation ~ith an adjuvant, in
particular neuramin;dase (6erman Offenlegungsschr;ft
-- 5
2,~20,649).
A varcine of th;s type is preferably used for cer-
tain stages of colon carcinoma (Duke C) and for other tumors
wh;ch carry anti~ens or ep;topes wh;ch are present in the
5 vaccine. Other tumors of th;s type are solid tumors, for
example carc;nomas of the pancreas~ of ~he stomach, of the
breast and of the lungs. The vaccine can be adm;nistered
parenterally or orally. The ant;gens can be administered
dissolved or suspended ;n phys;ological sal;ne, preferably
intrader~ally in PBS.
Two tests were carr;ed out to assess the stab;lity
of the antigenic compos;tion of the vaccine:
a) the Terasaki IIF Assay (ind;rect immunofluorescence
using tumor cells ~hich grow in the welLs of the Terasaki
microt;ter plate) ~;th monoclonal ant;bodies of various
specificiti~s~ It ;s pcssible by mea ns of this ~est to
measure the expression of membrane 3ntigens on in~act tumor
cells, against which a number of moncclonal ant;bod;es are
available tCancer Detec~;on and Prevent;on 6, 181-184,
1983). It was possible by this means to detect drastic
changes to the D~TA cell ~embrane during cul~iYation;
b) solubil;zation of the total cellular proteins us;ng a
detergen~ (Hybrndo~a 1, 413-421, 1982) follo~ed by S~S-
polyacrylam;de gel electrophoresis combined ~;th a silver
sta;n (Anal. 8;ochem. 105, 361-363,1980). The comb;nat;on
of these techniques ensures that no s;gnificant changes in
the total protain content of the DE-TA cell line have
occurred.
The examples wh;ch follo~ illustrate the invention.
Example 1
The carc;noma cell line ~ X was cultivated ;n a
cell culture ;n plastic bottles, growing as a monolayer in
RPMI-1640 med;um ~Moore, G.E., Gerner, R.E., Franklin, H.A.,
Culture of normal human leukocytes, J.A.M.A. 199, 519-524
(1967)) with 10X fetal calf serum. The adheren~ cells
growing ;n confluent cultures ~ere separated mechanically
or using trypsin wh;ch ~as d;ssolved ;n RPMI-1640 med;um
conta;n;ng no fetal calf serum, the collagenase was ;n-
act;vated by add;tion of fetal calf serum d;ssolved ;n
! 6 -
RPM1~1640, and then the ~ells were detached from the t;s-
sue culture bottles and washed 3 times in phosphate-buffered
sal;ne (PBS) at 37C.
About 107 cells, the major part of wh;ch as in
the form of aggregates, were incubated at 37C for 1 hour
in 1 ml of P3S ~h;ch contained 100 yg of m;tomyc;n C.
Then the cells thus inactivated ~ere ~ashed 3 ti0es ~;th
P~S and
a) washed 3 t;mes ;n 0.18 molar ammonium bicarbonate buffer
wh;ch had been adjusted to pH 7.4 w;th acetic acid. A
cell sed;ment corresponding to 107 cells was taken up ;n
100 ~l of the same ammon;um bicarbonate buffer and frozen
at -70C. The frozen mater;al ~as then freeze-dr;ed and
stored ;n a small glass bottle, which was closed air-t;ght,
in a refrigerator at ~4C. The cell material thus treated
can, after havin~ been taken up in ~BS, be used for vaccina-
tion of patients.
Alternatively
b) incubated w;th 0.1X glutaraldehyde in PBS at ~4C for
5 minu~es, the excess glutaraldehyde be;ng removed by wash-
ing 3 times ~;th PBS, and then incubated with 2X BSA
(~ovine Serum Albumin) at +4C for S ~inutes and washed
3 times in P~S. The cells thus treated can be stored a~
~4C and used for the vaccinat;on of pat;ents.
Or
c) incubated in formalin according to Lilly ~enno Rome;s
~1968), page 65, section 266, Oldenburg Verlag, Munich) at
25C overnight~ shak;ng occasionally. The cells ~about
108) were centr;fuged w;th decantation of the supernat~ t
(10 minutes at 800 x 9), and the cell sediment was suspen-
ded ;n 7 ml of double-d;stilled water (~ 1st wash). Th;s
washing process was repeated 4 times at intervals of 1 hour.
The cell sed;ment was then washed 3 t;mes, at ;ntervals
of 1 hour, in 7 ml of 70X ethanol eash t;me. The cell sedi-
ment was then washed 3 t;mes, at ;ntervaLs of 30 M;nutes,;n 7 ml of 80X ethanol each t;me~ The cell sed;ment was
then washed 3 times, at ;ntervals of 30 minutes, in 7 ml
of 96% ethanol each time. The cell sediment was then
washed 3 t;mes, at intervals of 30 minutes, ;n 7 ml
1~
76~9
- 7 -
of 99X ethanol each timeO The cell sediment was then
washed 3 t;mes, at intervals of 30 minutes, in 7 ml of
sterile P~S each time, and was stored sterile at 4C.
The cell~ thus treated can be stored at 4C and used for
the vacc;nat;on of pat;ents.
~e~
In order to isolate antigens from tumor cells by
immunoadsorption chromatography, purified monoclonal anti-
bodies which unambiguously react with antigens on the tumor
cells which are to be used as vaccine are covalently bound
to CN3r-act;vated sepharose 48. The process was that of
the Pharmac;a book "Aff;nity Chromatography", Princ;ples
and Methods, 12-18 ~1979), in particular page tS. The
carr;er-bound monoclonal ant;bod;es were then ;ncubatecl
with cell solubilizates at ~4C for 2 hours, shaking
occasionally. The latter were obtained from cultured cells,
which had been mechanically removed from the culture bot-
tles~ by means of extract;on with lys;s buffer ~5 9/ l
sodium deoxycholate, û.5 mmol/l PMSF - phenylmethylsulfonyl
fluoride, PBS, pH 8O3~ as described in Hybridoma 1,
413-421 t1982), in particular on page 414.
The loaded carrier was centrifuged and suspended
in lys;s buffer-SDS ~2a mM tris.HCl pH s.n, 1 mmoltl
PMSF, 5 gll Nonidet P-40 ~~ octylphenyl ethylene oxide;
Fluka A6), 5 g/l sod;um deoxycholate~ 1 mmol/l ethylened;-
am;netetraacetate and 1 g/l sodium dodecyl sulfate ~SDS))
for ~ashing.
Th;s washing process was repeated 3 times. The
carrier was then washed twice ;n lysis buffer without the
30 addit;on of SDS and then washed once with a washing buffer
~2 M tris.HCl~ pH s.a, 10 mmol/l NaCl, 0.1 mmol/l
EDTA and 0.5 g/l NP-40). The ant;gens thus purified were
removed from the solid carrier either by heating at +95C
for 5 m;nutes or by incubat;on in 6 mol/l NH4SCN at +4C
for 30 m;nutes.
