Note: Descriptions are shown in the official language in which they were submitted.
s~
I-IIGHLY-PURIFIED INFO~TORY RIBONUCL~IC ACID
(i-RNA), A PROCESS OF PRODUCING SA~E
AND THE USE THEREOF
The present invention relates to an i-RNA
(denoted as "informatory", and previously also as
"immune" ribonucleic acid) which is specific towards
antigens and is obtained in a cell-free system, and
which has so highly been purified that it is toler-
ahle for human beings and animals when parenterally
administered, with an intravenous application being
preferred. The invention further relates to a pro-
cess of producing thus highly purified i-RNA and the
use thereof.
Normally, i-RNA is formed by various immune-
competent body cells upon contact with an antigen.
It contains, and transfers, the informations ~or the
systems of antib~dies, regulator protein, and cell-
bound~immunity. That is, i-RNA iS always found in
the peripheral blood and in lymphatic organs, when
the macroorganism contends with -an antigen. A multi-
plicity o~ different types of i-RNA is transferred
by each blood transfusion, while the antigens react-
ing with the antibodies having respectively been
synthesized are unknown. The concentrations of the
individual i-RNA species, however, are ~ low for
detection.
5~
It is, therefore, required, in order to re-
cover a definite i-RNA, to e~perimentally separate
the reco~nition of an antigen from the synthesis of
an antibody. Said procedure will be most successful-
ly carried out in cell-free biosynthetic systems
which are capable either of only recognizing the
selected antigen or of only synthesizing antibodies.
There has been known from the work of Ja-
chertz et al that in antigen-recognition in a cell-
free system the first product to be isolated is
i-RNA (cf. Jachertz, Zeitschrift fur medizinische
Mikrobiologie und Immunologie 154, 245 ~1968); An-
nals of the New York Academy of Science 207, 122
(1973); Zeitschrift fur Immunitatsforschung und ex-
perimentelle Therapie 144, 260 (1972); and Journal
of Immunogenetics 1, 355-362 (1974)). Yet, the pre-
parations described by Jachertz of the i-RNA have
been considerably contaminated and have, therefore,
not been suitable for an application to human
beings. An improvement of the purification procedu
res was published by Jachertz in Molecular and Cel-
lular Biochemistry 24, 93 (1979). However, the expe-
rimental section of said publication, as well as of
the other papers, was so incomplete that repeated
attempts to reproduce his findings failed and the
results in total were taken for being dubious. This
resulted in that neither the results nor the inter-
pretations of said results have been accepted by the
art.
Attempts have also been made to prepare
i-RNA in vivo in order to subsequ~ntly recover it
_
5;~
~ 3 --
from the spleen ancl the lymph nodes of an animal
(cf. Liu Shi-Shan et al, "The Lancet", Jan. 23,
19~2, p. 197). However, said attempts did not suc-
ceed in a complete purification either, apart from
the expensive mode of production (horses were used
which, in each case, had to be killed).
It is the object o~ the present invention to
provide a highly~purified i-RNA, which is specific
towards antigens and is tolerable for human beings
when administered, more particularly by way of in-
jection, by producing said i-RNA in a cell-free sy-
stem. Quite surprisingly, this problem has success-
fully been solved, and the i-RNA thus produced is
compatible with parenteral, more particularly intra-
venous, applications to human beings and animals,
with warm-blooded animals and mammals being prefer-
red.
The process of producing a highly-purified
i-RNA, which is specific towards antigen and is to-
lerable for human beings when parenterally, more
especially intravenously, administered, and which
has been obtained in a cell-free system, by way of a
reaction of antigen, DNA (including i-DNA~, nucleo-
tides, and an enzyme system having been recovered
from leucocytes, lymphocytes, other cells or cell
components, extraction with an aqueous solution of
phenol and sodium dodecylsulphate, and further puri-
fication, is characterized in that purification is
achieved by density gradient centrifugation, more
preferably centrifugation with about 130,000 g em-
ploying a density gradient of cesium chloride,
5~
-- 4
saccharose and l'ris buffer. Thls centrifugation will
take about four hours. It is preferred to subse-
quently carry out another centrifugation step using
cesium sulphate. More preferably, an enzyme system
is used which has been recovered from the leucocytes
or lymphocytes of a spleen homogenate by means of
density gradient centrifugation. Employing a density
gradient of saccharose in Tris buffer with a saccha-
rose content of from 30 to 60 per cent at about
100,000 g has proven to be particularly suitable
therefor. It will be beneficial to previously stimu-
late the immunosystem of the animal, the spleen ho~
mogenate of which is to be used for recovering the
cell-free system. Said stimulation may be effected
in a conventional manner by the use of Freund's ad-
juvant, BCG, and, more particularly, Pind-Avi (ra-
diation-inactivated chicken pox viruses).
Another procedure for a mild purification is
affinity chromatography. For example, poly(U)sepha-
rose 4B or oligo-desoxy-thymidine may be used as the
matrix.
The highly-purified i-RNA, which is specific
towards antigen and is tolerable for human beings
and animals when parenterally, more especially in-
travenously, administered, and which has been ob-
tained in a cell-free system, according to the in-
vention, is indeed suitable to be directly used in
therapy~ It actually effects the syntheses of anti-
bodies, regulator protein and cell-bound immunity.
It may further be used to generate an active immuni-
zation and to produce anti-sera and antibodies in
~9~1L52
-- 5
vivo ancl ln vitro. The anti-sera and antibodies
having thus been obtained may, in turn, be used to
generate a specific passive immunization. Besides,
they may be used for analytical purposes, since they
are highly specific towards the respective antigens.
Thus, the i-RNA according to the invention i9 a key
substance, which enables various problems to be sol-
ved or the solutions to which to be improved in an
extraordinarily simple and elegant way and without
after-effects. It may, of course, also be applied
together with other compatible substances, e.g. as
an additive to transfusions of full blood, preserved
full blood, plasma, plasma fractions, plasma expan-
ders etc..
Typical antigens are, for example, bacteria,
viruses, viruids, fungi, tumors, and parasites. How-
ever, they may also be higher molecular weight che-
mical substances such as steroids, plant poisons and
animal venoms, as well as allergens. Radiation-inac-
tivated viruses are especially beneficial, as they
ceased to be pathogenic while retaining their anti-
genicity.
There has been found that the i-RNA accord-
ing to the invention is also capable of synthesizing
specific antibodies in a cell free system. It is a
finding of major importance that the i-RNA induces
the synthesis of a regulator protein in cells, which
regulator protein induces the new synthesis of cell-
autogenous i-RNA in the receptor cell. The injected
i-RNA and the newly formed i-RNA are, in turn, cap-
able of inducing the formation of antibodies. Thus,
~2~52
the i-~N~ is excellently suitable for a parenteral,
more particularly an intravenous, application for
the purpose of a specific therapy, in order to the-
reby rapidly and purposefully mobilize the body-in-
herent defense abilities. In so far as the body does
not yet contain the antigen, an active immunization
occurs which proceeds faster and with less compli-
cations than does the conventional vaccinatlon with
antigen. Since considerable quantities of antibodies
will as well be formed some time after the injection
of the i-RNA according to the invention, at some
later time the serum may be used for the passive
immunization or the recovery of antibodies. Said
sera and antibodies are, of course, excellently
suitable for special diagnostics and the recognition
of antigens.
The production according to the invention of
the i-RNA does not involve any manipulations of the
genetic material in the meaning of a recombination.
Notwithstanding the synthesis in cell-free systems,
the i-RNA according to the invention is understood
to be a regular component of the peripheral blood.
The criter~a of acceptability of an application of
i~RNA for the treatment of human beings may, there-
fore, rest on the criteria for-and the experiences
with blood transfusions. Consistently, those inject-
ions of i-RNA having so far been administered to
human beings have not shown any of the slightest
side-effects in all of the cases. On the other hand,
the biological activities as desired were recorded
in approximately all of the cases. This allows for
the conclusion that the i-RNA, which has been formed
in a cell~free system of immune-competent upon sti-
mulation with antigen, actually contains the -total
information required for the synthesis of antibodies
and for the occurence of cell-bound immunity. Fur-
thermore, the i-RNA is free from antigens and anti-
gen-fragments and other impurities such as proteins,
and information contents different from DNA and RNA,
which may, for example, be verified by means of
agarose-gel-electrophoresis and thin layer chromato-
graphy.
For the purpose of direct therapy, it is
generally sufficient to administer the highly-puri-
fied i-RNA according to the invention in the form of
one single injection. It only appeared to be indi-
cated in a few cases to repeat the injection about
10 days later.
The present invention is further illustrated
by way of the following examples.
Z~5~
EXAMPI,E 1
a) Preparation of the Enzyme System
Leucocytes or lymphocytes may be used for
the preparation of the appropriate enzyme system.
The preparation from spleen is particularly easy. In
this procedure, 1 to 2 g deep-frozen fresh spleen
(e.g. pig spleen~ are crushed in a mortar with dry
ice and liquid nitrogen. The powder is added to
1.5 ml of Tris buffer A in a centrifuge tube, and
the suspension is froæen at -70C. This procedure is
repeated for about 10 times. Then, the macerated
cells are centrifuged in the rotor SW 50.1 of a
Beckman ultracenti~uge with 30,000 g for 30 min.
Subsequent to the centrifugation, 1 to 1.5 ml of the
red clear supernatant is taken and put on a discon-
tinuous saccharose gradient. Said gradient comprises
1 ml of a 60 per cent solution of saccharose in Tris
buffer A, above which 2.75 ml of a 30 per cent sac-
charose solution in Tris buffer A are presentO Cen-
trifugation is effected in a Beckman centrifuge,
rotor SW 50.1, at 0C with 100,000 g. After 4 hours,
the desired fraction is obtained as a reddish clear
layer on the top of the 30 per cent saccharose. The
layer is withdrawn by means of a pre-cooled pipette
and diluted with chilled Tris buffer A in the ratio
of 1 : 1. The solution may be stored in liquid ni-
trogen.
L52
b) DNA (Deoxyribonucleic Acid) including 1-DNA (In-
formatory Deoxyribonucleic Acid)
1 to 2 g deep-frozen spleen are slowly thaw-
ed in 5 ml of Tris buffer A and homogenized in a
homogenizer by means of about 30 piston cycles. The
suspension is dropwise added to 20 ml of an emulsion
of a 2 per cent aqueous solution of sodium dodecyl-
sulphate with freshly distilled phenol (ratio 1 : 1)
contained in an Erlenmeyer flask, while the flask is
vigorously swirled in order to obtain a homogeneous
distribution. If the mixture is too viscous, a 3 per
cent saline solution may be added. After 24 hours,
the opaque milky emulsion is centrifuged at 2,000 g
for 15 min, in which step two phases are separated.
To the upper aqueous phase there is added the same
volume (+ 10 %) of anhydrous ethanol, so that the
DNA is precipitated. The DNA is transferred into 10
ml of a sterile 0.85 per cent sodium chloride solut-
ion by means of a thoroughly glowed platinum loop.
5 ml of a 1 per cent sodium dodecylsulphate solut-
ion, 5 ml of a 0.05 mole/l Tris A and 0.05 mole/l
EDTA buffer of a pH 7.6, and 2 mg proteinase K are
added and admixed. Dissolution of the DNA will take
about one day, after which it may again be precipi-
tated with ethanol. The steps of dissolution and
subsequent precipitation are repeated from three to
seven times, until the ratio of E260 to E280 has
reached a value between 1.9 and 2.0 and the hyper-
chromic effect amounts to an increase in the ex-
tinction at 260 nm of at least 20 %. A DNS absorp-
tion at 260 nm of 1 o.d. corresponds to a concentra-
~9~
-- 10 -
tion of about 40 ~g/ml. By use of that relation, the
DNA solution is diluted so that a concentration of
1 ~g/ml is obtained. This ready-to use solution is
frozen in portions of 0.5 ml each and is stored at
-20C.
c) Synthesis of i-RNA (Informatory Ribonucleic Acid)
The purified enzyme system and the antigen
are added to Tris buffer A at a pH 8~0 in an ice
bath, and a mixture of the nucleotides ATP, GTP,
CTP, and UTP is added. A smaller batch to which
tritium-labelled UTP has been added is prepared for
control. Pipetting is always carried out by using
chilled pipettes. The optimum concentrations of the
individual components are determined by several
tests. Each sample is incubated at 37C in a water
bath, and an a~ueous solution of 1 per cent sodium
dodecylsulphate and 1 per cent aqueous phenol is
added after only from 3 to 6 minutes. The ~ op~
period of incubation will depend to some extent on
the concentration and the respective antigen and may
be previously determined by means of some offhand
tests. Cooling is effected in an ice bath, and cen-
trifugation is carried out with 2,000 g. The super-
natant contains the synthesized ~crude i-RNA.
d) Purification of the i-RNA
The i-RNA is isolated from the supernatant
via a discontinuous cesium chloride/saccharose gra-
dient. To this end, 1 ml of a cesium chloride solut-
ion having a density of 1.9 g/ml is covered with a
2~2
layer of 0.75 ml of a 30 per cent saccharose solut-
ion in Tris buffer A having a pH 8.0 in each centri-
fuge tube. 2 ml of the samples are placed on the
gradients, respectively, and the tubes are sealed
wi.th sterile paraffin. After a centrifuge run at
18~C in a Beckman Rotor SW 50.1 with 130,000 g for
4 hours, the cesium chloride layer is withdrawn.
This is done by piercing the bottom of the respect-
ive centrifuge tube with a thoroughly glowed needle
and transferring the total cesium chloride solution
up to the saccharose layer into tubes. The layer
thus obtained is diluted 1 : 2 with sterile water
and may then be fro~ed and stored at -20C. Further
purification is effected by sedimenting over a ce-
sium sulphate gradient. Therefor, 4O8 ml of a cesium
sulphate solution having a density of 1O5 g/ml are
chaxged into centrifuge tubes made of cellulose ace-
tate (Roto.r Beckman SW 50.1). 0.3 ml of the sample
are placed as a layer on the top thereof, and then
centrifugation is carried out at 18C with 130,000 g
for at least 4 hours (preferably from 12 to 19
hours). After the run, the tubes are pierced through
the bottom, and the contents is cut into 13 to 14
fractions, each of which comprises 9 drops (= about
0.4 ml). 0.1 ml of each fraction is used for the
determination of radioactivity, while the remaining
0.3 ml is diluted with the same volume of water and
stored at -20C. The fractions exhibiting the high-
est radioactivity having been determined, the corre~
sponding fractions of the main batch are selected,
which fractions co~tain the highly-purified i-RNA.
231 S2
- 12 ~
The fractions may immediately be injected
without further purification steps, if desired.
It is possible to detect the i-RNA by elec-
tron optics, whereby molecular lengths of between
1,500 and 2,000 nm were observed. The degree of pu-
rification may be determined, for example, by way of
agarose gel electrophoresis.
Another method of identifying the i-RNA com-
prises synthesizing antibodies in a cell-free sy-
stem. This is achieved by mixing together a sample
of the i-RNA, highly-purified ribosomes, the above-
mentioned enzyme system, leucine, and valine in Tris
buffer A pH ~Ø The mixtures are incubated at 7C
for 45 seconds, and then the protein synthesis is
stopped b~ freezing at -20C. The antibodies may be
separated and purified by affinity chromatography on
immunoadsorption columns and be detected analytical-
ly .
The highest concentrations of antibodies are
obtained upon addition of only 10 4 i-RNA. This
finding shows that one molecule of i-RNA is capable
of synthesizing at least 104 molecules of antibody
in a cell-free system.
It is further possible by using a RNA repli-
case, to increase the amount of i-RNA by a factor of
to 10 . Repetition of said steps for several
times results in a clonization. As the i-RNA thus
obtained has retained its properties relative to
antibody synthesis, there can be excluded that the
~2~L5~
antibodies have been synthesized by remainders of
antigens that might have been carxied throuyh.
Analysis of the i-RNA resulted in that the
molecular weight thereof is about 1 to 2 x 106. As
the antigens, there were used Aujeszky virus, ~erpes
simplex virus, influenza virus, MSK virus, Varicella
virus, measles virus, HBs antigen, E. coli O-anti-
gen, sheep erythrocytes, ADH, tetanus toxin, and
systems with tumor cells such as LC2 of guinea-pigs,
p815 of DBA2 mice, and the polioma virus-induced
tumor of Lewis rats. First signs of success were
also observed with attenuated measles virus "Mora-
ten" and HBS antigen. Respective tests revealed that
no cross reactions occur. Thus, highly specific
i~RNA are formed dependent on the respective antigen
used.
EXAMPLE 2
~ h~pQ-~r~us
An i-RNA specific towards ~a~ it~ B was
prepared, and its activity and compatibility were
tested with more than 200 patients. The i-RNA con~
centration was 10 5 moles/ml. In most of the cases,
the doses thereof administered to the patients for
one time were from one to two ml, while in rare,
more serious cases a second injection was applied.
The healing quota as presently surveyable is approx-
imately 100 %.
52
- 14 -
~XA~lPLE 3
BALB/c mice were three times injected with
complete Freund's adjuvant applying 100 ~l at a time
ln one week intervals. The commercially available
complete Freund's adjuvant was rnixed with physiolo-
gical saline in ratio of 1 : 1. The animals were
kept for four weeks after the last injection. Then,
their spleens were removed and processed to recover
the cell-free system.
The yields of i-RNA obtained by using said
cell-free system were increased by the factor of 10
Iten).
