Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
lOS630S
Background of the Invention
The thymus has been recognized as an endocrine gland
having a major function in the immunological defense system
of the body. Recently, crude extracts of calf thymus have
been shown to contain a family of hormones, referred to as
thymosins, having molecular weights ranging from approximately
3,200 to 70,000 daltons. See for example, Trainin, N.,
Physiological Reviews, Vol. 54, p. 272 (1974) and White, A.
Ann. N.Y. Acad. Sci., Vol. 29, p. 253 (1975).
_ _
These hormones are believed to act by regulating the
rate of maturation of incompetent, as yet unidentified,
precursor cells to competent lymphocytes (T-cells) and thus
to become effective fighters against foreign invaders. In -~
addition, precursor cells of other types, e.g., erythroid,
are stimulated in their production and maturation by thymosin
preparations. Recently, a relatively crude calf thymus
extract containing thymosin was used to increase the immuno-
logic competence of a young girl born with impaired immunity.
Wara, D.W., et al, New Eng. J. Med., Vol 292, P. 70 (1975).
Also a recent report has described improvement in the total
hematological status (increase in number of both white and
red cells) in patients treated with a thymosin preparation.
See Alexsandrowicz J., et al, Proc Intl. Congress Immunol.,
Brighton, England, July 22-24, 1974.
There have also been reports, see for example Bach, J.F.,
et al, Immunology, Vol. 25, p. 353 (1973), that thymosin-like
activity has been detected in fresh pig blood. The factor
responsible therefor is believed to have a molecular weight
of approximately 1,000 daltons.
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DESCRIPTION OF THE INVENTION
. .
The present invention is concerned with methods and
pharmaceutical compositions useful for increasing immuno-
logic competence.
For purposes of understanding this invention, the ~-
following is further background on its development. We
had isolated and characterized a protein having thymus ~ -
hormone-like properties of increasing immunologic competence.
This protein had a molecular weight of approximately
56,700 daltons and as having the following approximate
amino acid ratio:
Amino Acid Mole %
-
Aspartic Acid 6.6
: Threonine 9.5
Serine 9.3
Glutamic Acid 10.3
: Proline 6.6
Glycine 8.3
Alanine 9.7
1/2 Cystine 0.9
Valine 9.5
; Methionine 0.7
Isoleucine 4.0
Leucine 5.9
: 25 Tyrosine 2.3
-, Phenylalanine 3.9
, Tryptophan 1.0
Lysine 6.3
Histidine 3.1
Arginine 3.2
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as having glycine and histidine as N~terminal amino acids;
as having an isoelectric point of about 5,0 (now determined
as ~eing about 4.5); and ~eing composed of four sub-units.
It has now been found that our previously described
protein is identical with human serum prealbumin, a protein
occuring naturally in human serum, which has previously
been isolated, purified and identified by amino acid
sequencing. (Cf, Kauda, Y., J. Biol, Chem., 249, 6796-6805,
1974).
Identification oE the present protein as human pre-
albumin was made by a comparison of its physical and chemi-
cal properties with a commercially available sample and by
cross reactivity with human prealbumin antibody.
While human prealbumin has previously been described,
it has been thought to act merely as a specific carrier for
several selected smaller molecules and ha~ not been recognized
as exhibiting any intrinsic biological activity. We have
discovered, surprisingly, that human prealbumin has power-
ful thymic hormone-like properties of increasing immunologic
competence. Examination of commercial samples of human
prealbumin in selected bioassays, as described below, indeed
shows that these materials possess the aforementioned
biological activity, heretofore unrecognizea.
Accordingly, one aspect of the present invention is
concerned with a method of increasing immunologic competence
by administering an effective amount of human serum pre-
albumin to a subject in need of said treatment,
A second aspect of the present invention is concerned
with pharmaceutical compositions useful for increasing
immunologic competence comprising a therapeutically effective
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amount of human serum prealbumin in admixture with a
pharmaceutically acceptable non-toxic carrier.
A third aspect of the present invention is concerned
with methods of isolation and purification of human serum i
prealbumin from human blood fraction Cohn IV-l. I
As used in the specification and the appended claims
the expression "immunologic competence" connotes the degree
of responsivi~y of those physiologic mechanisms comprising
immunity. Immunity endows the host with the capacity to
neutralize, eliminate or metabolize foreign materials,
e.g., bacteria, viruses and fungi, as well as cells of
other animal species, without injury to the host.
This expression is well known and accepted in the
immunology art as illustrated, for example, in Bellanti,
G.A., "Immunology", W.B. Saunders Co., Philadelphia, Pa.,
1971. Two broad types o-f immunity are recognized, namely,
humoral immunity based upon the production of soluble,
circulating antibodies, and cell~mediated immunity based
Ipon the production and functioning of specific types o -
lymphoid cells (lymphocytes). Mature lymphocytes partici-
pate in both humoral and cell-mediated immunity (cf.,
Gell, P.G.H. and Coombs, P.R.A.j Clinical Aspects of
Immunology, 2nd ed., F. A. Davis Co., Philadelphia, 1968;
Miller, J.F.A.P. and Osaba, D~,Physiological Reviews, 47:
137, 1967; Trainin, N., Physiological Reviews, 54: 272,
1974; White, A., Ann. N.Y. Acad. Sci., 29, 253, 1975).
While not intending to be bound by any mechanism of
action, it is believed that the present material acts by
increasing both the number and rate of maturation of
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immunologically competent lymphocytes from precursor
incompetent cells.
The enhancement of immunologic competence may be
- demonstrated by various indicia utilizing both in vitro
and in vivo small animal bioassays well known in the
immunology art. For example, the following assays may be
especially mentioned:
1. In vitro a~athioprine sensitive rosette assay
(in vitro rosette assay)
2. In vivo azathioprine sensitive rosette assay
3. Antibody synthesis in vivo
4. Antibody synthesis in vitro
5. Proliferation of l~mphoid tissue
6. Mixed lymphocyte reaction
7. Blastogenesis with concanavalin A
8. In vitro spontaneous rosette assay
9. Production of cytotoxic lymphocytes
10. Lymphocyte auto-sensitization in vitro
11. Lymphocyte auto-sensitization in v _ ~
We have utilized each of the above bioassays to
demonstrate the enhancement of immunologic compe,ence by
human prealbumin, as detailed in the Examples.
The above mentioned assays relate to one or more of
the following general classes of clinical significance
for which immunologic competence is believed to be a
factor:
stimulation of antibody synthesis (especially
assays 3 and 4)
replacement and restoration therapy (especially
assays 5 - 9)
autoimmune diseases (especially assays 10 and 11).
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The assay of choice for a rapid and accurate indication
of activity for increasing immunologic competence is the
well known above mentioned in vitro rosette assay as
~ described by Bach, J.P., Proc. Natl. Acad. Sci. U,S.A.,
Vol. 68, p. 2334 (1971), wherein the sensitivity of spleen
rosette forming cells to azathioprine [6-(1-methyl-4-nitro-
5-imidazolyl)mercaptopurine] is measured.
This assay shows good correlation with the number of
immunologically competent lymphocytes of the T-class. It
is this class of lymphocytes that is involved in functioning
cooperatively with B lymphocytes in antihody synthesis, and
which are the prime cells that regulate cell-mediated
immunity. A large volume of evidence supports the signifi-
cance of this ass~y for assessing immunological status
(cf. Bach. J.F., "The Mode of Action of Immunosuppressive
Agents", North Holland/American Elsevier Publishing Co. r
; Amsterdam/New York, 1975)~
Accordingly, human prealbumin may be useful clinically
for human treatment in situations where immunologic competence
is believed to be an important fac~ol-, for ~xample, auto-
immune diseases, te.g., lupus erytl)ematosus, ulcerative
; colitis, autoimmune hemolytic ancmia, thyroto~icosis,
rheumatoia arthritis, hepatic cixrhosis) thymic aplasia
and dysplasia, au~mentation of immunity in in~ectious
~e.g., bacterial, viral and fungal~ disorders, Hod~kin's
disease, hypogammaglobulinemic syndrome, aberrant cell
proliferative conditions, decrease in immunologic compe-
tence due to temporal decline in thymic hormone production,
in chemical or radiologically induced immuno-suppressed
states, and so forth.
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It has been found that, when essentially free from
impurities, human preal.bumin prepared as described below
exhibits activity in the in vitro rosette assay at levels
below nanogram.
While it is desirable for many purposes to utilize
essentially pure human prealbumin in a suitable composition
for therapeutic administration, the tedious purification
and preparation of the pure material in large quantities,
necessitating considerable loss of material and the
attendant expense and effort, make it desirable to utilize,
for many therapeutic purposes, less pure fractions con-
taining human prealbumin, providing that such fractions are
free of cytotoxic impurities. Thus, it has been found that
partially purified fractions exhibiting activity in the
in vitro rosette activity at approximately 0.01 to 0.20
micrograms, are highly useful for therapeutic purposes. ~ :
Human prealbumin, either in essentially pure form or
as a component of a partially purified fraction free of
. cytotoxic impurities, may be made up in the form of con-
ventional pharmaceutical or medicinal preparations by
admixture with pharmaceutically acceptable, non-toxic
excipients. For example, the material can be mixed with ~:~organic or inorganic inert pharmaceutical carriers suitable
for parenteral administration, for example, intramuscularly,
-- 25 subcutaneously, or intravenously in the form of, for : -
example, liquid solutions, suspensions, and the like, in
unit or divided dosages.
. .,
The pharmaceutical compositions containing the present .~
material may be subjected to conventional pharmaceutical
expedients such as sterilization (e.g. by Millipore* filtration
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and can contain conventional pharmaceutical excipients such
as preservatives, stahilizing agents, emulsifying agents,
bulking/binding agents, salts for the adjustment of osmotic
pressure, or buffers. The compositions may also contain
other therapeutically useful materials,or materials which
prolong the duration of action of the present compound.
Actual methods of preparing such dosage forms are known or
will be apparent to those skilled in the art. An extensive
compilation of such formulation techniques may be found,
for example, in "Remington's Pharmaceutical Sciences" by
E. W~ Martin. One preferred method for preparing formula-
tions containg the present material is by reconstitution of
lyophilized human prealbumin. Thus, human prealbumin
prepared as described hereinbelow may be sterilized and
lyophilized either solely or with other solid excipients~
and stored in a sterile vial until needed. Immediately
prior'to administration, the desired amount of solvent,
e.g. water, water containing preservatives, or a solution
of various excipients in water, is added to dissolve the
human prealbumin.
In any event, the pharmaceutical composition to be
administered will contain a quantity of human prealbumin
in a therapeutically effective amount for treatment of the
particular condition of concern.
The dosage regimen may consist of unit or divided
dosages, but in any event will necessarily be dependent upon
the needs of the subject being treated and the judgment
of the attending medical practitioner. However, as a broad
guideline for mos~ purposes,'the present essentially pure
material will be administered in the range of from about
_g_
_~ ~"~.~Pr~.~ eP~.
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10 pg/kg/day to about 20 ~g/kg/day, preferably from about
100 pg/kg/day to about 3 ~g/kg/day. Expressed in alternate
terms ~or an average (70 kg) adult human subject this would
be from about 0.7 ng/day to about 1.4 mg/day, preferably from
about 7 ng/day to about 0.2 mg/day or on a schedule determined
by results of initial daily treatment at the above level. A
less pure fraction will necessarily be administered in
correspondingly higher dosage.
Human prealbumin may be isolated either as a component
of a partially purified fraction, free of cytotoxic
impurities, or in essentially pure form, starting with
human blood or a readily available human blood fraction by
means of a multi-step purification procedure.
A particularly valuable source of human prealbumin
is the human blood fraction known as the Cohn IV-l fraction-,
which is obtained, and general]y discarded as a waste
fraction, during the fractionation of human blood. This
fraction is thus available in large quantities from blood
sources and is relatively inexpensive. On the average,
the Cohn IV-l fraction exhibits activity in the in vitro
rosette assay at approximately 24 micrograms. In terms of
activity in the rosette assay, the Cohn IV-1 fraction
-~ represents approximately a ten-fold purification from crude
human serum. Thus, pure human prealbumin exhibiting
2S activity in the rosette assay below 1 nanogram, typically
from 0.2 to 1.0 nanogram, represents a 24,000 to 120,000-
fold enhancement of activity as compared with the Cohn IV-l
fraction, and a 240,P00 to 1,200,000-fold enhancement of
aetivity as compared with crude human serum. This enhanee-
3n ment in activity is believed due, in part, to the removal of
an inhibitory fraetion normally present in serum. The com-
plex puri~ication procedure necessary to achieve such a
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remark~b]e degree o~ enhancement of activity, and to rcnder the
resultant material essentially pure, is described below.
The procedure for the purification of the hllman pre-
- albumin, starting with the Cohn IV-l fraction involves
the following steps:
l.a. Molecular filtration to exclude high
molecular weight material and low mole-
cular weight material, or
b. ammonium sulfate fractiollation,
2. Gel chromatography,
3. Repeat of step 2, or free-flow electro-
phoresis,
4. Preparative gel electrophoresis, and
5. Gel chromatography.
During each stage of the purification procedure the
various fractions which are generated are routinely assayed,
preferably using the ln v tro rosette assay, to determine
the thymus hormone-like activity. The fraction or fractions
containing the major part of such activity are further
processed in the later stages, thus concentrating the
active fractions into smaller and smaller volumes, while
separating the active component from inactive impurities,
until the desired material is obtained in essentially pure
form.
While preferred embodiments of the purification steps
outlined above are described in Examples 1 and 9, a brief des-
cription of the purification procedure is presented below:
The crude Cohn IV-l fraction may optionally be lyo-
philiæed, if desired. This is conveniently done by sus-
pendin~ the crude Cohn IV-l fraction in distilled or
deionized water, stirring to break up lumpst and then
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removing solvent and other volatiles under reduced pressure.
The lyophilized material has greater stability and
may be stored in the cold for extended periods. Prior
to purification, the crude or lyophilized Cohn IV-l -
fraction is made up as an aqueous solution and preferably
centrifuged to remove sidement.
For molecular filtration, distilled or deionized water
is used as a solvent, preferably adjusting the pH to about
7.0, and for ammonium sulfate fractionation, an aqueous
buffer is utilized.
In one embodiment of the present process Cohn IV-l
fraction (crude or, preferrably lyophilized) in solution in
distilled or deionized water, is subject to two molecular
weights substantially above and below that of the desired
component. A suitable molecular filtration technique
involves, initially, the passage of the above-mentioned
solution through a hollow fiber fractionator with a suitable
membrane filter to remove components of high molecular
weight (greater than about 60-70,000 daltons, depending
upon filtration pressure. Suitable hollow fiber fraction-
ators are (depending upon uolume of substrate) the Amicon
DC-2 and DC-30 fractionators. In using the DC-2 fraction-
ator, a suitable membrane filter is the Amicon HIDX-50.
With the DC-30 fractionator, a suitable filter is the
Amicon* HIOX-50.
The filtrate from the above molecular filtration is
subjected to another molecular filtration to remove low
molecular weight materials. Thus, passage through a
similar hollow fiber fractionator fitted with a membrane to
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retain material of molec lar weight of approximately 10,000
daltons or highcr, results in the dcsired material, having
a molecular weight of approximately 56,700 daltons, being
isolated from the retentate. A suitable filter for this
purpose with the DC-2 apparatus is the Amicon HIDP-10, and
with the DC-30 apparatus, the Amicon ~iIOSM.
Alternatively, the filtration procedure may be reversed
so that first low molecular weight, then high molecular
weight, impurities are removed. The former procedure is
preferred.
In a second, and preferred, embodiment, the crude or,
preferably lyophilized, Cohn IV-l fraction is subjected
to ammonium sulfate fractionation. By this proceclure a
much higher (approximately g-fold) yield of desired material
is obtained cornpared to the molecular fractionation techni-
-~ ques described above.
The Cohll IV-l fraction is first dissolved in a suita~le
buffer having a pH between about 7.8 and 8.2 such as Tris
[commonly known as tris(hydroxymethyl)aminomethane]-sodium
azide and preferably centrifuged to remove sediment. The
solution is then treated at a temperature between about 0
and 5C., sequentially with poxtions of ammonium sulfate,
to "salt out" various fractions. After each addition of
ammonium sulfate, the precipitate formed is separated from
thè supernatant, which is then further treated with ammonium
sulfate.
The fraction containing the desired material is "salted
out", o~ precipitated, when ammonium sulfate is added such
as to bring the ammonium sulfate content of the solution up
from about 40 to about 60 percent af saturation. The
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quantity of ammonium sulfate necessary to reach the appro-
priate degree of saturation may be readily determined from
solubility tables.
The precipitate containing the desired material is
preferably desalted by techniques well known in the art,
for example by diafiltration or preferably by dialysis of a
solution of the precipitate in distilled water. The desalted
material is then concentrated in preparation for the next
step by, for example, lyophilization from the frozen state.
In the next step, the desired material from the pre-
vious molecular filtration or ammonium sulfate fractionation
is subjected to chromatography on a polysaccharide gel
column which fractionates components applied thereto by
molecular weight. Suitable columns are those prepared
from cross-linked destrans such as Sephadex* G-75, G-100,
G-150 or G-200 (Pharmacia Fine Chemicals). A preferred
material is Sephadex* G-150, as it affords an optimum
separation of components as determined by examination of
the K versus log molecular weight curve (see Andrews,
Biochem. J., Vol. 91, p. 222, 1964). Such a gel chromato-
graphy is suitably carried out by passing through the
loaded column an aqueous elution buffer having a pH of
approximately 7.8 to 8.2, preferably about pH 8Ø A suit-
able buffer is Tris-NaCl-NaN3. The temperature should be
between about 0 and 10C., preferably about 5C. Eluent
fractions are collected from the gel chromatography and the
fraction(s) containing the desired component may be deter-
mined by ultraviolet absorption, total protein eluted and/or
bioassay. The column may be calibrated so that the eluent
fraction containing the desired molecular weight range may
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be readily determined (see ~ndrcws, ci~cd above).
The desircd fraction(s) is preferably desalted by
dialysis or preferably by diafiltration and concentrated
in preparation for the next step by, for example, lyophili~
zation. The diafiltration is carried out under pressure
(e.g. 70-80 psi nitrogen) through a membrane with the
appropriate pore sizes, so that the desired mater;al is
retained. Suitable membranes that may be mentioned are
the ~micon UM-05 and UM-10, having molecular weight cut-off
ranges of 500 and 10,000, respectively. Other membranes
that could be used are the Amicon UM-2, PM-10 or UM-20E.
In the next step, the above ma~erial from the gel
chromatogra~hy is either recycled through the same or
similar gel column or, alternatively, is subjected to
free--~low electrophoresis. The free-flot~7 electrophoresis
is carried out according to procedures known per se in the
art. The p~l should be between about 5.0 and 5.5, preferably
about 5.25. A preferred buffer for establishing such pH
is sodium acetate-acetic acid. The location of the desired
component may again be established by the use of ultra-
violct absorption, total protein and/or bioassa~ methods,
and/or by use of a calibrated column.
The desired material obtained from the previous step
is then subjected to preparative polyacrylamide disc gel
electrophoresis, in the usual manner for such procedure.
It is preferred that such electrophoresis be carried out in
a manner such that the separator gel and the elution bufer
are at a pH between a~out 8.5 and 9.5, preferably about 8.9.
A preferred buffer for establishing such pH is Tris-HC1.
30 - Normally, after this procedure, there is obtained
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electrophoretically homogeneous human prealbumin. The
homogenity of the component obtained may be determined by
standard analytical techniques such as the use of analytical
disc-gel electrophoresis or isoelectric focusing, and the
hormonal activity may be determined by bioassay. As a
final step, the material is freed from polyacrylic acid and
residual salts by chromatography as described above, on a
micro-polysaccharide column, preferably of Sephadex G-75.
The first peak that elutes from the column contains the
active material as judged by the above-mentioned criteria,
and the product itself may be obtained by lyophilization.
In the following specific examples there are described
the preparation of both essentially pure and partially
purified human prealbumin, as well as descriptions of the
bioassays utilized and the results obtained therefrom. It
should be recognized by those skilled in the art that the
descriptions contained herein are illustrative only of
the invention and should not be construed as limiting the
scope or spirit of the invention in any manner.
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EX~MPLE 1
Purification of human prealbumin from Cohn fraction IV-l.
Four kilos of Cohn human blood fraction IV-l (Cutter
~aboratories) were stirred in 20 1 of double distilled
water at 5C for four hours and lyophilized to afford 1105 g.
of dry material which was suspended in ten volumes of deionized
water. The pH was adjusted to 7.0 with ammonium hydroxide
and the mixture stirred for one hour at room temperature.
The sediment was removed and the supernate passed through
an HIOX-50 cartridge in an Amicon DC-30 apparatus. The
lower molecular weight fraction from this step was then
passed through an HIOSM cartridge in the same apparatus.
After lyophilization of the retentate there was obtained
50 g. of material which was chromatographed, in one gram
portions, (after dissolution in the below-mentioned buffer)
on a 5 x 90 cm. G-75 Sephadex (Pharmacia Fine Chemicals) -
column equilibrated with 50 mM Tris-100 mM NaCl-.02% sodium
azide buffer, pH 8.0, at 5C. The chromatography was
monitored by optical density measurements at 280 nm, the
contents of each tube were lyophilized and diafiltered to
determine total protein, and then subjected to an in vitro
rosette assay. Over 90% of the activity eluted was
recovered in a fraction slightly retarded on this column
with a K v value of 0.055, corresponding to an approximate
molecular weight range of about 50,000 to 70,000 daltons.
This material (total from all one gram runs - 20 g.) was
; then subjected to free-flow electrophoresis at pH 5.25 in an
acetate buffer system (25 mM NaOAc-7 mM HOAc). As above,
the progress was monitored by optical density, total protein
i 30 and bioassay. The material resulting from combination of
active fractions was further purified by preparative poly-
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acrylamidc ~el clcctrophorcsis using a 10% cross-linked gel.
The spaccr and sample gels were 0.060 M in Tris, adjusted to
"r~ 6.6-6.~ with HCl. The separator gel was 0.375 M in Tris,
' adjusted to pll 8.9 with IICl. The-electrode vessel'buffer was
0.024 M in Tris and 0.192 M in glycine, pH 8.2-8.4. The
elution buffer was 0.375 M in Tris, adjus~ed to pH 8.8-9.0
with HCl. The activity was eluted in a ~raction migrating
between the tracking dye (Bromophenol Blue) and the albumin
fxaction. This material (approximately 100 mg. total) was
electrophoretically homogeneous when run in an analytical
system at pH 8.9.
~epeat of the Sephadex chromatog-aphy, as described
above, on a micro (0.8 x 76 cm.) column ~fforded essentially
pure prealbumin as a white solid.
The S20W value determined by sedimentation velocity in
the Spinco model E ultracentri~uge was fo,und to be ~.07 and
the molecular weight of the molecule determilled ~y s~dimen-
tation equilibriurn was found to be 56,700 daltons. After
- treatmen~ with 5M guanidinium-hydrochloride - 0.8% dithio-
threitol -O.OlM phosphate buffer pH 5.2 oJlowed ~y 5M urea -
0.1~ sodium clodecyl sulfate - 1~ dithiothreitol -O~OlM phos-
; .
phate buffer pH 7.4 followed by electrophoresis at pH 7.0 in
a sodium dodecylsulfate ~ containing acrylamide gel, the
molecular weight of the undissociated material was ~ound to
be S2,000 daltons and there was partial dissociation into
four sub-units with a molecular weight of approximately
13,500 daltons each.
The protein was homogeneous upon isoelectric focusing
in polyacrylamide ~el containing ampholines with a p~I range
o~ 3-10, and possesscd an approximate isoelectric point o
4.5. N-terminal analysis obtaincd hy dansylation ind~cated
the presence o derivativcs corresponding to dansyl
i
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glycinc a~ dansyl histidinc. ~mino ~cid composition w~s
de~crmined ~y hydrolysis o~ the protcin and chroma~ogra~hy
on a Durrum ~mlno acid analyzer undcr standard conditions
and af~ordcd the following comp~sition:
~mino ~cid . Mole
~spartic ~cid - . -6.6
Threonine 9 5
Serine - 9.3
Glutamic Acid 10.3
Proline . 6.6
Gl~cine 8.3 ' . ,
Alanine -9.7
- lJ2 Cystine 0.9
. Valine 9.5
- 15 Methionine 0.7
- Isoleucine - ~.. o
Leucine 5.9
~yrosine 2.3
, Phenylalanine 3.9
, Tryptophan 1.0
Lysine 6.3
istidine 3-1
~rgi.nine 3-~
Identity was further established by comparison with
authentic samples of human prealbumin.
~,
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EXAMPLE 2
,
In vitro rosette assa~ during purification of human prealbunlin.
. . .............. _ . .... . . . .
~ The ln vitro rosette assay, was performed essentially
as described by Bach, J.F., et al, Proc. Natl. ~cad. Sci.
. . _
U.S.A., Vol. 68, p. 2734 (1974). The exact protocol was
as follows:
A. Prcparation of azatllioprine sodium salt stock solution.
277 Mg. of azathioprine free acid, (Imuran, Burroughs
Wellcome), was dissolved in 25 ml. of water in a volumetric
flask. Approximately 1 ml. of lN NaOH was added dropwise
with stirring to dissolve all of the powder. The final solu-
tion was diluted 1:100 in Hank's balanced salt solution, pH
7.2, prepared from powdered medium (Difco Labs., Detroit, Mich.)
to afford a solution containing 0.106 mg./ml. of azathioprine
sodium salt, pH 8.2. This solution was filtered through a
millipore filter to effect sterilization. The final solution
~as stored away from light in a refrigerator. Stability was
checked by measuring optical density at 280 and 330 nm.
B. ~or the assay itself, spleen cells from G-8 week
old male C57Bl/6 Simonsen mice thymectomized 7-30 days pxior
to sacrifice ~ere used. Individual spleens were homogenized
; and washed in cold Hank's balanced salt solution. The cells
were pelleted at 200 x g for ten minutes in a refrigerated
centrifuge. A inal pooled suspension of 40-60 x 106
~S nucleated cells per ml. was prepared.
Control series: to determine the sensitivity of
thymectomi~ed spleen cells to azathioprine sodium salt, the
i azathioprin~ sodium salt was ~itrated, ranging from 25 ~g./tube
to 1.56 ~./tul~e, usin~ 2-fold serial dilutions of 0.2~ ml. of
8~0ck solution (part A)in llank's. Spleen cell suspension
.
~` -2~-
. . .
1C)5ti305
.
p~cpared ~ovc (0-1 Inl.) was added to eacll dilution
~4.~ x 106 ccll/tube),
Te~t series: test fractions of human prealbumin (0 125 ml
.
aliquots) were serially diluted 2-fold in Hank's. 2.5 Micrograms
a7~athioprine sodium salt in 0.1~5 ml, of Ikll~k's; (see abovc)
~nd 0.1 ml. o~ s~lc~l~ ccll suspension, were addcd to each
dilu~ion of thc test raction.
Both the control and the test series were incubated at
37C. in a water bath for 60 minutes. 0.2 Ml. of 50% sheep
erythrocytes (SR~C) in Alsever solution (Grand Island Biological
Co., "Gibco", Grand Island, N.Y.), prepared 2 days previously,
was diluted in 15 ml. Hank's. 0.125 M1. of this SRBC suspension
was added to each tube in both series, and the cells were
pelleted in a refrigerated centrifuge at 200 x g for 5 minutes.
Pelleted cells were refrigerated at 4C. for 90 minutes,
gently resuspended on a rotator for five minutes and the
rosettes counted in a ~alessez hemocytome~er.
The specific activity was determined by the minimum
quantity of proteill in the test fractions that inhibited
rosette formation by 50% or more in the presence of
2.5 ~g./tube of azathioprine sodium salt. The specific
activity of the fractions obtained during purification
(Example 1) is shown in the following chart, as is a
relative purification factor, using the Cohn IV-l fraction
as a reference.
,
- 30
-21-
.,
.. ~ , . . .
105~305
~cl:ivity in
~osettc ass;lyruri~ication
Stcp Fraction (~IcJ of pro~cill? factor
1. Cohn fraction IV-l 24
2. DC-30 l-7IOSM 3 8
cartri.dge retentate
3. Chromatography on 0.1 240
Sephadex G-75, pll 8,
fraction 2
4. Fxee-flo~ electro- 0.01 2,400
phoresis, pH 5.25,
fraction 2
5. Preparative polyacxyl- 0.002 12,000
amide gel electro-
. phoresls pH 8.9,
fraction 1
6. Chromatography on micro- 0.0004. 60/000
Sephadex G-75, -
. fraction
The above chart demonstrates the use of the in vitro
rosette assay to monitor the puri~icati.on of human pre- -
albumin, and demonstrates that a 60,000-fold puxification
tbased on activity) ~rom Cohn fraction IV~l has been
~0 achieved.
.
.
.
. 2S
, . .
.. .
.~ . . .
. 30
.
~ -22-
. ~ ,
105~305
EXAMPLE 3
Stimulation of antibod~ synt:hes_ .
~ When normal animals, or animals that are experimen- ¦
tally depressed in their immunological responsivity, are
injected with a foreign antigen~ e g ~ a protein obtained
from another species, stimulation occurs of an immunologi-
cal defense mechanism, e.g., synthesis of antibodies that
have the capaclty to "neutralize" the foreign material.
Protocol: Normal mice (C57Bl/6 strain) were trcate~,
by a single intravenous injection, with sheep erythrocytes
(SRBC) as an antigenic stimulant. At the same time the
test mice were injected intravenously ~itll a quantity of human
prealbumin (an appropriate fractiQn as depicted on the
chart ln ~xample 2)~ Control mice received the antigen
and bovine serum albumin (BS~). Seven days later the
animals were killed, the spleens di~sect~d out and each
spleen utilized for antibody assay ~n vitro acc:ording to
the method o ~erne, I-I.K., et al, Science. Vol. 1~0,
p. ~05 (19G3). The antibody response is ~xpressed as the
number o plaque forming cells (PFC~ per 106 spleen cells.
A. The following chart illustrates antibody orma.ion
utilizing a human prealbumin fraction-("hormone") corres- j
ponding to step 2 from the chart in Example 2. Both l9S(IgM~
and 7S(IgG) antibodies were assayed.
, .
-23-
~, I
i ,... .
. - . :
1056305
. ` PFC/10~ Spleen Cells
Material Ir~ectcdl9S(IcJM) 7S(IgG)
Saline 33 2
SRBC 105 114
SRBC -~ 10 ~g. hormone 193 112
SRBC ~ 100 1l~. hormone249 173
SRBC + 400 ~. hormonc 276 310
Saline ~ 400 llg. hormone
Saline ~ 400 ~g. BS~ 3
o sr~c + lo ~J. BS~ 120 119
SR13C ~ 100 1l~. BS~ 154 76
SR~C ~ ~00 l~. BS~ 15~ 152
The data show that the human prealbumin administration increased
` 15 significantly the capacity of spleen cells to synthesize 7S and
l9S antibody.
B. The effect of human prealbumin ("hormone") Gn antibody
synthesis can also be demonstrated in an in vitro assay. In this
case spleen cells from C57Bl/6J mice were cultured and were then
~, 20 treated either with sheep erythrocytes, sheep erythrocytes plus
hormone, or hormone alone. The hormone utilized in this
experiment was from step 3 from the chart in ~xample 2.
The antibody response was measured after five days of
culture. Only the 19S antibody was measured. The results
are expresscd in the ~ollowing chart.
,~ .
~ .
.
-24-
1056305
Mat~rial added to19S ~ntibody response- :
pleen ce].l c~lture(P~C/106 spleen cclls)
_ 46
SRBC 1332
SRBC ~ hormone (10 ~g.)2097
SRBC ~ hormone ~50 ~g.~1964
Hoxmone (50 ~g.) 58
. . ~
The abo~e results demonstrate that the human prealbumin
-preparation was active in stimulating IgM(19S) antibody
synthesis when added i.n vitro.
.
'.
. ,
-25-
'
lOS6305
X~ I.E 4
~fect o~ }lumall pl-ealbumill in neonatal~, thvmectomized mice
It is well est:ablislled that neonatal thymectomy
~ ~1oducer. all "imm~u~ologic cripple". .The operatcd animals
~ail to ~row normally, show was~in~ and havc little
rexistaoce to incctions, call not synt-llesizc antibody in
rcsponsc to challengc by specific antigens, and have no
c~11 m~diatcd immunity, i.e., cannot reject a skin graft
from an allo~onic strain of the-same species. Gcnerally
~o such operated animals die within five to ten wee}is-post-
operatively from generalized infections, depending upon
the degree to which the environment in which they are housed
is free of infectious agents. This neonatal thymectomy
syndrome has been d~monstrated in a variety of species and
is also seen in children born either without a thymus or
with thymic aplasia or dysplasia, Miller, J.F.A.P., et al,
Physiol. Revs., Vol. 47, p. 137 (1967) and Trainin, N.,
Physiol. Revs., Vol. 54, p. 272 (1974).
Protocol: C57Bl/Ka mice were thymectomized within
24 hours of birth utilizing surgically sterile technigues
and housed under conditions free of specific infectious
pathogens. Groups of ten each of these neonatally thymecto-
mized mice were treated as indicated below. Bovine serum
albumin (BSA) was used as a control protein.
~. Antibody synthesis.
Beginning nine weeks post-thymectomy, each mouse
was injected intraperitoneally on alternate days (total of
eight injections~ with 1 mg. per injection per mouse of
either a humal prealbumin preparation ("hormone") or BSA.
The hormone utilized for this experiment was that from step 2
-26-
.~ '
- 105~305
from thc char~ in ~xample 2. The animals were sacrificed
one wee~ after the ]ast injection or hormone and ~ive days
after a single intrapcritoneal injection of the antigen,
sheep erythrocytes. Antibody titers (19S) were determined
according to the method of Mishell, R. I., J. Exp. Med.,
Vol. 126, p. 423 (1967). The results are expressed in the
table below.
l9S J~n~ o~ly ~,s~ ~ 106 ~plcen Cclls
l~ormonc Trc~ated Mice BSA Trcated Mice
. _
58,650 9,500
The above cha1t demonstrates that the administration of
the human prealbumin preparation restored to neonatally
thymectomized mice the ability to synthesize l9S antibody
in a response to a challenge of sheep erythrocytes, a
thymic dependent antigen.
B. The mixed lymphocyte interaction.
When lymphoid cells of mice of a specific strain
are mixed ln vitro with lymphoid cells of mice from an
unrelated strain, the "foreign" nature, from an immunolosi-
cal point of view, of the cells to one another results in
a proliferative response of each cell. As indicated below,
one of the two cell populations can be prevented from
proliferating, or undergoing blastogenesis by adding to
one cell population prior to mixing, an agent that inhibits
cell division. The ability of cells to recogni~e foreign
cells is a reflection of a cell-mediated, immunological
response. This is the so-called "one way" mixed lymphocyte
reaction.
~27-
105~305
L~nphoid c~lls from n~onatally thymectomized mice or
from gell~tically thymusless (nude) mice are incapable of
this typ~ o~ recogni-tion, i.e., do not react in the mixed
- lymphocyte reaction when incubated with lymphoid cells from
adult m~ce of another strain.
The protocol used was that described by Goldstein,
A.L., et al, J. Immunol., Vol. 106, p. 773 (1971).
The data shown below were obtained with lymph node cells
from neonatally thymectomized C5~Bl/Ka mice treated at four
wee~s of age wiLh sin~le subcutaneous injections, over a
two week period, of 1 mg. each of"hormone"corresponding to
Step 2 from the chart in Ex~mple 2. A control group of
neonatally thymectomized C57B16/~a mice rcceived similar
doses of 1 mg. of bovine serum al~umin. The animals ~7ere then
sacri~iced and the lymph nodcs (mesentellc, axillary and
inguillal) and splcens dissect:ed out. Cell susl~ension~ of
lymph nodes of each mouse were tcsted in thc mixcd lym~hocyte
; rcaction, usin~ cqual numbers of C~7BlG/I~a lymph node cclls
(~) and allogenic (BL~) cells (B). "Background'incorporation
for each cell type was obtained separately and subtracted
from the mixed cell reaction value to afEol-d the difexellce
in counts per minute (~CPM), result;n~ from the interacLion
of the two cell types.
The data obtained were as follows:
.~ .
. ' .
-28-
`
:1 '
1056305
~ymi.clin~ Incor~r.~tion
Treatment ~CIM St.~mulation Index
Bovine Serum Albumin 11,596 28.
Hormone - -1~,358 48.8
~CPM - A X B - ~A + B)
2Stimulation index A + B
It is clear that the lymph node ceils ~rom the animals treated
with the human prealbumin preparation showed an appro~imately
two-fold increase in the sti.mulation index. These data
indicate that the cells from the neonatally thymectomized mice
treat-ed ~ith this preparat:ion were significantly enhanced in
thei.r-immunolo~ical competence as reflected in their capacity
to "xecognize" lyrnph nod~ cells of an histoincompatible strain
of mice.
,
.
.
, 25
., .
' 30
;
, -29-
1'
1056305
EX~MPL 5
~rolieration o Lymplloicl Tissue
. Mice deprived of their thymus gland within 24-48 hours
of birth (neonatal thymectomy) or genetically athymic
(nude) mice fail to develop normal lymphoid tissue. I'hat
is, their lymph nodes and spleen (the lymphoid organs) are
smal]. and show a paucity of lymphocytes. This is a reflec- !
tion of the fact that early in life, the thymus produces
large numbers of lymphoid cells that are exported to, and
ln seed, the peripheral lymphoid organs when these cells pro-
liferate. This normally results in growth and maturation
of normal lymphoid structures.
Protocol: Genetically athymic (nude) mice 4 weeks of age
were given 8 daily subcutaneous injections of 1 mg. of human
prealburnin preparation ("hormone") corresponding to Step 2 ~rom
the chart from Example 2 over a two week period prior to sacri-
'ice. Control mice received injec-tions of 1 mg. bovine serum
albumin. The lymph nodes (mesenteric, inguislal and axillary)
were dissected out, blotted, pooled and the number of total
lymphoid cells counted. The data are as follows:
~ .
;~ Bovine Seru~ ~lb_min ~ormone
Tis.sue No. of Cells - No. of Ce]ls
Spleen 1.02 Y~ 108 1.8 ~ 108
~S Lymph Nodes21.3 x 106 41.5 x 10
"
- It may be noted that proliferation of lymphold cells
in normal mlce is known to be under thymic regulation.
The genetically thymusless (nude) mouse has small, undevelopecl
lymphoid tissue. The above data indicate an increase in
human prealbumin treated mice of numbers of lymph node
cells by a factor of ~2 x 102, i.e., a 200-fold increase,
~30~
1056305
EX~M~'LE 6
Stim~llati(>n b~y Mitoqens: Blastogen~sis on Exposure to
Concanavalill A.
Lympho;d cells from nude mice, because they are immuno-
logically unresponsive, do not respond to concanavalin A,
a mitogen ~nown to act on immunologicaliy competent
T-cells, to accelerate their matura-tion and differentiation.
Protocol: Treatment of nude mice was as described
for proliferation of lymphoid tissue (Example 5) using
the same preparation and quantity of human prealbumin. The
assay used is described by Claman, H. N., J. Immunol., Vol. 112,
p. 960 (1974) based upon the incorporation of 3H-thymidine
into lymphoid cells incubated in vitro with and without
addition of concanavalin A (Miles-Yeda, Kankakee, Ill.)
}5
Data obtained: CPM*
Cells from BSA treated mice ~ phosphate-ringer 110
buffer -t ccncanavalin A
Cells from hormone treated mice ~ phosphate-ringer 419
buffer + concanavalin A
*Avera~e of 3H th~midine incorporated per sin~le incubation
tube. j-
:1 I
Injection o the human prealbumin preparation increase~
by approximately ~-fold the mitogenic response of the cells
to concanavalin A~ Thus, administration of this material
to nude mice increased the number of host cells exhibiting ~ `
T-cell properties in their responsivity to the mitogcn.
.~ I
-31-
' 1-
. !
.- .,...................................................................... I
- , - - ; . . -. ... ` . - .. . .- . . ., ~
.: . .. .- ` ` .. ~ .... ` .. ` :
... . . . .. . . .
lOS6305
~ MI~rJI~ 7
In ~;t:~o Mat~ ;lt:i~n o~ Lyml)hocytcs to Immurlolo(lically
Compe~cnt: T-cells.
Th~ numbcrs o~ spont~neous erythrocytc (~) rosette
fo~-ming cells in periplleral blood lymphocytes is one index
of host immunolo~ical competcnce since this index r~flects
the number of circulating, immunologic~ll.y competent T-cells.
In normal individuals, the number of E rosette cells in
the peripheral blood generally represents 65-80% of the total
lymphocyte population. Values below this range are often
observed in i~nunologically deficient states, including
malignant diseases, th~mic aplasia or dysplasia, rheumatoid
arthritis, etc.
Protocol:. Lymphocytes were separated from human
peripheral ~lood on a Ficoll-Hypaque ~radient, Boyun, ~. r
Scan. J. C~in Lab. Invest~, Vol. ~1: Supl. ~7, (19~
T lymphocytes were identified by spontaneous rosette foxma-
. tion with sheep erythrocytes, Bentwich, Z. r et al, Cl;n.
Immunol. Immunopath., Vol. 1, p. 511 (1973). A chan~e of -
2p 10~ (on a scale of 100~) is consi.dered significant. The
data.obtained are as ~ollows:
.
'~ ' ' "' ' '' .
~ 30
... . ... ...
. ~ ' ~ ,~ , ' - '
105~305
INI~'I.UENCI~ OF lli]~N rr~AL~UMIN (llOr~MONE CORRE~rONDIN~ TO ST~P 3,
Cll~l~T, F,X~lrLrJ 2) ]N V[rl'~~O ON PI~CE:NT OF SPONT~NL,OIJS ERYTII~OCYTE
(E) ROSETTL I;'ORMrN~. CLL,].S IN PER]:PIIER~L BLOOD LYM~IIOCYTLS OF
NOI~MAI. INDIVII)U~LS ~N~ TIEN'rS WITII IIODG~IN'S DISl'ASE
-~Inl~ it or~
Lylnphocytcs from . Splcen +RIS(l)
Scve~ llormal Initial ~ ~50 ~. Extract ~ 50 1~
5 Individuals E Rosettes llormone (RIS)(l) Hormonc
~.D.* N.D.*
2 65 65 N .D . N . D .
3 61 68 N.D. ~.D.
4 . 66 5~ N.D. N.D.
S ~ 6G 60 47 68
6 613 69 4~ 6~
7 72 69 S~ 71
Lymphocytes from
Six Patients
with Hodgkin's
D.isease
_
67 66 N.D. N.D.
2 . 6.~ 72 N.3. N.D.
3 ~ 48 70 N.D. N.D.
4 55 70 N.D. N~n~
62 - 78 43 66
- 6 43 70 ~ 54
~-N.D. = Not done ~1) 10 ~Ig. protein/assay tube
The above results suggest that in v:Ltro incubation wit.h -the
human pr.eal-~umin preparation of separated, peri.pheral
~lood lymphocytes from individuals that. have lower tharl normal
percentages of spontaneous ~ rosette formin~ cells results in
an incre2s~ in the percentage of these c~lls. Such data have
been interpreted to indicate that inadequate thymic hormone
concentration is the basis for the lower numbcrs o~ E roset-tc
formin~ cells in these cli.nical conditions. In contrast,
precursor cells arc apparcntly prescnt in these individuals
sincc incubation o~ t-.hcsc cells with added human prealbumin
prcparatioll incrcases tllc numbc!r oi spont~ncous ~ rosctte
forn)i.n~ cclls.
~ -33-
: ~ ,
- ' ' '. ' ' ~: ' - ' ' ' -
- 105f~3V5
llocl(Jkin'; ~is(,lse ~-~nticnts frcqucnt].y show lowcr thcln
no~ al num~er~ of ;pon~ancolls E rosct~e ~03^mincJ cclls. Th2
ahovc~ data 1eveal ~11at peri~l~eral lymphocytes of S out oE 6
o thc llodcJkil1's patients studies showcd, on incubatioJ1
with human prealbumin preparation, a slgnific~nt increase
in thc perccn~ of spontaneous E rosctte forming cells.
.~ In addition, the human prealbumin preparation
counteracted the inl1.~bi~ory effect of an eY.tract o. spleen
from Hodgkin's patients on the numbers of spontaneous ~
rosette formi11c~ cells in the peripheral blood lymphocytes of
n~rmal indi~ric1uals and in one pat~.ent with Hodgkin's disease.
5
-34-
105~305
I~X~ IPL~: B
_ _
~ct; vi tx_f lluln.~n Pr~
The above sheep roset~e forming cell assay referred to
in Example 2, ~as applied also in assays in which a highly
purified human pr~albumin preparation (correspondiny to Step 6
from the Cha~t in Example 2) ~as administered in vivo. The
protocol was as follows:
The preparation to be assayed was administered to normal
adult C57B1/6 mice (5-~ ~eeks of age) that had been thylnecto-
mized two wee~is prior to use. Daily injections oneach of three successive days were gi~en intraperitoneally
in doses of 2-4 ~g. per mouse.
Twcnty--four hours ~ollowin~J the last in~ction~ the
animals ~cre sacriiced, the splecns quicl;ly rem~ovecl, cell
15 suspensions prepared and used in the rosette ass~. Th~
data are represented be]ow:
End Po~nt
Norrnal mouse, not thym~ctomized 0.78 ~
aza1;hioprine
Thymectomized mouse, injected with 25 ~g.
saline azathioprine
Thymectomized mouse, injected with O.lg5 ~.
2 ~y. preparation az~t~lioprine
Thymectomized mouse, injected with 0.098 ~g
~ ~g. preparation azathioprine
The above data show that immuno~o~ical competence ~as
restored to the spleen cells of thymectomized mice by the
administration of the preparation. It should be noted that
iniection of the purified human prealbumin preparation
restoxed to spleen cells of tllymectomi~d mice a sensitivity
~3
to the inhibitory effec~s of azathioprine, bascd upon numbcrs
-35-
,
. :
:, ` . : ' '
1~5~31)5
oi~ spl~en ros~t:te for.millrl c~lls, equal ~o t ha~ of norlnal
mouse s~ n c~lls. Th~ absolutc ac~:ivity of 1:hc pr~paratior
uscd i.n thi.s assay is, in t:hc ln vitro assay, 0.4 ny.
.
-36-
~ . . . . . . ..
`. ' ' ~ ' . ' ''
105~305
_Xl~ r. I~ 9
Alternate purificatiorl of human prea]bl1min from Cohn fraction ~V-l
__.. _ _ ..................................... :. ... . .
100 G. of Cohn frac~ion IV-l (wet weigllt) is stirred
-- with 500 ml. of distilled water f~.r 4 hours at 2-5~C. The
suspension is then lyophilized, yielding approximately
40 g. of a dry powder. This material is then dissolved in
1,000 ml. of 50 m~1 Tris-100 mM sodium chloride - 0.02%
sodium azide buffer, p~l 8.0, and stirred for 3 hours at
ro~m temperature. The solution is then centrifuged at
13,000 x g.for 30 minutes at 2-5C. Tne clear greenish
supernatallt in a suita~le container surrounded by ice is
then made 40% saturated with a~monium sulfate by the additiorl
of 243 g. o~ ammonium sulfate to 1,000 ml. of the supernatant.
The suspension is t~llowed to settle at 5~C. o~ernight and
the precipitate removed by cent~ifugation. The supexnatant
is then made 60~ saturated with ammonium sulfate by the
addition of 132 g. oE ammonium sulfate to 1,000 ml. of the
supernatant and the precipitate collec~:ed as before. ~the
precipitate is then dissolved in tlle minimum volume o~ cold
distilled water and dialyzed exhaustively against distilled
; water in the cold an~ lyophilized to afford 17 g. of material.
The above material, in 1 g. aliquots~ is applied to a
5 x 90 cm. column of Sephadex G-150 (total bed volume of
1,760 cubic centimeters~, equilibrated with 50 mM Tris -
100 mM sodium chloride - 0.02% sodium azide, pll 8.0, and
- the column eluted with the same buffer. The material eluting
in the molecular weight range of 40,000-70,000 daltons is
collected, desalted by diafiltration through a UM-10 ~micon
membrane at 70-80 psi nitrogen pressure, and lyophilized.
i 30 The total material obtained after all o~ the 1 g. runs on
,~' ' .
, 3,--
. ~
.., ..
.i -
1~5~30~
t:he ~;el?~ ]c,Y col.umn is ~ y.
~tcr recllromatography of this mate~i.al, in 1 g.
ali.quots as desribed abov~, usin~ the same Sephadex
- column, followed by diafiltration and lyophilization as
above, th~re is obtained 2 g. o~ material.
The chart below illustrates the weight of material
and the activity in the in vitro rosette assay, as described
in e~ample 2,
Dry Weight Activity - Rosette
10 Material (Grams) Assay (~gs.)
_ . _ . . ..
Cohn IV-l ~0 12
~0-6~ ammonium 17 1.6
sulfate precipitate
G-150~ fracticn 3 4 0.2Q
G ].50, fractioll 3, 2 0.02
repeat
The material from the above pxocedure may be.subjected to
preparative polyacry:Lamide gel electrophoresi~ and chroma-
. tography on a micro column as dessribed in Example 1to afford the subject material, essentially rree of i.mpuri-
ties, exhibiting activity in the in vitr.o rosette assay
comparable to that c~escribed in Example 2, i.e., from
about 0.2 to about 1~0 ng.
. .
'., " ' ' ' ',
`, 30
~ 8
., ~ . . . .. . . . . . . . . . . . . . . . ..... . . . .. ... ... . . ...... . .. . ... . .
... .
~ ' . ' '
1(~5~305
~XAMPL~ 10
EEfe.ct_o _lum_rl Prealbulni.n In Vitro on Promoti.ng Maturation
of Immunoloc~i.cally Competent Cells
~ Inmature thymocytes from norm.al mice are not normally
T cells with significant cytotoxic activity. Acceleration
of the maturation of these thymocytes to lymphocytes
-~ exhibiting cytotoxic activity is an indication of the
enhancement of immunologic competence.
Protocol: 1 x 107 thymocytes from C57Bl/6 mice are cul-
tured with 5 x 106 irradiated Balb/c mouse spleen cells, with
and without 5 ~g material corresponding to the last step in
the chart in Example 9, or 5 ~g BSA, for five days. Cyto-
tox.i.city is measured on P815 Y(H-2d) cells labeled with
-. 51Cr
% 1Cr Release
, Thymocytes + Ba].b/c 1 5 ~g BSA 15
!j Thymocytes ~ Balb/c -~ 5 ~g human pre- 45
albumin preparation
The da-ta show that ].ymphoid cells that normally do
not exhibit cytotoxic activity show, in the above data,
the development of significallt cytotoxicity as a resulr of
their exposure in vitro to the human prealbumi.n preparation.
.,
::,
:
' 30
.~ .
~ -39-
:`
:
,,,~
' ~ ' ~'-' ` ' , '` '
105~;305
EX~MPI.E 11
As intlica~ed above in ~ample 5, lymphoid cells from
nude mice are cJenerally immunologically unresponsive. A
study was conducted of the effect of a human prealbumin
preparation on eliciting an immunological response from
nude mouse spleen cells incubated in vitro. The mixed
lymphocyte reaction (see E~ample 4, above) was the a,ssay
~' system.
Protocol: 5 X 105 spleen cells of nu/nu mlce (Balb/c
background) were cultured with 5 x 105 irradiated Fl (Balb x
C57/K6) mouse spleen cells for three days, with tritiated
thymidine added for the last four hours. A concentration
of 2 ~g/tube of human prealbumin preparation (hormone)
corresponding to the ~ast step in the chart in Example 9
was utilized. Values are means + S.E. for three experiments.
, ' .
Cell Source Syngeneic Allo~eneic
Hormone BSA Hormone BSA
nu/nu spleen 1241+74 1027+~5 6725+75 1567~r67
1 20
'~ The data show that, whereas the nude mouse spleen cells
¦ incubated with bovine serum albumin did not respond in the
~ mixed lymphocyte reaction, similar cells incubated with
-3, 2 ~g of the hormone preparation per 5 x 105 spleen cells
did effectively endow these cells with the capaci-ty to
- function in the mixed lymphocyte reaction. Thus the hormone
converted immunologically non-responsive cells to cells
~'~ , with immunological activity.
.ii . .
~' 30
-40-
,~
.,
'' -: ': , . ` , - ' , ' : . -.
105~305
EX~M~ E 12
Lymphocy~e ~utosellsitization In Vitro
When lylnpiloid cells Or an anima] are allowed to incu-
bate either ln vitro or in an enclosed chamber, with non-
lymphoid cells of the same animal, a phenomcnon occurs that
is terme~ autosensitization. That is, the immunologically
active ]ymphoid cells are capable of reco~nizing that
another type of cell is not identical but, in this sense,
is foreign. The fact that this OCCUl-S can be assessed by
evaluating the degree of cytotoxicity of these sensitized
lymphocytes. This is done by measuring the extent of lysis
or "killing" of cells (target cells~ to which the lympho-
cytes have been sensitized by mixing the sensitized cells
with the tar~et cells labeled with an isotope, generally a
radioactively labeled chromium salt. l`ollowing incubation,
and as a result of lysis of the target cell, the radio-
activity of labeled cells is r~leased into the medium
where its quantity can be measured. The percent of total
radioactivity released is taken as a measure of the degree
of cytotoxicity of the sensitized lymphoid cells
(Takagusi, M. and Klein, E., TransplaII~atlon, 9:219, 1970)~
Protocol: Preparation of peripheral blood lymphocytes:
Lymphocytes were separated from whole blood by the method
of Ficoll Isopaque gradient. Boyoum, ~., Scand. J. Clin.
Lab. Inv., Vol. 21 (supp. 97), p. 77 (1968).
Culture media: Fibroblast monolayers and target cells
were cultured in Earle's medium plus 10% fetal calf serum
3 (Gibco). Iymphocyte sensitization and the-cytotoxicity assay
- was carried out in RPMI 1640 medi~m (Gibco) supplemented
-41-
~,.
-
. ~
1056305
with l0'~ huma1l ~13 positive serum, 2 mM glutamine and ~ mM
1~epes buf~er (Gibco).
In vitro senslt;zation of l~~phoc~tes: Normal skin
- fibroblasts, used between their 3rd and l0th in vitro
passage, were seeded in plastic tissue culture flasks
(25 cm2, Falcon, Oxnard, USA). Shortly before they reached
confluency1 the monolayers were irradiated (4000 R) with
an X~ray machine (Siemens, stabilipan ]5 mA, 22 KV,
Filter Al l mm, dose rate 362 R~min, distance: 40 cm).
Aliquots of the lymphoid cells (from 107 to l.5 x 107) were
poured into the flasks with ancl also without the monolayers.
Culture medium was replaced on day 3. On day 6 the lympho--
cytes were recovered by pipetting and washing with fresh
medium. Only a srnall percentage of the lymphoid cells
remained firmly attached to the sensi-tizlng monolayer. The
; co]lected lymphocytes were washed twice and counted in the
~ presence of trypan blue to assess via';~ility.
; Cell-mediated cytotoxicity assay: Lymphocytes were
,_
tested by the method of Takasugi, M. and Klein, E.,
Transplantation,Vol. 9, p. 219 (1970). Four hundred target
cells (skin fibroblasts) were seeded in a v~lume of 20 ~l
per well. After overnight incubation, 50% of the cells
were attached. The lymphocytes were added in a volume of
20 ~l and the reactio~ was stopped after 4~ hours. The
number of remaining target cells was recorded after washing
with phosphate buffered saline, pH 7.4, fixing with methanol
and staining the plates with Giemsa (Gibco)~ The cell nu~,ber
in wells exposed to lymphocytes cultured previously without
the sensitizing monolayers, was taken as the base line for
3~ evaluation of cytotoxicity. The number of ce]ls in wells
,s
-~2
; i
1056305
seeded with sensitized lymphocytes and in medium control
wells was therefore compared to this value.
The effect of "hormone" (corresponding ~o the prepara-
tion in step 4 in the chart in Example 2) on the above
described autosensitization was tested as follows:
10 ~g. of hormone in 0.1 ml. of RPMI 164nmedium was
added to sensitization flasks containing 4 ml. culture
medium.
In the first two studies (subject C.C.) the hormone
was present during the six days of sensitization. In the
remaining experiment (subject G.K.) the hormone was present
only during the first three days,a1 which time the medium
was replaced. The lymphocytes were removed and tested for
- their cytotoxic activity as described above.
~ 15 The results are shown in the following table.
~ ~ .
, 20
. j .
.
j~ 25
.~ ' .. . .
~ 30
s 43
1 .
" 1056305
r
(~ x xx
a~ I ~ ~J N C~\ O t~ t~
~ I ~
r ~li ~ ~ ro co d r~ t~ rn
n ~" N N r-- N ~ N ~ ~~ N
~ ~1 . ~1 +1 ~ 1 +1
r In ~ ~ Ct ~D et N rn cn ~t a; ~ r
_ a~ o ~ ~-r m ~J ~ In cn ~n t~ v~ r x
~ Lr> Ln n In ~ Ln Ln ~ ~ ~t a~ aJ ~
ci r~.~- r
;-l X u~ ~ ns
x x x t~s ~
S_ ~-- O O N N t~ O Ll-- .r-
N ~) I ~ ~
a~ ~ LIJ t~ m t~ cs c)~ LD CO D ~ ~ ~ I~ r~ r- '
r- 1~ C~ l ~ Ct N ~ r~ r~ ~ ~t t~l ~a
E ~1 I +1 +l+1+l +1 +l 11 +, ,, .+_ ~, ,, ~
3 ~~ r. t~ ~ N ~r.~ r,~ r~ cn LD Vl L S_ ~a 1l
rS N d- CtCS~) O cn N ~J t~ CJ aJ {~ r.
~ d- Ln LnLnIY) Ln c~ N N r.~ c~
C~ ' ~ o ~ s_
r~ 5
O ~ ~ ~ I
o E ~ ) 3
c~s Ln r~J o ~ r~l ~ r-- E
~_ 1~ N r,~ a ~ ~ ~ ~ ~
o Lli t~ r- r,r~ rnr~ cn ~D CO t~ ' ' +~
N I~) , ~ ~ t~ r- ~ _
. +l +l +l +l +l +l -~-I +l +l -~ tl * * ~ .
t~ o o LD O ~cn O N ~
J ~a C~ ~ D fl LD O N<~ ~r) C
~- Ln t~ t~ t~ ~I n Ln ~ ct ~
. . ~
~LI._ ~
-- O E ~ I ~ I I
W o o ~ Ln Ln
o o o o
V V V V
e~
t~ X X X
,- ~
t~ ~,s . . . . . . ..
.o ~ ~ t >C~
. ,~ O . ~ t~ ~ Cs
a) ~ 4-
.~ . , r.
.~
~ ~ .
:i . . . ~ '
O r ~S ,U)
. a~ t~- . . . . . . . . . ~.
U C~ ~> ~ ~ ~ ~ ~ ~ ~ ~ ~ ,
.
_44_ : .
: . .
105f~305
It is appal-cellt from th(~ data that additions to the
sensi,ti,zation flasks of 10 ~g of the-~ hormone preparation
either for the entire 6 days of sensitization or only
duri.ng the first 3 days of the 6-d~y sensitization period
preVellted itl both cases the appearance of cyto-toxic cells,
That is, the hormone preparation blocked the auto-sensitizci-
tion phenomenon. The data indicate the potential utility
of the preparation in preventing or ameliorating an ongoinc,
autosensitization process that may be an etiologic factor
in autoimmurle diseases.
:
.~ .
'~ 30
.~
-~5~
~. '
1056305
_XAMP.L~ ~3
L~mpl~_cvl-e Au~os(ns.iti.7at-ion in Vivo
Protocol: The techn.que utilized in Example 12 was
used to examinc the effect of the hormone preparation on
autosensitization in vivo~ In addition, the cardinal role
._ _
of the th~nus gland, and its hormones, influencing this
autosensitization was examined. The procedure d;ffers
; from the above in vitro autosensitization in that auto-
sensitization was achiev-ed by placing the taryet cells
(MC57M fibrosarcoma tumor cells from mice of same strain)
mixed with host spleen cells, in a cell impermeable milli~
pore chamber and th.en inserting the chamber into the
peritoneal cavity of C57Bl/6 normal mice or mice of this
strain that had been thymecto~llized at one month of age. The
lS hormone preparation was ad~inistered i.n vivo; 20 ~g were
~ injected one hour before and six hours after chamber implanta-
` tion and then every day until day four of sensitizatiGn.
An.imals were sacrificed on the fifth day; the cells, mostly
lymphocytes, were recovered from each chamber and assayed
for cytotoxi.city, using 51Cr-labeled fibroblasts (from mice
of the same strain) as target cells.
The data obtained are the followiny:
Chamber host Cell no/well % Reductiona
Mean + S.E.
- Tx -~ hormone* 429 -~ 13 -2
. 25
. Tx 294 + 7 30
-j Intact
(Litter mates) 393 _ 9 6
b 420 ~ 20
a ++~-, P ~ < 0.001; +-~, P <0.01; +, P >0.05
.^ b Freshly prepared spleen cells used as controls.
--. * Correspondinq to SteP 4 from Chart, ~xamPl.e 2
~ .
-~6-
~ ' '
1~56305
'l'he above data inc'licate that injection of thc hormone
prepaL~cltion ~.n VIVO in thymectomizcd mice effectively
bloclced the clcvel.opment of autosensiti.zati,on of the chamber
containing lymphocytes by the mouse fibrosarcoma cells.
These data, together ~Jith the above example of blocking by
the hormone of autosensitization in vltro, further indicate
the potential utility of the hormone preparation in human
diseases in ~hich an etiologic component is autoimmunization,
i.e., the inability to recognize "self".
.
'
',
'
' 20
-47-
'~
. .
'
105630S
EX~ PI.I~
The following illustrates representative ~harmaceutic.ll
compositions (per dose) of the present invention, illustra-ted
for a preparation corresponding tc~ the last step in the
chart in Example 9.
A. Human prealburnin preparation 1.0 mg.
Sodium chloride 9.0 mg.
Water foL injection q.s. 1.0 ml.
B~ Human prealbum:in preparation 1.0 mg.
Monobasic sodium phosphate
monohydrate 5.4 mg~
Dibasic sodium phosphate 8.66 mg.
Sodium c~hloride 2.5 mg.
Wat:er for injection q.s~ 1.0 ml.
C. I-luman prealbumin preparation l.Q mg.
Mannitol 100 mg.
- Water for injection q.s. 1.0 ml.
..
D. Human prealbumin prepaL~tiol^~ 1.0 my.
Monobasic sodium phosphate
monohydrate 5.4 mg.
Dibasic sodium phosphate 8.66 mg.
Mannitol 25 mg.
Water for injection q.s. 1.0 ml.
All o~ the solid ingredients are disso]ved in water
and lyophilized in a sterile vial. Prior to administering,
J~ ,' water is added to dissolve the solids. For vials to be
used for multiple dosing, it is preferred that water con-
taining a preservative, e.g., 1.2 mg. methyl paraben/ml. and
0.1~ mg. propyl paraben/ml., be used. Rcconstituted compo-
sitions may b~ storecl at 4C- for up to two wee]cs.
-4~-
`` ` ' . "''
..,~1
,~' . . ' ~ .
