Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~2~ 3~ `
PR~ FSS FOR THE SEPARATION OF LYMPHOC~TE-ACTIVATING
E`AC~ FR~" r~cuBATIoN MEDIA OF CELL POP~LATIONS,
PARTICULARLY FROM A GRANUL.OCYTO-MACROP~IAGIC LINE, BY AN
ANTlGEN-ANTIBODY REACTION UTILIZING ANTI-MDP ANTIBODIES
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ... _ _ ~ .. _ _ _ _ _ _ ,. _ _ _ _ _ . _ _ ._ . _ _ _ ._ _ _ _ _ _ _ _ _ _ _
The invention relates to a process ~or se~arating
lymphocyte activating factors, among which factors having
pyrogenic-endogenous proper-ties, from an incubation
medium of cell populations, o~ a granulocyto-macrophagic
line, by an antigen-antibody reac-tion utilizing anti-
MDP antibodies.
It is known that the administration of endotoxins
and of numerous other bacterial products is capable of
inducing the produc-tion by leucocytes of lymphocyte
activ~ting- or stimulating- factors, more par-ticularly
1~ T lymphocyte-activating factors (LAF factors). Pyrogen-
endogens (PE) obtained from leucocytes are included amona
the fac-tors having that type of activity. These factors
are held responsible to a great extent for the rise in
temperature ~nduced in vivo by -the administration of
endotoxins and other bacterial products.
N-acetylmuramyl-L-alanyl-D-isoglutamine, or MDP
(for muramyl dipeptide), has been identified as being the
minimum s-tructure of the bacterial peptidoylycans capable
of replacing whole mycobacteria in the Freund Complete
adjuvant (FCA). This adjuvant, which may be obtained by
synthesis, has also been found capable of producing hyper-
thermia in rabbit, either by liberating circulating leuco-
cyte pyrogens, or by direc-t action on the centres of
thermoregulation. It is to be noted that the minimum pyro-
3~ genic dose oE MDP is 30 micrograrns/kg, when the produc-t is
injected intravenously, and the dose of 0.1 nanogram, i~e.
about 20 picomoles, when administered intracerebroventri-
c~llarly. This dose is close to that at which the pyrogen endogen
- '~ ?~~
~2~S~
is -theore-tically ac-tive~
Factors having activity of the LAF type, and
particularly such factors which possess pyrogen.ic-endo-
genous properties, may also be induced 1n _tro . In
particular, the above-said cell populations, particularl~
those belonging to -the monocyte and lymphocy-te series, are
capable of liberating factors or cytokins, for example
l-ymphokins or monokins, when they are incubated in a culture
medium containing an immunostimulant agent. These factors,
which are liberated in the culture medium, may be isolated
from the latter. Pyrogen-endogens,particularly those which
are also known under the name Interleukins 1 (IL-l) may
also be included among these factors.
Numerous immuno-stimulant agents may be utilized
to induce the in vitro production of said interleukins 1.
For example lectins, natural products particularly of
viral, bacterial, fungal or parasitic origin, more parti-
cularly products derived from bacterial walls such as
products of the bacterial lipopolysaccharide type (LPS)
may be mentioned.
Whole micro-organisms may also be used. Il-l'S may
be induced in human mononucleated cell cul-tures of the
peripheral blood, stimulated for an hour by means of hea-t-
killed staphylocci. Adherent cells, previously washed, are
incubated for 24 hours, whereby a culture supernatant liquor
containing leucocyte pyrogens, deno-ted below by the abbre-
viation "PL"is obtained after centrifugation at 25.000 G. This
li~uor can then be stored at 4C.
It has also been shown that ~DP and numerous
homologs of MDP could be used to induce the i.n vi-tro produc-
tion of the above-mentioned IL-l's. The LAF fractions which
can thus be isolated from the stimulated culture medla have
also pyrogenic acti-~ity. However,. when a preferred class
of MDP homologs, more particularly ~-ester derivatives of
N-acetyl-muramyl-L-alanyl-D-glutamine, are used, it is pos-
sible to induce in said cell cultures the prodllction of
monokins having the characteris-tic immunos-timulan-t acti-
3~ `
3vities of IAF, however bein~ devoid of endogenous-~yroa,enicity. Such
~SDP homologs are ~-esters of N-acetyl-muramyl-L-alanyl~ luta~une,
described in ~opean pa-tent n 6068, Dec. 12, 197g. The use of
~-butylester of N-acetyl-muramyl-L-alanyl-D-ylukamlne is
most preferred. Advantageous conditions under which such
induction can be carried out, are described in French patent
N 2,519,553.
Separation processes, even purification processes
utilizing antigen-antibody reactions have already been des-
cribed. Thus a process has already been described for obtai,-
ning a fraction of anti-pyrogenic immunoglobulins, by
immunisation of rabbit with a supernatant liquor from a
culture of human leucocytes previously stimulated and
endowed with pyrogenic activity (DINA~ELLO, C.A., RENFER,L.
and WOLFF, S.M~, J~ Clinv Invest~, vol. 60, 1977, p,465-
a, 7 2 ) n
In the same way the use of these antibodies to
constitute affinity chromatography colums, by fixing of said
antibodies to an immunoabsorbant material known under the
designation SEPHAROSE 4B, previously activated with cyanogen
bromide has also been described by ANFINSEN et coll. in
"Proc. Natl. Aca~. Sci. USA", vol. 71. p. 3139-314~. These
antibodies will be denoted below as "human anti-PL".
These antibodies fixed to SEPHAROSE ~B have been
used in a known process for purifyin~ PL, or more generally
pyrogen-endogens (PE) derived from any other source. This
process comprises contacting the previously concentrated
supernatant liquors, for e~ample under the conditions
already indicatedl with an immunoabsorbant column of
SEPHAROSE 4B activated with cyanogen bromide and carrying
anti-PL-human rabbit an~ibodies, eluting the PL, subjec-
ting the eluate to chromatography on a column of the
molecular sieve marketed under the name SEPHADE~ G50, and
collecting the fractions having a molecular weight of
the order of 15 000, providing a single band on polyacry~
lamide gel containing 7O5~ of sodium-dodecyl sulfate (SDS),
and endowed with pyrogenic activity and LAF activity~
.
The PL fractions thus ob-tained, thou~h concentrated
in leucocyte-pyro~ens,however s-till contain additional subs-
tantial amounts of o-ther factors, particula~]-y lvmphokins,
monokins, and in-terferoll, whose separation by conven-tional
processes is hardly permissible.
It is an object of the invention to overcome these
difficulties, at least to a large ex-tent, more particularly
to provide a process ~or the selective separation from the
abovesaid media, or, as the case rnay be, purificatiGn of
factors having LAF-type activity hereafter designated,as
"LAF factors", of PL's or, more generally, pyrogen-endogen
fractions, denoted below by the abbreviation PE, also endowed
with LAF properties.
lt is also an object of -the invention to provide
a process for the removal of PE,s from any medium which may
contain it, with a view to the production of a medlum subs-
tantially devoid of pyrogen-endogens. Other active principles
essentially devoid of PE can then be obtained from said
medium.
The invention arised from the discovery that
factors having LAF type properties, and among -them pyrogen-
endogen factors, can be reco~nized selectivelv by specific
anti-MDP antibodies.
The process according to -the invention for -the
separation or purification of the factors having a LAF type
activityl and particularly PE, starting from a ]iquid
biological medium containing them in a dissolved state,
comprises contact:ing this medium with an-ti-MDP antibodies,
to form a complex of PE fac-tor and o-E the anti-MDP an-tibodies
and recoverin~ the P~ factor from said complex.
Anti-MDP antibodies capable of being used in -the
purification process according to the invention may be
obtained in any manner known per se, particularly by
immunisation of an animal, for example rabbit, with an MDP
or MDP derivative previously fixed to a carrier molecule,
such as bovine serum-albumin, polylysines or any other
rolecule havin~ a sufficiently high molecular wei~ht to
~75~3~
confer on the conjugate obtained the required immunogenicity.
As re~ards -the general conditions for the production of
anti-MDP antibodies, reference may be made to the article
of REICHERT C.M. ancl coll. published in Molec. Immun. 17,
357-363 (1980) Reference may also be made to -the article
of BAHR G.M. ancl coll. published in Molec. Immun. l9, N 5,
737-745 (1982).
The antibodies contained in the anti-sera obtained
may be purified, for example by affinity chroma-tography on
a column of resin carrying molecules of MDP or MDP deriva-
tives fixed thereto. Advantageously, recourse is had to acolumn of the insoluble suppor-t material marketed under
the name SEPHAROSE 4B with groups of MDP-lysine (N-acetyl-
muramyl-L-alanyl-D-isoglutaminyl-L-lysine)fixed thereon,
after prior activation of the support material with cyanogen
bromide.
The anti-MDP antibodies (polyclonal anti-MDP
antibodies) fixed to such a material may then be eluted,
for example, by acidification of the medium, particularly
at a pH less than 4, or by increasing the ionic concentra-
tion of the medium, by means of ionic salts highly solublein water , such ,as sodium thiocyanate~
However, according to an important preferred
feature of the invention, `'selective" anti-MDP antibodies
are used which recognise specifically the N-acetyl-muramyl-
L-alanyl-D-isoglutamine structure as a whole, particularly
to the exclusion of either the isolated N-acetyl-muramic
acid structure, or that of isola-ted L-alanyl-D-isoglutamine.
The latter selective anti-MD~ antibodies, or
preferably monoclonal antibodies, will enable a subs-tantial-
ly more thorough degree of purification than polyclonalantibodies, since the latter are capable of fixing other
cons-ti-tuents of the biological medla concerned J for example
numerous other glycopep-tide structures also present in these
media Nonetheless, polyclonal anti-~lDP antibodies may be
used advantageously to produce a first enrichment of the
PE factor, more thorough purifications being -then, if
necessary, repeated with monoclonal "selective" ankibodies.
A technique for producing hybridomas secreting s~lch mono-
clonal antibodies and for recovering the latter from the
culture media of the hybridomas, as well as the conditions
in which they may be applied, will be indicated below,
exclusively by way of example.
The process according to -the invention therefore
comprises ~orming complexes bet~een the IAF factorsland
particularly the PE factors, on the one hand, and the
anti-MDP antibodies, preferably "selective" antibodies, on
the-other hand, and recovering the LAF factors and, as the
case may be, PEs themselves, from these complexes, after
dissociation thereof.
Advantageously selective anti-MDP antibodies borne
on a support insoluble in aqueous media are used. Any
support, preferably in powdery form, on which anti-MDP
antibodies can be fixed without loss of their capacity to
form complexes with isolated LAP factors can be used .
It is preferred to use selective anti-MDP antibodies fixed
to the support materia~ marketed under the name S~PHAROSE~
4B, after activation of the latter with cyanogen bromide.
Of course ,ot~er supports may be envisaged, such as glass
beads, agarose and SEPHADEX~ it being naturally understood
that it will be up to the specialist to proceed with
testing of the selective anti~MDP antibodies fixation and
assaying whether the complexing activity of the fixed anti-
MDP antibodies with res~ect to PE or LA~ is malntained.
The recovery operations of the PE factor from the
complex previously formed between the latter and the anti-
MDP selective antibodies may be carried out in any knownmanner, for example by acidifying the medium, particularly
to a pH less than 4, or by increasing the ionic concen-
tration of the medium, by means of highly soluble ionic
salts, for example sodium thiocyanate, sodium chloride or
ammonium thiocyanate. These methods are particularly
advantageous, especially when the anti-MDP antibodies have
12(~5030
previouslv been fixed to a suitable insoluble support. It
is advantageous to use an anti-MDP antibody obtained from
a solution of polyclonal anti-MDP antibodies, purified
beforehand by affinity chromatography on a SEPHAROSE 4B-
MDP- lysine column, whereby the anti-MDP antibodies may
then be eluted from the column, particularly under the
above indicated conditions. The anti-MDP antibodies can
be fixed to an insoluble support by any conventional anti-
body fixation technique, for example that described in
lG "Affinity Chromatography : Principles and Methods", edited
in PHARMACIA
The medium containing said LAF factors from which
they may be extracted by the process of the invention, may
be constituted by the supernatant liquor of any culture
of cell lines capable of producing LAF,IL-l or PE, when
immunostimulated as defined above. It relates principally
to monocytes, macrophages capable of beins obtained parti-
cularly from peripheral blood, lung, peritonium and bone
marrow.
Cells of any origin may be employed, particularly
human, mouse, rat guinea pig, monkey, rabbit or dog cells,
in primary c~ltures or continuous lines.
A source Gf activable cells is also constituted by
the above-mentioned cells, from which have been p_eviously
separated the elements not adhering to culture bottles of
the type marketed under the trade mark NUNCLON or FALCON.
It is possible particularly, for sources of activatable
cells, to refer again to the article of I. GERY and B.H.
WAKSMAN, entitled "Potentiation of the T-lymphocyte
Response to Mitogens", published in "The Journal of Ex-
perimental Medicine", volume 136, 1972, p. 143-155, and
to the book edited by E. PICK wi~h the collaboration of
M. LANDY, entitled "Lymphokine Reports 1", Academic Press,
Inc.
The media containing said LAF and, as the case
may be, PE to which the process according to the invention
is applicable may also be constitu-ted by enriched frac-
t.ions of said supernatant ]iquors, such as obtained by
dialysis of the latter to eliminate therefrom -the conten-t
of inoryanic salts and of "small molecules", for e~ample
those having molecular weights less than about 12,000.
The invention will be s-till bet-ter illustra-ted by
the description of the techniques which have permitted
the determination of the capacity of recognition of the
LAF, PE factors or of similar factors by anti-MDP selec-
tive antibodies.
1.. Production of monoclonal antibodies
a) Il~lunisation of mice
Two groups of 2 month-old fernale BALB/c mice were
immunised with MDP previously fixed to bovine serumalbumine
(MDP-BSA). The first group received two intradermal injec-
tions separated from each other by a three-week interval,
of 100 ,ug of MDP-BSA emulsified with -the FREUN~ complete
adjuvant(FCA). The second group underwen-t the same -treatment,
in-traperitoneally, and wi-th doses of lOOJug of MDP-BSA in
PBS buffer.
b) Fusion of the cells and culture of cell hybridS
(hybridomas)
Nine weeks after the last immunisation, the mice
were boosted twice at a one-day in-terval, with a solution
of 100 ~g of MDP-BSA, in a saline medium, -this -time intra-
peritoneally . Three days after the las-t booster, the
mice were sacrificed, the spleen cells recovered and cell
fusion of these spleen cells was carried out with a line
of myeloma cells, for example myeloma NSO,/l. It goes
naturally without saying that it is possible to use any
other accessible type of myeloma cells capable of formlng,
by fusion with spleen cells, ce:Ll hybrids capable of
inducing the production of asci-tes in animal, said
ascites then being capable of being used as a source of
antibodies having the characteristics of those initial]y
produced by the spleen cells involvecd in the fusion.
c~
Cell fusion of lO0 million spleen cells of each yroup
of mice with 107 ~SO/l cells in -the presence of a 41~ poly-
ethylene glycol solution l 500, may be carried ou-t by -the
technique descri~ed by Z. ESH~IAR et coll. in J. immunol.l24,
775, 1980. After fusion, -the celIswere dis-tributed in three
to four microplates with 96 wells, selected in an EAGLES
medium, modified by DuLsEcco~ con-taining the characteris-
tic constituents of HAT medium, having a content of hypoxan~
tine, aminopterine and thymidine (HAT-DMEM) and a high
con-tent of glucose, said medium being further completed with
up to 15% of horse serum, 50 uni-ts per ml of penicillin
and of streptomycin, 1 m~l sodium pyruvate and 2 nM of
glutamine.
After two weeks of cul-ture, the cell hybrids were
transferred into a HT-DMEM medium. I'he cells which deve-
loped, could then be cultivated routinely in a medium based
only on DMEM and horse serum.
Sorting the clones_~.roducing selective anti-MDP an-tibodies
From lO to 15 days after the fusion operation, sorting
of the hybrids capable of producing anti-MDP antibodies was
done, by resorting to the technique called "radioimmunoassay"
in solid phase, on microplates of poly-(vinyl chloride)
whose wells had previously been coated with a solution of
MDP-A--L, i.e. with MDP fixed on or conjugated to A--L,
-that is multi(poly-D-L-alanyl)-(poly-L-lysine) (lOO~ul of
a solution of 25~ug/ml of MDP-A--L per well). After incuba-
tion at ambiant temperature, for one hour, the microplates
were washed three -times with a solution of 1% horse serum
in PBS buffer (PBS-HS). 50~ul of the supernatant liquors
of the hybrldoma cultures were then added to each of the
wells and incubated for 2 hours at ambiant tempera-tu:re.
After three washings with PBS~HS, 50 ~l of goat Ig anti-
mouse antibodieslabelled ~ith iodine 125 (125I) (105 counts
per minute : cpm) was added. It was left to incubate over-
night at ~C. The radioactivity level was de-termined in a
Gamma radiation counter after 4 washings, dryings and
cuttings out of the wells.
3~3 `
The table which follows surnmarizes the "fusion"
efficiencies" of the two fusion tests mentioned above.
___ _ _ ~er o~ cultures
5Group Immunization of hybrid cells Posi-tive
which have gr~n hybridciras
1 MDP-BSA in FCA 384/384 72/384
2 MDP-BSA in PBS 288/288 29/288 .
_
1~ Tnose hybridomas which led to detection of 5 000
to 15 000 cpm, having reyard to a background noise of 500
cpm in tests carried out with supernatants which did not
contain anti-M3P antibodies, were considered as providing
a positive response.
A second selection was carried out under similar
conditions employing a second series of tests in microplates
whose wells had been coatéd respectively with ~lDP-A--L,
with M-A--L and with DP-A--L, ~,e, conjugates of A--L and
of MDP's basic constituents: muramic acid (M) and L-alanyl-
D-isoglutaminel(DP) respectively~-The monoclonal antibodies
which were active against MDP-A~-L, yet which had only
little or no activity with respec-t to DP-A--L or M-A--L,
were selected.
To produce larger amounts of antibodies, the selec-
ted hybridomas were administered intraperi-toneally atrates
of 10 hybrid cells to BALB/c x DBA/2)~1 mice. The anti-
bodies secreted were then recovered in known manner from
the fluid ascitic tumors induced by these hybridomas in
these mice.
Monoclonal antibodies capable of specificallv
recognizing the carrier molecule, that is to say BSA~
were also selected, under the above indicated conditions.
These monoclonal antibodies were used as controls.
To produce larger amounts of antibodies, -the
selected hybridomas were administered intraperitoneally
3~ `
in the propor~ion of 10 x 10 hybrid cells to BALB/c x DBA/2)Fl
mice. The antibodies secreted were then recovered in known
manner from fluid ascitic tumors induced by these hybridomas
in these mice, particularly by ractionating the ascite on
a gel, particularly a SEPI-IACRYL~200 gel, and reeovering
the band containing the immuno-globulin which cross-reacts
with MDP antigen (in a zone corresponding to a ~olecular
weight of approximately 160,000).
2. Detection of the eapacity of_anti-MDP antib~dies to canbine with
the PE factors
-
To demonstrate the capacity of anti-MDP rnonoclonal antibo-
dies to ccmbine selectively with the PE's, recourse was had to the
study of the capacity of these anti-MDP antibody preparations of inhi-
biting the increase in the incorporation of thymidine3H (specific acti-
~ity 1 Ci~m~ol) b~ thymocytes of 6 week old C57Bl/6 mice stimulated by
the PE in the presence of PHA (phytohemagglutinin), employed at thedose of 1 ~g/ml, under the conditions described by ~SENW~SSER, L.,
DrN~RELL0, C.A. in "Cell. Immunol." vol. 63, p.l34 - 142.
The tests based on the detec~ion o~ the LAF acti-
vity, based on the measured increase of the incorporation
of the tritiated thymidine) are extremely sensitive and in-
volve much sm311er doses of PE th~l-those which would be required for
the establishment of the inhibition by the anti-MDP anti~odies of the
pyrogenic activity itself m vivo in the rabbit.
In the experiments of inhibition of the LAF acti-
vity, the above said purified PL fractions, diluted to
1/100 was preincubated for 1 h at 37C, then for 12 h. at
4C with the different antibodies, that is to say :
a) either a l/20th dilution of the different anti-~P
monoclonal antibodies obtained in the mouse;
b) or a l/200th dilution o the rabbit anti-PL immunoglo-
bulin fraction or the normal rabbit immunoglobulin fraction.
The mixture was then added to the thymocyte
culture in the presence of PHA.
As indicated by the results shown in table 1,
the incorporation of thymidine H is slightly increased only
12
by the addition of PHA. The addi-tion of anti-MDP mono-
clonal antibodies to PHA does not modify -this effect.
In the presence of PL and P~-IA, an increase in the proli-
ferative respGnse (P ~0.0l) was observed. The addition Gf
monoclonal antibodies to -the mixture of these -two subs-
tances results in inhibi-tion by 80% of the increase so
observed.
In a following series of experiments, the effects
of anti-MDP monoclonal antibodi.es on the LAF activity
of the purified preparation of PL were compared with those
of other antibodies: anti-BSA monoclonal antibodies,
specifically anti-PL rabbit immunoglobulins and normal
rabbit immunoglobulins.
As is shown by the results gi~en below, none of the
control antiserums was capable of modifying significantly
the incorporation of the thymidine 3H induced bv the
PHA + PL association. On the o-ther hand, -the specifically
anti-PL rabbit antiserum used at the dilution of 1/2GOth
resulted in an inhibition by 48% to 38%. In all the ex-
perimen-ts, the specifically anti-~DP mouse monoclonal
antibodies inhibited the LAF activity of the purified PE
preparation very significantly (53, 34, 59 and 28%).
In order to check that the inhibition induced by
the anti-MDP monoclonal antibody was specifically l.inked
with an anti-MDP activity, 100 ~g/ml of MDP were added at
the moment of incubation of the PL with the different
antiserums, before the stimulation of the -thymocytes by
the PHA. In this experiment, repeated three times, the
addition of MDP did no-t modify the inhibition of -the LAF
activity induced by the specifically anti~PL rabbit serurn.
In the same way, -the responses observed wi-th the two other
control antisera, normal rabbit immunoglobulins and
anti-BSA monoclonal immunoglobulins, were not modified. To
the contrary, the inhibition produced by the anti-MDP
monoclonal antibodies was significantly reduced (41 to
1 0 0 g6 ) ( P C O . 0 1 ) .
The mean resul-ts are collec-ted in -the table hereafter:
5~3~3
INHIBIT-ION BY AN ANTI-MDP MONOCLONAL A~TIBODY
OF THE IN VITRO LAF ACTIVITY OF PE
Control 317 + 38
PHA (1 ~g/ml)615 - 49
PHA + anti-MDP (1/20) 569 - 57
PHA ~ PL 2079 + 237
PHA + PL + anti-MDP921 - 33
This selective complexing reaction may be brought into
play for separating the PL factor from a medium containing
it, in admixture with other peptides, glycopeptides and par-
ticularly peptidoglycan fragments. It is in -this respect
remarkable to note that the latter do not lead tG any immu-
nological reaction with the anti-MDP antibodies
The PL factor as involved in the PL factor - anti-
MDP antibody complexes, may be recovered by dissociation of
these complexes. ~owever ,as has been indicated above, this
dissociation can be carried out much more simply, when -the
anti-~DP antibodies are conjugated with an insoluble
carrier.
A procedure Eor separating larger amounts either of
LAF factor and/or of PE from the supernatant of a cul.ture
of cells of a suitable g.ranulocyto-macrophagic line and
using a column of a cvanogen bromide activated SEPHAROSE
~ and of anti.-MDP monoclonal. antibodies cou~led there-to
is described hereafter.
a) Immuno-absor~t_on of the LAF factor
The supernatant liquor, possibly concen-trated before-
hand, is contacted with anti-MDP conjugated to an immuno-
absorbant material After sufficient contact to enable theselective fixation of the LAF factor, the immuno absorbant
material is washed to remove the non-specific products
retained. Advantageously the operation is carried out on
a column and the rinsings are carried out by the passage
of the rinsing liquid through -the colurnn. The molecules
~2~D3~
14
retained on the antibodies of the column, that is to say
the LAF factor and possibly other monok~nes or mediators
ha~ing distinct molecular wel~hts and capable of containing
the MDP structure in a form accessible to the antibodies,
5 are eluted by a low pH buffer, for e~ample a solution based
on glycine/HCl, of pH 3.2, or by a solution with a high
salt concentration, for example lM sodium thiocyanate.
b~ Should other molecules be eluted at the same
time as the LAF factor it should be desirable to separate
10 these molecules, such as by chromatography on a molecular
sieve enabling separations of molecules according to their
molecular weights.
Among the molecules possibly present, are mentioned
factors having yenerally smaller molecular weights.
15 Consequently, it may be desirable,to separate mo`e ~arti-
cularly molecules having molecular weights less than 12 000.
Consequently, the process according to the invention
can also be applied to a!separation and rapid purification
of monokines with L/~F activity, devoid o pyrogens-endogens,
20 such as obtained from the supernatant of cell populations of
a granulocyto-macrophagic line, in the presence of an
ester of N-acetyl-muramyl-L alanyl-D-glutamine, particular-
ly as described in French Patent N 2,519,553.
~Two experiments were done in order to prove that
the antibody is binding to the endogenous pyrogen and,
therefore, blocking its activity~ A macrophage super-
natant (rich in endogenous pyrogen)was passed on an
anti-MDP column after previous st~mulation with an
analog~of ~Dp consi~ting o~ ~ -methylester o~ N-acetyl-
30 muramyl-L-alanyl-D-glutamine activity. When injected by
the intracerebroventricular route the abovesaid analog
does not induce fever. However it can stimulate macro-
phages to produce endogenous pyrogen~
The following experimental procedure was used.
Cyanogen bromide-activated sepharose beads were
coupled to t~o monoclonal distinct anti-MDP antibodies
prepared as indicated above. A control column of mono-
clonal anti-dinitrophenol antibodies of anti-DNP was also
prepared. Flnally, a colurnn o~ monoclonal anti-MDP antibody
was prepared ~nd bloc'k~d with murabutide ( ~ -bu-tylester
of N-acetyl-muram~,~l-L-alanyl-D-glutamine) (20 rng). The
colums were calibrated -to pH 7.2. 10 mg of BSA was passed
over each column followed by washing with PBS and then with
0.lM glycine/HCl. The colums were reequilibrated to pH.7.2
before passage of the supernatants.
Experience I
2 colums of monoclonal anti-MD~ were used. The
volume of each was 5 ml, containing 23.5 mg of Ig immuno-
globulin anti-MDP. One of the colums was blocked wi-th 20 mg
murabutide (MB).
2.5 ml of macrophage supernatant was passed over
each column and recycled 3 -times. The supernatant was
collected in 5 ml and the colu~,ns were washed firs-t with
10 ml PBS, then another 20 ml PBS, then with 20 ml of
0.lM glycine/HCl~ The first 5 ml collec-ted af-ter passages
o~er the colums as well as the 10 ml of washing were tested
(each on 3 rabbits) for induction of fever by the i.c.v.
route
Results
__
Column 5 ml 10 ml
~ ~ _ _ _ _
an-ti-MDP ~.67 + 0.15 0.00
anti-MDP
blocked with~B 1.07 ~ 0.15 0.00
Thus, -there was a significant reduc-tion in the
pyrogen content of -the supernatants af-ter passage over an
anti-MDP column~
Experience II
4 colums were pre~ared carrying :
1. anti--MDP blocked with MB
2. anti-DNP
3. anti-MDP
4. anti-MDP.
16
The size of each column was 10 ml wi-th approximately
50 mg of im~unoglobulin coupled on -the beads.
5 ml of macrophage supernatant was passed over each
column, cycled twice and collected in a final volume of
20 ml. The collected supernatant was tes-ted for -the abili-ty
thereof to induce fever (injection of 1 ml of supernatant
i.v. into a rabbit~O
Xesults
Column ~~
1 0 ----
- 0.65 0.05
anti-MDP
blocked with MB O.65 0.05
anti-DNP 0.7 0.05
anti-MIDP 0.01 0.01
anti-MDP 0.01 0.01
Thus the pyrogenic activity of the supernatant was
absorbed out by passage over the anti-MDP column but
not over the anti-DNP or over the anti MDP blocked with
murabutide (MB)
lhus -tne process accor~ing to the inven-tipn enables
a very selective separation of the LAF factors and more
particul.arly PE factors and consequen-tly has the great
~5 advantage of facilitating the study of other factors which
may initially be contained in the abovesaid media, in
admixture with the pyrogen-endogens.
In par-ticular, the invention also enables the pro-
duction of distinct monokins or lymphokins, essentially
freed or devoid of pyrogens-endogens, where~y the study
of said monokins or lymphokins is then considerably fa-
cilitated.
The process according to the invention i.s Gf
particular advan-tage when applied to any extraction
3~D `
process of active principles from any media also con-tai-
ning PE or factors having similar biological properties
then capable of contamina-tiny the final extraction pro-
ducts sought.
For example it has been shown tha-t interferon ~
can be produced by human leucocytes, after stimulation
by different agents, among which are mumps virus (TSUNEO,
K. and MINAGAWA, T., ~. Gen Virol., 54 (1981), pages 293
to 299) or the Sendal virus (MOGENSEN, K.E., Blood Trans-
fusion and Immunohematology, volume ~3, N 3, (l980),
pages 373 to 397) The induction sys-tem of in-terferon is
similar to that by which other monokins and lymphokins are
induced too. It comprises taking up the buffy coat of
centrifuged human blood and storing it for 2A hours a-t
4C or, alternatively, separating the mononuclear cells
of the peripherial blood on a FICOLL-PACK gradient.
Leucocytes freed from red blood cells can be suspended
in a culture medium and incubated with paramyxoviruses
(generally Sendal virus) for 24 hours. The cells and the
debris can then be centrifuged, whereby a supernatant
liquor is obtained which then contains interferon~ .
The latter can then be purified and concentrated. However,
most of these in-terferon preparations still contain
numerous mediators and proteins freed from the leucocytes
during these operations even after purification. Fur-ther
interferon preparations used clinically may induce
severe hyperthermies (STRA~DER, H., Texas Rep. Biol. Med.
vol. 35 (1977), page 429). These authors have suggested
that these hyperthermias could be due to contamination
of the interferon preparations by leucocy-te pyrogens
produced by similar s-timulations in vitro~ Consequently,
the method of the invention may be used for the removal
of these pyrogens, to provide an interferon of great
value for therapeutic tes-ts. The me-thod of selec-tive
separation of the pyrogen-endogens is also applicable
to the production of interferon freed from pyrogenic
1~ .
activity starting from -the supernatan-t liquor separated
from the leucocyte cultures, by passage thereof over
eolums containing anti-MDP antibodies.
As is self-evident and as emerges moreover already
from the foreyoing, the invention is in no way limi-ted -to
the examples contemplated hereahove, it encompasses on
the con-trary all modifications, particularly those emplo-
yiny monoelonal antibodies obtained from hybridomas
resulting from the fusion of myeloma cells with splenoeytes
from the mouse or other animals immunised wi-th any MDP
homolog, sueh as for e~ample,-~ -ester ~-amide derivatives
of N-acetyl-muramyl-L-alanyl-D-glutamic acid, to the
extent that these antibodies permit, like those which have
been mentioned above, of proceeding with separations
which have been described, PE, PL or LAF or of factors
having similar biologieal properties. It is again self-
evident that the polyclonal or monoclonal antibodies so
obtained, and more particularly their use for effecting such
such separations consti-tute equivalents of those which
have been more particularly described and, whereby the
use of said antibodies would not depart from -the scope
of the invention as claimedv
In general,-the monoclonal antibodies capable of
being used in the proeess according -to the invention
may be consti-tuted by any monoclonal antibody selectively
recognising MDP homologs, characterised by the presence
o-f a peptide chain linked to the N-acetyl-muramic acid
group, the first aminoacyl of the ~e~tide chain, tha-t is
to say the aminoacyl ensuring the linkage with the N-acetyl
muramic group, being selected from among L-alanyl, L-seryl
or L-valyl. The second aminoacyl of the peptide chain
hardly intervenes in the specificity of the important
antigen determlnant to be ta}cen into consideration, as
regards suitable monoclonal antibodles for use in -the
process of this invention. Par-ticularly suitable mono-
clonal antibodies are those which do not recognise the
3~3
19N-aeetyl muramic acid structure not linked to the above-
indleated peptide group, even at coneentrations lO0 times
greater than t.he minimum eoneentration neeessary for the
formation of eomplex ~etween the MDP derivative (or MDP)
5 and the eorresponding monoelonal antibody.
Conversely, suitable monoclonal antibodies may
also be obtained by employing initially in the realisatio
of the eell fusions for the production of hybridomas
produeing monoclonal antibodies, spleen cells from animals
lO previously immunized against MDP homologs .in which the
D~ and \~-earboxyl funetions ean bear different substitu-
ents. In partieular, it is possible to resort to monoelo-
nal antibodies obtained starting from animals initially
immunized against N-aeetyl--muramyl-L-alanyl-D)glutamie
aei.d diesters or again N-aeetyl-muramyl-L-alanyl-D-gluta-
mine ~-esters, as deseribed in E:uropean patent n 6068,
Dec. 12, 1979.
Any type of myeloma suitable for production of
hybridomas ean be used. In partieular, reeourse may be had
to any myeloma described in the technical literature or
in published patent applieations or patents. There may
be mentioned, by way of example, the myelomas deseribed
in the published French patent N 78 17545/2 394 607.
It is recalled that the process disclosed in French
patent No. 2,519,553, for obtaining non-pyrogenic
30 LAF from eell-lines eapable of producing it, partieularly
from granuloeyto-macrophagic cells, eit.her tumoriyenic or
not in a medium comprising the appropriate nutrients,
eomprises incubating said cell-lines in the presence of a
non- pyrogenie MDP~erivative, particularly murabutide, and there-
35 after recovering the non-pyrogenic lymphocyte activating :Eactor then
released in said ~dium.
