CA1205030A - Process for the separation of lymphocyte activating factors from incubation media of cell populations, particularly from a granulocyte-macrophagic line, by an antigen-antibody reaction utilizing anti-mdp antibodies - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

PROCESS FOR THE SEPARATION OF LYMPHOCYTE ACTIVATING
FACTORS FROM AN INCUBATION MEDIUM OF CELL PO-PULATIONS, PARTICULARLY FROM A GRANULOCYTE-MACROPHAGIC LINE, BY AN ANTIGEN-ANTIBODY REACTION UTILIZING ANTI-MDP ANTIBODIES

The invention relates to a process for the separation or purification of a factor, having lymphocyte activating properties and, as the case may be, pyrogenic-endogenous properties. It comprises contacting said medium with anti-MDP antibodies and recovering either the complex then formed of said factor and of the anti-MDP antibodies or the medium then substantially freed from said factor.

Description

~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.

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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.

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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

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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 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~

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.

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 ~~

- 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.

Claims (13)

1 - Process either for the separation or for the purification of a factor,having either lymphocyte-activa-ting properties, or pyrogenic endogenous properties or both, said factor being contained initially in a liquid biological medium which comprises contacting said medium with anti-MDP antibodies to form a complex between said factor and said anti-MDP antibodies and recovering either said factor or the medium then substantially freed from this factor, or both.

2 - The process of claim 1 which comprises recovering said complex and separating said factor from said complex.

3 - The process of claim 2 wherein said factor is LAF factor.

4 - The process of claim 1, wherein the anti-MDP
antibodies are selected from among those which specifically recognize the N-acetyl-muramyl-L-alanyl-D-isoglutamine structure as a whole, to the exclusion of the isolated N-acetyl-muramic acid structure or of the isolated L-alanyl-D-isoglutamine structure.

5 - The process of claim 1 wherein the anti-MDP
antibodies are fixed to a water-insoluble support.

6 - The process of claim 3 wherein the anti-MDP
antibodies are fixed to a water-insoluble support.

7 - The process of claim 3 which comprises contacting said complex with a buffered medium acidified at a pH less than 4.

8 - The process of claim 3 which comprises contacting said complex with a concentrated solution of an ionised salt.

9 - The process of claim 1 wherein said biological medium consists of a supernatant liquor from cell cultures of an immunostimulated line of granulocytomacrophages.

10 - The process of claim 3 wherein said biological medium consists of a supernatant liquor from cell cultures of an immunostimulated line of granulocytomacrophages.

11 - The process of claim 9, wherein said biological medium is free of proteins having a molecular weight less than about 12 000.

12 - The process of claim 1 wherein said liquid biological medium contains both a biological principle to be extracted and a pyrogen-endogen factor having also LAF
properties, which comprises separating the formed complex of said pyrogen-endogen factor and of said anti-MDP antibodies and recovering said biological medium containing said biological principle substantially free of pyrogen-endogen contamination.

13 - The process of claim 12 wherein said biological principle is interferon.