Human complement factors and their therapeutic use

~ ~\ ~Z~4~72 This invention relates to human complement Factor I and Factor H, both of which have now been found to be effective for the therapy of autoimmune diseases.

Antibody-antigen complexes are generated when antibodies bind to their specific alloantigens or autoantigens in vivo. Most of these complexes react with serum complement components (Cl, C4, C2 and C3), and thus so-called "immune complexes", consisting of antigen, antibody and the complement components including C3b, are generated. These immune complexes further interact with C5-C9 components, generating a membrane attack complex, C5b-C9, and an anaphylatoxin C5a, one of the most potent chemical mediators of inflammation. Moreover, such immune complexes are often deposited on the inside surfaces of blood vessels and also in the connective tissues around the vessels. These sequential events eventually cause inflammatory reactions and tissue damage.

on the other hand, there is a metabolic process for inactivating such active immune complexes. Factor I, one of the complement regulatory proteins, can ~ ' , 1~9~1 37Z inactivate C3b molecules in the immune complexes by converting the C3b to C3bi or C3d in cooperation with Factor H. C4b-binding protein and the complement receptors (CRl, CR2 5 and CR3) on erythrocytes and phagocytes.

The immune complexes bearing such C3bi and C3d cannot further activate C5-C9 components and are eventually processed and cleared/ mainly by the hepatic tissues.

The level of immune complexes in the blood, therefore, is the result of a balance between 10 the processes resulting in their generation and their inactivation (degradation).

Factors H and I and the ability of endogenous Factors lI and I to inactivate C3b molecules in the normal course of the mammalian metabolism are known.

Indeed, Factor I was 15 hitherto known as "C3b inactivator" ~= "C3bINA") or 'IC4b inactivator", [see e.g.

Hsiung et al.

Biochem. J. (1982) 203, 293-298].

Factor H was hitherto known as ",BlH" [see e.g.

Weiler et al.

Proc.

Natl.

Acad.

Sci. USA 73 3268-3272 (1976~].

However, their value in the treatment of autoimmune 20 diseases has not hitherto been suspected, nor has any casual connection between Factors H and I and such autoimmune diseases as systemic lupu5 erythematosus (SI.E), rheumatoid arthritis (RA) and glomerulonephritis been proved or suggested.

<'=;+t L~ L~ 1 4~4 4'~ ~ .

~L29~ 2 These autoimmune di ease~, 0u~h a~ ~L~. ~A and ~lomerulonephriti~, ~hat are not eff~ctiVel~ cuxed by curren~ ~onv~ntional ~her~py are con~i~ered t~ be va~cula~ di~ea~e~, and we hsliev~ that ~hey are malnly caused by the depasi~ion of immune complexe~ ~nGide ~nd outside ~he blood v~8~ 0~ ~rioU~ ue~, although the p~e~i#e me~hani~m o~ the depo#ition of ~he~e imm~n~ aomplex~8 haB no~ ~a~ been ~lucid~ted. ~e~ently, an inYer~e rela~ionshil? b~twe~n ~he frequ~ncy of oacurrenae of S~E and the genetiaally controlled den~ity o~ complement recepto~ 1 ~CRl) on e~ythro~yte~ (site~cells) has been ~ugge~ed. ~hl~ al~o ~uyge~ that the oscurrence and ~x~erba~ion of SL~ i~, at lea~t in part, due to a reduced capability to p~oce~-or inacti~a~e the immuns com~l~xe~. ~oWe~er. ~t iG not ~oc~ible to exyl~i~ a~or~ ~U~h o~U~ence or ~xac~r~atio~ o~l~ b~ it~ on erYthrocYtes. beaau~e (1~ about 40~ of SLE patients have normal level~ of CRl ~e~ (2~ a~ou~ lZ% o~ normal ~ubj~ct~ h~Ye low le~el~ o~ CRl sit~ (~) the~e are no dif~erences in CRl ~ b~tw~en the exaaerbatio~ and regres~on G~ye~ in the ~am~ SLE pa~ien~.

We h~ve b~en searchlng ~or ~ac~ors other ~han CRl to aorrelate wi~h the oacurrence and ~x~c~b~tion o~ and have now found that th~ eompl3me~t ~omponen~ F~c~or ' ;A~ ' .

",~ ` ' I`IU`~ b lt~ M I I~IH~K~+I_L~K L-JI`I 1~1:L 41~4 4Yl~ ~2~ 2 I aAd Fa~tor ~I, both of which are ~entlal ~o~ the inac~ivae~ on of immune cornplexee, decrea~ed in the pLeor ea~ly pha~e~ o~ the exaaer~ation stage. ~ut not durin~ moQt of the regre~sio~ Yt~ge.

Thi~ ~ug~e~t~ th~t ~he temporary decrease in ~ac~or I and~or Factor H m~y be one cau~e o~ the aacumulation of immune ~om~l~xa~ thAt ~xacerbata ~he clini~al condition~ of SLE pa~ien~, The present invention is ~hus ba~ed upon the discovery that ~he adminis~ratlon o~ ~xo~enou~ ~a~or H andfor Faat~r I to a mammalian, e.g. human. patien~ ~uffering ~om one or more of ~he autoimmune di~ease~, e~pecially SLE, RA and glome~ulone~hri~is, will cure or all~viate ~h~ symptons of th~t d~ sea~e .

In accordanae with the pre~ent inven~ion, there i~, therefore, pro~ided a pharmaceutical aompo~ition ~ompri~ing Fa~or E~ and/or Facto~ I in admix~ure with a pharmaceu~ically acceptable ~ar~ier, diluent or adjuvant~ P~ferably t~e ca~rier ~r diluent comprises a pyroqen-f~ee liquid, such as saline, pho~hats-~u~ered saline or Rin~er'~ ~olution, all in ~ ~rm pr~er~bly s~it~ble ~or in~ ion~. ~oWe~ e~red. ot~qr li~uids pref2rablr i~otonic to h~man blovd may ~Q em~loy~.

1`10~ 5 ~æ6 1~ 5~ I~MT M~I~KS+CLEFJ~ LON 01 q04 4310 P. 07 ~.. ~ In the a~companyin~ drawing~:

~ igure 1 i8 a g~aph showin~ the variation o~ urinar~ prot~in lev~l~ (o~dinate) a~ain~t ~ime in day~ ~s~i~sa) in mice ~u~erlng from ~pontaneous autoimmune di~ea~e~, one ~oup ~hown by l~xl~ on the graph) ha~ing been given a ~ingle administra~iun of Fa~tor ~ and ~h~ other ~roup ~hown by ~o~l on the ~raph~ bein~ a aontro~:

and Figure 2 i~ a graph ~imilar to Figur~ 1 but ~h~w~ng the ef~ct o~ Factor I.

Th~ e~ct~ o~ ~actor~ ~ and ~ on autoimmune di~ea~e~ ~ere inve~tigated by admini~terin~ the e compound~ to an anim~l ~u~fering 6pontaneou~ au~oimmu~e di~eases. As a re~ult, Faotor I and~or FactoL ~ were demon~tra~ed to have a po~ent the~peutic and pre~en~i~e e~ect ~n the autoimmune d~ease~ in thi~ animal. Tbe te~t animals u~ed were mic~ he MRL~l~r ~tc~in.

These ~nimals d~v~}o~ s~ontan~ou~ly A yroup 0~ ptOnl~ ~lo~oly ~e~embling ~ho~e of human S~E, ~ and glomerulone~hriti~. They are aom~only reaogniz~d a~ ~ro~idin~ a model ~o~ ~hd lnve~tiga~$o~ and ~re~me~t o~ these autolmmune di~order~ in hu~an~ ~nd o~her mammals ~ee e.g.

Andrew~ e~ a~ xp.

Med. l~.q, 11~ 15 s7e)J We ha~e ~ound that th~ ~o-called a~lve immune aomplexe~ whiah de~elop the autoimmune di~eas~s are . . ~ NO~/.E15 '~6 17~ GI~T 11~KS-~CLEF~K LOI`I 01 41~'1 q~L0 P.0~3 ~L299L~372 inactivated by the admini~tra~ion of exogenou6 Factor I and/o~ Factor H. FactOr I and ~ac~o~ ~ are known to be proeent ~n blood pl~æma. and aan be separated and ~urifled by a comblna~lon o~ seYeral method~. ~or ~xampl~, F~ct~r I can be purified to homogeneity fro~ ~re~h huma~ plasma by column chromaCography u~in~ ly~ine-Sepharo~e, QAE-~ephadex, C3b-Sepharo~ and Sephadex G-15Q (SEPH~ROSE ~nd ~EP~A~X are t~ade ma~ks).

In add~tlon. F~ctor I c~n be purifi~d from human plasma by u8ing an agarose column ~oupled with a mou~e monoclonal ~nt~ n~i~ody.

Factor ~I can be puri~i~d co homogeneitY ~rom ~re~h human pla~ma b~ u~ing lys~n~-S~pharo~e, ~A~eph~dex, DEAE-Toyop~arl.

Sephacryl S-30~ ~nd hyd~oxyapati~e aolumn chrom~toqr~h~ (TOYOPE~RL and SEPHACRYL are tradQ ma~ks)~ al~o ~06~lble to ob~ain FAC~O~ I and~or Faator ~1 ~y u~ing genetic engineerin~ in which the aompl~ment~rY ~nd~or genomic DNA ~or Facto~ ~ and/or ~ctor ~l a~ ~xpre~ed in, for exampl~, E. coli, y~as~, A~ o~yçes or mammalian cell~.

FUl l dQtail~ Of ~he ~paration and puri~iaation vf Fac~or I (C3bIN~) and Fac~or H ~lH? ~re given by ~earon ~J.

Immunol. 119, 124~ 52 (~977)~, ~ro~ley et al. tBiocham. J. ~ls~o) 191, 173~ , N~ga~awa ~t al.

tJ.

Immunol, 12S, ~7~-5B~ B0)~, We~ler et al. ~P~OQ, Natl.

Ac~d. 9~i. USA ~, 3~ 3272 (1~76)] and Whaley al. ~J.

Exp. ~Sed. 1~4. 1147~ 3 ~1976)~r_~h4~ \ .

rlO~.~5 ~36 17:~11 GMT MRR'KS+~LERI< LC'~`~ 01 404 43113 P.0~ ~ 2~ ~ acto~ H and ~ are pre~erably emp~o~ed in the pre~ent in~ention in a ~orm ~rèe (or essen~ially or ~ub8tantially free~ from native blood pla~ma cont~minants (such as -transferrin, plasmin, Iq~ or Factor B) ~o ~ to hav~ ~on~i~tent and predicta~le phy~ioal and bioahemiaal propertieB~ However. as noted he~eaf~r, o~h~r ~xo~enous m~tecial~ may be ~dded ~0 the pharmaceutical fo~mulation ~ontai~ing Fac~or H and/or I, i~ desired, to achie~e particular re~ult~.

Factors I and H axe pre~erably admini~tered-ln the fo~m Of a ~olution ill 8aline or p~o~ph~te-bu~fered sallno (PBS), pre~erabl~ ha~in~ a pH value of ~rom 7 to de~ired, human tran~errin and/or s~rum al~umin o ~ ) m~ y o ~ a ~ ue E3 ~ u r ~ U ~ c ~ u ~ Fa~or I and/o~ Factor H.

Othe~ adjuvants, e.g. a~ino acids such as glycine, may, if de~ired, b~ adminis~er~d ins~ead or a~ w~ lthough t~e ~ac~or~ ~n b~ admin~ r~d o~all~ or non-orally, non-oral admini~ration, ~uch a~ avenou~ in~ec~ion, i~ pre~e~red. The dosase o~ the drug will de~e~d on th~ age, ~linical conditions and body w~igh~ of th~ ~ati~t~ as well a~ the nature and ~e~ity of ~he di~order. In ~eneral, Fac~o~ I or Factor ~I i given in a do~s of from S0 to ~00~ m~ o~ o~ ~rom 50 ~o 600~ mg. ~e~pe~iv~ ~, ~ I`IOY.~5 'C10 1~ Z C;MT Mrl~l<51CLCr~l~ L01`1 E31 4~14 401~ ~ 7 ~ d~ r fox ~n adult human.

This may admlni~t~red in a single do~e or in divid~d do~e~.

In one preferred em~odimen~ o~ the in~ention, ~he com~o6ieion ~ p~ovided in uni~ dosag~ form. E~h unlt do~e p~ef erably contairL~ f rom 50 tO 1, 000 mg 9f Factor and/or from 50 tO ~.000 mg of Factor ~I.

Wher~ th~ unit dose contain~ ~o~h Fa~tor I and Fact~r E~, the total amount th~eo~ iB pre~e~ably ~om 100 ~o 7.04Q mg. ~ore p~ef ~ably, each unit do~e com~ o~ ~rom lOP to 590 mg o~ Faotor I and/or f rom ~00 ~o 2, 000 m~ o~ Faator H and mos~ pr~ecably f rom 100 tO 300 mg o~ Factor I and/or from ~00 to 1.000 mg of Fac~or H.

Althou~h ~actor I and/or Factor ~ it~elf can admini~tet~d di~ectly ~ th~ ~re~entive and therapeu~l~ drug fo~ au~oimmune diae~e~, ~t 1~ p~e~rred to admini~t~r them in ~80ciation with variou~ ~nown pharmac~utical carrierB. dil~n~s or adju~ntR ap~rvpri~ee eo ~he formulaeion, e.~. cap~ul~s or inje~tion~. Wi~h regard to th~ ~oxi~lty o Fa~eor I ~nd Fac~o~ H, the L~5a valus~ on ~n~r~venou~ injection in m~ce were g~te~ ~han 2 g~k~ and 3 ~/k~, ~e~pe~iYely.

The eff~ctive~e~ of ~cto~s H and I i~ d~mo~tr~d ~y the ~ollowin~ Experiments 1 to 3, ~hil~t ~he composition~ o~ ~he invention ar~ u~trat~d by ~u~saque~t ~a~ple~ 1 to 5.

NO~,/. 05 ~ 17: ~3 GI~T i1:1F~KS-~CLERK LON Ell 40'1 ~gl0 P. ~1 9~ Exp~iment l Effect~ o~ Factor I and/or ~actor ~ on rnice ~ h MRL~l~r mi~ With ~on~aneoU~ autoimn)une disea~e~ e~:e u~ed in ~his experiment.

Sinae these micQ beqarL to show ~rot~inuri~ wlth ~geing due to the renal glomerular defect ~hat i8 induced ~y the de~o6ition o~ th~ immun~ complexes on th~ ~lom~r~ h~ir pathologiCal condition8 we ~ ~udg~d by the protein contents of theiL urine. ~a~tor I and Fa~tor ~I, u~ed in thi~ experimon~, had boen purif i~d to homo~n~it~ e~ed by ele~trophore~ B in the pre~en~e of ~PS ~odiu~ ~odeayl sulfat~).

Twelve weeks old MRL/lpr mice were divided in'co ~o~lt group~ (5 mi~e per ~roup), de~ignated a0 grou~ P2, ~43 and ~. P'actor I di~olved in ~BS wa~ aclnl~nl~t~red in~rav~nousl}~ ~co the mice o~ qroup ~ItZ at a dosa~e of 6 m~/kg once every four ~ay~ for 4 weeks. Faa~o~ H dis~olved in PBS was adminis~r~d intrav~nou~1Y to th~ mie~ of grsup ~ a do~age o~ 100 mg/k~ at th~ same in~erval~. Faato~ I a~d Faa~or ~ ai~o~v~d in P~, were administered intravenously ~o ~he mice ~ o~ ~4 at dosag~ o~ 6 ~nd 100 m~/kg, ~Gp~c~ively. ~t ~he ~Ame int~rvals, To the mice of ~roup ~1 ~control)~ only PBS wa3 admini~tered intravenously as abo~e. On~ d~y a~er ~he ~in~l ad~ini~ra~ion~ ea~h group o~ mi~e wa~ , ,~ . ., ~`~OV.

El5 '~.6 17:13~1 C~l1T I~RK~;+CLER'K LOI`I 01 '10'1 '1310 P. lZ ~2~87;~ maintained fo~ ~4 hou~ in metabolic ca~e~, ~nd the uri~e was coll~cted. Urinary protein le~els Were determined by u6iny a protein a~a~ kit produced by Bio-Ra~ L~boratoriea o~ Riahmond, California, U8A, UBi~ bovin~ serum albumin as th~ ~Candard protein. Th0 ~oll~wing Table show~ the urln~ry protein level~ (mg pro~in/24 hourB~5 mi~e) o~ the~e four group~. A~ ~hown, compared with the ~ontrol ~g~oup ~1), thele were ma~ked de~reABe~ in the urin~ry pro~ level~ in group~ ~, #3 and ~4.

Thus, Faator I, ~a~tor ~ and the mixture of the~e factor~ were all ef~ectlve in impro~ing or preventinq the prote~nU~i~ t~at repre~ent~ ~h~ pathologiadl oondi~ion~ o~ ~he aU~oimmune di~ea~es.

Tabl0 .. . . . . .. .

Grou~ Urinary prvtein 1e~1B mg~24 hour~S mi~ 1.

Con~rol 14.g .

Adm1n.i6tration o~ ~actor I g.7 3.

Ad~inist~ation o~ Faator ~ 7, 4 4~ Administration of Fac~or8 I ~nd ~ 6~5 .... .... . ..

1`10~ . 05 ' ~ 1't GMT ITll: F'I<S+CLEl~'K LO~I 01 '104 4~1EI P . 13 ,, 8 ~:~ ~1 ExperilEle~lt 2 The~p~ut~ ~ o~ca~ o~ ;1 o~ n~ , ~am~ n~ c~ ati~n o~ ator 16 we~k~ old MRL~lpr mice that had ~lready manifast~d nephritis were divi~d into two ~roup8 t5 mi~e per group), ~ na~ed a~ groups #l ~nd ~, a~d housed in ~parate cages. On day 1, e~ch grou~ o~ mice wa~ maintsined ~or 17 hou~ in meta~olic ca~ nd their u~ine was colle~t~d. On day 2I Factor ~I d~olved in P~S wa~ adminietered iAtr~venou~ly to the miae o qroup *2 at a doeage o~ ~ m~/kg. To the mi¢e o~ group ~1 (aontrol)l only PBS wa~ admini~ered intravenou~ly. On d~y~ 3~ 6l 10, 14, ZZ and 31, the urine o~ eaah group wa8 colleated by maintaining the mice for 17 hour~ in metabolic aages as above. Urln~ prot~in level~ wer~ determined ~y usin~ a protein as~ay kit produc~d by 3io-Rad La~orato~ies as de~ri~ed in Experlm~ne 1.

Yig~rR 1 ~how~ the urinar~ pro~ein le~el~ (m~ prot~in/hou~mouse) o~ ~he ~Wo ~oUp~.

The resul~ o~ the ~rou~ ~1 a~e m~ked "o" whil~t ~ho~ o~ grou~ ~2 ar~ ma~k~d "xl'. As ~hown. a ~t~gle admini~tr~tion o~ ~a~tor B~rongly and time-dependentl~ redu~ed the urinary protein level~ iD grou~ ~, and thi~ ~edUa~ian wa3 ~aintained ~o~ about 20 ~a~ In con~ra~t. the ~ont~ol mic~ in gr4up ~1, to whi~h only P~ wa6 ~dmi~ ere~, ~ime-dependently incrfla~d thei~ urinary protein l~v~ ~ ` ~ ... ..... .. . .

I`IUV . ~ b l f l~ YlH~ tl L~ l< LUN 1~11 41~14 4Y~ 1 1'. 14 1~ ExP~-~im-~nt-3 Effec~ ~ Factor I on ronal qlome~ular.funCtion-for .

~ 3 w~k~ old ~RL~lpr mice ~ha~ had already mani~e~d nephritls were d~ided into ~o group~ ~5 mi~e per grou~), design~ted ~roUp~ #1 and ~2, and t~en houBed in separa~o cages. On days 2 and 6. PBS alo~e and ~aator I di~solved in PBS at a doga~ o~ 1 m~k~ were admlnistered tO ~he mice in group~ #1 and ~2, re~p~ctl~ely, On days 1, 5 and 9 a~er admini~ra~ion, eaoh grou~ of mlce was maintained for 17 hours in me~a~olic ~age~ and thei~ urlne wa~ collected a~ ~bo~.

Total urinary p~o~ein lev~l~ were deee~mi~ed by usinq a ~roeein assay ~it ~rodu~ed b~ Bio-Rad Laborata~i~s as described ~ Experiment 1.

In addi~ion~ the urinary p~oCei~ of each group wa~analyzed by 6DS-polyacrylamida~ :

gel ele~troRho~e~s. After staining the ~el wi~h coomassie brilliant blue and ~*-~aining w~ ~h m2th~nol-aaetic acid solu~ion, t~e ~mount~ of p~otein in ` each band in the:g~l w~re det~rmined by a densitom~t~r ~CS-900~0, 5himaz~)- From the amountB o~ total u~in~ry :

p~ote1n and of fractionat~d pr4tein ~a~ds on ~h~ ~el, the amoun~ usin~ry protein~ of highe~ (~or~ th~n 40K~ and lower lle~ than ~OK) molecular w~i~h~ w~r~ calc~late~ ~igure ~ ~hows the ~ounts of urinar~ pro~ins lmg pro~ein/hour/mouse) with khe high~r and low~r moleaul~r~ w~ight of ~he two gro~p~ of mice. Th~ :

. , , . .:

5 '~6 17:0~, G~1T M~RKS~CLE~K LCil`l 01 404 ~ P.l5 13 amount~ Of hi~her molecula weighe pro~ein ~re show~ br a ~olid line; the amounts o~ lowor mol~cul~r ~eight protein are shown by a dot~ed line. As in ~xperimen~ 2, the resultB o~ groUp #l are m~rked "o~ and tho~e Of group ~2 a e mar~ed"x". A~ ~hown, the admini~tra~ion a~ ~aator I Btrangly and time-d~pendently red~ed th~ amount~ o~ urin~ry protein e~pecially in the higher molecular wQight ~ra~ions ~mainly con~i6ting 0~ ~W 6~K p~ot~in~ th~t are pre~n~ in M~/lpr mlce with Rev~re glome~ulon~phrit1~, without ~feating ~h~ amount~ o~ lowar molecular weight f~action~ inly con~i~ting of ~W 14K and 2ZK prot~in~). ThU~. ~actor ~ wa~ ef~ti~e ~ proving the renal ~lomerula~ ~Unc~ion fO~ filtration. On the othe~ h~n~, adminiBtration Of PBS ~lone did n~t inhibit a time-dq~ndent increaRe o~ th~ ur~nary prot~in level~ with ~igh~r molecUlar w~ight, leading to the renaI glom~rular da~c~ in M~L~1Pr mice.

I'c i~ well known that normal glomeruli in tl~e kidney can ~ilter only protein~ o~ MW le~ than ~OK.

50 g o~ ~aetor I wer~ di~olved in ~. 9 litre~ o~ s~erillz~d pho~phat~-~uf~er~d ~aline of FE~ 7. 5.

Th~ pE~ value o~ the re~ul~in~ Bolution W~51B ~;h~n ad ju6~ed ~a~k ~o ~ value o~ 7. 5 by th~ addi~i~n o~ sGdium h~droa~ide, af ter whlch ~he ~olu~ion w~ mad~ up ~o a tot~l volume .

,, .

NOV. 05 '~ 17: ~6 ~;11T !~lRR~KS+t~LERI~ LClIl 01 404 4~10 P. 16 t'".~ 14 oP Z litre~ by the addition o~ ~urther phoephate-~uff~red ~aline ~or injection~. Th~ ~olution wa~ ~ilter~d under ~tsrile condition~ and tAen ~oure~ into ~ial~, each aontaining 250 mg o~ ~he ac~ive ingredi~n~.

~xdmple 2 In~e~t~ble ~ ~ Th~ prooedure d~aribed in Exampl~ 1 was rep~ate~, exaQpt th~t the pho8~hate-bu~red ~aline additionallr cont~ined 0.1 ~ glycln~: ~he p~ waB ~till 7.5.

~.

The p~oc~dure de~cribed in EXample 1 wa~ rep~ d, eXc~t that the pho~phate-buf~red salin~ contaln~d 2 m~/ml of human ~an~fer~in as a ~abilizer: ~he pH ~till 7.5.

~m~ 200 ~ of Fac~or ~ wer~ diR~ol~d in 4.5 li~ree oP ~t~rilized pho~phate-bUf~ar~d ~line o~ pH 7.5. ~nd ~he pH of the r~u~ting 901ution ~a~ adju~ b~k t~ a ~lue of 7.5 by ~he a~dltion o~ sodium hydroxid~. The ''' ~ .

. ' , :

NO~.05 '~, 17:~7 ~;.MT l~lRF~l<S-tCLERI< LON ~1 404 ~1310 P,17 2 ~ ~ ~7 ~ ~olution was made up to A ~otal volume of S litre~ by ~he addl~ion of furtheL pho~phat~-buffered saline for in~ection~ .

The ~olu~ion was filte~ed under ~t~rile aonditions a~d then pou~ed in~o vials, ~ach ~ontaining aoo mg of the ~o~iYe in~redient.

The ~rooeduro de~c~ibod in Exam~}~ 4 wa~ ~pea~ed, exaept tha~ the pho~phate-bu~fered ~aline contained 2 ~g~ml of human tr~n~errin as a s~a~ ze~; th~ pH wa~ still 7.5.

q, ~ .

, ~ .