CA2147600A1 - Production of human monoclonal antibodies active against hepatitis b surface antigen - Google Patents

Abstract

Monoclonal antibodies effective for the diagnosis and treatment of hepatitis B have been prepared from a cell line ob-tained by fusing a xenogeneic hybridoma designated SPAZ 4 with blood cells of a patient immunized with hepatitis B vaccine.

Description

21~7600 PRO~UCTION OF ~MAN MONOCLONAL ANTIBODIES
ACTIVE AGAINST ~EPATITIS B SURFACE ANTIGEN

The present invention concerns hybridoma cell lines which produce humzn antibodies which neutralize the hepatitis B virus, methods for producing the cell lines, antibodies produced by the cell lines, and uses o, the antibodies, pzrticul2rly therapeutically.

The making of hybridom2 cell lines for the purpose of producing monoclonal znti~odies is in general well known at thls time to researchers in this art. The presen~ in~-ention concerns the obtaining of humzn monoclonal antibodies effective in pzrticul2r asainst hepatitis B surfzce zntigen (HBsAg), such antibodies being prepzred according to a generzlly applicable method described by the zpplic2nt in Hvbridomz 2(4):361 ~1983) 2nd United Xingdcm Patent Application 2,113,715A, publisned ~.ugust 10, lg83. ~fore particularly, it has been found that 2 hybridomz cell line comprising a parent rodent immortalizing cell, such 2s 2 murine myeloma cell, e.g. SP-2, fused to z human pzrtner cell results in zn immortalizing xenogeneic hybridoma cell. This xenogeneic hybridomz cell mzy be ~used to z cell cz~zble of producing an anti-HB5P.g human zntibody, resul.inc in z no~el SUBSTlTlJTE SHEET

WO 94/ll495 21~7600 triom2 cell line capable of gener2ting hum2n entibody effective ag2inst such antigen in the human. Alternztely, when sreat2r s.zbility is desired, 2 triomz cel; line which prefe_ably no longer h2s the c2pzbility of producing its own antibody is m2de and this trioma is then fused with z further cell cap2ble of producing useful 2g2inst said antigen so as to ob;ain a still more stable hybridoma (qu2àrom2) which ~roduces antibody against the 2ntigen.

The 2pplicant's publications earlier re'erred to describe the preoaration of a xenoseneic hybridom2 re erred to 2s SPAZ 4, prepzred from drug resist2nt cell line SP-2 obtain2ble, e.g., from the NIG~S ~uman Genetic Mut2nt Cell.Repository Ref. GM.35669A (see V.S. DRHS 1982 Catalog of Cell Lines). Prepar2tion of SPAZ 4 is summzrized as follows. .The SP-2 cell line is fused with norm2l hum2n peripher21 lymphocytes by conventionzl techniques. A lcrge number of hybrids is obt2ined and, after zooroximately five ~ee~s, five clones zre selected wnich show fast growth 2nd no 2ntibody ~roduction. These cells 2re selec;ed for resist2nce to 8-2zaguianine 2nd with three of these lines it is possible to obtzin mutants which zre resistznt to 20 ~g/ml of 8-2zzgu2nine.
Ihese cells are 2t the s2me time sensitive to ~y?oxanthine-~minopterin-Thymidine (RAT) medium which showed th2t they h2d lost their ability to produce hypoxanthine phos?horibosyl tr2ns_ _ase.
One of these cell lines is SPAZ 4.

Cell line SPAZ 4 mzy be fused with cells ob;2inod from the blood of persons immunized with hep2titis ~ v2ccine tO obtain hybridoma cell lines which provide positive cultures when s.andard selection procedures ~re used involving binding of antibodies to relev2nt viral antigons. It is preferred that s2id positiv2 SU8SlllUTE SHEE~

WO 94/1149S PCT/US~2/09749 - 21~76oo cultures be placed through a s2cond selection process in which different subtypes of the virus zre used for antigen preparation.
This provides zn opportunity to pinpoint the exact antigenic dete~min~nt recoanized by the antibody.

The cell lines resulting from the fusion of 2 xenogeneic hybridoma znd the hum2n monoclon21 2ntibody producing cell (trioma) zre therefore useful-in providing monoclon21 antibodies cap2ble of effective 2ctivity in neu-r21izing a virus czusing hepatitis, and said 2ntibodies can therefore prevent the sprezd o' hep2titis through e-g. blood tr2nsfusion . They c2n 21so be used to aive initi21 protection to newborn babies or exposed individualS ezrlier th2n 2 vzccine could be effective.
Anti-hepatitis 2ntibodies may be used to protect immunosuppressed patients, including trznsplzntation patients, from recurrent hepztitis. This is most signific2n; in cases of hepatitis B
positive liver recipients. Further, the antibodies czn be used in diagnostic 2ssays.

It hzs 21so been found thzt zntibody frzgments, such 25 }2b fraGmnnts c2n ~lso bind to hep2titis B virus sur'ace antiaen.
These fr2gments ~lso m2ke up part of this inven.ion.

Specific 2ntibodies wnich h2ve been made cccording to this invention include PEl-l, Z~.l-l, Z~ 2, MD3-4 and L03-3, each o' these zntibodies being of the IgGl class.

Tne cell line producing PEl-l ~-2s deposited 2t .he American Type Culture Collection on Oc.ober 16, 1986 2nd given accession number ATCC H~ 9234; the cell line producing Z~1-l was deposi.ed as ATCC ~B 919.1 on September ~, 1986 znd the cell line producing SUBSTITUTE SHEET

21~7600 ZMl-2 w2s deposited zs ATCC ~B 9192. The zddress o' the ~meric2n Type Culture Collection is 12;01 P2r~1zwn Drive, Rockville, Mzryland 20852.

The cell lines of the present invention 211 beh2ve ~s typical (mouse x hum2n) x human hybridom2s 2nd produce.their respective antibodies in concentrztions r2noing up to 25 mg/l in s.znd2rd sus?ension culture.

DLSCRIPTION OF TH~ FI~URE5 Figure 1 shows the results or 2 direct binding enzyme linked immuno2sszy comp2ring binding kinetics of 2ntibody PEl-l (shown by the single line) znd antibody ZM1-2 (double line). Details 2re -given in Ex2mple ~

Eigure 2 sho~-s the serum levels of antibody Prl-l in rhesus monkey serum determined 2t v2rious times post-dosage. Det2ils zre given in E~2mple 4C.

Throughout the specification and cl2ims, the same design2tion is given to both the cell line 2nd the 2ntibody it proàuces, i.e.
cell line PE1-1 produces monoclonzl 2ntibody PEl-1; cell line ZMl-l produces monoclon21 2ntibody Z~l-l, etc. I, is felt tn2;
one OL ordinzry s~ill in the 2rt will understznd whetnc- the ce71 line or the zntibody is being discussed.

~ !onoclon21 zntibody and cell line PEl-l h2s also been refe~red to by the inventor 2nd the inventor's 2ssignee zs OS~ ;77 znd 64-577. Likewise, monoclor.zl 2ntibody 2nd cell line Z~1-2 hzve zlso been referred to 2s 26S-695, 2nd monoclonal 2ntibody znd cell line L03-3 hzve been refe_red to 2s 266-215.

SUBSTITUTE SH EET

WO 94/1149~ PCT/US92/09749 ,. 214760~

The 2ntibodies ~nd zntibody fragments obt2ined 2ccordina to this invention have good specificity for hepatitis B surface 2ntigen in in vitro ELISA binding zss2ys.

As the 2ntibodies of the present invention 2re of humzn origin, they are advantageously used in human therzpy, as no allergenic response develops with repeated ther2py, 2S ocCurs with murine or ovine 2ntibodies- Thus, another 2spect of this invention is a me;hod of treating hepztitis B through the 2dministr2tion of one or more of the 2forementioned ~ntibodies.
It hzs been found that repezted doses of 2pproxim2tely 10-.0 mg - ~ntibody will substzntially reduce the zmount of circulating H~sAg. Additionzl doses were found to decrease the 2mount of HBsAg levels to below the de.ectable limits o, 2ntigPn tests.

~ nother zspect o. this invention is z coc~t2il of two o, more monoclonal 2ntibodies. This mixture is pzr;icul2-ly suite~ for 2aministration to patients who carry 2 non-wild ty?e s.rain o' hep2titis B virus which does not bind well wi;h z siven single monoclonal antibody. For exPmple, one comp2ssion2te need p2tien;
who suffered from hepztocellul2r czrcinom2 2nd ch_onic hepatitis E
w2s given 2ntibody P~l-l prior tc liver tr2nsplzn;2tion, znd repez-ed doses there2fter- (Det2ils 2re given below in Ex2mple 5). A'ter zpproxim2tely four 2nd one-h21f months o. tre2tm_n., low levels of serum HBsAg could be de.ected with 2 polyclon21 antibody, but not with PL1-1. Polyme-zse ch2in rP~c;ion (FC~) D~.
znzlysis of the 230 bzse pair region of the ~3sAg gene corresponding to the ~ut2tive monoclonzl ~ntibo~y binding domzin was performed. The PCR DNA was cloneà into M13 bzc;eriophzGe 2n~
the resulting D~JA wzs sequenced. Anzlysis of clones from e~ch o.
the serum s2mples rev~zled two v2ri2nt sequences when comp2red to SUBSTITUTE SHEET

WO g4/11495 PCT/VS92109749 214 76 o~ -6-PC~ DNA from the origin2l liver 2r.d pre-antibody th~r2py. The v2riznt DNA codes for t~o different 2mino 2cids in the S protein of H~sAg znd zlso codes for 2 stop codon (U~G) in the viral polymPr2se gene. Both v2riant Genes contzin 2n amino zcid chzng2 resulting in the substitution or 2rginine ror glycine in a conserved peptide domain.

Since the monoclon21 zntibodies PL1-1, Z~!1-2, ZMl-l, MD3-4 and ~03-3 hzve been shown to bind to different e~itopes, 2nd 2t leas~
one of the monoclonal 2ntibodies h2s been found to bind to every vzriant ~irus tested to date to z sufficient e~tent to render it clinically useful, znother 2spect of this inven~ion is a coc~t2il of two or more of t~e monoclonzl antibodies selected rrom the group consisting o.: PEl-l, Z~1-2, ZMl-l, ~ID3-4 2nd L03-3. Pzrticularly preferred 2re cocktails of two monoclonal 2ntibodies, especially the mixture of PEl-1 2nd ZMl-2 znd the mixture of PEl-l 2nd L03-3.
The rz.io o. the monoclon21 2n;ibodies present in the mixture may ry depending on many factors 2pp2rent to one of crdinary skil in tne zrt 2nd include: the genotype o' the hepatitis virus o~
viruses present in the p2tient~s serum, the relati~-e binding strenaths o. the antibodies chosen, the epitopes ~o which the chosen zntibodies bind, 2nd economic considerations. Generally, the 2ntibodies will be present in a ratio ranging from l gc, ~ore ~ypically fro.m 25:75, and preferably in a substan.i211y eGu21 amoun;.
Sections o. the PEl-l, ZMl-l, ZMl-2 and MD3-4 ~-ere sequence~
using standard techniques. The sequence obtzinec for the V~ region of PEl-l is given in T~ble 8-1, and 2re2s corresponding to the CDRl, CDR2, and CDR3 tDH 2nd J~ ) are noted. As the CDR regions 2re particul2rly import2n- regions in determining the bindin~ properties of an SUBSTiTUTE SHEET

~ rCT/US92/09749 antibody, this invention includ~s 2n 2ntibody thzt h2s 2n 2mino acid seauence of its CDRl reaion which is subst2ntially sim~lar to thzt of PEl-l, as set forth in ~able 8-1. This invention also includes an antibody that h2s an amino 2cid sequence in its CD~2 region which is subst2ntially similzr to th2t o' PEl-l, 2s set forth in T2ble 8-1. Further, this invention also comprises zn antibody that hzs an zmino acid seauence of its CDR3 region which is subst2nti211y simil2r to the CDR3 region O r PEl-l, as set 'or.h in T~ble 8-1.

Likewise, the Vn region of Z~l-l was seauenced as is given in Table 8-2 ~re2s corresponding to its CDRl, CDR2, znd CDR3 ( D~
znd JH~) 2re also indicated. This invention includes 2n 2ntibody which h2s zn 2mino acid sequence of its CDRl region which is subst2ntially sim~l2r to thzt of ZMl-l 2s set forth in T2ble 8-2.
Also, this invention includes 2n 2ntibody which h2s 2n zmino 2cid sequence of its CD~2 region which is subs.an,i311y simil2r to thc.
OI ZMl-l 2s set forth in T2ble 8-2, znd further this in~-ention zlso comprises 2n 2ntibody thzt hzs an zmino 2cià seauence o. its CDR3 region which is substantially simil2r to thzt of Z~l-1 2s set forth in T~ble 8-2.
The DNA sequences ~hich code for the regions of ZMl-2 2nd MD3-4 are given in T2ble 8-3 2nd 8-4 respectively. This inventicn also includes 2ny 2ntibody which has amino acid sequences which are subst2nti211y simil2r to that of the regions of 2Ml-2 2na MD3-4 as set for;h in Tables 8-3 and 8-4.
The DNA sequences which code for the VH regions of P~
ZMl-l, zMl-2 2nd MD3-4 were determined and 2ppe2r in Tables 8-1 znd 8-2, 8-3 znd 8-A respectively. These seauences or ~ppro?ria~e fr2cments may be used in cloning antibodies (or modified 2ntibocieS) or as probes. Antibodies which are produced through gene;ic W O 94/11495 P~T/US92/09749 2147~0D

engineering proc2sses (rather th2n conventional hzrvesting fro~
hybriàom2s) czn be ~2de using cloning techniaues ~hich 2re known in .he zrt D~A from other sources may be used to procuce a synthetic antibody molecule ~hich retains the binding ch2:2_teris-~cs of PEl-l, ZMl-l, Z~11-2 2nd ~D3-~ by virtue of h2ving substznti2l1y similzr CDRl, CDR2, and/or CDR3 regions Such zntibodies zre ~ithin the scope of this invention The D~A sequences ~hich code fo_ the VL liSht ch2in v2ri2~1e regions of El-l, ZMl-l, 2Ml-2 ~nd ~ID3-4 zre gi~en in ~2bles 9-1, 9-2, 9-3, znd 9-4, respectively This invention 21so includes 2ny 2ntibody ~hich has 2mino acid sequences ~hich zre subst2ntially similar to th2t o. the regions of P~l-l, ZMl-l, ZMl-2 and MD3-4 as set forth in Tables 9-1, 9-2, 9-3 and 9-4 Also ~ithin the scope of this invention are the DN~ sequences ~hich code for the V8 region, the VL region, the C~Rl regions, the CDR2 regions 2nd/or the CD~3 regions of ~El-l, ZMl-l, Z~1-2 2nd MD3-4. Also included is DNA ~hich ~ould hybricize to any of the 2forementioneà sequences under stringent hybridization conditions This DNA is subst2ntially free from other DNA of the donor m2mm21, and ~ay contain introns or it may be cDNA.
As used throughout the specific2tion and _lzims, the following ce-initions are intended An zmino zcid sequence is nsubstzntizlly simil2rn to znother amino zcid se~uence if their amino acid homology is z- le2st 80~ Referring to D~ s.ringen;
hybridiz2tion conditions~ are those in wnich hyoridization is effected zt 60C in 2 5 X szline citr~te buffer (SSC? followed merely by rinsing zt 3?C zt z reduced buffer concentrztion wnich will no- 2ffect the hybridizatio~s ~-hich tzke plzce r~ssoci2ted mzmmalian DNA~ mezns DNA present in the mzmmzl ~-hich is the sourc_ OI the VH zntibody chzin, but which is not in~olved in coding for zn 2n,ibody or zn.ibody fr2gmen;

The invention is more fully exemplified in the following non-limiting exzmples SUBSTITUTE SHE~T

WO 94/11495 21~ 760~ PCTrUS9~09749 PRObu~llON OF ANTIBODY CE~L LINES

~ um2n volunLeers zre i ~izeà with hep2titis B v2ccir.e. MD3-¢, ZMl-2, Z~l-l, and PEl-1 hybridoma cell lines zre derived from lymphocy.es of individu21s immunized with Heptzv2x~ (~erc~ ~ Co.).
Cell line L03-3 is developed from cells of 2n individual injecte~
severzl times with Hept2v2x~ znd just preceding the fusion, Recombiva $ (Merck & Co-)- Peripher21 blood lymphocytes ar2 purified by density gradient centrifugztion on a cushion of Percoll ~Pharm2ci2 Inc.), density 1.08S g/ml. The isolated lymphocytes 2re w2shed three times in Hank~s ~21anced Salt Solution and mixed with 2n equ21 numDer of cells .rom (mouse x hum2n) cell line SPAZ-~. The cell mixture is pelleted 2t room tempe ature with ~C0 x g for 5 minutes. After removing the medium, the cell pellet is treated with a 50~ soLution o. PEG-1000 in Dulbecco's ~inimzl Essenti21 ~.edium (PIFM) fo- 1 minute zt 37CC
after which the medium was slowly diluted with Dulbecco's ~EM.
The cells 2re collected by cen;rifugztion znd resuspended into Dulbecco's MEM containing 20% fetal bovine serum. The cells 2re seedeà at 2pproximztely 2 X 10 ce~ls per ml into microwell plates. On the following day fresh medium cont2ining the components of ~T medium (hypoxanthine 2minopterin thymidine) is 2dded in order tG select ag2inst non-fused S?AZ-~ cells. On d2y zfter fusion the medium is replaced with fresh mcdiu~ con;ain~nS
only ~T as all cells sens,ti~-e to H~T-selec.ion had been killed by that time.

A.ter 3 to ~ weeks, when g~od grow.h of hybridoma-like cells could be seen microscopically, supernat2nts a-e tested for the SUBSTlTlJTE SHEET

PCTtUS92/09749 , presence of anti-hepztitis B surfzce zntigen an-iboày.' Pn ELIS.~-2sszy using 2 1/100 dilution of Heptav2 ~ on the solid ph2se is used. After incubztion wi~h the supe nzt2nts the pl2tes 2re de~eloped with 2 kit of biotinyl2ted go2t znti-h~m2n immunoglubulin 2nd zvidin-coupled horser2dish peroxidzse (Vec;zs.zin~, Vector Laboratories Inc.). The enzyme is detected by the color re~ction with phenylenedizmine. Positive cultures are picked into new wells 2nd 2 pzrt of the cells is cloned by limiting dilution in Dulbecco's ~!E~ containing 20~ fe.al bovine serum znd 10 mouse thymocytes per milliliter. The cloning plztes zre tested by the szme ELISA method as described 2bove and positive cultures zre expznded znd frozen.

All the cell lines behzve zs typiczl (mouse x hu~zn') ~. hum2n hybridomzs znd produce their respective an;ibod'es in concentrztionS rznging U? to 25 mg~l in st2ndzrd suspension culture.

E ~LE 2 I~UNOC'nr MICi'.L Ci;~ CTERIZ~TION

ntibodv Clzss/Subcl2ss TAe ~munoglobulin c12ss of 2ntibodies pF~ , ZMl-1, Z~-2, ~ID;-.
znc L03-3 is determined using ELTSA methocology. Each antibody is ca?turzd on zn 2ntigen-co2ted pl2.e 2nd e2ch zss2y is devzlc ed ~ith subclzss specific, pero.Yidase-conjug2;ed anti-humzn Ig (T2go). EZch of the 2ntibodies 2~e cle2rly I~Gl.

.

SUBSTiTUTE StlEET

21~7600 B. Light Chain Type Using ELISA methods similzr to those described in A, 2bove, each antibody is tested with 2nti-~ or 2nti-~ light cn~in reag2~;s (Taao). The following results zre obtained.

PEl-l lambd2 - Z~ l kz~pa ZMl-2 kzppa L03-3 lambdz MD3-4 lambda C. Isoelectric Focusina (IEF) ~ . szmple of zntibody L03-3 or PEl-l is a~plied to gel. E2ch is found to behzve zs z basic protein.

D. S~ecificitv Purified HBsAg o, sub;ypes 2dw znd 2yr zre purch2sec fro~
Scripps Lzborztories, Szn Die~o, C21iforni2. H3sAg subty?e zyw is obt2ined from Connzught L2bor2tories (Willowdzle, Ont2rio). ELlsi 2ss2ys 2re performed essentizlly 2s àescrlbed by Ostberg, e; 21.
(1983) Hvbridoma 2:361-367.

PEl-l rezcts ii;h both 2yr 2nd zdw, b~t i; reac;s sligh;ly better wi;h the 2d-.~ subtype. L03-3 re2cts substznti211y equally well with 2yr znd zdw. Z~!l-l shows h~gher rezct~`vity wi~h 2c~, bu; ZMl-2 binds slightly better to zyr. These results zr2 confirmed for PEl-l 2nd L03-3 by Scztchzrà znalysis in solid pnase RIA ~ith solid 2dsorbed 2yr o- 2dw zntigen. Thus, zlthouah these SUBSTtTUTE SH EET

WO 94/1 149~ PCI-/USg2/09749 monoclonal antibodies appzrently do not bind ~o the subtypic deter~inznt, their re2ction with HBsAg czn be significzntly affected by the subtype.

G. Allo.~e Deter~ination Allotypes 2re determined usin~ rezgents supplied by the Centr21 Laboratory of .he Netherl2nds Red Cross T,ansfusion Ser~ice. Inhibition ELISA or direct binding ELISA 2re used.
Results are presented in Tabie 1, below. ~.s c2n bs seen, there is no zpp2rent restriction on high a'finity 2nti-~BsAg antibodies with respect to light chzin or allotype.

.Allot~es of Anti-H~sA~ Monoclon21 Antibodies AllotYoes Antibodv z f z Km(3 PEl-1 - + - *
Z~1-2 +
L03-3 _ + _ ZM1-1 ND ND ND -.

ND = Not determined ~ = Antibody h2s ~ liqht ch~in which does not h2~ie ~m zlloty -s C-. A. nit~

The affinity for solid 2dsorbed HBsAg is detcrmined ror e2ch zntibody using r2diolabelled zntibodies essenti211y as described SU8STITUT~ SHEET

WO 94/11495 PCT/US92~09749 _ : ,.21q76~0 by Wznds, et al. (i981) Gastroenteroloav 80:225-232, whicn is hereby, in~orpor2ted by reference. A~tibodies 2re 12beled ~-ith 125I with Iodogen (Pierce). For e2ch monoclonzl exce;s L03-3, the solid phase absorbed HBsAg is 2yw. L03-3 is 2ss2yed with both 2yr 2nd zdw with essenti211y the s2me results. Antibody-2ntiyen incub2tion occurs 2t room temper2ture.

The relztive 2ffinity is 21so de;ermined using 2n inhibition ELISA in which ~z~ying concentrations of soluDle H3sAg (z~
sub;ype) 2re pre-incub2ted with monoclon21 2ntibody znd ths mixture is then incubated at 37C in a microtiter ~-ell co2ted with HBs~g. Results z-P p-esented below in T2ble 2.

TA~LE 2 Affinit~ of Monoclon21 Pntibodies for HBsAq .
~ntibody Solid Phzse RIA, ~ 1 Inhibition ELISA, M 1 PE1-13.6 X 10' ~2 X 10' ZMl-21.5 X 109 ~7 X 108 L03-31.7 X 109 ~1 X 108 Z~l-l S X 109 ~1 X 108 .

As c2n be seen from the t2ble zbove, for bo.h pr 1-1 2nd ZM'-2 the ELIS results 2-o c~pro~im2;sly ;wo--old lowe- ;h2n t;.e F~
resul.s, which is ~i;hin the r2nge of ex?^rimen;2l error.
sc2tch2rd plot o. the results of the RIA perLon~cd on 8~-1 indic2tes thzt there might be 2 low zffinity bir.ding site. It i5 thus possiole thzt the ELIS~ is me2surinG this low zfr'inity binding site, 2s thg ELISA results are some 50-fold lo~r ;han ;ne SUE~ 111 UTE SH~ET

WO g4/11495 PCT~US92/0974g 4,,~,60~ -RIA. In 2ddition, Scatchard plots also indic2te thzt there 2re considerzbly less high zffinity ZMl-l sites thzn Z~1-2 or PEl-l high 2ffinity si;es. While not wishing to be bound by theory, it zppe2rs that Z~ l m2y have the highest 2ffinity for H~sAg of the four 2ntibodies compared, but only for ~BsAg in 2 cert2in sp2ti21 2rrzngement. This arrangement is only mznifested in a s~zll percentage of HBs~g moiecules. It is 21so possible that ;his ~zy be àue to bivalent binding of ZMl-l to HBs~g while the low affinity site is monov21ent.

E~MPLE 3 Potency of PEl-l Antibody PEl-l is tested for po;ency in the ~USAB
radioimmunoassay (Abbott). Tests are performed agains; the Bureau of Biologics Reference ~epati;is B immune globin, 2nd se~er21 commerci21 hep2titis B immune globulin pre~z_2;ions (H-Bis Im~une Globin~ Hep B G2~m2gee I~mune Globin~ 2nd Hyper Hep I~"une C-lobin~, 211 purch2sed from 2~pharm2ceu;ic21 supply house).
Despite the f2ct that the immune globulin prep2rations 2re polyclonzl 2nd PEl-l is monoclon21, ;he binding datz 2re within the criteria of the Bure~u of Eiologics ror comp2ring immune globul~n prepzr2tions, i.e., the lines were p2r211Ql 2t a prob2bility level less th2n or eGu21 to 0.01.

De;ermination of potency is 2s follows. ~repar2tions are comp2red on a weiGht basis (an absorb2nce ct 208 r~ OL 1.~ iS
2ssumed equal to 1 mg/ml). Prep2rztions of PEl-l which have becn stored 2; 5C 2re then compered with the 2bove polyclon21 prep2r2tions which h2ve 21so been s;ored 2t 5C. The log2rit:~m of SUBSTiTUTE Stl EET

-15- ~ ~600 1000 divided by ~g/ml in the prepzrztion (i.e. the log of a number thzt is inversely proportionzl to the concen;rztion of the i~munoglobin, simil2r to the log of the dilution f2ctor) is then plot;ed vs. log counts per minute (Zverzge o triplicates). The hyoo.Aesis that the fitted lines zre par211el is tested using znalysis o ~-2riznce. It is found th2t the lines 2re p2rallel z.
z probability level of less than or ecu21 to 0.01. Lines of 211 prep2r2tions zre p2rzllel and z common slope is determined. The x-in;ercepts zr2 c21cul2ted from the common slope znd the difference in intercep's us~d to determine the difference in potency. By this procedure, monoclon21 antibody PEl-l is some 435 times more potent th2n the Burezu of Biologics reference hepatitis B immune globin. Since the commercial hepztitis B immune globulin preo2rztions were found to be two-fold (or less) more po;ent ;hzn the 8ureau o, Biologics reference prep2rztion, ~cl-l is at least 200 times more po;ent ;han the commercizl hep2titis B immune globulin prep2rations on 2 weight bzsis.

E ~M~LE

.. Bindinq Xinetics Direct binding enzyme linked i~muno2ss2ys z~e used to compare the kinetics of binding to ~Bs~g of zntiboàies P-l-l 2nà ZMl-2.
ELISA microtiter plztes are coated with Hept3v2Y~ zt 1 ~g/ml.
Wells are then incub2ted z; 37C with 2~ fetal calf se-um in phosphate buffered saline.- ~!onoclon21 zn.ibody FE1-1 or ZMl-2 at 0.5 ~g/ml in 2~ fetal calf serum are incuba;ed in tne wells for vzrious times. A; the indic2ted times tne antibody solùtion is removed 2nd the well is rinsed three times with fresh 2~ fet21 SUBSTITUTE SH EET

W O 94ill495 PC~r/US92/09749 ~ 6~ -16-calf serum. The well is then incubzted with 2~ fet21 c21f serum until the wells for the 90 minute time point con.zin 2~ fe;zl calf serum. Thus, soiu;ion is then replaced with either peroxiàase conjugated Go2t 2nti-lambda chzin (PEl-l wells) or goat 2n;i-k2po~
ch2in (ZMl-2 wells). Quantitation of peroxid2s2 conjugate bound to pl2stic is accomplished with tne ~ddi;ion OL- O-PhenY1ene~iZ~m~ e and H202 Results zre presented in Fig. 1, ~here 2 single line is PEl-1 and 2 àouble line is ZMl-2.

As czn be seen in Fig. 1, at 2 concent-ztion 2t which ptl-l is zlmost completely reacted in S minutes, the reaction of Z~1-2 with solid zdsorbed ~sAg i5 not completed in 30 minutes znd may continue to react for 90 minutes or more. Thus, PEl-1 binds significzn;ly faster to antigen in this assay. ~.ssu~ing this 21so occurs in vivo, PEl-l is likely to be more efficient in neutralizing ~irzl particles before they can infect the ll~-er.

. fiel2tive Posi.ion of Epitopes The relative position of the epitopes of zntibodies PL1-1 L03-3, 2nd Z~1-2 are determined. A simult2neous s2ndwich i~m~tno2ss2y with a solid-zdsorbed monoclonzl antibody is used.
The s2me zntibody is radiol2belled 2nd incub2ted in ~ microtite~
well with the inhibitor znd serum from z hep2titie E posit~Ye p2tient. Rzdiol2belled PL1-1 F2b frzgment is used ~-hile radiol2belled L03-3 is int2ct Ig~. P~esults 2re oree2nted in Tc~le 3, below.

SUBSIIIUTE SHEET

2t~76o~

Inhibition of Binding of Radiolabelleà Monoclon21 Antibody to B sAg by Unl2belled Monoclor.al Antibodies Solid-.~bsorbed Iodin2ted Inhibitor IC50 ngtml m2B maB m29 ~03-3 L03-3 L03-3 10 L03-3 L03-3 PEl-. >22,500 PEl-l PEl-l PE1-1 8 PEl-l PEl-l ZMl-2 76 PEl-l PEl-l L03-3 ~22,500 Monoclon21 antibody ZMl-2 is only approxim2tely nine times less effecti~e in inhibiting 125I-PEl-l's binding to ~Bs.~g thzn unl2belled PE1-1, wherezs L03-3 is thousands OL times le~s e'fective. Thus, the epitopes of ZMl-2 2nd PEl-l 2_e probzbl~
ne2r e2ch other on the H~sAg molecule while the L03-3 e~itope is prob2bly on 2 dif,erent p2_t of the molecule. The reciproc2l e~periment, PEl-l inhibition of r2diolabelled L03-3, provides further e~idence that PEl-l 2nd L03-3 bind to epltopes th2t zre not o~erl2pping.

The similzrity of PEl-l 2nd Z~!1-2 epito?es znd their dif'erence from the L03-3 is confirmed by immuno2ss2y wi;h resuced 2nd zl~ylzted H~sAg. L03-3 c2n bind to den2tured zn.ic2n whi}e both Z~!1-2 znd PEl-1 cznnot so blnd. It should be no~ed th2t PEl-l 2nd ZMl-2 hzve distinc. epitopes since their rezction wl;h dif'erent subtypes ~2ries.

SUBSTITUTE SH F~

WO 94/1149~ PCT/US92/09749 ~ 6~ -18-C Phzrmokinetics of PE1-1 in Rhesus ~onkevs The pharmokinetics of PEl-l is s;udied in two rhesus mon~eys E2ch 2nim21 receives z single intrzvenous bolus injec,ion (0 5 mg/kg) of monoclon21 2ntibody P~l-l Serum levels of pr 1-1 2re determined 2t vzrious times post-dose using 2n ELTSa bzsed szndwich immuno2ssay with Heptzv2x~ co2ted on ELISA plztes 2nd rz~bit anti-idio.ypic zntibodies to PEl-l Results 2re ~hown in Figure 2 serum levels of PEl-l in the two rhesus mon~eys zre char2cterized by z biphasic decline (t 1/2~ = 1 2nd 1 ~ dzys;
t 1/2~ = 11 and 16 dzys) with the shorter h21f-life possibly associzted with the distribution ph2se o' the monoclon21 zr,tibody The voiume of distribution zt stezdy stzte (Vdss) is czlculcted to be 114-144% of the pl2sn2 volume, which suggests little distribution of PEl-l to 2 tissue comp2rtmcnt iQ the ~n;icen-fre-monkey E ~PLE 5 CLINICAI TRIALS

A Comoassion2te use of PEl-l in two p2tients ~-ith end-st2ce liver disease second2r~ to chronic 2ctive heo2titis B 2nd heo2tocellul2r c2rcinomz underqoinq liver tr2nSOl2ntctiOQ

P~1-1 w2s provided on 2 comp2ssionz,e ne3d b2sis to two p2tients with end-stzge li~er dise2se unde-goinc liver tr2nspl2ntation Pztient 1 W2S Z 56 yezr old m21e with c 20 ycar history of chronic 2CtiVe hep2;itis 2nd 2 di2gnosis of SUBSmUTE SHEET

~7600 hepatocellulzr czrcinomz. The second pz;ient ~-zs z 10 ye2r old mzle thought to hzve been infected with hepztitis B 2t birth.
P2tient -2 wzs initizlly ev21u2ted for 2 12rge mzss in the right lobe of the liver, which z biopsy confirmed was hepztocellulzr c2rcinom2.

Preoperztive àoses or PEl-l were 2dministered to these patients znd signific2ntly reduced their circul2ting ~BSAG lev21s befo~e the trznsplznt procedure. Ezch patient zlso recei~-ed t~o 20 mg doses of PEl-l durin5 trznsplzntztion. Postoperztive dosing then begzn on the second dzy following surgery.

Patient -1 never beczme H~sAg negztive, 21though his circul2ting HBsAg levels did diminish markedly from their pretrzatment level. Pztient ~2 beczme ~3sAg negztive, firs, r.oted on post-trznspl2nt dzy 9. P2tient 1 received 2dditionzl doses o~
PEl-l rznging from 5-40 mg zt 2-20 dzy inte-vzls. Pztient ~2 recei~-ed ei;her 5 or 10 mg doses on zverzce OL evey 21-2S c2ys.

No zdverse events were reported for either of these pztier.ts during the period they received PEl-l. However, z?proximately four weeks zf.er Pztient ~1 w2s disch2rged from tr.s hospitzl, it w2s determined thz, he h2d metzst2tic m21ign2ncy. ~e e~pired on post-tr2ns?1znt czy 139. No evidence of recurre.-,; hepz;itis wzs noted during his post-trznspl2nt course despite ;he prese?.ce of detectzble circulzting ~;~s.g. P.lthouah z hepztitis ~ -irus D~.
zss2y w2s neg2tive preo^er2tively, z single positi~-e ~21ue w2g detected 60 d~ys post-trznsplznt.

SUBSTITUTE 5HE~

~ 6 -20-On post-trznspl2nt day 143, P2tient 72 w2s first seen to be positive for ~sAg. ~he HBsAg level fluctuzted for 2 short time before it then stzbilized at a level signicantly below his pretreztment levels. Isolztes of this patient~s hep2titis B virus obtzined before treatment with PEl-l 2nd 2t lzter times were znzlyzed for th2ir binding zbili-y to PEl-l. PE1-1 w~s found to be 2ble ;o bind to the vari2nt virus, bu; not zs well 25 it hzd to the wild-type virus.

Genetic zn21ysis of the two virzl isol2;es indic2ted single nucleotide differences in z highly conserved region of the mzjor virzl surf2ce protein. Such differences, when comp2red to the pre-tre2tment virus, c.ould potentizlly encode for 2 single amino acid difference which would reduce the binding zbility of PEl-l to the hepz.i;is B vir21 binding p2rticle.

~. Use of PEl-l in Dztients with chronic 2ctive he~2titis 3 underooina liver trzns~l2nt2tion (not com~lic2ted bv he~ztocellul2r c2rcinom2) This study involved five p2tients who were ~.Bsi.g positive (but did not have hepatocellulzr c2rcinomz) znd wno underwent liver trznspl2nt2tion. E2ch pztient w2s 2~minis.ered three d2ily preoperztive doses o P~l-l, (10, 20 2nd ~0 mg, respectively) over z three d2y period. ~he liver tr2nspl2nts were ;hen perfon~ed from 2 minimum of two d2ys ;o 2 m2ximum of 32 G ys following their preoperative dose of ;he study drug. An zdditional ~0 mg dose o~
PEl-l w2s 2dministered during the oper2tion. P.ll fi~-e tr2ns?12r.ts ~ere success'ully completed.

SUBSTITUTE StlEET

WO 94/1149~ PCT~US92/09749 21~7~,g The patients' H3sAg titers, liver enzymes, 2nd other clinicel p2rameters were closely monitored during their hospitzl st2ys.
Follow-up ev21uztions znd a~rinistr2tion of PL1-1 by each patients' private physicizn continued on z regul2r basis (approxim2tely every 1 to 3 wee~s). Dosing 2nd other par2me~ers v2ried from p2tient to patient.

Two patients (:5 and ~6) h2d simil2r results ;o P2tient ~2, zbove, in th2t z vari2nt virus 2ppezred 2f.er 2 period o negztive HBsAg screening results. The sera of these pztients rem2ined zctive with PEl-1. Seauence 2nalysis indic2ted the presence of single nucleotide differences between tAe v2ri2nts in the pztients' ser2 2nd wild type virus. Two v2riznts we~e detected in ezch patient. T ~02ss2ys znd seauence znzlysis indic2ted th2t the vzri2nts in e2ch patient were different znd they zlso diffe-ed from the v2riants of Pztient ~2.

Pztient ~3 is 2 39 yezr old C2ucasi2n mz1e who had end-s;ago liver dise2se second2ry to 2 16 yezr history of chronic hep2titis B. The three preoperative doses of PEl-1 thzt were 2oministered to Pztient ~3 c2used 2 subst2ntial reduction in his HBsAg titer.
On post-trznspl2nt dzys 2 2nd 3, he received 20 mg of Prl-l 2nd W25 first noted to be HBsAg neg2tive on post-trznspl2nt czy 2.
For t~-o months there2fter, P2tient .~3 received 10 m~ pr 1-1 on an zve-2ge of eve~y 1 tO 7 dzys. Since then, ho hzs -ecei~-ed 7.5 o-10 mg doses of PE1-1 eve~y 14 ;o 43 dzys. Histo?2.hological ev21uztion of z liver biopsy performed in Febru æ~y, 19~9 W2S
neg2tive for both H3sAg 2nd HBcA~. Pztient 53 rem2ins H.BsAg negztive 582 d2ys 2fter tr2nsplant. In 2ddition to receiving PEl-l, he h4s zlso received three consecutive monthly injections o Recombiv2 ~ in July, August and September, 1989.

SUBSr`~UTE SHEET

WO 94/l1495 PCT/US92/09749 ~ 22-.

Pztient ~4 wzs 2 40 ye2r old P.r2~ic femzle who hzd end-stzge liver disezse second2ry to a 10 year history of chronic active ~:ep2titis B. The three preoperative doses of Pcl-l g-ven to Pzti-nt ~4 czused 2 subst2ntizl reduction in her H~sAg level.
P2tient '2 received 20 mg of PL1-1 on post-tr2nsplant d2ys 1 znd 2, znd wzs found to be HBsAg neg2tive on post-tr2nsplznt d2y 6.
For t-~o months there2fter, she received 10 mg PEl-l on zver2ge of every 3 to 8 days. Since then, she received 10 mg P~l-l every 5 to 26 d2ys. ~pproxim2tely 1 ye2r zfter he_ trans?l2nt, the p2tient developed hep2tic zrtery thrombosis, but rem2ined H3sAg negative, and wzs re-trznspl2nted. Three dzys lzter, due to ischemiz, 2 third trznsplznt wzs performed. Twenty d2ys follo-iing, 2 fourth transpl2nt w2s perrormed due to in'ec;ion.
The pztient expired 18 days after the fourth tr2nsplant (404 d2ys zfte- her initizl tr2nsplant), second2ry to liver _2ilure znd b2cterial sepsis. ~istop2thologic21 ev21uation of 2 liver bioosy from her first trzns?lznted liver showed th2t she wzs ~3S,~.G
negz;ive.

Pztient r5 is z 38 yezr old Czuc2si2n m21e who h2d end-st2ge liver disease secondz_y to chronic zctive hepztitis B. The preoperztive doses o~ PEl-l 2~ministered to the pztien;
substznti211y lowered his circula;ing ~BsAg level. P2tiQnt 5 rec-ived 20 mg PEl-l on post-trznsplznt d,3ys 2 2nd 3, ~nd t~s found to De ~:~sAg nec,~tive on post-transplant d2y 3. During tr.e firs. two months post-trznsplzn;, he received 10 mg PEl-l on ~ver2se o. every 3-7 dzys. L2ter, he received 10 mg F~ very 9 to 26 dzys. The pztient wzs noted to be ~:~sAg positive on post-;rzns?12nt dzy 252, although his 2ntigen level is subst2n~ially lower than his pre-transpl2n~ level.

SUBSTITUTE SHEET

WO 94/11495 ~1~7~ PCT/US92/09749 His.op2thologic21 evalu2tion or a liver biopsy perrormed in J2nuzry 1990 is positive for H~sAg znd H3c~.g.

P2tient -6 is z 38 yezr old Czuczsian mzle wno h2d end-s;2go liver disease secondzry to chronic 2ctive hepztitis fi 2nd 21cohol 2buse. This patient zcquired his initi21 infection vi2 z blood tr2nsfusion. Prio- to the tr2nsplznt, he W25 positive for both ~8sAg 2nd H3eAg. E2ch preoperative dose of PEl-l c2used 2 decre2se in the le-~el of the p2tient's H~s.~g titer. P2tien; -6 received 20 mg of PEl-l on post-trznspl2nt dzys 1 znd 2 2nd was noted to be H~sA.g negztive on post-tr2nsplant dzy 1. For two months thereafter, Patient r6 received 10 mg of Pcl-l on 2verae of every 3-14 days. Subseauently he h2s received 10 mg PEl-l every 7 to 63 d2ys on 2n outp2tient basis. The first HBs~g positive response wzs noted on post-;ranspl2nt d2y 251 2nd occurred zfter his longest durztion (63 days) between doses of ~cl-l. Althougn 2. present P2tient ~6 is positive ror HBs~g, his tlter remzins significzntly lower tn2n pre-tr2nspl2n. levels.

Pztient .7 is a 38 ye2r old Czuc2si2n fem21e with 2 histo~y of Iv drug.zbuse. This pztient hzd end-st2ge liver dise2se secondzry to chronic 2ctive hep2;itis B. Prior to trznspl2nt2tion, the patient w2s positive for }:3sAg and ~EeAa.
Ezch preoperztive dose of PEl-l czused 2 decre2se in the pctient's HBsAg titer. Tne first month pos.-tr2nsplznt, ~2.ien; ~7 re_ei~ed between 10 2nd 40 mg of Pcl-l on the cve-zc2 OL every 1-7 à2ys, znd w2s noted to be HBsAg negz.ive on pos.-trcns?12nt d2y 15.
~ Subse~uently~ she recei~-ed 10 mg PEl-l every 15 ,o 29 d2ys.
~istopathologic21 ev21u2tion of 2 liver biopsy performoc in July, 1989 wzs negztive for ~.3s g 2nd H3cAg. P2tient -~7 re~-i~s HEsAg negative 464 dzys post-trznsplznt.

SUBSTlTlJTE SHEET

WO ~4/11495 PCT/US92/09749 ?~L4rl6~

EXA~PLE 6 P~EACTIVITY WITH V~IANT VI~USUS

The re2ctivity o, the monoclon21 2n;ibodies P_l-l, Z~!1-2, end L03-3 with variant hep2titis B viruses isolzted from pztients described in Ex2r.ple 5 is investig2ted.. Rzdioi~munozsszys 2re performed by determining the radio2ctivity bound to z solid phzse zdsorbed-2ntibody. A solution of z monoclonzl zntiboay 2t 2 concentrztion of 20~g/ml in phosphzte-buffered saline con;2ining 0.02~ NaN3 is incub2ted for zt least 18 hours in U-bottom wells (Fzlcon MicroTest III Flexible Assay Plates). The solution is le...o~ed from the wells znd the wells zre then w2shed three times with distilled w2ter. Fet21.c21f serum at 2 concentrztion o. 2~
in phosph2te-bufrered saline is 2dded 2nd incub2ted overniaht z;
room temperature with solutions of serum HBsAg or controls 2nd l25I-r2diolzbelled antibody (2pproximztely 4,000 cpm in l~ fetzl czlf serum). Wells 2re then w2shed with distilled w2ter three times. Individu21 wells 2re excited 2nd counted. P~esults 2~e presented in Tzble a, below.

Rel2tive Re2ctivitv of Serum-Derited Vzri2nt ~3saa with H~sAa-S~ecific ~.onoclonzl ~.ntibodies S2m~1e~ L03-3:L03-3~ PEl-l:Z~1-2 Z~1-2:ZM1-2 Control 1.000 1.000 1.000 P2tient ~'2 (234) 0.013 0.070 0.233 P2tient 1~ (251) 0.007 0.02~ 0.010 P2tient ~3 (264) 0.043 0.173 0.179 SUBSTlTUTE SHEET

WO g4/11495 2 PCT~US92/09749 _ 1176oO

~Representztive ~sAg-positive serum szmples deri~-ed from p2tionts after liver trznspl2nt2tion znd tre2tment ~ith 2n znti-HBsAg therzpeutic monoclon21 antibody P~l-l. Numbers in pzrentheses denote dzys zfter trznspl2nt2tion.

~L03-3:L03-3 indicztes a rzdioimmuno2ssay composed OL both solid-zdsorbed znd r2diolzbelled human mor.oclon21 2ntibody L03-3. PL1_1:ZM1_2 indicates a rzdioimmuno2sszy composed of humzn monoclon21 2ntibody PEl-l solid-zdsorDed znd hum2n monoclon21 2n.ibody Z~1-2 rzdiol2belled. ZMl-2:ZMl-2 indi ca;es 2 r2dioimmunozss2y composed of both solid-2dsorbed 2nd r2diolzbelled human monoclon21 2ntibody Z~Il-2. Con;rol HBsAg-positive serum reacted well with 2ntibodies L03-3, Pr,1-1 and ZMl-2.

EXAM~LE 7 ~RÇE SC~1E PRODUCTION Or ~NTI~ODIES

To initiate a production run with cells, one or more zmpule(s) o~ frozen cells is removed from liquid nltrogen. After rapidly hezting in a 37C wzter b2th un;il most OL th.e iC2 h2s melted, the empule is opened inside a vértic21 12min2r flow hood.
The contents of the 2mDule zre mixed with z 1 ml ~olume OL
Dulbecco's ~!EM/Hzm's F12(1:1) (DM~M/F12) to whicn ferric szl;s h2ve been 2dded to 2 fin2l concentrz.ion OL' 50 ~M of Fe'~ ter mixing, the tube is filled UD to 2pproximztel~ 10 ml with ;he s~me medium znd the cells zre collected by cen;rifu52;ion. The c-ll pellet is resuspended into 5 ml OL the zbove mentione.a medium with 20~ fet21 bovine serum 2nd seeded into 1 well o_ z 6-well tissue cuIture plzte. ~he cells zre incubated in a 37C incuD2tor in c SUBS I l I UTE SH~ET

W O 94/11495 PC~r/US92/09749 ~,6~-26-5% C02-ztmosphere. When the cells h2ve estzblished themsel~-es in cultu.e 2nd start to multiply an~ have zn 2pproxLm2te cell concentr2tion of 106/ml, the cells znà the medium zre moved into c ;issue culture flzsk with z surf2ce zre2 of 80cm2 znd diluted to 40 ml using DMEM/F12 (without s~-um). Wnen the cells hzve once again re2ched a concentrztion of 106/ml, they, 2r.d the medium, zre mo~-ed into 2 tissue culture flzsk with z surf2ce zre2 of 17;cm2 znd further diluted to z ~olume of 100 ml using DI~EM/F12. -.inen the cells have once again re2ched optimal concentr2tion, the cells 2nd the medium 2re tr2ns erred into 2 roller bot~le with 2 8;0Cm2 surf2ce zrea 2nd diluted to a finzl volume OL 500 ml. When this roller bottle hzs rezcned optimzl cell concentr2;ion, it is split 1/3 into nEw roller bottles using the szme medium 2s be'ore. This splitting process o' the roller bottles is continued until 2 sufficient number of bottles hzve been ob;2ined in order to gi~-e a desired number of cells to seed into ;he verzx System 200 reac.o~.

The ver2x Svstem 200 The Yerz.~ System 200 re2ctor is 2 closed cell cultur2 system wh=re cells 2re cultivated in stainless s;eel ~eighted microspheres (density 1.6g/mL) composed o' cross-lined type ~
bovine collzgen. The microspheres are lo ded }r,to z vertical tr2ns?2rent glass tube ;hrough which ;he culture m~dium (szm- ~S
above ) ia pumped, entering at the bo.;om. The inlet to ;ne tu~2 is o-~ed in such 2 f2shion thc; the microspheres ~ill es;z~lish a fluidi ed bed configur2tion when ;he medium is pumpe~ ;hrou~h z; a suitzble ~elocity. During operztion, fresh mediu,~ is const2n~1y zdded and conditioned medium ~ ~ved zt z rzte ce.e~inea D~ .he cell qro~-.h zs monitored by glucose consumo;ion. ~emperztu~e is SUBSmUTE SHF~

WO 94/1149~ 76 PCT/US92/09749 mzintzined zt 37C; p~ is mzintzined zt i.l 2nd oxyyen/nitrogen rztio is 21so controlled.

~ .fter lozding of the microspheres in 1~ fet21 bovine serum contzining medium, the reactor is run for zt le2st three dzys without cells to zscertain thzt the microsphere 1O2ding did not contzminzte the System. During this time the reactor is fed with protein-free medium to reduce the priming dose o fetal bovine serum. If all systems 2re operzting satisfzctorily, the cells from the roller bo;tles are inoculated into the re2ctor.

The verax Svstem 20~0 This eauipment uses the szme type of microspheres 2s the System 200, znd its controls cnd oper2tions 2re essenti211y the s2me zs for the sm211er system. The S~stem 2000 represen;s cn ap~roximately lS-fold sc21e-u~ comp2red to the System 200.

~!onitorina of Yield of AntibodY from Full-Sczle Culture The conditioned medium is monitored, ezch .ime the h~rvest tznk is emptied, for the level of humzn immunoolobulin in the supe-nzt2nt usina 2n ELISA-type zssay. The results are con~rmed using a Protein A H?LC method.

.zr~es;ina of Cell Culture ~!edi2 znd Production of Harvest ~ool The conditioned m~dium is continuously being removed from tho Ver2x equipment into a re riaerzted h2rvest tank. ~his medium is 12ter unloaded (using the ni.rogen p~essure in the Verzx system~
in;o z mobile stzinless steel tznk for further processing.

SUI~STITUTE SHEET

PCT~US92~09749 6~

Cell Culture ~!edi2 The mediz routinely used is a 1:1 mixture o, Dulbecco's ~I,;.t H21 2nd ~:am's F12 (~ediztech). The medium is purch2sed 2s 2 po~der sufricient for 50 liters o' finished medium. Two such containers of e2ch medium powder,are 2dded into 2 stainless steel tznk cont2ining appro.Yimately 190 liters o' w2ter. The powde- is suspended with 2n impeller un.il 211 h2s been dissolved. Sodium bic2rbon2te is 2dded 2s recommended by the m2nufzcturer and the p:~.
of the medium is set to 7.4. Sodium selenite is 2dded to a finæ~
concentra;ion of 17.3 ~g/l and the volume is topped up to 2001 with ~-ater. The medium is also supplemented with ferric ions in the form of.ferric nitrate/sodium citrate to 2 fin21 concen~r2tion of 50 ~M Fe+'+. The medium is'immediztely added to the medium tznk or the ,verzx System 5200 through the built-in sterilizztion filter. ~o protein is 2dded to the medium. No cntibiotics Oc 2ny type 2re ever used.

PURIr ICATION Or T~ ONOCLONAl P~2Ic~ODY

Description of ~,ethodology o' H2rvesting znd Purific2tion o,' End Product The monoclon21 ~ntibody is produced in cell cul;ure 'rcm ~
hy`ridom2 cell line in the 2bsence o' serum. Tnis m-ans that ~e hzve 2 n^ed to remove from the finzl product only componen;s fron the cellul2r m2teri21. As hum2n monoclon21 cn;1bodies zre not in themselves expected to be immunogenic, it becom.es very im?ort2nt to r^move zll poten;i211y immunogenic components.

SUBSIlIUTE SHEET

WO 94tl1495 ~7 PCT/US92/09749 The go21 of the purification procedures is z final product thzt is more thzn 99.9~ pure, using affinity chrom2tography. We depend he2vily on the biological specificity of 2ffinity chrom2togr2phy. Each step of the purification process (summzrized in T2ble 5) is discussed in more det2il, SuDrz.

PurifiCation Summ2ry Ste~ Conditions ~2terizls Cell RemovalRoom Temp. Polyvinylidene difluoride filters, 0.65/O.gS ~m absolute.
Concentration Microfiltration~4C Polysulfone filter nominal 30,000 daltons. Polyester (0.8~m) and cellulose zcetzte (0.2~m) 2bsolute filters.
Protein A 4C Agzrose couDled Sta~h~loccocus ch-omatogr2phy 2ureus Protein 2,.
Concentration 4C Cellulose tri2cet2te filter, nomin21 20,000 dzlton.
Gel ~C Seph2cryl S-300, Ringer's chrom2togr2phy- L2ct2ted Solution Ion exch2nge 4C SeDh2cryl S-300, Ringer~s L2ctated Solution Cell H2rvest znd Remov21 of Particul2te ~2teri21s 'rom the Conàitioned Medium Even though most of the cells are reteined by the microspheres, 2 sizable number of cells 2re present in the harvested supern2tant. To 2void gross contamination of the medium SUBSTITUTE SHEET

WO 94/11495 PCT/US92/09~49 ?,'~ 4~16~ -by cell components the supern2;2nt is filtered through a polyvinylidene difluoride 0.65~m Prostzck~ filter (~illipore), immediztely after remo~zl from the Verax harvest t2nk. This type of filter unit works in 2 t2ngenti21 flow mode wnich 2110ws filtration of 12rge zmount of p2rticul2te m2terial withou.
clogging the filter. The clezred medium is collec~ed into z refriger2ted stzinless steel t2nk.-Concentration of Conditioned Medium The conditioned medium is concentrated using a nomin21 30,000dalton polysulfone spir21 wound membrzne supplied by ~illioorz.
After concentr2tion, the pH is set to 7.0 using 1~ acetic zcid.
The mzterial is sterile filtered through 2 Sartobr2n-PH 0.8/0.2~m (52rtorius) filter (the 0.8~m component is polyes~er, the 0.2~m component is cellulose zcetate) before being stored 2t 4C. The m2terizl is microfiltrzted (0.22 ~M ~!illipore) 2nd filled into polypropylene vessels.
.

Protein A Chromatoar20hv The extremely powerful purificztion steo utilizes the high 2ffinity of the humzn IgGl zntibody to-St2~hvlococcus zureus Protein A.

The Protein A is purch2sed 21re_dy coupled c0~;2lont}y by an 2mid2 bond to 2gzrose. P.'ter p2cking tne ccl in 2 COlUmA, the column with its contents znd att2ched tubing is s2nitized b-y treztment with 70~ ethznol in wzter for 24 hours. The column is then eauilibr2ted with PBS, pH 7Ø

SUBSTITUTE SHEET

2l~7~

Performing the zffinity chrom2tography sep2r2tion on the Protein A column in~ol~es the following sequentizl steps:

A) Loadina. The concentrated conditioned medium is 102ded on the column with 2 pump. The effluent from the column is collected 2nd monitored for the presence of 2ntibody by the humzn immunoglobulin ELISA. The column is 102ded to such 2 dearee thzt a mezsur2ble amount of zntiboày-contzining fluid p2sses through the column. The overload f~zction is sepzr2tely recovered znd recycled if it contzins more th2n 20 mg/ml znti~ody.

B) Washinq. To ~ ~ve unbound materials the column is e~tensi~ely washed with phosphate buffered saline, pH 7 with sodium chioride added to 2 fin21 concentrztion o, O.SM. This ~sh is followed by z second w2shing step using z buffer of 0.02~
sodium citrzte, pH 5.6, contzining 0.5~5 sodium chloride. Thls ~sh rele2ses sm211 2mounts of the humzn zn.ibody.

C) Elution. The bound monoclonzl zntibodies zr2 eluted from the column using a buffer composed of 0.02~5 sodium citrate, p~ 3.0, contzininq 0.5~5 sodium chloride. The eluted mzteri21 is continuously diluted into ~ volume of 1~. Tris-~Cl, pH 8.0 to rz~idly restore nezr-neutrzl cond~tions.
.
The Protein A purific2tion is performed in z closed s-ys. m utilizing z ~zters 650 Protein Purificztion Sys~em.

SU8STi7UTE SHEET

WO 94/11495 ?CT/US92/09749 ~ 6~Q -32-Concentrztion of Protein A Column Eluzte .
In order to m2ke the following purification step more e fe-tive znd convenient, the elu2te from the P~otein A column is concentrzted to zt lezst 5 mg/ml antibody. The concentr2te is sterile filtered through z 0.2 ym filter 2nd the sterile concentr2te is stored zt 4C until su~ficient materi21s-h2ve been collected for the next purification step.

Size Se~ar2tion b~ Gel Chromatoar2~h~ on Seoh2cr-vl S-300 ~iah Resolution The zntibody prepzr2tion is run on a Sephzcr-~l S300 High Resolution (Phzrmzci2) gel, pzcked in 2 Ph2rmaci2 B~113/120 column with a bed volume of approxim2tely 10 liters. The column is packed in L2c~ated Ringer's Irrigztion USP (Trzvenol Labor2to ies~. The elution of the column is moni;o-ed by z ~2te=s 650 Protein Purific2tion Sys;em.

The purpose of this step is not principzlly zddition21 purific2tion, but buffer change. After the elution of the Pro;ein A column the ~n;ibodies zre in a complex, h-~pe-tonic buffer comoosed o. sodium citrzte, sodium chlo-ide znd Tris-HCl. This bu,fer mi~;u-e czn not be used direc;ly as 2 ~-ehicle for an in;_z~-enous injec;ion- The bu.,er zf.er ;his s.ep is sui;able bo;h for in;rz~-enous injection znd for long tenm refrigerated s;orzge.

SUBSTITU~E SHEET

W O ~4/1149~ PCT/US92~09749 _33_ ~60~

P.emovzl of ~ost Cell DNA b~ P2ssace over zn lon Exchanoe Column Even zfter the ~rotein A chromatography, which removes the bulk of DN~ present in the concentrated supern2t2nt, and the Seph2cryl 5-300~R which Lt ~es DNA molecules thzt 2re eithPr significzntly lzrger or signific2ntly sm211er th2n _he monoclonz~
2ntibody product, there is a smzll; but detect2ble, presence of DNA in the antibody prepzration. We have selected tO remo~-e this cont2min2nt by 2n ion exch2nge step on 2 strong znion exch2naer, Q
SepA2rose (ph2rm2ciz Inc-). At the p~ of Lzct2ted Ringer's solution, zntibody proteins have a positive chzrge, 2nd 2re repelled by the znion exchznger. Nucleic zcids, however, have 2 neg2tive charge at this pH, 2nd will bind to the column.

The column wzs p2c~ed according to the manuf2cturer's suggestions. After decznting the 20~ ethanol solution the gel is delivered in, 100 ml of gel w2s suspended in 200 ml of Lzctatea Ringer's solution. The slurry is poured into z Ph2rm2ci2 F.50/30 column, znd when the gel has pzcked itself to 2 constznt ~olume, it is sznitized with 1 column volu~e of O.SN sodium hydroxide, followed by 3 column volumes of Dulbecco's PBS, ,ollowed by 5 column ~olumes of L2ct2ted Ringer's solution. l.~mediately ~rior to use the column w2s wzshed with zn 2ddition21 5 column ~-olumes of L2ctz;ed Rincer's solution. The sæmple is then passed th.rough - tAe column znd the p2ss-through is collected in c sterile cont2iner.

SUBSTITUTE SHEET

WO 94/1149~ ~ PCI-/US92/09749 EXAM.PLE 8 MOL~:CULA~ ANALYScS O~ PEl--1, ZMl-l, ZMl--2 AND MD3-~

The heavy vari2ble (V~) cnain of antibodies PEl-l, ZMl-l, Z~!1-2 and MD3-4 2re isolated and sequenced. Totzl ~NA is extrzc.ec from 107 hybridom2 cells of e~ch cell line using procecures cescribed in Sanz, et al. 1989 J. Immunol. i~2: 883, ~hich is hereby incorporated by reference. Single stranaed DNA is synthesizea usins A~V-reverse trznscript2se 2s the enzy~e 2nd oligo-aT as tne primer. The quantity of the synthesized ss-cD~lA
is assessed by ~easuring the incorporation o. 32p-dCT.

Polymer2se chain rezctions (PCR) 2re perlormed essenti211y as recommended by the mznuf2cturer ~Perkin Elmer Cetus, Nor~21k, Connecticut). One microgr2m of DMA is zdded to 2 200 ~m solu.-o~
of.e2ch of d3T?, dCTP, dGTP znd dTTP, wi;h 100 p moles ezcA o_ primer znd 5 units of T2q DNA poly~er2se. PCR cycles a~e zs follows: denztur2;ion 2t g8C for 3 minu.es, annealing at 55C for 2 minutes, znd extension 2t 72C for ;hree minutes, cont~olled in 2 D~ thermzl cycle- (Per~in Elmer Ce.us).

~ .~?lified D~IA is size selected on 2 1.0~ lo~ meltir.g 2gaEcce gel, ligzted into tAe EcoRV site of 2 fiLUESCP~IPT ph2gEm~d vector, 2r.d ;r2ns,0rmed in;o c2c12 comDetent BSJ72 b2cter~ 3. Singl st-cnded D~3 for s_s~encing is isolz.ed ~rom each FositiY_ c~ore 2';e_ su?erin.ec;ion ~ith ~!13:~07 zs descriDed by Sanz, et al , s~r2. Seauencino is zccomplished vi2 the dideoxy crain termin2tion me;hod zs described by 52nger, e. 21. 1980 J. Mol.
Eiol. 1~3:161, e:ccept 2 modified T7 DN3. pol~meras2 (Seouen~Se) is used zs described by TzDor, e. 21. 1987. P~AS (USA) 84:4767.
Results 2re giYen in Tzbles 8-1, 8-2, 8-3 and 8-4.

SUBSTITVTE SH EET

WO 94/11495 PCTrUS92/09749 - 21~760G~

Table 8-l DNA seauence OL the V~ region of PEl-l is sho~n below.
The leader, V~III, D, ana JH4 regions are denoted by the d~sned line, complement2rity-deter~ining regions CDRl 2nd C~R2 are indicated by the asteri5ks. Amino acids 2ppear 25 single le-ter 2bbrevi2tions below the DN~.

c- - LEAOER ~ ~
ATG 6A6 m G&6 CTG AGC TGG 6TT TTC CTC 6TT 6CT CTT TTA A6A 6CT 6TC CA6 T6T CA6 6T6 CA6 H E F G L S ~ Y F L Y A L L R 6 V Q C a v CA
Y~III
CTG 6T6 GA6 TCT GG6 G6A 6GC 6T6 GTC CA6 CCT GG6 A6`6 TCC CT6 AGA CTC TCC T6T 6CA ~CC TCT
L- V E S & 6 G V Y Q P 6 R S L R L S C A A S
AAAAAAACDRlAAAAAAAA
GGA TTC ACC TTC AGT AGG TAT GGC ATG CAC TGG GTC CGC CA6 GCT CCA 6~C AA6 6G6 CT6 6A6 T66 G F T F S R Y 6 H H ~ V R Q A P 6 K 6 L E
AAAA~AAAAAAAAAAA~AAAAAAAAAAAA COR2 AAAAAAA~AA~AAAAAAAAAAAAAAAAAAAAA~
GT6 GCA 6T6 ATA TCA TAT 6AT 6&A A6T AAT AAA TG6 TAT 6CA GAC TCC 6T6 AA6 6&C C6A TTC ACC
Y A V I 5 r D 6 S H K ~ Y A D S V K 6 R F T

ATC TCC A6A 6AC AAT TCC AA6 AAC ACT CT6 m CT6 CAA AT6 CAC A6C CT6 A6A 6C~ 6C6 6AC AC6 I S R D ~ S K N T L F L a H H S L R A A D T
> ~- D > `~ -66T 6TA TAT TAC T6T 6C6 AAA 6AT CAA CTT TAC m 66T TC6 CA6 A6T CCC 6~6 CAC TAC T66 6TC
6 Y Y r c A K D Q L Y F 6 S Q S P 6 H Y ~ Y
J~4 _ ~

5UBSmUT~ 5t~EET

6~

Table 8-2 DNA sequence of the VH region of ZMl-l is sho~n below.
The leader, VHIII, D and JH4 regions 2re denoted by the d2shed line; comDlement2rity-determining regions CD~l 2nd CDR2 are indicated by the asterisks- Amino acids 2Dpear 2s single letter zbbreviations belo~ the DNA.

< - - LEADER ~ <
AT6 6A6 m 6GG CT6 AGC TG6 GTT TTC CTT GTT 6CT ATA TTA 6M 66T 6TC CA6 T6T 6A6 6T6 CA6 H E F 6 L S S~l V F L V A I L E 6 Y Q C E Y Q
- Y"III

~AAAAACDRlAAAAAA`AAA-- _ _ 66A TTC ACC TTC AGT AG6 TAC 6AC AT6 TAC TG6 6TC C6C CAA 6CT ACA 66A AAA 6~T CT6 6A6 TE~

AAAAAAAA~AAAAAAAAAAAAAAAAAAAAcDR2AAAAAAAAAAAAAAAAAAAAAAAA~AAAAA--6TC TCA 6CT ATT G&T CCT ACT 6&T &AC ACA TAC TAT. 6CA 6AC TCC 6T6 M6 66C C6A TTC ACC ATC
V S A I 6 P T 6 D T r Y A D S V K 6 R F T

TCC A6A 6AA MT 6CC M6 MC TCC TT6 TAT CTT AUA AT6 MC 6~C CT6 A6A 6CC 6~6 6AC AC6 6cr _ > <------D------~ < J~4 6T6 TAT TAC T6T 6CA A6A EAT TTA 6AA CTC T66 6~;C CA6 6~A ACC CT6 6TC ACC 6TC TCC TU.
v r Y c A R D L E L ~1 6 Q 6 T L Y T V S S

SU8STlTlJTE SHEET

WO 94/11495 PCT/USg2/09749 ~1~7~o Table 8-3 DNA sequence of the VH region o~ ZMl-2 is shoun belou.
The le2der, VH IV, D 2nd J~4 regions are aenoted by the d2shed line; complementarity-determining regions CDRl and CDR2 are indicated by the asterisks. Amino zcids 2ppear as sing}e letter abbrevi2tions belou the DNA.

~ LE~DER > <
AT6 AAA CAC CT6 TG6 TTC TTC CTC CT6 CT6 6T6 6~ A 6TT CCC A6A TG6 6TC 6T6 TCC CA6 6T6 CA6 H K H L 1~ F F L L L V A Y P R ~I Y Y S Q Y
Y"IY

~AAAAAAACDRl~AAAAAA
CGT GGC TCC TTC AGT GAT TAC TTC TGG AAT T~6 TTC C66 CA6 CCC ~CC 6éi6 AA6 C6C CT6 6A6 T66 R G S F S D Y F Sl N ~ F R Q P A 6 K R L E
_ ~ A A A A ~ A A A A A A A A ~ A A A A ~ ~ A A ^CDR2^^^~^^A^^^ A A A A A ~ ~ A ~ A A ~ ~ A ~ ~ A A ~ A A A A ~ A--CTT 6G6 C6T 6TC TAT ACC A6T 6~iA AGT 6TC 6AC TAC MC CCC TCC CTC M6 A6T C6A 6TC ACC 6T6 L 6 R Y r T S 6 S Y D r H P S L K S R Y T V

TCA 6T6 6AC AC6 TCC AA6 AA6 CA6 TTC TCC CT6 A~i6 CT6 A6C TCT 6T6 ACC 6TC 6C6 EAC AC6 ~iCC
S Y D T S K K a F S L R L S S Y T Y A D T A
~ ~____-D > ~ J14 6T6 TAT TAT T6T 6C6 A6A 66A CT6 TCC 66T m 6AC TAC T66 6~7C CA6 6~iA 6CC CT6 6TC ACC 6TC
Y Y r C A R 6 L ~ S 6 F D r ~ 6 Q 6 A L Y T Y
______~
TCC CCA
S P

SUBSTITUTE SHEET

Table 8-DNA sequence of the YH region of MD3-4 is sho~n belo~.
The le2der, V~V, D and JH3 regions 2re denoted by the d2shed line; comDlement2rity-determining regions CD~l 2nd CD~2 2re indic2ted by the 2sterisks. Amino zcids 2ppezr 2s single letter abbrevi2tions below the DNA.

. . . .. . .
c- - LEADER > c AT6 6&6 TCA ACC 6CC ATC CTT 6GC CTC CTC CT6 6CT 6TT CTC CAA 66A 6TC T6T OEC 6AA 6T6 CA6 Y~Y

*~CDR1^^^^^^^-GGA TAC AGC m ACC AGC TAC TGG ATC AGC T66 6T6 C6C CA6 AT6 CCC 6~i6 AA 6cC CT6 6A6 T~;
G Y S F T S Y ~l I S ~I Y R Q H P 6 K 6 L E ~l A ~ cDR2 ~ A * * A ~
AT6 6~; A6~ CTT 6AT CCT AGT 6CC TCC TCT 6CC ATC TTC A6C CC6 TCC CTC CM ~iC C~C 6TC lCC
H 6 R L D P S A S S A I F S P S L ~ 6 H Y T

ATC TCA 6TT 6AC M6 TCC AT6 A66 ACT 6CC TAC 6T6 CA6 T~6 A6A A6C CT6 M6 6CC TC6 EAC ACC
S Y D K S H R T A Y Y Q ~I R S L K A S D T
~ ~ D
6CC AT6 TAt TAC T6T 6C6 A6A CAT 6TC Cl;C 6AA M6 A6T AT6 6TT CA6 66A 6TC ATT ATA AA6 6AC

.c J;,3 >
6CT m 6AT ATC T66 6~C CM 6~6 ACA AT6 6TC ACC 6TC TCT TCA
A F D I ~ 6 ~ 6 T H Y T Y S S

SUBSmUTE SHEET

W O 94/1149~ P~r/US92~09749 - 21~7~

Following the procedures of Example 8, the light v2riable (VL) chain of antibodies PEl-l, ZMl-l, ZMl-2 2nd MD3-4 are isolated and sequenced. Results are given.in Tables 9-1, 9-2, 9-3 and 9-4.

SUBSmUTE S~EET

Table 9-1 DNA sequence of the VL region of PEl-l is sho~n belou.
The VV and J3 regions are denoted by the dashed line;
complement2rity-determining regions CD~l, CDR2 and CD~3 are indicated by the asterisks. Amino acids appe2r as single lette~ 2bbreviations belo~ the DNA.

CA6 TCT CA6 CT6 AC6 CA6 CCG CCC TC6 6T6 TCA 6T6 6CC CCA 6~6 CA6 AC6 6CC A6~ ATT ACC T6T
s a L T O P P S Y S Y A P 6 . 0 T A R I T C
~ AAAAAAAAA~AA~AACDRlAAAAAAAAAAAAAAAAAAA
6~6 66A 6AC AAC ATT 6&6 AGT AAA AGT 6T6 MC T66 TTC CA6 CA6 AA6 CCA 6erC CA6 6CC CCT 6TC
6 6 D N I 6 S lC S Y H ~ F ~ O K P 6 Q A P V
I~A~A~ ACDR2~A~ A~A

L v v r D D H E R P S 6 I S E R F S 6 5 H S

6~;6 AAC AC6 6CC ACC CT6 ACC ATC A~C A66 6TC 6AA 6CC 6~6 6AT 6A6 6CC 6AC TAT TAC T6T CA6 6 H T A T L T I S R V E A 6 D E A D Y Y C ~1 ~AAAAAAAAAAACDR3AAAAAAAAAA> <AAAAAA J 3_ >
6T6 T66 6AT A6T A6T A6T 6AT CAT 6T6 6TA TTC 6~C 66A 6~i6 ACC AA6 CT6 ACC 6TC CTAV ~ D S S S D H Y V F 6 6 6 T K L T V L

SUBSTITUT~ SHEET

W O 94/11495 PC~r/US92/09749 - 21~7~o Table 9-2 DNA sequence of the VL region of ZM1-1 is shoun below.
The leader, YII and J5 regions are denoted by the dashed line; comple~ent2rity-determining regions CDRl, CDR2 and CD~3 are indiczted by the 2sterisks. Amino acids 2ppear as single letter abbreviations belou the DNA.

< LEADER -AT6 6AC AC6 AG6 6TC CCC 6CT CA6 CTC CT6 6G6 CT6 CTA AT6 CTC Tc6 6TC CCA 66A TCC A6T
H D T R Y P A Q L L 6 L L H L ~I Y P 6 S S 6 < V,II

TGC A6A TCT A6T CTA AGC CTC 6T6 6AC AGT 6AC 66A AAC ACC TAC TT6 AAT T6~ m CTC CA6 A66 C R S S L S L Y D S D 6 N T Y L N ~I F L Q R
^^^^^^^~^~^CDR2^^^^^^^~
CCA 66C CAA TCT CCA AG6 CGC CTA ATT TAT CA6 ~.11 TCT A6C C66 6AC TCT 666 6TC CU~ 6AC A6A

TTC A6C 6~C A6T 6C-6 TCA 6GC ACT 6AT TTC ACT CT6 AAA ATC A6C A66 6T6 6A6 6CT 6A6 6AT 6TT

^^^^^^^^^^~CDR3~ ,. <~ _ J 5 6~C 6TT TAT TAC T6C AT6 CAA 66T ACA CAC T66 CC6 ATC ACC TTC 6 C CAA 6~i6 ACA C6A CT6 6Ac 6 Y Y Y C H tl 6 T H 1~ P I T F 6 Q P T R L E
>
AT~ MA C6A
K R

SUBSTIl UTE SHEET

WO 94/1149~ PCT/US92/09749 Table 9-3 DNA sequence of the VL region of ZMl-2 is sho~n belo~.
The leader, VI and J regions are denoted by the dashed line:
complementarity-determining regions CDRl, CDR2 znd CDR3 are indicated by the 2sterisks. Amino acids 2ppe2r as single letter abbrevi2tions belou the DNA.

~ LEADER > <------H R P Y A Q L L G L L L L ~ F P G S R C D
V,I ~tD
CA6 AT6 ACC CAG TCT CCA TCT TCC 6T6 TCT 6 U TCT 6T6 6~A 6AC AGA 6TC ACC 6TC ACT T6T C66 Q H T Q S P S S V S A S V G D R V T Y T C R
AAAA A A AAA A ~ A A~ CDRl^^AA^^A^A A~ A A~ A ~
6C6 h6T CA6 6GT ATT AGC AGT TG6 TTA 6CC TE6 TAT CA6 CA6 AAA CCh 6&6 AAA ~CC CCT A M CTC
A S Q G I S S ~ L A ~ Y Q Q K P 6 K A P K L
~A~A`~CDR2^^^^^^^^^^^^^^^
CT6 ATC CAT 6CT GCA TCC AGT TTG CAA AGT 666 6TC C U T U AG6 TTC ATC E~C A6T 66A TCT 6~6 . 2~[C
A U EAT TTC ACT CTC ACC ATC ACC A6C CT6 CA6 6CT 6AA 6AT m 6 U ACC TAC TAT T6T CAA CA6 T D F T L T I T S L Q A E D F A T Y .Y C ~ Q
~ COR3**~**~ '^^^^^^- J~ >
6CT 6AC A6T CTC CCT m ACT TTC 6&C E~A 666 ACC AA6 6T6 6AC TTC AAA C6A

W O 94/11495 1~7600 PC~r/US92/09749 T2ble 9-4 DNA sequence of the VL region of MD3-4 is shown below.
The VIII znd J3 regions are denoted by the dashed line;
comDlementarity-determining regions CDRl, CDR2 and CDR3 are incicatec by the asterisks- Amino acids aDpe2r as sinsle letter 2bbrevi2tions below the D~A.

<_ V~
CA6 TC~ CAG CT6 AC6 U G CCT GCC TCA 6T6 TCC 6T6 TCC CCA 66A CA6 ACA 6CC A6C ATC ACC TGC
~ S ~ L T Q P A 5 Y S Y S P 6 Q T A S I T C
~AAAAAAAAAAAAAAACDRlAAAAAAAAAAA~AAAAAAA--TCT 66A 6AT A6A TT6 6G6 6AT 6AA 11~ 6CT TCC T66 TAT CA6 CA6 M 6 CCA 6~C CA6 TCC CCT ATT
S 6 D R L 6 D E F A S ~ Y ~ CA K P 6 ~ S P
A~AA~A~AAAACDR2AAAAAAAAA-Al~
CT6 6TC ATC m GA6 6AT M C M 6 AG6 CCC TCA 6~6 ATC CCT 6 M C6A TTC TCT 6~C TCC M C TCT
L V I F E D ~ K R P S 6 I P E R F S 6 S ~ S

6u6 AAC ACA 6CC ACT CT6 ACC ATC AGC 666 ACC CA6 6CT AT6 6AT 6A6 6CT 6AC TAT TAC T6T CT6 6 ~ T .A T L T I S 6 T ~ A H D E A D Y Y C L
AAAAAAACDR3AAAAAAAAAAA> <AAAAAA J~3 >
6C6 T66 6CC A6C A6C CTT T66 6T6 TTC 66C 66A 66~ ACC M 6 CT6 ACC 6TC TT6 A ~ A S S L ~ Y F 6 6 6 T K L T Y L

SUBSrl~UTE SHEET

Claims (34)

What is claimed is:

1. A human monoclonal antibody which neutralizes hepatitis B virus.

2. An antibody according to claim 1 which is IgG1.

3. An antibody according to claim 2 wherein the hepatitis B
virus which is neutralized is non-wild type.

4. An antibody according to claim 2 which is selected from the group consisting of PE1-1, ZM1-1, ZM1-2, MD3-4 and LO3-3.

5. A monoclonal antibody cocktail comprising a mixture of at least two human monoclonal antibodies, each of which antibodies is neutralizing toward hepatitis B virus, and each of said monoclonal antibodies specific for a different epitope of hepatitis B surface antigen.

6. A cocktail according to claim 5, wherein one of said monoclonal antibodies is PE1-1.

7. A cocktail according to claim 6, wherein the cocktail further comprises ZM1-1.

8. A cocktail according to claim 6, wherein the cocktail further comprises ZM1-2.

9. A cocktail according to claim 6, wherein the cocktail further comprises LO3-3.

10. A Fab fragment of a human monoclonal antibody of claim 1.

11. A Fab fragment according to claim 10 wherein the monoclonal antibody is selected from the group consisting of:
PE1-1, ZM1-1, ZM1-2, MD3-4 and LO3-3.

12. A hybridoma cell line comprising a xenogeneic immortalizing cell fused to a human cell producing an antibody which neutralizes hepatitis B.

13. A cell line according to claim 12 wherein the xenogeneic immortalizing cell comprises a hybridoma fused from a parent immortalizing cell and a human partner cell.

14. A cell line as claimed in claim 13 wherein the parent immortalizing cell is a murine myeloma or hybridoma.

15. A method of making the hybridoma cell line of claim 12 comprising making a xenogeneic immortalizing cell line drug resistant, fusing the resulting drug resistant immortalizing cell to a human antibody-producing cell, and selecting the desired hybrid.

16. An antibody produced by the cell line of claim 12.

17. An antibody produced by the cell line of claim 13.

18. An antibody produced by the cell line of claim 14.

19. A cell line according to claim 12 which produces an antibody designated PE1-1.

20. A cell line according to claim 12 which produces an antibody designated ZM1-1.

21. A cell line according to claim 12 which produces an antibody designated ZM1-2.

22. A cell line according to claim 12 which produces an antibody designated LO3-3.

23. A method for combatting a hepatitis B virus comprising administering to a subject in need of such treatment a hepatitis B
combatting effective amount of human monoclonal antibody which neutralizes hepatitis B.

24. A method according to claim 23 wherein the monoclonal antibody is selected from the group consisting of: PE1-1, ZM1-1, ZM1-2, MD3-4, LO3-3 and mixtures thereof.

25. A method of reducing the level of circulating hepatitis B surface antigen in a patient comprising administering to the patient a HBsAg-reducing effective amount of human monoclonal antibody which neutralizes hepatitis B.

26. A method according to claim 25 wherein the monoclonal antibody is selected from the group consisting of: PE1-1, ZM1-1, ZM1-2, MD3-4, LO3-3 and mixtures thereof.

27. A monoclonal antibody which has a VH region which is substantially similar to that shown in either of Tables 8-1, 8-2, 8-3 or 8-4.

28. A monoclonal antibody which has a VL region which is substantially similar to that shown in either of Tables 9-1, 9-2, 9-3 or 9-4.

29. A cell line which produces an antibody according to claim 27.

30. A cell line which produces an antibody according to claim 28.

31. DNA substantially free from associated mammalian DNA comprising a DNA sequence which hybridizes under stringent conditions to a strand of DNA substantially similar to that selected from the group consisting of:
a) a strand of Table 8-1;
b) a strand of Table 8-2;
c) a strand of Table 8-3;
d) a strand of Table 8-4;
e) a strand of Table 9-1;
f) a strand of Table 9-2;
g) a strand of Table 9-3; and h) a strand of Table 9-4.

32. A cell line having DNA comprising the DNA of claim 31.

33. A cocktail according to claim 6, wherein the cocktail further comprises MD3-4.

34. A cell line according to claim 12 which produces an antibody designated MD3-4.