AU684455B2 - Production of human monoclonal antibodies active against hepatitis B surface antigen - Google Patents

Description

OPI IATE 08/06/94 APPLN. ID 31775/93 I ilili I li AOJP DATE 11/08/94 PCT NUMBER PCT/US92/09749 111 IllillI I m H 111111111 11111111111 AU9331775 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 94/11495 C12N 5/20, 5/28, C07K 15/28 Al C12P 21/08, A61K 39/395 (43) International Publication Date: 26 May 1994 (26.05.94) (21) International Application Number: PCT/US92/09749 Published With international search report.

(22) International Filing Date: 6 November 1992 (06.11.92) (71) Applicant (for all designated States except US): SANDOZ, LTD. [CH/CH]; CH-4002 Basle (CH).

(72) Inventor; and Inventor/Applicant (for US only): OSTBERG, Lars, G. [SE/ US]; 19 Catalpa Road, Convent Station, NJ 07961 (US).

(74) Agents: BATTLE, Carl, W. et al.; Sandoz Corporation, 59 Route 10, East Hanover, NJ 07936 (US).

(81) Designated States: AT, AU, BB, BG, BR, BY, CA, CH, CS, 4 DE, DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, MN, MW, NL, NO, PL, RO, RU, SD, SE, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LJ, MC, NL, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG).

(54) Title: PRODUCTION OF HUMAN MONOCLONAL ANTIBODIES ACTIVE AGAINST HEPATITIS B SURFACE

ANTIGEN

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

WO 94/11495 PCT/US92/09749 -1- PRODUCTION OF HUMAN MONOCLONAL ANTIBODIES ACTIVE AGAINST HEPATITIS B SURFACE ANTIGEN The present invention concerns hybridoma cell lines which produce human antibodies which neutralize the hepatitis B virus, methods for producing the cell lines, antibodies produced by the cell lines, and uses of the antibodies, particularly therapeutically.

The making of hybridoma cell lines for the purpose of producing monoclonal antibodies is in general well known at this time to researchers in this art. The present invention concerns the obtaining of human monoclonal antibodies effective in particular against hepatitis B surface antigen (HBsAg), such antibodies being prepared according to a generally applicable method described by the applicantyin Hybridoma 2(4):361 (1983) and United Kingdom Patent Application 2,113,715A, published August 1983. More particularly, it has been found that a hybridoma cell line co'mprising a parent rodent immortalizing cell, such as a murine myeloma cell, e.g. SP-2, fused to a human partner cell results in an immortalizing xenogeneic hybridoma cell. This xenogeneic hybridoma cell may be fused to a cell capable of producing an anti-HBsAg human'antibody, resulting in a novel SUBSTITUTE SHEET WO 94/11495 PCT/US92/09749 -2trioma cell line capable of generating human antibody effective against such antigen in the human. Alternately, when greater stability is desired, a trioma cell line which preferably no longer has the capability of producing its own antibody is-made and this trioma is then fused with a further-cell capable of producing useful against said antigen so as to obtain a still more stable hybridoma (quadroma) which produces antibody against the antigen.

The applicant's publications earlier referred to describe the preparation of a xenogeneic hybridoma referred to as SPAZ 4, prepared from drug resistant cell line SP-2 obtainable, from the NIGMS Human Genetic Mutant Cell.Repository Ref. GM35669A (see U.S. DHHS 1982 Catalog of Cell Lines). Preparation of SPAZ 4 is summarized as follows. The SP-2 cell line is fused with normal human peripheral lymphocytes by conventional techniques. A large number of hybrids is obtained and, after approximately five weeks, five clones are selected which show fast growth and no antibody production. These cells are selected for resistance to 8-azaguianine and with three of these lines it is possible to obtain mutants which are resistant to 20 gg/ml of 8-azaguanine.

These cells are at the-same time sensitive to Hypoxanthine- Aminopterin-Thymidine (HAT) medium which showed that they had lost their ability to produce hypoxanthine phosphoribosyl transferase.

One of these cell lines is SPAZ 4.

Cell line SPAZ 4 may be fused with cells obtained from the blood of persons immunized with hepatitis B vaccine to obtain hybridoma cell lines which provide positive cultures when standard selection procedures are used involving binding of antibodies to relevant viral antigens. It is preferred that said positive SUBSTITUTE SHEET P:\WPDOCS\UR\SPCSi549283.SPC 8/97 -3cultures be placed through a second selection process in which different subtypes of the virus are used for antigen preparation. This provides an opportunity to pinpoint the exact antigenic determinant recognized by the antibody.

The cell lines resulting from the fusion of a xenogeneic hybridoma and the human monoclonal antibody producing cell (trioma) are therefore useful in providing monoclonal antibodies capable of effective activity in neutralizing a virus causing hepatitis, and said antibodies can therefore prevent the spread of hepatitis through e.g. blood transfusion. They can also be used to give initial protection to newborn babies or exposed individuals earlier than a vaccine could be effective. Anti-hepatitis antibodies may be used to protect immunosuppressed patients, including transplantation patients, from recurrent hepatitis. This is most significant in cases of hepatitis B positive liver recipients. Further, the antibodies can be used in diagnostic assays.

9 It has also been found that antibody fragments, such as Fab fragments can also bind I: to hepatitis B virus surface antigen. These fragments also make up part of this invention.

SUMMARY OF THE INVENTION According to one embodiment of the invention there is provided a cell line which produces an antibody designated PE1-1, which antibody binds to hepatitis B surface antigen.

According to another embodiment of the invention there is provided a cell line which produces an antibody designated ZM1-1, which antibody binds to hepatitis B surface antigen According to another embodiment of the invention there is provided a cell line which produces an antibody designated ZM1-2, which antibody binds to hepatitis B surface antigen ':'\WjV(oS(JISPCS\U \S493.S.SFC -M/97 -3a According to another embodiment of the invention there is provided a monoclonal antibody which binds to hepatitis B surface antigen and has a V, region which is substantially similar to that shown in either of Tables 8-1, 8-2, 8-3 or 8-4.

According to another embodiment of the invention there is provided a monoclonal antibody which binds to hepatitis B surface antigen and is substantially similar to that shown in either of Tables 9-1, 9-2, 9-3 or 9-4, According to another embodiment of the invention there is provided a monoclonal antibody which binds to hepatitis B surface antigen and has a V, region comprising three CDR regions of Table 8-1.

According to another embodiment of the invention there is provided a monoclonal antibody which binds to hepatitis B surface antigen and has a VL region comprising three CDR regions ofTable 9-1.

According to another embodiment of the invention there is provided a monoclonal antibody which binds to hepatitis B surface antigen and comprises the mature V region of Table 8-1 and the mature V region of Table 9-1.

According to another embodiment of the invention there is provided a method for combatting a hepatitis B virus comprising administering to a subject in need of such treatment a heoatitis B combatting effective amount of human monoclonal antibody as defined herein which neutralizes hepatitis B.

According to another embodiment of the invention there is provided 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 as defined herein which neutralizes hepatitis B.

herein which neutralizes hepatitis B.

P:\WpDU OChLJR\SPCS\5.14SPC PC 0/8)9 -3b According to another embodiment of the invention there is provided DNA in an isolated form comprising a DNA sequence which hybridizes under stringent conditions to a straid 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 Specific antibodies which have been made according to this invention include PE1-1, ZMI-1, ZM1-2, MD3-4 and L03-3, each of these antibodies being of the IgG class.

The cell line producing PE1-1 was deposited at the American Type Culture Collection on October 16, 1986 and given accession number ATCC HB 9234; the cell line producing ZM1-1 was deposited as ATCC HB 9191 on September 4, 1986 and the cell line producing S C ,ip WO 94/11495 PCT/US92/09749 -4- ZM1-2 was deposited as ATCC HB 9192. The address of the American Type Culture Collection is 12301 Parklawn Drive, Rockville, Maryland 20852.

The cell lines'of the present invention all behave as typical (mouse x human) x human hybridomas and produce.their respective antibodies in concentrations ranging up to 25 mg/l in standard suspension culture.

DESCRIPTION OF THE FIGURES Figure 1 shows the results of a direct binding enzyme linked immunoassay comparing binding 'inetics of antibody PE1-1 (shown by the single line) and antibody ZM1-2 (double line). Details are given in Example 4A.

Figure 2 shows the serum levels of antibody PE1-1 in rhesus monkey serum determined at various times post-dosage. Details are given in Example 4C.

Throughout the specification and claims, the same designation is given to both the cell line and the antibody it produces, i.e.

cell line PE1-1 produces monoclonal antibody PE1-1; cell line ZM1-1 produces monoclonal antibody ZM1-1, etc. It is felt that one of ordinary skill in the art will understand whether the cell line or the antibody is being discussed.

Monoclonal antibody and cell line PE1-1 has also been referred to by the inventor and the inventor's assignee as OST 577 and 64-577. Likewise, monoclonal antibody and cell line ZM1-2 have also been referred to as 265-695, and monoclonal antibody and cell line L03-3 have been referred to as 266-215.

SUBSTITUTE SHEET WO 94/11495 PCT/US92/09749 The antibodies and antibody fragments obtained according to this invention have good specificity for hepatitis B surface antigen in in vitro ELISA binding assays.

As the antibodies of the present invention are of human origin, they are advantageously used in human therapy, as no allergenic response develops with repeated therapy, as occurs with murine or ovine antibodies. Thus, another aspect of this invention is a method of treating hepatitis B through the administration of one or more of the aforementioned antibodies.

It has been found that repeated doses of approximately 10-40 mg antibody will substantially reduce the amount of circulating HBsAg. Additional doses were found to decrease the amount of HBsAg levels to below the detectable limits of antigen tests.

Another aspect of this invention is a cocktail of two or more monoclonal antibodies. This mixture is particularly suited for administration to patients who carry a non-wild type strain of hepatitis B virus which does not bind well with a given single monoclonal antibody. For example, one compassionate need patient who suffered from hepatocellular carcinoma and chronic hepatitis B was given antibody PE1-1 prior to liver transplantation, and repeated doses thereafter. (Details are given below in Example After approximately four and one-half months of treatment, low levels of serum HBsAg could be detected with a polyclonal antibody, but not with PE1-1. Polymerase chain reaction (PCR) DNA analysis of the 230 base pair region of the HBsAg gene corresponding to the putative monoclonal antibody binding domain was performed. The PCR DNA was cloned into 13 bacteriophage and the resulting DNA was sequenced. Analysis of clones from each of the serum samples revealed two variant sequences when compared to SUBSTITUTE SHEET WO 94/11495 PCT/US92/09749 -6- PCR DNA from the original liver and pre-antibody therapy. The variant DNA codes for two different amino acids in the S protein of HBsAg and also codes for a stop codon (UAG) in the viral polymerase gene. Both variant genes contain an amino acid change resulting in the substitution of arginine for glycine in a conserved peptide domain.

Since the monoclonal antibodies PE1-1, ZM1-2, ZM1-1, MD3-4 and L03-3 have been shown to bind to different epitopes, and at least one of the monoclonal antibodies has been found to bind to every variant virus tested to date to a sufficient extent to render it clinically useful, another aspect of this invention is a cocktail of two or more of the.monoclonal antibodies selected from the group consisting of: PE1-1, ZM1-2, ZM1-1, MD3-4 and L03-3. Particularly preferred are cocktails of two monoclonal antibodies, especially the mixture of PE1-I and ZM1-2 and the mixture of PE1- and L03-3.

The ratio of the monoclonal antibodies present in the mixture may vary depending on many factors apparent to one of ordinary skill in the.art and include: the genotype of the hepatitis virus or viruses present in the patient's serum, the relative binding strengths of the antibodies chosen, the epitopes to which the chosen antibodies bind, and economic considerations. Generally, the antibodies will be present in a ratio ranging from 1:99, mo.e typically from 25:75, and preferably in a substantially equal amount.

Sections of the PE1-1, ZMI-1, 2M1-2 and MD3-4 were secuenced using standard techniques. The sequence obtained for the V H region of PEI-1 is given in Table 8-1, and areas corresponding to the CDRl, CDR2, and CDR3 (DH and JH4) are noted. As the CDR regions are particularly important regions in determining the binding properties.of an SUBSTITUTE SHEET WO 94/11495 PCT/US92/09749 -7antibody, this invention includes an antibody that has an amino acid sequence of its CDR1 region which is substantially similar .to that of PE1-1, as set forth in Table 8-1. This invention also includes an antibody that has an amino acid sequence in its CDR2 region which is substantially similar to that of PE1-1, as set forth in Table 8-1. Further, this invention also comprises an antibody that has an amino acid sequence of its CDR3 reaion which is substantially similar to the CDR3 region of PE1-1, as set forth in Table 8-1.

Likewise, the region of ZM1-1 was sequenced as is given in Table 8-2. Areas corresponding to its CDRI, CDR2, and CDR3 (D

H

and J.

4 are also indicated. This invention includes an antibody which has an amino acid sequence of its CDR1 region which is substantially similar to that of ZM1-1 as set forth in Table 8-2.

Also, this invention includes an antibody which has an amino acid sequence of its CDR2 region which is substantially similar to that of Z!1-1 as set forth in Table 8-2, and further this invention also comprises an antibody that has an amino acid sequence of its CDR3 region which is substantially similar to that of ZH1-1 as set forth in Table 8-2.

The DNA sequences which code for the regions of ZMI-2 and MD3-4 are given in Table 8-3 and 8-4 respectively. This invention also includes any antibody which has amino acid sequences which are substantially similar to that of the regions of ZM1-2 and MD3-4 as set forth in Tables 8-3 and 8-4.

The DNA sequences which code for the VH regions of PE1-1, Zml-l, ZM1-2 and MD3-4 were determined and appear in Tables 8-1 and 8-2, 8-3 and 8-4 respectively. These sequences or appropriate fragmen t q may be used in cloning antibodies (or modified antibodies) or as probes. Antibodies which are produced through genetic S UB SIYN1TE SH FT WO 94/11495 PCT/US92/09749 -8engineering processes (rather than conventional harvesting from hybridomas) can be made using cloning techniques which are known in the art. DNA from other sources may be used to produce a synthetic antibody molecule which retains the binding characteristics of PE1-1, ZMl-1, ZM1-2 and'MD3-4 by virtue of having substantially similar CDRI, CDR2, and/or CDR3 regions. Such antibodies are within the scope of this invention.

The DNA sequences which code for the VL light chain variable regions of PE1-1, ZM1-1, ZM1-2 and MD3-4 are given in Tables 9-1, 9-2, 9-3, and 9-4, respectively. This invention also includes any antibody which has amino acid sequences which are substantially similar to that of the regions of ?E1-l, ZMl-1, ZmN-2 and MD3-4 as set forth in Tables 9-1, 9-2, 9-3 and 9-4.

Also within the scope of this invention are the DNA sequences which code for the V H region, the VL.region, the CDR1 regions, the CDR2 regions and/or the CDR3 regions of PEl-1, ZM1-l, ZM1-2 and MD3-4. Also included is DNA which would hybridize to any of the aforementioned sequences under stringent hybridization conditions.

This DNA is substahtially free from other DNA of the donor mammal, and may contain introns or it may be cDNA.

As used throughout the specification and claims, the following definitions are intended. An amino acid sequence is "substantially similar" to another amino acid sequence if their amino acid homology is at least 80%. Referring to DNA, "stringent hybridization conditions" are those in which hybridization is effected at 601C in 2.5 X saline citrate buffer (SSC) followed merely by rinsing at 37°C at a reduced buffer concentration which will not affect the hybridizations which take 9 lace. "Associated mammalian DNA" means DNA present in the mammal which is the source of the VH antibody chain, but which is not involved in coding for an antibody or antibody fragment.

The invention is more fully exemplified in the following non-limiting examples.

SUBSTITUTE SHEET WO 94/11495 PCI/US92/09749 -9- EXAMPLE 1 PRODUCTION OF'ANTIBODY CELL LINES Human volunteers are immunized with hepatitis B vaccine. MD3-4, ZM1-2, ZM1-1, and PE1-1 hybridoma cell lines are derived from lymphocytes of individuals immunized with Heptavax' (Merck Co.).

Cell line L03-3 is developed from cells of an individual injected several times with Heptava and just preceding the fusion, Recombivax (Merck Peripheral blood lymphocytes are purified by density gradient centrifugation on a cushion of Percoll (Pharmacia Inc.), density 1.085 g/ml. The isolated lymphocytes are washed three times in Hank's Balanced Salt Solution and mixed with an equal number of cells from (mouse x human) cell line SPAZ-4. The cell mixture is pelleted at room temperature with 400 x g'for 5 minutes. After removing the medium, the cell pellet is treated with a 50% solution of PEG-1000 in Dulbecco's Minimal Essential Medium (MEM) for 1 minute at 371C after which the medium was slowly diluted with Dulbecco's MEM.

The cells are collected by centrifugation and resuspended into Dulbecco's MEM containing 20% fetal bovine serum. The cells are seeded at approximately 2 X 106 cells per ml into microwell plates. On the following day fresh medium containing the components of HAT medium (hypoxanthine aminopterin thymidine) is added in order to select against non-fused SPAZ-4 cells. On day 4 after fusion the medium is replaced with fresh medium containing only HT as all cells sensitive to .AT-selection had been killed by that time.

After 3 to 4 weeks, when good growth of hybridoma-like -ells could be seen microscopically, supernatants are tested for the SUBSTITUTE S WF FT WO 94/1 1495 PC1r/US92/09749 presence of anti-heoatitis B surface ahtigen antibody.' An ELISA-,assay USing a 1/100 dilution of HeptavaPon the solid Dhase is used. After incubation with the s upernat ants' the plates are developed with a kit of biotinylated goat anti-human immunoglubulin and avidin-coupled horseradish peroxidase (VectastainP, Vector Laboratories *Inc.) The enzyme is detected by the color reaction with phenylenediamine. Positive cultures are picked into new wells and a part of the cells is cloned by limiting dilution in Dulbecco's M4EM containing 20% fetal bovine serum and 10 mouse thymocytes per milliliter. The cloning plates are tested by the same ELISA xethoas described above and positive cultures are expanded and frozen., All the cell lines behave as typical (mouse x human) x human hybridomas and produce their respective antibodies in concentrations ranging up to 23 mg/l in standard suspension culture.

EXAMLE 2 IMUN~OCHERI CAL C 1.RACTE.I ZATI ON A.Antibody Class/Suhbclass The inumunoolobulin class of antibodies MI-1, ZMl-2, MD3-4 and L03-3 is determnined using ELISA methodology. Each antibody is captured on an antigen-coated plate and each assay is developed with subclass specific, perox -Is e-conj ugated anti-human Ig (Tago). Each of the antibodies are clearly IgG 1 S U B -iUTE S H EET WO 94/11495 PCrIUS92/09749 B. Licht Chain TVoe Using ELISA methods similar to those described in A, above, each antibody is tested with anti-r. or anti-X light chain reagents (Tago). The following results are obtained.

PEl-1 lmd ZILl-l' kappa Z141-2 kappa L03-3 lambda MD3-4 lambda C. Isoelectric Focusing (IEF) A sample of antibody L03-3 or PEI-l is applied to gel. Each is fLound to behave as a basic orotein.

D. Soecificiltv Purified H.BsAg of subtypes and avzr axe purchased from Scripps Laboratories, San Diego, California. hBsAg subtype ayw; is obtained from Connaught Laboratories (Willowdale, Ontario). EL SA assays are performed essentially as described by Ostberg, et a!.

(1983) Hybridona 2:361-367.

PEl-- 1 reacts with both ayr and adw, but it reacts slightl.y better with the adw subtype, L03-3 reacts substantially equally weli~ with ayr and adw. ZMI2-I shows hicher reactivity with adl, but Z?!l-2 binds slightly better to ayr. These results are con-Firmed for PEI-1 and L02-3 by Scatchard analysis in solid phase RIA with solid' adsorbed ayr or ad-4 antigen. Thus, although these SUBSTrrUTF SHEFT WO094/11495 PCT/US9 -12monoclonal antibodies apparently do not bind to the subtypic deterMinant, their reaction ith HBsAg can be significantly affected by the subtype.

G. Allotv-oe Deternination 2/09749 Allotypes are determined usin& reagents suppli ed by the Central Laboratory of the Netherlands Red Crotss Transfusion Service. Inhibition ELISA or direct binding ELISA are used.

Results are presented in Table 1, belo-w.' As can be seen, there is no apparent restriction on high af f inity anti-iBsAg antibodies with respect to light chain or allotype.

TABLE I.

Allotvnes of Anti-HBsAa Monoclonal. Antibodies Antibody PEl-l ZlM!l-2 L03-3 Al lotvDes fz Km (3) ND =Not deterinined Antibody has X light chain which does not ha,7e Rm allotvcss Cz. Affinity The affinity for solid adsorbed HBsAg is determined for each antibody using radiolebe2Iled antibodies essentially as described SUBSTITUTE SHEET WO 94/11495 PCT/US92/09749 -13by Wands, et al. (1981) Gastroenteroloov 80:225-232, which is hereby incorporated by reference. Antibodies are labeled with 125I with lodogen (Pierce). For each monoclonal except L03-3, the solid phase absorbed HBsAg is ayw. L03-3 is assayed with both ayr and adw with essentially the same results. Antibody-antigen incubation occurs at room temperature.

The relative affinity is'also determined using an inhibition ELISA in which varying concentrations of soluble HBsAg (ayw subtype) are pre-incubated with monoclonal antibody and the mixture is then incubated at 37 0 C in a microtiter well coated with HBsAg. Results are presented below in Table 2.

TABLE 2 Affinity of Monoclonal Antibodies for HSsAa Antibody Solid Phase RIA, M 1 Inhibition ELISA, -1 PE1-1 3.6 X 109 -2 X 0 ZMi1-2 1.5 X 10 -7 X L03-3 1.7 X 109 ~1 X 108 ZM1-1 5 X 109 ~1 X 108 As can be seen from the table above, for both PE1-1 and ZM1-2 the ELISA results are approximately two-fold lower than the FIA results, which is within the range of experimental error. A Ecatchard plot of the results of the RIA performed on Z41-1 indicates that there might be a low affinity binding site. It is thus possible that the ELISA is measuring this low affinity binding site, as the ELISA results are some 50-fold lower than the SR T1TUTF C;IFFT WO 94/11495 PCT/US92/09749 -14- RIA. In addition, Scatchard plots also indicate that there are considerably less high affinity ZMI-1 sites than ZM1-2 or PE1-1 hign affinity sites. While not wishing to be bound by theory, it appears that ZH1-l may have the highest affinity for HBsAg of the four antibodies compared, but only for HBsAg in a certain spatial arrangement. This arrangement is only manifested in a small percentage of HBsAg molecules. It.is also possible that this may be due to bivalent binding of ZM1-1 to HBsAg while the low affinity site is monovalent.

EXAMLE 3 Potency of PE1-1 Antibody PEl-1 is tested for potency in the AUSAB radioimmunoassay (Abbott). Tests are performed against the Bureau of Biologics Reference Hepatitis B immune globin, and several commercial hepatitis B immune globulin preparations (H-Bic Immune Globin Hep B Gammagee Immune Globinr and Hyper Hen Immune Globin all purchased from a pharmaceutical supply house).

Despite the fact that the immune globulin preparations are polyclonal and PE1-1 is monoclonal, the binding data are within the criteria of the Bureau of Biologics for comparing immune globulin preparations, the lines were parallel at a probability level less than or equal to 0.01.

Determiination of potency is as follows. Preparations are compared on a weight basis (an absorbance at 208 Pnm of 1.4 is assumed equal to 1 mg/ml). Preparations of PE1-1 which have been stored at 5rC are then compared with the above polyclonal preparations which have also been stored at 50C. The logarithm of SUBSTiTUTE SHEET WO 94/1 1495 5 PCT/US92/09749 1000 divided by .g/ml in the preparation the log of a number that is inversely proportional to the concentration of the immunoglobin, similar to the log of the dilution factor) is then plotted vs. log counts per minute (average of triplicates). The hypothesis'that the fitted lines are parallel is tested using analysis of variance. It is found that the lines are parallel at a probability level of less than or equal to 0.01. Lines of all preparations are parallel and a common slope is determined. The x-intercepts are calculated from the common slope and the difference in intercepts used to determine the difference in potency. By this procedure, monoclonal antibody PE1-1 is some 435 times more potent than the Bureau of Biologics reference hepatitis B immune globin. Since the commercial hepatitis B immune globulin preparations were found to be two-fold (or less) more potent than the Bureau of Biologics reference preparation, PE1-1 is at least 200 times more potent than the commercial hepatitis B immune globulin preparations on a weight basis.

EXAmLE 4 A. Bindina Kinetics Direct binding enzyme linked immunoassays are used to compare the kinetics of binding to HBsAg of antibodies PE1-1 and Z!1-2.

ELISA microtiter plates are coated with Heptavax at 1 pg/ml.

Wells are then incubated at 370C with 2% fetal calf serum in phosphate .buffered saline.. Monoclonal antibody PE?-1 or M21-2 at pg/ml in 2% fetal calf serum are incubated in the wells for various times. At the indicated times the antibody solution is removed and the well is rinsed three times with fresh 2% fetal SUBSTITUTE SHEET WO 94111495 C17US92/O9749 -16calf serum. The well is then incubated with 2% fetal calf serum until the wells for the 90 minute time point contain 2% fetal calf serum. Thus, solution is then replaced with either peroxidase conjugated goat anti-lambda chain (PE1-1 wells) or goat anti-kappa chain (ZM1-2 wells). Quantitation of peroxidase conjugate bound to plastic is accomplished with the addition of 0-phenylenediamine end H 2 0 2 Results are presented in Fig. 1, where a single line is PEI-1 and a double line is ZM1-2.

As can be seen in Fig. 1, at a concentration at which PEl-1 is almost completely reacted in 5 minutes, the reaction of ZM1-2 with solid adsorbed HBsAg is not completed in 30 minutes and may continue to react for 90 minutes or more. Thus, PEi-1 binds significantly faster to antigen in this assay. Assuring this also occurs in vivo, PE1-1 is likely to be more efficient in neutralizing viral particles before they can infect the liver.

B. Relative Position of Eoitones The relative position of the epitopes of antibodies PE1-1, L03-3, and ZM1-2 are determined. A simultaneous sandwich immunoassay with a solid-adsorbed monoclonal antibody is used.

The same antibody is radiolabelled and incubated in a microtiter well with the inhibitor and serum from a hepatitis B positive patient. Radiolabelled PEl-i Fab fragment is used while radiolabelled L03-3 is intact IgG. Results are presented in Table 3, below.

SUBSTITUTE SHEET WO 94/11495 PCr/US9/09749 -17- TABLE 3 inhibition of Binding of' Radiolabelled MIonoclonal Antibody to HBsPg by Unlabelled 2ionoclonal Antibodies Solid-Absorbed Iodinated Inhibitor IC 50 ng/znl inaB maBma L03-3 L03-L-3 L03-3 L03-3 LO.3-3 PEl-. >22,500 PE1-l PE1-l PE1-1 8 PE1-1 PEl-1 Z14 -2 76 PEl-1 PEl-. L03-3 >22,500 mohoclonal antibody ZMl-2 is only approximately nine times less effective in inhibitino 125 1I-PE1l1s binding to liBsAg than unlabelled PEl-., whereas L03-3 is thousands of times less e ffective. Thus, the' epitopes of ZMl-2 and PE1-l are probably near e~ch other on the HBsAg molecule while the L03-3 epitope is probably on a different part of the molecule. The reciprocal exoeriment, PEl-l inhibition of radiolabelled L03-3, provides furtLher evidence that PEl-l and L03-3 bind to epitopes that a--e not overlapping.

The similarity of PEl-l and ZMI-2 epitopes and their diffAer,,-nce from the L03-3 is confirned by i!ri-nunoassay with reduced and al.Kylated Has.Ag. L03-3 can bind to denatured anticen while both ZIXil-2 and PEI1 cannot so bind. It should be noted that.

PE1-1 and Z141-2 have distinct eoitoDes since their reaction with different subtypes varies.

SUBSTITUTE SHEET WO094/11495 PCf/US92/09749 C. Pharmokinetics Of PE1-2 in Rhesus Yonkevs T he pharmokinetics of PE1-i is studied in two rhesus nw'nkeys.

Each animal receives a single intravenous bolus injection mg/kg) of monoclonal antibody PE1-I.' Serum levels of PE51 are determined at various times post-dose using an ELISA based sandwich immunoassay with keptava.x coated on ELISA plates and rabbit anti-idiotypic antibodies to PE1-i. Results are shown in Figure 2.

Serum levels of P:U'-l in the two rhesus monkeys are characterized by a biphasic decline (t 1/2a 1 and 1.4 days; t /2P 11 and 16 days) with the shorter half-life possibly associated with the distribution phase of the inonoclona3 artibody.

-The volume of distCribution at steady state (Vdss) is calclvlated to be.114-144% of the plasma volume, which suggests little distribution of PE1-1 tc a tissue compartment in the anticen-free monkey.

EXIOLPLE CLINICAL TRIALS A. Compassionate use of ?EI-1 in two vatients with end-stace liver disease-secondarv to chronic active hgoatitis B and heoatocellular carcinoma underaoino liver transalantation PE1-I was provided on a compassionate need basis to two oatients with end-stage liver disease undergoing liver transplantation. Patient 1_1 was a 56 year old male with a 20 year history of chronic active hepatitis and a diagnosis of S UB8STI'TUTE S H EET WO 9411495 PCT/US92/09749 -19hepatocellular carcinoma. The second patient was a 10 year old male thought to have been infected with hepatitis B at birth.

Patient #2 was initially evaluated for a large mass in the right lobe of the liver, which a biopsy confirmed was hepatocellular carcinoma.

Preoperative doses of PE1-1 wdre administered to these patients and significantly reduced their circulating HBsAg levels before the transplant procedure. Each patient also received two mg doses of PE1-1 during transplantation. Postoperative dosing then began on the second day following surgery.

Patient #1 never became HBsAg negative, although his circulating HBsAg levels did diminish markedly from their pretreatment level. Patient #2 became HBsAg negative, first noted on.post-transplant day 9. Patient #1 received additional doses of PE1-1 ranging from 5-40 mg at 2-20 day intervals. Patient F2 received either 5 or 10 mg doses on average of every 21-25 days.

No adverse events were reported for either of these patients during the period they received PE1-I. However, approximately four weeks after Patient #1 was discharged from the hospital, it was determined that he had metastatic malignancy. He expired ci: post-transplant day 139. No evidence of recurrent hepatitis was noted during his post-transplant course despite the presence of detectable circulating HBsAg. Although a hepatitis B virus DNA assay was negative preoperatively, a single positive value was detected 60 days post-transplant.

SUBSTITUTE SHEET WO 94/11495 WO 94/149 PCT/US92/09749 On post-transplant day 143, Patient #2 was first seen to be posittL- for HBsAg. The HBsAg level fluctuated for a short time" before it then stabilized at a level signicantly below his pretreatment levels. Isolates of this patient's hepatitis B virus obtained before treatment with PE1-1 and at later times were analyzed for their binding ability to PEI-1. PE1-1 was found to be able to bind to the variant virus, but not as well as it had to the wild-type virus.

Genetic analysis of the two viral isolates indicated single nucleotide differences in a highly conserved region of the major viral surface protein. Such differences, when compared to the pre-treatment virus, could potentially encode for a single amino acid difference which would reduce the' binding ability of PE1-1 to the hepatitis B viral binding particle.

B. Use of PE- 1-in patients with chronic active heatitis 3 undercoina liver transolantation (not comolicated by heoatocellular carcinoma) This study involved five patients who were HBsAg positive (but did not have hepatocellular carcinoma) and who underwent liver transplantation. Each patient was administered three daily preoperative doses of PE1-1, (10, 20 and 40 mg, respectively) over a three day period. The liver transplants were then performed from a minimum of two days to a maximum of 32 days following their preoperative dose of the study drug. An additional 40 ma dose of PE1-1 was administered during the operation. All five transplants were successfully completed.

SUBSTITUTE

SHEET

WO 94/11495 PCT/US92/09740 -21- The patients' HBsAg titers, liver enzymes, and other clinical parameters were closely monitored during their hospital stays.

Follow-up evaluations and administration of PE1-1 by each patients' private physician continued on a regular basis (approximately every 1 to 3 weeks). Dosing.and other parameters varied from patient to patient.

Two patients and had similar results to Patient 42, above, in that a variant virus appeared after a period of negative HBsAg screening results. The sera of these patients remained active with PE1-1. Sequence analysis indicated the presence of single nucleotide differences between the variants in the patients' sera and wild type virus. Two variants were detected in each patient. Immunoassays and sequence analysis indicated that the variants -in each patient were different and they also differed from the variants of Patient #2.

Patient #3 is a 39 year old Caucasian male who had end-stage liver disease secondary to a 16 year history of chronic hepatitis B. The three preoperative doses of PE1-1 that were administered to Patient #3 caused a substantial reduction in his HBSAo titer.

On post-transplant days 2 and 3, he received 20 rg of PE1-1 and was first noted to be HBsAg negative on post-transplant day 2.

For two months thereafter, Patient #3 received 10 mg PE1-1 on an average of every 1 to 7 days. Since then, he has received 7.5 or mg doses of PE1-1 every 14 to 43 days. ristopathologic&.

evaluation of a liver biopsy performed in February, 1989 was negative for both HBsAg and HBcAg. Patient #3 reme .s EBsAg negative 582 days after transplant. In addition to receiving PE1-1, he has also received three consecutive monthly injections of Recombivax in July, August and September, 1989.

SUBSTITUTE SHEET WO 94/11495 PCTUS92/09749 -22- Patient #4 was a 40 year old Arabic female who had end-stage liver disease secondary to a 10+ year history of chronic active Hepatitis B. The three preoperative doses of PE1-1 given to Patient #4 caused a substantial reduction in her HBsAg level.

Patient #4 received 20 mg of PEI-1 on post- ransplant days 1 and 2, and was found to be HBsAg negative on post-transplant day 6.

For two months thereafter, she received 10 mg PE1-1 on average of every 3 to 8 days. Since then, she received 10 mg P21-1 every to 26 days. Approximately 1 year after her transplant, the patient developed hepatic artery thrombosis, but remained HBsAg negative, and was re-transplanted. Three days later, due to ischemia, a third transplant was performed. Twenty days following, a fourth transplant was performed due to infection.

The patient expired 18 days after the fourth transplant (404 days after her initial transplant), secondary to liver failure and bacteria. sepsis. Histopathological evaluation of a liver biopsy from her first transplanted liver showed that she was HBsAg necative.

Patient #5 is a 38 year old Caucasian male who had end-stage liver disease secondary to chronic active hepatitis B. The preoperative doses of PE1-1 administered to the patient substantially lowered his circulating HBsAg level. Patient received 20 mg PE1-1 on post-transplant days 2 and 3, and was found to be HBsAg negative on post-transplant day 3. During the first two months post-transplant, he received 10 mg PE1-1 on average of every 3-7 days. Later, he received 10 mg PE1-1 every 9 to 26 iays. The patient was noted to be HBsAg positive on post-transplant day 252, although his antigen level is substantially lower than his pre-transplant level.

SUBSTITUTE SHEET WO 94/11495 PCT/US92/09749 -23- Histopathological evaluation of a liver biopsy performed in January 1990 is positive for HBsAg and HBcAg.

Patient #6 is a 38 year old Caucasian male who had end-stage liver disease secondary to chronic active hepatitis B and alcohol abuse. This patient acquired his initial infection via a blood transfusion. FP:-r to the transplant, he was positive for both HBsAg and H3eAg. Each preoperative dose of PE1-1 caused a decrease in the level of the patient's HBsAg titer. Patient #6 received 20 mg of PE1-1 on post-transplant days 1 and 2 and was noted to be HBsAg negative on post-traneplant day 1. For two months thereafter, Patient #6 received 10 mg of PE1-1 on average of every 3-14 days. Subsequently he has received 10 mg PE1-1 every 7 to 63 days on an outpatient basis. The first HBsAg positive response was noted on post-transplant day 251 and occurred after his longest duration (63 days) between doses of PE1-1. Although at present Patient #6 is positive for HBsAg, his titer remains significantly lower than pre-transplant levels.

Patient #7 .s a 38 year old Caucasian female with a history of IV drug.abuse. This patient had end-stage liver disease secondary to chronie active hepatitis B. Prior to transplantation, the patient was positive for H3sAg and HBeAg.

Each preoperative dose of PE1-l caused a decrease in the patient's HBsAg titer. The first month post-transplant, Patient #7 received between 10 and 40 mg of PE1-I on the average of every 1-7 days, and was noted to be HBsAg negative on post-transplant day 16.

Subsequently, she received 10 mg PE1-1 every 15 to 29 days.

Histopatholoaical evaluation of a liver biopsy performed in July, 1989 was negiative for .HBsAg and HScAg. Patient #7 remains HBsAg negative 464 days post-transplant.

SUBSTITUTE SHEET WO 94/11495 PCT/US92/09749 -24- EXAMPLE 6 PEACTIVITY WITH VARIANT VIRUSUS The reactivity of the monoclonal antibodies PS1-1, ZM1-2, and L03-3 with variant hepatitis B viruses isolated from patients described in Example 5 is investigated. Radioimmunoassays are performed by determining the radioactivity bound to a solid phase adsorbed-antibody. A solution of a monoclonal antibody at a concentration of 20jg/ml in phosphate-buffered saline containing 0.02% NaN 3 is incubated for at least 18 hours in U-bottom wells (Falcon MicroTest III Flexible Assay Plates). The solution is removed from the wells and the wells are then washed three times with distilled water. Fetal .calf serum at a concentration of 2% in phosphate-buffered saline is added and incubated overnight at room temperature with solutions of serur HBisAg or controls and 12 5I-radiolabelled antibody (approximately 4,000 cpm in.1% fetal calf serum). Wells are then washed with distilled water three times. Individual vells are excited and counted. Results are presented in Table 4, below.

TABLE 4 Relative Reactivity of Serum-Derived Variant HBsAo with HBsA-Soecific Monoclonal Antibodies Samole* L03-3:L03-3** PE1-1:ZM1-2 ZM1-2:ZM1-2 Control 1.000 1.000 1.000 Patient #2 (234) 0.013 0.070 0.233 Patient #4 (251) 0.007 0.024 0.010 Patient =3 (264) 0.043 0.173 0.179 SUBSTITUTE SHEET WO 94/11495 WO 9411495PCT/US92/09749 *Reoresentative Ii~sAg-positive serum samples derived f rom patisnts after liver transplantation and treatment with an anti-H~sAg therapeutic monoclonal antibody PEl-l. Numbers in parentheses denote days after transplantation.

**LO3-.3:LO3-3 indicates a radioixnmunoassay composed of both solid-adsorbed and radiolabelled*human monoclonal antibody L03-3. PEl-l:Z241-2 indicates a radio immunoas say composed of human monoclonal antibody PEl-1 solid-adsorbed and human monoclonal antibody ZM1-2 radiolabelled. ZmIl-2:Z11-2 indicates a radjoimmunoassay composed of both solid-adsorbed and radiolabelled human monoclonal. antibody ZI-2. Control HBsAg-positive serum reacted well with antibodies L03-3, PEl-1 and ZMl-2.

EXAXLE 7 LARGE SCALE PRODUCTION OF AIBODIES To initiate a productiva run with cells, one or more ampule(s) of frozen cells is removed from liquid nitrogen. After rapidly heating in a 37 0 C water bath until most of the ice has melted, the ampule is opened inside a vertical laminLar flow hood.

The contents of the am-pule are mixed with a I1nl volume o: Dulbecco's MEM/I~am's F12(1:1)(N/1) to which ferric salts have been added to a final concentration of 50 gM of Fe-+-!.Afe mixing, the tube is filled up to approxcimately 10 ml with the same medium and the cells are collected by centrifugation. The cell pellet is resusbended into 5 ml ofl the above mentioned mediunl with fetal bovine serum and seeded into I well of a 6-well tissue culture plate. The cells are incubated in a 370C incubator in a SUBSTiTUTE SHEET WO 94/11495 PCT/US92/09749 -26- C0 2 -atmosphere. When the cells have established themselves in culture and start to multiply and have an approximate cell concentration of 10 6 /ml, the cells and the medium are moved into a tissue culture flask with a surface area of 80cm 2 and diluted to ml using DMEM/F12 (without serum). When the cells have once again reached a concentration of 10 /ml, they, and the medium, are moved into a tissue culture flask with a surface area of 175cm 2 and further diluted to a volume of 100 ml using DMEM/F12. When the cells have once again reached optimal concentration, the cells and the medium are transferred into a roller bottle with. a 850cm 2 surface area and diluted to a final volume of 500 ml. When this roller bottle has reached optimal cell concentration, it is split 1/3 into new roller bottles using the same medium as before. This splitting process of the roller bottles is continued until a sufficient number of bottles have been obtained in order to give a desired number of cells to seed into the Verax System 200 reactor.

The Verax System 200 The Verax System 200 reactor is a closed cell culture system where cells are cultivated in stainless steel weighted microspheres (density 1.6g/mL) composed of cross-lined type I bovine collagen. The microspheres are loaded into a vertical transparent glass tube through which the culture'medium (same as above) is pumped, entering at the bottom. The inlet to the tube is formed in such a fashion that the microspheres will establish a fluidized bed configuration when the medium is pumped through at a suitable velocity. During operation, fresh medium is constantly added and conditioned medium removed at a rate determined by the cell growth as monitored by glucose consumption. Temperature is SUBSTITUTE SHEET WO 94/11495 PMcTUS92/0749 -27maintained at 37 0 C; pH is maintained at 7.1 and oxygen/nitrogen ratio is also controlled.

After loading of the microspheres in 1% fetal bovine serum containing medium, the reactor is run for at least three days without cells to ascertain that the microsphere loading did not contaminate the System. During this time the reactor is fed with protein-free medium to reduce the priming dose of fetal bovine serum. If all systems are operating satisfactorily, the cells from the roller bottles are inoculated into the reactor.

The Verax System 2000 This equipment uses the same type of microspheres as the System-200, and its controls and operations are essentially the same as for the smaller system. The System 2000 represents an approximately 15-fold scale-up compared to the System 200.

Monitorina of Yield of Antibody from Full-Scale Culture The conditioned medium is monitored, each time the harvest tank is emptied, for the level of human immunoglobulin in the supernatant using an ELISA-type assay. The results are confirmed using a Protein A HPLC method.

Harvestinc of Cell Culture Media and Production of Harvest Pool The conditioned medium is continuously being removed from the Verax equipment into a refrigerated harvest tank. This medium is later unloaded (using the nitrogen pressure in the Verax system) into a mobile stainless steel tank for further processing.

SUBSTITUTE SHEET WO 94/11495 WO 941495 PC/US92/09749 -28- Cell Culture Media The media routinely used is a 1:1 mixture of Dulbecco's MEM .H21 and Ham's F12 (Mediatech). The medium is purchased as a powder sufficient for 50 liters of finished medium. Two such containers of each medium powder, are added into a stainless steel tank containing approximately 190 liters of water. The powder is suspended with an impeller until all has been dissolved. Sodium bicarbonate is added as recommended by the manufacturer and the pH of the medium is set to 7.4. Sodium selenite is added to a final concentration of 17.3 ug/1 and the volume is topped up to 2001 with water. The medium is also supplemented with ferric ions in the form of.ferric nitrate/sodium citrate to a final concentration of 50 Mi The medium is'immediately added to the medium tank of the Verax System S200 through the built-in sterilization filter. -No protein is added.to the medium. No antibiotics of any type are ever used.

PURIFICATION OF T:-HE MONOCLONAL ANTIBODY Description of Methodology of Harvesting and Purification of End Product The monoclonal antibody is produced in cell culture from a hybridoma cell line in the absence of serum. This means that we have a need to remove from the final product only components from the cellular material. As human monoclonal antibodies are not in themselves expected to be immunogenic, it becomes very important to remove all potentially immunogenic components.

SUBSTITUTE SHEET WO 94/11495 PCr/US92/09749 -29- The goal of the purification procedures is a final product that is more than 99.9% pure,'using affinity chromatography. We depend heavily on the biological specificity of affinity chromatography. Each step of the purification process (summarized in Table 5) is discussed in more detail, supra.

Purification Summary Step Cell Removal Concentration Miczofiltration Protein A chromatography Concentration Gel chromatography- Ion exchange Conditions Room Temp.

,4 0

C

4 C 4 0

C

4 °C 4 0

C

Materials Polyvinylidene difluoride filters, 0.65/0.45 pm absolute.

Polysulfone filter nominal 30,000 daltons. Polyester (0.8pm) and cellulose acetate (0.2pm) absolute filters.

Agarose coupled Staohvloccocus aureus Protein A.

Cellulose triacetate filter, nominal 20,000 dalton.

Sephacryl S-300, Ringer's Lactated Solution Sephacryl S-300, Ringer's Lactated Solution Cell Harvest and Removal of Conditioned Medium Particulate Materials from the Even though most of the cells are retained by the microspheres, a sizable number of cells are present in the harvested supernatant. To avoid gross contamination of the medium SUBSTITUTE

SHEET

WO 94/11495 WO 94/11495 PCT/US92/09749 by cell components the supernatant is filtered through a polyvinylidene difluoride 0.65pm Prostack filter (Millipore), immediately after removal from the Verax harvest tank. This tvpe of filter unit works in a tangential flow mode which allows filtration of large amount of particulate material without clogging the filter. The cleared medium is collected into a refrigerated stainless steel tank.

Concentration of Conditioned Medium The conditioned medium is concentrated using a nominal 30,000 dalton polysulfone spiral wound membrane supplied by Millipore.

After concentration, the pH is set to 7.0 using 1M acetic acid.

The material is sterile filtered through a Sartobran-PH 0.8'0.2gm (Sartorius) filter (the 0.8pm component is polyester, the 0.2pm component is cellulose acetate) before being stored at 4°C. The material is microfiltrated (0.22 gM Millipore) and filled into polypropylene vessels.

Protein A Chromatocraohv The extremely powerful purification step utilizes the high affinity of the human IgG1 antibody to Staohvlococcus aureus Protein A.

The Protein A is purchased already coupled covalently by an amide bond to agarose. After packing the gel in a column, the column with its concents and attached tubing is sanitized by treatment with 70% ethanol in water for 24 hours. The column is then equilibrated with PBS', pH SUBSTITUTE SHEET WO094/11495 PCI'/US92/09749 -31- Performing the affinity chromatography separation on the Protein A column involves the following sequential steps: A) Lcadina. The concentrated conditioned medium is loaded on the column with a pump. The effluent from the column is collect2d and monitored for the presence of antibody by the human immunoglobulin ELISA. The column is loaded to such a degree that a measurable amount of antibody-containing fluid passes 'through the column. The overload fraction is separately recovered and recycled if it contains more than'20 mg/ml antibody.

B) Washin. To'remove unbound materials the column is extensively washed with phosphate buffered saline, pH 7 with sodium chloride added to a final concentration of 0.5M. This wash is followed by a second washing step using a buffer of 0.02M sodium citrate, pH 5.6, containing 0.5M sodium chloride. This wash releases small amounts of the human antibody.

C) Elution. The bound monoclonal antibodies are eluted from the column using a buffer composed of 0.02M sodium citrate, pH containing 0.5M sodium chloride. The eluted material is continuously diluted into a volume of IM Tris-HCl, pH 8.0 to rapidly restore near-neutral conditions.

The Protein A purification is performed in a closed system utilizing a Waters 650 Protein Purification System.

SUBSTiTUTE SHEET WO 94/11495 PCT/US92/09749 -32- Concentration of Protein A Column Eluate In order to make the following purification step more effective and convenient, the eluate 'rom the Protein A column is concentrated to at least 5 mg/ml antibody. The concentrate is sterile filtered through a 0.2 pm filter and the sterile concentrate is stored at 4°C until sufficient materials'have-been collected for the next purification step.

Size Seoaration by Gel ChromatocraDhv on Sephacryl S-300 Hich Resolution The antibody preparation is run on a Sephacryl S300 High Resolution (Pharmacia) gel, packed in a Pharmacia BP113/120 column with a bed volume of approximately 10 liters. The column is packed in Lactated Ringer's Irrigation USP (Travenol Laboratories). The elution of the column is monitored by a Waters 650 Protein Purification System.

The purpose of this step is not principally additional purification, but buffer change. After the elution of the Protein A column the antibodies are in a complex, hypertonic buffer composed of sodium citrate, sodium chloride and Tris-HC1. This buffer mixture can'not be used directly as a vehicle for an intravenous injection. The buffer after this step is suitable both for intravenous injection and for long term refrigerated storage.

SUBSTITUTE SHEET WO 94/11495 PCT/US92/09749 -33- Removal of Host Cell DNA by Passace over an Ion Exchance Column Even after the Protein A chromatography, which removes the bulk of DNA present in the concentrated supernatant, and the Sephacryl S-300HR which removes DNA molecules that are either significantly larger or significantly smaller.than the monoclonal antibody product, there is a small; but detectable, presence of DNA in. the antibody preparation. We have selected to remove this contaminant by an ion exchange step on a strong anion exchanger, Q SSepharose (Pharmacia Inc.). At the pH of Lactated Ringer's solution, antibody proteins have a positive charge, and are repelled by the anion exchanger. Nucleic acids, however, have a negative charge at this pH, and will bind to the column.

The column was packed according to the manufacturer's suggestions. After decanting the 20% ethanol solution the gel is delivered in, 100 ml of gel was suspended in 200 ml of Lactated Ringer's solution. The slurry is poured into a Pharmacia K50/30 column, and when the gel has packed itself to a constant volume, it is sanitized with 1 column volume of 0.5N sodium hydroxide, followed by 3 column volumes of Dulbecco's PBS, followed by column volumes of Lactated Ringer's solution. Immediately prior to use the column was washed with an additional 5 column volumes of Lactated Ringer's solution. The sample is then passed through the column and the pass-through is collected in a sterile container.

S U STrTtITF rIWFFT WO094/11495 PCr/US92/09749 -34.- EXAMPLE 8 MOLECULAR ANALYSES OF PEI1, ZMl-l, ZMl-2 AND MD3-4 The heavy variable chain of antibodies PEI-11 ZM1-2, zNl-2 and MD3-4 are isolated and sequenced. Total RNA is extracted from 107 hybridoma cells of each cell line using procedures described in Sanz, et al. 1989 Z. Immunol. 142:883, which is hereby incorporated by reference. Single stranded DNA is synthesized using AMV-reverse transc_.-:otase as the enzyme and oligo-dT as the primer. The quantity of the synthesized ss-cDNA is assessed by measuring the inoprt o o'3p-dC'rP.

Polymerase chain reactions (PCR) are performed essentially as recommended by the manufacturer (Perkin Elmer Cetus, Norwalk, Connecticut). One microgram of DNA is izdded to a 200 g.m solution of -each of dATP, dCTP, dGTP and dTTP, with 100 p moles each of primer and 5 units of Tag DNA polymerase. PCR cycles are as follows: denaturaion at !SIC for 3 minutes, annealing at 55*C for 2 minutes, and extension at 72*C for thr'ae minutes, controlled in a DNA thermal cycler (Perkin Elmer Cetuz).

Aiolified DNlA is sizze selected on a 1.0% low melting acarose gal, ligated into the EcoRV site of a BLUESCRIPIT phagemi.id vector, and transformed into CaCl 2 competent BSj72 bactert-ia. Sinc-19 stranded D.A for sequencing is isolated from each positive clone after superinfection with M13'07 as described by Sanz, et al., Sequencing is accomplished via the dideoxv chain termination method as described by Sanger, et al. 1980 J. Mol.

Biol. 143:161, except a modified T7 DNA poJ.ymerase (Secuenase) Is used as described by Tabor, et, al. 1987. PNAS (USA) 84:4767.

Results are given in Tables 8-1 8-2, 8-3 and 8-4.

-MStJI I I ITF r,-4FFT WO 94/11495 WO 9411495PCTJUS92/09749 Table 8-1 DNA sequence of the V. region of PEl-1 is shown below.

The leader, V HIII, D, and J H4 regions are denoted by the dashed line; complementarity-determ-ining regions CD.R1 and CDR2 are indicated by the asterisks. Amino acids appear as single letter abbreviations below the DNA.

LEADER ATG GAG TTT GGG CTG AGC TGG GTT TTC CEC GTT GCT CTT TTA AGA GGT GTC CAG TGT CAG 6TG CAG H E F G L S W V FLYV A LL R G V Q C Q V Q CTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GCA 6CC TT L- V E S G G G V V Q P G R S L R L S C A A S GGA TTC ACC TTC AGT AGG TAT GGC ATG CAC TGG GTC CGC VG GCT CCA GGC MAG 666 CTG GAG TE-G G F T F *S R Y G M H W V R (I A P G K G L E W GTG GCA GTG ATA TCA TAT GAT GGA AGT MAT MAA TGG TAT GCA GAC TCC GT6 MAG GGC CGA TTC ACC V A V I S Y D G S NHK W Y AD S V K G R F T ATC TCC AGA GAC MAT TCC MAG MAC ACT CTG iTT CTG CAA ATG CAC AGC CTG AGA GCT GCG GAC ACG R D H S K N T L F L Q M H S L R A A D T GGT GTA TAT TAC TGT GCG AAA GhT CAA CTT TAG TTT GrGT TCG CAG AGT CCC 6.66 CAC TAC TGG GTC G V Y Y C A K VI Q L Y F 6 S Q S P 6 H Y W V CAG GGA ACC CTG GTC ACC GTC TCC ITA Q0G T L V T V S S SU RS=1 t ITF r,14 -7 WO 94/11495 PCT/US92/09749 -36- Table 8-2 DNA sequence of the V H region of ZMl-l is shown below.

The leader, V HIII, D and J H 4 regions are denoted by the dashed line; corplementarity-determining regions ODRI and CDR2 are indicated by the asteri~sks. Amino acids a~pear as single letter abbreviatio.z below the DNA.

LEADER ATG GAG MT GGG CTG AGC TGG GTT TTC CTT GT[ GCT ATA TTA GMA GiST GTC CAG TGT GAG GTG CAG H E F G L S 14 V F L V A I *L E G V 0 C E V (I CTG GTG GAG TCT 666 GGA GGT TTS GTA CAG CCT 666 666 TCC CTG AGA CT C TCC TGT GCA GCC TCT L V E S G G G L V Q P G G S L R L S C A A S GGA TTC ACC TTC AGT AGG TAC GAC ATG TAC TGG GTC CGC CAA GCT ACA GiGA AMA GGT Cr6 GAG TGG G F T F S R Y 014 Y W V R Q AT G KG L E 14 GTC TCA GCT ATT GGT CCT ACT GGT GAC AC, TAC TAT. CA GAC TCC GTG MAG GGC '6A TTC ACC ATC VS A I6 P T G D T Y Y A D S V K G R F T I TCC AGA GMA MT GCC MAG MAC TCC rT~r TAT Crr ACA ATG MAC GGC CTG AGA 6CC GGG SAC ACG GCT S 'R E H A K~ M S L Y L T H M G L R A 6 D T A 6TG TAT TAC TGT GCA AGA GAT TTA GMA CrC V Y Y C A RD L E L TGG GGC GAG GGA ACC CTG GTC ACC GTC TCC TCA 14 GX Q6 T L V T V S S SUBSTITUTE SHEET 0O94/11495 PCr/US92/09749 -37- Table 8-3 DNA sequence of the V H re gi,:n of ZMl-2 is shown below.

The leader, VHIV, D and J H 4 regions are denoted by the dashed line; comolementarity-determining regions CDRI and CDR2 are indicated by the asterisks. Amino acids appear as "ngle letter abbreviations below the DNA.

ATG AAA CAC CTG TGG TTC TTC CTC CTG CTG GTG GCA GTT CCC AGA TG GTC GTG 7CC tAG GTG CAG M K H L W F F L L L V A V P R W V V S Q V Q VXIV CTG CAG GAG TCG GGC CCA GGA CTG GTG PAG GCT GCG GAG ACC CTG 7CC CTC ACC TGC ACT GTC TCC L Q E S G P G L V K A'S E T L S L T C T V S CGT GGC TCC TTC AGT GAT TAC TTC TGG MAT TGG TTC CGG CAG CCC GCC GIS MG CGC CTG GAG TG.G R G 5 F S D Y F W4 N WF R (I P A G K R L E W CTT GGG CGT GTC TAT ACC AGT GGA AGT GTC GAC TAC MAC CCC TCC CTC MAG AGT CGA GTC ACC GTG L G R V Y T S G S V D Y N P S L K S R V T. V TCA 676 GAC ACG TCC AAG AAG CAG TIC ICC CTG AGG CTG AGC TCT GIG ACC GTC GCG F-AC ACG 6CC S V D T S K K Q F S L R L S S V T V A D T A GTG TAT TAT TGT GCG AGA V Y Y CA R TCC CCA S P GGA CTG TCC GGT TTT GAC TAC TGG GGC CAG GGA 6CC CTG GTC ACC GTC G L 5 6 F D Y HW 6 G A L V T V SUBSTITUTE SHEET .W091411495 PCT/US92/09749 -38- Table 8-4 DNA sequence of the V H region of MD3-4 is The leader, V ,D and J H 3 r egions are denoted line; corplementarity-deterrnining regions CDR!.

shown below.

by the dashed and CDR2 are indicted by the asterisks.~ Amino acids appear as single letter abbreviations below the DNA.

LEADER AT6 GGG TCA ACC GCC ATC CTT GGC CTC CTC CTG GCT GTT CTC CMA GGA GTC TGT GCC FAA GTG CAG M G S T A I L G L L L A V LQG G V C A E V Q CTG GTG CMA TCT MG GCA CiNG GTG AMA MG CCC GGG GAG TCT CTG AGG AlT TCC TGT MAG GOT iT L V QS G A E V K K P G ES5 L R I S C K G S GGA TAC AGC TTACC AGC TAC TGG ATC AGC TGG GTG CGC CAG ATG CCC GGG AA GCOC CTG GAG Tw G G Y, S F T S Y W I S W V R H P G K G L E W ATG GGG AGO CTT GAT CCT AGT GCC TCC TCT GCC ATC TTC AGC CCG TCC CTC CMA EGC CA,'C GTC ACC H G R L' D P S A S S A I F S P S L Q G M V T ATC TCA GTT GAC MAG TCC ATG AGO ACT GCC TAC GTG CAG TG AGA AEC CTG MAG GCC TCG GAC ACC I S V fl K S M R T A Y V Q W R S L K A S D, T G6.C ATG TAT TAC TGT GCG AM Y Y C A GCT TTT GAT ATC TGG GGC A F D I G AGA CAT GTC CGC GMA MG AGT ATG GiT CAG GGA GTC ATT ATA AAG GAC RHM V R E K S H V a G V I I K D CAA GG ACA ATG GTC Qs Gr M V ACC GTC TCT TCA T V S S SUBSTITUTE SHEET I WO 94111495 WO 94/ 1495PCf/US92/09 749 -39- EXAMPLE 9 Following the procedures of Examp~le 8, the light variable (Vchain of antibodies PE1-i, ZMl-l, ZM1-2 and MD3-4 are isolated and secuenced. Results are given, in Tables 9-1, 9-2, 9-3 and 9-4.

SUBSTrIUTE SHEET IWO94/11495 PCr/US92/09749 Tabl.e 9-1 DNA sequence of the V Lregion of PEl-1 is shown below.

The VV and J3 regions are denoted by the dashed lime; compleinentarity-determining regions CDRl, CDR2 and CDR3 are indicated by the asterisks. Amino acids aoo~ear as single letter abbreviations below the DNA.

VV GAG -TCT GAG CTG ACG GAG CCG CCC TCG GTE TCA GTG GCC CCA GG GAG ACE ECG AGG ATT ACC TET a S QL T C1 P~ P. SVS V A P G ,Q T AR I T C G-GG GGA GAG MAC AUT GGE AGT AMA AGT GTG AAC TGGG TTC GAG GAG MAG CCA GGC GAG GCC CCT ETC G G D N I GES K S V N W F Q C1 K P G ti A P v CTG ETC ETC TAT SAT GAT MAC GMA GE CCC TCA GG ATT TCT GAG GA TTC TCT EEC TCC AAC TCT L V V Y D DN E R P S G IS5 ER F S ES 5 S SEE AAG ACE GCC ACC CTG ACC ATC AEC AGG ETC GMA GCC EGG GAT GAG GCC GAG TAT TAC TGT GAG GEN TA T*L ,T I S R V E AG D E A D Y Y C Q GTG TEE EAT AGT AET AET EAT CAT V W D S 5 S D H .3 GTE GTA TTC GEC SEA SEE ACCMAG CGE ACC ETC CTA V V F G EE T K L T V L SUBSTITUTE SHEET IWO094/11495 PCr/US92/09749 -41- Table 9-2 DNA secuence of the V Lregion of ZMl-l is shown below.

The leader, VII and JS regions are denoted by the dashed line; comolernentarity-detertnining regions CDRl, CDR2 and CDR3 are indicated by the asterisks. Amino acids appear as single letter abbreviations below the DNA.

ATG GAC ACG AGG GTC CCC GCT CAG CTC CTG GGG CTG CTA ATG CTC TE-G GTC CCA GGA TCC AGT GG D T R V P A Q L L G L L M L W V P G S S G GAT GTT GTG GTG ACT CAkG TCT CCA CTC TCC CTS CCC GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC D V V V T Q S P L S L P V T L SQC P A S I S TGC AGA TCT AST CTA C R 55 L CC-A 6SC CAA TCT CCA P S G C S P AGC CTC GTG SAC AGT SAC GGA MAC ACC TAd 'TTG AAT TGG MT CTC CAG AGG S L V D S D G IN T Y L H W F L CQ R ASS CGC CTA ATT TAT CAG C17 TCT AGC CGG GAC TCT 555 GTC CCA SAC AGRA R R L* I Y (I L S S R 0 S G V P 0 R TTC AGC GS-C AGT 555 TCA GGC ACT SAT TTC ACT CTG AAA ATC AGC AEG GTG GAG GCT GAG SAT IT F S G S G S5 G T D F T L K I S R V E A E D V X SSC GTT TAT TAC TGC ATG CMA GGT ACA CAC T9G CCG ATC ACC TTd GGC CMA 55 ACA CGA CTG GAG 6 V Y Y d M 0 G T H W P1 I F SQC P T R L E ATT AAA CGA I K R SUBSTiTUTE SHEET IWO 94A 1495 PCrIUS92/09749 -42- Table 9-3 DNA sequence of the V, region of Z?1l-2 is shown below.

The leader, VI and J regions are denoted by the dashed line; complementarity-determining regions CORi, CDR2 and CDR3 are indicated by the asterisks. Amino acids appear as single letter abbreviations below the DNA.

LEADER ATG AG CCC GTC GCT CAG CTC CTG GGG CTC CTG CTG CTC TGG TTC CCA GGT TCC, AGA TGC GAC ATC M R P V A Q L L G L L L. L W F P G S R C D I' V CAG ATG ACC CAG TCT CCA TCT TCC GTG TCT GCA TCT GTG GGA GAC AGA GTC ACC GTC ACT TGT CGG Q M T Q. S P S 5 V S A S V G D R V T V T C R GCG AGT CAG GGT AlT AGC AGT TGG TTA GCC TGG TAT CAG CAG MAA CCA GG6 AMA GCC CCT AAA CTC A S Q G I S S W L AW V Q Q K P G K A P K L I C(rG ATC CAT GCT GCA TCC AGT TTG CMA AGT GG GTC CCA TCA AGG TTC ATC G-C AGT GGA TCT GGG L I H A A SS L QS GYVP S R F I GS GS .2q'o ACA CAT 'ITC ACT CTC ACC ATC ACC AGC CTG CAG GCT VM GAT M~ GCa ACC TAC TAT TGT CMA CAG T D F T L T I T S L Q A E D F A T Y .Y C 0 (1 3 GCT GAC AGT CTC CCT MI ACT TTC GGC GGA GGG ACC MAG GTG GAC TTC AAA CGA A D0S L P F T F G666 T K V D F K R SUBSTITUTE SHEET WO 94/1 1495 PCr/US92/o9749 -43- Table 9-4 DNA sequence of the V region of MD3-4 is shown below.

The VIII and J3 regions are denoted by the dashed line; cornlementarity-determining rcgions CDRl, CDR2 and CDR3 are indicated by the asterisks. Amino acids appear as single letter abbreviations below the DNA.

VIII CAG TCT CAG CTG ACG CAG CCT GCC TCA GTG TCC GTG TCC.CCA GGA CAG ACA GCC AGC ATC ACC TGC a S QL TOQP A S V S V S P G Q T A S IT C TCT GGA GAT AGA TTG GGG GAT GM TM GCT TCC TGG TAT CAG CAG MAG CCA GGC CAG TCC CCT AT S 6 D R L G D E F A S W1 Y Q Q K P G Q S P I~ CTG GTC ATC TTT GAG GAT AAC AAG AGG CCC TCA 606 ATC CCT GAA CGA TTC TCT GCC TCC MAC TCT L VI F -E D H K R P S G I P ER F S G S H' *S 606 AAC ACA 6CC ACT CTG ACC ATC AGC 606 ACC CAG GCT ATG GAT GIAG GCT GAC TAT TAC TGT 6 H T *A T L T I S G' T Q A M D E A D Y Y C I.

GCG TGG GCC AGC AGC Cli' TG-G 6TG TTC ESOC GGA 666G ACC MAG CTG ACC GTC TTG A W4 A S S L W4 V F G G G T* K L T V L S UB8STiTUTE S HEET

Claims (13)

1. A cell line which produces an antibody designated PE1-1, which antibody binds to hepatitis B surface antigen.

2. A cell line which produces an antibody designated ZM1-1, which antibody binds to hepatitis B surface antigen.

3. A cell line which produces an antibody designated ZM1-2, which antibody binds to hepatitis B surface antigen.

4. A monoclonal antibody which binds to hepatitis B surface antigen and has a VH region i V. which is substantially similar to that shown in either of Tables 8-1, 8-2, 8-3 or 8-4. A monoclonal antibody which binds to hepatitis B surface antigen and is substantially similar to that shown in either of Tables 9-1, 9-2, 9-3 or 9-4.

6. A monoclonal antibody which binds to hepatitis B surface antigen and has a V, region comprising three CDR regions of Table 8-1.

7. A monoclonal antibody which binds to hepatitis B surface antigen and has a VL region comprising three CDR regions of Table 9-1.

8. A monoclora. antibody which binds to hepatitis B surface antigen and comprises the mature VH region of Table 8-1 and the mature VH region of Table 9-1.

9. 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 as defined in any one of claims 4 to 8 which neutralizes hepatitis B. A method according to claim 9 wherein the monoclonal antibody is selected from the group consisting of: PE1-1, ZM1-I and ZM1-2 and mixtures thereof.

11. A method of reducing the level of circulating hepatitis B surface antigen i' a patient comprising administering to the patient a HBsAg-reducing effective amount of human monoclonal antibody az defined in any one of claims 4 to 8 which neutralizes hepatitis B.

12. A method according to claim 11 wherein the monoclonal antibody is selected from the group consisting of: PE1-1, ZM1-1 and ZM1-2 and mixtures thereof.

13. A cell line which produces an antibody according to claim 4. o V.. S* 14. A cell line which produces an antibody according to claim

15. DNA in an isolated form 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.

16. A cell line having DNA comprising the DNA of claim SANDOZ LIMITED By Its Patent Attorneys DAVIES COLLISON CAVE