CZ116495A3 - Production of human monoclonal antibodies 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 obtained by fusing a xenogeneic hybridoma designated SPAZ 4 with blood cells of a patient immunized with hepatitis B vaccine.

Description

The present invention relates to hybrid cell lines that produce human antibodies that neutralize the hepatitis B virus surface antigen, the production of cell cultures, the antibodies that produce the cell cultures, and the use of antibodies, particularly in therapy.

BACKGROUND OF THE INVENTION

The generation of hybrid cell cultures for the production of monoclonal antibodies is currently a well-known invention in the art. The present invention relates to the production of human monoclonal antibodies which are particularly effective against hepatitis B virus surface antigen, which antibodies are prepared according to the generally applicable method described by Applicants in Hibridoma 2 (4): 361 (1983) and in United Kingdom Patent Application 2,113,715 A, published

More specifically, hybrid cell cultures that contain native rodent immortalized cells, such as murine myeloma cells SP-2, when associated with a human partner cell, have been found to produce an immortalized xenogeneic hybrid cell. This xenogeneic hybrid cell may be associated with a cell capable of producing human anti HBsAg antibodies, resulting in a new trioma cell culture capable of producing human antibodies effective against these antigens in a human organism. Or, if greater stability is required, a trioma cell culture is produced that best no longer has the ability to produce its own antibodies, and this trioma is then coupled to another cell that is capable of usefully generating antibodies against said antigen to obtain an even more stable one. a hybridoma (quadrome) that produces antibodies against antigens. <

Previously, applicant publications related to the preparation of a xenogeneic hybridoma referred to as SPAZ 4, which is prepared from a drug-resistant cell culture that can be obtained, for example, from a NIGMS human genetically mutant Reposit cell. Ref. GM 35669A (in the US DHHS 1982 Catalog of Cell Lines). The preparation of SPAZ4 is described as follows. SP2 cell culture is coupled to normal human peripheral lymphocytes by conventional techniques. A large number of hybrids were obtained, and after about five weeks, 5 clones were selected that showed rapid growth and no antibody production. These cells are selected for resistance to 8 azaguanine

2 and 3 of these cultures yield azaguanine-resistant mutants at a dose of 20 µg / ml. These cells are also sensitive to hypoxanthine-aminopterinthymidine (HAT) medium, indicating that these cells have lost their ability to produce hypoxanthine-phosphoribosil transferase. One of these lines is SPAZ-4.

SUMMARY OF THE INVENTION

SPAZ 4 cell culture can be associated with cells that are obtained from the blood of a person immunized with Hepatitis B vaccine to obtain hybrid cell cultures that provide positive cultures when standard selection procedures are used to develop binding of antibodies to major viral antigens. It is recommended that said positive cultures be subjected to a second selection process in which different virus subtypes are used to prepare antigens. This makes it possible to emphasize the exact antigenic determinants that are recognized by antibodies.

Cell cultures that result from the fusion of xenogeneic hybridoma and cell

and.

producing human monoclonal antibodies (trioma) are therefore useful in detecting monoclonal antibodies that are capable of effectively neutralizing the hepatitis B-causing virus, and said antibodies may therefore prevent the spread of hepatitis by e.g. blood transfusions. They can also be used as an initial protection for newborns or exposed individuals before a vaccine can be effective. Hepatitis antibodies can be used to protect immunosuppressed patients, including patients undergoing transplantation, from recurrent hepatitis. This is most significant in the case of hepatitis B positive liver recipients. Furthermore, antibodies can be used in diagnostic assays.

It has also been found that antibody fragments such as Fab fragments can bind to the hepatitis B virus surface antigen. These fragments also form part of the invention.

Specific antibodies produced according to the invention include PE1-1, ZM1-1, ZM1-2, MD3-4 and LO3-3. All of these antibodies belong to the IgG class. ,

The cell culture that produces PE1-1 is deposited in American Type - Culture

Collection October 16, 1986 under ATCC accession number HB 9234; a cell culture that produces ZM1-1 is deposited under ATCC HB 9191 on Sep. 4, 1986, and a cell culture that produces ZM1-2 is under ATCC HB 9192. Address American:

Type Cultur Collection's address is 12301 Parklawn Drive, Rockville, Maryland 20852.

The cell cultures presented in the present invention all behave as typical (mouse x human) x human hybridomas and produce their respective antibodies at a concentration of 25 mg / L upwards in a standard suspension culture.

Description of the picture

Figure 1 shows the result of the assay using direct binding of labeled enzyme and compares the binding kinetics of PE1-1 (shown by a single line) and the binding kinetics of ZM1-2 (shown by a double line). Details are described in Example 4A.

Figure 2 shows the values of PE1-1 antibodies in rhesus monkey sera, which are determined at different dose intervals. Details are in Example 4C.

The specifications and claims equally refer to both the cell culture and the antibody produced by the cell culture, i.e., the PE1-1 cell culture produces the monoclonal antibody PE1-1, the ZM1-1 cell culture produces the monoclonal antibody ZM1-1, etc. It is clear that one one of ordinary skill in the art is to understand whether it is a cell culture or an antibody.

The inventor and the inventor have also mentioned monoclonal antibody and PE1-1 cell culture such as OST 577 and 64-577. Similarly, the monoclonal antibody and ZM1-2 cell culture is referred to as 265-695 and the monoclonal antibody and LO3-3 culture is referred to as 266-215.

The antibody and antibody fragments obtained according to the invention have good specificity for the hepatitis B surface antigen in an in vitro ELISA.

Because the antibodies of the present invention are of human origin, they are preferably used in human therapy, since there are no allergic responses to repeat therapy, such as using murine and sheep antibodies. Thus, another aspect of the invention is a method of treating hepatitis B by administering one or more of the aforementioned antibodies. Repeated doses in the range of 1Q-4Q mg of antibody were found to significantly reduce the amount of circulating HBsAg. Additional doses reduce the amount of HBsAg below the detectable limit of antigen assays.

Another aspect of the invention is a mixture of 2 or more monoclonal antibodies. This composition is particularly suitable for administration to patients who are carriers of a non-wild type hepatitis B virus that does not bind well with a particular single monoclonal antibody. For example, a patient affected simultaneously

- 4 hepatocellular carcinoma and chronic hepatitis B antibody PE1-1 was administered prior to liver transplantation followed by repeated doses (more detailed description is in Example 5). After approximately 4.5 months of treatment, low levels of viral HBsAg were detected with polyclonal antibodies, but not with PE1-1. DNA analysis was performed by polymerase chain reaction (PCR) of 230 base pair regions corresponding to the binding domain of the monoclonal antibody. PCR DNA is cloned in M13 bacteriophage and the resulting DNA is sequenced. Analysis of the clone from each serum sample revealed 2 different sequences when compared to the original liver PCR-DNA prior to antibody therapy. The variant DNA encodes 2 different amino acids in the HBsAG S protein and also encodes a stop codon (UAG) in the viral polymerase gene. Both variant genes contain an amino acid chain that results from the replacement of agrinin by glycine in the conserved peptide region.

The monoclonal antibodies PE1-1, ZM1-2, ZM1-1 and LO3-3 bind to different epitopes. There is a monoclonal antibody discovered that binds to all variants of the virus and is already sufficiently clinically tested. As a result of this another aspect of the invention, a mixture of 2 or more monoclonal antibodies selected from the group of PE1-1, ZM1-2, ZM1-1, MD3-4 and LO3-3, a mixture of 2 antibodies, especially a mixture of PE1 -1 and ZM1-2 or mixtures of PE1-1 and LO3-3. Ratio *

The monoclonal antibodies in the composition may vary depending on many factors that are known in the art, including: genotype of hepatitis virus or viruses present in the patient's serum, relative binding power of selected antibodies, epitopes to which the antibodies bind, and economic aspects . Generally, the antibodies will be present at a ratio of 1:99, more often at a ratio of 25:75, and preferably at about the same amount. .

The sections PE1-1, ZM1-1, ZM1-2 and MD3-4 were sequenced using standard techniques. Table 8-1 lists the sequences obtained from the PE1-1 V1 region and indicates the regions corresponding to CDR1, CDR2 and CDR3 (D (- | and Jh4)). Because CDR regions are particularly important for determining binding properties. antibodies, the invention includes an antibody having the amino acid sequence of its CDRI region substantially identical to that of the PE1-1 region as set forth in Table 8-1. The invention also includes an antibody having the amino acid sequence of its CDR2 region substantially identical The invention also includes an antibody having an amino acid sequence in its CDR3 region substantially identical to that region of PE1-1 as shown in Table 8-1.

Similarly, the arrangement Vh of the region ZM1-1 is described in Table 8-2. The regions corresponding to CDR1, CDR2 and CDR3 (D1- and Jh4) are indicated. The present invention encompasses an antibody having the amino acid sequence of its CDR1 region substantially identical to that region of ZM1-1 as shown in Table 8-2. The invention also encompasses an antibody having an amino acid sequence in its CDR2 region substantially identical to that region at ZM1-1 as shown in Table 8-2. The invention also encompasses an antibody having an amino acid sequence in its CDR3 region substantially identical to that region on ZM1-1 as shown in Table 8-2.

Tables 8-3 and 8-4 contain the DNA sequences that encode the ZM1-2 and MD3-4 regions. The invention also encompasses any antibody having an amino acid sequence substantially the same as those in the ZM1-2 and MD3-4 regions as set forth in Tables 8-3 and 8-4.

Tables 8-1, 8-2, 8-3, and 8-4 identify and appear DNA sequences that encode the VH region of PE1-1, ZM1-1, ZM1-2, and MD3-4. These sequences or suitable regions can be used to encode antibodies (or modified antibodies) or as probes, antibodies that are produced by genetic engineering (rather than conventional hybridoma acquisition) can be generated using cloning techniques known in the art. DNA from other sources can be used to produce synthetic monoclonal antibodies that retain the binding characteristics of the PE11, ZM1-2, ZM1-1 and MD3-4 antibodies by having substantially the same CDR1, CDR2 and / or CDR3 regions. These antibodies are also within the scope of the invention.

Tables 9-1, 9-2, 9-3, and 9-4 contain the DNA sequences that encode different regions of PE1-1, ZM1-2, ZM1-1 and MD3-4 for the Vβ light chains. The invention also encompasses any antibodies having an amino acid sequence that is substantially the same as that in the PE1-1, ZM1-2, ZM1-1 and MD3-4 regions as set forth in Tables 9-1, 9-2 below, 9-3 and 9-4.

The invention also encompasses DNA sequences that encode a Vh region, VnnDd mm nn, d, h, λ ΚΛ ΚΛ ΚΛ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4, -., ζ_ινι i - ia

MD3-4. It also includes DNA that could hybridize to any of the foregoing sequences under stringent hybridization conditions. This DNA is substantially free from other mammalian donor DNA and may contain introns or may be cDNA.

In view of what is used in the specifications and claims, it is appropriate to make the following definitions. The amino acid sequence is almost the same as the other sequence

- 6 amino acids if their amino acids are at least 80% homologous. Referring to stringent hybridization conditions means those in which hybridization is effective at 60 ° C in 2.5 x citrate buffered saline (SCC), followed by only increasing the temperature to 37 ° C in a reduced buffer concentration that does not affect the hybridization that is taking place . Attached mammalian DNA means DNA present in mammals that is a source of V 1 - antibody chains, but which do not arise when encoding an antibody or antibody fragment.

The invention is better illustrated in the following non-limiting examples.

Examples of embodiments of the invention

Example 1

Production of antibodies by cell cultures

Human volunteers are immunized with hepatitis B vaccine. MD3-4, ZM12, ZM1-1, and PE1-1 hybrid cell cultures are made from lymphocytes of individuals immunized with Heptavax® (Merck & Co). LO3-3 cell culture is generated from cells of an individual several times immunized with Heptavax® and thus prior to joining, Recombivax® (Merck & Co). Peripheral blood lymphocytes are purified by density gradient centrifugation on a Percoll plate (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) SPAZ4 cell culture. The cell mixture was granulated at room temperature with 400 x g for 5 minutes. After removal of the medium, the cell granules are treated with a 50% solution of PEG-1000 in Dulbecco's Minimum Essential Medium (NEM) for one minute at 37 ° C, then the medium is slowly diluted with Dulbecco's NEM. Cells are harvested by centrifugation and then ground in Dulbecco's NEM containing 20% fetal bovine serum. Cells are seeded in micro-hole plates at a concentration of 2 x 10θ cells per ml. The next day, fresh medium containing HAT medium components (hypoxanthine aminopterin thymidine) is added to separate unbound SPAZ-4 cells. On the fourth day after the coupling, the medium is replaced with fresh medium containing only HT, since all cells sensitive to HAT selection are already destroyed.

After 3 to 4 weeks, when microscopically good growth of hybridoma cells is seen, supernatants are tested for the presence of antibodies against hepatitis B surface antigen. A solid phase ELISA using Heptavax ® diluted 1: 100 is used. After incubation, the plates develop in kits of biotinylated goat anti-human immunoglobulin and avidin-binding peroxidase from horseradish (Vectastatin®, Vector Laboratories Inc.). The enzyme is detected by color reaction with phenylenediamine. Positive cultures are plated in new wells and part of the cells are cloned by partial dilution in Dulbecco's NEM containing 20% fetal bovine serum and 107 mouse thymocytes per milliliter. The cloning plates were tested by the same ELISA method described above and the positive cultures were enlarged and frozen. All

- 8 cell cultures behave as typical (mouse x human) x human hybridomas and produce their respective antibodies at a concentration ranging from 25mg / L above in standard suspension cultures.

Example 2

Immunochemical characteristics

A. Antibody classes and subclasses

The immunoglobulin classes of antibodies PE1-1, ZM1-1, ZM1-2, MD3-4 and LO3-3 are determined using an ELISA methodology. Each antibody is captured on an antigen coated plate and each assay is performed with an anti-human immunoglobulin that is specific for subclass and peroxidase-conjugated (cue). All antibodies are apparently IgG class.

B. Types of light chains

Using an ELISA method similar to that described in Example A, each antibody is tested with anti kappa and anti lambda light chain reagents (Cue). The results are as follows:

PE1-1 lambda ZM1-1 kappa ZM1-2 kappa LO3-3 lambda MD3-4 lambda

C. Isoelectric focusing

A sample of LO3-3 and PE1-1 is applied to the gel. Everyone acts as an alkaline protein.

D. Specified

Purified HBsAg subtypes of adw and ayr are purchased from Scripps Laboratories, San

Diego, California. The HBsAg subtype ayw is obtained from Connaught Laboratories (Willowdale, Ontario). The ELISA assay is performed essentially as described by Ostberg, et al (1983) Hibridoma 2: 361-367.

- 9 PE1-1 reacts with both adw and ayr, but responds slightly better with adw. LO3-3 reacts essentially equally well with both ayr and adw. ZM1-1 shows higher reactivity with adw, but ZM1-2 binds slightly better to ayr. These results were verified for PE1-1 and LO3-3 by solid phase scatchard RIA analysis with ayr or adw tightly bound antigen. Thus, although these monoclonal antibodies do not appear to bind to subtype determinants, their response to HBsAg may significantly affect the subtype.

G. Determination of allotypes

Allotypes are determined using antigens supplied by the Dutch Red Cross Transfusion Service Laboratory. Inhibitory ELISA or direct binding ELISA is used. Table 1 shows the results. As can be seen, there is no apparent limitation of the high affinity of anti HBsAg antibodies in connection with the light chain allotype.

Table 1

Allotypes of anti HBsAg monoclonal antibodies

Antibody a f of Km (3)

PE1-1 - + -

ZM1-2 + * - + +

LO3-3 - + -

ZM1-1 NDNDND *

ND = unspecified * = antibody has lambda light chain that does not have Km allotypes

H. Affinity

The affinity for tightly adsorbed HBsAg is determined for each antibody using radiolabeled antibodies as essentially described by Wands, et al. (1981) Gastroenterology 80: 225-232, incorporated herein by reference. Antibodies are labeled 425 idogen (Pierce). For each monoclonal antibody, except LO3-3, the solid phase is absorbed by HBsAg ayw. LO3-3 is tested with both ayr and adw with almost the same results. The incubation of the antigens with the antibody takes place at room temperature.

The relative affinity is also determined using an inhibitory ELISA method in which different concentrations of dissolved HBsAg (ayw subtype) are incubated with a monoclonal antibody and this mixture is further incubated at 37 ° C in well-coated HBsAg tubes. The results are shown in Table 2.

Table 2

Affinity of monoclonal antibodies for HBsAg

Antibody solid phase RIA M ^_ 1 inhibition ELISA, M ^_1

PE1-1 3.6x10 ⁹ '~ 2x10 ⁹

ZM1-2 1.5 χ 10 ⁹ ~ 7x10 ⁸

LO3-3 1.7 χ 10 ⁹ ~ 1 χ 10 ⁸

ZM1-1 * 5x10 ⁹ ~ 1 χ 10 ⁸

As can be seen from the table above for the PE1-1 and ZM1-2 antibodies, the ELISA results are approximately 2-fold lower than the results using RIA assays, which is in the range of experimental error. The Scatchard plot of the RIA results generated for ZM1-1 shows that there could be low binding site affinity, since the ELISA results are 50 times lower than the RIA results. Furthermore, the Scatchard plots also show that there are substantially less high affinity ZM1-1 regions than ZM 1-2 or PE1-1 high affinity regions. Although not wishing to be limited by theory, it appears that ZM1-1 may have the greatest affinity for HBsAg of the 4 antibodies compared, but only for HBsAg in a particular spatial arrangement. This arrangement manifests only in a small percentage of HBsAg molecules. It is also possible that this may be due to bivalent ZM1-1 with HBsAg, while the low affinity regions are monovalent.

Example 3

Potency PE1-1

PE1-1 antibodies are tested for potency by AUSAB radioimmunoassay (Abbott). The tests are conducted against the immunoglobulin Bureau of Biologics Reference Hepatitis B, and against several commercially produced hepatitis B immunoglobulin preparations (H-Big Imune Globin®, Hep B Gammagee Imune Globin® and Hyper Heplmmune Globin®, all obtained from pharmaceutical supply companies). Despite immunoglobulin preparations being polyclonal and PE1-1 monoclonal, binding data

if they fit into the criteria of comparing immune globin preparations, i.e. the lines are parallel at a probability level of less than or equal to 0.01.

. Potency is determined as follows. The preparations are compared on a weight basis (absorbance at 280 nm 1.4 equals 1 mg / ml). The PE1-1 preparations are stored at 5 ° C and then compared to the preparations mentioned above and also stored at 5 ° C. The logarithm 1000 divided by µg / ml in the preparations (i.e., the log number that is inversely proportional to the immunoglobulin concentration, similar to the log of the dilution factor) is then plotted against the log counts per minute (3 times the average). The hypothesis that the corresponding lines are parallel is tested by analysis of variance. The lines were found to be parallel at a probability level of less than or equal to 0.01. The lines of all slides are parallel and the slope is determined. The X-sections are calculated from a common slope and the difference in sections is used to differentiate the force differences. In this way, the monoclonal antibody is found to be about 435 times more potent than hepatitis B immunoglobulin from the Bureau of Biologics reference. Because commercially produced hepatitis B immunogiobulins are 2x (or less) more potent than the Bureau of Biologics reference, PE1-1 is at least 200 times more potent than commercially produced hepatitis B immunoglobulin preparations on a weight basis.

Example 6

A. Coupling kinetics

Immunoassay utilizing direct binding of labeled enzyme is used to compare the kinetics of binding of PE1-1 and ZM1-2 to HBsAg. ELISA microtiter plates are coated with Hepavax ® at a concentration of 1 µg / ml. These are then incubated with 2% calf fetal serum in phosphate buffered saline. Monoclonal antibodies PE1-1 or ZM1-2 at 0.5 µg / ml in 2% calf fetal serum are incubated in the wells several times. At the indicated times, the antibody solution is removed and the well is rinsed three times with fresh 2% fetal calf serum. The well is then incubated with 2% fetal calf serum until the wells contain 2% fetal calf serum for a 90 minute period. Then the solution is removed by either peroxidase-conjugated goat anti lambda chains (PE1-1 wells) or goat anti kappa chains (ZM1-2) wells. The amount of peroxidase-conjugated bond is stabilized

- 12 by adding O-phenylenediamine and H 2 O 2. The results are shown in Figure 1, where the single line is for PE1-1 and the double line for ZM1-2.

As can be seen from Table 1, at concentrations where PE1-1 reacts almost completely within five minutes, the reaction of ZM1-2 with tightly adsorbed HBsAg is not complete after thirty minutes and may continue for ninety minutes or more. Thus, PE1-1 antibodies bind to antigen significantly faster in this assay. Assuming this is done in vivo, PE1-1 are probably more effective in neutralizing viral particles before they infect the liver.

B. Relative position of epitopes

The relative position of the epitopes of the antibodies PE1-1, LO3-3 and ZM1-2 is determined? Simultaneous sadwitch immunoassays with tightly adsorbed antibodies are used. The same antibodies are radiolabeled and incubated in microtiter wells with inhibitor a *.

The serum of a patient positive for hepatitis B. A radiolabeled Fab fragment of antibody PE1-1 is used, while radiolabeled LO3-3 is an intact IgG. The results are shown in Table 3, below.

Table 3

Inhibition of Binding of Radiolabeled Monoclonal Antibodies to HBsAg by Unlabeled Monoclonal Antibodies

Strongly adsorbed lodinated Inhibitor ICsong / ml maB maB maB and LO3-3 LO3-3 LO3-3 10 LO3-3 LO3-3 PE1-1 *> 22,500 PE1-1 PE1-1 PE1-1 8 PE1-1 PE1-1 ZM1-2 76 PE1-1 PE1-1 LO3-3 > 22,500 Monoclonal ZM1-2 antibody is approximately only 9 times less potent in inhibition 1-PEI-1na bonds HBsAg than unlabeled PE1-1, while LO3-3 is 100x less effective. Thus, epitopes of ZM1-2 antibodies and PE1-1 are on HBsAg molecule probably close, while the LO3-3 antibody epitope is about on another part of the molecule.

Reciprocal experiment, inhibition of PE1-1 by radiolabeled LO3-3, provides additional evidence that PE1-1 and LO3-3 bind to non-overlapping epitopes.

The similarity of PE1-1 and ZM1-2 epitopes and their difference from LO3-3 is confirmed by immunoassays with reduced and alkylated HBsAg. LO3-3 can bind to denatured antigen, while both PE1-1 and ZM1-2 cannot bind. It should be noted that PE1-1 and ZM1-2 have different epitopes because their reactions with different subtypes differ.

C. Pharmacokinetics of PE1-1 antibody in rhesus monkeys

PE1-1 pharmacokinetics were studied in two rhesus monkeys. Each animal received a bolus of PE1-1 antibody in a single intravenous injection (at a dose of 0.5mg / kg). Serum PE1-1 levels were monitored at various times post-dose using an ELISA method based on a sandwich immunoassay with Heptavax® coated on ELISA plates and using rabbit anti-idiotypic antibodies to PE1-1. The results are shown in Table 2. '

The serum level of PE1-1 in two rhesus monkeys is characterized by a biphasic decrease (t 1/2 λ = 1 and 1.4 days; t 1/2 β = 11 and 16 days) with a shorter half-life apparently associated with the distribution phase of the monoclonal antibody. The volume at which steady state distribution (Vdss) occurs was calculated to be 114144% of the plasma volume, indicating a small distribution of PE1-1 into the tissue compartment of monkeys in which no antigen is present.

Example 5

Clinical studies

A. Administration of PE1-1 in two patients with end-stage liver disease (chronic active hepatitis B and hepatocellular carcinoma) who have undergone liver transplantation

PE1-1 was administered to 2 patients with end-stage liver disease who underwent liver transplantation. Patient # 1 was a 56 year old man with a history of 20 years of chronic active hepatitis and a diagnosis of hepatocellular liver cancer. The second patient was a 10 year old boy who was

- 14 infected hepatitis B at birth. Patient # 2 was initially examined for a large focal point in the right liver lobe that the biopsy identified as hepatocellular carcinoma.

The preoperative dose of PE1-1 given to these patients significantly reduced circulating HBsAg levels prior to transplantation. Each patient also received 20mg of PE1-´1 'during transplantation? Post-administration of ΪΕΪ-1 began the second day after surgery.

Patient # 1 was never negative for HBsAg, although circulating HBsAg levels were significantly reduced relative to preoperative values. Patient # 2 became HBsAg negative, first registered on day 9 after transplantation. Patient # 1 received additional doses of PE1-1 in the 5-40mg range at 2-20 day intervals. Patient # 2 received either 5 or 10 mg doses on average every 21 to 28 days.

None of these patients experienced any side effects during the period when they received PE1-1. However, about 4 weeks after the release of Patient # 1 from the hospital, he was found to have a malignant metastatic process. He died 139 days after the transplant. There were no signs of recurrent hepatitis during the post-transplant period, although circulating HBsAg was still detectable. Although the hepatitis B virus DNA test was preoperatively negative, one positive value was detected 60 days after transplantation.

143 days after transplantation, HBsAg positivity was first observed in patient # 2. The HBsAg level fluctuated for a short time and then stabilized at a level substantially lower than the pre-treatment value. Hepatitis B virus samples from this patient were collected prior to PE1-1 treatment and later analyzed for binding ability with PE1-1. PE1-1 has been found to be able to bind to a variant virus, but not as much as a wild type virus.

Genetic analysis of the two viral isolates revealed a single nucleotide difference in the highly stable region of the major viral surface protein. This difference, when compared to the pre-treatment virus, could cause different coding for one amino acid, which reduced the binding ability of PE1-1 to the binding particles of hepatitis B virus.

- 15 B. Use of PEI-1U in patients with chronic active hepatitis B (uncomplicated hepatocellular carcinoma) who have undergone liver transplantation

This study included 5 patients who were HBsAg positive (but not hepatocellular carcinoma) and who had a liver transplant. Each patient received pre-operative doses of PE1-1, (10, 20 and 40 mg) 3 times daily within 3 days. Liver transplants were performed between 2 and 32 days after the preoperative dose of study compound. An additional 40 mg dose was administered during surgery. All 5 transplants were successfully completed.

HBsAg titers, liver enzymes, and other clinical parameters of patients were closely monitored during hospitalization. Subsequent monitoring and administration of PE1-1 were performed by the patient's private physicians as standard (approximately every one to three weeks). Dosage and other parameters varied from patient to patient.

Two patients (# 5 and # 6) had similar results to patient # 2 of the previous example, i.e., after a period of negative HBsAg screening results, a variant virus appeared. The sera of these patients remained active with PE1-1. Sequence analysis revealed the presence of one nucleotide difference between the variants present in the patient sera and the wild-type virus. Two variants were detected in each patient. Immunoassay and sequence analysis that the variants differ from patient to patient and also from those present in patient # 2 (in the previous example).

Patient # 3 is a 39-year-old Caucasian who has a final stage of liver disease caused by a 16-year history of chronic active hepatitis B. Patient # 3 received 3 preoperative doses of PE1-1 which caused a substantial reduction in HBsAg titers. On the second and third day after transplantation he received 20mg PE1-1 and for the first time in the second posttransplantation day HbsAg was negative. Over the next 2 months, Patient # 3 received 10mg PE1-1 on average every 1 to 7 days. After that he received 7.5mg or 10mg PE1-1 every 14 to 43 days. In February 1989, histopathological examination was performed with negative results for both HBsAg and HBcAg. Patient # 3 remained negative 582 days after transplantation. In addition to PE1-1, patient # 3 received 3 Recombivax ® injections at monthly intervals in July, August and September 1989.

Patient # 4 is a 40 year old Arab woman who has terminal stage of liver disease due to 10 years of chronic active hepatitis B. Three preoperative doses of PE1-1 given to Patient # 4 resulted in a significant reduction in HBsAg levels.

Patient # 4 received 20 mg of PE1-1 on the first and second postoperative days and HBsAg negative was on the sixth day after transplantation. Over the next 2 months, she received 10mg PE1-1 on average every 3 to 8 days. After that, she received 10 mg of PE1-1 every 5 to 26 days. About a year after transplantation, the patient developed hepatic arterial thrombosis and the patient remained a 'HBsAg'negative' and 'subdivided' lipid for 3 days due to ischemia, 3 transplants were performed. After 20 days she underwent a fourth transplant due to the onset, infection. The patient died of hepatic failure and bacterial sepsis on the eighteenth day after the fourth transplant (404 days after the first transplant). Histopathological examination of liver biopsy from the first transplanted liver showed that HBsAg was negative.

Patient # 5 is a 38-year-old Caucasian who had terminal-stage liver disease due to chronic active hepatitis B. Pre-operative doses of PE1-1 given to this patient significantly reduced circulating HBsAg levels. Patient # 5 received 20mg PE1-1 on days 2 and 3 post transplant and was HBsAg negative from day 3 post transplant. During the first two months after transplantation he received 10 mg of PE11 on average every 3 to 7 days. He later received 10 mg of PE1-1 every 9 to 23 days. HBsAg positivity was recorded 252 days after transplantation in the patient, although the HBsAg level is significantly lower than before transplantation. Histopathological examination of liver biopsy was performed in January 1990 and was positive for both HBsAg and HBcAg.

Patient # 6 is a 38-year-old Caucasian who was in terminal stage of liver disease because of chronic active hepatitis and alcohol abuse. This patient was infected by blood transfusion. Prior to transplantation, it was positive for both HBsAg and HBcAg antigens. Each preoperative dose of PE1-1 caused a decrease in HBsAg titers in this patient. Patient # 6 received 20 mg PE1-1. on the first and second day after transplantation and was negative for HBsAg on the first day after transplantation. Over the next 2 months, Patient # 6 received 10mg PE1-1 on average every 3 to 14 days. PE1-1 administration was followed on average every 7 to 63 days out-patient. The HBsAg positive response was first recorded 251 days after transplantation and occurred after the longest interval between PE1-1 administration (63 days). Although patient # 6 is now positive for HBsAg, HBsAg titres remain significantly lower than before transplantation.

Patient # 7 is a 38 year old Caucasian woman with a history of intravenous drug abuse. This patient has terminal liver disease as a result of ongoing chronic active hepatitis B. Prior to transplantation, she was positive for both HBsAg and HBcAg. each preoperative dose of PE1-1 caused a decrease in HBsAg titers. First month after

- 17 transplantation Patient # 7 received 10 to 40 mg of PE1-1 on average every 1 to 7 days and HBsAg negativity was first reported 16 days after transplantation. This was followed by administration of PE1-1 at a dose of 10mg every 15 to 29 days. Histopathological examination of liver biopsy was performed in July 1989 and was negative for both HBsAg and HBcAg. Patient # 7 remained HBsAg negative 464 days after transplantation.

Example 6

Reactivity with various viruses

The reactivity of the monoclonal antibodies PE1-1, ZM1-2 and LO3-3 with various hepatitis B viruses isolated from the patients of Example 5 was investigated. A 20 µg / ml solution of monoclonal antibodies in phosphate-buffered saline containing 0.02% ΝθΝβ was incubated for at least 18 hours in U-bottom wells (Falcon Microtest III Flexibile Assay Plates). After removing the solution from the wells, the wells were washed 3 times with distilled water. Calf fetal serum at 2% concentration in phosphate buffered saline was added and incubated with with HBsAg serum or with controls and antibody labeled ¹ 25 | run overnight at room temperature (approximately 4,000 cmp in 1% fetal calf serum). The wells were then washed three times with distilled water. Individual wells were excited and counted. The results are shown in Table 4.

Table 4

The relative reactivity of various serum-derived HBsAg with HBsAg-specific monoclonal antibodies

Sample* LO3-3: LO3-3 PE1-1: ZM1-2 ZM1-2: ZM1-2 control 1,000 1,000 1,000 Pac.2 (234) 0.013 0,070 0.233 Pac.4 (251) 0.007 0.024 0.010 Pac.3 (264) 0.043 0.173 0.179

- 18 ‘representative HBsAg positive serum samples obtained from liver transplant patients and treatment with anti HBsAg therapeutic monoclonal antibodies PE1-1. The numbers in parentheses indicate the days after transplantation.

[-03-3- stands for RadioIunoassay with 1 'of' Dbbd 'Human-Long-Term' Human LO3-3 Antibodies, strongly adsorbed and radiolabeled. PE1-1: ZM1-2 means radioimmunoassay composed of human monoclonal antibody PE1-1 strongly adsorbed and human monoclonal antibody ZM1-2 radiolabeled. ZM12: ZM1-2 means radioimmunoassay composed of both ZM1-2 antibodies, both strongly adsorbed and radiolabelled. Control HBsAg positive serum reacts well with antibodies LO3-3, PE1-1 and ZM1-2.

Example 7

Large-scale production of antibodies

To initiate cell production, remove one or more ampoules with frozen cells from liquid nitrogen. After rapid heating in a 37 ° C water bath until most of the ice melts, the ampoule is opened in a vertical laminar flow hood. The contents of the ampoule are mixed with 1 ml of Dulbecco's NEM / Ham F 12 (1: 1 ratio) to which ferric salt is added to a final concentration of 50 µM Fe ⁺⁺⁺ . After mixing, the tubes are filled into 10 ml with the same medium and collected after centrifugation. The cell pellets are resuspended in 0.5 ml of the previously mentioned medium with 20% fetal bovine serum and seeded in one of the six wells of the tissue culture plate. Cells are incubated in an incubator at 37 ° C in a 5% CO 2 atmosphere. When the cells in the culture stabilize and start to divide and the approximate cell concentration is 10 6 / ml, the cells and medium are placed in a 80 cm ² tissue culture flask and diluted with 40 ml of DEMN / F12 (serum-free). When the cell concentration is again 10 / / ml, the medium is transferred to a 175 cm ² tissue culture flask and further diluted with 100 ml of DMEM / F12 medium. After reaching the optimal cell concentration, the cells and medium are transferred to a cylindrical flask with a surface area of 850 cm ² and diluted to a final volume of 500 ml. When the optimum cell concentration is reached in the cylinder bottle, the volume is divided into thirds into other cylinder bottles using the same medium as before. This process of dispensing into new cylinders continues until it is obtained

- a sufficient number of cylinders to accommodate the required number of cells in a Verax System 200 reactor.

Verax System 200

The Verax System 200 is a sealed cell culture system where cells are cultured in stainless steel microspheres (1.6g / ml specific gravity) composed of bovine type I collagen. The microspheres are loaded into vertical transparent glass tubes through which the culture medium is passed. (above) so that it reaches the bottom. the inlet of the tube is of such a shape that the microspheres are placed in a configuration such that they can liquefy when the medium is passed at a suitable speed. During the operation, fresh medium is constantly supplied and the conditioned medium is removed at a rate that is dependent on cell growth, which is monitored by glucose consumption. The temperature is maintained at 37 ° C; The pH is maintained at 7.1 and the oxygen / nitrogen ratio is also controlled.

After loading the microspheres into a medium containing 1% fetal bovine serum, the reactor runs for at least 3 days without cells in order to verify that no contamination of the System has occurred during the loading of the microspheres. During this time, the reactor is filled with protein-free medium to reduce the fill dose of fetal bovine serum. If the system works satisfactorily, the cylindrical flask cells are placed in the reactor.

Verax System 2000

This device uses the same type of microspheres as system 200, and its operation and control is exactly the same as a smaller system. System 2000 is approximately 15 times larger than system 200.

Monitoring of complete culture antibody yield

The conditioned medium is monitored for human immunoglobulin levels by ELISA each time the container is emptied. The results were confirmed by Protein A HPLC. Extract from cell culture medium and formation of mined stock

The conditioned medium is continuously removed from the Verax to the freezer. This medium is then emptied (using nitrogen pressure in the Verax system) into a stainless steel mobile container for further processing.

- 20 Cell culture media

The media routinely used is a 1: 1 mixture of Dulbecco's NEM H12 solution and HAM F12 (Mediatech). Medium is supplied in powder form, which is sufficient - 50 liters of the "final-mediar" -tank-this-powder-is-poured-in— ”»

a stainless steel tank that contains about 190 liters of water. The powder is mixed with the rotor until dissolved, sodium bicarbonate is added as recommended by the manufacturer, and the pH of the medium is adjusted to 7.4. sodium selenite is added to a final concentration of 17.3 µg / l and made up to 200 liters with water. Ferric ions in the form of ferric nitrate / sodium citrate are also added to the medium to a final concentration of 50 µM Fe ⁺⁺⁺ medium is immediately fed to the Verax 200 reservoir through a built-in sterilization filter. No proteins are added to the medium. No antibiotics are ever used.

Monoclonal antibody purification

Description of the method of recovery and purification of the final product

Monoclonal antibodies produce cell cultures from hybrid cell lines in the absence of serum. This means that we only need to remove cellular material components from the end product, since human monoclonal antibodies are not themselves to be immunogenic, it is very important to remove all potentially immunogenic components.

The ultimate goal of the purification process is the final product more than 99.9% pure using affinity chromatography. We are strongly dependent on the biological specificity of affinity chromatography. All steps of the purification process (shown in Table 5) are described in more detail below.

- 21 Table 5

Summary of cleaning

Step Condition ‘ Material removal cells $ temperature rooms polyvinylidene difluoride absolute filters 0.65 / 0.45um absolute concentration microfiltration Low: 14 ° C polysulfone filters nominal 30000 daltons Polyester (0.8 µm) and cellulose acetate (0.2um) absolute filters Protein A chromatog raffia Low: 14 ° C gold-linked agarose protein A staphylococcus Concentration 3 ° Ct triacetate cellulose filters, nominally 20000daltons gelová chromatog raffia Low: 14 ° C Sefacryl S-300, Ringer-Lactate solution ion exchange Low: 14 ° C Sefacryl S-300, Ringer-Lactate solution

Obtaining cells and removing particulate material from the conditioned medium.

Although most cells are retained by microspheres, a significant number of cells are in the supernatant obtained. To avoid large contamination of the media with cellular components, the supernatant is filtered through a Prostack® polyvinylidene difluond 0.65 µm filter (Milipor) immediately after removal from the Verax reservoir. This type of filter operates in a tangential flow mode that allows the filtration of large amounts of particulate material without clogging the filter. The cleaned medium is collected in a stainless steel freezer.

Concentration of conditioned media

The conditioned medium is concentrated with a nominal 30000 dalton spiral aperture polysulfone membrane supplied by Millipore. After concentration, the pH

- 22 was adjusted to 7.0 by addition of 1M acetic acid. The material is sterile filtered through a Sartobran-pH 0.8 / 0.2 µm filter (Sartorius) (0.8 µm is polyester, 0.2 µm is cellulose acetate, then stored at 4 ° C. The material is microfiltered (0 , 22 µM Millipore) and filled into polypropylene containers.

Protein A chromatography

A particularly important purification step utilizes the high affinities of the human IgG1 antibody to gold staphylococcal protein A.

Protein A is delivered already covalently bound by an amide bond to agarose. The gel was packed into columns and the columns containing this content and the attached tube were treated with 70% ethanol in water for 24 hours. The column is then equilibrated with PBS, pH 7.0.

Performing separation by affinity chromatography on a protein A column comprises the following sequential steps:

A / Performance. The concentrated conditioned medium is loaded onto the column by a pump. The effluent from the column is collected and monitored for the presence of antibodies by ELISA using human immunoglobulins. The column is filled to such a degree that a measurable amount of fluid containing the antibodies passes through the column. The overfilled portion is separately collected and recycled if it contains more than 20mg / ml antibody.

B / Washing. To remove unbound material, the column was extensively washed with phosphate buffered saline pH 7 with sodium chloride delivered to a final concentration of 0.5M. This wash is followed by a second wash step using 0.2 M sodium citrate buffer pH 5.6 containing 0.5 M sodium chloride. This washing releases a small amount of human antibodies.

C / Eluce. Bound monoclonal antibodies are eluted from the column with a pH 3.0 buffer of 0.1 M sodium citrate containing 0.5 M sodium chloride. The eluted material is continuously diluted in a volume of 1M tris-HCl at pH 8.0 to quickly restore near neutral conditions.

Protein A purification is performed in a closed system using the Watwrs 650 Protein Purification System.

Concentration of the eluate obtained from the protein A column

To make the next purification step more efficient and more convenient, the protein A column eluate is concentrated to a concentration of at least 5 mg antibody per ml.

The concentrate is sterile filtered through 0.2 µm filters and the sterile concentrate is stored at 4 ° C until sufficient material has been collected for the next purification step.

Size separation by gel chromatography on Sephacryl S-300 high resolution

Antibody preparation is performed on a Sephacryl S-300 High Resolution gel (Pharmacia), which is packaged into Pharmacia BP113 / 120 columns containing approximately 10 liters. The column is packaged in a Ringer-Lactate USP pickle (Travenol Laboratories). Each elution in the column is monitored by the Waters 650 protein purification System.

The reason for this step is not a further purification, but a buffer change. After elution in a protein A column, the antibodies are in a complex, hypertonic buffer consisting of sodium citrate, sodium chloride and tris-HCl. This buffer mixture cannot be used as a vehicle for intravenous injection. The buffer after this step is suitable for both intravenous administration and long-term storage.

Removal of host cell DNA by passage through an ion exchange column

Even after protein A chromatography, which removes large amounts of DNA present in the concentrated supernatants, and Sephacryl S300HR chromatography, which removes DNA molecules that are either significantly larger or smaller than monoclonal antibodies, a small but detectable amount of DNA remains in the antibody preparation. A strong anion exchanger, Q Sephacrose (Pharmacia inc.), Was selected to remove this contamination. At the pH of the Ringer lactate solution, the antibody proteins have a positive charge and the anion exchanger repels them. However, the nucleic acid has a negative charge at this pH and binds to an ion exchange column.

Pack the column according to the manufacturer's recommendations. After decanting the 20% ethanol solution, the gel is removed and 100 ml of the gel is dissolved in 200 ml of Ringer-Lactate solution. The suspension is poured into Pharmacia K50 / 30 columns and when the gel has a constant volume is treated with 0.5 N sodium hydroxide in an amount equal to that of the column, then three times with Dulbecco PBS and then five times with Ringerlactate. Just prior to use, the column was washed with a 5-fold amount of Ringer-Lactate solution. The sample is then passed through the column and what has passed is collected in a sterile container.

- ²⁴ Example 8

Molecular analysis of PE 1 -1, ΖΜ 1 -1, ZM1 -2 and MD3-4

The variable heavy chains of the PE1-1, ZM1-1, ZM1-2 and MD3-4 antibodies are isolated and sequenced. cells, are extracted from all cell-line RNAs by the method of Sanz et al. 1989 J. Immunol. 142: 883, (incorporated herein by reference). Single stranded DNA is synthesized using AMV reverse transcriptase and oligo dT as a matrix. The amount of ss-DNA synthesized is estimated by measuring the incorporation of 32β-CTP.

The polymerase chain reaction (PCR) is performed exactly as recommended by the manufacturer (Perkin Elmer Cetus, Norwalk, Connecticut). One microgram of DNA is added to a 200 µm solution containing dATP, dCTP, dGTP, dTTP with 100 µmol of each matrix and 5 units of Tag DNA polymerase. PCR cycles are performed as follows: denaturation for 3 minutes at 98 ° C, tempering at 55 ° C for 2 minutes, and extension at 72 ° C for 3 minutes, controlled in a DNA thermal cycler (Perkin Elmer Cetus).

The amplified DNA was sized on a 1.0% dissolved agarose gel that was bound to the EcoRV sites of the BLUESCRIPT phagemid vector and transformed into CaCl2 competent BSJ72 bacteria. A single-stranded DNA sequencing is isolated from each positive clone after superinfection with M13K07 as described by Sanz at all. Sequencing is completed by the dideoxy chain termination method as described by Sanger et al. 1980 J.Mol. Biol. 143: 161, except for the modified T7 DNA polymerase (Sequentase) which is used as described by Tabor et al. 1987 PNAS (USA) 84: 4767. The results are shown in Tables 8-1, 8-2, 8-3 and 8-4.

- 25 Table 8-1

The DNA sequences of the Vu region of the PE1-1 antibody are shown below. Conductor, Vj-jlt1, D and J (- (4 regions are indicated by the dashed line; CDR1 and CDR2 complementarity determining regions are indicated by asterisks). DNA.

ATG Z “1 T7T Gcu CTG AGC i CJ GTT TTC CTC GTT GCT CTT TTA AGA G * j i GTC TIME TGT CAG G15 TIME H ^Ξ - G and_ WITH w In F L. IN AND L L R C IN Q C and Y and -In her___ CTG 5 l Q Limo T.CT GGG ..Gw \ GGC G1G GTC CAG CCT GGG AGS TCC CTG AGA CTC TCC TGT GCA GCC TCT L 1 - with G G G v v and p G R S L R L with C 1 Λ AND £

GGA G l · C T TTC cr nu: with AGu R MELT Y GGC G ATG H CAC ri TGG w GTC IN CGC CAG GCT ui_A p G \ j \ - G i »r * TO GGG G CTG GAS TGG * R and G i G CT- Q J c ATA TCA 7A7 5A7 C-jA rva i A.AT t <1 r-run TGa MELT G_A GAC TCC G 1 G i r · - r \ j - and - and • - * with with n .Λ J - 3 ATC 7GG AGA GAC ΑΛΤ TCC AAG AAC <Γ ”* · r \ L. t C i G TTT CTG CAA ATG CAC AGC CTG AGA GC i GCG GAC ACG £ £ 0 H with . < H 7 L r L Q 7. £ L 0 AND at GGT G t A MELT TRAY TGT GCa AAA GA i CAA CTT TRAY TTT GG i TCG CAG AGT ccc Crvtl CAC <- « t AC TGG GTC and t IN 1 IN C AND TO D and £ IN F G WITH Q £ □ with in W • j t CAG GGA ACC CTG GTC ACC GTC TCC TCA and 5 7 1 IN 7 in 5 with

-. . . _ -26 - _

Table 8-2

The DNA sequences of the Vβ region of the ZM1-1 antibody are shown below. regions complementary to CDR1 and CDR2 are indicated by asterisks. Amino acids are indicated by the abbreviation (initial letter) below the DNA.

-LEADERATG GAG ΤΓΤ GGG C7G AGC TGG GTI TTC GTT GCT ΤΤGTA GA GGT GTC CAG TGT GAG GTG CAG Η Ξ F 5 L S Μ V r L V A Z C Ξ ~ 7 Q C Ε V G

ETG GTG SAS TCT SGS GGA GST iGG CTA CCT GGG GGG TCC CiG AGA CTC i CC; ST Gi-λ cCC;

L S C

• CGR2 ·

-TC TCA GCT Aii GGT CCT ACT GGT GAC AND CA TAC TAT. IC Giá G_A GAC AAG OOV i. ^ -C mod λ i ^1. y <; AC 6? TS 0 Y YYAD ς VK and R- ¹

AGA GAA AAT GCC AAG AAC TCG ii G TAT lti ACA ATG AAC GGG CTG AGA Gera aAC ACG Gi with rehakhs --------------- ύ, ί ------------------- G G G T ---------------- ---------------- ---------------- ---------------- GT ---------------- GT AT GT AT AT GGA ACC GTC GTC ACC GTC TCC TCA • / YYCARDi-ELW

Example 9

Following the procedure of Example 8, the light variable chains (V1) of the antibodies PE1-1, ZM1-1, ZM1-2 and MD3-4 are isolated and sequenced. The results are shown in Tables 9-1,9-2,93 and 9-4. Table 9-l

The DNA sequence of the V1 region of the PE1-1 antibody is shown below. The VV and J3 regions are indicated by the dashed line; regions complementary to CDR1, CDR2, and CDR3 are indicated by asterisks. Amino acids are indicated by the abbreviation (initial letter) below the DNA

..................... — ........ v, v —...........

CAS TCT CAG CTG ACS CAG CCG CCC TTS GTS TCA GTS QSQLT QP FSVSV

................ + .... CDZ1 ........................

G «G SGA AAC ATT GGG AGi AAA AGT GTS AAC TSG GGDNIGSK SVHW

..................... * »» »» CDR2.Tr »«.

CTG GTC GTC TAT GAT GAT AAC GAA CGC CCC TCA R P S S

GCC CCA GGG CAS ACG GCC AGG ATT ACC TGT APGQ.TARI7C

TTC CAS CAS AAG CCA GGC CAS GCC CCT GTC FQQKPSQAPY

ATT TCT SAS CGA TTC TCT GGC TCC AAC TCT

GGG AAC ACS GCC ACC CTS ACC ATC AGC AGG GTC GAA T I S R V E G 0

SAS GCC GAC TAT TAC TST CAS A A Τ Ϊ C Q

- «•» w »w» * »» wwww »C0R3 *

STS TGG AST AST AST GAT CAT V ». 0 with S $ D H

GTS STA TTC GGC GGA GGG ACC

V V F with G e T

3 ----------------->

AA6 CTS ACC STC CTA C L Τ V L c

- 30 Table 9-2

The DNA sequences of the V (_ regions of antibody ZM1-1) are shown below. The conductor, VII and J5 regions are indicated by the dashed line; the regions conditional on the complementarity of CDR1, CDR2 and CDR3 are indicated by asterisks.

LEADER

ATS SAC ACS 6C CCC SCT CAS CTC CTS MS CTS CTA ATS CTC TM STC CCA MA TCC AST SM

HD TRVP AQLLGL LHLWVPSS S 6 · <----------------------------------- V, II .... ............-.........................>

SAT STT 6TS STS ACT TCT CCA CTC TCC CTS CCC STC ACC CTT MA CAS CCS S-CC TCC ATC TCC

DYVVTQSPLSL PVT LSQPASIS

-—-- **** »** · **» CDR1

TGC ASA TCT AST CTA AGC CTC STS SAC AGT SAC KA AAC ACC TAC TTS AAT TM TTT CTC CAS AM Vrsslslvdsds ktylhwflůr

---—--------------------—--—--—-— · * «τ« »» »» »CCR2» »» »« «» »» «» »————————————————————————————————————————————————————————————————————————————————————————

P SQSP_RR L-IY QL.SSR OSSVP'DR

TTC AGC KC AST KS TCA SGC SAT SAT TTC ACT CTS AAA ATC ASC AM STS SAS SCT SAS SAT STT F S S.SSSSTDFTLKISRYEASOV

----- i ------------------ CQR3 *.-..........-.........— J 5 .............

esc STT TAT TAC TSC ATS CAA K7 ACA CAC TSS CCC ATC ACC TTC KC CAA KS ACA CSA CTS SAS SVYYCHQS. T.HWP ITPSQPTRLE

ATT AAA CSA

1 . 1 O _ i • - 1 LU. O / i X O á i F and K Y LO: cn

i cr.

zcz oc

-J 'Τ-

Claims (12)

1. Human monoclonal hepatitis B. 2. Human monoclonal groups consisting of 3. A mixture of monoclonal antibodies comprising a mixture of at least two monoclonal antibodies, each of which neutralizes hepatitis B virus and each of which is specific for different epitopes of the hepatitis B virus surface antigen, wherein the antibody is selected from the group consisting of consisting of PE1-1, ZM1-1, ZM1-2 and MD3-4. * 4. A monoclonal antibody Fab fragment selected from the group consisting of PE1-1, ZM1-1, ZM1-2 and MD3-4. The human monoclonal antibody of claim 1 for use as a medicament for lowering the level of a circulating surface antigen in hepatitis B virus. The human monoclonal antibody of claim 5, which is selected from the group consisting of PE1-1, ZM1-1, ZM1-2, MD3-4, and mixtures thereof. ♦ * A monoclonal antibody, characterized in that the VH region is nearly the same as that shown in any of Tables 8-1, 8-2, 8-3 or 8-4. A monoclonal antibody, characterized in that the VL region is nearly the same as that shown in any of Tables 9-1, 9-2, 9-3 or 9-4. an antibody that neutralizes the virus of claim 1 which is z = PE1-1.ZM1-1. ZM1-2. MD3-4. 9. A cell culture characterized in that it produces the antibody of claim 7. 10. A cell culture which produces an antibody according to claim 8. 11. DNA nearly free of attached mammalian DNA, which comprises a DNA sequence that hybridizes under stringent hybridization conditions to a DNA strand nearly the same as this selected from the group consisting of: and) fiber according to tables 8-1, (b) v1ákno according to tables 8-2, C) fiber according to tables 8-3, (d) fiber according to tables 8-4, E) v1ákno according to tables 9- 1, F) v1ákno according to tables 9-2, G) fiber according to tables 9-3, (h) fiber according to tables 9-4 12. A cell culture having DNA comprising the DNA of claim 11.