HUT72546A - Production of human monoclonal antibodies active against hepatitis b surface antigen - Google Patents

Abstract

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

Description

Preparation of human monoclonal antibodies to hepatitis B surface antigen

Sandoz Ltd., BASLE, SWITZERLAND

Inventor: OSTBERG Lars G., CONVENT STATION, NJ,

USA

Date of filing: 06.11.1992

International Application Number: PCT / US92 / 09749

International Publication Number: WO 94/11495

The present invention relates to hybridoma cell lines that produce human antibodies that neutralize hepatitis B virus, and to methods of producing cell lines, antibodies produced by cell lines, and the use of antibodies, particularly for therapeutic purposes.

The preparation of hybridoma cells for the production of monoclonal antibodies is generally well known to those of ordinary skill in the art. The present invention relates to

Preparation of human monoclonal antibodies effective against -2 hepatitis Β surface antigen (HBsAg) according to the method described by the Applicants (Hybridoma 2: (4), 361 (1983); United Kingdom Patent Application, 2,113,715A, Aug. 1983

10.] can be produced. Specifically, it has been found that a hybridoma cell line containing a parent rodent immortalizing cell, i.e., a murine myeloma cell (e.g., SP-2), fused to a human partner cell results in an immortalizing xenogeneic hybridoma cell line. This xenogeneic hybridoma cell can be fused with a cell capable of producing an anti-HBsAg human antibody, resulting in a novel trioma cell line capable of producing human antibodies effective against such antigens in humans. Alternatively, if greater stability is desired, a trioma cell line is produced that is preferably no longer capable of producing its own antibody, and this triome is fused to an additional

with a cell that can the said antigen against usable antibodies produce, and so one even more stable hybridomas (Quadromas) we get, which is mentioned antigen antibodies produce.

Applicants have previously described in their publications the production of a xenogenic hybridoma, referred to as SPAZ 4, starting from the drug-resistant cell line SP-2, for example from the NIGMS Human Genetic Mutant Cell Repository (Ref. GM35669A, see U.S. DHHS 1982 Catalog of Cell Lines). to get. The production of SPAZ 4 can be summarized below. The SP-2 cell line was fused to standard human peripheral lymphocytes using standard techniques. A large number of hybrids were available, and about five weeks later five clones were selected which grew rapidly and produced no antibody. These cells were selected on the basis of their resistance to 8-azaguanine, and it was possible to obtain mutants that were resistant to 20 pg / ml of 8-azaguanine in three of these cell lines. At the same time, these cells are sensitive to hypoxanthine aminopterin thymidine (HAT) medium, indicating that they have lost their ability to produce hypoxanthine phosphoribosyl transferase. One of these cell lines is SPAZ 4.

The SPAZ 4 cell line is fused with cells obtained from the blood of subjects immunized with hepatitis B vaccine to obtain hiridoma cell lines that result in positive cultures using standard selection procedures, including binding of antibodies to the appropriate viral antibody. Preferably, said positive cultures are subjected to a second selection procedure using different subtypes of virus to produce antigen.

This gives us the opportunity to point out the exact antigenic determinant recognized by the antibody.

Cell lines derived from a fusion of a xenogeneic hybridoma and a cell producing a human monoclonal antibody (triome) are therefore useful in the production of monoclonal antibodies which are capable of effectively neutralizing a hepatitis-causing virus and therefore of preventing the transmission of hepatitis, e.g. through. They can also be used to provide initial protection for newborns.

-4 or affected individuals before a vaccine is effective. Anti-hepatitis antibodies can be used to protect immunosuppressed patients, including transplant patients, against a new occurrence of hepatitis. This is most important in patients with hepatitis B positive liver transplantation. Antibodies can also be used in diagnostic tests.

Antibody fragments, such as Fab fragments, have been found to be capable of binding to hepatitis B surface antigen. These fragments are also part of the present invention.

Each specific antibody produced according to the present invention (including PE-1, ZM1-1, ZM1-2 and LO3-3) is of the IgG1 class.

The cell line producing PE1-1 was deposited with the American Type Culture Collection on October 16, 1986, accession number ATCC HB 9234; the cell line producing ZM1-1 was deposited at ATCC HB 9191 on September 4, 1986, and ZM1-1 was deposited at ATCC HB 9192. The American Type Culture Collection is located at 12301 Parklawn Drive, Rockville, Maryland 20852.

The cell lines of the present invention behave as typical (mouse x human) x human hybridomas and produce appropriate antibodies at concentrations up to 25 mg / ml in standard suspension culture.

The figures in the appendix are briefly described below.

Figure 1 shows the results of a direct enzyme-linked immunoassay in which the binding kinetics of the PE1-1 antibody (indicated by a single line) and the binding of ZM1-2 antibody are shown.

kinetics (indicated by double line) are compared, details are shown in Figure 4A. is exemplified.

Figure 2 shows the serum levels of PE1-1 antibody in Rhesus monkeys, determined at different times after dose administration. See Figure 4C for details. example.

In the specifications and claims, the same label is used for both the cell line and the antibody it produces, i.e., the PE1-1 cell line produces the monoclonal antibody PE1-1; the ZM1-1 cell line produces the ZM1-1 monoclonal antibody, etc. We believe that one skilled in the art will recognize that the cell line or antibody is being discussed.

The monoclonal antibody PE1-1 is also referred to by the inventor and is designated by the inventors as IST 577 and 64-577. Similarly, the ZM1-2 monoclonal antibody and cell line was referred to as 265-695, and the LO3-3 monoclonal antibody and cell line was referred to as 266-215.

The antibodies and antibody fragments obtained by the method of the present invention have good specificity for hepatitis B surface antigens in in vitro ELISA binding assays.

because the antibodies of the present invention are human origin, therefore they are advantageous in human therapy,

because they do not develop an allergic response to repeated treatment as with rodent or bovine antibodies. Thus, the present invention also relates to a process

hepatitis B is one or more of the foregoing antibody administration. It turned out to be about 10-40 mg antibody Repeated doses significantly reduce the circulatory system

Amount of HBsAg. Further doses revealed that HBsAg

levels of antigen tests detection limit a below reduced. The present invention object furthermore you are two more

monoclonal antibody cocktail. This mixture is particularly useful for administration to patients carrying a non-wild type of hepatitis B virus that does not bind well to a particular monoclonal antibody. For example, a compassionate patient suffering from hepatocellular carcinoma and chronic hepatitis B was given PE1-1 prior to liver transplantation and then repeated with multiple doses. (Details are given below in Example 5). After about four and a half months of treatment, low levels of serum HBsAg could be detected with polyclonal antibody but not with PE1-1. A 230 base pair region polymerase chain reaction (PCR) analysis of the putative monoclonal antibody binding domain of the HBsAg gene was performed. The PCR DNA was cloned into bacteriophage M13 and the resulting DNA was sequenced. Analysis of the clones obtained from each serum sample revealed the presence of two variant sequences when compared to the PCR DNA from the original liver and pre-antibody therapy. The variant DNA encodes two different amino acids in the HBsAg S protein and one encodes a stop codon (UAG) in the viral polymerase gene. Both variant genes contain an amino acid change that results in the exchange of an arginine for glycine in a conserved peptide domain.

Since the monoclonal antibodies PE1-1, ZM1-2, ZM1-1, MD3-4 and LO3-3 have been shown to bind to different epitopes and at least one of the monoclonal antibodies

all of the viral variants tested to date have been found to bind to a sufficient degree and are therefore well suited for use in the clinic, and thus the present invention further provides a cocktail comprising two or more monoclonal antibodies from the group: -1, MD-3 and LO3-3. Particularly preferred are cocktails containing two monoclonal antibodies, especially mixtures containing PE1-1 and ZM1-2 as well as PE1-1 and LO3-3. The ratio of monoclonal antibodies in the mixture may vary depending on a number of factors, as will be apparent to those skilled in the art, including, for example, the hepatitis B virus or viruses genotype present in the patient's serum, the relative binding strength of the antibodies selected economic considerations. Generally, the antibodies will be present in a 1:99 ratio, more typically 25:75, and preferably substantially the same amount.

Sections PE1-1, ZM1-1, ZM1-2 and MD3-4 are sequenced using standard methods. The sequence obtained for the Vp1 region of PE1-1 is given in Table 8-1 and the regions corresponding to CDR1, CDR2 and CDR3 (D1 and J1) are indicated. Because CDR regions are particularly important regions in determining the binding properties of an antibody, the present invention relates to an antibody having the amino acid sequence of the CDR1 region substantially similar to the amino acid sequence of PE1-1 as shown in Figure 8-1. shown in the table. The invention further provides an antibody having a CDR2 region having an amino acid sequence substantially similar to that of PE1-1 as shown in Figure 8-1. shown in the table. THE ·

The invention further provides an antibody having

The amino acid sequence of the CDR3 region is substantially similar to

ΡΕ1-1

The amino acid sequence of the CDR3 region as in a

8-1.

shown in the table.

Similarly, the ZM1-1 region was determined as shown in Figure 8-2. is shown in Table. The CDR1,

CDR2 and

Areas corresponding to CDR3 (Dfl and Jh4) are also indicated.

The present invention comprises an antibody having

The amino acid sequence of the CDR1 region is substantially similar to

ZM1-1 as shown in Figures 8-2. shown in the table.

In addition, this invention includes an antibody having

The amino acid sequence of the CDR2 region is substantially similar

ZM1-1 as shown in Figures 8-2.

The present invention comprises an antibody having

The amino acid sequence of the CDR3 region is substantially similar to

ZM1-1 as shown in a

8-2. shown in the table.

The DNA sequences encoding the ZM1-2 and MD3-4 regions are shown in Figures 8-3.

and 8-4. table. The invention further relates to any antibody having substantially the amino acid sequence of the ZM1-2 and MD3-4 regions.

sequence as in the 8-3. and 8-4. table show. A PE1-1, ZM1-1, ZM1-2 and MD3-4 Vf] regions coding DNA sequences determined and 8-1 ., 8-2, 8- • 3rd and 8-4.

These sequences are either shown in the table.

suitable fragments can be used to clone antibodies (or modified antibodies) or as probes. As usual, instead of extracting from a hybridoma • ·

Antibodies produced by genetic engineering techniques may be prepared by cloning techniques known to those skilled in the art. DNA from other sources may be used to produce a synthetic antibody molecule that retains the binding properties of PE1-1, ZM1-1, ZM1-2, and MD3-4, with substantially similar CDR1, CDR2 and / or CDR3 regions. contain. Such antibodies are within the scope of the invention.

DNA sequences encoding the variable regions of PE1-1, ZM1-1, ZM1-2 and MD3-4 _VL light chain, 9-2. 9-3 to 9-1.. 9-4. tables. The present invention further provides any antibody having an amino acid sequence substantially similar to the amino acid sequence of regions PE1-1, ZM1-1, ZM1-2 and MD3-4 as shown in Figures 9-1, 9-2, 9-3. 9-4. is shown in Table.

The invention encompasses DNA sequences which encode the V1, Vp, CDR1, CDR2 and / or CDR3 regions of PE1-1, ZM1-1, ZM1-2 and MD3-4. Also included are DNAs capable of hybridizing to any of the aforementioned DNA sequences under stringent hybridization conditions. This DNA is substantially free of other donor mammalian DNA, may contain introns, or may be cDNA.

The following definitions are used throughout the specification and the claims. An amino acid sequence is substantially similar to other amino acid sequences if the amino acid homologue is at least 80%. For DNA, stringent hybridization conditions mean conditions for hybridization at 60 ° C, 2.5X saline

in citrate buffer (SSC) and then simply washed at 37 [deg.] C. with reduced buffer concentration, which does not affect the resulting hybridization. Linked mammalian DNA refers to DNA that is present in the mammalian source of the antibody chain but which is not involved in the encoding of an antibody or antibody fragment.

The invention is further illustrated by the following non-limiting examples.

Example 1

Production of antibody cell lines

Volunteers are immunized with hepatitis B vaccine. The MD34, ZM1-2, ZM1-1 and PE1-1 hybridoma cell lines are derived from human lymphocytes immunized with Heptavax (Merek & Co.). The L03-3 cell line was developed from human cells injected with Heptavax® several times and immediately before fusion with Recombivax®. Peripheral blood lymphocytes were purified by density gradient centrifugation on a Percoll Pillow (Pharmacia Inc., density 1.085 g / ml). The isolated lymphocytes were washed three times with Hank's balanced saline and then mixed with an equal number of cells from the (mouse x human) SPAZ-4 cell line. The cell mixture was pelleted by centrifugation at 400 x g for 5 minutes at room temperature. After removal of the medium, the cell pellet is treated with 50% PEG-1000 in Dulbecco's Minimum Essential Medium (MEM) for 1 minute at 37 ° C and the medium is slowly diluted with Dulbecco's MEM. Cells were harvested by centrifugation and resuspended in Dulbecco's MEM containing 20% fetal calf serum. The cells are approximately 2χ10θ cells per ml

«··

Concentrate is added to wells of microtiter plates. The next day, fresh medium containing the components of HAT (hypoxanthine, aminopterin, thymidine) medium was added to suppress the growth of non-fused SPAZ-4 cells. On the 4th day after fusion, the medium is replaced with fresh medium containing only HT, since cells sensitive to HAT selection have so far been killed.

Three to four weeks later, when the good growth of the hybridoma-like cells is already visible microscopically, the supernatants are examined for anti-hepatitis B surface antigen antibody. A solid phase ELISA assay using Heptavox® 1/100 dilution is performed. After incubation with the supernatants, the plates were developed with a kit containing biotinylated goat anti-human immunoglobulin and horseradish peroxidase (Vectastasin ^< - ') linked to avidin.

Vector Laboratoires Inc.). The enzyme is detected by color reaction with phenylenediamine. Positive cultures are transferred to new wells and a portion of the cells are cloned by limiting dilution in Dulbecco's MEM containing 20% bovine serum and 10 ⁷ mouse thymocytes per ml. The cloning plates were assayed using the same ELISA method as described above, and positive cultures were propagated and frozen.

Each cell line behaves as typical (mouse x human) x human hybridomas and produces the appropriate antibodies at concentrations up to 25 mg / ml under standard suspension conditions.

• ·

Example 2

Immunochemical characterization

A. Antibody class / subclass

The immunoglobulin class of PE1-1, ZM1-1, ZM1-2, MD3-4 and L03-3 antibodies was determined by ELISA. Each antibody was captured on antigen-coated plates and each assay was developed with subclass-specific peroxidase-conjugated anti-human Ig (Tago). Each antibody was clearly IgG] _.

B. Type of light chain

Each antibody is assayed with anti-κ or anti-λ light chain reagents (Tago) using ELISA methods similar to those described in section A. The following results were obtained.

PE1-1

ZM1-1 lambda kappa

ZM1-2 kappa

L03-3

MD3-4 lambda lambda

C. isoelectric focusing (IEF)

One sample of L03-3 or PE1-1 antibody is applied to the gel. All of them turned out to behave as a basic protein.

D. Specificity

Purified HBsAg of the adw and ayr subtype were purchased from Scripps Laboratories (San Diego, California). The

HBsAg -13ayw subtype was obtained from Connaught Laboratories (Willowdale, Ontario). Elisa assays are performed essentially as described by Ostberg et al., Ostberg et al. : Hybridoma, 2: 361-367 (1983)].

PE1-1 responds to both ayr and adw, but responds slightly more to the adw subtype. L03-3 reacts essentially the same with ayr and adw. ZM1-1 shows greater reactivity with adw, but ZM1-2 is slightly more bound to ayr. These results are confirmed by Scatchard analysis on PE1-1 and L03-3 using solid-phase RIA adsorbed on sol to ayr or adw. Thus, although monoclonal antibodies do not appear to bind to the subtype determinant, their reaction with HBsAg may be significantly affected by the subtype.

G. Allotype determination

Allotypes are determined using reagents available from the Central Laboratory of the Netherlands Red Cross Transfusion Service. Inhibition ELISA or direct binding ELISA is used. The results are shown in Table 1 below. As can be seen, there is no apparent restriction on the high affinity anti-HBsAg antibodies for the light chain or allotype.

-14 ·· ·· • · · »♦ · · •« * ···· · ♦

Table 1

Allotypes of anti-HBsAg monoclonal antibodies

allo types

antibody

Km (3)

PE1-1

ZM1-2

L03-3

ZM1-1

ND

ND

ND

ND = not determined * = The antibody has a light chain that has no Km allotype

G. Affinity

The affinity for solid adsorbed HBsAg is determined for each antibody using a radio-labeled antibody, essentially as described by Wands et al., (1981) in Wands et al., Gastroenterology 80, 225-232. Antibodies 125j_ _ve j_ labeled using Iodogen (Pierce). All monoclonal antibodies, except L03-3, are of HBsAg ayw subtype adsorbed to the solid phase. Examining L03-3 with both ayr and adw subtypes yields essentially the same results. The antibody-antigen is incubated at room temperature.

Relative affinity was also determined using an inhibition ELISA in which various concentrations of soluble HBsAg (ayw subtype) were preincubated with the monoclonal antibody and incubated at 37 ° C for a <···············································

microtiter plate covered with HBsAg. The results are shown in Table 2 below.

Table 2

The affinity of the monoclonal antibodies for HBsAg

antibody Solid phase RIA, M Inhibition PE1-1 3.6x10 ⁹ ~ 2x10 ⁹ ZM1-2 1.5x10 ⁹ ~ 7x10 ⁸ L03-3 1.7x10 ⁹ ~ lxl0 ⁸ ZM1-1 5 x! 09 ~ lxl0 ⁸

As shown in the table above, both PE1-1 and

In the case of ZM1-2

ELISA results are approximately twice lower than

RIA results that are within the margin of experimental error.

Scatchard representation of the results of the RIA experiment on ZM1-1 shows that a low affinity binding site may exist. Thus, it is possible that the ELISA measures this low affinity binding site, since the ELISA results are about fifty times lower than the RIA results. In addition, the Scatchard representations also confirm that there are significantly fewer high affinity ZM1-1 sites than high affinity ZM1-2 or PE1-1 high affinity sites. While not wishing to be bound by theory, it appears that of the four antibodies compared, ZM1-1 may have the highest affinity for HBsAg, but only for HBsAg of certain spatial structure. This arrangement manifests only a small percentage of HBsAg molecules. It is also possible that this is due to the fact that ZM1-1 is bivalent to HBsAg, whereas the low affinity site is monovalent.

ο ··

Example 3

Potential of PE1-1

The potential of PE1-1 antibody is assayed by AUSAB radioimmunoassay (Abbott). The investigations were conducted by the Bureau of

Biologics Reference Immunoglobulin Hepatitis B is used and many commercially available hepatitis B immunoglobulin formulations (H-Big Immune Globin®, Hep B Gammagee Immune Globin®, and Hyper Immune Globin®, all purchased from a drug company). Although the immunoglobulin formulations are polyclonal and the PE1-1 monoclonal binding data are within the criteria set by the Bureau of Biologics for comparing immunoglobulin formulations, ie, the lines are parallel to a probability level of 0.01 or less .

The potential is determined as follows. The formulations are compared on a weight basis (an absorbance of 1.4 at 208 nm corresponds to 1 mg / ml). The PE1-1 formulations stored at 5 ° C were then compared to the above polyclonal formulations, which were also stored at 5 ° C. The logarithm of 1000 pg / ml of the formulation (i.e., the logarithm of the number inversely proportional to the concentration of the immunoglobulin is similar to the dilution factor logarithm) as a function of the number of logs per minute (average of three measurements). The assumption that the fitted lines are parallel is examined by analysis of variance. The lines were found to be parallel at a probability level of 0.01 or less. The lines of each preparation are parallel and common

-17 slope can be determined. The X-axis sections are calculated from the common slope and then the difference between the axial sections is used to determine the potential difference. By this method, the monoclonal antibody PE1-1 is about 435 times more potent than the Bureau of Biologics reference hepatitis B immunoglobulin. Since commercially available immunoglobulin formulations have been found to be twice (or less) more active than the Bureau of Biologics reference formulation, PE1-1 is at least two hundred times more potent than commercially available immunoglobulin formulations by weight.

Example 4

A. Binding kinetics

Direct binding enzyme-linked immunoassays were used to compare the binding kinetics of HBsAg to PE1-1 and ZM1-2. ELISA microtiter plates were coated with Heptavax® at a concentration of 1 μρ / ιπΐ. The wells are then incubated at 37 ° C with 2% fetal calf serum in phosphate buffered saline. Monoclonal antibodies PE1-1 or ZM1-2 were incubated in the wells at 0.5 pg / ml in 2% fetal calf serum for various times. At the indicated times, the antibody solution is removed and the wells are rinsed three times with fresh 2% fetal calf serum. The well is then incubated with 2% fetal calf serum until the 90-minute wells contain 2% fetal calf serum. The solution is then replaced with either a peroxidase-conjugated goat anti-lambda chain (PE1-1 wells) or a goat anti-kappa chain (ZM1-2 wells). Quantitation of plastic bound peroxidase was performed by addition of O-phenylenediamine and hydrogen peroxide. The results are shown in Figure 1, where the single line represents the results obtained with PE1-1 and the double line represents the results obtained with ZM1-2.

As shown in Figure 1, at the concentration at which PE1-1 was almost completely reacted within five minutes, the reaction of ZM1-2 with HBsAg adsorbed on a solid support was incomplete for 30 minutes and 90 minutes or more. responds further. Thus, PE1-1 binds significantly more rapidly to the antigen in this assay. Assuming that this occurs in vivo, PE1-1 is likely to more effectively neutralize viral particles before they infect the liver.

B. Relative position of epitopes

The relative positions of epitopes on PE1-1, L03-3 and ZM1-2 antibodies were determined. Simultaneous sandwich immunoassay was used with a monoclonal antibody adsorbed to a solid phase. The same antibody is radiolabelled and then incubated in the wells of a microtiter plate with the inhibitor and serum from a hepatitis B positive patient. The radioactive isotope labeled PE1-1 Fab fragment is used while the labeled L03-3 is intact IgG. The results are shown in Table 3 below.

Table 3

Inhibition of the binding of radiolabeled monoclonal antibody to HBsAg by unlabeled monoclonal antibodies

Carrier adsorbed with Iodized Inhibitor IC5Q ng / ml maB maB maB

L03-3 L03-3 L03-3 10 L03-3 L03-3 PE1-1 > 22,500 PE1-1 PE1-1 PE1-1 8 PE1-1 PE1-1 ZM1-2 76 PE1-1 PE1-1 L03-3 > 22,500

The monoclonal antibody ZM1-2 is only approximately nine times less than required to inhibit ¹² 5i_p _E i_i binding to HBsAg than unlabelled PE1-1, L03-3 is thousands of times smaller while inhibited the same process. Thus, the epitopes of ZM1-2 and PE1-1 are probably located close to each other on the HBsAg molecule, while the epitope L03-3 is probably located on the other side of the molecule. The reverse assay, inhibition of PE1-1 by radioactive isotope labeled L03-3, provides further evidence that PE1-1 and L03-3 bind to non-overlapping epitopes.

The similarity of the PE1-1 and ZM1-2 epitopes and their difference from L03-3 was confirmed by immunoassay with reduced and alkylated HBsAg. L03-3 is capable of binding to denatured antigen, whereas both ZM1-2 and PE1-1 are unable to bind. It should be noted that PE1-1 and ZM1-2 have tolerant epitopes since their response to different subtypes is different.

-20 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

C. Pharmacokinetics of PE1-l in Rhesus monkeys

The pharmacokinetics of PE1-1 were examined in two Rhesus monkeys. Each animal receives a single intravenous bolus injection (0.5 mg / kg) of PE1-1 monoclonal antibody. Serum levels of PE1-1 were determined at various time points after administration by ELISA-based sandwich immunoassay, Heptavax® with ELISA plates and rabbit anti-idiotypic PE1-1 antibodies. The results are shown in Figure 2.

Serum levels of the two Rhesus monkeys are characterized by a biphasic decline (t1 / 2a = 1 and 1.4 days; t1 / 2β = 11 and 16 days), with a shorter half-life associated with the monoclonal antibody distribution phase. The steady-state volume of distribution (Vdss) is calculated as 114-144% of the plasma volume, suggesting that PE1-1 is only slightly introduced into tissue sections in the antigen-free monkey.

Example 5

Clinical trials

A. Exceptional use of PEl-1 in two patients with end-stage liver disease following liver transplantation after chronic active hepatitis B and hepatocellular carcinoma

PE1-1 is administered to two patients who undergo liver transplantation with end-stage liver disease on an exceptional basis. The first patient is a 56-year-old man who has had chronic hepatitis for 20 years and is diagnosed with hepatocellular carcinoma. The second patient is a ten-year-old boy who was infected with hepatitis B at birth. The second • · · · · ·

-21 patients first observed a large mass in the right lobe of the liver, which turned out to be hepatocellular carcinoma.

These patients received preoperative doses of PE1-1 and significantly reduced their circulating HBsAg levels prior to transplantation. In addition, each patient received a dose of 20 mg of PE1-1 at the time of transplantation. Postoperative injections began on the second day after surgery.

The first patient never became HBsAg negative, although his circulating HBsAg levels dropped significantly below pre-treatment levels. The second patient became HBsAg negative and was observed for the first time on day 9 after transplantation. The first patient received additional doses of ΡΞ1-1, ranging from 5 to 40 mg in 2-20 day intervals. The second patient received doses of 5 or 10 mg on average for each 21-28 years. on the sun.

No patients experienced adverse side effects while receiving PE1-1. However, about four weeks after the first patient was discharged from the hospital, she had cancer metastases. He died on the 139th day after transplantation. No recurrence of hepatitis was observed after transplantation in this patient, despite the presence of circulating HBsAg. Although a hepatitis B virus DNA test was negative before surgery, a single positive value was obtained 60 days after transplantation.

At day 143 after transplantation, the second patient was first observed to be HBsAg positive. HBsAg levels first fluctuated for a short time before stabilizing well before treatment. Hepatitis B isolated from this patient before and at a later time during treatment with PE1-1.

viruses were tested for their ability to bind to PE1-1. PE1-1 has been shown to bind to the variant virus, but not as well as to the wild-type virus.

Genetic analysis of the two viral isolates shows a single nucleotide difference in the major viral surface protein. Such differences, when compared to the pre-treatment virus, have the potential to reduce the ability of PE1-1 to bind to the hepatitis B viral binding moiety.

B, Use of PE1-1 in patients with active chronic hepatitis B who undergo liver transplantation (do not interfere with hepatocellular carcinoma)

This study included five patients who were HBsAg positive (but not hepatocellular carcinoma) and underwent liver transplantation. Each patient received three preoperative doses of PE1-1 daily (10, 20, and 40 mg) over a three-day period. Liver transplants were then performed at least two days and up to 32 days after the preoperative dose of test substance. During the operation, additional doses of 40 mg were given. All five transplants have been carried out smoothly.

Patient HBsAg titers, liver enzymes and other clinical parameters were closely monitored during the hospital stay. Further evaluations and administration of PE1-1 were performed regularly by the patient's GP (approximately 1 to 3 weeks). Dosage and other parameters varied from patient to patient.

Two patients (# 5 and # 6) had the same situation as in the previous experiment with patient # 2 when a variant virus appeared with a negative HBsAg test result.

After -23. The sera of these patients remained active with PE1-1. Sequence analysis showed a nucleotide difference between the variants in the patient's serum and the wild-type virus. Two variants could be detected in each patient. Immune assay and sequence analysis showed that the variants in each patient were different and also the variants found in patient # 2.

Patient # 3 is a 39-year-old Caucasian male who, after 16 years of chronic hepatitis B, is in the final stages of liver disease. Three doses of PE1-1 preoperatively administered to patient # 3 caused a significant decrease in HBsAg titer. On days 2 and 3 after transplantation, he received 20 mg PE1-1 and was initially considered HBsAg negative on day 2 after transplantation. Two months later, Patient # 3 received 10 mg PE1-1 every 1-7 days. Thereafter, he received doses of 7.5-10 mg PE1-1 every 14-43 days. Histopathological analysis of a February 1989 liver biopsy showed that it was negative for both HBsAg and HBcAg. Patient # 3 remained negative for HBsAg 582 days after transplantation. In addition to receiving PE1-1, he received injections of Recombivax® for three consecutive months (July, August, and September 1989).

Patient # 4 was a 40-year-old Arab woman with end-stage liver disease after about 10 years of active hepatitis B. In # 4. Three preoperative doses of PE1-1 administered to patients resulted in a significant decrease in HBsAg levels. Patient # 4 received 20 mg of PEl-1 on days 1 and 2 postoperatively and was HBsAg negative on day 6 after transplantation. Two months later, he received 10 mg of PE1-1 every 3-8 days. From now on, 5-26

received 10 mg of PE1-1 daily. Approximately one year after transplantation, the patient developed hepatic arterial thrombosis but remained HBsAg negative and underwent a new liver transplant. Three days later, a third transplant was required due to ischemia. Twenty days later, a fourth transplant had to be done because of an infection. The patient died 18 days after the fourth transplant (404 days after the first transplant) due to liver failure and bacterial infection. The first biopsy of the transplanted liver showed that HBsAg was negative.

Patient # 5 is a 38-year-old Caucasian male with end-stage liver disease following chronic active hepatitis. PE1-1 administered to the patient prior to surgery significantly reduced circulating HBsAg levels. Patient # 5 received 20 mg of PEl-1 on days 2 and 3 after transplantation and tested negative for HBsAg on day 3 after transplantation. During the first two months after transplantation, he received 10 mg of PE1-1 on average every 3-7 days. Later, he received 10 mg of PE1-1 every 9-26 days. The patient was HBsAg positive at day 252 post-transplant, although antigen levels were significantly lower than pre-transplant.

A 1990.

A liver biopsy in New Jersey showed that it was positive for both HBsAg and HBcAg.

Patient # 6 was a 38-year-old Caucasian male with end-stage liver disease following chronic active hepatitis B and alcoholism. This patient received the first infection due to blood transfusions. Prior to transplantation, both HBsAg and HBeAg were positive. Each dose of PE1-1 before surgery caused a decrease in the patient's HBsAg titer. Patients • · # 6 received 20 mg PEl-1 on days 1 and 2 after transplantation and were HBsAg negative on day 1 after transplantation. Two months later, patient # 6 received an average of 10 mg PE1-1 every 3-14 days. He then received 10 mg of PE1-1 as a 763-day cadaveric patient. The first HBsAg positive response was measured 251 days after transplantation and occurred after the longest interval (63 days) between the two doses of PE1-1. Although patient # 6 is currently HBsAg positive, her titer remained significantly lower than before transplantation.

Patient # 7 is a 38-year-old Caucasian woman who was injecting drugs. This patient has end-stage liver disease following chronic active hepatitis B. Prior to transplantation, the patient was positive for both HBsAg and HBeAg. Pre-transplant PE1-1 doses caused a decrease in the patient's HBsAg titer. In the first month after transplantation, Patient # 7 received 10 and 40 mg PE1-1 every 1-7 days, and was found to be HBsAgnegative on day 16 post-transplant. Thereafter, he received 10 mg of PEl-1 every 15-29 days. Histopathological evaluation of a liver biopsy performed in July 1989 indicated that it was negative for both HBsAg and HBcAg. Patient # 7 remained HBsAg negative for 464 days after transplantation.

Example 6

Reactivity with variant viruses

Monoclonal antibodies to PE1-1, ZM1-2 and L03-3 are shown in Figure 5.

The reactivity of hepatitis virus variants isolated from the patients described in Examples 1 to 5 is tested.

radioimmunoassay is performed by measuring the radioactivity bound to an antibody adsorbed to a solid phase. A solution of a 20 pg / ml monoclonal antibody in phosphate buffered saline containing 0.02% NaNg is incubated for at least 18 hours in U-bottom wells (Falcon Microtest III Flexible Assay Plates). The solution was removed from the wells and the wells were washed three times with distilled water. PBS with 2% fetal calf serum in saline was added and incubated at room temperature for serum HBsAg or kontroliokkal and 125j_ i _ve labeled antibody (approximately 4,000 cpm in 1% fetal bovine serum) overnight. The wells are then washed three times with distilled water. Each well is excited and measured. The results are shown in Table 4 below.

. Spreadsheet

Relative reactivity of the serum-derived variant HBsAg with HBsAgspecific monoclonal antibodies

Sample * ^** __________ L03-3: L03-3 * * PEl-1: ZMl-2 ZMl-2: ZMl-2

control 1000 1000 1000 # 2 Patient (234) 0,013 0,070 0.233 # 4 patients (251) 0, 007 0,024 0, 010 # 3 patients (264) 0,043 0, 173 0, 179

* Representative HBsAg-positive serum samples were obtained from patients who received liver transplantation and treatment with an anti-HBsAg therapeutic monoclonal PE1-1 antibody.

** L03-3 Represents a radioimmunoassay that is both absorbed on solid phase and labeled with radioactive isotope L03-3

Contains -27human monoclonal antibody. PE1-1: ZM1-2 refers to a radioimmunoassay containing a solid phase adsorbed human monoclonal antibody and a radioactive isotope labeled human monoclonal antibody ZM1-2. ZM1-2: ZM1-2 is a radioimmunoassay containing both a solid phase adsorbed and a radioactive isotope labeled human monoclonal antibody ZM1-2. Control HbsAg-positive serum responded well to L033, PE1-1 and ZM1-2 antibodies.

Example 7

Production of large quantities of antibodies

To initiate cellular production, one or two ampoules of frozen nitrogen are removed from frozen cells. In a 37 ° C water bath, heat rapidly until most of the ice melts, then open the vial in a vertical laminar box. The contents of the ampoule were mixed with 1 mL of Dulbecco's MEM / HAM F12 (DMEM / F12) to which 50 µM Fe ²⁺ salts were added. After mixing, the tube is filled to about 10 ml with the same medium and the cells are harvested by centrifugation. The cell pellet is resuspended in 5 ml of the above-mentioned medium containing 20% fetal calf serum and placed in a well of a 6-well culture plate. The cells were incubated at 37 ° C in a 5% carbon dioxide atmosphere. Once the cells have settled in culture and have begun to proliferate and the cell concentration is about ΙΟθ / ml, the cells and medium are transferred to an 80 cm 2 tissue culture flask and diluted to 40 ml with DMEM / F12 (no serum). When <44% cells again reached 10 µg / ml, the cells and media were transferred to a 175cC tissue culture flask and further Dilute further with DMEM / F12 to 100 ml.

When the cells reach the optimal concentration again, the cells and the medium are transferred and diluted to a final volume of 500 ml.

When the cells reach the optimum cell concentration in this rotated flask, they are transferred to three new rotated flasks, divided into three equal portions, using the same medium as previously used. This is the distribution of the rotated flasks!

continue to live until we receive enough rotated bottles to have enough cells to inoculate the Verax System 200 reactor.

The Verax System

200

The Verax System 200 Reactor is a closed cell culture system in which cells are cultured in stainless steel cross-linked type I collagen microspheres.

The microspheres are filled into a vertical, transparent glass tube through which the culture medium (same as above) is pumped in, entering the bottom of the container.

The entry into the tube is configured such that the microspheres create a floating bed configuration when the medium is pumped through the needles at a suitable rate.

During operation, fresh medium is continuously added and conditioned media is removed at a rate determined by cell growth. This parameter is maintained for glucose utilization and the oxygen / nitrogen ratio is also monitored.

. The temperature was maintained at 37 ° C; pH at 7.1

4 · ··

After loading the microspheres in medium containing 1% fetal calf serum, the reactor was run without cells for at least three days to ensure that loading of the microspheres did not infect the system. During this time, the reactor was fed with protein-free medium to reduce the starting dose of fetal calf serum. When each system functions satisfactorily, cells in the rotated vials are inoculated into the reactor.

A Verax System 2000

This device uses the same microspheres as the System 200, and controls and operates essentially the same as the smaller system. System 2000 represents an increase of approximately 15 times that of System 200.

Monitor antibody production of the final size culture

The conditioned medium is monitored by measuring the level of immunoglobulin in the supernatant by ELISA each time the collection tank is emptied. The results are confirmed by a Protein A HPLC method.

Recovery of the cell culture medium and production of the harvested medium

The conditioned medium is continuously fed from the Verax into a cooled collection tank. This medium is then transferred (using nitrogen pressure in the Verax system) to a mobile stainless steel container for further processing.

-30Cell culture medium

Routine media used are 1: 1 Dulbecco's MÉM H21 medium and Ham's F12 (Mediatech). The medium is purchased in powder form in an amount sufficient for 50 liters of medium. Two dishes containing each of these volumes of media were drained into a stainless steel container containing about 190 liters of water. The powders are stirred until completely dissolved. Add sodium bicarbonate as recommended by the manufacturer and adjust the H to 7.4. Sodium selenite was added

17.3 pg / l and then make up to 200 liters with water. The medium is further supplemented with Head ions in the form of Head Nitrate / Sodium Citrate to a final concentration of 50 pmol / L Head. The medium is added immediately to the medium in the Verax System S200 via the built-in sterilizing filter. No protein is added to the medium. No antibiotics are used.

CLEANING THE MONOCLONAL ANTIBODY

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

The monoclonal antibody is produced with a hybridoma cell line in the absence of serum. This means that the final product should only be purified from the cellular material. Since mutein monoclonal antibodies are not expected to be immunogenic per se, it is important that all potentially immunogenic components are removed.

The purification process is intended to provide a final product which is more than 99.9% pure. Affinity chromatography was used. We rely heavily on the biological specificity of affinity chromatography. Each step of the cleaning procedure (see section 5).

Table 1).

Table 5

Step

Cell extraction Concentration Micro filtering at room temperature 4 C Protein A chromatography 4 C concentration 4 C Gé1kromatográfia 4 C Ion exchange 4 C

Summary of cleaning

Conditions Materials

Polyvinylidene difluoride filters, 0.65 / 0.45 μη, absolute

Polysulfone filter, rated 30000 Dalion.

Absolute filters for polyester (0.8 μπι) and cellulose acetate (0.2 μπι)

Connected to agarose

Staphylococcus aureus

Protein A

Cellulose triacetate filter sheet, nominally

20000 Dalton

Sephacryl S-300, Ringer's lactate solution

Sephacryl S-300, Ringer's lactate solution

Extraction of cells and removal of debris from the conditioned medium

Although most of the cells are retained by microspheres, a significant number of cells remain in the supernatant recovered.

To avoid cross-contamination of the medium with the cellular components, the supernatant was filtered through a polyvinylidene difluoride filter plate (Prostack®, Millipore) immediately after removal from the Verax collection tank. This type of filter unit operates in a tangential flow mode, which allows a larger volume of solids to be filtered without clogging the filter plate. The clear medium is collected in a cooled stainless steel container.

Concentration of the conditioned medium

The conditioned medium is concentrated using a nominal 30,000 Dalton spirally wound polysulfone membrane available from Millipore.

After concentration, the pH is adjusted to 7.0 with 1 M acetic acid. The material was filtered through a 0.8 / 0.2 µιη Sartoban-PH (Sartorius) filter pad (0.8 µm component is polyester, 0.2 µιη component cellulose acetate) before 4 °

We would store it on C. The material was microfiltrated (0.22 µm,

Millipore) and then filled into polypropylene containers.

Protein A chromatography

This highly efficient purification step utilizes the high affinity of human IgG1 antibody for Staphylococcus aureus Protein A.

Protein A is purchased so that it is already covalently linked to agarose by an amide bond. After loading the gel into a column, the column, with its contents and the conductors attached thereto, was soaked in 70% ethanol for 24 hours.

-JJ> disinfect.

The column then

With PBS.

To carry out affinity chromatography,

(pH 7.0)

The following steps are performed on the Protein A column:

A) Application The concentrated conditioned medium is applied to the column using a pump. The column effluent was collected and tested for human antibody by human ELISA. Apply the solution to the column at such a rate that a measurable amount of antibody fluid is removed from the column. The fraction containing material was recovered separately and reprocessed if it contained more than 20 mg / ml of antibody.

B) Washing To remove unbound material, the column was thoroughly washed with phosphate buffered saline (pH 7) to which was added sodium chloride at a final concentration of 0.5 M. This washing is followed by a second washing step with 0.02 M sodium citrate (pH 5.6), 0.5 M sodium chloride. This wash was used to wash a small amount of human antibody from the column.

C) Elution Bound monoclonal antibodies were eluted from the column with 0.02 M sodium citrate (pH 3.0), 0.5 M sodium chloride buffer. The eluted material was diluted continuously in larger volumes of 1 M TRIS-HCl buffer (pH 8.0) to rapidly restore near neutral conditions.

Protein A purification is done in a closed system,

Waters 650 Protein Purification System.

-34 * · · · · · · · · · · · · · · · · · · · · · · · · · ·

Concentration of the Protein A column eluate

To perform the following purification steps more efficiently and conveniently, concentrate the eluate from the Protein A column to a concentration of at least 5 mg / ml antibody. The concentrate was filtered through a 0.2 µm filter sheet and the sterile concentrate stored at 4 ° C until sufficient material for the next purification step was obtained.

Size Separation by Gel Chromatography Sephacryl S-300-οη High Resolution

The antibody preparation is a high-resolution Sephacryl

It was run on an S-300 gel (Pharmacia) loaded onto a Pharmacia BP113 / 120 column having a bed volume of about 10 liters. The column was loaded onto Lactated Ringer's Irrigation USP (Travenol Laboratories). Elution from the column is monitored with a Waters 650 Protein Purification System.

The purpose of this step is not primarily purification, but rather buffer replacement. After elution of the Protein A column, the antibodies are in a complex hypertonic buffer containing sodium citrate, sodium chloride and TRIS-HCl. This buffer mixture cannot be used directly as a vehicle for an intravenous injection.

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

Even after Protein A chromatography, which removes most of the DNA present in the concentrated supernatant, and Sephacryl S-300HR chromatography, which removes DNA molecules that are significantly larger or smaller than the monoclonal antibody product, small but detectable amounts of DNA remain in the antibody formulation. To remove this impurity, an ion exchange step was performed on a strong anion exchanger, Q Sepharose (Pharmacia Inc.). At the pH of the Lactated Ringer solution, the antibody proteins are positively charged and repelled by the anion exchanger. However, nucleic acids have a negative charge at this pH η and thus bind to the column.

Fill the column as recommended by the manufacturer. After decanting the 20% ethanol solution into which the gel is delivered, 100 ml of the gel is suspended in 200 ml of Lactated Ringer's solution. The suspension was loaded onto a Pharmacia K50 / 30 column and, once a constant volume of gel had settled, was disinfected with 1 column volume of 0.5 M sodium hydroxide, followed by 3 column volumes of Dulbecco's PBS and 5 volumes of Lactate. on. The sample is then passed through the column and the effluent is collected in a sterile container.

Example 8

Molecular analysis of PE1-1, ZM1-1, ZM1-2 and MD3-4

The heavy variable (Vg) chains of the PE1-1, ZM1-1, ZM1-2 and MD3-4 antibodies were isolated and sequenced. Are each cell line 10 ”? total RNA was isolated from the cell by using the methods described by Sanz et al., J. Immunol. 142, 883 (1989)], which is incorporated herein by reference. Single-stranded DNA with AMV reverse transcriptase as enzyme and oligo-dT as primer

Synthesized with -36 molecules. The amount of single-stranded cDNA synthesized was measured by incorporation of 32p_bCTP.

Polymerase chain reactions (PCR) are performed essentially according to the manufacturer's specifications (Perkin Elmer Cetus, Norwalk, Connecticut). One microgram of DNA is added to a 200 pmol solution of each nucleotide (dATP, dCTP, dGTP and dTTP) and 100 pmol of each primer molecule and 5 units of Taq polymerase are used. PCR cycles include denaturation at 98 ° C for 3 minutes, annealing at 55 ° C for 2 minutes, and extension at 72 ° C for 3 minutes, control by DNA temperature controller (Perkin Elmer Cetus).

The amplified DNA was sized by size exclusion on a 1% low melting point agarose gel and ligated to the EcoRV cleavage site of a BLUESCRIPT phagemid vector and transformed into CaC12 competent BSJ72 bacteria. For sequencing, single-stranded DNA was isolated from each positive clone after being superinfected with M13KO7 [Sanz et al., J. Immunol. 142, 883 (1989)]. Sequencing was performed by the dideoxy chain termination method (Sanger et al., Journal of Molecular Biology 143: 161 (1980)), except that modified T7 DNA polymerase (Sequenase) was used as described by Tábor et al., Proceedings. of the National Academy of Sciences, USA 84, 4767 (1987)]. The results are shown in Tables 8-1, 8-2, 8-3 and 8-4.

8-1. Spreadsheet

The DNA sequence of the Vfj region of PE1-1 is shown below. The leader, V _H III, D, and regions are indicated by the dotted line; the CDR1 and CDR2 complementarity determining regions are indicated by an asterisk. The amino acids appear below their DNA in the form of a one-letter code.

ATG GAG I i i GGG CTG AGC • ADL £ L \ ---------------- TGG GTT TTC CTC GTT GCT CTT TTA AGA GCT GTC CAS Tot CAG GíG CAö H r G L_ s W V F L \ ! THE L L R G V Q c v the V TTT CTG G i a GAG TCT GGU GCA GGC S i G G i C CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GCA GCC TCT L V E 5 G G G V V a f ¹ G R S L R L s c The A s GGA TTC not rtü i AGG TAT GGC ATG CAC TGG GTC CGC CAG GCT CCA Gw- AAG G? \ G CTG GAG TGG G r T F  s R Y G H H W V R Q THE P G K G S ' G i G a i a OVER THE TCA TAT GA i tGA Aaa AAT AAA TGo TAT GCA GAC TCC G i G AAG GGC C = A i u u. - Λ t - - * 0 - - z · · s Π V .THE 0 V % □ G r ATC AGA GAC AAT TCC AAG AAC ACT CTG Γ ~ CTG CAA ATG CAC AGC CTG AGA GC i GCG GAC ACG T 5 R D H s K Η T L! - L the H H S L R THE A □ 7 GGT G i A TAT TAC TGT GCG AAA GAT CAA CTT TAC TTT & a i TCG CAG AGT ccc GGo CAC TAC TGG G i C G V Y Y C THE K d a f F G S Q S G H Y w r CAG GGA ACC CTG GTC ACC GTC TCC TCA Q C T L V T V s s 8-2. Spreadsheet The in the following ZM1-1 region DNS s sequence

we introduce it. The leader, VpjIII, D and regions are indicated by the dashed line; the CDR1 and CDR2 complementarity determining regions are indicated by an asterisk. The amino acids appear below their DNA in the form of a one-letter code.

<----------------------------- ------------------- LEADER -------------------- <--------- ATG GAG lii GGG Cio AGC TGu Gi ii iC CTT GTT GCT ΑΤΑ I i A GAA GGT GTC CAG TGT GAG GTG CAG hefglswyflvai.legvqceyq

-------------------------------- ν, ΙΙΙ

GiG nity TCi GGG GGA Gui iG GTA CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT VESGG GLVdPGGSLRLSCAAS

AGa TAC S Λ Y

M

A i G

CcC CAA Gv.i ACA GcA AAA Get C.G cAG i - .Λ

------------------------------ »*« ----------- siC Tl-A GCT ATT GGi Cui ACT Gc> GAC ACA TAC TAT. GuA GAC TCC GTG AAG e-C CGA TTC ACC ATC

V S A I G P T G □ T Y Y A 0 Ξ 7 <S R F T 1

TCC AGA GAA AAT GCC AAG AAC TCC i i G TAT CTT ACA ATG AAC CTG AGA G ^ v U C GAC ACG Gü. i s 'R E H The K H S L Y L T Μ H G L R The G □ T A GTG TAT TAC TGT GCA AGA DAM TTA GAA CTC T & a Got * CAG GcA ACC CTG G i C ACC GTC TCC TCA V Y Y c The R D L E L W G Q G T 1 u. V T Y 5 S 8 • 3. Spreadsheet The in the following ZM1 -2 region DNS s sequence

we introduce it. The leader, V _H III, D, and J 4 regions are indicated by the dashed line; the CDR1 and CDR2 complementarity determining regions are indicated by an asterisk. The amino acids appear below their DNA in the form of a one-letter code.

<------------------------- ----------------------- LEADER -------------------- <-------- ATG AAA CAC CTG TGG TTC TTC CTC CTG CTG GTG GCA GTT CCC AGA TGG GTC GTG TCC CAG GTG CAG HKHLWFFLLLYAYPRWVYSQYQ

--------------------------------- ν, ΙΥ -------------- ----------------------------------- CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG GCT GCG GAG ACC CTG TCC CTC ACC TGC ACT GTC TCC

LQESGPGLVKA'S ETLS LTCTYS * 'Τ **** -' --- ^--- -------------------------------- ------ CGT GGC TCC TTC AGT GAT TAC TTC TGG AAT TGG TTC CGG CAG CCC GCC & GG AAG CGC CTG GAG TGG RGSFSDYFWHWFRQPAGKRLEW

----------------------- CTT GGG CGT GTC TAT ACC AGT GGA AGT GTC GAC TAC AAC CCC TCC CTC AAG AGT CcA GTC ACC GTG

LGRVYTSGSVDYHP5LKSRYT V

TCA GTG GAC ACG TCC AAG AAG CAG TTC TCC CTG AGG CTG AGC TCT GTG ACC GTC GCG GAC ACG GCC 5VDTSKKQFSLRLSSVTYADTA

----------------------> <-----0-------> GTG TAT TAT TGT GCG AGA GGA CTG TCC GGT

VYYCARGL'SG <------------------------------------------ TTT GAC TAC TGG GGC CAG GGA GCC CTG GTC ACC GTC FDYWGQGALVTV

TCC CCA S P • ·

-398-4. Spreadsheet

The DNA sequence of the Vp region of MD3-4 is shown below. The leader, V _H III, D, and J _H 3 regions are indicated by the dashed line; the CDR1 and CDR2 complementarity determining regions are indicated by an asterisk. The amino acids appear below their DNA in the form of a one-letter code.

_{_} ___________________________ LEADER ----------------------------------------> ------- --- ATG KG TCA ACC GCC ATC CTT GGC CTC CTC CTG GCT GTT CTC CAA GGA GTC TGT KC GAA GTG CAG HGSTAILGLLLAVLaGVCAEVQ

---------------------------------- V, Y ------------- --------------------------------- CTG GTG CAA TCT GGA GCA GAG GTG AAA AAG CCC GGG GAG TCT CTG AGG ATC TCC TGT AAG GGT TCT lvqsgaeykkpgeslrtsckgs

GGA TAC AGC TTT ACC AGC TAC TGG ATC AGC TGG GTG CGC CAG ATG CCC GGG AA GGC CTG GAG TGG gysftsywismyrqhpgkglew _________ —... --------------- .------ -------- ATG KG AGG CTT GAT CCT AGT GCC TCC TCT KC ATC TTC AGC CCG TCC CTC CAA KC CAC GTC ACC hgrl dpsassaifspslqghvt

ATC TCA GTT GAC AAG TCC ATG AGG ACT KC TAC GTG CAG TGG AGA AGC CTG AAG KC TCG GAC ACC ISYOKSMRTAYVQWRSL WHO DT ___-___________________—> <-------—----------- ----- 0 ------------------------------>

GCC ATG TAT TAC TGT GCG AGA CAT GTC CGC GAA AAG AGT ATG GTT CAG GGA GTC ATT ATA AAG GAC amyycarhvrekshvqgyiíko <------------------------ J, 3 —------------------------ GCT TTT GAT ATC TGG GK CAA GGG ACA ATG GTC ACC GTC TCT TCA

AFDIWGQGTHVTVSS

Example 9

Using the procedures described in Example 8, PE1-

The light chain variable region of 1, ZM1-1, ZM1-2 and MD3-4 are isolated and determined. The results are 9-1, 9-

2, 9-3 and 9-4.

9-1. Spreadsheet

The DNA sequence of the Vp region of PE1-1 is shown below. The VV and J3 regions are indicated by the dashed line; CDR1, CDR2, and CDR3 complementarity determining regions

with an asterisk. The amino acids appear below their DNA in the form of a one-letter code.

22G AAC ACG GCC ACC CTG ACC ATC AGC AGu GTC GAA GCC GGG GAT GAG GCC GAC TAT iAC iGi C-.a

Η T A T L. i -iRVEAGDEA2YY2_

---- <* · »'------------------- j.3 ----------------- *' TG TGG GAT AGT AGT AGT GAT CAi G i G GTA i - GaC GGA GGG ACC AAG CTG ACC Gn. CTA '7 ·, · 2 S S S 2 Η V y Γ G G G 7 K! _ T V L

9-2, Table

The DNA sequence of the V _L region of ZM1-1 is shown below. The leader, VII and J5 regions are indicated by the dashed line; the CDR1, CDR2 and CDR3 complementarity determining regions are indicated by an asterisk. The amino acids appear below their DNA in the form of a one-letter code.

<------------------------------------ LEADER ------------ --------------------------------

ATG GAC ACG AGG GTC CCC GCT CAG CTC CTG GGG CTG CTA ATG CTC TGG GTC CCA GGA TCC AGT KG

MDTRVPAQLLGLLMLWYPGSSG <----------------------------------- vjl ------------ ------------------------------------

GAT GTT GTG GTG ACT CAG TCT CCA CTC TCC CTG CCC GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC

DYVVTQSPLSLPVTLGQPASIS

-------------------- "wwCDRl.» »» -------------------- TCC AGA TCi AGT C : AGC Cit. GiG GAC AGT GAC EGA AAC ACC TAC TTG AAT TGG 1 1 1 CTC CAG AGG

C R Ϊ S L S Y 0 S 0 G Η T Y L H - 1 C '

------------------------------------—--- »w» - <- wwwwww »CuR2 - --------------------------------- CCA GGC CAA TCT CA AGG CGC CiA ATT iAT CAG CTT TCT AGC COMPANY GAC TCT GGG GTC CCA GAC AGA

P G a S? R R L · I Y Q L S S P. D S G V? 0 .7 i iC AGC GGC AGT GGG ICA GGC ACT GAT TTC ACT CTG AAA ATC AGC AGG GTG GAG Gi_ 1 GAG GAT Gi 1 FSGSGSGTDFTLKISRVEAEDY

-----—---—————--- * ·· * »w -» * w *? »- * CDR3» ** »** ♦ **» «>

GGC GTT TAT TAC TK ATG CAA GG7 ACA CAC TGG CCG

GVYYCMQGTHWP

A1C ACC 1 i C ctrC CAA A CA C CA CT a GA c

ITrSQPTRLE

ATT AAA CGA I K P.

• ·

9-3. Spreadsheet

The DNA sequence of the ZM1-2 region is shown below. The leader, VI, and J regions are indicated by the dashed line; the CDR1, CDR2 and CDR3 complementarity determining regions are indicated by an asterisk. The amino acids appear below their DNA in the form of a one-letter code.

<--------------- --------------------------------- LEADER ------------------------ <----- ATG AGG CCC GTC GCT CAG C <C CG GGG CTC CTG CTG CTC TGG TTC CCA GGT TCC AGA TGC GAC ATC HRP '/ AQLLGLLLLWFPGSRCD I

CAG ATG ACC CAG TCT CCA TCT TCC GTG'TCT GCA TCT GTG GCA GAC AGA GTC ACC GTC ACT TGT CGG QHTaSPSSVSASVGORVTVTCR

---- -----—- CD R2 * »**» · »w» »· * '» ---—————— P— —--------——— ———————------- CTG ATC CAT GCT GCA TCC AGT TTG CAA AGT GGG GTC CCA TCA AGG TTC ATC GGC AGT GGA TCT GGG LZHÁA5S'_QSGYPSRrI = S = SG 2 ^ - - - - - —- - - - - —— - - - —— - - - —— - - - —— - - —— - - - - —— - - - - - - - _ - _ - - - - —W » »» »» »

ACA GAT HC ACT CTC ACC ATC ACC AGC CTG CAG GCT GAA GAT TTT GCA ACC TAC TAT TGT CAA CAG Tűr i L i x i SLQAEDFA T ϊ. YCQC * »» ··> <»» »». »--------------- j -------------------- ------ GCT GAC AGT CTC CCT TTT ACT TTC GGC GGA GGG ACC AAG GTG GAC TTC AAA CGA

ADSLPFTFGGGTKVDFKR

9-4. Spreadsheet

The DNA sequence of the V _L region of MD3-4 is shown below. Regions VIII and J3 are indicated by the dashed line; the CDR1, CDR2 and CDR3 complementarity determining regions are indicated by an asterisk. The amino acids appear below their DNA in the form of a one-letter code.

__________________________________ V.III ----------------------------------------------- CAG TCT CAG CTG ACG CAG CCT GCC TCA'GTG TCC GTG TCC CCA GGA CAG ACA GCC AGG ATC ACC TGC

QSQLTQPASVSVSPGQTASITC

TTTTTjT.w.TtTr.T.CÜ! --------— - - —- - —----- _- ----—— -

T GGA GAT AGA TTG GGG GAT GAA i i 1 GCT TCC TGG TAT CAG CAG AAG CCA GCC CAG TCC CCT A. i.

GDRLGDcrASWYQQKPGQSPC

--------------------- »» · »» »» »« «CDR2 * ♦♦ **» **** ▼ ------- --— --------—- 27C GTC ATC> ι i GAa GAi AAC AAG AGa CvC, CA G \ ra ATC CCT GAA CGA TTC TCT GGC TCC AAC> -.

(J ι / ι

GC-G AAC ACA GCC ACT CTG ACC ATC AGC GGG ACC CÁS GHTATLISG'TG

() (1)

GGG i GuC AGx. Gi. L11 A A S S L <** ♦ * »* -------------- G.3 ---------------—————>

TGG GTG TTC GGG GAA GGa ACC AAG CTG ACC GTC TTG W V F G G G T 'L T V L

Claims (15)

A human monoclonal antibody, characterized in that it neutralizes hepatitis B virus. 2. The antibody of claim 1, which is of the IgG1 type. The antibody of claim 2, wherein the hepatitis B virus neutralized by it is not of the wild type. The antibody of claim 2, wherein said antibody is selected from the group consisting of PE1-1, ZM1-1, MD3-4 and L03-3. 5. A monoclonal antibody cocktail comprising a mixture of at least two human monoclonal antibodies, each of which is capable of neutralizing hepatitis B virus, and each of said antibodies is hepatitis B virus. The cocktail of claim 5, wherein one of said monoclonal antibodies is PE1-1. A cocktail according to claim 6, characterized in that the cocktail also contains ZM1-1. 8. A cocktail according to claim 6, wherein the cocktail further comprises ZM1-2. A cocktail according to claim 6, characterized in that the cocktail also contains L03-3. The human of claim 1 Fab fragment. The monoclonal antibody of claim 10 Fab fragment, characterized in that the monoclonal antibody is selected from the group consisting of PE1-1, ZM1-1, ZM1-2 and L03-3. · · · 12. A hybridoma cell line comprising a xenogeneic immortalizing cell fused to a human cell producing a hepatitis B neutralizing antibody. 13. The cell line of claim 12, wherein the xenogeneic immortalizing cell comprises a hybridoma fused to a parent immortalizing cell and a human partner cell. 14. The cell line of claim 13, wherein the parent cell is a rodent myeloma or hybridoma. A method for producing a hybridoma according to claim 12, wherein the xenogeneic immortalizing cell line is made drug resistant, the resulting drug drug resistant immortalizing cell is fused to a human antibody-producing cell and the desired hybrid is selected. 16th Antibody, characterized by that the 12th claim of cell line we produce it. 17th Antibody, characterized by that the 13th claim of sej with a tone we produce it. 18th Antibody, characterized by that the 14th claim of sej with a tone we produce it. 19th 12 . A cell line according to claim 1, with characterized in that one PE1-1 is produced. 20th 12 . A cell line according to claim 1, with characterized in that one ZM1-1 . 21st 12 . A cell line according to claim 1, with characterized in that one ZM1-2 . 22nd 12 . A cell line according to claim 1, with characterized in that one L03-3 . • · • · · t '· * · · e · · ·· * · ··· ··· 23. A method of controlling a hepatitis B virus, comprising administering to a patient in need of such treatment an amount of a human monoclonal antibody neutralizing hepatitis B sufficient to combat hepatitis B. 24. The method of claim 23, wherein the monoclonal antibody is selected from the group consisting of PE1-1, ZM1-1, ZM1-2, MD3-4, L03-3, and mixtures thereof. 25. A method of reducing the amount of circulating hepatitis B surface antigen in a patient, comprising administering to the patient an amount of a human monoclonal antibody neutralizing hepatitis B sufficient to reduce HBsAg. 26. The method of claim 25, wherein the monoclonal antibody is selected from the group consisting of PE1-1, ZM1-1, ZM1-2, MD3-4, L03-3, and mixtures thereof. 27. A monoclonal antibody, having a region substantially similar to that of 8-1, 8-2, Refer to Tables 8-3 or 8-4. 28. A monoclonal antibody characterized in that it is Has a Vl region that is essentially similar to 9-1, 9-2, You are 9-3 Refer to Tables 9-4. 29th Cell line, characterized by that the 27th claim of antibodies produce. 30th Cell line, characterized by that the 28th claim of antibodies produce. 31st From related mammalian DNA sequences essentially rescue DNA, with that characterized by so DNS sequence • · • »· 9 • 99 9 ·· ···· · » Contains -46 which, under stringent conditions, hybridizes to a DNA strand substantially similar to that selected from the group consisting of: the) the 8-1 spreadsheet one thread; b) the 8-2 spreadsheet one thread; c) the 8-3 spreadsheet one thread; d) the 8-4 spreadsheet one thread; e) the 9-1 spreadsheet one thread; f) the 9-2 spreadsheet one thread; g) the 9-3 spreadsheet one thread; h) the 9-4 spreadsheet one strand. 32. A cell line comprising the DNA of claim 31. 33. The cocktail of claim 6, wherein the cocktail further comprises MD3-4. 34. The cell line according to claim 12, wherein the MD3-4 antibody is produced.