RU2146706C1 - Monoclonal antibody binding with hepatitis b virus surface antigen, fab-fragment and method of decrease of level of circulating surface antigen of hepatitis b virus antigen in patients - Google Patents

Abstract

FIELD: medicine, immunology, virology. SUBSTANCE: invention relates to monoclonal antibody showing effectiveness for diagnosis and treatment of patients with hepatitis B. Monoclonal antibodies are obtained from the cells strain prepared by fusion of xenogenic hybridoma SPAZ-4 with blood cells of patient immunized with hepatitis B vaccine. Monoclonal antibodies eliciting the antihepatitis effect are used for protection of patients showing depressed immunity and for diagnosis of hepatitis B. EFFECT: enhanced effectiveness of monoclonal antibody. 5 cl, 2 dwg, 13 tbl, 9 ex

Description

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

 The creation of hybridoma cell lines for the purpose of producing monoclonal antibodies is now, in general, a matter well known to researchers working in this technical field. The present invention relates to the production of human monoclonal antibodies, effective, in particular, against hepatitis B surface antigen B / HBsAg /, the antibodies being prepared in accordance with the generally accepted method described by the applicant in Hybridoma, 2 (4): 361 (1983), and in the application for United Kingdom Patent 2113715A, published August 10, 1983. More specifically, a hybridoma cell line containing a rodent parental immortalizing cell, such as a mouse myeloma cell, for example, SP-2 fused to a human partner cell, was found to result in tate immortalized xenogenic hybridoma cell. This xenogenous hybridoma cell can be fused to a cell capable of producing a human anti-HBsAg antibody, resulting in a new triomic cell line capable of generating a human antibody effective against such a human antigen. On the other hand, when greater stability is desired, a triomic cell line is created, which preferably further does not have the ability to produce its own antibody, and this trioma is then fused with an additional cell capable of producing something useful against said antigen, and thus an even more stable hybridoma is obtained. / quadroma /, which produces an antibody against antigen.

 Referring to the applicant's earlier publications, to describe the preparation of a xenogenic hybridoma called SPAZ4 obtained from a drug-resistant cell line SP-2, which can be obtained, for example, from the NIGM Human Genetic Mutant Cell Repository Ref. GM35669A / cm. U.S. DHHS 1982 Catalog of Cell Lines. Obtaining SPAZ4 is as follows. The SP-2 cell line is fused to normal human peripheral blood lymphocytes by conventional techniques. A large number of hybridomas are obtained, and after about five weeks, five clones are selected that grow rapidly and do not form antibodies. These cells are selected for resistance to 8-azaguanine and with three of these lines, mutants that are resistant to 20 μg / ml of 8-azaguanine can be obtained. At the same time, these cells are sensitive to the environment with hypoxanthine-aminopterin-thymidine / GAT /, which indicates that they have lost their ability to produce hypoxanthine-phosphoribosyltransferase. One such line is SPAZ4.

 The SPAZ4 cell line can be fused with cells obtained from the blood of individuals immunized with the hepatitis B vaccine to obtain hybridoma cell lines that produce positive cultures using standard selection techniques, including the binding of antibodies to the viral antigens in question. Preferably, said positive cultures are again included in the secondary selection process in which various subtypes of the virus are used to produce the antigen. This provides an opportunity to accurately pinpoint the correct antigenic determinant recognized by the antibody.

 The cell lines resulting from the fusion of a xenogenic hybridoma and a human monoclonal antibody / trioma / producing cell are therefore suitable for producing monoclonal antibodies capable of acting effectively to neutralize the hepatitis causing virus, and said antibodies can therefore prevent the spread of hepatitis through, for example, a blood transfusion. They can also be used to initially protect newborns or exposed people before the vaccine can act. Anti-hepatitis antibodies can be used to protect a patient with a suppressed immune system, including patients with a tissue transplant, from repeated hepatitis. This is most important in the case of hepatitis B - positive hepatic recipients. In addition, antibodies can be used in diagnostic tests.

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

Specific antibodies that are constructed in accordance with the present invention include PE1-1, ZM1-1, ZM1-2, MD3-4 and L03-3, each of these antibodies being an IgG _i class antibody.

 The cell line producing PE1-1 was deposited in the American Type Culture Collection on October 16, 1986 under the ATCC HB 9234 index; the cell line producing ZM1-1 was deposited under the ATCC index HB 9191 on September 4, 1986 and the cell line producing ZM1-2 was deposited under the ATCC index HB 9192. The American Type Culture Collection is 12301 Parklawn Drive, Rockville, Maryland 20852.

 The cell lines of the present invention all behave like typical hybridomas (mouse x humans) x humans and produce their corresponding antibodies at concentrations in the range of up to 25 μg / L in standard suspension culture.

 In FIG. 1 shows the results of an enzyme-linked immunosorbent assay of direct binding, comparing the kinetics of binding of antibodies PE1-1 / shown by a single line / and antibodies ZM1-2 / double line /. Details are given in Example 4A.

 In FIG. Figure 2 shows serum levels of antibody PE1-1 in the serum of rhesus macaque, determined at different times after dosing. Details are given in Example 4C.

 Monoclonal antibody and cell line PE1-1 are also described by the inventor and named by the inventor OST 577 and 64-577. Similarly, the monoclonal antibody and cell line ZM1-2 are designated as 265-695 and the monoclonal antibody and cell line L03-3 are designated 266-215.

 Antibodies and antibody fragments obtained in accordance with the invention have good specificity for hepatitis B surface antigen in in vitro ELISA binding assays.

 Since the antibodies of the present invention are of human origin, they are mainly used to treat people, since they do not cause an allergic reaction during repeated treatment, as is the case with antibodies of murine and sheep. Thus, another aspect of the present invention is a method for treating hepatitis B by administering one or more of the aforementioned antibodies. It was found that repeated doses of approximately 10 to 40 mg of the antibody will significantly reduce the amount of circulating HBsAg. Additional doses were found to reduce the amount of HBsAg to levels below the detection limit in antigenic assays.

 Another aspect of the present invention is a mixture of two or more monoclonal antibodies. Such a mixture is particularly suitable for administration to patients who carry a non-wild type of hepatitis B virus strain that does not bind well with this single monoclonal antibody. For example, a patient in need of help suffering from malignant hepatoma and chronic hepatitis B is given PE1-1 antibody before liver transplantation and repeated doses after transplantation (details are given below in Example 5). After approximately four and a half months of treatment, low serum HBsAg levels can be determined in the case of a polyclonal antibody, but not in the case of PE1-1. DNA analysis of the polymerase chain reaction (PCR) of the 230 bp HBsAg gene region corresponding to the putative monoclonal antibody binding domain is performed. PCR DNA is cloned into the bacteriophage M13 and the resulting DNA is sequenced. Analysis of clones from each of the serum samples reveals bivariate sequences when compared with PCR DNA from the original liver and antibody before treatment. Variant DNA encodes two 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 change leading to the replacement of arginine with glycine in the conserved region of the peptide.

 Since it has been shown that monoclonal antibodies PE1-1, ZM1-2, ZM1-1, MD3-4 and L03-3 bind different epitopes and it was found that at least one of the monoclonal antibodies binds to each variant of the virus tested so far enough to make it clinically useful. Another aspect of the present invention is a mixture of two or more monoclonal antibodies selected from the group consisting of PE1-1, ZM1-2, ZM1-1, MD3-4 and L03-3. Particularly preferred are mixtures of two monoclonal antibodies, especially a mixture of PE1-1 and ZM1-2 and a mixture of PE1-1 and L03-3. The ratio of monoclonal antibodies present in the mixture may vary depending on many factors that are obvious to specialists in this field of technology and including the genotype of the hepatitis virus or the virus present in the patient’s serum, the relative binding efficiency of the selected antibodies, the epitopes to which the selected antibodies bind, and economic considerations. Typically, antibodies will be present at a ratio of 1:99, typically 25:75, and preferably substantially equal amounts.

Sections PE1-1, ZM1-1, ZM1-2, and MD3-4 are sequenced using standard techniques. The sequence obtained for the V _{H region of} PE1-1 is given in table. 8-1 and regions corresponding to CDR1, CDR2 and CDR3 / D _H and J _H4 / are marked. Since CDR regions are particularly important in determining the binding properties of an antibody, the present invention includes an antibody that has the amino acid sequence of a CDR1 region that is substantially similar to the sequence of PE1-1, as shown in Table 1. 8-1. The present invention also includes an antibody that has the amino acid sequence of the CDR2 region, which is essentially similar to the sequence of PE1-1, as shown in table. 8-1. In addition, the present invention also includes an antibody that has the amino acid sequence of the CDR3 region, which is essentially similar to the CDR3 region of PE1-1, as shown in table. 8-1.

Similarly, the V _H region of ZM1-1 is sequenced, as given in Table. 8-2. Areas corresponding to its CDR1, CDR2 and CDR3 / D _H and J _H4 / are also indicated. The present invention includes an antibody that has the amino acid sequence of the region of CDR1, which is essentially similar to the sequence of ZM1-1, as shown in table. 8-2. In addition, the present invention includes an antibody that has the amino acid sequence of the CDR2 region, which is essentially similar to the sequence ZM1-1, as shown in table. 8-2, and further the present invention also includes an antibody that has the amino acid sequence of the CDR3 region, which is essentially similar to the sequence ZM1-1, as shown in table. 8-2.

 DNA sequences that encode the ZM1-2 and MD3-4 regions are given in Tables 8-3 and 8-4, respectively. The present invention also includes any antibody that has amino acid sequences that are substantially similar to the sequences of the ZM1-2 and MD3-4 regions indicated in Tables 8-3 and 8-4.

The DNA sequences that encode the V _H regions of PE1-1, ZM1-1, ZM1-2 and MD3-4 are determined and presented in Tables 8-1 and 8-2, 8-3 and 8-4, respectively. These sequences or corresponding fragments can be used in cloning antibodies / or modified antibodies / or as probes. Antibodies that are obtained by genetic engineering methods / rather than conventional selection from hybridomas / can be created using cloning techniques that are known in the art. To obtain an artificial antibody molecule, DNA can be used from other sources, which retains the binding properties of PE1-1, ZM1-1, ZM1-2 and MD3-4 due to the presence of regions similar to essentially CDR1, CDR2 and / or CDR3. Such antibodies are included in the scope of the present invention.

DNA sequences that encode the variable regions V _{L of the} light chain PE1-1, ZM1-1, ZM1-2 and MD3-4 are given in tables 9-1, 9-2, 9-3 and 9-4, respectively. The present invention also includes any antibody that has amino acid sequences that are substantially similar to the sequences of regions PE1-1, ZM1-1, ZM1-2 and MD3-4 indicated in tables 9-1, 9-2, 9-3 and 9-4.

Also included in the scope of the present invention are DNA sequences that encode the V _H region, V _L region, CDR1 regions, CDR2 regions and / or CDR3 regions PE1-1, ZM1-1, ZM1-2 and MD3-4 . Also included are DNAs that will cross with any of the above sequences under stringent hybridization conditions. Such DNAs are essentially free of other mammalian donor DNA and may contain introns or may be cDNA.

In the description and claims, definitions are used that have the following meanings. An amino acid sequence is a “substantially similar” other amino acid sequence if their amino acid homology is at least 80%. With respect to DNA, “stringent hybridization conditions” are conditions when hybridization is carried out at 60 ^° C. in 2.5 x saline - citrate buffer / SSC /, followed by only washing with a reduced concentration buffer at 37 ^° C. which will not affect hybridization that occur. "Associated mammalian DNA" means DNA that is present in a mammal, which is the source of the V _H chain of the antibody, but which is not included in the coding of the antibody or antibody fragment.

 The present invention is supplemented by the following examples, which are not limiting.

Example 1
Obtaining cell lines of antibodies
Volunteers immunized with vaccine hepatitis B. Hybridoma cell lines MD3-4, ZM1-2, ZM1-1 and PE1-1 lymphocytes obtained from individuals immunized with Heptavax ^® / Merck & Co./. The cell line L03-3 was detected from cells of individuals injected several times with Heptavax ^® , and immediately before the fusion - Recombivax ^® / Merck & Co./. Peripheral blood lymphocytes are purified in a density gradient on a Percoll gradient / Pharmacia Inc./, density 1,085 g / ml. The isolated lymphocytes are washed three times with Hanks balanced saline and mixed with an equal number of cells from the cell line (mouse x human) SPAZ-4. The cell mixture was pelleted by centrifugation at room temperature at 400 x g for 5 minutes. After removing the medium, the cell pellet is treated with a 50% PEG-1000 solution in Dulbecco's minimum supporting medium (MEM) for 1 minute at 37 ^° C, after which the medium is slightly diluted with Dulbecco MEM. Cells are harvested by centrifugation and resuspended in Dulbecco MEM containing 20% fetal bovine serum. Cells are plated - approximately 2 • 10 ⁶ cells per ml - in microcellular plates. The next day, fresh medium was added containing the components of the GAT medium / medium with hypoxanthine-aminopterin-thymidine / to select non-fused SPAZ-4 cells. On the 4th day after the fusion, the medium is replaced with fresh medium containing only HT, since all cells sensitive to GAT selection were killed during this time.

After 3-4 weeks, when good hybridoma-like cells are observed in the microscope, the supernatant is checked for the presence of antibodies against the hepatitis B surface antigen. An ELISA assay using a 1/100 dilution of Heptavax ^® on the solid phase is used. After incubation, the plates were treated with supernatants set protivochelovecheskogo biotinylated goat immunoglobulin and horseradish peroxidase mixed with avidin / Vestastain ^®, Vector Laboratories Inc./. The enzyme is determined by the staining reaction with phenylenediamine. Positive cultures dive into new cells and some of the cells are cloned by a limiting diluent with Mul Dulbecco containing 20% fetal bovine serum and 10 ⁷ mouse thymocytes per milliliter. Cloning plates are checked by the same ELISA method as described above, and positive cultures are propagated and frozen.

Example 2
Immunochemical characterization
A. Class / subclass of antibody
The class of immunoglobulins of antibodies PE1-1, ZM1-1, ZM1-2, MD3-4 and L03-3 is determined using the ELISA methodology. Each antibody is captured in a coated antigen plate and each assay is performed with a specific, peroxidase-conjugated anti-human Ig / Tago /. Each of the antibodies is clearly IgG _I.

B. Type of light chain
Using ELISA methods similar to those described above in A, an antibody with a reagent with anti-k or anti-λ light chains / Tago / is tested. The following results are obtained.

 PE1-1 - lambda, ZM1-1 - kappa, ZM1-2 - kappa, L03-3 - lambda, MD3-4 - lambda.

C. Isoelectric focusing / IEF /
The antibody sample L03-3 or PE1-1 is introduced into the gel. Everyone is found to behave like a basic protein.

D. Specificity
Purified subtypes of HBsAg adw and ayr are purchased from Scripps Laboratories, San Diego, California. The HBsAg ayw subtype is obtained from Connaught Laboratories (Willowdale, Ontario). ELISA assays are performed essentially as described by Osterg et al. (1983) Hybridoma 2: 361 - 367.

 PE1-1 interacts with both ayr and adw, but with the adw subtype it reacts somewhat better. L03-3 reacts essentially the same with both ayr and adw. These results are confirmed for PE1-1 and L03-3 by Scatchard analysis in solid phase RIA with ayr and adw antigen adsorbed in the solid phase. Thus, although these monoclonal antibodies do not explicitly bind to the subtype determinant, their interaction with HBsAg can be significantly affected by the subtype.

E. Allotype determination
Allotypes are determined using reagents supplied by the Central Laboratory of the Netherlands Red Cross Transfusion Service. An inhibition ELISA or direct binding ELISA is used. The results are given in table. 1. As can be seen, there is no explicit restriction on high affinity anti-HBsAg antibodies against the light chain or allotype.

G. Affinity
The affinity for HBsAg absorbed in the solid phase is determined for each antibody using antibodies labeled with radioactive isotopes, essentially as described in Wands et al., (1981) Gastroenterology 80: 225-232, which is incorporated herein by reference. Antibodies are labeled ¹²⁵ I with iodogen / Pierce /. For each monoclonal body, with the exception of L03-3, HBsAg absorbed in the solid phase is ayw. L03-3 is analyzed with both ayr and adw, with essentially the same results. Incubation of the antibody-antigen is carried out at room temperature.

Relative affinity is also determined using an ELISA of inhibition in which soluble HBsAg / ayw subtype / at varying concentrations is pre-incubated with monoclonal antibody and the mixture is then incubated at 37 ^° C in a HBsAg-coated microtiter cell. The results are given in table. 2.

 As can be seen from the table. 2, the ELISA results for both PE1-1 and ZM1-2 are approximately two times lower than the RIA results, which is within the experimental error. The Scatchard distribution of the RIA results performed for ZM1-1 shows that a binding center with low affinity is possible. Thus, it is possible that ELISA measures this binding site with low affinity, as ELISA results are 50 times lower than RIA. In addition, Scatchard's distribution also shows that there are ZM1-1 centers with significantly lower affinity than sites with high affinity ZM1-2 or PE1-1. Although theoretically there is no desire for binding, it turns out that ZM1-1 may have the highest affinity for HBsAg of the four antibodies being compared, but only for HBsAg with a specific spatial arrangement. This arrangement is found only in a small percentage of HBsAg molecules. It is also possible that this may be due to the divalent binding of ZM1-1 and HBsAg, while the low affinity site is monovalent.

Example 3
Efficiency PE1-1
The PE1-1 antibody is tested for efficacy by AUsAB / Abbott / radioimmunoassay. The tests are carried out with respect to hepatitis B immunoglobulin from the Bureau of Biologic Reference and some commercial hepatitis B / H-Big Immune Globin ^® immunoglobulin preparations, Hep B Gammagee Immune Globin ^® and Hyper Hep Immune Globin ^® , all procured from pharmaceutical companies / . Despite the fact that immunoglobulin preparations are polyclonal and PE1-1 is monoclonal, the binding data meet the Bureau of Biologic criteria for comparing immunoglobulin preparations, i.e. the lines are parallel at a probability level less than or equal to 0.01.

Determination of effectiveness is as follows. Drugs are compared by weight / absorbance at 208 nm 1.4, presumably corresponding to 1 mg / ml /. PE1-1 preparations, which were stored at 5 ^° C, are then compared with the above polyclonal preparations, which were also stored at 5 ^° C. A logarithm of 1000 divided by μg / ml in the preparations is prepared. the logarithm of a number that is inversely proportional to the concentration of immunoglobulin, like the logarithm of the dilution factor /, from the logarithm of counts per minute / average of three measurements /. The hypothesis that the corresponding lines are parallel is checked using analysis of variance. The straight lines are found to be parallel at a probability level less than or equal to 0.01. The lines for all drugs are parallel and determine the overall slope. The x-segments are calculated from the total slope and the difference in the segments is used to determine the difference in efficiency. By this technique, the PE1-1 monoclonal antibody is approximately 435 times more effective than the Bureau of Biologic hepatitis B immunoglobulin. Since it has been found that commercial hepatitis B immunoglobulin preparations are two / or less / times more effective than Bureau of Biologic, PE1-1 is at least 200 times more effective than commercial hepatitis B immunoglobulin preparations relative to weight.

Example 4
A. Binding Kinetics
Direct binding enzyme-linked immunosorbent assay is used to compare the binding kinetics of HBsAg with antibodies PE1-1 and ZM1-2. On microtiter EI IA Heptavax ^® applied at 1 ug / ml. The cells are then incubated at 37 ^° C. with 2% fetal calf serum in phosphate buffered saline. Monoclonal antibody PE1-1 or ZM1-2 at a content of 0.5 μg / ml in 2% fetal calf serum is incubated in cells for a different period of time. At a specific time, the antibody solution is removed and the cell washed three times with fresh 2% fetal calf serum. The cell is then incubated with 2% fetal calf serum until the cells for 90 minutes show 2% fetal calf serum. The solution is then replaced with peroxidase conjugated with either goat anti-lambda chain antibodies / cells with PE1-1 / or goat anti-kappa chain antibodies / cells with ZM1-2 /. Quantification of the plastic bound peroxidase conjugate is carried out with the addition of O-phenylenediamine and H ₂ O ₂ . The results are shown in FIG. 1, in which a single line corresponds to PE1-1 and a double line corresponds to ZM1-2.

 As can be seen from FIG. 1, at a concentration at which PE1-1 almost completely interacts within 5 minutes, the interaction of ZM1-2 with HBsAg adsorbed on the solid phase does not end in 30 minutes and can last up to 90 minutes or longer. Thus, in such an analysis, PE1-1 binds to the antigen much faster. Assuming that this also occurs in vivo, PE1-1 is probably more effective in neutralizing virus particles before they can infect the liver.

B. Relative position of epitopes
The relative position of the epitopes of antibodies PE1-1 L03-3 and ZM1-2 is determined. A simultaneous sandwich immunoassay with a monoclonal antibody adsorbed on a solid phase is used. The antibody is labeled with a radioactive isotope and incubated in a well of a hepatitis B serum microtiter plate. A Fab fragment of labeled PE1-1 is used, while labeled L03-3 is whole IgG. The results are given in table. 3.

The ZM1-2 monoclonal antibody is only about nine times less effective in inhibiting the binding of ¹²⁵ I-PE1-1 to HBsAg than unlabeled PE1-1, while L03-3 is less than thousands of times more effective. Thus, the ZM1-2 and PE1-1 epitopes are probably close to one another on the HBsAg molecule, while the L03-3 epitope is probably on the other side of the molecule. A reciprocal experiment — inhibition of PE1-1 labeled L03-3 — provides further evidence that PE1-1 and L03-3 bind to epitopes that do not overlap.

 The similarity of the PE1-1 and ZM1-2 epitopes and their difference from L03-3 are confirmed by immunoassay with reduced and alkylated HBsAg. L03-3 can bind to the denatured antigen, while ZM1-2 and PE1-1 cannot bind. It should be noted that PE1-1 and ZM1-2 have different epitopes, since their interaction with different subtypes diverges.

C. Pharmacokinetics of PE1-1 in rhesus monkeys
The pharmacokinetics of PE1-1 are studied on two rhesus monkeys. Each animal receives a single intravenous injection of a loading dose of 0.5 mg / kg / monoclonal antibody PE1-1. Serum levels of PE1-1 determined at various times after dosing, using ELISA based sandwich immunotest with Heptavax ^®, coated on ELISA plates and rabbit anti-idiotypic antibodies to PE1-1. The results are shown in FIG. 2.

 Serum levels of PE1-1 in two rhesus monkeys are characterized by two slopes / t 1/2 α = 1 and 1.4 days; t 1/2 β = 11 and 16 days / with a shorter half-life, probably associated with the distribution phase of the monoclonal antibody. The volume of distribution in steady state (Vdss), which is 114 - 144% of the plasma volume, is calculated, which suggests the weak distribution of PE1-1 to the tissue site in a monkey free of antigen.

Example 5
A. The use of PE1-1 in sympathy with two patients in the final stage of liver disease derived from acute chronic hepatitis B and malignant hepatoma undergoing liver transplantation
PE1-1 is given to two patients in the final stage of liver disease who are undergoing liver transplantation. Patient No. 1 is a man aged 56 years, suffering from chronic acute hepatitis for 20 years, with a diagnosis of malignant hepatoma. Another patient, a 10-year-old boy, is believed to be infected with hepatitis B at birth. Patient N 2 initially had a large mass of the right lobe of the liver, which, as the biopsy confirms, is a malignant hepatoma.

 Preoperative doses of PE1-1 are administered to these patients and their circulating HBsAg levels are significantly reduced prior to the transplant procedure. Each patient also receives two 20 milligram doses of PE1-1 during transplantation. Postoperative doses are then started on the day after surgery.

 Patient N 1 never gets a negative reaction to HBsAg, although his level of circulating HBsAg significantly decreases relative to the level before treatment. In patient No. 2, the response to HBsAg becomes negative, which is first observed on the 9th day after transplantation. Patient No. 1 receives additional doses of PE1-1 in the range of 5 to 40 mg with an interval of 2 to 20 days. Patient No. 2 receives either 5- or 10-milligram doses on average every 21 to 28 days.

 An adverse event is not reported in any of these two patients at the time they receive PE1-1. However, approximately four weeks after patient No. 1 was discharged from the hospital, it was established that he had a metastatic malignant tumor. He died on the 139th day after surgery. There was no apparent recurrent hepatitis after the transplant, despite the presence of a marked amount of circulating HBsAg. Although the DNA test for hepatitis B virus before surgery is negative, one positive value is determined 60 days after transplantation.

 On day 143 after the transplant, it was first discovered that patient N 2 was positive for HBsAg. HBsAg levels fluctuated for a short time before settling at a level significantly lower than levels before treatment. This patient’s hepatitis B virus isolates obtained before treatment, PE1-1 and more recently, are analyzed for their ability to bind to PE1-1. It was found that PE1-1 is able to bind to a variant of the virus, but not as well as with the wild-type virus.

 Genetic analysis of two viral isolates shows single nucleotide differences in the highly conserved region of the main surface protein of the virus. Such differences when compared with the virus before treatment can potentially encode a single amino acid difference that will reduce the binding ability of PE1-1 to the hepatitis B virus binding particle.

B. Use of PE1-1 for patients with chronic acute hepatitis B who undergo a liver transplant / uncomplicated malignant hepatoma /
This study includes five patients who tested positive for HBsAg (but did not have a malignant hepatoma) and who undergo a liver transplant. Three daily doses of 10, 20 and 40 mg, respectively, are administered to each patient within three days before surgery. A liver transplant is performed at least two days and a maximum of 32 days after the administration of preoperative doses of the study drug. An additional dose of 40 mg PE1-1 is administered during surgery. All five transplants ended successfully.

 During hospitalization, patients closely monitor HBsAg titers, liver fragments, and other clinical parameters. Regular observation and administration of PE1-1 / approximately every 1-3 weeks / by the attending physician of each patient continues. Dosage and other parameters vary from patient to patient.

 Two patients (Nos. 5 and 6) have similar results to those of Patient No. 2 mentioned above in that the variant virus appears after a period of negative HBsAg data. The sera of these patients remain active with PE1-1. Sequence analysis reveals the presence of individual nucleotide differences between variants from patient sera and wild-type virus. Each patient has two options. Immunoassays and sequence analysis show that the options for each patient are different and they also differ from the varieties of patient N 2.

Patient No. 3 is a 39-year-old Caucasian patient with a terminal stage of liver disease as a result of a 16-year-old chronic hepatitis B. Three preoperative doses of PE1-1 administered to patient No. 3 cause a significant decrease in his HBsAg titer. On the 2nd and 3rd day after the transplant, he receives 20 mg of PE1-1 and for the first time a negative reaction to HBsAg is noted on the 2nd day after the transplant. Within two months, patient N 3 receives 10 mg of PE1-1 on average every 1 to 7 days. He then receives 7.5 milligrams or 10 milligram doses of PE1-1 every 14 to 43 days. A histopathological evaluation of a liver biopsy performed in February 1989 is negative for both HBsAg and HBcAg. Patient No. 3 remains negative for HBsAg for 582 days after transplantation. In addition to PE1-1, he also receives three consecutive monthly injections of Recombivax ^® in July, August and September 1989.

 Patient No. 4 is a 40-year-old woman of Arab origin in whom the terminal stage of liver disease after 10 years of chronic acute hepatitis B. Three preoperative doses of PE1-1 given to patient No. 4 cause a significant decrease in HBsAg. Patient No. 4 receives 20 mg of PE1-1 on the 1st and 2nd day after the transplant, and a negative reaction to HBsAg is detected on the 6th day after the transplant. Within two months after this, she receives 10 mg of PE1-1 on average every 3 to 8 days. She then receives 10 mg of PE1-1 every 5 to 26 days. About 1 year after the transplant, the patient develops hepatitis arterial thrombosis, but the response to HBsAg remains negative, and the transplant is repeated. Three days later, due to ischemia, a third transplant is performed. After 20 days, a fourth transplant is performed due to infection. The patient died 18 days after the fourth transplant / 404 days after the first transplant / due to liver failure and bacterial sepsis. A histopathological evaluation of a biopsy from a liver transplanted for the first time shows a negative reaction to HBsAg.

 Patient No. 5 is a Caucasian 38 years old with a terminal stage of liver disease after chronic acute hepatitis B. Preoperative doses of PE1-1 administered to the patient significantly reduce his level of circulating HBsAg. Patient No. 5 receives 20 mg of PE1-1 on the 2nd and 3rd day after transplantation and a negative reaction to HBsAg is detected on the 3rd day after transplantation. During the first two months after transplantation, he receives 10 mg of PE1-1 on average every 3 to 7 days. He later receives 10 mg of PE1-1 every 9 to 26 days. The patient has a positive reaction to HBsAg on day 252, although his antigen levels are significantly lower than before transplantation. A histopathological evaluation of a liver biopsy performed in January 1990 was positive for both HBsAg and HBcAg.

 Patient N 6 is a Caucasian 38 years old with a terminal stage of liver disease after chronic acute hepatitis B and alcohol abuse. This patient initially contracted a blood transfusion. Prior to transplantation, he has a positive reaction to both HBsAg and HBeAg. Each preoperative dose of PE1-1 causes a decrease in HBsAg titer in the patient. Patient No. 6 receives 20 mg of PE1-1 on the 1st and 2nd day after transplantation and a negative reaction to HBsAg is noted on the 1st day after transplantation. After this, for two months, patient No. 6 receives 10 mg of PEI-1 on average every 3 to 14 days. Subsequently, he receives 10 mg of PE1-1 every 7 to 63 days on an outpatient basis. The first positive reaction to HBsAg is observed on the 251st day after transplantation and takes place after the longest / 63 day / break between doses of PE1-1. Although Patient N 6 currently has a positive response to HBsAg, its titer remains significantly lower than before transplantation.

 Patient No. 7 is a 38-year-old woman of Caucasian descent with an IV degree of substance abuse. This patient has a terminal stage of liver disease due to chronic acute hepatitis B. Prior to transplantation, the patient has a positive reaction to both HBsAg and HBeAg. Each preoperative dose of PE1-1 causes a decrease in the titer of HBsAg in the patient. In the first month after transplantation, patient N 7 receives from 10 to 40 mg of PE1-1 on average every 1 to 7 days and a negative reaction to HBsAg is noted on the 16th day after transplantation. She then receives 10 mg of PE1-1 every 15 to 29 days. A histopathological evaluation of a liver biopsy performed in July 1989 is negative for both HBsAg and HBcAg. Patient N 7 remains negative for HBsAg on day 464 after transplantation.

Example 6
Variant Reactivity
The reactivity of monoclonal antibodies PE1-1, ZM1-2 and L03-3 with various hepatitis B viruses isolated from the patients described in Example 5 is being investigated. Radioimmunoassay is carried out by determining the radioactivity of the boundary of the antibody absorbed on the solid phase. A solution of monoclonal antibody with a concentration of 20 μg / ml in phosphate-buffered saline containing 0.02% NaN ₃ , incubated for at least 18 hours in cells with a U-shaped bottom / Falcon Micro Test III Flexible Assay Plates /. The solution was removed from the cells and the cells were then washed three times with distilled water. Fetal calf serum was added at a concentration of 2% in phosphate buffered saline and incubated overnight at room temperature with solutions of serum HBsAg or control samples and labeled with ¹²⁵ I antibody / approximately 4000 imp. in minutes in 1% fetal calf serum. The cells are then washed three times with distilled water. Individual cells are energized and counted. The results are given in table. 4.

Notes to the table. 4:
^* Presented serum HBsAg positive samples were taken from patients after liver transplantation and treatment with anti-HBsAg monoclonal antibody PE1-1. The numbers in parentheses indicate the number of days after transplantation.

^** L03-3: L03-3 denotes a radioimmunoassay that is performed for human monoclonal antibody L03-3 both adsorbed on the solid phase and labeled with a radioactive isotope. PE1-1: ZM1-2 denotes a radio immunoassay for a monoclonal human antibody PE1-1 adsorbed on a solid phase and labeled with a radioactive isotope of a human monoclonal antibody ZM1-2, ZM1-2: ZM1-2 denotes a radioimmunoassay for a monoclonal human antibody ZM1-2 as adsorbed on a solid phase, and labeled with a radioactive isotope. Control samples positive for HBsAg serum interact well with antibodies L03-3, PE1-1 and ZM1-2.

Example 7
Large-scale antibody production
To initiate a production cycle with cells, one or more ampoules of frozen cells are removed from liquid nitrogen. After rapid heating in a water bath at 37 ^o C until almost all the ice has melted, the ampoule is opened in a laminar box. The contents of the ampoule are mixed with 1 ml of a mixture of MEM Dulbecco and Ham's F12 / 1: 1 by volume / (DMEM / F12), to which iron salts are added to a final concentration of Fe ⁺⁺⁺ 50 μM. After mixing, the tube is filled to approximately 10 ml with the same medium and the cells are collected by centrifugation. The cell pellet is resuspended in 5 ml of the above medium with 20% fetal bovine serum and plated in 1 well of a 6-cell tissue culture plate. Cells are incubated in an incubator at 37 ^o C and in an atmosphere with 5% CO ₂ . When the cells are fixed in the culture and begin to multiply and their concentration becomes approximately 10 ⁶ / ml, the cells and the medium are transferred to a tissue culture vial with a surface area of 80 cm ² and diluted to 40 ml using DMEM / F12 (without serum). When the cell concentration reaches 10 ⁶ / ml again, they and the medium are transferred to a tissue culture vial with a surface area of 175 cm ² and further diluted to a volume of 100 ml using DMEM / F12. When the cells reach optimal concentration again, they and the medium are transferred to a roller bottle with a surface area of 850 cm ² and diluted to a final volume of 500 ml. When the optimal concentration of cells is achieved in this roller bottle, its contents are divided into 3 parts and transferred to new roller bottles using the same medium as before. This process of dividing the contents of the roller vials is continued until the number of vials sufficient to have the desired number of cells for plating in the Verax System 200 fermenter is obtained.

Verax System 200
The Verax System 200 fermenter is a closed cell cultivation system of periodic action, in which cells are cultured on stainless steel-weighted microspheres (density 1.6 g / ml), consisting of cow collagen of longitudinal transverse type I. Microspheres are loaded into a vertical transparent glass tube, through which the culture medium is pumped (the same as mentioned above), coming from below. The entrance to the pipe is designed in such a way that the microspheres will create a fluidized bed configuration when the medium is pumped at the appropriate speed. During the process, fresh medium is constantly added and the conditioned medium is removed at a rate determined by cell growth, which is controlled by glucose consumption. The temperature is maintained at 37 ^° C., the pH is maintained at 7.1, and the oxygen / nitrogen ratio is also regulated.

 After loading the microspheres containing 1% fetal bovine serum, the fermenter works for at least three days without cells to ensure that the loading of the microspheres does not contaminate the system. At this time, a protein-free medium is supplied to the fermenter to reduce the priming dose of fetal bovine serum. If all systems work satisfactorily, cells from roller bottles are inoculated into the reactor.

Verax System 2000
This equipment uses the same type of microspheres as the System 200, and in it the control and operation methods are essentially the same as in the smaller system. System 2000 is approximately 15 times larger than system 200.

Control of antibody exit from natural culture
The conditioned medium is monitored, each time emptying the collection, to the level of human immunoglobulin in the supernatant using an ELISA assay. The results are confirmed using HPLC with protein A.

Collection of cells grown in the culture medium, and the formation of the collected fund
The conditioned medium is continuously withdrawn from the Verax equipment to a cooled collection. This medium is subsequently discharged / using the nitrogen pressure in the Verax system / into a mobile stainless steel collector for further processing.

Control cell medium
The medium that is usually used is a 1: 1 mixture of MUL Dulbecco H21 and Ham F12 / Mediatech /. The medium is purchased in the form of a powder sufficient to prepare 50 l of the final medium. Such a medium powder from two such containers is introduced into a stainless steel container containing approximately 190 liters of water. The powder is suspended with a rotating mixer until it is completely 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 the volume is adjusted to 200 L by adding water. The medium is also supplied with iron ions in the form of iron nitrate with sodium citrate to a final concentration of Fe ^{+++ of} 50 μM. The medium is immediately loaded into the medium tank in the Verax System 200 through an integrated sterilization filter. No protein is added to the medium. Never use any antibiotics.

 Monoclonal antibody purification.

Description of the methodology for the collection and purification of the final product
A monoclonal antibody is obtained in cell culture from a hybridoma cell line in the absence of serum. This means that only the components of the cellular material must be removed from the final product. Since it is not expected that human monoclonal antibodies themselves are immunogenic, it becomes very important to remove all potentially immunogenic components.

 Purification operations using affinity chromatography are aimed at the final product with a purity of more than 99.9%. It all depends on the biological specificity of affinity chromatography. Each stage of the cleaning process / briefly shown in Table 5 / is discussed in more detail below.

 Cell collection and removal of microparticle material from the conditioned medium.

Even though most of the cells are held in place by microspheres, a fair number of particles are present in the collected supernatant. To avoid contamination of large cellular components, the supernatant was filtered through a polyvinylidene diftoridny filter, 0.65 micron, Prostack ^® / Millipore / immediately after removal from the Verax collection installation. This type of filter unit operates in tangential flow mode, which allows you to filter a large amount of material with microparticles without clogging the filter. The purified medium is collected in stainless steel containers with cooling.

Air conditioning concentration
The conditioned medium is concentrated using a Millipore polysulfone spirally wound membrane of 30,000 daltons. After concentration, the pH was adjusted to 7.9 using 1M acetic acid. The material for sterilization is filtered through Sartobran-PH, 0.8 / 0.2 μm / Sartorius / / component 0.8 micrometer is a polyester, 0.2-micron component is cellulose acetate / before storage at 4 ^o C. The material is subjected to microfiltration / 0.22 μm, Millipore / and poured into vessels made of polypropylene.

Protein A Chromatography
The extremely deep purification step utilizes the high affinity of the human IgGl antibody for Staphylococcus aureus A protein.

 Protein A is purchased already linked covalently amide bond with agarose. After packing the column with gel, it and its contents and connecting tubes are disinfected by treatment with 70% ethanol in water for 24 hours. The column is then balanced by EGF, pH 7.0.

The implementation of affinity chromatographic separation on a column of protein A includes the following sequential steps
A / Download. Concentrated conditioned medium is charged to the column using a pump. The effluent from the column is collected and monitored for the presence of antibodies by ELISA with human immunoglobulin. The column is loaded to such an extent that a measurable amount of antibody-containing liquid passes through the column. The overloaded fraction is recovered separately and returned to the cycle if it contains more than 20 mg / ml of antibody.

 B / Flushing. To remove unbound materials, the column is washed extensively with phosphate-buffered saline, pH 7, with sodium chloride added to a final concentration of 0.5M. After such washing, a second washing step follows, using a buffer of 0.02 M sodium citrate, pH 5.6, containing 0.5 M sodium chloride. Such flushing releases small amounts of human antibodies.

 C / Elution. Bound monoclonal antibodies are eluted from the column using a buffer composed of 0.02 M sodium citrate, pH 3.0, containing 0.5 M sodium chloride. The eluted material is continuously diluted in a volume of 1 M Tris-HCl, pH 8.0, to quickly restore conditions close to neutral.

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

Protein A column eluate concentration
To make the next purification step more efficient and convenient, the protein A column eluate is concentrated to at least 5 mg / ml of antibody. The concentrate is sterilized by filtering it through a 0.2 μm filter, and the sterile concentrate is stored at 4 ^° C. until sufficient material is collected for the next purification step.

Size separation by gel chromatography on Sephacryl S-300 high resolution
The antibody preparation is passed through high-resolution gel Sephacryl S-300 (Pharmacia), which is filled with a Pharmacia BP113 / 120 column with a bed volume of approximately 10 L. The column is filled in Lactated Ringer's Irrigation USP / Travenol Laboratories /. Column elution is monitored by a Waters 650 Protein Purification Systems.

 The goal of this stage is mainly not additional purification, but buffering. After elution from the protein A column, the antibodies are in a complex hypertonic buffer consisting of sodium citrate, sodium chloride and Tris-HCl. Such a buffer mixture cannot be directly used as a vehicle for intravenous injection. After this step, the buffer is suitable for intravenous injection as well as for longer storage at low temperatures.

Removal of host cell DNA by passage through an ion exchange column
Even after chromatography with protein A, which removes the bulk of the DNA present in the concentrated supernatant, and after treatment with Sephacryl S-300, which removes DNA molecules that are either significantly larger or significantly smaller than the molecules of the monoclonal antibody, a small amount is present in the antibody preparation. but a noticeable amount of DNA. To remove this contamination, an ion exchange step on a strong anion exchange resin, Q-Sepharose / Pharmacia Inc./, is selected. At the pH of the lactated Ringer's solution, the antibody proteins have a positive charge and are repelled by the anion exchange resin. Nucleic acids, however, have a negative charge at this pH, and will bind in a column.

 The column is filled in accordance with the proposals of the manufacturers. After decanting with a 20% ethanol solution, the gel is added to the lactated Ringer's solution - 100 ml of the gel is suspended in 200 ml of the solution. The mixture is poured into a Pharmacia K50 / 30 column and, when the gel itself has settled to a constant volume, it is disinfected with a volume of 0.5 N sodium hydroxide equal to the column volume, then a volume equal to three column volumes, Dulbecco PBS, then a volume equal to 5 column volumes , lactated Ringer's solution. Immediately before use, the column is washed with an additional 5 column volumes of lactated Ringer's solution. Then, a sample is passed through the column and the material passed through the column is collected in a sterile container.

Example 8
Molecular Analysis of PE1-1, ZM1-1, ZM1-2 and MD3-4
The heavy variable chain (V _H ) of antibodies PE1-1, ZM1-1, ZM1-2 and MD3-4 was isolated and sequenced. Complete RNA is extracted from 10 ⁷ hybridoma cells of each cell line using the techniques described in Sanz et al., 1989, J. Immunol, 142: 883, and this work is incorporated by reference. Single-stranded DNA is synthesized using AMV reverse transcriptase and oligo-dT as a primer as an enzyme. The amount of synthesized single-stranded cDNA is determined by measuring inclusions of ³² p-dCTP / deoxycytidine-5'-triphosphate /.

Polymerase chain reactions / PCR / carried out essentially as recommended by the manufacturer / Perkin Elmer Cetus, Norwalk, Connecticut /. 1 μg of DNA is added to a 200 μm solution of each of dATP, dCTP, dGTP and dTTP with 100 pmoles of each of the primers and 5 units of Taq DNA polymerase. The PCR cycles are as follows: denaturing at 98 ^° C for 3 minutes, renaturing at 55 ^° C for 2 minutes and elongating at 72 ^° C for 3 minutes under control in a thermal cell / Perkin Elmer Cetus /.

Amplified DNA was selected by size on a 1.0% low-melting agarose gel, ligated into the EcoRV site of the phagemid vector BLUES-CRIPT and transformed into CaCl ₂ -competent bacterium BSJ 72. For sequencing, single-stranded DNA was isolated from each positive clone after superinfection with C13K07 described by Sanz et al., see above. Sequencing is performed by dideoxy chain termination as described by Sanger et al., 1980, J. Mol. Biol. 143: 161, with the exception of iodified T7 DNA polymerase / sequenase /, which uses the method described by Tabor et al., 1987, PNAS (USA) 84: 4767. The results are given in tables 6, 7, 8 and 9.

Example 9
Following the procedures of Example 8, the light variable chain (V _L ) of the PE1-1, ZM1-1, ZM1-2, and MD3-4 antibodies was isolated and sequenced. The results are given in table. 10, 11, 12 and 13.

Claims (5)

 1. A monoclonal antibody that binds to the surface antigen of hepatitis B virus and neutralizes the hepatitis B virus, said antibody containing the variable regions of the heavy and light chains of the mature PEI-1 antibody shown in Table 1. 8 and 10.  2. The antibody according to claim 1, characterized in that it is IgG1.  3. The antibody according to claim 1, characterized in that it is produced by the ATCC HB 9234 cell line.  4. The Fab fragment of the monoclonal antibody according to claim 1, which binds to the hepatitis B virus surface antigen and neutralizes the hepatitis B virus, said antibody comprising the heavy and light chain variable regions of the mature PEI-1 antibody shown in Table 1. 8 and 10.  5. A method of reducing the level of circulating surface hepatitis B virus antigen in a patient, comprising administering to the patient an effective amount of a monoclonal antibody that binds the surface antigen of hepatitis B virus and neutralizes the virus, thereby reducing the level of circulating surface antigen, the antibody comprising variable regions of the heavy and light chains mature PEI-1 antibodies are shown in table. 8 and 10.

Images