Monoclonal antibody binding to the surface antigen of hepatitis b virus, fab-fragment and the way to reduce circulating levels of surface antigen of hepatitis b virus in a patient

 

(57) Abstract:

The invention relates to monoclonal antibodies that are effective for the diagnosis and treatment of hepatitis C. Monoclonal antibody obtained from cell line, obtained by merging xenogenic hybridoma marked SPAZ-4, with the blood cells of a patient immunized with hepatitis b vaccine Century Monoclonal antibodies protivogerpetical actions used to protect patients with depressed immunity and for the diagnosis of hepatitis C. 3 S. and 2 C.p. f-crystals, 13 tab., 2 Il.

The invention relates to a line of hybridoma cells that produce human antibodies that neutralize the virus of hepatitis B, methods of obtaining cell lines, antibodies produced in cell lines, and use of antibodies, particularly for medicinal purposes.

Create lines of hybridoma cells for the purpose of producing monoclonal antibodies currently, in General, is a fact well known to researchers working in this field of technology. The present invention relates to the obtaining of monoclonal antibodies, highly effective, in particular, against the surface antigen of hepatitis B /HBsAg/, and antibodies to receive in accordance with generally accepted with the Anna 10 August 1983. More specifically, we discovered that the cell line of hybridoma containing parent immortaliserwow cage rodent, such as cell myeloma mouse, for example, SP-2, merged with partner cell, resulting in immortalsouls xenogeneic hybridoma cell. This xenogeneic hybridoma cell can be merged with a cell capable of producing the anti-HBsAg antibody man, resulting in a new line trionyx cells capable of generating antibody, highly effective against this antigen person. On the other hand, when the desired high stability, create a line trionyx cells, which preferably has no further ability to produce its own antibodies, and this Trimo then merge with the extension cage, capable of producing something useful against the above-mentioned antigen, and thus get even more stable hybridoma /quadroma/, which produces the antibody against the antigen.

Refer to earlier publications of the applicant to describe the receipt of xenogenic hybridoma called SPAZ4 derived from drug-resistant cell line SP-2, which may be obtained, for example, from NIGM Human Genetic Mutant Cell Repository Ref. GM35669A /see U. S. DHHS 1982 Catalog Oh the blood of conventional techniques. Get a large number of hybrid and approximately five weeks selected five clones that grow quickly and do not form antibodies. These cells are selected for resistance to 8-azaguanine and with three of these lines can be used to obtain mutants that are resistant to 20 μg/ml 8-azaguanine. At the same time, these cells are sensitive to the environment gipoksantin-aminopterin-thymidine /GAT/, which shows that they have lost their ability to produce gipoksantin-phosphoribosyltransferase. One of these lines is SPAZ4.

Cell line SPAZ4 may be fused with cells obtained from the blood of persons ionizirovannykh vaccine hepatitis B, to obtain a cell line of hybridoma that yield a positive culture using standard methods of sampling, including antibody binding to viral antigens in question. Preferably, the aforementioned positive culture was again involved in the secondary process of selection in which to obtain the antigen used different subtypes of the virus. This provides an opportunity to accurately detect the correct antigenic determinant recognized by the antibody.

Cell lines resulting from the merger to the consequently, to obtain monoclonal antibodies, are able to act effectively neutralising the virus that causes hepatitis, and these antibodies may, therefore, prevent the spread of hepatitis through, for example, a blood transfusion. They can also be used for primary protection of the newborn or exposed individuals before will be able to act the vaccine. Protivogelmintnye antibodies can be used to protect patients with depressed immunity, including patients with transplantation of tissue from repeated hepatitis. This is the most important in the case of hepatitis B - positive liver recipients. In addition, antibodies can be used in diagnostic tests.

Also discovered that fragments of antibodies such as Fab fragments, can also be contacted with the surface antigen of hepatitis B. These fragments are also part of the present invention.

Specific antibodies, which were designed in accordance with the present izobreteniem include PE1-1, ZM1-1, ZM1-2, MD3-4 and L03-3, with each of these antibodies is an antibody of class IgGi.

The cell line producing PE1-1, deposited in the American Type Culture Collection, October 16, 1986 under the index ATCC HB h ZM1-2 deposited under index ATCC HB 9192. Address American Type Culture Collection 12301 Parklawn Drive, Rockville, Maryland 20852.

Cell line of the present invention, all behave as typical hybridoma (mouse x human) people and produce the corresponding antibodies at concentrations in the range of up to 25 µg/l in the standard suspension culture.

In Fig. 1 shows the results of ELISA analysis of direct linking, comparing the binding kinetics of antibodies PE1-1 /shows a single line and antibodies ZM1-2 /double line/. Details are given in example 4A.

In Fig. 2 shows the serum levels of antibodies PE1-1 in the serum of rhesus macaques identified through various times after dosing. Details are given in example 4C.

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

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

As the antibodies of the present invention have the policy response with repeated treatment, as is the case with antibodies mouse and sheep. Thus, another aspect of the present invention is a method of treatment of hepatitis B by introducing one or more of the above-mentioned antibodies. Found that repeated doses of approximately 10 to 40 mg of antibody will significantly reduce the number of circulating HBsAg. It is found that additional doses reduce the amount of HBsAg to levels below the detection limit in antigenic analyses.

Another aspect of the present invention is a mixture of two or more monoclonal antibodies. This mixture is particularly suitable for administration to patients who are Medici type strain of hepatitis B virus, which does not bind well with the data of a single monoclonal antibody. For example, in need of assistance to a patient suffering from malignant hepatoma and chronic hepatitis B, give the antibody PE1-1 to a liver transplant and re-dose after transplantation (details are provided below in example 5). Approximately four and a half months of treatment can be determined low levels of HBsAg in the serum in the case of polyclonal antibodies, but not in the case of PE1-1. Perform DNA analysis by polymerase chain reaction /PCR/ participation is. PCR DNA clone in the bacteriophage M13 is sequenced and the resulting DNA. Analysis of clones from each of the serum samples detects dual sequence when compared with PCR DNA from the original liver and antibodies before treatment. Variant DNA encodes two different amino acids in the S-HBsAg protein and also encodes a stop codon /UAG/ viral polymerase gene. Both variants of the gene contain amino acid change that leads to the replacement of arginine by glycine in a conservative region of the peptide.

As it was shown that monoclonal antibodies PE1-1, ZM1-2, ZM1-1, MD3-4 and L03-3 bind different epitopes and found that at least one of the monoclonal antibodies is associated with each variant of the virus, tested still 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. Especially preferred is a mixture of two monoclonal antibodies, particularly a mixture of PE1-1, 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, obvious to specialist, the relative efficiency of binding of the selected antibodies, epitopes, which bind the selected antibodies, and economic considerations. Typically, the antibody will be present in the ratio of 1:99, a typical 25:75, and preferably essentially equal amounts.

Section PE1-1, ZM1-1, ZM1-2 and MD3-4 is sequenced using standard techniques. The sequence obtained for VHarea PE1-1, is given in table. 8-1 and marked the areas corresponding to CDR1, CDR2 and CDR3 /DHand JH4/. As CDR region are particularly important areas when determining the binding properties of the antibodies, the present invention includes an antibody that has an amino acid sequence of CDR1 region, which is essentially similar to the sequence of PE1-1, as shown in the table. 8-1. The present invention also includes an antibody that has an amino acid sequence region CDR2, which is essentially similar to the sequence of PE1-1, as shown in the table. 8-1. In addition, the present invention also includes an antibody that has an amino acid sequence of the CDR3 region, which is essentially similar to the region CDR3 PE1-1, as shown in the table. 8-1.

This way is sequenced and JH4/. The present invention includes an antibody that has an amino acid sequence region CDR1, which is essentially similar to the sequence ZM1-1, as shown in the table. 8-2. In addition, the present invention includes an antibody that has an amino acid sequence region CDR2, which is essentially similar to the sequence ZM1-1, as shown in the table. 8-2, and further the present invention also includes an antibody that has an amino acid sequence of the CDR3 region, which is essentially similar to the sequence ZM1-1, as shown in the table. 8-2.

DNA sequences that encode region ZM1-2 and MD3-4, are given in tables 8-3 and 8-4, respectively. The present invention also includes any antibody that has an amino acid sequence that is essentially similar to the sequences of areas ZM1-2 and MD3-4 listed in tables 8-3 and 8-4.

Determine the DNA sequences that encode the VHarea PE1-1, ZM1-1, ZM1-2 and MD3-4, and present them in tables 8-1 and 8-2, 8-3 and 8-4, respectively. These sequences or relevant fragments can be used in cloning the antibody /or modified antibodies/ or as probes.created using the techniques of cloning, which are known in the art. To obtain molecules artificial antibodies can be used with DNA from other sources, which retains the properties of the binding PE1-1, ZM1-1, ZM1-2 and MD3-4 due to the presence of regions that are 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 VLlight 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 an amino acid sequence that is essentially similar to the sequences of areas PE1-1, ZM1-1, ZM1-2 and MD3-4 specified in tables 9-1, 9-2, 9-3 and 9-4.

Also in the scope of the present invention include DNA sequences that encode the VHthe region VL-the region CDR1 region, a CDR2 region and/or CDR3-region PE1-1, ZM1-1, ZM1-2 and MD3-4. Also includes DNA that will hybridize with any of the foregoing sequences under conditions of stringent hybridization. Such DNA essentially free of other DNA mammal donor and may contain introns or can be cDNA.

In the description and the claims are definitions that are shadowsoftoronto, if their amino acid homology is at least 80%. Relative DNA "tough hybridization conditions" are conditions where hybridization is carried out at 60oC 2.5 x saline - citrate buffer /SSC/ followed by only washing at 37oC the reduced buffer concentration, which will not affect the hybridization that occurs. "Associated DNA mammal" means DNA that is present in a mammal, which is the source of VH-chain antibodies, but which is not included in the encoded antibody or antibody fragment.

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

Example 1

Obtaining cell lines antibodies

Volunteers are subjected to immunization vaccine hepatitis B. hybrid Cell lines MD3-4, ZM1-2, ZM1-1 and PE1-1 is obtained from lymphocytes of persons immunized with Heptavax/Merck & Co./. Cell line L03-3 state of the cells of individuals injected several times Heptavaxand immediately before the merger - Recombivax/Merck & Co./. Peripheral blood lymphocytes clear in the density gradient on the gradient of Percoll /Pharmacia Inc./, density 1, the first number of cells from the cell line (mouse x human) SPAZ-4. The mixture of cells is then precipitated by centrifugation at room temperature at 400g for 5 minutes. After removal of the cellular environment the residue is treated with 50% solution of PEG-1000 in a minimum maintenance medium (MEM) Dulbecco for 1 minute at 37oC, after which the environment is slightly diluted with MEM, Dulbecco. Cells are harvested by centrifugation and resuspended in the MEME of Dulbecco containing 20% fetal bovine serum. Cells were seeded at approximately 2106cells per ml in microequities tablets. The next day, add fresh medium containing components GAT-environment /environment gipoksantin-aminopterin-thymidine/ to select naslite cells SPAZ-4. On the 4th day after the merger environment replaced with fresh medium containing only GT, since all cells are sensitive to GAT-selection, during this time killed.

After 3 to 4 weeks, when the microscope see good growth hybridogenic cells, the supernatant tested for the presence of antibodies against the surface antigen of hepatitis B. Apply ELISA analysis using dilution 1/100 Heptavaxon the solid phase. After incubation with supernatants tablets process a set of biotinylated goat protivoallergicheskogo immunoglob the work of painting with phenylenediamine. Positive culture dives into new cells and some cells of clone by limiting dilution with the MEME of Dulbecco containing 20% fetal bovine serum and 107the mouse thymocytes / ml. Tablets clone check the same ELISA method as described above, and a positive culture propagation and freeze.

Example 2

Immunochemical characterization

A. Class/subclass antibodies

Determine the class of immunoglobulins antibodies PE1-1, ZM1-1, ZM1-2, MD3-4 and L03-3, using the ELISA methodology. Each antibody capture the covered antigen tablet and each analysis is performed with a specific, conjugated with peroxidase protivoallergicheskim Ig /Tago/. Each of the antibodies is clearly IgGI.

B. Type of light chain

Using ELISA methods such as those described above in A, have the antibody reagent with anti-k or anti-light chain /Tago/. Get the following results.

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

C. Isoelectric focusing /IEF/

Sample antibodies L03-3 or PE1-1 is introduced into the gel. To find that everyone behaves as the main protein.

D. Specificity

Purified HBsAg subtypes adw and ayr buy at Sc, as described Osterg et al. (1983) Hybridoma 2: 361 - 367.

PE1-1 interacts with both ayr and adw, but with subtype adw he responds a little better. L03-3 reacts essentially the same as with ayr and adw. These results are confirmed for PE1-1 and L03-3 analysis of Scatchard in solid-phase RIA with adsorbed in the solid phase ayr - adw-antigen. Thus, although these monoclonal antibodies are not explicitly associated with subtitulos determinant, on their interaction with HBsAg significantly can influence the subtype.

E. Determination of allotype

Allotype determine, using reagents supplied by the Central Laboratory of the Netherlands Red Cross Transfusion Service. Using ELISA inhibition or ELISA direct linking. The results are given table. 1. As you can see, there is no explicit restriction on high-affinity anti-HBsAg antibodies against the light chain or the allotype.

G. Affinity

The affinity for carbon in the solid phase HBsAg determined for each antibody using antibodies labeled with radioactive isotopes, essentially as described by Wands et al., (1981) Gastroenterology 80: 225 - 232, which is included in this as a reference. Antibodies mark 125I iodophenol /Pierce/. For each monoclonal body, except L03-3, and the same results. Incubation of the antibody-antigen was performed at room temperature.

The relative affinity also determined using ELISA inhibition, in which soluble HBsAg /subtype ayw/ at varying concentrations pre-incubated with a monoclonal antibody, and the mixture is then incubated at 37oC in the cell microtitration coated with HBsAg. The results are given in table. 2.

As you can see from the table. 2, the results of ELISA for PE1-1, and ZM1-2 is about two times lower than the results of the RIA, which is well within experimental error. Distribution of Scatchard results of the RIA performed for ZM1-1, indicates the possible existence of linking the centre with low affinity. Thus, it is possible that the ELISA measures the binding with low affinity, as the results of ELISA in 50 times lower than the RIA. In addition, the distribution of Scatchard also shows that there are centers ZM1-1 with significantly less high affinity, than sites with high affinity ZM1-2 or PE1-1. Although theoretically there is no desire to bind, it turns out that ZM1-1 may have the highest affinity to HBsAg of four compared antibodies, but only to HBsAg with a certain space is but it may be due to the divalent linking ZM1-1 and HBsAg, while a site with low affinity is monovalent.

Example 3

The effectiveness of PE1-1

Antibody PE1-1 check on the effectiveness of radioimmunoassay analysis AUsAB /Abbott/. Tests are carried out in relation to the immunoglobulin hepatitis B from Bureau of Biologic Reference and some of the commercial preparations of immunoglobulin hepatitis B /H-Big Immune Globin, Hep B Gammagee Immune Globinand Hyper Hep Immune Globinall purchased in the firms supplying pharmaceuticals/. Although immunoglobulin products are polyclonal, and PE1-1 is a monoclonal, data binding match the criteria of the Bureau of Biologic for comparison of immunoglobulin products, i.e., a line parallel with the level of probability less than or equal to 0.01.

Determining the effectiveness carried out as follows. Drugs compare in weight /absorption at 208 nm 1,4 presumably corresponds to 1 mg/ml/. Drugs PE1-1, which was stored at 5oC, then compare with the above polyclonal preparations, which were also stored at the 5oC. Build a graph of the logarithm of 1000 divided by µg/ml of preparatory breeding/, from the logarithm of the counts per minute /the average of three measurements/. The hypothesis that the corresponding direct are parallel and verify, using analysis of variance. Find that the lines are parallel when the level of probability less than or equal to 0.01. Direct all drugs are parallel and define a common slope. From the General slope of the calculated x-cuts and the difference in lengths used to determine differences in efficiency. According to this method monoclonal antibody PE1-1 is approximately 435 times more effective than immunoglobulin hepatitis B from the Bureau of Biologic. As discovered that commercial preparations immunoglobulin hepatitis B in two and /or less/ of times more effective than the drug Bureau of Biologic, PE1-1 is at least 200 times more effective than commercial preparations of immunoglobulin hepatitis B, relative to the weight.

Example 4

A. Kinetics of binding

Using enzyme-linked immunosorbent assay with direct linking to compare the binding kinetics of HBsAg antibodies PE1-1, ZM1-2. On titration microplates EI I put A Heptavaxwith a concentration of 1 µg/ml the Cells are then incubated at 37oC with 2% fetal calf serum in saferoom calf serum incubated the cells for various periods of time. At certain times the antibody solution is removed and the cell three times washed with fresh 2% fetal calf serum. The cell is then incubated with 2% fetal calf serum until then, until the cells for 90 minutes does not show the content of 2% fetal calf serum. The solution is then replaced by a peroxidase conjugated or goat antibody lambda chain /cell PE1-1/, or with goat antibodies to Kappa-chain /cell ZM1-2/. Quantitative determination of peroxidase conjugate associated with plastic, carried out with the addition of O-phenylenediamine and H2O2. The results are presented in Fig. 1, on which a single line corresponds to PE1-1 and double line - ZM1-2.

As can be seen from Fig. 1, at a concentration at which PE1-1 is almost fully interacts within 5 minutes, interaction ZM1-2 adsorbed on the solid phase HBsAg is not the end of 30 minutes and can last up to 90 minutes or longer. Thus, this analysis PE1-1 binds to the antigen is significantly faster. Assuming that it takes place in vivo, PE1-1 is probably more effective to neutralize virus particles before they can infect the liver.

B. Relative Polo is the first sandwich immunotest with adsorbed on the solid phase monoclonal antibody. The above-mentioned antibody have been labelled with a radioactive isotope and incubated in the wells of microtiter plate with the serum of a patient with hepatitis B. Use Fab-fragment labeled PE1-1, while labeled L03-3 is a whole IgG. The results are given in table. 3.

Monoclonal antibody ZM1-2 only approximately nine times less effective at inhibiting binding125I-PE1-1 with HBsAg than unlabeled PE1-1, while L03-3 less effective in the thousands. Thus, the epitopes ZM1-2 and PE1-1 is located on the HBsAg molecule is likely close to each other, while the epitope L03-3 is probably on the other side of the molecule. Reciprocal experiment, inhibition of PE1-1 labeled L03-3 - serves as an additional proof that PE1-1 and L03-3 contact epitopes that do not overlap.

The similarity epitopes PE1-1, ZM1-2 and their difference from L03-3 are confirmed by immunoassay with restored and alkilirovanny HBsAg. L03-3 can communicate with denatured antigen, while ZM1-2 and PE1-1 cannot be contacted. It should be noted that PE1-1, ZM1-2 have different epitopes, as their interaction with different subtypes differs.

C. Pharmokinetic PE1-1 in rhesus monkeys

Pharmokinetic PE1-1 g/ monoclonal antibodies PE1-1. Levels of PE1-1 in serum determined at various times after dosing, using ELISA-based sandwich immunotest with Heptavaxapplied to the tablets ELISA and rabbit antiidiotypic antibodies to PE1-1. The results are shown in Fig. 2.

Levels of PE1-1 in the serum of two rhesus monkeys are characterized by two slopes /t 1/2 = 1 and 1.4 days; t 1/2 = 11 and 16 days/ with a shorter half-life, probably related to the phase distribution of monoclonal antibodies. Calculate the volume of distribution at steady state /Vdss/, which is 114 - 144% of plasma volume, which suggests a weak distribution of PE1-1 to a tissue site at a monkey-free antigen.

Example 5

A. Application of PE1-1 of sympathy for the two patients in end-stage liver disease, derived from acute and chronic hepatitis B and malignant hepatoma, carrying a liver transplant

PE1-1 give two patients in end-stage liver disease who have a liver transplant. Patient No. 1 - a man aged 56 years, supporting over 20 years of chronic acute hepatitis diagnosed with malignant hepatoma. Another patient, a boy of 10 years, it is assumed that infected with hepatitis B at birth. P is camping malignant hepatoma.

These patients administered preoperative dose of PE1-1 and significantly reduce their levels of circulating HBsAg prior to the transplant procedure. Each patient receives two 20-milligramme dose PE1-1 during transfer. Postoperative dose then start typing on the next day after surgery.

Patient No. 1 never becomes negative for HBsAg, although the level of circulating HBsAg he significantly reduced relative to the level before treatment. Patient N 2 reaction becomes negative for HBsAg, which was first celebrated on the 9th day after transplantation. Patient No. 1 gets an extra dose of PE1-1 in the range of 5 to 40 mg every 2 to 20 days. Patient # 2 gets either 5-or 10-milligramme dose on average every 21 - 28 days.

Not reported adverse events in any of these two patients at a time when they get PE1-1. However, approximately four weeks after the patient No. 1 was discharged from the hospital, it was established that he has metastatic malignant tumor. He died on 139 th day after the operation. No obvious recurrent hepatitis after transplantation, despite the presence of appreciable quantities of the circus is the determined 60 days after transplantation.

On the 143-day post-transplant for the first time discovered that the patient N 2 is positive for HBsAg. The level of HBsAg briefly hesitated before established at a level much lower levels before treatment. Isolates of hepatitis B virus in this patient obtained before treatment, PE1-1 and recently examined for their ability to associate with PE1-1. Discovered that PE1-1 is able to communicate with the variant of the virus, but not as good as with wild-type virus.

Genetic analysis of the two viral isolates shows single nucleotide differences in a 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, which will reduce the binding ability of PE1-1 and the binding particle of hepatitis B.

B. Application of PE1-1 for patients with chronic, acute hepatitis B, which are subjected to liver transplant /not complicated malignant hepatoma/

The present study included five patients with positive HBsAg /but no malignant hepatoma/ and which undergo a liver transplant. Each patient in techinian two days and a maximum of 32 days after the introduction of preoperative doses of the investigational medicinal product. An additional dose of 40 mg PE1-1 is administered during surgery. All five transplants were successful.

During hospital stay in patients carefully control the titers of HBsAg, fragments of the liver and other clinical parameters. Continue regular monitoring and the introduction of PE1-1 /approximately every 1-3 weeks/ attending physician of each patient. Dosage and other parameters vary from patient to patient.

Two patients /N 5 and 6/ have results similar to the results of the patient N 2, referenced above, in that the variant virus appears after a period of receiving negative data on HBsAg. The serum of these patients remain active with PE1-1. The sequence analysis reveals the presence of single nucleotide differences between the variants from patients ' sera and wild-type virus. In each patient, there are two options. The immunoassays and sequence analysis show that the variants of each patient are different and they are also different variants from patient No. 2.

Patient # 3 - Caucasian 39 years of age, whose end-stage liver disease in the 16-year-old chronic hepatitis B. Three preoperative dose of PE1-1, which receives 20 mg PE1-1 and the first negative response to HBsAg is celebrated on the 2nd day after transplantation. Within two months the patient N 3 gets 10 mg PE1-1 on average every 1 to 7 days. Then he gets a 7.5-milligramme or 10-milligramme dose PE1-1 every 14 - 43 days. Histopathological evaluation of biopsy of the liver, carried out in February 1989, is as negative for HBsAg and HBcAg. Patient No. 3 remains with a negative response to HBsAg for 582 days after transplantation. In addition PE1-1, it also receives three consecutive monthly injections Recombivaxin July, August and September 1989

Patient # 4 - 40-year-old woman of Arab origin, which has end-stage liver disease after 10 years of chronic acute hepatitis B. Three preoperative dose of PE1-1, which receives the patient N 4, cause a significant reduction in the level of HBsAg. Patient # 4 gets 20 mg PE1-1 in the 1-St and 2-nd day after transplantation, and a negative response to HBsAg is found on the 6th day after transplantation. Within two months after that she gets 10 mg PE1-1 on average every 3 to 8 days. Then she gets 10 mg PE1-1 every 5 to 26 days. Approximately 1 year after transplantation, the patient develops hepatitis arterial thrombosis, but the response remains negative for HBsAg, and repeat transplant. Three days posi ienda died 18 days after the fourth transplant /through 404 days after first transplant/ due to liver failure and bacterial sepsis. Histopathological evaluation of the biopsy from the liver, transplanted for the first time, shows negative reaction to HBsAg.

Patient No. 5 - Caucasian 38 years of age, whose end-stage liver disease after chronic acute hepatitis B. Preoperative dose of PE1-1, enter the patient reduce his level of circulating HBsAg. Patient No. 5 receives 20 mg PE1-1) on 2nd and 3rd day after transplantation and a negative response to HBsAg is detected on day 3 after transplantation. During the first two months after transplantation, he gets 10 mg PE1-1 on average every 3 to 7 days. Later he gets 10 mg PE1-1 every 9 to 26 days. The patient has a positive response to HBsAg on the 252-day, although the level of antigen it is significantly lower than before the transplant. Histopathological evaluation of liver biopsies performed in January 1990, is as positive for HBsAg and HBcAg.

Patient N 6 - Caucasian 38 years of age, whose end-stage liver disease after chronic acute hepatitis B and alcohol abuse. This patient was initially infected by blood transfusion. Before transplanting a positive reaction on HBsAg and HBeAg. Each pre-operative dose of PE1-1 causes a decrease in the patient who is noted on day 1 after transplantation. After that within two months the patient N 6 receives 10 mg PEI-1 on average every 3 to 14 days. Later, he gets 10 mg PE1-1 every 7 to 63 days on an outpatient basis. The first positive response to HBsAg is celebrated on the 251-day after transplantation and occurs after the longest /63 days/ interval between doses of PE1-1. Although at present the patient N 6 positive HBsAg, his title remains considerably lower than before the transplant.

Patient # 7 - 38-year-old woman of Caucasian origin with IV degree of substance abuse. This patient's end-stage liver disease due to chronic sharp hepatite B. Before transplanting the patient the positive reaction of both HBsAg and HBeAg. Each pre-operative dose of PE1-1 causes a decrease of HBsAg titer in the patient. In the first month after transplantation the patient N 7 receives from 10 to 40 mg PE1-1 on average every 1 to 7 days and observed a negative response to HBsAg on the 16th day after transplantation. Then she gets 10 mg PE1-1 every 15 - 29 days. Histopathological evaluation of liver biopsies performed in July 1989, is negative for both HBsAg and HBcAg. Patient # 7 remains negative for HBsAg at 464 day after transplantation.

Example 6

Reactionist is that B, selected patients, which was discussed in example 5, is investigated. The radioimmunoassay is carried out, determining the radioactivity of the border sorbed on the solid phase antibody. A solution of monoclonal antibodies with a concentration of 20 μg/ml in phosphate buffered saline solution containing 0.02% of NaN3, incubated for at least 18 hours in cells with a U-shaped bottom /Falcon Microtest III Flexible Assay Plates/. The solution is removed from the cells and the cells are then washed three times with distilled water. Add fetal calf serum at a concentration of 2% in phosphate buffered saline solution and incubated overnight at room temperature with solutions of serum HBsAg or control samples and tagged125I antibody /approximately 4000 imp. in minutes in 1% fetal calf serum/. The cells are then washed three times with distilled water. Individual cells are excited and counting. The results are given in table. 4.

Notes to the table. 4:

*Presents positive for HBsAg serum samples taken from patients after liver transplantation and the treatment of anti-HBsAg therapeutic monoclonal antibody PE1-1. Numbers in parentheses indicate the number of days political person L03-3 adsorbed on the solid phase, and tagged with a radioactive isotope. PE1-1:ZM1-2 indicates radioimmunoassays for monoclonal antibodies person PE1-1 adsorbed on the solid phase, and tagged with a radioactive isotope monoclonal antibodies person ZM1-2, ZM1-2:ZM1-2 denotes a radioimmunoassay for monoclonal antibodies person ZM1-2 as adsorbed on the solid phase and labeled with radioactive isotope. Control samples positive for HBsAg serum interact with antibodies L03-3, PE1-1, ZM1-2.

Example 7

Large scale production of antibodies

To initiate the production cycle with cells, remove one or more vials of frozen cells from liquid nitrogen. After a quick warm in a water bath at 37oC up until almost all the ice melts, the ampoule is opened in a laminar box. The contents of capsules mixed with 1 ml of a mixture of MEM, Dulbecco and Ham's F12 /1:1 by volume)/ (DMEM/F12), to which was added iron salts 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 harvested by centrifugation. Cellular precipitate resuspended in 5 ml of the above medium with 20% fetal cow sivaram> and in an atmosphere with 5% CO2. When cells are entrenched in the culture and begin to multiply and their concentration becomes approximately 106/ml, the cells and the medium is transferred into a bottle for tissue culture with a surface area of 80 cm2and diluted to 40 ml using DMEM/F12 (without serum). When the cell concentration again reaches 106/ml, and the medium is transferred into a bottle for tissue culture with a surface area of 175 cm2and further diluted to a volume of 100 ml using DMEM/F12. When cells are again achieved optimal concentration, and the environment is transferred to a roller bottle with a surface area of 850 cm2and diluted to a final volume of 500 ml. When the roller bottle with an optimal concentration of cells, its content is divided into 3 parts and transferred into a new roller bottles using the same environment as before. This process of dividing the content of roller bottles continue until, until you get a number of bottles, enough to have the desired number of cells for sowing in the fermenter Verax System 200.

Verax System 200

The fermenter Verax System 200 is a closed system cell culture batch, in which cells are cultivated on a weighted stainless steel is crosfire load in the vertical transparent glass tube, through which pumped cultural environment /the same as mentioned above/ coming from below. The entrance to the pipe is arranged so that the microspheres will create a fluidized bed configuration, when the medium is pumped at an appropriate speed. In the process, constantly add a fresh environment and remove the air-conditioned environment with a speed determined by the growth of cells, which control the flow of glucose. Maintain the temperature of the 37oC, maintain pH 7.1 and is also regulated by the ratio of oxygen/nitrogen.

After loading of the microspheres containing 1% fetal bovine serum medium fermenter works at least for three days without cells to ensure that the loading of the microspheres is not contaminated system. At this time in the fermenter is served free of the protein environment to reduce the priming-dose fetal bovine serum. If all systems are working satisfactorily in the reactor inoculant cells from roller bottles.

Verax System 2000

This equipment use the same type of microspheres that System 200, and the methods of control and operation is essentially the same as in the smaller system. System 2000 is approximately 15 times more compared with the system 200.

The collection of cells grown in culture medium, and the formation of the collected Fund

Air-conditioned environment is continuously removed from Verax equipment to be cooled in the collection. The medium was subsequently discharged /using nitrogen pressure in the system Verax/ mobile collection stainless steel for further processing.

Control of cellular environment

Environment, which usually is a mixture of 1:1 MEM, Dulbecco H21 and Ham F12 /Mediatech/. Wednesday buy in powder form, is sufficient for the preparation of 50 l of final medium. This powder medium of two such containers is introduced into the tank of stainless steel containing approximately 190 liters of water. The powder is suspended in a rotating mixer up until it all dissolves. Add sodium bicarbonate, as recommended by the manufacturer, and establish the pH of the medium is 7.4. Add sodium Selenite to the final concentration of 17.3 ág/l and the volume was adjusted to 200 l by adding water. The environment also supply iron ions in the form of iron nitrate with sodium citrate to a final concentration of Fe+++50 μm.add protein. Never use any antibiotics.

Purification of monoclonal antibodies.

Description of the methodology of collection and purification of the final product

Monoclonal antibody receive in cell culture of cell line hybrid in the absence of serum. This means that the final product you only need to remove components of the cellular material. Since it is not expected that monoclonal human antibodies are immunogenic, it becomes very important to remove all potentially immunogenic components.

The purpose of the cleanup operations, using affinity chromatography, is the final product with a purity of more than 99.9%. It depends greatly on the biological specificity of affinity chromatography. Each stage of the treatment process /briefly shown in the table.5/ is discussed in more detail below.

The collection of cells and removal of particulate material from kondicionirovanie environment.

Even though most of the cells retained microspheres, a decent number of particles present in the collected supernatant. To avoid a large pollution cellular components, the supernatant filtered through a polyvinylidene-differeny filter, 0.65 micron, Prostackin the mode of the tangential flow, that allows you to filter large amount of material particles without clogging the filter. Clean environment is collected in stainless steel tanks with cooling.

The concentration of the air-conditioned environment

Air-conditioned environment focus, using the supplied Millipore polysulfone spiral wound membrane with nominal value of 30,000 daltons. After concentrating establish a pH of 7.9, using 1M acetic acid. The material to be sterilized filtered through a Sartobran-PH, 0,8/0,2 μm /Sartorius/ /component of 0.8 micrometer is a polyester, 0,2 µm component is a cellulose acetate/ before you put it on storage at 4oC. the Material is subjected to microfiltration /0.22 μm, Millipore/ and poured into a vessel made of polypropylene.

Chromatography with protein A

Stage extremely deep cleaning uses high affinity antibody IgGl person to protein A of Staphylococcus aureus.

Protein A purchase covalently linked by an amide bond with agarose. After stuffing gel column and its contents, and the connecting tube disinfected by treatment with 70% ethanol in water for 24 hours. The column then balance EGF, pH 7.0.

The implementation of the Hai

A/ Download. Concentrated conditioned medium is loaded into the column by a pump. Stemming from the column flow is collected and controlled for the presence of antibodies by ELISA with human immunoglobulin. Column load to such an extent that through the column were measured quantity containing the antibody of the liquid. Overloaded fraction extracted separately and return in the cycle if it contains more than 20 mg/ml antibody.

B/ Washing. To remove unbound materials, column copiously washed with phosphate buffered saline, pH 7 with sodium chloride added to a final concentration of 0.5 M. After this washing should stage a second washing step using buffer of a 0.02 M sodium citrate, pH of 5.6, containing 0.5 M sodium chloride. Such washing releases small amounts of human antibodies.

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

Purification with protein A carry BTA from the column with protein A

To make the next stage of cleaning more efficient and convenient, the eluate from the column with A protein concentrate until at least 5 mg/ml antibody. The concentrate is sterilized by filtering it through a filter of 0.2 μm, sterile concentrate stored at 4oC as long, until you collect a quantity of material sufficient for the next stage of purification.

Separation by size by gel-chromatography on sephacryl S-300 high resolution

The drug antibodies passed through the gel-sephacryl S-300 high resolution /Pharmacia/, which fill column Pharmacia BP113/120 with the volume of the layer is approximately 10 l Column is filled in Lactated Ringer's Irrigation USP /Travenol Laboratories/. Elution of the column control system Waters 650 Protein Purification Systems.

The purpose of this phase is mainly no additional purification and buffer adjustment. After elution from the column with protein A antibodies are complex hypertonic buffer consisting of sodium citrate, sodium chloride and Tris-HCl. This buffer mixture cannot be directly used as a carrier for intravenous injection. After this stage, the buffer is suitable for intravenous injection, and for longer storage demoted when the ven 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 much larger or much smaller molecules, monoclonal antibodies, drug antibodies is a small but noticeable amount of DNA. To remove this contamination is elected stage ion exchange strong anion-exchange resin - Q-sepharose /Pharmacia Inc./. At pH Laktionova of ringer's solution proteins antibodies have a positive charge and repel anion-exchange resin. Nucleic acids, however, have a negative charge at this pH, and will be contacted in the column.

The column is filled in accordance with the proposals of the manufacturers. After desantirovaniya 20% solution of ethanol gel make aktirovannye of ringer's solution, 100 ml of gel are suspended in 200 ml of solution. The mixture is then poured into the column, Pharmacia K50/30 and, when the gel itself will fit up to a constant volume, it is disinfected with 0.5 N sodium hydroxide equal to the volume of the column, then the volume equal to three column volumes, SFR Dulbecco, then the amount equal to 5 column volumes, Laktionova of ringer's solution. Directly before use, the column prom is passed to the sample and passed through a column of material is collected in a sterile container.

Example 8

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

Isolated and is sequenced heavy chain variable /VH/ antibodies PE1-1, ZM1-1, ZM1-2 and MD3-4. Total RNA extracted from 107hybridoma cells of each cell line using the techniques described in Sanz et al., 1989, J. Immunol, 142: 883, and this work is included as a reference. Single-stranded DNA synthesized using the AMV enzyme-reverse transcriptase and oligo-dT as primer. The number of synthesized single-stranded cDNA is determined by measuring inclusions32p-dCTP /deoxycytidine-5'-triphosphate/.

Polymerase chain reaction /PCR/ carry out essentially as recommended by the manufacturer /Perkin Elmer Cetus, Norwalk, Connecticut/. 1 µg of DNA added to 200 μm solution of each of dATP, dCTP, dGTP and dTTP 100 Polemi of each primer and 5 units of Taq DNA polymerase. The PCR cycles as follows: denaturing at 98oC for 3 minutes, resaturate when 55oC for 2 minutes and elongation at 72oC for 3 minutes under the control of the DNA-thermoacetica /Perkin Elmer Cetus/.

Amplified DNA are selected based on the size of 1.0% gel low-melting agarose, are ligated into the EcoRV site fiamengo vector BLUES-CRIPT and transform in CaCl2-competent bacteria M13K07, as described by Sanz et al., see above. Sequencing performed by the method of termination of detoxicate, as described by Sanger et al., 1980, J. Mol. Biol. 143:161, except iodevicereader T7 DNA polymerase /sequenase/, for which use 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, is isolated and is sequenced variable light chain (VL) antibodies PE1-1, ZM1-1, ZM1-2 and MD3-4. The results are given in table. 10, 11, 12 I.

1. Monoclonal antibody that binds to the surface antigen of hepatitis b virus and neutralizes the virus of hepatitis b, with the indicated antibody contains a variable parts of the heavy and light chains of the Mature antibody PEI-1, are given in table. 8 and 10.

2. The antibody under item 1, characterized in that it is an IgG1.

3. The antibody under item 1, characterized in that it is produced by the cell line was ATSS HB 9234.

4. Fab-fragment of monoclonal antibodies under item 1, which binds to the surface antigen of hepatitis b virus and neutralizes the hepatitis b virus, and the said antibody comprises the variable parts of the heavy and light chains of the Mature antibody PEI-1, are given in table. 8 and 10.

5. is giving the patient an effective amount of a monoclonal antibody which binds to the surface antigen of hepatitis b virus and neutralizes the virus, thereby reducing the level of circulating surface antigen, the antibody comprises the variable parts of the heavy and light chains of the Mature antibody PEI-1, are given in table. 8 and 10.

 

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