JPH04505099A - Human monoclonal antibodies against human immunodeficiency virus - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Human monoclonal antibodies against human immunodeficiency virus Background of the invention The U.S. government is supporting this research project through a research grant (AI-72658) from the National Institutes of Health. All rights reserved.

Human immunodeficiency virus (IIIV) is a contributing factor to acquired immunodeficiency syndrome (AIDS). It is considered that.

Two types so far, HrV-1 and HIV-2.

has been identified.

-II, those infected with BM will eventually die from AIDS. However, this infection fW Currently, there are approximately 15 million people in the United States alone.

There are several methods for treating patients with AIDS or +111. For example, the antiviral preparation azidothymidine (AZT) has been developed. ) has been administered short-term to patients suffering from AIDS and ARC (AIDS-related complex disease). It shows clinical and immunological disease-modifying effects, reducing mortality rate and This can contribute to reducing the frequency of infection.

This disadvantage of AZ-drug administration is always associated with the problem of significant drug toxicity. Despite this, AZT is widely used in clinical settings from an economical perspective. . To reduce this disadvantage, blood transfusion and/or It may be necessary to reduce the dose or stop continuous administration of AZT and administer it intermittently. Seem. At present, there are no known AIDS drugs that can replace AZT.

Other therapeutic methods include administering one or more lymphokines. I'm researching.

Among them, interferon (especially gamma interferon) and interleukin Kin-2 was found to be effective in treating IIIV infection. However, these Based on the results of clinical trials, this does not necessarily imply that Ptl1I is effective. , and patients treated with lymphokines often experience hypotension, nausea, and Side effects such as nausea and diarrhea were observed.

Recently, monoclonal antibodies with specificity for the Nu protein have been used for treatment. It has been proposed to be administered as a medicine to infected patients. This HI’V protein is A component of the virus particle and expressed by irv infected cells. Also, this ■Iv proteins are named p24 and gp4+ (identification of 941 and For single-layer coatings, U.S. Time Permit No. 4.725.669 (by Ella Seven, 1988) February! Published on the following day)! j! ! Shown.

Regarding the identification of p24, please refer to the scientific journal “Science (5cieIIce) J No. 228 (19115), page 1091, AIlam, J. It is described in the paper by S., et al. However, currently, there is no model for R11 infection treatment. It is difficult to utilize clonal antibodies. Because 1 (IV protein There are limited mouse and rat monoclonal antibodies available for , and the available human-derived anti-[11V monoclonal antibodies are currently unknown. That's because there aren't any.

Furthermore, when mice-derived antibodies are administered to humans, they cause a life-threatening immune response. and in humans, such antibodies target HIV proteins. There is also the problem that it does not effectively combine with quality.

human cell lines may be useful in diagnosing and/or treating patients infected with the virus. However, monoclonal antibodies of human origin, especially against Ma III It is more difficult to produce monoclonal antibodies than those derived from rats or mice. It has been proven that there is. Reasons for this include the following, for example: . In other words, the reason is: (crystal) In most cases, human lymphocytes are used as a collection source. There is synthetic peripheral blood, but this blood contains only a small number of antibody-producing cells. (b) Transformation of antibody-producing cells is performed using Epstein-Perr virus (Ell). antibodies by those transformed cells. Production must be low and unstable: (e) By fusing the EBV transformation system with mouse myeloma cells, This type of fusion can stabilize the antibody production ability as the body's production level increases. Heterohybridoma caused by human chromosome deletion leads to loss of immunoglobulin production ability. is often lost; and (d) Human lymphoblastoid lineage or heteromyeloma and normal or transformed BIII Fusion with I cells stabilizes antibody production, but until recently there was no satisfactory parent for this cell type. Few cell lines were obtained.

Purpose of the invention The first object of the present invention is to provide monoclonal antibodies against protein components of 1IIV. The object of the present invention is to provide a human lymphoblastoid cell line that produces .

The second objective of the present invention is to provide a drug with low non-specific toxicity to HIV-infected patients. Diagnostic and therapeutic agents containing human monoclonal antibodies against V protein The goal is to provide the following.

The third object of the present invention is to use HIV proteins to treat HIV infection. To provide a method for administering human monoclonal antibodies directed against protein components of That is.

A fourth object of the present invention is to provide a pharmaceutical formulation for treating HIV-infected patients. It is to be.

These and other objectives will be readily understood by those skilled in the art from the following description. Will.

Summary of the invention For the diagnosis, treatment, and prevention of human immunodeficiency virus (HIV), discovered a new monoclonal antibody. This monoclonal antibody.

Directly reacts with HIV proteins KD41 and p24 by infected cells 6 The human monoclonal antibodies of the present invention can be used in patients infected with or exposed to [lIV]. It is used as a diagnostic agent, as a direct therapeutic agent, or as a vaccine. in the form of a combination by covalent coupling with a base or a cellular preparation. indirectly as a therapeutic agent or as a specific anti-BIV agent for treatment and diagnosis or Radioactive liquid species (antibody/said combination will be referred to here as immunotoxin) It is available as. The present invention relates to HIV proteins "p41 and p24". Provides a stable human lymphoblastoid cell line that secretes human clonal antibodies against It is something to do. Also, does the present invention apply to BI? for protein, 24 and B41 The present invention provides human monoclonal antibodies.

From another aspect of the invention, the invention provides B! How to treat mammals infected with V. With what we offer.

This method involves the administration of human anti-HIV monoclonal antibodies necessary for the treatment. be.

From a further aspect of the invention, the invention! Human monoclonal antibody against 111 It consists of a pharmaceutical formulation containing an effective amount.

Detailed description of the diagram FIG. Obtained from the collected serum or a part of the human monoclonal antibody-producing cell line of the present invention This shows a radioimmunoprecipitation test in which antibodies are reacted.

Figure 2 shows a Western plot of human serum and anti-p24 cloned antibody of the present invention. This shows the analysis of the results.

Figures 3(a-r) show human strains of the present invention producing monoclonal antibodies against HIV. A study of the proliferation rate and immunoglobulin production of some lymphoblast cell lineages. It's rough.

FIG. 4 is a graph showing inhibition testing of monoclonal antibodies 126-I6 and 71-31. It's rough.

Detailed description of the invention References to publications and patents in this specification are provided where they are cited. Described.

The present invention provides monoclonal antibodies against proteins encoded by the HIV gene. A novel stable human lymphoblastoid cell line that produces! Four strains can be isolated. The resulting antibodies of the present invention can be used to prevent 1llV infection, diagnose and treat HIV-infected patients. The HrV proteins H<+ and D24 (and their This is a monoclonal antibody directed against the precursor and fragment of the protein.

gp41Fi is a membrane glycoprotein that appears on the surface of infected cells, and is responsible for the transmission of HIV. gene product (Essex, Sponsored, U.S. Pat. No. 4.725.669 (198 Published on February 16, 2013)), p24 is the core protein of the virus, and one) The human monoclonal antibodies of the invention can be used to determine whether one has been exposed to or infected with HIV. It could be used as an antibody to form a diagnostic test that is commonly used. Example 6 below demonstrates the use of antibodies of the invention in diagnostic tests. to humans When administered, the antibody confers passive immunity to [lrV-infected patients. Also, one of the invention People who have not been infected but are in a high-risk environment (e.g. Patients are at risk of being infected with IIIV by needles that are side-layered with grv4@. It can also be administered prophylactically to medical personnel.

Furthermore, the antibodies of the present invention target epitopes of HIV proteins B41 and p24. It can also be used for ping.

A particularly important use of the human monoclonal antibody of the present invention is to treat pregnant women infected with HIV. All antibodies of the present invention are immunoglobulin G (IgG) 7. It's rotaibu. Since IgG reaches the fetus from the placenta via the uterus, there is no sense of FHV. By administering it to pregnant women, there is no passive administration to the fetus, making it more effective. Treatment may be possible.

A human cloned antibody is combined with a cytotoxic agent to produce an immunotoxin (Vitex). References: Vitetta et al, 5science 2 38.1098-11114.1917), or as an anti-irradiation agent. or by attaching to the surface of toxin-containing liposomes, infected cells can be Or it becomes possible to target drugs. Here the term immunotoxin .

Conjugation of an antibody with one or at a toxin, drug, radionuclide or cytotoxic agent 6 Cytotoxic agents that can bind to the antibodies of the present invention include ricin, sifrelia These include toxins, and radionuclides.

Lysine is a very strong toxin, and soybean (Rieinms cos-snts) Typical human monoclonal antibody immunotoxin of the present invention produced from For therapeutic use, antibodies (antibodies to proteins expressed by HIV-infected cells) bind) to a toxin (e.g., ricin) that develops toxicity to [IV-infected cells]. Match. The combination of antibodies and cytotoxic agents has very low nonspecific toxicity. A human monochrome that may be highly toxic to target cells. By means of cloned antibodies, such toxic agents are directed against target cells (in this case, HIV-infected cells). ), treatment can be carried out without exposing uninfected cells to toxic agents. It would be possible.

As a general method for binding monoclonal antibodies, including those of the present invention, are described in detail in the literature by Vitetta et al. (cited above) for cytotoxic agents. . In addition, Genetic's European patent application II (No. 279 JIS, 198 @August 24, 2016) k is also listed.

The human lymphoblastoid cell line (which produces the monoclonal antibodies of the invention) is HIT - Lymphocytes obtained from seropositive patients were infected with Nibstein-Pearl virus (EmaB). invitro (in vitro) system to immortalize the lymphocytes. Therefore, it was formed.

First, blood was collected from a 5gm body that was seropositive for HIV, and then Peripheral blood mononuclear cells were collected and cultured with EVB overnight. Next, EVB infection Cells were cultured for 3 to 4 weeks. In this case, each well of the microtiter well The cells are cultured so that the number of cells is 10,000. After culturing, irv harvest Not-for-sale products (non-commercial type) and commercially available products using beads ( Commercial type) Using ELISA (enzyme-linked immunosorbent assay) The anti-HIV antibody production was examined. Each sample that tested positive by ELISA The specificity of the reaction was tested with identical beads coated with bovine serum albumin (BSA). confirmed by.

Approximately 9% of lymphoblastoid cell lineages are positive in non-commercial ELISA. It was sex. Cells in this positive well were divided into multiple wells, The cells were further cultured for 2 weeks or more. In the ELISA assay, HIV Among the proteins, 2.4% were positive. In addition, 0.67% for BSA beads It was found that it had specificity for IIIV by non-reactivity. anti-11 1V antibody production is directed against 1p41 or p24, and EL ISA, Western blot, radioimmunoprecipitation and/or immunofluorescence It was found that it showed remarkable reactivity in resence. Therefore, these things The characteristics of the cloned monoclonal antibodies are that all of the cloned antibodies in Example 5 below are It is applicable to Menoa7i for the diagnosis of IV and IIIV. Release a stable loan 10 or more per well with irradiated human lymphoblast feeder cells. After subcloning once or twice to obtain +00 cells, transfer to a tissue culture flask. I moved it to

In the second round of immortalization-selection, peripheral Blood mononuclear cells were collected and immortalized by EBY infection, and as described above. Anti-HIV antibody production was assayed. As a result, anti-gp4+ and We were able to obtain four lymphoblastoid cell lines that produced anti-p24 monoclonal antibodies. Done (+2O-1ft, 12B-6, 12fi-50, 167-7 and + 91-3 line).

In addition, one cell line (9g- 4,3).

Obtained from positive media derived from the first group of SSa bodies. This medium After several failures, the culture was subcultured twice and stabilized.

The histoplasm produced from the lymphoblastoid cell line of the present invention is a very important discovery. Animal retroreferences are listed in Table 3.

The present invention f provides several epitopes related to the human monoclonal antibodies of the present invention. For example, as is clear from the data shown in Example 5, Cloned antibodies 50-69 and 9to43 have the same epitope cluster (i.e. , the amino acid sequence missing between residues 579 and 613); Antibody 98-6 has a different region (amino acids missing the gap between residues 642 and 692). array). However, monoclonal antibodies 50-69 and 98-43 group specificity is different. Because 50-69 binds to peptide 599-613 However, 911-43 binds to peptide 579-GO4. three anti-92 Four antibodies were tested (e.g. 71-31, 91-5 and 9l-6), and the results Fruit.

All? -24 (+3l-198) bound to the same region.

As shown in Table 3 below, the human antibodies of the present invention against gp4I antibody (i.e. 50-69, 98-6.9143, 120-16, 128-6, 126-50, 167-7, 191-3) are antibody-dependent cells serum therapy (e.g., passive immunotherapy) Epidemiology) and seroprophylaxis are dominated by specific antibodies achieved by ADCC. and of effector cells (cytotoxic T-cells and monocytokiller cells). Increasing the ^DCC response with migration to specific target cells is B! V feeling It is thought that this may be important for anti-inflammatory serum therapy and serum prophylaxis.

Therefore, the 14 lymphoblastoid cell lines obtained are stable in the culture medium and can be used in infected patients. The aim is to produce human monoclonal antibodies that target HIV expressed in the human body.

The human monoclonal antibody of the present invention is a monoclonal antibody of all rgG hepterotypes. Bodily produced phosphorus (recovered from the supernatant of blast cell culture medium and purified by known 1.G purification methods) It is purified by As such a method.

Protein A 7) 10-Scout matograph Ren Affigel Blue (Bio-Rad, Richmond.

C) and protein A chromatography; There is chromatography (IIPLC).

The 11 stable lymphoblastoid cell lines described in Examples 1-6 below are unique Generate human monoclonal antibodies directed against the epitope. This unique epitope is It is expressed in HIV-infected patients. Therefore, some epitopes Further epitope mapping was performed (see Table 3 in Example 4 below). Tuping uncovers the exact specificity of each monoclonal antibody and develops a vaccine. We identified targets on Il+vH4+ and l+24 protein molecules that may be related to the product. Make it clear.

The human monoclonal antibodies of the present invention contain gp41 and 924 viral proteins. reacts with immunodominant (common) or nondominant epitopes of child In the specification, immunodominant refers to the fact that most S people are responsible for producing antibodies. Anti-Ill 50-69 against gp41, which refers to antigenic determinants that respond and In-18a immunodominant epitope. These two types of antibodies are It is used in dynamic immunotherapy and as a diagnostic agent. For antibodies against mountains-3 1 and 91-5 are non-dominant epitopes.

Lymphoblastoid cell lines 91-5 and 120-16 (mountain and B<t, respectively) produced human monoclonal antibodies against the American type culture (^TC C.

Rockville, MD), each with CRLI0038 td. and CRLIDD3 files.

The classification of the antibodies of the present invention as immunodominant or non-dominant is as follows. This was done based on the results of the inhibition test described in Example 6 below.

In this assay, serum collected from a body with a positive blood pressure is biotinylated. The anti-HIV protein monoclonal antibody was used to inhibit the binding of anti-HIV protein monoclonal antibodies.

For monoclonal antibodies against p24 and gp<+.

Only 141 gave antibody-dependent cellular cytotoxicity (^OCC). Ingeitoro From the results of cytotoxicity tests.

The lowest dose that showed a significant lethal effect was found to be 115-25 mg antibody/ml. It was. ^ In the DCC assay, 1. peripheral blood mononuclear cells as effectors. CEM (MIIC) and IITLV-111B infected, NIRjll cell line 0 and RF (virus strains) were used as targets.

Two types of monoclonal antibodies against gp4+ and a monoclonal antibody against Mt. It was purified and combined with the diglycosylated A chain of lysine.

Anti-p24 immunotoxin (IT) up to 20 μg Ab/■l can be applied to infected or uninfected It had no lethal effect on H9 or 0937J11 cells. however , IT of the monoclonal antibody against gp4+ is a protein of infected H911I vesicles. The synthesis was suppressed, and the 50% inhibition (IC50) concentration was 500++g/w+1.

The IC50 concentration of anti-B4++i in infected U937 cells is 100Q++g/ml Met. In the presence of chloroquine (chloroqwrIIe), these im Notoxin IC50 concentration a S-10 mg/ml "C".

When used for the treatment of patients suffering from AIDS or AIDS, the human monomer of the present invention may be used. Cloned antibody (IIV 17) 624 and specificity for p41 protein (having Ils properties and binding Ils properties) can be used as a passive immunizing agent within a wide concentration range (approximately 200 About 15. The range is preferably 50 mg to 1. can be administered within the range of Antibodies of the invention may be administered parenterally, or preferably via the intravenous route.

A typical treatment method involves administering a palliative dose of 111 doses over a period of 6 months. However, various treatment methods may be adopted depending on the patient's symptoms. The total dose required in each case of treatment may be divided into several doses or - Achieved with 1 administration. Single administration of human monoclonal antibodies according to patient symptoms or other anti-HIV treatments such as ALT. anti-HIV treatment The drug company will administer the drug once or twice a week, or more times depending on the patient's health status and condition. It could be administered in numbers.

The human monoclonal antibodies of the present invention can be used to treat HIV infection based on conventional pharmaceutical formulations. The present invention can be administered to patients with disease or those at high risk of infection. The pharmaceutical formulations of the human monochromes of the present invention are shown in Examples 1 to 4 below. Contains an effective amount of anti-[11V] consisting of approximately 200 mg to 15 g of the antibody. . When an effective dose is administered by multiple injections, the amount for each injection is It doesn't matter how much, because ultimately what matters is reaching that effective dose. It is. Additionally, in such formulations, pharmaceutically acceptable carriers, diluents, It is also possible to use salts and other known substances 0 isotonic salts, water free, 10% Maltose, Human Serum Albumin, Glycine or Ugly Pharmaceutically Acceptable Substances In pharmaceutical formulations of the human monoclonal antibodies of the present invention, diluents, carriers, etc. Or it could be used as a solvent.

Note that the present invention is not limited to the following examples.

(Hereafter, margin) Example 1 Preparation of human B-cells 58 seropositive individuals who are addicted to drugs or homosexual by intravenous injection Blood was taken from. Commercially available enzyme-linked immunosorbent ARA7I ( ELISA) (Organon-Technica Bioenzabee F [ITLV- 111EL [SA, Dahem, MC] was used to determine the amount of antibodies against HIV in the blood. Investigate the existence and further Western Plot (Nove Apass Immunoplot Atsuse) Jin Biorad.

Hercules, and Bittic DuPont, DuPont, Wilmington, Confirmed by DE). The medical condition of Kanfu is based on the literature by Zallapatzner et al. -Patner et al, Proe, 1lat, Ac1d, Set .

■S＾　84：　5404゜ Evaluated by the Immunologic staging system described in 1987). Ta.

Scale score T4/Tll ratio #T4/■-#phosphorus/1° sphere/maO> 1.0 > 5 (IQ > 15001 < 1.0 > 500 > 150 02, <1.0 (500) 1500 3　＜　1.0　＜　SOD　＜　1!100 Heparin-treated sample taken from patient record Blood was diluted with RPMr-1640, blood: RPMI-1640 was 1 :1, then add this diluted blood to Histopark (Sigma, Centrol). Peripheral blood mononuclear cells were isolated by centrifugation at 300 x 1.30 min on a The cells were collected. Restore the cell-Histopaque interface, wash three times, and After adjusting the concentration to 2 x 1 G' cells/mI, EBV transformation Mark Z cell line I3-1 (22, υ stop, ^cad lucher; Lexi request , ^TCC, aTsukwil, MD, available as CRL1812 II Rika The sample was soaked overnight in the filtered supernatant obtained from the above. Lymphocytes were prepared using 10% fetal bovine serum (Hyclo). NLab, Logan, UT), 2■ML-Glutamine, 100U/servingl Penicillium and RPkl containing 100 micrograms/ml Stref0 tomycin. l-1640 medium (M, A, Bioproducts, Walkersville, MD) After washing the surface with (complete medium).

Go to 96 large microtiter plate (= Star, Cambridge, M^), After the cells were adjusted to oooo cells/well, they were cultured in the medium for 4 weeks.

Example 2: Isolation and screening of linoblastoid cell lines for anti-HIV antibody production ng Positive after screening for anti-HIV antibody production by non-commercial ELISA The cultured strains were inoculated onto 24 large tissue culture plates (=stars) and cultured for over 2 weeks.

The pre- and post-seeding cultures were fed with complete medium every half hour. HI The medium (including supernatant) that showed specific reactivity to V was treated with radiation (gamma 30 GK-5 human lymphoblastoid cells (G1115GO mutant strain) ;5atoh, J, et al, +Il, EIIg, J, 1led , 309:217.1983) on the feeder layer and diluted 1,2 times. It was cultivated as a substitute.

Place stable lawns on feeder cells at a concentration of 10 to IaO#l cells/well. The cells were then subcultured 1 to 3 times and further transferred to flasks for proliferation.

Furthermore, an immortalized BJI cell culture line (hereinafter referred to as lymphoblastoid cell line) has been established. was established, and its characteristics were investigated through the following experiments.

Screening of strains cultivated by 96 large plates is non-commercial This was done using ELISA. Imron 2 plate (Dynatic, Cente 4 mg/sr diluted with carbonate buffer (j[I9.8) (F) [ITLV-1116 Light Seven (ElectronucleoX, I++c, Silver Springs, MD) and left overnight at 4°C. .

The plate was then washed with phosphate buffer (pH 7.2, o, oos% surfactant). (Contains Twee+20) (PBS-Tween) three times. culture Add supernatant (0.1 ml/well) and incubate at 37°C for 90 min. I turned on.

Next, the plate was washed with PBS-Tweell and treated with sheep anti-human immunoglobulin. Phosphorus alkaline phosphatase (Organon Technica Cavell, Marvuan, De The ink was carefully incubated at 31°C for 90 minutes.

Thereafter, the plate was washed with PBS-Tween M, and the substrate (p-Nitro Elf) was washed with PBS-Tween M. phosphate 7 (10% jetanolamine) was added and reacted for 30 minutes. child The reaction was stopped by adding 25 ml of IN IIaOH, Automatic ELISA reader (MR600 microplate reader, Dynatic ) and calculated the absorbance at 4.5++m.

The specificity of antibody binding was determined by using culture supernatant as a sample and using BIV-bound beads (Pioen). Zabeed) and BSA (Sigma Chemical Co, ) coated (1° 25% BS^/PBS, room temperature, 1 hour treatment) III-Je coated heat (or This was determined by examining the reactivity to Ganon Technica Kabel). [I The reactivity of the Iv coated beads was used as a criterion for specificity. The specificity of monoclonal antibodies Other methods of assay include commercially available kits ( It can be done in a western pm using Orad, Richmond, C^). Radioimmunoprecipitation (RIP: Radioi-sm++ oprsci) pitation) is also possible.　RIP Assassin 7i is Binter and Ho Nen's method CPinter & [lo+++e++, J, Immmnol -Methods, in press). 30μg HTLV-11 1. Use Lymort (obtained from Organon Technica) as Polton Hunter reagent. 115 (11 marks, unmarked) using England Nuclear, Boston, MA). What is known and what is labeled?

Separation was performed using a Biogel P-4 column (Bio-Rad). Culture supernatant 50p 1 15 x 10’ cps (F) 1111 with Lysade for 1 hour at 37°C Incubated. After that, 10% fixed stephlococcus (5tephlococcus) cocc hots awesome) (pansolvin, karpaiochem, radisho , C^) SOμl was added. The immunoprecipitate was washed three times by centrifugation and air-dried. The pellet was resuspended in buffer, boiled for 3 minutes, and washed with 0% SDS polyacrylate. Electrophoresis was performed on a lylamide gel.

After electrophoresis, the gel was transferred to X-Omat S film (:I) for 1 to 3 days. Duck, Ronchester.

IIy) exposed on top.

Anti-H! v Antibody class and L mirror type were determined by ELISA. This ara seven In step B, the microtiter plate (Immunon 2) was heated to 4 mg/ml HIT. Coated with Isade (electro-nucleonic) and mixed with culture supernatant and ink. I let him bet. The type of antibody binding to HIT is the following alkaline phosphatase goat anti-human IgG (gamma specificity), goat anti-human kappa chain and goat anti-human IgG (gamma specificity) using the anti-human lambda chain (Organon Handica Capel, Marvan, PA)) Decided. Monoclonal antibody subtype fumohuman IgG (Jimdo, Sanfranc Alkaline phosphatase-labeled mice for four subtypes of Cisco, C^) The results were analyzed using a super clonal antibody ELISA.

Quantification of immunoglobulin was also performed using ELISA. Imron 2 plate Goat anti-human 1. Coat with G (gamma specificity), culture supernatant and inkinibasi It was heavy. Bound IgG to alkaline phosphatase labeled goat human 1. G (Gun) Detected by specificity). Human IjG purified based on IN affinity Pel) was used to obtain the wIs curve.

5 Yukito AT-derived cells cultured in microtiter wells14,329 An individual culture has been established. Approximately half of them have - patients (scale score 1) was obtained by three blood draws. remaining wells was established by selecting cells from 57 J#s with scale scores of O to 3. It is something that was given. The results of this method are shown in Table 1.

(Hereafter, margin) G Table 2 A scale-up of the characteristics of cell cultures from patients at specific stages of HrV infection. number of patients, number of wells, number of positive wells, HTv-specific antibody clamp (e)/Wellsco A. Number of wells [11V seronegative patient 1: 3 63) O87,3 111V seropositive patients: 0 4 725 18 (2.4%) 6 (6.8%) □, 921 13 8. 70 180 (2%) 1i6 (0.7%) 0.8!12 20 2.792 54 (1.9%) +6 (0.5%) (1,653 companies 2.023 88 ( 4.3%) 9 (0.4%) Law 58 14.329 340 (2.4%) 9 7 (0,67%>0.66(1): those having a+v-specific reactivity, Including those with non-specific reactivity.

The specificity of anti-HIV antibodies was demonstrated by comparing 1IfV-coated beads and 8SA-coated beads. , = determined by Marshall ELfS^.

(2): B cells transformed by EBV come into contact with each other and form a class. form a tar. The number of clusters was determined by microscopic observation.

The results in Table 2 show that cultured cells derived from patients with a scale score of 3 have a scale score of 3. showed higher antibody production ability than those derived from patients with low A. However, stage 3 Only 10% (9/8g) of wells from patients showed reactivity; On the other hand, in wells from stage 0-2 patients, 3137% of wells were resistant to H+V. showed specific reactivity. Therefore, 6 weeks after culture, patients with low scale scores We were able to obtain a high percentage of Iv-specific antibody-containing wells using cells derived from individuals. .

Analysis of antibodies from expanded cultures of ELISk positive wells was performed by RIP. provided. Only 59% of these culture supernatants showed positive results in RIP analysis. Ta. RIP analysis shows that 44 other than 57 supernatants are caused by the enma gene. Showing reactivity to the encoded protein, 111% g day protein, mo 2 showed reactivity for reverse transcription**.

Thus, specific lymphoblastoid cell lines were isolated and cloned as described below. It was.

Example 3: Specificity and reactivity of some human monoclonal antibodies of the present invention The above 57 cell lines were diluted 2-fold from 10,000 to 10111I cells/well. Cloned. Select the well with the lowest concentration of antibody-producing cells and heat the well. Each cell was cloned as 100 or 10 cells. By using this method , three cell lines producing anti-p41 antibodies and four cell lines producing anti-p24 antibodies. Total of 7 cell lines (100 or 2 at IO cells/well) - cloned three times) was established. of antibodies obtained from these cell lines. Regarding reactivity, it is shown in 1st [and 2nd g].

As shown in Figure 1, all seven cell lines in this example exhibited fG subtype resistance. gave birth to a body. In Figure 1, [12!1] mark 11 [11V Lysade was received. (Lane 1), when reacted with antibody of cell line 50-69 (Lane 2), When reacting with antibody of cell line 916 (lane 3), all cell line 98-4 When reacting with antibody 3 (lane 4), when reacting with antibody 31 for cell line piles (Lane 5), when reacting with antibody of cell line 91-5 (Lane 6), cell line 91-5. When reacting with antibody 91-6 (lane 7), reacting with antibody 9g-4,9 (Lane 8). The molecular weights of major viral proteins are shown in the figure. It is shown in kilodaltons on the left. Three cell lines (5G-69, 98-6 and 9g-43) antibodies are proteins encoded by the -esy- gene. protein bound to gp4t. Four cell lines (71-31, 91-51, 91-6, 98-4,9) antibody is a protein p24 encoded by the gat gene. Combined. Cell lineage determined by RIP? +-31,! +-5,91-6 antibody reacted with p24.

9g-4,9 antibody does not react with RIP because the subtype of this antibody IgG, which does not bind to protein A, does not precipitate.

Antibodies from all three of these anti-G&L cell lines were also Figure 2) was tested.

In Figure 2, normal serum (lane 1), serum from a uIv-infected patient (lane 2) ), cell line supernatant Nia 1-31 (lane 3), 91-51 (lane 4), 91-6 (Lane 5), 9g-4,9 (Lane 6) reaction Gender was expressed by bands in the Western plot. This western plot solution According to the analysis, all three cell lines reacted with 924, and the straddle a "precursor" p55 and p4(l). All four antibodies reacted with p24 degradation products. It moved with a mobility of about 22 kilodaltons (kd). Four anti-υma monoclonal anti- Three of the bodies were added to an intermediate precursor (indicated by a mouse monoclonal antibody). The mobility was approximately 37) 1,28 kd.

The growth characteristics and antibody production of each cell line were investigated. each detail The sublineage was seeded in replicate wells at 0.6 x 10' cells/liter, and 1 The cells were cultured for 8 to 8 days. Figure 3 shows changes in cell number and immunoglobulin production. showed that.

In Figure 3, multiple wells were picked up at each time point to determine the number of viable cells. (marked with *) and the secreted amounts of human T and G (marked with O) were measured. Figure 3 a-g are , respectively, show the results of the same experiment conducted with different cell lines. i.e. , Fig. 3a shows cell line 5G-69, Fig. 3 shows cell line 9B, 6. Figure 3 C is thin. Cell line 911-43, Figure 3d is cell line 71-31. Figure 3C is cell line 91 -5, Figure 3 f shows cell line 91-6, and Figure 3 g shows cell line 9g-4,9. It was used as a sample.

The cell density reaches its peak on the fourth day, and the highest density at that time is +, ox toe The L was 2.4 K and 106 m1i/ml. Cells in logarithmic growth phase Doubling time was 40 to 61 hours. Immunoglobulin production depends on the cell line. Although the pattern differs, it generally reaches its peak on the 5th day of culture, and the production amount at that time is Rinashi II 2ug/sl Tea Tsuta.

Notably, cell line 9g-4,9 has recently been developed with human monoclonal antibodies. lost its production ability.

Example 4: Lymphoblastoid cell line producing human monoclonal antibodies against HIV further prepared.

Peripheral blood heavy fluid cells were collected from 39 other HIV-seropositive individuals and collected. Cells were immortalized by EBV infection. Then, the method shown in Examples 1 to 3 Screening and selection were carried out based on the following. Positive cultures were grown and 2x Depending on the dilution, layer 1 to 3 times with 10 to 100 ml cells/well. repeated. Four stable lymphoblastoid cell lines producing human monoclonal antibodies I gained control.

These strains have 120-16.12G-6,12 for 1p41 protein. 6-50, +34-F6 for p2t protein.

Each monoclonal antibody in this example was commercially prepared in the same manner as in Example 3. ELISA (supernatant has reactivity towards HIT-coated beads, BS^ - No reactivity to coated beads (I/X), radioimmunoprecipitation and Their properties were investigated by stamp plot. The results are shown in Table 3 below. did.

(Hereafter, margin) Example 5: Human monoclonal antibody properties of the present invention Biological activity of the monoclonal antibody of the present invention (using a recombinant antibody with separate layers) Ebitopsi '. 1lENV9 is a clone B4+tan surrounding residues 461 to 71i1. (obtained from DuPont, Wilmin, DE), PE3 is IP+ The 2367 myic acid sequence of 20 Force 1 et al. (from DuPont, Wilmington, DE The obtained), )12+ contains residues 561-649 of p41 (separate Call me! Leverun, PA).

As is clear from Table 3 above, 1. anti-gp41 human monoclonal antibody of the present invention All of 1. G-7 rotib has antibody-dependent cellular cytotoxicity (ADD). Ras.

Additionally, epitope mapping allows binding to different epitopes of viral proteins. Five anti-H41 antibodies are shown. Monoclonal antibody 50-69 is 599-6 Binds to 13 residues. The cloned antibody 9g-43 surrounds residues 642-692. Reacts with peptides. Monoclonal antibody 911-43 has H4 of 579-f04. Reacts with the peptide surrounding the residue. Monoclonal antibody 120-16 is 644-61 Binds to i3 residue. And monoclonal antibody 116-7 is 6fil-6B3 Binds to residues. The numbering system for p41 peptides is based on the literature (see, Vi ro+, -Lee: 570.1987).

All three anti-p24 clone antibodies (71-31, 91-5, 9l-6) +38-198 Binds to the 84 Vp24 peptide surrounding the remains (Na The encoding system is based on the method of Wayne-Poplin et al. @taL Ce1140: 9. +9117) (Table 3).

Finally, the monoclonal antibodies of the invention are not able to neutralize HIV infection. Ta. Additionally, the 120-clonal antibody 120-Is increased the pathogenicity of the virus.

Example 6: Inhibition test regarding monoclonal antibodies of the present invention This example describes the application of the monoclonal antibody of the present invention to an assay for the diagnosis of HIV. This is an example.

Imron 2 plate (Dynatic) was mixed with 0.05M bicarbonate buffer (1119 Coat with HXv lysate (0.5 μg/well) diluted in After treatment at 7℃ for 2 hours, treatment at 4℃ overnight), 0.05% Tweea-containing phosphoric acid Play with 1 liter of buffer salt IWI solution (P[17,4) (PBS-Twee++) After washing the plate, add serotype IIIV positive or negative individuals to each well of the plate. (0.5 μg/well, 11 G to l:lo.

After sample addition, the plate was incubated overnight at room temperature, followed by P. Washed three times with PS-Tween. Pre-diluted biotinylated monoclonal anti- - Add the HIV antibody (see below) to a volume of 100 ml and add another 37 mL of the plate. Incubate for 2 hours at ℃. The wells were then treated with PBS-Tween 3 times. Wash twice and use the avidin-biotinylated horseradish peroxidase complex (vector). PBS-Tvee was added with PBS-Tvee and incubated at 37°C for 2 hours. After washing for 5 hours with Add [ABTS] as one substance and incubate for 30 minutes at room temperature. I turned on. The absorbance of each well was determined by ELISA using a wavelength of 410!1-. I read it with a reader.

Biotinylation of monoclonal antibodies that react with BIV was performed as follows. So Each monoclonal antibody was precipitated with ammonium sulfate and/or protein Partial purification was performed using a base column. After dialysis with 0.1M sodium bicarbonate, 1ml of antibody (5 g/ml) to 75 l of hydroxyl-succinimide biotin (DM 5-g) per SOI. By stirring for 3 hours at room temperature The reaction was run and dialyzed against PBS (pH 7,4). The obtained biotiny Store monoclonal antibodies in 50% glycerol at -25°C until use. did.

The results of the inhibition test are shown in FIG. In Figure 4, HIV seven rotib positive blood The positive serum was represented by (+) and the negative serum was represented by (-).

The data shown in Figure 4 shows that antibodies 120-16 and 71-31 each Reacts with dominant and non-dominant epitopes It shows. Therefore, [17 rotib-positive serum is labeled monoclonal MIV lysate to determine the epitope of antibody +20-16 as immunodominant can compete with that antibody for binding to the code. Antibody 71-31 is HIV with an epitope that does not compete with serotype-positive serum and was established.

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Claims (21)

[Claims] 1. A human monoclonal antibody that combines human immunodeficiency virus protein p24. A human monoclonal antibody characterized by its ability to react. 2. The human monoclonal antibody according to claim 1, wherein said monoclonal antibody A human monoclonal antibody characterized in that the antibody is an IgG serotype. 3. A lymphoblastoid cell line that produces human monoclonal antibodies, the human monoclonal antibody comprising: The cloned antibody is one that reacts with the human immunodeficiency virus protein p24. A lymphoblastoid cell line that produces human monoclonal antibodies characterized by: 4. The human lymphoblastoid cell line according to claim 3, wherein the cell line is Human lymph buds characterized by being derived from humans infected with human immunodeficiency virus Globular cell lineage. 5. A method for treating a mammal infected with human immunodeficiency virus, the method comprising: The method comprises a pharmaceutically acceptable carrier and human immunodeficiency virus protein p24. comprising a human monoclonal antibody against an effective amount necessary for said treatment. A method for treating mammals infected with human immunodeficiency virus. 6. 6. The method of claim 5, wherein the effective amount is from 200 mg to 15 m A method for treating a mammal infected with human immunodeficiency virus, characterized by being between g. . 7. 6. The method according to claim 5, wherein the human monoclonal antibody is administered maternally. A method for treating a mammal infected with human immunodeficiency virus, the method comprising administering via . 8. Pharmaceutical formulations for treating mammals infected with human immunodeficiency virus The formulation comprises a human monochrome against the human immunodeficiency virus protein p24. A human immunodeficiency virus-infected mammalian animal comprising an effective amount of a human immunodeficiency virus antibody. Pharmaceutical prescriptions for treating things. 9. A pharmaceutical formulation according to claim 8, wherein said formulation is pharmaceutically acceptable. treatment of a mammal infected with human immunodeficiency virus, characterized in that the carrier comprises a carrier that is Pharmaceutical prescription for. 10. A human monoclonal antibody, the antibody comprising a human immunodeficiency virus protein. human body characterized by reacting with protein gp41 and giving antibody-dependent cell-mediated cytotoxicity Monoclonal antibodies. 11. The human monoclonal antibody according to claim 10, wherein the monoclonal antibody A human monoclonal antibody characterized in that the main antibody is an IgG serotype. 12. A lymphoblastoid cell line that produces human monoclonal antibodies, said human monoclonal antibody Noclonal antibodies react with the human immunodeficiency virus protein gp41. A lymphoblastoid cell line that produces human monoclonal antibodies characterized by: 13. The human lymphoblastoid cell line according to claim 12, which cell line is a human lymphocyte characterized by being derived from a human infected with human immunodeficiency virus. Blast cell lineage. 14. A method for treating a mammal infected with human immunodeficiency virus, the method comprising: , the method comprises a pharmaceutically acceptable carrier and the human immunodeficiency virus protein gp. containing a human monoclonal antibody against 41 in an effective amount necessary for said treatment; The human monoclonal antibody described above is a human monoclonal antibody characterized by conferring antibody-dependent cell-mediated cytotoxicity. A method for treating mammals infected with immunodeficiency virus. 15. 15. The method according to claim 14, wherein the monoclonal antibody is an IgG Treatment of mammals infected with human immunodeficiency virus characterized by serotype Method. 16. 15. The method of claim 14, wherein the effective amount is from 200 mg to 1 Treatment of mammals infected with human immunodeficiency virus, characterized in that the dose is between 5 mg. Method. 17. 15. The method according to claim 14, wherein the human monoclonal antibody is Treatment of mammals infected with human immunodeficiency virus, characterized by administration via the body Method. 18. Pharmaceutical treatments for treating mammals infected with human immunodeficiency virus and the formulation is a human immunodeficiency virus protein gp41. The claimed human monoclonal antibody contains an effective amount of human monoclonal antibody, and the requested human monoclonal antibody is Human immunodeficiency virus-infected mammals are characterized by causing cytotoxicity. Pharmaceutical prescriptions for treatment. 19. 19. The pharmaceutical formulation according to claim 18, wherein the formulation is A human immunodeficiency virus-infected mammal comprising an acceptable carrier. Pharmaceutical prescriptions for treatment. 20. A human monoclonal antibody, which has ATCC acquisition number 10038. A human monoclonal antibody characterized by being produced by No. 21. A human monoclonal antibody, which has ATCC acquisition number 10037. A human monoclonal antibody characterized by being produced by No.